BRAF V600E and Molecular Testing in Anaplastic Thyroid Cancer: A Patient Guide to Precision Treatment

Abstract

Background

BRAF V600E anaplastic thyroid cancer is a molecularly defined form of anaplastic thyroid cancer in which the tumor carries the BRAF V600E mutation. Identifying this mutation can directly influence treatment because eligible patients may be considered for BRAF- and MEK-targeted therapy. Molecular testing can also reveal other genetic alterations that may affect treatment selection, clinical-trial eligibility and the overall precision-oncology strategy.

Molecular testing must not delay urgent care. While the laboratory is analysing the tumor, the medical team should continue evaluating the airway, swallowing, tumor extent, distant spread and possibility of surgery or radiation. The American Thyroid Association recommends prompt BRAF V600E assessment and comprehensive molecular profiling because clinically relevant targeted treatments may be available.[1] 

Objective

In this clinical review, we explain what the BRAF V600E mutation means, how it is detected and how the result may influence treatment decisions. We also examine immunohistochemistry, PCR, DNA-based next-generation sequencing, RNA fusion testing and liquid biopsy.

If you have already received a molecular pathology report, this article will help you understand terms such as pathogenic variant, variant allele frequency, actionable alteration, gene fusion and variant of uncertain significance. It will also explain why a BRAF-negative result does not necessarily mean that the tumor has no other treatment-relevant molecular finding.

Evidence Reviewed

This review is based on the American Thyroid Association ATC guideline, current FDA prescribing information, molecular profiling studies and major clinical investigations of BRAF-directed treatment.[1,15–20] The phase II ROAR study evaluated dabrafenib plus trametinib in 36 patients with unresectable or metastatic BRAF V600E-mutant ATC. The investigator-assessed objective response rate was 56%, including three complete responses, while median progression-free survival and overall survival were 6.7 and 14.5 months, respectively.[17]

These findings demonstrate meaningful clinical activity, but they must be interpreted carefully because the study was small, open-label and non-randomized.[17] 

Clinical Conclusion

Rapid BRAF V600E testing and comprehensive DNA- and RNA-based molecular profiling should generally be initiated early and, where feasible, performed in parallel. A confirmed BRAF V600E-positive result may support treatment with dabrafenib plus trametinib in eligible patients with locally advanced or metastatic ATC when satisfactory locoregional treatment options are unavailable.[15] 

A mutation result does not determine the entire treatment plan. The final decision should combine molecular findings with pathological confirmation, stage, airway safety, tumor anatomy, resectability, distant metastases, general health, treatment tolerance and the patient’s informed preferences.

What Molecular Testing Adds to an ATC Diagnosis

Anaplastic thyroid cancer is first confirmed through biopsy and examination by a pathologist. Molecular testing then studies the tumor more deeply to identify genetic changes that may be helping the cancer grow. When we examine these changes, we are not replacing the diagnosis. We are looking for information that may guide targeted treatment, clinical-trial selection or the order in which different treatments are considered.[1]

Two patients may both have ATC, yet their tumors may not contain the same molecular alterations. One tumor may carry BRAF V600E, while another may have a RAS alteration, a rare gene fusion or no currently actionable molecular target. This is why treatment cannot be based on the cancer name alone.[3,6]

What a BRAF V600E-Positive Result May Mean

If your report states that BRAF V600E was detected, it means that the tumor contains a specific alteration in the BRAF gene. This alteration can keep an important cell-growth pathway continuously active. In an eligible patient, the result may support treatment with the BRAF inhibitor dabrafenib together with the MEK inhibitor trametinib.[15,17]

This result does not guarantee that the tumor will shrink, that surgery will become possible or that the disease will be cured. It identifies a treatment opportunity, but the oncologist must still consider the stage, tumor location, airway risk, distant spread, general health and previous treatments. Selected patients have experienced meaningful responses, and some initially unresectable tumors have later been reconsidered for surgery, but this does not occur in every case.[17–19]

What a BRAF-Negative Result Means

A BRAF-negative result does not mean that molecular testing has found nothing important. Broader DNA- and RNA-based testing may identify NTRK, RET, ALK or other alterations that could influence treatment or clinical-trial eligibility.[6,23–25]

The meaning of a negative result also depends on the sample. A test may be less reliable when the biopsy contains extensive dead tissue, very few tumor cells or degraded genetic material. A negative liquid biopsy may also occur when the tumor does not release enough DNA into the blood. In these situations, the medical team may review another tissue block, repeat the biopsy or combine tissue and blood-based testing.[1,12,13]

What Should Happen While Results Are Pending

Molecular testing should begin early, but urgent medical care must continue at the same time. The team should assess breathing, swallowing, vocal-cord function, tumor spread, nutritional status and whether surgery or radiation is feasible. If you develop increasing breathlessness, noisy breathing, inability to swallow liquids, bleeding or rapidly worsening neck swelling, these symptoms require immediate medical attention rather than waiting for a molecular report.[1]

The molecular result is one part of the treatment decision. The safest plan combines what the tumor contains with what the patient urgently needs and what treatment the body is reasonably able to tolerate.

The Five Things to Know Immediately After an ATC Diagnosis

BRAF V600E Testing Should Begin Early

BRAF V600E testing should be requested as soon as anaplastic thyroid cancer is strongly suspected or confirmed. ATC can progress quickly, and a positive result may identify a targeted-treatment option that could influence the early treatment plan. Rapid BRAF testing is therefore clinically important rather than an investigation to be considered only after other treatments have failed.[1]

You should ask whether the tumor has been tested specifically for BRAF V600E and which method was used. Immunohistochemistry may provide a rapid preliminary result, while PCR or next-generation sequencing can confirm the alteration at the molecular level.[1,10,11]

Rapid BRAF Testing Does Not Replace Comprehensive Molecular Profiling

A focused BRAF test answers one urgent question: whether BRAF V600E is present. Comprehensive DNA- and RNA-based profiling answers a broader question by examining the tumor for other mutations, fusions and biomarkers that may affect treatment or clinical-trial eligibility.[1,6]

We should not assume that a negative BRAF result completes the molecular investigation. When enough tissue is available, rapid BRAF assessment and broader molecular profiling should proceed in parallel so that valuable time is not lost by ordering one test only after another test has returned negative.[1]

A Positive Result Creates an Opportunity, Not a Guarantee

If BRAF V600E is detected, the result may support treatment with dabrafenib and trametinib in an eligible patient. Clinical studies have shown meaningful tumor responses in some patients with BRAF V600E-positive ATC, and selected patients have later undergone surgical reassessment after targeted treatment.[17–19]

However, a mutation cannot predict the complete outcome. A positive result does not guarantee tumor shrinkage, long-term control, operability or cure. The oncologist must still consider the cancer stage, airway risk, tumor anatomy, distant metastases, previous treatment, general health and the patient’s ability to tolerate therapy.[1,15]

A Negative or Inconclusive Result May Need Further Review

A report stating that BRAF V600E was not detected may represent a true negative result, but the reliability of that conclusion depends on the sample and the method used. Extensive tumor necrosis, low tumor-cell content, degraded DNA or RNA, and an exhausted tissue block can reduce the quality of molecular testing.[1,9]

A negative liquid biopsy also does not always exclude a mutation within the tumor because some cancers release only small amounts of detectable DNA into the bloodstream. In such cases, the medical team may review another tissue block, repeat tissue sampling or combine tissue testing with plasma-based analysis.[12,13]

Urgent Clinical Care Must Continue While Testing Is Underway

Molecular testing is important, but it must not delay treatment of immediate problems. The patient may require urgent assessment of breathing, swallowing, vocal-cord function, bleeding, nutrition, tumor extent and resectability while the molecular report is still pending.[1]

When I explain this to a patient, I describe molecular testing as one part of a larger treatment pathway. The laboratory identifies what may be driving the tumor, while the clinical team determines what the patient needs immediately and which treatment can be delivered safely. The strongest plan combines pathology, molecular findings, imaging, stage, airway safety, surgical opinion, treatment tolerance and the patient’s informed preferences.

Why Molecular Testing Has Changed the Treatment of Anaplastic Thyroid Cancer

ATC Was Traditionally Treated Mainly According to Stage and Resectability

For many years, treatment decisions in anaplastic thyroid cancer were based mainly on where the tumor had spread, whether it could be removed surgically and whether the patient was strong enough to receive radiation or systemic treatment. These factors remain essential because ATC can rapidly affect the trachea, oesophagus, major blood vessels and nearby nerves.[1]

However, stage and anatomy do not explain why two patients with apparently similar tumors may respond differently to the same treatment. One patient may have a tumor driven by BRAF V600E, while another may have a RAS alteration, a gene fusion or several changes associated with aggressive tumor behaviour. Molecular testing has added this deeper biological information to the traditional clinical assessment.[3–6]

Precision Oncology Asks What Is Driving the Tumor

Precision oncology aims to identify molecular changes that are helping a particular cancer grow. The purpose is not simply to produce a long genetic report. The important question is whether a finding can guide treatment, support clinical-trial eligibility or explain why a tumor has become resistant to therapy.

In ATC, BRAF V600E is especially important because it may identify a patient who could be considered for combined BRAF and MEK inhibition. Other less common alterations, including NTRK, RET or ALK fusions, may also have treatment relevance in selected patients.[1,6]

When we study the tumor at this level, we move from asking only, “Where is the cancer?” to also asking, “What is helping this cancer grow?” Both questions are necessary, and neither should replace the other.

Tumors That Look Similar May Be Molecularly Different

Under the microscope, two tumors may both be classified as anaplastic thyroid carcinoma, but their molecular profiles may differ significantly. Some tumors carry BRAF V600E, while others are associated with RAS alterations. Additional changes involving TP53, the TERT promoter or the PI3K–AKT–mTOR pathway may contribute to dedifferentiation, rapid growth and treatment resistance.[3–5]

This molecular diversity helps explain why ATC should not be treated as one completely uniform disease. It also explains why a treatment that is appropriate for one patient may not be suitable for another. The diagnosis gives the disease a name, while molecular testing may reveal a more individual treatment pathway.

Molecular Results May Influence Early Treatment Decisions

In slower-growing cancers, molecular testing may sometimes be performed after several other investigations. In ATC, the process should begin early because the disease can progress over a short period. A rapid BRAF V600E result may influence whether targeted therapy is considered, while broader DNA- and RNA-based profiling may identify additional actionable findings or clinical-trial options.[1]

The medical team should not wait passively for the molecular report. Airway assessment, imaging, staging, surgical review, radiation planning, nutritional support and symptom management should continue at the same time. Molecular testing is most useful when it is integrated into urgent multidisciplinary care rather than treated as a separate laboratory exercise.

Molecular Testing Does Not Replace Pathology, Imaging or Clinical Judgment

A molecular alteration cannot confirm the full diagnosis by itself. BRAF V600E may also occur in papillary thyroid carcinoma and other cancers, so the result must be interpreted together with tumor morphology, immunohistochemistry and the patient’s clinical presentation.[1]

Similarly, a molecular report cannot independently determine whether surgery is possible, whether the airway is safe or how much treatment a patient can tolerate. If you receive a report showing an actionable mutation, the finding may open an important treatment opportunity, but it does not replace evaluation by the oncologist, surgeon, radiation oncologist, pathologist and radiologist.

Modern ATC care is therefore based on several layers of information. Pathology confirms what the tumor is, imaging shows where it is located, molecular testing identifies possible biological drivers, and the multidisciplinary team determines how those findings should be converted into a safe treatment plan.[1,30]

What Exactly Is Molecular Testing?

Molecular Testing Looks for Changes Inside the Tumor

Molecular testing examines the genetic and biological changes within cancer cells. In anaplastic thyroid cancer, it may identify mutations, gene fusions, copy-number changes or other biomarkers that help explain how the tumor is growing. Some of these findings may influence treatment, while others are mainly useful for understanding the biology of the disease.[1,6]

A pathologist first determines whether the tissue is consistent with ATC. Molecular testing then adds another layer of information. It does not replace the biopsy diagnosis, imaging or clinical examination, but it may reveal a target that cannot be identified by looking at the tumor under a microscope alone.

If you receive a molecular report, it may contain many gene names and technical terms. Not every finding is equally important. The medical team must determine whether an alteration is truly disease-driving, whether it has an approved treatment, whether the evidence comes from ATC or another cancer, and whether the treatment is suitable for that particular patient.

Molecular Testing, Genomic Testing and Biomarker Testing

The terms molecular testing, genomic testing, mutation testing and tumor profiling are often used interchangeably, but they are not always identical.

Molecular testing is the broadest term. It can include tests for DNA mutations, RNA fusions, protein expression and other biological markers. Genomic testing usually refers more specifically to the examination of multiple genes or large regions of tumor DNA. Mutation testing may focus on one defined alteration, such as BRAF V600E. Tumor profiling generally refers to broader testing designed to identify several potentially important alterations at the same time.[1,6,14]

Biomarker testing is also a broad term. A biomarker may be a DNA mutation, a gene fusion, a protein such as PD-L1 or a feature such as microsatellite instability. This means that not every biomarker is a genetic mutation.

Tumor Genetics Is Different From Inherited Genetics

Most molecular testing in ATC examines changes that developed within the tumor. These are called somatic alterations. They are present in the cancer cells but are usually not present in the rest of the body.

A germline alteration is different. It is present from birth and can potentially be inherited by children or shared with biological relatives. Finding BRAF V600E in an ATC specimen generally indicates a somatic tumor alteration rather than an inherited family condition.[1]

This distinction matters because patients may become anxious when they see the word “genetic” on a cancer report. A tumor mutation does not automatically mean that the cancer was inherited. Germline testing is considered separately when the personal history, family history or molecular finding raises a genuine hereditary concern.

A Mutation Is a Change in the DNA Sequence

A mutation is an alteration in the genetic code. Some mutations change the structure or activity of a protein, while others may have little or no effect on tumor behaviour.

BRAF V600E is a specific mutation that changes one amino acid in the BRAF protein. This can cause persistent activation of the MAPK growth pathway. Other mutations may affect tumor-suppressor genes such as TP53 or genes involved in the PI3K–AKT–mTOR pathway.[3,6]

A report may classify a mutation as pathogenic, likely pathogenic or of uncertain significance. A pathogenic alteration has sufficient evidence linking it to disease biology. A variant of uncertain significance does not yet have enough evidence to be treated as an established cancer driver or treatment target.

A Gene Fusion Joins Parts of Two Different Genes

A gene fusion occurs when parts of two genes become abnormally connected. The resulting fusion may produce a protein that continuously stimulates cancer-cell growth.

Fusions involving NTRK, RET or ALK are uncommon in ATC, but they can be clinically important because targeted treatments may be available for selected patients. These alterations are one reason why broader molecular profiling should not stop after a negative BRAF result.[6]

RNA-based testing can be especially helpful for detecting gene fusions because it examines the abnormal transcript produced by the fused genes. Some DNA-only panels can detect certain fusions, but their ability depends on how the panel was designed and which genomic regions it covers.

Amplification Means That a Gene Has Too Many Copies

Normally, cells carry a controlled number of copies of each gene. In a gene amplification, the tumor contains many additional copies of a particular gene. This may cause excessive production of the protein encoded by that gene and may contribute to cancer growth.

Amplification is different from a mutation. A mutation changes the genetic sequence, while amplification increases the number of copies. Molecular reports may also describe deletions, which mean that part or all of a gene has been lost.

These findings are not automatically actionable. Their significance depends on the gene involved, the strength of the evidence and whether a suitable treatment has been clinically validated.

Gene Expression Shows Which Genes Are Active

Gene-expression testing examines how actively certain genes are being used by the tumor. DNA can be compared to the stored instructions, while RNA reflects which instructions are being copied and used at that time.

This is why DNA and RNA testing provide different but complementary information. DNA sequencing may identify mutations and some structural changes, while RNA analysis can help confirm that a gene fusion is actively expressed. Integrated DNA and RNA profiling can therefore provide a more complete molecular picture in selected patients.[4,6]

Protein Testing Is Also Part of Molecular Pathology

Not every clinically useful test directly examines DNA or RNA. Immunohistochemistry uses antibodies to detect specific proteins in tumor tissue. For example, BRAF V600E immunohistochemistry can identify expression of the altered BRAF protein, while PD-L1 immunohistochemistry evaluates a protein involved in immune regulation.[1,10]

Protein testing may be faster and more widely available than comprehensive sequencing. However, it answers a narrower question and may require molecular confirmation when the result is unclear, unexpected or inconsistent with the rest of the pathology.

The Purpose Is to Find Clinically Meaningful Information

A long genomic report is not necessarily better than a shorter report unless the findings can be interpreted correctly. The most useful molecular test is one that examines adequate tumor tissue, includes the alteration types relevant to ATC and returns results within a clinically useful period.

When we review a report, we should ask three separate questions. First, is the alteration real and technically reliable? Second, is it helping drive the cancer? Third, is there a treatment supported by evidence for this alteration in ATC or in a broader tumor-agnostic setting?

Molecular testing is therefore not only about identifying genetic changes. Its real value lies in converting reliable tumor information into a practical and individualized treatment decision.[1,6,14]

What Is BRAF?

The BRAF Gene and the BRAF Protein

BRAF is a gene that provides instructions for producing the BRAF protein. This protein helps transmit growth signals from the surface of a cell to its nucleus. Under normal conditions, the pathway becomes active only when the cell receives an appropriate signal to grow, divide or respond to its environment.[2,27]

The BRAF protein belongs to a group of enzymes called kinases. A kinase helps control cell activity by adding a phosphate group to another protein. This process may sound highly technical, but the basic idea is simple: BRAF helps pass a message from one part of the cell to another.

In a healthy cell, these messages are tightly regulated. The pathway turns on when it is needed and becomes less active when the signal is complete. When BRAF is altered by certain mutations, the growth message may remain active even when the cell has not received a normal external signal.

Where BRAF Sits in the MAPK Pathway

BRAF is part of a signalling system commonly called the MAPK pathway. The main sequence can be simplified as:

RAS → RAF → MEK → ERK

This pathway helps regulate cell growth, division, survival and differentiation. BRAF is one of the RAF proteins positioned between RAS and MEK. When RAS becomes active, it can activate BRAF. BRAF then activates MEK, and MEK activates ERK. ERK carries the signal deeper into the cell and can influence which genes are switched on.[2,3]

This pathway is necessary for normal cell function. The problem is not that the pathway exists, but that it may become abnormally and continuously active in a cancer cell.

When I explain this to a patient, I describe the MAPK pathway as a chain of messengers. Each messenger normally passes the instruction to the next only when the body gives a proper signal. A BRAF V600E mutation can allow one messenger to keep sending the growth instruction without waiting for permission.

How BRAF V600E Changes Cell Signalling

The BRAF V600E mutation changes the structure of the BRAF protein. This altered protein can remain active and repeatedly stimulate MEK and ERK. The cancer cell may then continue receiving signals that support growth and survival.[2,27]

This does not mean that BRAF V600E is the only abnormality in the tumor. ATC commonly contains several molecular alterations, and changes involving TP53, the TERT promoter or the PI3K–AKT–mTOR pathway may contribute to dedifferentiation, aggressive growth and treatment resistance.[3–6]

BRAF V600E may be an important driver, but the behaviour of the tumor still depends on the complete molecular environment. This is why one mutation can guide treatment without fully explaining the cancer.

Why BRAF Matters in Anaplastic Thyroid Cancer

BRAF is clinically important in ATC because BRAF V600E can be targeted with specific medicines. Dabrafenib inhibits altered BRAF signalling, while trametinib inhibits MEK further down the same pathway. By blocking two connected points, the combination aims to reduce the persistent growth signal created by the mutation.[15,17]

This treatment differs from conventional cytotoxic chemotherapy. Chemotherapy generally affects rapidly dividing cells through broader mechanisms, whereas targeted therapy is selected because the tumor contains a particular molecular feature.

A positive BRAF V600E result may therefore change the treatment discussion. It may support the use of BRAF- and MEK-directed therapy in an eligible patient and may also influence whether the tumor is reassessed for surgery after an initial response.[17–19]

However, BRAF positivity does not automatically determine the entire plan. The treating team must still assess the airway, tumor anatomy, stage, metastases, resectability, previous treatment and the patient’s overall condition.

BRAF V600E Is Not Found Only in ATC

BRAF V600E can also occur in papillary thyroid carcinoma and in several non-thyroid cancers. Its presence therefore cannot confirm ATC by itself. The diagnosis still depends on the microscopic appearance of the tumor, immunohistochemical findings and the overall clinical context.[1,27]

Some BRAF-positive ATCs may arise alongside or evolve from a differentiated papillary thyroid carcinoma. During this transformation, the tumor may acquire additional molecular changes that contribute to loss of normal thyroid-cell features and more aggressive behaviour.[3–5]

For this reason, the pathologist may examine whether differentiated and anaplastic components are present within the same specimen. The molecular result must always be interpreted together with the tissue findings.

A Positive BRAF Result Identifies a Target, Not a Predetermined Outcome

If your tumor is BRAF V600E-positive, the result may provide an important treatment opportunity. It does not tell you with certainty how much the tumor will shrink, how long the response will last or whether surgery will become possible.

Clinical trials have shown that some patients experience substantial responses to dabrafenib plus trametinib, while others have a limited response or later develop resistance.[17] Additional molecular alterations, tumor heterogeneity and differences in the patient’s clinical condition may all influence the result.

BRAF testing is therefore valuable because it identifies a specific biological weakness that may be targeted. Its meaning becomes clinically useful only when the result is combined with pathology, imaging and multidisciplinary medical judgment.

What Does “V600E” Actually Mean?

The Letters and Number Describe One Specific Protein Change

The term BRAF V600E may look like a laboratory code, but each part has a clear meaning. The letter V represents valine, an amino acid normally present at a particular position in the BRAF protein. The number 600 identifies that position, and the letter E represents glutamic acid, which replaces valine after the mutation occurs.[2,27]

The same finding may appear on a molecular report as BRAF p.V600E, BRAF p.Val600Glu or BRAF c.1799T>A. These expressions describe the same underlying alteration at different biological levels. The protein notation describes the amino-acid change, while the DNA notation describes the change in the genetic sequence that produces it.

For most patients, the most important point is simple: BRAF V600E is a specific mutation that can keep growth signalling active inside the cancer cell.

Why One Amino-Acid Change Can Matter So Much

Proteins depend on their three-dimensional shape to work correctly. Replacing one amino acid with another can change the structure and activity of the protein, especially when the change occurs in an important regulatory region.

In BRAF V600E, valine is replaced by glutamic acid at position 600. This change can make the BRAF protein behave as though it has received a continuous activation signal. It can then repeatedly stimulate MEK and ERK further down the MAPK pathway, even when the cell has not received a normal instruction to grow.[2,27]

This persistent signalling can contribute to uncontrolled cell division, survival and tumor progression. However, the mutation does not work in isolation. ATC often contains additional molecular abnormalities that may influence how aggressively the tumor behaves and how it responds to treatment.[3–6]

V600E Is Not a Cancer Stage or Severity Score

The number 600 does not indicate the cancer stage, tumor size, percentage of cancer cells or severity of the disease. It only identifies the location of the amino-acid change within the BRAF protein.

Similarly, a BRAF V600E-positive result does not mean that the patient has “600 mutations” or that the cancer is 600 times more aggressive. Molecular notation is descriptive rather than a measure of disease burden.

If you see a percentage next to the mutation on a sequencing report, that percentage may represent the variant allele frequency. It describes how often the altered sequence was detected within the DNA analysed from that sample. It is not the percentage of the body affected by cancer and does not directly predict the chance of response to treatment.

Not Every BRAF Alteration Is BRAF V600E

A molecular report may contain the word BRAF without showing the V600E mutation. Other BRAF alterations include different substitutions, insertions, deletions, amplifications and gene fusions. Their biological and therapeutic significance can be very different.

This distinction is important because the strongest ATC-specific clinical evidence for combined BRAF and MEK inhibition relates to tumors with a confirmed BRAF V600E alteration.[15–17] A different BRAF finding should not automatically be treated as though it were V600E.

When the report identifies an uncommon BRAF alteration, the oncology team should review whether it is classified as pathogenic, whether it activates the same pathway and whether any treatment evidence applies to that exact alteration. The presence of the BRAF gene name alone is not enough to determine treatment.

Why Laboratories Use Precise Molecular Notation

Precise notation helps the oncologist, pathologist and molecular laboratory confirm that they are discussing the same genetic change. It also allows the result to be matched accurately with clinical-trial eligibility, regulatory indications and published treatment evidence.

For example, an FDA-authorized treatment or companion diagnostic may specify BRAF V600E rather than all possible BRAF alterations. This is why the exact wording on the report matters. The difference between “BRAF mutation detected,” “BRAF V600E detected” and “BRAF variant of uncertain significance” can significantly change the clinical interpretation.[14,15]

A complete report should ideally identify the gene, exact variant, classification, testing method and any limitations related to the sample.

What the Result Can and Cannot Tell You

A confirmed BRAF V600E result can identify a molecular treatment target. In eligible patients with ATC, it may support consideration of dabrafenib plus trametinib and may influence the broader multidisciplinary treatment pathway.[15,17]

The result cannot independently determine whether surgery is possible, whether the airway is safe or how long treatment will remain effective. It also cannot predict with certainty whether the tumor will shrink or whether resistance will later develop.

When we interpret BRAF V600E correctly, we treat it as a specific and clinically important biological finding. It is neither the complete explanation for the cancer nor a guaranteed answer to it. Its value comes from combining precise molecular information with pathology, imaging, staging and the patient’s overall clinical condition.

Is BRAF V600E a Driver Mutation?

What Is a Driver Mutation?

A driver mutation is a genetic alteration that actively contributes to the development, growth or survival of a cancer. It gives the tumor cells a biological advantage, allowing them to divide more easily, avoid normal growth controls or survive under conditions that would damage healthy cells.

BRAF V600E is generally regarded as an oncogenic driver because it can continuously activate the MAPK signalling pathway. Instead of waiting for a normal growth signal, the altered BRAF protein can repeatedly stimulate MEK and ERK, sending ongoing instructions that support cancer-cell growth.[3,27]

When we describe BRAF V600E as a driver, we do not mean that it is always the only cause of the tumor. ATC usually contains several molecular alterations. BRAF V600E may start or maintain one important growth pathway, while other abnormalities help the cancer become more aggressive, lose normal thyroid-cell features or resist treatment.[3–6]

What Is a Passenger Mutation?

A passenger mutation is an alteration found within the tumor that does not appear to play a major role in driving its growth. Cancer cells may accumulate many genetic changes as they divide, but only some of these changes provide a meaningful survival advantage.

A sequencing report may therefore contain several mutations, yet only one or two may have clear biological or treatment relevance. The remaining findings may be passengers, harmless inherited variants or variants whose significance is not yet known.

This is why a long molecular report does not automatically mean that the patient has many useful treatment targets. Each finding must be interpreted according to its biological function, its classification and the quality of clinical evidence supporting a treatment.

BRAF V600E May Be an Early Event in Thyroid Cancer Evolution

In some thyroid cancers, BRAF V600E may develop during an earlier differentiated phase, such as papillary thyroid carcinoma. If the tumor later becomes anaplastic, additional alterations may have accumulated during its evolution.[3–5]

Changes involving TP53, the TERT promoter, CDKN2A or the PI3K–AKT–mTOR pathway may contribute to loss of differentiation, rapid progression and treatment resistance. This helps explain why blocking BRAF can be highly important but may not fully control every part of the disease.

A patient may therefore have a tumor that remains dependent on BRAF signalling while also using other biological pathways. This molecular complexity can influence the depth and duration of the treatment response.

A Driver Mutation Is Not Automatically a Treatable Mutation

Not every driver alteration has an approved medicine. Some mutations clearly contribute to tumor growth but cannot yet be targeted effectively with available treatment.

BRAF V600E is especially important because medicines have been developed to inhibit the altered BRAF pathway. Dabrafenib targets BRAF, while trametinib targets MEK further down the same signalling pathway. This creates a clinically meaningful treatment opportunity for selected patients with BRAF V600E-positive ATC.[15,17]

By contrast, alterations such as TP53 may have major biological importance but do not currently provide the same established mutation-specific treatment pathway in ATC. A molecular report must therefore distinguish between a driver that explains tumor biology and a driver that can guide an available treatment.

Why Some BRAF-Positive Tumors Respond Differently

Even when two patients have BRAF V600E-positive ATC, their tumors may not respond in exactly the same way. One tumor may remain strongly dependent on the BRAF–MEK pathway, while another may also rely on parallel growth pathways or contain resistant tumor-cell populations.

The amount and distribution of the mutation may also differ between tumor regions. Additional mutations, tumor heterogeneity, previous treatment and the patient’s general condition can all influence the clinical result.[4–6]

This is why BRAF V600E should be viewed as an important treatment target rather than a guaranteed prediction of response. The mutation may help the oncology team select a therapy, but it cannot independently predict how much the tumor will shrink or how long control will continue.

What This Means for the Patient

If your report identifies BRAF V600E as a pathogenic driver mutation, it means that the alteration is believed to contribute meaningfully to the cancer’s growth. It may also make a targeted-treatment pathway available, depending on the stage, resectability and complete clinical situation.

The result should still be reviewed with the full molecular profile. The oncologist needs to know whether other important alterations are present, whether the sample was reliable and whether the proposed treatment fits with surgery, radiation, airway management and the patient’s ability to tolerate therapy.

BRAF V600E may be one of the most important findings on an ATC molecular report, but it remains one part of a larger biological and clinical picture.[1,3,6]

Is BRAF V600E Inherited?

Most BRAF V600E Mutations in ATC Are Acquired Within the Tumor

In anaplastic thyroid cancer, BRAF V600E is usually a somatic mutation. This means that the alteration developed within the tumor cells during the patient’s lifetime. It was generally not present at birth and is not usually found in every cell of the body.[1,27]

When a molecular laboratory tests an ATC biopsy, it is mainly examining the genetic material inside the cancer. A BRAF V600E-positive result therefore usually describes the tumor rather than the patient’s inherited genetic makeup.

This distinction is important because the word “genetic” can create unnecessary fear. A genetic change in the tumor does not automatically mean that the disease was passed down through the family.

Somatic and Germline Mutations Are Different

A somatic mutation develops in a particular group of cells after birth. It may contribute to the formation or progression of a tumor, but it is usually not present in the patient’s egg or sperm cells. For that reason, it is generally not passed to children.

A germline mutation is present from conception and is found in almost every cell of the body. Germline alterations can sometimes be inherited from a parent and passed to the next generation.

BRAF V600E detected in ATC tissue is ordinarily interpreted as a somatic tumor alteration unless there is unusual evidence suggesting otherwise. In routine practice, the finding does not usually indicate a hereditary BRAF-related cancer syndrome.[1,27]

Does a Positive BRAF Result Mean My Children Are at Risk?

A BRAF V600E-positive tumor result does not usually mean that the patient’s children, siblings or parents carry the same mutation. Family members generally do not need BRAF testing solely because BRAF V600E was identified in an ATC specimen.

If you are reading your report and are worried about your children, the most important question to ask is whether the test was performed on tumor tissue or as a dedicated germline test. In most ATC cases, it is a tumor test.

The oncology team may recommend genetic counselling for other reasons, but a somatic BRAF V600E result alone does not usually establish an inherited family risk.

Why Family History Still Matters

Even though BRAF V600E is usually acquired, the medical team should still ask about the patient’s family history. A history of several relatives with thyroid cancer, unusually early cancers, multiple primary cancers or a known hereditary cancer syndrome may justify additional evaluation.

In such situations, the concern may not be BRAF V600E itself. The clinician may be looking for another inherited alteration that increases cancer risk.

A patient can therefore have both a somatic tumor mutation and a separate inherited risk factor, but these are different questions and require different tests.

When Germline Genetic Testing May Be Considered

Germline testing may be considered when the patient’s personal or family history suggests a hereditary cancer syndrome. It may also be appropriate when tumor sequencing identifies an alteration that is known to occur in inherited conditions or when the variant appears at a level that raises concern for a germline origin.

This assessment should ideally involve a clinical geneticist or genetic counsellor. They can review the family history, explain the possible benefits and limitations of testing, and help the patient understand what a positive or negative germline result may mean.

Tumor sequencing is not always designed to confirm inherited disease. If a report raises a germline concern, a separate blood or saliva test may be required in a certified laboratory.

A Tumor Mutation Is Not Caused by Something the Patient Did

Patients sometimes blame themselves after learning that their cancer contains a mutation. They may wonder whether food, stress, medicines or one particular event caused the genetic change.

In most cases, it is not possible to identify one exact cause of a somatic mutation. Cancer develops through a complex process involving aging, random DNA errors, environmental influences, tissue biology and the accumulation of multiple molecular changes.

A BRAF V600E result should therefore be understood as information about the cancer, not as evidence of personal fault.

Why the Distinction Matters for Treatment

Whether BRAF V600E is inherited does not determine whether targeted therapy may be useful. The treatment decision is based mainly on whether the mutation is present in the tumor and whether the patient meets the clinical criteria for BRAF- and MEK-directed therapy.[1,15]

The result may guide treatment, but it does not usually change the genetic risk for the patient’s relatives. These two questions should remain separate: one concerns how the tumor may be treated, while the other concerns whether the family may carry an inherited cancer predisposition.

For most patients with BRAF V600E-positive ATC, the practical message is reassuring. The mutation is usually an acquired feature of the tumor, not a mutation that has been passed through the family.[1,27]

How Common Is BRAF V600E in Anaplastic Thyroid Cancer?

Published Estimates Vary Between Studies

BRAF V600E is one of the most frequently identified actionable alterations in anaplastic thyroid cancer, but it is not present in every tumor. The American Thyroid Association guideline describes BRAF V600E in approximately 40%–70% of reported ATC series. However, large genomic studies have sometimes reported lower or more variable frequencies.[1,3,6]

For this reason, we should not present one percentage as though it applies to every patient or population. A practical interpretation is that BRAF V600E is common enough that every patient with suspected or confirmed ATC should be tested, but it is not common enough to assume that the mutation is present before the result is available.

If you are diagnosed with ATC, your age, symptoms or tumor size cannot reliably predict whether the cancer is BRAF-positive. The answer must come from testing the tumor.

Why Different Studies Report Different Percentages

Several factors can change the apparent frequency of BRAF V600E. One important factor is the population included in the study. A specialist thyroid-cancer centre may receive a different group of patients from a general hospital, and molecular patterns can also vary between geographic and ethnic populations.[6,7]

The testing method also matters. Some studies use immunohistochemistry, while others use PCR or next-generation sequencing. These methods have different detection limits, and older studies may have used less sensitive techniques than modern laboratories.

Sample quality can further influence the result. ATC tissue frequently contains extensive necrosis, meaning that part of the tumor is already dead. If the biopsy contains very few viable cancer cells, the molecular test may fail to detect a mutation that is actually present.[1]

Differences in pathology can also affect reported rates. Some tumors contain both an anaplastic component and a differentiated thyroid-cancer component, particularly papillary thyroid carcinoma. The proportion of these mixed tumors within a study can influence the measured prevalence of BRAF V600E.[3,26]

The Relationship With Papillary Thyroid Cancer

BRAF V600E is commonly associated with papillary thyroid carcinoma. In some patients, ATC may arise within or alongside a previously differentiated papillary thyroid cancer.

In such cases, BRAF V600E may have been present during the earlier differentiated stage. Additional molecular alterations may then accumulate as the tumor becomes less differentiated and more aggressive.[3–5]

This does not mean that every BRAF-positive papillary thyroid cancer will become anaplastic. Most papillary thyroid cancers do not undergo anaplastic transformation. Transformation usually involves a more complex series of molecular events rather than the presence of BRAF V600E alone.

BRAF-Positive and BRAF-Negative ATC Are Not the Only Categories

It is useful to distinguish BRAF V600E-positive from BRAF-negative ATC because the result may affect treatment. However, ATC cannot be divided into only two biological groups.

A BRAF-negative tumor may contain a RAS alteration, a rare gene fusion, changes in the PI3K–AKT–mTOR pathway or several abnormalities for which no established targeted treatment currently exists. A BRAF-positive tumor may also carry additional alterations involving TP53, TERT or other genes.[3–6]

Therefore, a positive rapid BRAF result should not automatically end broader molecular testing. Comprehensive profiling may reveal additional information that helps explain tumor behaviour, identify clinical-trial options or guide future treatment if resistance develops.

Prevalence Cannot Predict an Individual Patient’s Result

Population statistics describe groups, not individuals. Even when a mutation is present in a substantial proportion of ATC cases, no doctor can determine your BRAF status from probability alone.

When I explain this to a patient, I say that prevalence tells us why testing is important, but the laboratory result tells us what is present in that particular tumor. A 40% or 60% population frequency does not mean that an individual patient is partly positive or partly negative.

The clinically useful question is not simply how common BRAF V600E is. The more important question is whether the mutation is reliably detected in your tumor and whether that result can be converted into an appropriate treatment decision.[1,6]

How Can a Differentiated Thyroid Cancer Become Anaplastic?

Anaplastic Transformation Is a Process of Tumor Evolution

Some anaplastic thyroid cancers arise within or next to a pre-existing differentiated thyroid cancer, most often papillary or follicular thyroid carcinoma. The differentiated component may still resemble normal thyroid tissue, while the anaplastic component has lost many of those features and behaves far more aggressively.[3,31]

This change is called anaplastic transformation or dedifferentiation. It does not usually occur because of one new mutation alone. The tumor gradually accumulates additional molecular abnormalities that allow the cells to grow faster, invade surrounding structures and become less responsive to the normal controls that regulate thyroid-cell behaviour.[2–5]

When we study both components from the same tumor, they may share an early driver alteration such as BRAF V600E or RAS. The anaplastic component often contains further abnormalities that are absent or less prominent in the differentiated part. This supports the idea that ATC can develop through stepwise molecular evolution.[3–5]

Differentiated Thyroid Cells Lose Their Normal Identity

A normal thyroid cell has specialised functions, including the ability to produce thyroid proteins and take up iodine. Differentiated thyroid cancers may retain some of these features, even though the cells are malignant.

During anaplastic transformation, the cancer cells progressively lose this specialised identity. They may stop expressing proteins needed for iodine uptake and become less recognisable as thyroid cells under the microscope. This loss of differentiation is one reason radioactive iodine is generally ineffective against ATC.[1,31]

The process is not simply that the tumor becomes larger. Its biology changes. The cells may divide more rapidly, invade nearby tissues, spread earlier and use several growth pathways at the same time.

Early Driver Alterations May Remain Present

BRAF V600E or a RAS alteration may be present before the tumor becomes anaplastic. These changes can act as early drivers by activating growth pathways such as MAPK signalling.[3]

If the tumor later transforms, the original driver may remain detectable. This is clinically important because a BRAF V600E-positive ATC may still depend partly on BRAF–MEK signalling and may therefore respond to targeted inhibition.[17]

However, the presence of the original driver does not explain the entire transformation. Additional molecular changes are usually needed before a differentiated thyroid cancer develops the highly aggressive behaviour of ATC.

TP53 Loss Can Remove an Important Cellular Safeguard

TP53 is a tumor-suppressor gene. Its normal role includes detecting DNA damage, pausing cell division and helping severely damaged cells undergo controlled death.

When TP53 function is lost, genetically unstable cells may continue dividing instead of being repaired or removed. TP53 alterations are much more common in anaplastic thyroid cancer than in ordinary differentiated thyroid cancers and are considered important events in anaplastic progression.[2–5]

TP53 is biologically important, but it does not currently provide the same established targeted-treatment pathway as BRAF V600E. Its presence may help explain aggressive behaviour without directly identifying an approved mutation-specific treatment.

TERT Promoter Alterations May Support Continued Tumor Growth

TERT is involved in maintaining telomeres, which protect the ends of chromosomes. Most normal cells cannot divide indefinitely because their telomeres gradually shorten.

TERT promoter mutations may increase telomerase activity and help cancer cells continue dividing. These alterations are frequently associated with aggressive thyroid cancers and may cooperate with BRAF or RAS abnormalities during progression.[2–4]

A TERT promoter mutation should not be confused with a treatment target equivalent to BRAF V600E. It is currently more useful for understanding tumor biology and aggressive potential than for selecting a routine TERT-directed therapy.

The PI3K–AKT–mTOR Pathway May Add Another Growth Route

Some ATCs acquire abnormalities involving PIK3CA, PTEN, AKT or related genes. These changes can activate the PI3K–AKT–mTOR pathway, which supports cell growth, metabolism and survival.[2–4,26]

This pathway may operate alongside MAPK signalling. A tumor may therefore retain BRAF V600E while also developing another route that helps it survive. Such molecular complexity can contribute to aggressive behaviour and may influence resistance to treatment.

This is one reason why rapid BRAF testing should be accompanied by broader genomic profiling. A positive BRAF result identifies an important target, but the complete report may reveal additional changes that affect the tumor’s biology.

Not Every Differentiated Thyroid Cancer Becomes Anaplastic

Most papillary and follicular thyroid cancers do not transform into ATC. Even when BRAF V600E is present in papillary thyroid carcinoma, anaplastic transformation remains uncommon.

The transformation appears to require the accumulation of several additional molecular and cellular changes. The presence of one mutation should therefore not be used to tell a patient that a differentiated thyroid cancer will inevitably become anaplastic.[3,31]

If you have a history of papillary or follicular thyroid cancer and later receive an ATC diagnosis, the pathologist may look for both differentiated and anaplastic components in the tissue. Comparing these areas can help explain how the disease evolved and whether they share the same early driver alteration.

Why Understanding Tumor Evolution Matters for Treatment

Tumor evolution helps explain why a treatment may work initially and later become less effective. A BRAF-directed medicine may suppress cells that depend strongly on BRAF signalling, while another tumor-cell population may survive through a different pathway.

It also explains why repeat molecular testing may sometimes be considered after progression. A new biopsy or liquid biopsy may reveal changes that were absent, undetectable or uncommon at the time of the original diagnosis.

For the patient, the main message is that ATC is not simply a differentiated thyroid cancer that has grown larger. It has undergone a deeper biological transformation. The treatment plan must therefore address both the original molecular driver and the additional changes that have made the cancer more aggressive.[3–5]

Tumor Heterogeneity: Can Different Parts of the Same Cancer Have Different Mutations?

ATC May Contain More Than One Tumor-Cell Population

Anaplastic thyroid cancer is not always made of one genetically identical group of cells. As the cancer grows, different cells may acquire additional molecular changes. These related but genetically distinct groups are called tumor clones.[4,5]

An early driver alteration, such as BRAF V600E or RAS, may be present in many tumor cells. Other alterations may appear later and occur only in a smaller population. These later changes are called subclonal alterations because they are present in only part of the tumor.

This variation within a single cancer is known as intratumoral heterogeneity. It helps explain why one region may respond strongly to treatment while another region continues to grow.

Different Areas of the Primary Tumor May Not Be Identical

A large ATC may contain areas of viable anaplastic cancer, necrosis, inflammation and sometimes a remaining differentiated thyroid-cancer component. The molecular profile may differ between these areas.[4,5]

For example, the differentiated and anaplastic components may share BRAF V600E, suggesting a common origin. The anaplastic component may then contain additional abnormalities involving TP53, TERT, PIK3CA or other genes acquired during progression.

If a biopsy samples only one small area, it may accurately identify the dominant driver but still fail to capture every alteration present elsewhere in the tumor. This does not mean that biopsy-based testing is unreliable. It means that every molecular result must be interpreted in light of the sample’s size, location and tumor-cell content.

The Primary Tumor and Metastases May Also Differ

Cancer cells that spread to the lungs, bones, liver or other sites originate from the primary tumor, but they may continue evolving after they leave the thyroid. A metastatic lesion can therefore contain some of the same alterations as the neck tumor while also carrying additional changes.[26]

Early driver mutations may remain stable across several tumor sites, while later pathway alterations can vary. In some patients, this may create differences in treatment sensitivity between the primary tumor and individual metastases.

On imaging, the medical team may see this as a mixed response. One lesion may shrink while another remains unchanged or becomes larger. Such a pattern can suggest biological differences between tumor sites, although imaging alone cannot identify the exact molecular reason.

Why One Small Biopsy May Not Capture Everything

A biopsy represents the tissue that was sampled, not every cancer cell in the body. This is particularly important in ATC because the tumor may be large, rapidly changing and partly necrotic.

If the sampled area contains too few viable tumor cells, the test may miss an alteration. If a mutation is present only in a small subclone, it may fall below the assay’s detection limit. A negative result must therefore be interpreted together with tumor content, sequencing quality and the type of test used.[1,12]

When adequate tissue is available, comprehensive DNA- and RNA-based profiling increases the chance of detecting clinically meaningful alterations. In selected situations, testing a metastatic site or using liquid biopsy may provide additional information, but neither approach guarantees that every tumor clone will be detected.

Why Heterogeneity Can Affect Treatment Response

Targeted therapy is most effective when a large proportion of the cancer remains dependent on the targeted pathway. In BRAF V600E-positive ATC, dabrafenib and trametinib may suppress cells that rely strongly on BRAF–MEK signalling.[17]

However, another tumor-cell population may already use an alternative growth pathway or may later develop a resistance mechanism. Treatment can then reduce the sensitive cells while allowing resistant cells to survive and become more prominent.

This does not mean that targeted treatment should be avoided. It explains why a confirmed target can produce a major response without guaranteeing permanent control.

Why Heterogeneity Matters When the Cancer Progresses

If the disease begins growing after an initial response, the oncology team may consider whether the molecular profile has changed. A repeat biopsy can examine a progressing lesion, while a liquid biopsy may detect tumor DNA released from several disease sites.

Repeat testing is not required at every stage for every patient. It is most useful when the result could change treatment, identify a resistance alteration or support clinical-trial selection.

When we interpret molecular testing, we should therefore avoid thinking of the cancer as genetically frozen at the time of diagnosis. ATC can continue evolving, and its molecular profile may become more complex as the disease progresses or adapts to treatment.[5,12]

When Should BRAF V600E Testing Be Performed?

Testing Should Begin as Soon as ATC Is Suspected or Confirmed

BRAF V600E testing should be initiated early when anaplastic thyroid cancer is strongly suspected or confirmed by pathology. ATC can progress rapidly, and a positive result may identify a targeted-treatment option that could influence the initial treatment pathway rather than only later treatment.[1]

The medical team should not wait until surgery, radiation or chemotherapy has failed before requesting BRAF testing. In a patient with a rapidly enlarging thyroid or neck mass, suspicious imaging and pathology compatible with ATC, the biopsy material should be assessed promptly for BRAF V600E while the diagnosis is being completed.[1,9]

When I explain this to a patient, I make the purpose clear: early testing does not mean that targeted therapy will automatically be started. It means that the team will have the molecular information available when an urgent treatment decision must be made.

Rapid Testing May Be Started During Pathological Evaluation

BRAF V600E immunohistochemistry can sometimes provide an early result because it detects the altered BRAF protein in tumor tissue. This may be particularly useful when the clinical situation is urgent and the turnaround time for comprehensive sequencing is longer.[1,11]

A rapid positive result should still be interpreted together with the microscopic diagnosis and the clinical findings. When the staining pattern is unclear, weak or inconsistent with the pathology, confirmation using PCR or next-generation sequencing may be required.

The pathologist plays an important role in this process. They must identify viable tumor, select the most suitable tissue block and ensure that enough material remains for broader DNA- and RNA-based testing.

Comprehensive Profiling Should Not Be Delayed Until the BRAF Result Returns

Rapid BRAF testing and comprehensive molecular profiling answer different questions. The rapid test asks whether one immediately actionable mutation is present. Broader profiling examines the tumor for additional mutations, gene fusions and biomarkers that may influence treatment or clinical-trial eligibility.[1,6]

Whenever adequate tissue is available, these investigations should proceed in parallel. Waiting for a negative BRAF result before ordering comprehensive sequencing can create an avoidable delay, especially if the broader report later identifies an NTRK, RET or ALK fusion.

A positive BRAF result also does not make wider profiling unnecessary. Additional alterations may help explain the tumor’s aggressive behaviour, provide future trial options or become relevant if the disease later develops resistance.

Urgent Care Must Continue While Testing Is Pending

Molecular testing is important, but a patient should never be left waiting for a report while urgent clinical problems remain untreated. The team should continue assessing the airway, vocal-cord movement, swallowing ability, nutritional condition, tumor extent, distant metastases and possible resectability.[1]

If the patient has breathlessness, noisy breathing, bleeding, inability to swallow liquids or rapidly increasing neck swelling, immediate medical assessment takes priority. Airway protection, imaging, surgical review and radiation planning may need to proceed before the complete molecular report is available.

The molecular laboratory and clinical team should therefore work at the same time rather than one after the other. The laboratory investigates what may be driving the tumor, while the treating team protects the patient from the immediate consequences of the disease.

The Result Should Reach the Multidisciplinary Team Quickly

A molecular result has limited value if it remains unread in the laboratory system. Once BRAF V600E is detected or excluded, the finding should be communicated promptly to the oncologist, surgeon, radiation oncologist and pathologist involved in the case.

The team must then decide whether the result changes systemic treatment, surgical planning or the order of treatment. A positive result may support consideration of dabrafenib plus trametinib in an eligible patient, but the decision still depends on stage, tumor anatomy, available locoregional treatment, organ function and the patient’s overall condition.[1,15]

Early BRAF testing is therefore not simply about obtaining a faster laboratory answer. Its real purpose is to ensure that a potentially important treatment opportunity is identified before unnecessary time is lost.

Rapid BRAF Testing and Comprehensive Molecular Profiling Answer Different Questions

Rapid BRAF Testing Looks for One Urgent Treatment Target

Rapid BRAF testing is designed to answer a focused clinical question: does the tumor contain the BRAF V600E mutation? This result may be available sooner than a broad sequencing report and can influence early treatment planning in a rapidly progressing cancer.[1,11]

BRAF V600E immunohistochemistry examines tumor tissue for the altered BRAF protein. A focused PCR assay can look directly for the specific DNA change. These methods are useful because they concentrate on one clinically important alteration rather than analysing many genes at the same time.[1,10,11]

If the rapid result is positive and consistent with the pathology, the medical team can promptly evaluate whether BRAF- and MEK-directed treatment is appropriate. However, the result must still be considered alongside the cancer stage, airway safety, resectability, distant metastases and the patient’s ability to tolerate treatment.[1,15]

Comprehensive Profiling Examines the Wider Molecular Picture

Comprehensive molecular profiling has a broader purpose. It may examine dozens or hundreds of genes for mutations, fusions, amplifications, deletions and other biomarkers. Depending on the platform, it may use DNA, RNA or both.[1,6]

This wider analysis may identify alterations involving RAS, TP53, the TERT promoter or the PI3K–AKT–mTOR pathway. It may also detect uncommon but potentially actionable gene fusions involving NTRK, RET or ALK.[3,6]

Many findings on a broad report will not have an approved treatment. Some may mainly explain aggressive tumor biology, while others may support a clinical trial. The report therefore requires careful interpretation rather than simply counting the number of mutations identified.

One Test Should Not Be Used as a Substitute for the Other

A rapid BRAF test cannot provide the same information as comprehensive profiling. It may answer the most urgent BRAF question but will not reliably examine the full range of molecular findings that may be present in ATC.

Comprehensive sequencing also cannot always replace a rapid BRAF assessment. A broad report may take longer, especially when tissue must be transported to another laboratory or when both DNA and RNA analysis are required. In a disease that can progress quickly, waiting only for a large sequencing panel may delay recognition of an immediately relevant BRAF V600E result.[1]

The most effective approach is therefore not to choose between rapid testing and comprehensive profiling. When sufficient tissue is available, both can be initiated early because they provide different but complementary information.

Parallel Testing Can Prevent Avoidable Delays

Sequential testing can create a slow pathway. The team may order a rapid BRAF test, wait for the result and then request broad sequencing only after BRAF V600E is not detected. This can add another period of waiting before other clinically relevant findings become available.

Parallel testing reduces this delay. While the rapid test searches for BRAF V600E, DNA- and RNA-based profiling can begin examining the wider tumor genome. If BRAF is negative, the broader investigation is already underway rather than beginning from the start.[1,6]

If BRAF is positive, comprehensive profiling can still be valuable. The tumor may contain additional alterations that affect its biology, provide future clinical-trial options or become relevant if resistance develops.

Limited Tissue Requires Careful Planning

Small biopsies can contain only a limited amount of viable tumor. If the tissue is divided among too many separate tests without coordination, the sample may become exhausted before comprehensive profiling is completed.

The pathologist should therefore decide how the tissue will be used. Part of the specimen is needed to confirm the diagnosis, while sufficient material should be preserved for rapid BRAF assessment and broader molecular testing.[1,9]

When tissue is extremely limited, the team may select a combined assay, use another available tissue block or consider a repeat biopsy. Liquid biopsy may provide supplementary information, but a negative plasma result cannot always exclude an alteration within the tumor.

The Two Results Must Be Interpreted Together

Rapid and comprehensive results may sometimes disagree. BRAF immunohistochemistry may be positive while sequencing is negative, or sequencing may detect the mutation when staining is unclear. Such discordance should lead to review of the tissue, tumor content, assay sensitivity and technical quality rather than an immediate assumption that one method is wrong.[10,11]

When we combine both forms of testing, we gain speed and depth. The rapid test may identify an urgent treatment target, while comprehensive profiling provides a broader map of the tumor. Together, these findings can support a more informed and individualised ATC treatment decision.[1,6,14]

Fine-Needle Aspiration, Core Biopsy or Surgical Tissue: Which Sample Is Best?

The Best Sample Is One That Confirms the Diagnosis and Preserves Enough Tumor for Molecular Testing

There is no single biopsy method that is best for every patient with suspected anaplastic thyroid cancer. The ideal sample must provide enough viable tumor for the pathologist to confirm the diagnosis, perform immunohistochemistry and preserve material for BRAF testing and broader DNA- and RNA-based profiling.[1,9]

ATC often contains extensive necrosis, inflammation and blood. A sample may therefore appear large but contain only a small number of usable cancer cells. This is why the quality and location of the biopsy can be more important than the apparent size of the specimen.

When we plan tissue sampling, we should think beyond the immediate diagnosis. The patient may need rapid BRAF immunohistochemistry, molecular confirmation, comprehensive next-generation sequencing and possibly additional biomarker tests. Early coordination between the clinician, interventional radiologist and pathologist can reduce the risk of exhausting the tissue.

Fine-Needle Aspiration Can Provide a Rapid Initial Diagnosis

Fine-needle aspiration uses a thin needle to collect cells from the thyroid mass or an involved lymph node. It is commonly available, relatively quick and less invasive than a surgical biopsy.

In some patients, fine-needle aspiration provides enough characteristic cells for the pathologist to suspect or confirm ATC. The collected material may also be used for a cell block, immunohistochemistry and selected molecular tests.[1]

However, aspiration mainly collects individual cells rather than a larger piece of tissue architecture. If the tumor is very necrotic or the needle enters an area containing only inflammation and dead tissue, the sample may be non-diagnostic or insufficient for comprehensive profiling.

A fine-needle sample should therefore not be considered a failure simply because a second biopsy is needed. Its role may be to establish the initial concern rapidly, while a core biopsy provides the additional tissue required for complete pathological and molecular evaluation.

Core-Needle Biopsy Often Provides More Tissue

A core-needle biopsy uses a wider needle to remove small cylinders of tissue. This allows the pathologist to assess how the tumor cells are arranged and how they interact with surrounding tissue.

The additional material can improve the ability to distinguish ATC from other aggressive neck malignancies, including lymphoma, metastatic cancer and poorly differentiated thyroid carcinoma. It may also provide more reliable tissue for immunohistochemistry, BRAF testing and DNA/RNA sequencing.[1,9]

For this reason, core biopsy is often valuable when ATC is strongly suspected and the initial aspiration is limited. In some urgent cases, fine-needle aspiration and core biopsy may be obtained during the same procedure so that diagnosis and molecular testing can proceed without an avoidable second appointment.

Core biopsy is still not guaranteed to succeed. If the needle samples a necrotic region, the tissue may remain inadequate. Imaging guidance can help the operator target a viable and solid part of the tumor while avoiding major blood vessels, the trachea and other critical structures.

Surgical Tissue Can Provide the Largest Specimen

When surgery has already been performed or a diagnostic surgical procedure is necessary, the resected tissue may provide the most material for pathological examination. It allows the pathologist to evaluate different areas of the tumor, identify any coexisting differentiated thyroid carcinoma and select the most viable block for molecular testing.

Surgical tissue may also help demonstrate whether BRAF V600E or other alterations are shared between the differentiated and anaplastic components. This can provide useful information about tumor evolution.

However, surgery should not be performed only to obtain a larger molecular sample when a safer biopsy can establish the diagnosis. ATC may involve the trachea, oesophagus, nerves or major blood vessels, and an unnecessary operation can delay treatment or create significant complications.

The decision to operate must therefore be based on the patient’s anatomy, resectability and clinical needs rather than the laboratory’s preference for more tissue.

A Lymph Node or Metastatic Lesion May Sometimes Be Sampled

The thyroid mass is not always the safest or most informative biopsy site. A large cervical lymph node, lung lesion, liver lesion or other metastatic deposit may occasionally be easier to access and may contain more viable tumor.

Testing a metastatic site can confirm that the distant lesion belongs to the same cancer and may provide material for molecular profiling. It can also be useful when the primary thyroid tumor is extensively necrotic or difficult to approach safely.

The medical team must still consider tumor heterogeneity. A metastatic lesion may share the principal driver mutation with the thyroid tumor but contain additional molecular changes. The report should therefore identify exactly which site was sampled.

The Pathologist Should Select the Most Suitable Tissue Block

Not every part of a biopsy or surgical specimen is equally useful for sequencing. The pathologist reviews the slides and chooses the block containing the highest proportion of viable tumor with the least necrosis, inflammation and normal tissue.

The laboratory may require a minimum tumor-cell percentage before testing can proceed. In some cases, the pathologist can mark the tumor-rich area so that non-tumor tissue is removed before DNA or RNA extraction.

This selection process directly affects the reliability of the result. A technically advanced sequencing panel cannot overcome a specimen that contains almost no viable cancer cells.

Tissue Should Be Preserved Rather Than Exhausted by Repeated Small Tests

A limited biopsy can be consumed quickly if separate tests are ordered one by one. Diagnostic stains, repeated single-gene assays and unnecessary additional markers may leave too little material for comprehensive profiling.

The clinical team and pathologist should therefore agree on a tissue-use strategy from the beginning. Rapid BRAF testing should be performed, but sufficient material should also be protected for broader DNA- and RNA-based analysis whenever possible.[1]

If the tissue is inadequate, the report should state that the sample failed or was insufficient. It should not be described simply as mutation-negative. The next step may involve another block, repeat core biopsy, sampling of a metastatic lesion or supplementary liquid biopsy.

For the patient, the practical question is not whether the biopsy was performed with a thin or thick needle. The important question is whether it produced enough viable tumor to confirm the diagnosis and complete the molecular tests that may influence treatment.

Why Molecular Testing Sometimes Fails

The Sample May Contain Too Little Viable Tumor

A molecular test can only analyse the material that reaches the laboratory. In anaplastic thyroid cancer, a biopsy may contain blood, inflammatory cells, normal thyroid tissue and large areas of necrosis, with only a small amount of living tumor remaining.[1,9]

The specimen may look adequate in size but still contain too few cancer cells for reliable DNA or RNA analysis. If the proportion of tumor is below the laboratory’s required level, the test may fail quality control or produce a result with reduced sensitivity.

This is why the pathologist examines the tissue before sequencing. They identify the most viable area and may mark the tumor-rich portion so that unnecessary normal tissue can be removed before molecular analysis.

Extensive Necrosis Can Reduce the Quality of the Test

ATC can grow so quickly that parts of the tumor outgrow their blood supply and die. This dead tissue is called necrosis. A biopsy taken from a necrotic region may provide very little intact genetic material, even when the needle entered the visible tumor mass.[1,9]

Necrotic tissue can also contain degraded DNA and RNA. The molecular laboratory may therefore be unable to generate enough high-quality sequencing data to issue a reliable report.

Imaging guidance can help the radiologist target a solid, enhancing and apparently viable area. However, no biopsy technique can completely eliminate the possibility of sampling necrosis in a large and heterogeneous tumor.

DNA and RNA Can Be Damaged During Collection or Processing

DNA is generally more stable than RNA, but both can deteriorate. Delayed fixation, prolonged fixation, inappropriate storage, excessive heat and repeated handling may reduce sample quality.

RNA is particularly fragile. This matters because RNA-based testing can be valuable for detecting gene fusions involving NTRK, RET, ALK and other genes. A specimen may therefore produce usable DNA sequencing while failing RNA quality control.

Tissue obtained from a bone metastasis can present an additional difficulty. Some decalcification methods used to soften bone can damage nucleic acids and interfere with molecular testing. When a non-bone lesion is safely accessible, it may sometimes provide better material for comprehensive profiling.

The Tissue Block May Have Been Exhausted

A small biopsy may be used for routine microscopy, diagnostic immunohistochemistry, repeat stains and focused mutation testing before comprehensive sequencing is ordered. By the time the specimen reaches the molecular laboratory, too little tissue may remain.

This is known as tissue exhaustion. It does not mean that anyone necessarily made a mistake, because some stains may have been essential to confirm the diagnosis. However, early planning can reduce avoidable loss of material.[1]

When ATC is suspected, the pathologist and clinical team should consider the likely need for rapid BRAF testing and broader DNA- and RNA-based profiling from the beginning. This allows them to limit unnecessary testing while preserving enough tissue for clinically important investigations.

A Mutation May Be Present Below the Assay’s Detection Limit

Every molecular test has a limit of detection. If the mutation is present in only a small number of tumor cells or the specimen contains a large amount of normal tissue, the altered DNA may fall below the level that the assay can reliably identify.

Tumor heterogeneity can contribute to this problem. A mutation may be abundant in one part of the tumor but uncommon or absent in the sampled region. A negative result therefore needs to be interpreted together with the tumor percentage, sequencing depth, specimen quality and testing method.[11,12]

This does not mean that every negative result should be doubted. A high-quality negative result from an adequate specimen can be clinically reliable. The concern is greater when the report specifically mentions low tumor content, poor nucleic-acid quality, limited coverage or failed quality-control measures.

Technical Failure Is Different From a Negative Result

A report stating “BRAF V600E not detected” means that the test did not identify the mutation within the analysed sample and within the limits of that assay.

A report stating “quantity not sufficient,” “insufficient tumor,” “failed quality control” or “unable to obtain a result” means that the laboratory could not complete a reliable analysis. These statements should not be interpreted as proof that BRAF V600E or another alteration is absent.

When I review such a report, I first look at the specimen-quality comments before interpreting the mutation list. The technical notes may be as important as the main result.

What Can Be Done After an Inadequate or Failed Test?

The pathologist may review whether another tissue block contains more viable tumor. If no suitable material remains, the team may consider a repeat core biopsy or sampling an accessible lymph node or metastatic lesion.

A plasma-based liquid biopsy may also provide useful supplementary information, particularly when obtaining more tissue is difficult. However, a negative liquid-biopsy result cannot always exclude an alteration in the tumor because some cancers release very little detectable DNA into the bloodstream.[12,13]

The next step should depend on whether another result could change treatment. Repeating an invasive biopsy may be reasonable when it could identify an actionable alteration, but the risks must be weighed against the patient’s airway, general condition and urgency of treatment.

A failed molecular test is therefore not the end of the investigation. It is a signal to review the specimen, understand why the test failed and choose the safest method for obtaining more reliable information.

Tumor Cell Percentage, Tumor Purity and Why They Matter

A Biopsy Contains More Than Cancer Cells

A tissue sample taken from an anaplastic thyroid cancer does not usually contain tumor cells alone. It may also include normal thyroid tissue, blood vessels, immune cells, scar tissue, inflammatory cells and areas of necrosis. The proportion of viable cancer cells within the tested sample is called the tumor-cell percentage or tumor purity.[1,9]

Before molecular testing begins, the pathologist examines the specimen under a microscope and estimates how much of the selected area contains viable tumor. This estimate helps the molecular laboratory decide whether the sample is suitable for DNA or RNA analysis.

A sample with a higher tumor-cell percentage generally provides a clearer molecular signal. When the sample contains very few cancer cells, altered DNA may be diluted by DNA from normal cells and become more difficult to detect.

Low Tumor Purity Can Produce an Uncertain or False-Negative Result

Every molecular assay requires a minimum amount of tumor DNA. If the percentage of cancer cells is below the test’s detection threshold, a mutation may be present but remain undetected.

For example, BRAF V600E may exist within the tumor, but a biopsy containing mostly necrosis, inflammation or normal tissue may not provide enough altered DNA for the laboratory to identify it reliably. The risk is greater when the mutation is present only in a smaller tumor-cell population.[11,12]

This is why the technical section of the report matters. A statement such as “low tumor content may reduce sensitivity” means that a negative result should be interpreted cautiously. It does not automatically prove that the mutation is absent.

Tumor Percentage Does Not Measure Cancer Burden in the Body

Patients may misunderstand a report stating that the specimen contains 30% or 60% tumor. This percentage refers only to the tissue placed under the microscope and selected for testing.

It does not mean that 30% or 60% of the patient’s body contains cancer. It also does not describe the cancer stage, tumor size, chance of survival or expected treatment response.

When I explain this result, I tell the patient that tumor purity is mainly a laboratory-quality measure. It helps us judge how confidently the molecular findings can be interpreted.

The Pathologist Can Improve the Sample Before Testing

The pathologist may mark the most tumor-rich area of the slide so that the laboratory can remove surrounding normal tissue. This process, called tumor enrichment or microdissection, can increase the proportion of cancer DNA in the tested material.

However, enrichment cannot create viable tumor where none exists. If the sample contains extensive necrosis or very few cancer cells, another tissue block or repeat biopsy may still be required.

Tumor-cell percentage is therefore not a minor technical detail. It directly affects whether a molecular result can be trusted and whether a negative finding truly reflects the biology of the tumor.[1,9,12]

BRAF V600E Immunohistochemistry

What the VE1 Antibody Detects

BRAF V600E immunohistochemistry is a tissue-based test that uses an antibody known as VE1. This antibody is designed to recognise the abnormal BRAF V600E protein produced by tumor cells carrying the mutation.[1,10]

The pathologist applies the antibody to a thin section of the biopsy and examines the stained tissue under a microscope. When the altered BRAF protein is present, the tumor cells may show a characteristic staining pattern.

This test examines protein expression rather than directly reading the tumor’s DNA sequence. It can therefore provide a rapid indication that BRAF V600E is present while broader molecular testing is still being completed.

Why Immunohistochemistry Is Useful in ATC

Anaplastic thyroid cancer may progress quickly, and the medical team may need molecular information before a large sequencing panel is complete. BRAF V600E immunohistochemistry can be performed in many pathology laboratories using tissue that has already been collected for diagnosis.[1,11]

A clear positive result can alert the treating team that a clinically important target may be present. The oncologist can then begin evaluating whether BRAF- and MEK-directed treatment may be appropriate, while molecular confirmation and comprehensive profiling continue.

The advantage of immunohistochemistry is therefore mainly speed and accessibility. It does not provide a complete molecular profile and should not be treated as a substitute for broader testing.

How the Pathologist Interprets the Staining

The pathologist examines whether staining is present within the tumor cells and whether the pattern is strong, diffuse and consistent with true BRAF V600E expression. The result must be evaluated in the correct area of the specimen because inflammatory cells, blood, necrosis and normal tissue can complicate interpretation.[10,11]

A report may describe the result as positive, negative or equivocal. A positive result means that the expected staining pattern was observed. A negative result means that convincing tumor-cell staining was not seen. An equivocal result means that the pattern was too weak, incomplete or uncertain to support a confident conclusion.

The quality of the tissue matters. Poor fixation, extensive necrosis, low tumor-cell content or heavy background staining may reduce confidence in the interpretation.

A Positive IHC Result Should Fit the Pathology

BRAF V600E immunohistochemistry should not be interpreted separately from the rest of the diagnosis. The pathologist must first confirm that the stained cells are truly part of the tumor and that the overall findings support thyroid carcinoma.

BRAF V600E can occur in papillary thyroid carcinoma and several other cancer types. Its detection does not independently prove that the tumor is anaplastic thyroid cancer.[1]

If a specimen contains both papillary and anaplastic components, the pathologist may compare their staining. Shared BRAF V600E expression can support the possibility that the anaplastic component evolved from the differentiated tumor.

When Molecular Confirmation Is Helpful

A clear immunohistochemical result may be clinically useful, but molecular confirmation is particularly important when the staining is weak, patchy, technically difficult or inconsistent with other findings.[1,10,11]

PCR or next-generation sequencing can determine whether the corresponding BRAF DNA alteration is present. Confirmation may also be needed when a treatment decision depends on a validated molecular test or companion diagnostic.

When I review a BRAF result, I do not look only at the word “positive” or “negative.” I also consider which method was used, how much viable tumor was present and whether the pathologist described any technical limitation.

A Negative IHC Result Does Not End Molecular Testing

A negative immunohistochemical result may mean that the tumor does not contain BRAF V600E. However, it may also be influenced by poor tissue preservation, low tumor content or technical limitations.

If the clinical and pathological findings remain suspicious, a molecular assay may still be appropriate. Broader profiling is also necessary because a BRAF-negative tumor may contain another clinically relevant alteration.[1,6]

The most useful role of immunohistochemistry is therefore rapid screening. It helps identify an urgent target, but it does not replace comprehensive DNA- and RNA-based investigation.

PCR-Based BRAF Testing

PCR Looks Directly for a Specific DNA Change

Polymerase chain reaction, commonly called PCR, is a laboratory method used to identify selected DNA alterations. In ATC, a focused PCR test can search specifically for the genetic change responsible for BRAF V600E.[10,11]

The method copies a chosen region of DNA many times so that a small amount of altered genetic material becomes easier to detect. Some PCR assays are designed only for BRAF V600E, while others examine a limited group of common mutations.

Unlike immunohistochemistry, PCR tests the DNA sequence rather than the altered protein. This can provide direct molecular confirmation that the mutation is present.

Why PCR Can Be Fast and Sensitive

Because PCR examines a predefined region, it can often be completed more quickly than broad next-generation sequencing. It may also detect BRAF V600E when the amount of altered DNA is relatively small, depending on the assay and sample quality.

Focused PCR is therefore useful when the main urgent question is whether BRAF V600E is present. It may be performed on formalin-fixed tumor tissue, cell-block material or another suitable specimen.

However, sensitivity does not remove the need for adequate tissue. A sample containing almost no viable tumor may still produce an unreliable or false-negative result.

Different PCR Methods Do Not Have Identical Performance

PCR is not one single test. Laboratories may use allele-specific PCR, real-time PCR, digital PCR or other focused methods. Each assay has its own detection threshold, quality requirements and list of variants that it can identify.

A test designed specifically for BRAF V600E may detect that alteration very well but may not recognise an unusual BRAF mutation. The report should therefore state which variants were included and whether any technical limitation affected the result.

When a report says “BRAF negative,” it is important to know whether the laboratory tested only V600E or examined a broader range of BRAF alterations.

PCR Cannot Provide a Complete Tumor Profile

The main limitation of focused PCR is its narrow scope. It answers the question it was designed to answer, but it does not usually evaluate the full range of mutations, gene fusions, amplifications and biomarkers that may be present in ATC.

A negative BRAF PCR result does not exclude an NTRK, RET or ALK fusion. It also does not fully assess alterations involving RAS, TP53, TERT or the PI3K–AKT–mTOR pathway.[3,6]

For this reason, rapid PCR and broad molecular profiling should be viewed as complementary. PCR may provide speed, while next-generation sequencing provides greater molecular depth.

How PCR and Immunohistochemistry Can Support Each Other

Immunohistochemistry detects the abnormal protein, while PCR detects the corresponding DNA change. When both are positive, the result is usually straightforward.

If they disagree, the pathologist and molecular laboratory should review the sample. Possible explanations include low tumor content, tissue heterogeneity, technical limitations or difficulty interpreting the staining pattern.[10,11]

A third method, such as next-generation sequencing, may be useful when the results remain discordant. The aim is not to choose the test that produces the preferred answer. The aim is to determine which result most accurately represents the tumor.

Next-Generation Sequencing: What Patients Need to Understand

NGS Examines Many Genes at the Same Time

Next-generation sequencing, or NGS, allows a laboratory to analyse many regions of tumor DNA or RNA in one test. Instead of looking only for BRAF V600E, it can search for a wider range of molecular alterations that may help explain tumor growth or influence treatment.[6–8]

The number of genes examined depends on the panel. Some tests analyse a relatively small group of cancer-related genes, while others study several hundred genes and selected genomic biomarkers.

A larger panel is not automatically better. The test must include the genes and alteration types that are clinically relevant to ATC, and the laboratory must be able to interpret the findings accurately.

Targeted Gene Panels Are Common in Clinical Practice

Most clinical NGS tests use targeted gene panels. These panels focus on genes known to be involved in cancer rather than sequencing every part of the genome.

A targeted DNA panel may detect single-letter DNA changes, small insertions or deletions, selected copy-number alterations and some gene rearrangements. The exact capability depends on how the assay was designed.[6,14]

A well-designed targeted panel can provide clinically useful information with less tissue and a shorter analysis process than whole-genome sequencing. For a patient with ATC, the most important issue is whether the panel covers the molecular findings that could affect treatment.

DNA Sequencing Does Not Detect Every Type of Alteration Equally Well

DNA-based NGS is particularly useful for detecting mutations such as BRAF V600E, RAS alterations and changes involving TP53, PIK3CA or other cancer-related genes.[3,6]

It may also detect selected gene fusions, but fusion detection can be difficult when the relevant breakpoints occur in large or complex regions of DNA. Some DNA panels cover only specific fusion partners or selected intronic regions.

This is why a report described as “comprehensive DNA sequencing” may still not provide complete fusion assessment. RNA-based testing can offer additional value when NTRK, RET, ALK or another fusion is clinically important.

Sequencing Depth Refers to How Many Times a Region Was Read

During NGS, the same region of DNA may be read many times. This is called sequencing depth or read depth. Repeated reading helps the laboratory distinguish a true alteration from technical noise.

A deeper result may improve the ability to detect variants present at a low level, but depth alone does not guarantee accuracy. The sample must still contain enough viable tumor, the relevant region must be covered and the laboratory must use reliable quality-control methods.

When a report mentions low coverage, it may mean that one part of the gene was not read sufficiently well. An alteration in that region could therefore be missed.

Coverage Describes Which Regions the Test Can Examine

Coverage refers to the genes and genomic regions included in the assay. A panel may list a gene but test only selected portions of that gene.

Two laboratories can therefore test the same tumor and produce different reports because their panels cover different regions, use different detection methods and apply different interpretation rules.[6–8]

This does not necessarily mean that one laboratory is wrong. It means that molecular testing is shaped by assay design. The oncology team should know whether the panel included the alteration classes most relevant to the patient.

NGS Can Identify More Than One Important Finding

A broad report may identify BRAF V600E together with TP53, TERT promoter or other alterations. It may also show that the tumor lacks a commonly actionable mutation but contains a rare fusion or biomarker relevant to a clinical trial.[3–8]

Not every finding will guide treatment. Some alterations are considered pathogenic because they contribute to tumor biology but have no established targeted medicine. Others may be classified as variants of uncertain significance.

The report should separate established actionable findings from alterations that are mainly biological, prognostic or investigational.

Some NGS Panels Can Assess Additional Biomarkers

Depending on the platform, NGS may also estimate tumor mutational burden or evaluate microsatellite instability. These biomarkers may influence immunotherapy consideration in selected cancers.

However, not every NGS panel is validated for these purposes. A report should clearly state whether TMB or MSI was assessed and whether the result met the laboratory’s quality requirements.

PD-L1 is different because it is usually measured by immunohistochemistry rather than DNA sequencing. A comprehensive molecular report and a PD-L1 report therefore provide different types of information.

Turnaround Time Varies Between Laboratories

NGS usually requires several steps, including tissue review, DNA or RNA extraction, sequencing, quality control, bioinformatic analysis and clinical interpretation. The time required varies according to the laboratory, sample and panel.

A failed first extraction, poor RNA quality or the need to request another tissue block can extend the process. This is another reason why urgent BRAF testing may be performed separately while comprehensive NGS is underway.[1]

The clinical team should continue airway assessment, staging and treatment planning rather than waiting for every molecular result before acting.

The Final Report Requires Clinical Interpretation

NGS can produce a large amount of data, but the treatment decision cannot be made by software alone. The oncologist and molecular pathologist must determine whether the alteration is reliable, whether it is clinically actionable and whether the evidence applies to ATC.

When I interpret a sequencing report for a patient, I separate the findings into three groups. The first group contains alterations with established treatment relevance. The second contains findings that may support a clinical trial or selected off-label discussion. The third contains biological or uncertain findings that should not independently determine treatment.

The value of NGS is not the number of mutations listed. Its value is the ability to identify reliable information that can be converted into a safe and evidence-based treatment decision.[1,6,14]

DNA-Based Versus RNA-Based Molecular Testing

DNA and RNA Provide Different Types of Information

DNA contains the genetic instructions stored inside the cell. RNA is produced when the cell copies and uses part of those instructions. Molecular laboratories can study either material, but the two tests do not always identify the same types of alterations equally well.[4,6]

DNA-based sequencing is particularly useful for detecting mutations such as BRAF V600E, RAS alterations, TP53 changes and abnormalities involving genes in the PI3K–AKT–mTOR pathway. It may also identify small insertions, deletions, copy-number changes and selected gene rearrangements, depending on how the panel was designed.[3,6]

RNA-based sequencing is especially helpful when the medical team is searching for gene fusions. A fusion creates an abnormal connection between two genes, and RNA testing can show whether that abnormal genetic message is actually being produced by the tumor.

When we combine DNA and RNA analysis, we can often obtain a more complete view of the cancer than either test would provide alone.

What DNA Sequencing Does Well

DNA sequencing can reliably identify many common cancer-driving mutations. In ATC, this includes BRAF V600E and alterations involving NRAS, HRAS, KRAS, TP53, PIK3CA and other cancer-related genes.[3,6]

DNA is also more stable than RNA. This means that DNA testing may still succeed in a tissue specimen where the RNA has become too degraded for reliable analysis. Formalin-fixed biopsy material, which is commonly used in routine pathology, often preserves sufficient DNA for targeted sequencing.

A DNA panel may also estimate biomarkers such as tumor mutational burden or microsatellite instability when the assay has been specifically designed and validated for those purposes.

However, the phrase “DNA sequencing completed” does not mean that every possible molecular alteration has been excluded. The panel only examines the genes, regions and alteration types included in its design.

Why Gene Fusions Can Be Difficult to Detect With DNA Alone

Gene fusions occur when parts of two genes become abnormally joined. The exact joining point may be located within a large or technically complex region of DNA.

A DNA panel may detect a fusion when it covers the correct breakpoint region. However, some genes contain long introns or several possible fusion partners, making complete DNA coverage difficult. If the breakpoint falls outside the regions examined by the panel, the fusion may be missed.

This is particularly important for NTRK, RET and ALK fusions. These findings are uncommon in ATC, but they may have significant treatment relevance when present.[6,23–25]

A report that states “no fusion detected” should therefore be interpreted according to the method used. A negative result from a limited DNA panel is not always equivalent to a negative result from a comprehensive RNA fusion assay.

Why RNA Testing Is Useful for Fusion Detection

RNA testing examines the abnormal message produced after two genes have fused. Instead of searching through large regions of DNA for every possible breakpoint, the test looks for the expressed fusion transcript.

This can make RNA-based sequencing more effective for detecting clinically important fusions, especially when the DNA breakpoint is difficult to analyse. RNA testing may also help confirm that a fusion identified in DNA is actively expressed by the tumor.

For example, if an NTRK fusion is detected and confirmed as an expressed transcript, the finding may support consideration of a TRK-directed treatment in an appropriate patient. Similar reasoning applies to selected RET and ALK fusions.[23–25]

RNA analysis is therefore not simply a duplicate of DNA testing. It answers an additional biological question: is the tumor producing an abnormal fusion message that may be driving its growth?

RNA Is More Fragile Than DNA

The main limitation of RNA testing is that RNA degrades more easily. Poor fixation, old tissue, prolonged storage, necrosis and repeated processing can reduce RNA quality.

A sample may produce a successful DNA report but fail RNA quality control. This does not mean that the tumor has no gene fusion. It means that the laboratory could not analyse the RNA reliably.

When a report states that RNA sequencing failed, the medical team should review whether another tissue block is available. A repeat biopsy may be considered when fusion testing could materially influence treatment and the procedure is clinically safe.

The laboratory may also use an alternative method, such as targeted PCR or fluorescence-based testing, for a specific suspected fusion. However, these focused methods examine only predefined alterations and do not provide the same breadth as an RNA panel.

Combined DNA and RNA Testing Can Reduce Important Gaps

A combined panel can use DNA to detect mutations and copy-number changes while using RNA to improve fusion detection. This is often a practical approach in ATC because both mutation-driven and fusion-driven tumors can occur.[1,6]

If the tumor is BRAF-negative, RNA-inclusive profiling becomes particularly important. Without it, a clinically relevant NTRK, RET or ALK fusion may remain unidentified.

A combined test does not guarantee that every alteration will be found. Sample quality, tumor content and assay design still matter. However, it generally reduces the chance that the report will miss an alteration simply because the wrong type of genetic material was examined.

The Term “Comprehensive Profiling” Is Not Standardised

Different laboratories may use the term comprehensive genomic profiling for very different tests. One panel may examine hundreds of genes using DNA only. Another may include fewer genes but analyse both DNA and RNA. A third may include tumor mutational burden, microsatellite instability and selected fusion testing.

The oncology team should therefore review what the panel actually covers rather than relying on the word “comprehensive.”

When I examine a report, I look for whether BRAF V600E was assessed, whether the major ATC-related genes were included and whether RNA-based fusion testing was performed. I also check whether the report explains any failed component or area of insufficient coverage.

For you as a patient, the most useful question is not simply, “Was NGS performed?” The better question is, “Did the test examine both the mutations and gene fusions that may be relevant to my ATC?”

A Negative DNA Test May Still Require RNA Testing

If DNA sequencing does not identify an actionable mutation, the investigation should not automatically stop. RNA testing may still reveal an oncogenic fusion that was not detectable through the DNA panel.

This is particularly important when the report shows that fusion coverage was limited or when the tumor lacks the more common BRAF and RAS alterations. In such a situation, the absence of a DNA mutation does not mean that the cancer has no molecular driver.

DNA and RNA testing should therefore be viewed as complementary. DNA provides the stable genetic record, while RNA shows which abnormal messages the tumor is actively producing.[4,6]

Liquid Biopsy and Circulating Tumor DNA in ATC

What Is Circulating Tumor DNA?

Cancer cells can release small fragments of their DNA into the bloodstream. This material is called circulating tumor DNA, or ctDNA. A liquid biopsy examines blood plasma for these tumor-derived fragments.[12,13]

The blood also contains normal cell-free DNA released from healthy tissues. The laboratory must therefore separate a small tumor signal from a much larger background of normal DNA.

A liquid biopsy may search for BRAF V600E and other mutations without requiring another tissue procedure. This can be valuable when the tumor is difficult to biopsy, the original specimen is exhausted or the patient is not medically fit for another invasive procedure.

Why Liquid Biopsy Can Be Helpful in ATC

ATC can involve several sites at the same time. Tumor DNA released into the blood may come from the thyroid mass, lymph nodes and distant metastases. A plasma test may therefore capture molecular information from more than one tumor location.[12]

The test is minimally invasive and can usually be performed through a routine blood draw. It may also provide results when tissue is unavailable or insufficient.

In an urgent clinical situation, a liquid biopsy may be sent while the team attempts to obtain or review tissue. It can also supplement a tissue result by identifying an alteration not detected in a small biopsy.

However, liquid biopsy should not be described as automatically better than tissue testing. Its usefulness depends on how much tumor DNA is present in the bloodstream.

Not Every Tumor Releases Enough DNA Into the Blood

Some cancers release large amounts of ctDNA, while others release very little. The amount may depend on tumor size, location, blood supply, rate of cell death and extent of metastatic disease.

A patient may have an alteration within the tumor but an undetectable level of that alteration in plasma. This is sometimes called a non-shedding or low-shedding tumor.

For this reason, a negative liquid biopsy does not always prove that BRAF V600E or another molecular alteration is absent. The result may simply mean that the amount of tumor DNA in the blood was below the assay’s detection limit.[12,13]

This limitation is especially important when the plasma result is negative but the clinical suspicion remains high or tissue testing has not been completed.

A Positive Liquid-Biopsy Result Can Be Clinically Useful

When a pathogenic alteration such as BRAF V600E is clearly detected in plasma, the result can provide important molecular information. It may support rapid treatment planning, particularly when the tissue sample is inadequate.

The oncology team should still review whether the assay is validated for the intended clinical decision and whether tissue confirmation is required. A positive plasma result should also be interpreted with the pathology and imaging rather than used alone to establish the ATC diagnosis.

Liquid biopsy identifies molecular material in the blood, but it does not show the tumor’s microscopic appearance. Pathological confirmation remains essential.

Tissue and Plasma Results May Not Always Match

A tissue biopsy samples one location, while a liquid biopsy may detect DNA from several tumor sites. The two tests may therefore produce different results.

A mutation may be present in a metastatic lesion and detectable in plasma but absent from the sampled region of the primary tumor. The opposite can also occur when the tissue contains the mutation but insufficient tumor DNA reaches the blood.[12]

Technical factors can also contribute to discordance. Different platforms may have different gene coverage, sequencing depth and detection limits.

When results disagree, the team should review sample quality, tumor content, assay design and the patient’s disease distribution. The aim is to understand the reason for the difference rather than assuming that one result must be incorrect.

Liquid Biopsy May Help Monitor Treatment Response

If a measurable mutation such as BRAF V600E is present in plasma before treatment, its level may fall when the tumor responds. A later rise may suggest increasing molecular disease activity.

This creates a potential role for serial ctDNA testing. It may provide biological information before some changes become obvious on imaging, although this use is still developing in ATC.[12,13]

Liquid biopsy should not replace CT scans, clinical examination or symptom assessment. A falling ctDNA level does not prove that every tumor site is controlled, and a rising level should be confirmed within the full clinical context.

The most useful approach may be to combine ctDNA with imaging and clinical evaluation rather than use it as a single independent marker.

Liquid Biopsy May Help Identify Resistance

When a tumor initially responds and later progresses, the molecular profile may have changed. A repeat plasma test may detect new alterations associated with resistance or reveal that another tumor clone has become dominant.

This can be useful when a progressing lesion is difficult to biopsy. It may also guide clinical-trial selection if a new potentially actionable alteration appears.

However, a negative repeat liquid biopsy does not exclude resistance. The tumor may still be progressing through molecular mechanisms that are not covered by the assay or are not detectable in plasma.

Repeat testing should therefore be performed only when the result could influence the next treatment decision.

Liquid Biopsy Is Usually Complementary to Tissue Testing

Tissue remains important because it confirms the diagnosis, shows tumor morphology and allows immunohistochemistry. It can also provide DNA and RNA for broad molecular analysis.

Liquid biopsy provides a less invasive and potentially wider view of tumor-derived DNA in the bloodstream. Its main limitation is that a negative result may be falsely reassuring.

When we use both methods together, tissue provides pathological certainty and detailed local molecular information, while plasma may add speed, access and information from multiple tumor sites.[12,13]

For the patient, the practical message is clear. A positive liquid biopsy may be useful, but a negative liquid biopsy should not automatically end the molecular investigation when adequate tissue testing has not been completed.

Which Molecular Testing Strategy Is Most Useful in ATC?

No Single Test Answers Every Clinical Question

BRAF immunohistochemistry, PCR, DNA sequencing, RNA sequencing and liquid biopsy each have a different role. One method may provide speed, another may provide breadth and another may help when tissue is unavailable.

The best strategy is usually not to search for one “perfect” test. It is to combine the right methods according to the urgency, sample quality and treatment question.

A rapid BRAF test may identify an immediate target. DNA sequencing may reveal broader mutations and biomarkers. RNA testing may detect gene fusions. Liquid biopsy may supplement tissue analysis when another biopsy is difficult.[1,6,12]

A Practical Testing Approach Begins With Adequate Tissue

The first priority is obtaining enough viable tumor to confirm the diagnosis. The pathologist should then preserve tissue for rapid BRAF assessment and comprehensive molecular profiling.

Where feasible, rapid BRAF testing and broad DNA/RNA analysis should begin in parallel. This reduces the delay that occurs when one test is ordered only after another returns negative.

If tissue is inadequate, the team should review another block, consider a repeat biopsy or use an accessible metastatic lesion. Plasma testing may be added, particularly when further tissue collection is unsafe or impractical.

BRAF-Positive Tumors Still Benefit From Broader Profiling

Once BRAF V600E is detected, the immediate treatment opportunity becomes clearer. However, broader profiling may still identify additional alterations that help explain aggressive behaviour or become relevant if resistance develops.

The tumor may carry TP53, TERT promoter or PI3K-pathway abnormalities in addition to BRAF V600E. These findings may not change the first treatment, but they can provide important biological context.[3–6]

Broader profiling can also establish a baseline molecular record. If the disease later progresses, repeat testing can be compared with the original profile to identify newly emerging alterations.

BRAF-Negative Tumors Need RNA-Inclusive Profiling

When BRAF V600E is not detected, the quality of the sample and the testing method should first be reviewed. If the result is reliable, the next priority is comprehensive DNA- and RNA-based profiling.

RNA testing is important because a BRAF-negative tumor may contain an NTRK, RET or ALK fusion. Although these findings are uncommon, they may create a meaningful treatment opportunity.[23–25]

A BRAF-negative result should therefore be treated as the beginning of a wider molecular investigation rather than the end of precision oncology.

The Testing Plan Must Match the Patient’s Clinical Condition

A patient with airway compromise cannot wait for every molecular result before receiving urgent treatment. The medical team may need to protect the airway, begin radiation planning or make other immediate decisions while sequencing continues.

In a more stable patient, there may be enough time to complete broader profiling before finalising the systemic-treatment strategy.

When I explain this process, I tell patients that molecular testing should move quickly, but it should not control the entire clinical timeline. The test supports the treatment plan; it does not replace urgent medical judgment.

The Most Useful Test Is the One That Can Change a Decision

A technically impressive report has limited value when it arrives too late, uses an inadequate sample or fails to examine the relevant alteration types.

The most useful molecular strategy is one that provides reliable information within a clinically meaningful period. It should answer whether BRAF V600E is present, whether another actionable mutation or fusion exists, and whether the findings can guide treatment or clinical-trial selection.

For most patients with ATC, this means rapid BRAF assessment, tissue-based DNA and RNA profiling, and selective use of liquid biopsy. These methods work best when the pathologist, oncologist, surgeon and molecular laboratory plan them together.[1,6,12,14]

Understanding Your Molecular Pathology Report Line by Line

Start With the Specimen and the Confirmed Diagnosis

Before reading the mutation results, check which specimen was tested. The report may refer to a core biopsy from the thyroid mass, an involved lymph node, a distant metastasis or tissue removed during surgery.

This detail matters because the molecular findings describe the tested specimen. A metastatic lesion may share the main driver alteration with the thyroid tumor but may also contain additional changes acquired during progression.

The report should also confirm that the specimen contains anaplastic thyroid carcinoma or tissue consistent with the established diagnosis. Molecular testing cannot replace pathological confirmation because the same genetic alteration may occur in more than one cancer type.[1]

Check Whether the Sample Was Adequate

The report may state the estimated tumor-cell percentage, amount of viable tissue and quality of the extracted DNA or RNA. These details help you understand how reliable the result is.

A sample with sufficient viable tumor and successful quality-control testing gives the laboratory a stronger basis for identifying or excluding an alteration. A specimen with low tumor content, extensive necrosis or degraded genetic material may produce an incomplete or less sensitive result.

When I read a molecular report, I examine the specimen-quality section before focusing on the mutation list. A negative result from a high-quality sample means something different from a negative result accompanied by a warning that the sample was inadequate.

Identify Which Testing Method Was Used

The report should explain whether the laboratory used immunohistochemistry, PCR, DNA-based next-generation sequencing, RNA sequencing or a combined DNA/RNA panel.

Each method has a different purpose. Immunohistochemistry may rapidly detect the altered BRAF V600E protein. PCR may search for one predefined DNA mutation. Broad NGS may examine many genes, while RNA sequencing may improve the detection of gene fusions.[1,6]

You should not assume that a report described as “molecular testing” examined every relevant type of alteration. The test may have evaluated only BRAF V600E, or it may have analysed hundreds of genes. The assay description and coverage section show what was actually tested.

Read the Exact Gene and Variant

A molecular result should identify the gene and the precise alteration. For example, a report may state:

BRAF p.V600E detected

The gene is BRAF, and the specific protein alteration is V600E. The report may also provide the DNA-level notation, such as c.1799T>A.

The exact wording matters because not every BRAF alteration has the same biological or treatment significance. A rare BRAF fusion or non-V600 mutation should not automatically be interpreted as equivalent to BRAF V600E.

Understand the Variant Classification

The laboratory may classify an alteration as pathogenic, likely pathogenic, benign, likely benign or a variant of uncertain significance.

A pathogenic alteration has sufficient evidence showing that it contributes to disease biology. A likely pathogenic alteration has strong, but not fully conclusive, evidence of clinical importance.

A variant of uncertain significance, or VUS, does not yet have enough evidence to be considered a confirmed driver or treatment target. A VUS should not independently determine therapy merely because it appears on the report.[6,8]

Different laboratories may use slightly different classification systems. The molecular pathologist should interpret the finding according to current scientific evidence, tumor type and available clinical data.

Actionable Does Not Always Mean an Approved Treatment Is Available

A report may describe an alteration as actionable. This usually means that the finding could influence treatment, clinical-trial eligibility or further investigation.

However, actionability exists at different levels. One alteration may have an approved treatment specifically supported in ATC. Another may have a tumor-agnostic approval that applies across several cancer types. A third may be linked only to early clinical-trial evidence.

The report should therefore distinguish between established treatment relevance, possible off-label use and investigational evidence. The word actionable should not be interpreted as a guarantee that the medicine is appropriate, available or likely to work.

Understand “FDA-Approved,” “Off-Label” and “Clinical Trial”

An FDA-approved therapy has been authorised for a defined indication in the United States. The exact approval may depend on the cancer type, stage, mutation and previous treatment.

An off-label therapy is an approved medicine being considered outside its officially authorised indication. Off-label use may sometimes be supported by evidence, but it requires careful clinical judgment and may not be covered by insurance.

A clinical-trial option means that the alteration may meet the molecular eligibility requirements for a research study. It does not mean that the patient will automatically qualify because trials also consider age, organ function, previous treatment, stage and general health.

Regulatory approval also varies between countries. A medicine approved in the United States may have a different indication or access pathway in the UK, Canada, Australia, Europe or another region.

Read the Evidence Tier Carefully

Some reports place findings into tiers according to clinical significance. A high-tier alteration may have established treatment relevance, while a lower-tier finding may be based on limited studies or preclinical evidence.

These tier systems are not identical across laboratories. One report may use Roman numerals, while another may use labels such as strong clinical significance, potential clinical significance or uncertain significance.

The oncology team should therefore interpret the meaning of the tier rather than relying on the number alone. The strength of evidence must also be checked specifically for ATC.

Variant Allele Frequency Is a Laboratory Measurement

The report may show a percentage next to the mutation, known as the variant allele frequency. This percentage describes how often the altered sequence was detected among the DNA reads covering that position.

For example, if BRAF V600E has a variant allele frequency of 25%, approximately one quarter of the analysed DNA reads at that location contained the alteration. This does not mean that 25% of the body contains cancer or that there is a 25% chance of responding to treatment.

Variant allele frequency is influenced by tumor purity, normal cells in the sample, copy-number changes, tumor heterogeneity and technical factors. It must therefore be interpreted within the full report rather than used as a standalone measure.[6,8]

Coverage and Read Depth Affect Confidence

NGS reports may include sequencing depth or coverage. Read depth describes how many times a region of DNA was analysed. Adequate coverage helps the laboratory distinguish a genuine alteration from technical noise.

If an important region had poor coverage, the report may state that a mutation could not be reliably excluded. A negative result is more convincing when the relevant gene region was adequately covered and the sample passed all quality-control requirements.

Some reports provide a general statement such as “all reportable regions met minimum coverage.” Others identify individual genes or regions with limited analysis. These comments deserve attention because they define the boundaries of the test.

Read the Limitations Section Before Accepting a Negative Result

The limitations section may explain that the assay does not detect every type of mutation, fusion or copy-number change. It may also state that low tumor content reduced sensitivity or that RNA testing failed.

For example, a DNA panel may report no fusion detected while also explaining that only selected fusion breakpoints were covered. In that situation, a dedicated RNA fusion panel may still be appropriate.

A molecular report is not a complete map of every cancer cell. It is a technically defined analysis of a particular specimen using a particular assay. Understanding what the test could not evaluate is as important as understanding what it found.[1,6]

A Fictional Example of a BRAF-Positive Report

A simplified report may state that a core biopsy from the thyroid mass contained 50% viable tumor and passed DNA quality control. It may then report BRAF p.V600E, pathogenic, variant allele frequency 28%, with a note that the alteration has established treatment relevance in ATC.

This would mean that BRAF V600E was detected reliably in the tested specimen. It would not mean that the patient should automatically begin treatment without further review.

The oncology team would still consider stage, resectability, airway risk, previous therapy, organ function and the current regulatory indication before deciding whether dabrafenib plus trametinib is appropriate.[14,15]

The Report Is Data, Not the Complete Treatment Plan

A molecular report provides important evidence, but it cannot independently decide treatment. The oncologist, molecular pathologist, surgeon and radiation oncologist must translate the findings into a clinical plan.

When you receive the report, ask which finding is most important, how strong the evidence is and whether the result changes the immediate treatment pathway. A clearly interpreted report should help reduce confusion rather than add more technical language.

What Does Variant Allele Frequency Mean?

VAF Describes the Proportion of Sequencing Reads Carrying a Variant

Variant allele frequency, commonly shortened to VAF, is the proportion of DNA reads containing a particular alteration at the tested location.

If the laboratory reads one region of the BRAF gene 1,000 times and detects BRAF V600E in 300 of those reads, the reported VAF may be approximately 30%.

This is a measurement from the analysed specimen. It is not a direct measurement of the percentage of tumor in the patient’s body.[6,8]

VAF Is Influenced by Tumor Purity

A biopsy containing many normal or inflammatory cells may have a lower VAF because the tumor DNA is diluted by normal DNA. A tumor-rich specimen may show a higher VAF for the same alteration.

This means that a low VAF does not automatically indicate that the mutation is unimportant. A genuine BRAF V600E alteration detected at a lower level may still be clinically relevant if the assay is validated and the result is technically reliable.

The pathologist’s estimate of tumor-cell percentage helps the molecular team interpret whether the VAF is consistent with the composition of the sample.

Tumor Heterogeneity Can Also Affect VAF

If an alteration is present in nearly all tumor cells, its VAF may be higher than an alteration found only in a smaller subclone. However, the relationship is not exact because copy-number changes, normal-cell contamination and other biological factors can alter the percentage.

A lower VAF may sometimes suggest that the alteration is present in only part of the tumor, but this conclusion should not be made from the percentage alone.

The molecular pathologist must interpret VAF together with tumor purity, sequencing depth, copy-number status and the expected biology of the gene.

A High VAF Does Not Guarantee a Better Treatment Response

Patients may assume that a higher BRAF V600E percentage means that targeted therapy will work better. Current clinical practice does not support using VAF alone to predict the depth or duration of response.

A tumor with a high BRAF V600E VAF may still contain additional resistance mechanisms. A tumor with a lower but genuine VAF may still respond if an important population of cancer cells depends on BRAF signalling.

Treatment response is influenced by the entire molecular profile, tumor burden, disease distribution, previous treatment and the patient’s clinical condition.

VAF Does Not Indicate Cancer Stage

A 40% VAF does not mean stage IV disease, 40% body involvement or a 40% survival probability. Cancer stage is determined through anatomy, imaging and the presence or absence of distant metastases.

VAF should also not be confused with tumor-cell percentage. The pathologist estimates tumor percentage under the microscope, while VAF is calculated from sequencing reads.

These measurements are related but not identical.

Tissue VAF and Plasma VAF Are Not Directly Comparable

In tissue testing, VAF is influenced by the proportion of tumor cells in the biopsy. In liquid biopsy, VAF reflects the small amount of tumor DNA mixed with normal cell-free DNA in the bloodstream.

A plasma BRAF V600E VAF may therefore be much lower than the VAF reported from tumor tissue. This does not automatically mean that the mutation has become less important.

Changes in plasma VAF over time may provide useful information about molecular response, but they should be interpreted with imaging, symptoms and treatment history. Tissue and plasma percentages should not be compared as though they were measured in the same biological environment.[12]

VAF Is Helpful but Should Not Control the Decision Alone

Variant allele frequency can help the laboratory assess whether a finding is technically believable and whether it may be clonal or subclonal. It can also provide a baseline for selected monitoring strategies.

However, the most important question remains whether the alteration is pathogenic, actionable and supported by clinical evidence. VAF adds context, but it should not replace clinical interpretation.

What Does “BRAF V600E Not Detected” Actually Mean?

The Test Did Not Identify BRAF V600E in the Analysed Sample

A report stating BRAF V600E not detected means that the assay did not identify the mutation in the material that was tested and within the limits of that particular method.

In a high-quality specimen with adequate viable tumor and successful coverage, this result may strongly support that the tumor is BRAF V600E-negative.

However, the phrase does not always prove that the alteration is absent from every cancer cell in the body. The reliability of the conclusion depends on the sample and the test.[9,11]

The Sample Quality Must Be Reviewed

If the report also states that the specimen contained very little tumor, extensive necrosis or degraded DNA, the negative result may be less certain.

A test can only detect an alteration when enough tumor-derived genetic material is present. If the sample is mostly normal tissue or dead tumor, BRAF V600E may fall below the assay’s detection threshold.

In this situation, the team should not simply label the patient as definitively BRAF-negative. Another tissue block, repeat biopsy or alternative testing method may need to be considered.

The Method Used Also Matters

A negative immunohistochemical result means that convincing BRAF V600E protein staining was not seen. A negative PCR result means that the predefined DNA alteration was not detected. A negative broad NGS result means that the mutation was not identified within the sequencing assay’s coverage and sensitivity.

These are related but not identical statements. When immunohistochemistry is negative but the tissue is adequate, molecular testing may still be useful if the result would influence treatment.

Similarly, if a focused PCR test examined only V600E, the report should not be interpreted as proving that every possible BRAF alteration is absent.

A Negative Liquid Biopsy Requires Particular Caution

A plasma test may return negative because the tumor does not release enough detectable DNA into the bloodstream. This can happen even when BRAF V600E is clearly present in the tumor tissue.

A negative liquid biopsy is therefore less reliable for excluding a mutation than a positive result is for identifying one. If tissue is available, tissue-based testing should still be reviewed or completed.[12,13]

If tissue is inadequate and the plasma result is negative, the team must decide whether another biopsy is safe and likely to change treatment.

BRAF-Negative Does Not Mean No Molecular Driver Exists

A tumor without BRAF V600E may contain a RAS alteration, NTRK fusion, RET fusion, ALK fusion or another molecular abnormality. Some findings may have treatment relevance, while others may mainly explain tumor biology.[6,23–25]

This is why comprehensive DNA- and RNA-based profiling remains important after a negative rapid BRAF test. The investigation should not stop after one mutation has been excluded.

A BRAF-negative result changes the molecular pathway, but it does not end precision treatment assessment.

“Not Detected” Is Different From “Test Failed”

A successful negative test means that the assay worked but did not identify the alteration. A failed test means that the laboratory could not complete a reliable analysis.

Statements such as quantity not sufficient, insufficient tumor, failed quality control or RNA analysis unsuccessful should not be interpreted as negative mutation results.

The report should clearly distinguish between an absent finding and an unsuccessful test. When this distinction is unclear, the oncology team should contact the molecular laboratory for clarification.

What Should Happen After a Reliable Negative Result?

After a reliable BRAF V600E-negative result, the medical team should confirm that comprehensive DNA and RNA testing has been performed or is underway. The report should be reviewed for other actionable alterations and biomarkers.

At the same time, treatment planning should continue according to stage, resectability, airway safety, symptoms and overall health. The patient should not be left without a clinical plan simply because BRAF-directed therapy is not supported.

The correct interpretation is not that molecular treatment has failed. It is that one important target was not identified and the team must evaluate the remaining biological and clinical options.

What Is a Variant of Uncertain Significance?

A VUS Is a Genetic Change Whose Meaning Is Not Yet Clear

A variant of uncertain significance, usually shortened to VUS, is a change in a gene for which current scientific evidence is insufficient to determine whether it contributes to cancer growth. The laboratory has detected a genuine difference in the DNA sequence, but researchers do not yet know whether that difference is harmful, harmless or biologically relevant.[6,8]

A VUS is not the same as a pathogenic mutation. A pathogenic alteration has enough evidence showing that it affects the function of a gene and contributes to disease. A VUS remains uncertain because the available clinical, laboratory or population data are incomplete or conflicting.

Molecular reports may list one or several uncertain variants. Their presence does not mean that the cancer has several additional treatment targets. In many cases, these findings do not influence the immediate treatment plan.

Why Molecular Reports Contain Uncertain Findings

Every person has many natural differences in their DNA. Most of these differences are harmless. When a sequencing panel examines hundreds of cancer-related genes, it may identify rare variants that have not been studied extensively.

Some variants are uncommon even in large population databases. Others may affect a part of a protein whose function is not fully understood. A few may have been reported in cancer but without enough evidence to show that they actively drive tumor growth.

As molecular databases become larger, researchers may learn more about these alterations. Until then, the laboratory must avoid classifying them as pathogenic without sufficient evidence.

A VUS Should Not Be Treated as an Actionable Mutation

A variant of uncertain significance should not independently determine targeted treatment. The fact that a VUS appears in a well-known cancer gene does not mean that a medicine directed at that gene will help the patient.

For example, a report may list an unusual BRAF variant but classify it as uncertain. This finding should not automatically be treated as equivalent to BRAF V600E. The clinical evidence supporting dabrafenib and trametinib in ATC relates primarily to a confirmed pathogenic BRAF V600E alteration.[15–17]

The same principle applies to uncertain findings in RET, NTRK, ALK or other genes. A medicine should not be selected merely because the gene name appears on the report. The exact alteration, biological effect and evidence level must be reviewed.

A VUS Is Not Automatically a Passenger Mutation

A passenger mutation is understood to have little or no role in driving the cancer. A VUS is different because its biological effect is not yet known.

Some uncertain variants may later be shown to be harmless. Others may eventually be reclassified as pathogenic when more clinical or laboratory evidence becomes available.

Until that reclassification occurs, the safest approach is to treat the finding as uncertain. We should neither exaggerate its importance nor dismiss the possibility that its meaning may change in the future.

How Laboratories Classify a Variant

Molecular laboratories review several types of evidence before classifying a variant. They may examine whether the alteration has been reported in cancer databases, whether it occurs in healthy populations, whether it affects an important protein region and whether laboratory studies suggest that it changes protein function.[6,8]

They may also consider whether the alteration is repeatedly found in the same cancer type and whether patients with that finding have responded to a particular treatment. A computer prediction alone is not sufficient to establish clinical actionability.

Different laboratories may occasionally classify the same rare variant differently because they use different databases or interpret incomplete evidence in different ways. When the finding could influence treatment, review by a molecular pathologist or molecular tumor board may be helpful.

Can a VUS Be Reclassified Later?

Yes. A VUS can be reclassified when new evidence becomes available. It may be changed to pathogenic, likely pathogenic, likely benign or benign.

The original laboratory may issue an amended report, although recontact practices differ between institutions. Patients should not assume that every laboratory will automatically notify them many years later.

If an uncertain variant becomes clinically relevant during the course of treatment, the oncology team can ask the laboratory whether the classification has changed. Updated interpretation may also be considered when the disease progresses or when a new clinical trial targets that particular alteration.

Does a VUS Mean the Mutation Was Inherited?

A VUS identified through tumor sequencing does not automatically mean that the alteration is inherited. It may be present only in the cancer cells, or it may represent a normal constitutional variant present throughout the body.

Tumor-only testing cannot always distinguish these possibilities with certainty. If the variant occurs in a gene associated with an inherited cancer syndrome and the patient’s personal or family history raises concern, separate germline testing may be considered.

The result should be reviewed with a clinical geneticist or genetic counsellor when hereditary risk is a genuine possibility. Family members should not undergo testing solely because an unconfirmed VUS was listed on a tumor report.

What Should a Patient Ask About a VUS?

When your report contains a VUS, ask whether the finding has any established relevance to ATC, whether it changes treatment and whether the laboratory recommends future review. In most cases, the answer will be that the variant should be documented but should not control the present treatment plan.

When I explain a VUS to a patient, I describe it as a question identified by the laboratory rather than an answer. It may deserve future observation, but it should not be presented as a proven driver, treatment target or explanation for the entire cancer.

What If BRAF Immunohistochemistry and Sequencing Disagree?

Discordant Results Can Occur

BRAF V600E immunohistochemistry and molecular sequencing examine different biological features. Immunohistochemistry detects the altered protein within tumor cells, while PCR or NGS examines the underlying DNA sequence.[10,11]

Because these methods are different, their results may occasionally disagree. Immunohistochemistry may appear positive while sequencing does not detect BRAF V600E. In another case, sequencing may identify the mutation even though the tissue staining was negative or uncertain.

A disagreement does not automatically mean that one laboratory made an error. It means that the tissue, methods and technical limitations require careful review.

Low Tumor Content Can Affect Sequencing

If the specimen contains only a small number of viable tumor cells, the altered DNA may be heavily diluted by normal DNA. BRAF V600E may then fall below the sequencing assay’s detection limit.

In such a situation, the pathologist may see convincing staining in a small group of cancer cells, while the molecular test reports the mutation as not detected. Reviewing the tumor-cell percentage and sequencing quality can help explain the difference.[11]

A second tumor-rich block or another biopsy specimen may provide a clearer molecular result.

Poor Tissue Preservation Can Affect Immunohistochemistry

Immunohistochemistry depends on preservation of the protein within the tissue. Delayed fixation, extensive necrosis, damaged tissue or technical staining problems may weaken or eliminate the expected signal.

The DNA may still be sufficiently preserved for sequencing. A molecular assay can therefore detect BRAF V600E even when immunohistochemical staining is negative or equivocal.

This possibility is one reason why a negative rapid stain may require molecular testing when the result would affect treatment.

Weak or Patchy Staining May Be Difficult to Interpret

True BRAF V600E staining is generally expected within the cytoplasm of the tumor cells, but weak, patchy or uneven staining can create uncertainty. Background staining in macrophages, inflammatory cells or necrotic material may also complicate interpretation.[10,11]

The pathologist must confirm that the stained cells are malignant and that the pattern fits validated criteria. A few isolated stained cells should not automatically be called positive without considering the complete tissue appearance.

When the staining is equivocal, PCR or NGS can provide direct evidence from the DNA.

Tumor Heterogeneity May Produce Different Results

A mutation may be present in one part of the tumor but absent or less common in another. If immunohistochemistry and sequencing were performed on different tissue blocks, they may have analysed different tumor-cell populations.

For example, a specimen may contain both differentiated papillary thyroid carcinoma and anaplastic carcinoma. One component may show stronger BRAF V600E expression or a higher proportion of mutation-positive cells than the other.

The pathologist should therefore confirm which component was tested and whether the same tissue area was used for both methods.

Different Assays Have Different Detection Limits

Not all PCR and NGS tests have the same sensitivity. One assay may detect a low-level mutation that another method cannot identify. The panel may also have technical limitations related to sequencing depth or coverage.

The report should state the assay’s detection threshold and whether the specimen met quality requirements. These details help determine how much confidence should be placed in a negative result.

The exact BRAF alteration also matters. The VE1 antibody is designed for BRAF V600E. It does not reliably represent every other possible BRAF mutation or fusion.

The Pathology and Molecular Teams Should Review the Case Together

When results disagree, the most appropriate response is a coordinated review. The pathologist can re-examine the slides, confirm the stained tumor cells and estimate viable tumor content. The molecular laboratory can review sequencing depth, variant reads and quality-control findings.[10,11]

If enough tissue remains, the test may be repeated using another block or a different method. A focused high-sensitivity PCR assay may help resolve an uncertain NGS result, while broader sequencing may clarify an equivocal immunohistochemical finding.

The team should not select whichever result appears more convenient for treatment. The purpose of repeat review is to determine which interpretation is technically and biologically most reliable.

Treatment Should Be Based on a Confirmed and Clinically Valid Result

When BRAF-directed treatment is being considered, the oncology team should ensure that the mutation has been confirmed through a reliable and clinically accepted method. This is particularly important when the first result is weak, equivocal or technically limited.[14,15]

Urgent treatment decisions may occasionally need to begin before every testing question has been resolved, especially in rapidly progressive disease. Even then, the decision should involve the oncologist, molecular pathologist and wider ATC team.

For the patient, a discordant report does not necessarily mean that the treatment opportunity has been lost. It means that the evidence must be clarified before the result is used to guide a major clinical decision.

BRAF Is Important, but It Is Not the Entire ATC Genome

One Actionable Mutation Does Not Describe the Whole Tumor

BRAF V600E is one of the most clinically important molecular findings in ATC because it may guide a defined targeted-treatment pathway. However, ATC usually develops through the accumulation of several molecular abnormalities rather than one alteration alone.[3–6]

A BRAF-positive tumor may also contain changes involving TP53, the TERT promoter, CDKN2A, PIK3CA or other genes. These additional findings may contribute to rapid growth, dedifferentiation, invasion or resistance.

This is why comprehensive profiling remains useful even after BRAF V600E has been identified. The rapid result may guide the first treatment decision, while the broader profile provides a more complete biological picture.

BRAF-Negative ATC May Still Contain a Clinically Relevant Driver

When BRAF V600E is not detected, the tumor may be driven by another alteration. RAS mutations are found in a proportion of ATCs, and uncommon fusions involving NTRK, RET or ALK may also occur.[3,6]

Some of these findings may support an approved tumor-agnostic treatment or a selective targeted approach. Others may be relevant mainly for clinical trials.

A negative BRAF test should therefore lead to broader molecular investigation rather than the conclusion that precision oncology has no role.

Some Alterations Explain Aggressive Biology but Do Not Yet Provide a Treatment

TP53 and TERT promoter alterations are common in aggressive thyroid cancers. They may help explain how a differentiated thyroid cancer became anaplastic, but they do not currently provide the same routine mutation-specific treatment pathway as BRAF V600E.[2–6]

This distinction is important when reading a report. A pathogenic alteration may be biologically meaningful without being directly actionable.

Patients may feel disappointed when a major mutation has no matched medicine. However, understanding the tumor biology can still support prognosis research, trial selection and future treatment development.

Rare Findings Can Sometimes Be More Clinically Important Than Common Ones

A molecular alteration does not need to be common to matter. An NTRK or RET fusion may occur in only a small proportion of ATCs, yet its identification can substantially change the treatment discussion.[23–25]

This is one reason why RNA-inclusive profiling is valuable. A limited mutation panel may detect common DNA variants but fail to identify an uncommon fusion.

The clinical importance of a finding therefore depends on actionability and evidence, not only on prevalence.

The Molecular Report Should Separate Different Levels of Relevance

When we review a complete ATC report, the findings can be understood in several groups. Some have established treatment relevance, such as BRAF V600E in the appropriate clinical setting. Others may support a tumor-agnostic targeted treatment, a clinical trial or an off-label discussion.

A third group contains alterations that mainly explain aggressive tumor biology. A fourth group contains uncertain variants that should not determine treatment.

The report becomes useful when these levels are clearly separated. A long list of genes without clinical interpretation can create confusion rather than improve care.

The Whole Treatment Plan Still Depends on the Patient

Molecular profiling examines the cancer, but it does not measure breathing difficulty, swallowing ability, nutritional decline, pain, functional strength or the patient’s preferences. These clinical factors may be as urgent as the genetic findings.

If your tumor contains an actionable alteration, the result may create a valuable treatment opportunity. The oncology team must still determine whether that treatment fits with surgery, radiation, airway management, organ function and the overall goals of care.

The complete molecular profile helps us understand the tumor more accurately. The complete clinical assessment helps us decide what should be done for the person living with it.[1,30]

RAS Alterations in Anaplastic Thyroid Cancer

What Are RAS Genes?

RAS refers to a family of genes that includes NRAS, HRAS and KRAS. These genes produce proteins that help transmit growth signals from the cell surface toward pathways controlling division, survival and differentiation.[2,3]

Under normal conditions, a RAS protein becomes active for a limited period and then returns to an inactive state. A cancer-associated RAS mutation can interfere with this control, allowing the protein to continue sending growth signals even when the cell has not received a normal instruction to divide.

RAS can activate more than one signalling route. It may stimulate the MAPK pathway through RAF, MEK and ERK, while also influencing the PI3K–AKT pathway. This wider signalling effect helps explain why RAS-driven tumors may behave differently from tumors driven mainly by BRAF V600E.[2–4]

Which RAS Alterations Are Found in Thyroid Cancer?

The three principal RAS genes are NRAS, HRAS and KRAS. In thyroid tumors, NRAS alterations are commonly discussed, although mutations involving HRAS and KRAS can also occur.[2,3]

A molecular report may describe an alteration such as NRAS Q61R, HRAS Q61R or KRAS G12D. The letters and numbers identify the exact amino-acid change within the RAS protein, just as V600E identifies a specific change in BRAF.

The exact variant matters because not every RAS alteration has the same biological effect. A pathogenic activating mutation is different from an uncertain RAS variant that has not been shown to drive tumor growth.

RAS May Act as an Early Driver in Thyroid Tumor Evolution

RAS mutations can occur in differentiated thyroid cancers, particularly tumors with follicular-pattern biology. When such a tumor later becomes poorly differentiated or anaplastic, the original RAS driver may remain present.[2,3]

The anaplastic component may then acquire additional alterations involving TP53, the TERT promoter, PIK3CA, PTEN or other genes. These later changes can contribute to loss of differentiation, increased invasion and resistance to normal cellular controls.[3–5]

This pattern is similar to BRAF-positive progression in one important way: an early driver remains present while additional abnormalities make the tumor more aggressive. However, BRAF-driven and RAS-driven ATCs are not biologically identical.

RAS-Driven and BRAF-Driven ATC Often Represent Different Molecular Pathways

BRAF V600E and RAS mutations are often found in different molecular subgroups of thyroid cancer. A tumor driven by BRAF V600E usually has direct activation of the MAPK pathway at the level of BRAF, while a RAS-mutated tumor can signal through several downstream pathways.[3,4]

This distinction matters clinically because the established ATC-specific targeted-treatment pathway for dabrafenib plus trametinib applies to confirmed BRAF V600E-positive disease. A pathogenic RAS mutation should not be treated as though it were BRAF V600E.[15–17]

If your report shows a RAS alteration, the oncologist must interpret the exact gene, variant and available evidence. The presence of RAS may help explain the tumor’s biology, but it does not automatically identify an approved RAS-specific treatment for ATC.

Is RAS Directly Actionable in ATC?

At present, RAS alterations do not provide the same established mutation-specific treatment pathway in ATC as BRAF V600E. Several medicines that affect RAS or downstream pathways are being studied across different cancers, but their relevance depends on the exact RAS variant, tumor type and clinical-trial setting.

Some KRAS mutations have targeted medicines in other cancers, but those results should not be broadly applied to NRAS- or HRAS-mutated ATC. The gene name alone is not enough. The exact alteration and the clinical evidence in that disease must match.

For many patients with RAS-mutated ATC, the finding may therefore be more useful for molecular classification, research and clinical-trial evaluation than for selecting a currently established routine treatment.

Why Broader Profiling Still Matters in a RAS-Positive Tumor

A RAS mutation may be an important driver, but it does not describe the entire tumor. The report should still be reviewed for TP53, TERT promoter, PI3K-pathway abnormalities, gene fusions and immune-related biomarkers.[3–6]

Additional findings may help explain why the tumor has become anaplastic or why it is progressing rapidly. In some cases, another alteration may provide a more clinically useful treatment opportunity than the RAS mutation itself.

This is why molecular reports should not be interpreted by selecting only the first pathogenic gene listed. The complete profile and the evidence attached to each alteration must be considered together.

What a RAS-Positive Result Means for the Patient

If your report identifies a pathogenic RAS mutation, it suggests that abnormal RAS signalling is contributing to the cancer’s growth. It may help the medical team understand the molecular pathway of the tumor and consider suitable clinical trials.

The result does not mean that BRAF-directed treatment will work. It also does not determine whether surgery, radiation, immunotherapy or another systemic treatment is appropriate.

When I explain a RAS-positive result, I separate biological importance from treatment actionability. The mutation may be a genuine driver, but the treatment decision must still be based on the evidence available for that exact alteration and the patient’s complete clinical situation.

TP53: A Key Alteration in Anaplastic Transformation

TP53 Normally Protects Cells From Serious DNA Damage

TP53 is a tumor-suppressor gene that provides instructions for producing the p53 protein. This protein helps protect the body from cells that have developed serious genetic damage.

When DNA damage occurs, p53 may temporarily stop cell division so that repair can take place. If the damage is too severe, p53 can help direct the cell toward controlled death. This reduces the chance that a highly abnormal cell will continue dividing.

For this reason, p53 is sometimes described as an important guardian of genomic stability. The phrase is useful, but the biological process is more complex than one protein acting alone.

What Happens When TP53 Function Is Lost?

A pathogenic TP53 alteration can reduce or eliminate the protective function of p53. Damaged cells may then continue dividing instead of stopping for repair or being removed.

As these cells multiply, they may accumulate additional genetic abnormalities. This increasing instability can support faster growth, loss of normal thyroid-cell features and resistance to cellular control mechanisms.[2–5]

TP53 alterations are much more characteristic of anaplastic and poorly differentiated thyroid cancers than of many ordinary differentiated thyroid tumors. Their presence supports the concept that loss of p53 function is an important step in anaplastic transformation.[2,3]

TP53 May Be Acquired During Progression

A differentiated thyroid cancer may initially contain an early driver such as BRAF V600E or RAS. During progression, a population of tumor cells may acquire a TP53 alteration.

The differentiated and anaplastic components may therefore share the original BRAF or RAS mutation, while the TP53 change is more prominent or present only in the anaplastic component.[3–5]

This pattern helps explain how one tumor can contain areas with different levels of aggressiveness. It also shows why molecular evolution is usually a stepwise process rather than the result of one sudden genetic event.

How TP53 May Appear on a Pathology Report

TP53 may be evaluated through DNA sequencing, immunohistochemistry or both. Sequencing identifies a change in the TP53 gene, while immunohistochemistry examines the pattern of p53 protein expression within tumor cells.

Some TP53 mutations cause strong accumulation of abnormal p53 protein, producing intense staining. Other alterations lead to complete loss of detectable protein. A normal-looking staining pattern does not always exclude a TP53 mutation, and an unusual pattern must be interpreted by the pathologist.

DNA sequencing generally provides more precise information about the exact genetic alteration. Immunohistochemistry can add useful pathological context but should not always be treated as a perfect substitute for molecular testing.

TP53 Is Biologically Important but Not Routinely Actionable

A TP53 alteration may be one of the most important explanations for why a thyroid cancer has become highly aggressive. However, biological importance and treatment actionability are different.

There is currently no established TP53-directed treatment pathway in ATC comparable to dabrafenib and trametinib for BRAF V600E-positive disease. A report showing pathogenic TP53 should therefore not create the expectation that a proven p53-restoring medicine is routinely available.

Research is examining methods to reactivate altered p53, exploit vulnerabilities created by TP53 loss and combine treatment with other pathway inhibitors. These strategies remain investigational and should be described according to the level of clinical evidence available.

Does TP53 Predict the Exact Outcome?

TP53 alterations are associated with aggressive tumor biology, but they cannot independently predict how long a patient will survive or how the tumor will respond to treatment.

The outcome still depends on stage, tumor anatomy, distant metastases, airway involvement, resectability, molecular targets, performance status and access to timely multidisciplinary care.

A pathogenic TP53 result may help explain the biology of the disease, but it should not be used alone to give a precise prognosis. Population associations cannot determine the course of one individual patient.

Can TP53 Contribute to Treatment Resistance?

Loss of normal p53 function may allow genetically damaged tumor cells to survive under stress. This can contribute to genomic diversity and create populations that are more difficult to control.

However, resistance is rarely caused by TP53 alone. ATC may use several pathways, and treatment pressure can select resistant clones with additional abnormalities.

If a BRAF-positive tumor also contains TP53 loss, BRAF/MEK therapy may still produce a meaningful response. The TP53 alteration does not automatically cancel the value of the BRAF target, but it may form part of a more complex molecular background.[5,17]

What a TP53 Result Means for the Patient

If your report identifies a pathogenic TP53 alteration, it usually means that an important tumor-suppressor system has been disrupted. The finding may help explain anaplastic transformation and aggressive behaviour.

It does not usually identify an approved TP53-specific medicine. The oncology team should therefore focus on other actionable alterations, standard multimodality treatment and suitable clinical trials.

When we discuss TP53, we should be honest about both sides of the result. It is scientifically important, but its immediate treatment value is currently more limited than that of BRAF V600E.

TERT Promoter Mutations in Anaplastic Thyroid Cancer

What Is TERT?

TERT stands for telomerase reverse transcriptase. This gene provides instructions for producing a central part of the telomerase enzyme.

Telomerase helps maintain structures called telomeres at the ends of chromosomes. Telomeres protect chromosome ends, but they become shorter each time many normal cells divide.

When telomeres become critically short, normal cells usually stop dividing. This is one of the natural limits placed on repeated cell replication.

How Cancer Cells Use Telomerase

Cancer cells need to overcome normal limits on cell division. Increased telomerase activity can help maintain telomeres, allowing malignant cells to continue replicating for a longer period.

A TERT promoter mutation does not change the main protein-coding sequence of the gene. Instead, it alters a regulatory region that controls how actively the gene is used.

The mutation can increase TERT expression and support continued tumor-cell division. This does not make the cells literally immortal in every situation, but it can remove one important barrier to prolonged cancer growth.[2–4]

What Does “Promoter Mutation” Mean?

A promoter is a regulatory region of DNA located near a gene. It helps determine when and how strongly that gene is activated.

A mutation in the TERT promoter can create new binding sites for regulatory proteins, causing the tumor to produce more telomerase than it normally would.

The report may describe alterations such as TERT promoter C228T or C250T. These names identify specific DNA changes in the regulatory region. They are different from mutations occurring within the protein-coding portion of a gene.

TERT Promoter Mutations Are Associated With Aggressive Thyroid Cancer

TERT promoter alterations occur more often in aggressive thyroid cancers than in many low-risk differentiated tumors. They are frequently identified in poorly differentiated and anaplastic thyroid carcinoma.[2–4]

The mutation may be present together with BRAF V600E or RAS. When early growth-pathway activation and increased telomerase activity occur in the same tumor, they may contribute to a more aggressive biological pattern.

This association does not mean that every thyroid cancer with a TERT promoter mutation will become anaplastic. The transformation still depends on the wider molecular and clinical context.

BRAF and TERT May Cooperate Biologically

BRAF V600E activates MAPK signalling, while a TERT promoter mutation can support ongoing cellular replication. When both alterations are present, they may work through different biological mechanisms that together support tumor progression.[2–4]

In a differentiated thyroid cancer, this combination may be associated with more aggressive features than either alteration alone. In ATC, the finding may help explain how the tumor evolved, but it does not replace staging or direct measurement of the disease.

The presence of both BRAF V600E and TERT promoter alteration also does not mean that BRAF-directed treatment is ineffective. If BRAF V600E is confirmed, the mutation may remain clinically actionable despite the additional TERT finding.

Is a TERT Promoter Mutation Directly Treatable?

At present, a TERT promoter mutation does not provide an established routine targeted-treatment pathway in ATC. Medicines designed to inhibit telomerase or exploit telomere-related vulnerabilities remain areas of research.

A molecular report may classify the alteration as pathogenic because it contributes to tumor biology. Pathogenic, however, does not automatically mean that an approved targeted medicine exists.

For most patients, TERT promoter status is currently more useful for understanding tumor evolution and aggressive behaviour than for selecting a standard mutation-specific treatment.

Does TERT Determine Prognosis by Itself?

TERT promoter alterations are associated with aggressive thyroid-cancer biology, but the result cannot independently determine an individual patient’s outcome.

The stage, airway involvement, distant metastases, ability to perform surgery, response to systemic treatment and overall functional condition remain central to prognosis.

A molecular alteration may add context, but it should not be used to give a fixed survival prediction. The treating team must interpret the finding together with all other clinical information.

Why TERT Should Still Be Included in Comprehensive Profiling

Even though TERT is not routinely targetable, identifying the mutation can help reconstruct the molecular history of the tumor. It may show that the cancer has acquired mechanisms supporting prolonged growth in addition to an early BRAF or RAS driver.[2–4]

The finding can also contribute to research classification and may become relevant to future clinical trials. Recording it now creates a more complete molecular baseline if the disease later progresses or new treatments become available.

For the patient, the most important distinction is clear. A TERT promoter mutation may explain part of the tumor’s aggressive biology, but it does not currently offer the same direct treatment pathway as BRAF V600E.

PI3K–AKT–mTOR Pathway Alterations in Anaplastic Thyroid Cancer

What Is the PI3K–AKT–mTOR Pathway?

The PI3K–AKT–mTOR pathway is a network of proteins that helps cells regulate growth, metabolism, survival and the use of nutrients. Under normal conditions, the pathway becomes active only when the cell receives an appropriate signal.

The pathway can be simplified as:

PI3K → AKT → mTOR

PI3K receives and passes the growth signal. AKT then supports cell survival and metabolism, while mTOR helps regulate protein production, nutrient sensing and cellular growth.[2–4]

This pathway is necessary for healthy cells. The problem begins when a mutation, gene loss or other abnormality keeps the pathway continuously active within a cancer cell.

Which Genes in This Pathway May Be Altered?

ATC may contain abnormalities involving PIK3CA, PTEN, AKT, TSC1, TSC2 or other genes connected to PI3K–AKT–mTOR signalling.[2–4]

PIK3CA produces one part of the PI3K enzyme. An activating PIK3CA mutation may increase pathway signalling and support cancer-cell growth.

PTEN normally acts as a brake on the pathway. When PTEN is lost or inactivated, PI3K–AKT signalling may become more active because an important regulatory control has been removed.

Changes involving TSC1 or TSC2 can also affect mTOR regulation. However, the meaning of each alteration depends on the exact variant, the tumor type and the strength of clinical evidence.

These Alterations May Be Acquired During Tumor Progression

A differentiated thyroid cancer may initially be driven by BRAF V600E or RAS. During progression, some tumor cells may acquire an additional PI3K-pathway abnormality.[3,4,26]

This creates more than one growth route. A BRAF-positive tumor may use MAPK signalling while another group of tumor cells also relies on PI3K–AKT–mTOR activity.

The presence of parallel pathways can contribute to rapid growth and may help some tumor cells survive when one signalling route is blocked. This is one possible explanation for why a tumor may initially respond to targeted therapy and later progress.

PI3K-Pathway Alterations May Differ Between Tumor Sites

Some studies of advanced thyroid cancer have found that alterations involving PIK3CA or AKT can differ between the primary tumor and metastatic lesions.[26]

An early driver such as BRAF may remain present across several sites, while later pathway abnormalities appear only in selected tumor regions. This reflects tumor heterogeneity and ongoing molecular evolution.

If one lesion grows while other sites are responding, the oncology team may consider whether the progressing area has developed a different molecular profile. Repeat biopsy may occasionally be useful when the result could influence the next treatment decision.

Are These Alterations Directly Treatable?

Medicines that affect PI3K, AKT or mTOR have been developed and studied in several cancers. However, an alteration in this pathway does not automatically establish that one of these medicines will benefit a patient with ATC.

The exact gene and mutation must be considered. An activating PIK3CA mutation, PTEN loss and an uncertain AKT variant do not have identical meanings.

At present, PI3K–AKT–mTOR findings in ATC are often more useful for understanding tumor biology or identifying a clinical-trial opportunity than for selecting a routine mutation-specific treatment.[2–4]

A report should therefore avoid presenting every pathway alteration as an established target. The level of evidence must be stated clearly.

Why These Findings Still Matter

Even when no approved treatment is available, PI3K-pathway abnormalities may help explain why the cancer has become aggressive or why resistance has developed.

They may also support eligibility for a molecularly selected clinical trial. As new medicines and combination strategies are studied, a previously non-actionable alteration may become more relevant.

When I explain this result to a patient, I describe it as an important piece of the tumor’s biological map. It may not determine the immediate treatment, but it can help us understand the wider signalling system that is supporting the cancer.

What the Result Means for the Patient

If your report identifies a pathogenic alteration involving PIK3CA, PTEN, AKT or mTOR-related genes, the finding should be reviewed by a molecular pathologist or molecular tumor board.

The team should ask whether the alteration is known to activate the pathway, whether it is present in a large tumor-cell population and whether any clinical evidence supports treatment in ATC.

The result should not be interpreted in isolation. Its clinical meaning depends on the complete molecular profile, tumor stage, treatment history and available clinical trials.

NTRK Fusions in Anaplastic Thyroid Cancer

What Are NTRK Genes?

The NTRK gene family includes NTRK1, NTRK2 and NTRK3. These genes produce TRK proteins that normally help regulate the development and function of nerve cells.

In a healthy cell, TRK signalling is controlled by specific growth factors. A cancer-associated NTRK fusion can remove this normal control and cause the TRK protein to remain active.

The continuously active fusion protein can stimulate pathways involved in cell growth and survival. In this setting, the fusion may act as a true oncogenic driver.

What Is an NTRK Gene Fusion?

An NTRK fusion occurs when part of an NTRK gene becomes joined to part of another gene. The abnormal connection produces a fusion protein that can send repeated growth signals.

The molecular report may show a result such as:

ETV6–NTRK3 fusion detected

The first gene is the fusion partner, while NTRK3 provides the active kinase portion. Other NTRK fusion partners are also possible.

The exact fusion should be classified as pathogenic or likely pathogenic before it is used to guide treatment. An uncertain alteration involving an NTRK gene is not automatically equivalent to a confirmed oncogenic fusion.

NTRK Fusions Are Rare but Clinically Important

NTRK fusions are uncommon in ATC, but rarity does not reduce their importance when they are present. A confirmed NTRK fusion may identify a tumor that is highly dependent on TRK signalling.[6,23]

This can create a treatment opportunity with a TRK inhibitor in an appropriate patient. Such treatment is described as tumor-agnostic because eligibility may be based on the molecular alteration rather than only on the organ where the cancer began.

However, tumor-agnostic does not mean that every patient will respond in the same way. The evidence may include several cancer types, while the number of ATC patients within those studies may be small.

Why RNA Testing Is Important

RNA-based sequencing is particularly valuable for detecting NTRK fusions. It examines the abnormal fusion transcript produced by the tumor and can confirm that the fusion is actively expressed.

Some DNA panels can detect NTRK fusions, but their performance depends on which breakpoint regions and fusion partners are included. A DNA-only test may miss a fusion when the breakpoint lies outside the covered region.

If your report states that no actionable mutation was found, you should check whether RNA fusion testing was completed successfully. A negative DNA panel does not always exclude an NTRK fusion.

How NTRK Fusions May Be Confirmed

When an NTRK fusion is identified by NGS, the laboratory may consider whether further confirmation is needed. This may depend on the assay, fusion partner and quality of the sequencing result.

Pan-TRK immunohistochemistry can be used as a screening test because it detects TRK protein expression. However, staining alone may not identify the exact NTRK gene or fusion partner.

A positive screening result may therefore require confirmation through RNA sequencing, PCR or another validated molecular method before treatment is selected.

What Clinical Evidence Is Available?

Larotrectinib has shown meaningful activity in patients with TRK fusion-positive thyroid cancers.[23] These studies support the biological importance of confirmed NTRK fusions and the potential value of TRK-directed therapy.

However, the evidence should be interpreted carefully for ATC. Thyroid-cancer studies may include several histological types, and the ATC subgroup may be small.

We should therefore state that NTRK-directed treatment can be clinically relevant in selected fusion-positive patients without suggesting that it guarantees long-term control or cure.

Resistance Can Still Develop

A tumor may initially respond to a TRK inhibitor and later develop resistance. The cancer can acquire a new alteration within the NTRK kinase region or activate another growth pathway.

If progression occurs after an initial response, repeat tissue or liquid-biopsy testing may help identify the resistance mechanism. The result may support another treatment strategy or clinical-trial option.

Resistance does not mean that the original fusion result was incorrect. It means that the cancer continued evolving under treatment pressure.

What an NTRK Fusion Means for the Patient

If your ATC is BRAF-negative but a pathogenic NTRK fusion is detected, the finding may significantly change the treatment discussion. The oncologist can assess whether a TRK inhibitor is appropriate and available within the patient’s country and clinical situation.

The result must still be considered with airway safety, stage, resectability, organ function and other treatments. A molecular target does not remove the need for urgent multidisciplinary care.

For a patient with a rare cancer, this is one of the clearest examples of why broad RNA-inclusive testing matters. A rare molecular finding may provide information that a focused BRAF test could never reveal.

RET Fusions in Anaplastic Thyroid Cancer

What Is RET?

RET is a gene that produces a receptor protein involved in cell growth and development. Under normal conditions, the RET protein becomes active only after receiving an appropriate signal at the cell surface.

A RET fusion can remove this normal control. Part of the RET gene becomes joined to another gene, producing an abnormal protein that may remain continuously active.

This persistent signalling can stimulate MAPK and PI3K-related pathways and support tumor growth.

RET Fusion and RET Mutation Are Not the Same

A RET fusion occurs when RET becomes joined to another gene. A RET mutation is a change within the RET gene sequence itself.

This distinction is important because different thyroid cancers may contain different types of RET alterations. RET mutations are strongly associated with medullary thyroid carcinoma, while RET fusions are more commonly discussed in follicular-cell-derived thyroid cancers.

If an ATC report lists RET, the oncology team must identify whether the finding is a fusion, mutation, amplification or uncertain variant. The gene name alone does not determine treatment.

RET Fusions Are Uncommon in ATC

RET fusions are rare in ATC, but they may have substantial clinical importance when present.[6,24,25]

A fusion-positive tumor may be dependent on continuous RET signalling. Selective RET inhibitors have been developed to block this abnormal pathway.

Because the alteration is uncommon, it may be missed when testing is limited to BRAF, RAS and a small group of common mutations. Comprehensive DNA and preferably RNA analysis increases the chance of detecting it.

RNA Testing Can Improve RET Fusion Detection

RET can fuse with several different partner genes. The breakpoint may occur in a region that is difficult for some DNA panels to analyse completely.

RNA sequencing can detect the abnormal fusion transcript and show that the tumor is actively producing the altered RET message. This can strengthen confidence that the fusion is a genuine driver.

A report stating that RET fusion testing was negative should therefore be reviewed to determine whether RNA analysis was included and whether it passed quality control.

Why Selective RET Inhibition Matters

Earlier medicines affecting RET also inhibited several other kinases, which could increase off-target effects. Selective RET inhibitors were designed to block RET more precisely.

Selpercatinib has demonstrated activity in RET-altered thyroid cancers, including RET fusion-positive disease.[24,25] This evidence supports the importance of identifying a confirmed RET fusion.

However, the strength of evidence specifically in ATC may be more limited than in larger thyroid-cancer groups. Treatment decisions should therefore account for the histological type, regulatory indication, access and the patient’s complete clinical condition.

A RET Fusion Must Be Interpreted Precisely

The report should identify the fusion partner, classification and method of detection. A known in-frame fusion retaining the RET kinase region is more likely to be oncogenic than an uncertain rearrangement with unclear biological effect.

A molecular tumor board may be helpful when the fusion is unusual or when the treatment evidence is not straightforward.

The team should also review whether the result was obtained from the primary tumor or a metastatic lesion, especially if the disease shows mixed behaviour between different sites.

Resistance to RET-Directed Therapy May Occur

A RET fusion-positive tumor may respond to selective RET inhibition and later develop resistance. New alterations can affect the RET protein itself or activate another signalling pathway.

Repeat molecular testing may help identify these changes when the cancer progresses. This information may support a different RET inhibitor, another targeted approach or a clinical trial.

As with BRAF and NTRK treatment, a molecular response does not guarantee permanent control. Tumor evolution continues during therapy.

What a RET Fusion Means for the Patient

If a pathogenic RET fusion is detected, the finding may provide a meaningful targeted-treatment opportunity. The oncology team should confirm the exact alteration and review whether selective RET inhibition is appropriate within the patient’s treatment setting.

A RET fusion does not independently determine whether surgery, radiation or other systemic treatment is needed. It is one part of the complete treatment plan.

When I explain this finding, I emphasise that rare does not mean unimportant. The fusion may occur in only a small proportion of ATC tumors, but for the individual patient who carries it, the result may substantially change the available treatment discussion.

ALK and Other Rare Gene Fusions in Anaplastic Thyroid Cancer

What Is ALK?

ALK stands for anaplastic lymphoma kinase. It is a gene that produces a receptor protein involved in the growth and development of certain cells.

In normal tissue, ALK activity is carefully controlled. In some cancers, part of the ALK gene becomes joined to another gene, creating an abnormal fusion protein that remains active without the usual external signal.

This continuous activity can stimulate pathways such as MAPK and PI3K–AKT, allowing cancer cells to grow and survive. When a tumor depends strongly on this abnormal signalling, the ALK fusion may function as an oncogenic driver.[32]

What Is the STRN–ALK Fusion?

One of the ALK alterations identified in aggressive thyroid cancer is the STRN–ALK fusion. In this rearrangement, part of the STRN gene becomes connected to the region of ALK that produces its active kinase component.[32]

The fusion can create continuous ALK signalling and promote tumor growth. Foundational laboratory research showed that STRN–ALK can transform cells and may represent a potential therapeutic target in selected aggressive thyroid cancers.[32]

However, detecting the word ALK on a molecular report is not enough. The report must identify whether the finding is a confirmed fusion, a point mutation, an amplification or a variant of uncertain significance. These findings do not have the same biological or treatment meaning.

ALK Fusions Are Rare in ATC

ALK fusions occur in only a small proportion of aggressive thyroid cancers. Their rarity means that they may not be detected in smaller genomic studies and may be missed when testing is limited to common mutations such as BRAF and RAS.[6,32]

Although uncommon, a confirmed ALK fusion can be important for the individual patient. A rare alteration may provide more treatment relevance than a common alteration that has no matched therapy.

This is another reason why molecular testing should examine gene fusions rather than only point mutations. A BRAF-negative result does not exclude the possibility of a rare fusion-driven tumor.

Why RNA Testing Can Help Detect ALK Fusions

ALK can fuse with different partner genes, and the joining point may occur within a region that is difficult for some DNA panels to cover fully.

RNA-based sequencing examines the fusion message produced by the tumor. It can confirm that the abnormal ALK transcript is actively expressed and may provide a clearer identification of the fusion partner.

A DNA panel may report that no ALK rearrangement was detected, but the strength of that result depends on how the test was designed. If RNA analysis was not included, some fusions may remain undetected.

When I review a BRAF-negative molecular report, I therefore check whether RNA fusion testing was performed successfully rather than assuming that DNA sequencing alone excluded every possible rearrangement.

Can an ALK Fusion Be Treated?

ALK inhibitors are established treatments in some other cancers, particularly selected lung cancers with confirmed ALK rearrangements. Their role in ATC is much less clearly defined because ALK-positive ATC is rare and the available evidence is limited.

A confirmed pathogenic ALK fusion may support discussion at a molecular tumor board, referral to a specialist centre or consideration of an appropriately designed clinical trial. Selected off-label treatment may occasionally be discussed, but it should not be presented as a routine or proven ATC standard.

Evidence from laboratory studies or individual cases cannot establish that every ALK-positive ATC will respond. The treatment decision must consider the exact fusion, available clinical evidence, previous therapy, organ function and the urgency of the disease.

Other Rare Molecular Fusions May Also Occur

Comprehensive molecular profiling may occasionally identify other uncommon gene rearrangements. Some may activate known cancer pathways, while others may have uncertain biological significance.

A rare fusion should be evaluated carefully. The molecular team should confirm that the fusion is structurally capable of producing an active protein, that it is expressed by the tumor and that clinical evidence exists for targeting it.

A computer-generated report may list medicines associated with the same gene in other cancers. This does not prove that the treatment will work in ATC. Evidence from another tumor type must be separated from evidence directly obtained in thyroid cancer.

What an ALK or Rare Fusion Result Means for the Patient

If your report identifies a confirmed pathogenic ALK fusion, the finding may provide a possible treatment direction, particularly when no more established target such as BRAF V600E is present.

The result should be reviewed by a molecular pathologist and an oncologist experienced in ATC. They should confirm the test method, exact fusion partner, evidence level and whether an appropriate clinical trial or targeted-treatment pathway is available.

For the patient, the important message is that a rare finding is not automatically unimportant. However, rarity also means that the evidence may be limited, and the treatment decision must remain cautious and individualised.

Tumor Mutational Burden, Microsatellite Instability and Mismatch Repair

These Biomarkers Are Related but Not Interchangeable

Tumor mutational burden, microsatellite instability and mismatch-repair deficiency are sometimes discussed together because they may provide information about how a tumor interacts with the immune system. However, they describe different biological features.

Tumor mutational burden, or TMB, estimates the number of acquired mutations found within a defined amount of tumor DNA.

Microsatellite instability, or MSI, describes instability in short repeated regions of DNA.

Mismatch-repair deficiency, or dMMR, means that an important DNA-repair system is not functioning properly.

A tumor may show more than one of these features, but one result should not automatically be assumed from another.

What Is Tumor Mutational Burden?

Tumor mutational burden is usually reported as the number of mutations per megabase of DNA analysed. A megabase represents approximately one million DNA bases.

A tumor with many mutations may produce more abnormal proteins called neoantigens. These proteins may help the immune system recognise the cancer as foreign, which is one reason TMB has been investigated as a potential biomarker for immune checkpoint treatment.

However, TMB is not a direct measurement of immune activity. A tumor can have a higher mutation burden and still avoid immune attack, while another tumor with a lower burden may respond to immunotherapy.

TMB Results Depend on the Testing Platform

Different sequencing panels may analyse different genes and use different mathematical methods to estimate TMB. The result from one laboratory may therefore not be directly comparable with a result from another.

Sample quality also matters. Low tumor-cell content, poor sequencing coverage or previous treatment may affect the accuracy of the estimate.

A report should state whether the assay was validated for TMB measurement and which threshold the laboratory used. The term TMB-high should not be assigned casually from a mutation count alone.

ATC May Be Genomically Complex Without Always Being TMB-High

Anaplastic thyroid cancer commonly contains more genomic abnormalities than many differentiated thyroid cancers. However, this does not mean that every ATC is classified as TMB-high.[3–5]

Some tumors may meet a laboratory’s threshold, while many others will not. The result must be obtained from an appropriate validated assay rather than assumed from the aggressive nature of the disease.

Aggressive behaviour and high tumor mutational burden are not the same thing. A rapidly growing cancer may have several major driver abnormalities without containing the number of mutations required for a TMB-high classification.

What Is the Mismatch-Repair System?

The mismatch-repair system corrects certain errors that occur when DNA is copied during cell division. Important mismatch-repair proteins include MLH1, MSH2, MSH6 and PMS2.

When one or more of these proteins are lost, DNA errors may accumulate. This condition is called mismatch-repair deficiency.

Mismatch-repair status can be assessed through immunohistochemistry, which examines whether the proteins are present in tumor cells. It may also be assessed through molecular testing that looks for microsatellite instability.

What Is Microsatellite Instability?

Microsatellites are short repeated sequences of DNA. When mismatch repair is defective, these repeated regions may become longer or shorter as the cells divide.

A tumor with substantial instability may be classified as MSI-high. A tumor without this pattern may be described as microsatellite stable.

MSI-high status can have treatment relevance in several cancer types because it may indicate a greater likelihood of immune recognition. However, MSI-high and mismatch-repair deficiency appear to be uncommon in ATC and should not be presumed without testing.

MSI-High and TMB-High Are Not the Same Result

Mismatch-repair deficiency can produce a high number of mutations, so some MSI-high tumors are also TMB-high. However, the relationship is not universal.

A tumor may be TMB-high for reasons unrelated to mismatch-repair deficiency. Another tumor may have a mismatch-repair abnormality without producing the same TMB result on every assay.

The molecular report should therefore list MSI, mismatch-repair status and TMB separately when these biomarkers have been assessed.

Can These Findings Influence Immunotherapy?

MSI-high, mismatch-repair-deficient or TMB-high status may support consideration of immune checkpoint treatment under applicable clinical and regulatory circumstances.

However, the finding does not guarantee response. The oncology team must also consider the cancer type, previous therapy, immune-related risks, organ function and whether the biomarker was obtained through a validated test.

In ATC, immunotherapy decisions may also consider PD-L1 expression and emerging clinical evidence. No single immune biomarker provides a complete prediction of benefit.[20–22]

A Negative Result Does Not Automatically Exclude Immunotherapy

A microsatellite-stable or TMB-low result does not always mean that immunotherapy can never be considered. Clinical trials in ATC have reported responses in selected patients using immune checkpoint inhibitors, even though these biomarkers do not identify every responder.[22]

The decision may depend on PD-L1 expression, treatment history, combination strategies and the patient’s overall clinical situation.

Biomarkers help refine the probability of benefit, but they should not be interpreted as absolute yes-or-no tests unless a specific treatment indication requires that result.

What These Biomarkers Mean for the Patient

If your report includes TMB, MSI or mismatch-repair findings, ask which method was used and whether the test met quality standards.

A result such as TMB-high or MSI-high may open an additional treatment discussion, but it should be interpreted by the oncology team rather than read as a guarantee that immunotherapy will control the cancer.

If these biomarkers were not tested, the team can decide whether further assessment is clinically useful. The value depends on whether the result could change treatment or clinical-trial eligibility.

PD-L1 in Anaplastic Thyroid Cancer

PD-L1 Is a Protein Biomarker, Not a Gene Mutation

PD-L1 stands for programmed death-ligand 1. It is a protein that may be present on the surface of tumor cells and some immune cells.

PD-L1 can bind to the PD-1 receptor on immune cells. This interaction sends an inhibitory signal that reduces immune-cell activity. Some cancers use this pathway to protect themselves from immune attack.

A PD-L1 result is usually obtained through immunohistochemistry. It is not the same as DNA sequencing and should not be described as a tumor mutation.

How PD-L1 Is Tested

A pathologist applies a validated antibody to the tumor tissue and examines the staining pattern. The report may state the proportion of tumor cells showing PD-L1 expression.

Depending on the assay and cancer type, laboratories may use measurements such as the tumor proportion score or combined positive score. These methods are not interchangeable, and the threshold used in one cancer should not automatically be applied to ATC.

The report should identify the antibody clone, scoring system and percentage or category of expression. These details are necessary because different assays may produce different results.

Why PD-L1 May Be Increased in ATC

ATC can create a highly inflammatory tumor environment. Cancer cells and surrounding immune cells may express PD-L1 as part of the interaction between the tumor and the immune system.[4,22]

Some tumors may increase PD-L1 expression in response to inflammatory signals. Others may activate immune-escape pathways through molecular changes within the cancer cells.

High expression suggests that the PD-1/PD-L1 pathway may be active, but it does not prove that blocking this pathway will produce a clinical response.

What Clinical Studies Have Shown

The prospective spartalizumab study demonstrated that PD-1 blockade can produce responses in a proportion of patients with ATC. Responses were more frequently observed among tumors with higher PD-L1 expression, but PD-L1 was not a perfect predictor.[22]

Some PD-L1-positive tumors did not respond, and biomarker interpretation was limited by the relatively small number of patients. This means that PD-L1 may enrich the likelihood of benefit without guaranteeing it.

Emerging retrospective studies have also examined immune checkpoint treatment combined with BRAF- and MEK-directed therapy in BRAF V600E-positive ATC.[20,21] These results are encouraging but remain less certain than evidence from a large randomised trial.

A High PD-L1 Result Does Not Guarantee Response

A tumor can express high levels of PD-L1 and still resist immunotherapy. Other immune-suppressive mechanisms may be present, or the immune cells needed to attack the cancer may be absent or inactive.

The tumor may also contain molecular pathways that limit immune-cell entry or function. Treatment response therefore depends on the wider tumor microenvironment rather than one protein alone.

A high PD-L1 result should be viewed as one supportive biomarker, not as proof that immune checkpoint treatment will work.

A Low or Negative PD-L1 Result Does Not Always Exclude Benefit

PD-L1 expression can vary between different tumor regions and may change over time. A small biopsy may not represent the complete tumor.

Previous treatment can also influence expression. Radiation, targeted therapy and inflammatory changes may alter the relationship between the tumor and the immune system.

For these reasons, a negative PD-L1 test may reduce confidence in expected benefit but does not always prove that immunotherapy has no role. The decision should consider the complete evidence and the specific clinical setting.

PD-L1, TMB and MSI Should Be Read Separately

PD-L1 measures protein expression. TMB estimates mutation quantity. MSI and mismatch-repair testing assess DNA-repair instability.

A tumor may be PD-L1-high but microsatellite stable. Another may have a higher TMB but low PD-L1 expression. These results describe different aspects of tumor biology.

The oncology team should therefore avoid combining them into one general “immunotherapy-positive” label. Each biomarker has its own method, threshold and limitations.

PD-L1 Is Not a Substitute for BRAF Testing

PD-L1 expression does not determine whether the tumor contains BRAF V600E. A patient may have both findings, one finding or neither.

In BRAF V600E-positive ATC, BRAF/MEK-directed treatment has established clinical relevance.[15–17] The possible addition or sequencing of immunotherapy remains a more complex and evolving decision.[20,21]

In BRAF-negative ATC, PD-L1 may contribute to the immunotherapy discussion, but comprehensive DNA- and RNA-based profiling is still needed to search for other actionable alterations.

What a PD-L1 Result Means for the Patient

If your report shows PD-L1 expression, ask how it was measured, what percentage was reported and whether the result changes treatment in the context of your ATC.

The result may support discussion of immune checkpoint treatment, but it cannot independently determine the plan. The oncologist must consider stage, tumor growth, airway risk, previous treatments, autoimmune history and the possibility of immune-related adverse effects.

When I explain PD-L1, I describe it as one sign of how the tumor may be interacting with the immune system. It provides useful information, but it is not a complete prediction of whether immunotherapy will succeed.

BRAF/MEK Therapy and Immunotherapy

Why These Treatments May Be Combined

Dabrafenib and trametinib directly suppress BRAF–MEK signalling in BRAF V600E-positive tumor cells. Immunotherapy works differently by reducing signals that prevent immune cells from attacking the cancer. Researchers are therefore studying whether targeted therapy and immune checkpoint inhibition can provide stronger or more durable control when used together.[20–22]

Targeted therapy may rapidly reduce tumor activity, while immunotherapy may help the immune system recognise remaining cancer cells. This biological reasoning is promising, but it does not prove that every patient should receive all three medicines.

What the Current Evidence Suggests

A retrospective study evaluated pembrolizumab in addition to dabrafenib and trametinib for BRAF V600E-positive ATC. Patients receiving the combination showed encouraging survival outcomes compared with those receiving BRAF/MEK therapy alone.[20]

However, the study was not randomised. The patients may have differed in health, disease extent, treatment timing and access to surgery or radiation. The findings therefore support further investigation but do not establish the combination as the only correct treatment for every BRAF-positive patient.

Prospective evidence has also shown that PD-1 blockade can produce responses in a proportion of patients with ATC, particularly in some tumors expressing PD-L1.[22] PD-L1 remains an imperfect biomarker, and a positive result cannot guarantee benefit.

The Decision Must Be Individualised

Adding immunotherapy may increase the risk of inflammation affecting the thyroid, lungs, liver, bowel, skin, endocrine glands or other organs. A history of autoimmune disease, previous treatment and the urgency of tumor control may influence whether the combination is suitable.

If your oncologist recommends immunotherapy, ask whether it is being used within an established indication, a clinical trial or an individualised treatment strategy. We should present the combination as an emerging option supported by developing human evidence, not as a guaranteed improvement over BRAF/MEK therapy alone.[20–22]

What Patients Should Know About BRAF/MEK Treatment Safety

Fever Is One of the Most Important Problems

Fever, chills and weakness can occur during dabrafenib and trametinib treatment. Some patients may also develop dehydration, low blood pressure or changes in kidney function if fever is persistent or severe.[15]

A new fever should be reported promptly rather than managed only with home remedies. The oncology team may need to exclude infection, assess hydration and decide whether treatment should be temporarily interrupted.

Fever during targeted therapy does not always mean that the cancer is worsening. However, it should not be ignored because infection and treatment-related fever can initially appear similar.

Other Common Adverse Effects

Patients may experience fatigue, skin rash, reduced appetite, nausea, diarrhoea, headache, muscle discomfort or joint pain.[15] The severity varies, and many problems can be managed when they are reported early.

Skin changes require attention because BRAF-directed treatment can cause several forms of rash and may rarely be associated with serious reactions. New painful skin lesions, blistering, widespread redness or involvement of the mouth or eyes require urgent assessment.

Regular blood tests may be used to monitor liver function, kidney function, blood glucose and other treatment-related changes. The exact schedule depends on the patient’s condition and other medicines.

Cardiac, Eye and Lung Monitoring May Be Required

Trametinib can affect heart function in some patients. The oncology team may therefore arrange an echocardiogram or another cardiac assessment before and during treatment.[15]

New shortness of breath, chest discomfort, ankle swelling or marked reduction in exercise capacity should be reported. These symptoms can have several causes in ATC, including the cancer itself, so medical evaluation is needed.

Blurred vision, eye pain, flashing lights or sudden visual changes also require prompt assessment. Although serious eye toxicity is uncommon, delay could increase the risk of lasting injury.

New or worsening cough and breathlessness may indicate infection, tumor progression, airway involvement or drug-related lung inflammation. The cause cannot be determined safely without clinical review.

Drug Interactions Must Be Checked

Dabrafenib can affect enzymes involved in the metabolism of other medicines, and other drugs may alter dabrafenib exposure.[15] The oncology team should therefore receive a complete list of prescription medicines, over-the-counter products, vitamins, herbal products and Ayurvedic formulations.

A product described as natural is not automatically free from interaction risk. Multi-ingredient preparations may affect liver enzymes, bleeding, glucose, blood pressure or the absorption of cancer medicines.

When I review complementary treatment during targeted therapy, I prefer ingredient-level documentation and planned laboratory monitoring. The purpose is to support the patient without reducing the effectiveness or safety of oncology treatment.

Why Patients Should Not Stop or Restart Targeted Therapy Independently

Treatment Interruptions May Be Necessary, but They Must Be Supervised

The oncology team may temporarily interrupt dabrafenib, trametinib or both when a patient develops fever, cardiac changes, eye toxicity, severe skin reactions or other clinically important adverse effects.[15]

A temporary pause does not always mean that treatment has permanently failed. The team may investigate the cause, treat the complication and restart therapy when it is safe. In other situations, dose modification or permanent discontinuation may be required.

The correct response depends on the type and severity of the adverse effect. Patients should therefore not use a general internet instruction to alter their treatment.

Stopping Treatment May Allow Rapid Tumor Progression

ATC can progress quickly, and a tumor controlled by BRAF/MEK inhibition may begin growing when treatment is interrupted for too long. This does not mean that every missed dose will cause immediate progression, but unnecessary gaps can reduce continuity of disease control.

At the same time, continuing treatment through a serious adverse effect may create avoidable harm. The safest approach is rapid communication with the treating team rather than choosing independently between continuing and stopping.

Restarting Without Assessment Can Also Be Unsafe

A patient who has recovered from fever or another symptom may feel ready to restart treatment. However, blood tests, cardiac assessment or another clinical review may still be required.

The oncology team may restart both medicines together, restart them at a modified dose or delay one component depending on the problem. These decisions should follow the current prescribing guidance and the patient’s individual condition.[15]

For you as a patient, the practical rule is simple: report significant symptoms early and obtain direct instructions before interrupting, reducing or restarting BRAF/MEK therapy.

FAQs

Reference

1. ATA Clinical Guideline

Bible, K. C., Kebebew, E., Brierley, J., Brito, J. P., Cabanillas, M. E., Clark, T. J., Jr., Di Cristofano, A., Foote, R., Giordano, T., Kasperbauer, J., Newbold, K., Nikiforov, Y. E., Randolph, G., Rosenthal, M. S., Sawka, A. M., Shah, M., Shaha, A., Smallridge, R., & Wong-Clark, C. K. (2021). 2021 American Thyroid Association guidelines for management of patients with anaplastic thyroid cancer. Thyroid, 31(3), 337–386. https://doi.org/10.1089/thy.2020.0944

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Brief: The principal clinical guideline for ATC diagnosis, staging, airway evaluation, pathological assessment, molecular testing, surgery, radiotherapy, systemic treatment, supportive care, and multidisciplinary management.

2. Genomic Landscape Review

Xu, B., & Ghossein, R. (2016). Genomic landscape of poorly differentiated and anaplastic thyroid carcinoma. Endocrine Pathology, 27(3), 205–212. https://doi.org/10.1007/s12022-016-9445-4

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Brief: Reviews major driver and progression-associated alterations in aggressive thyroid cancer, including BRAF, RAS, TP53, TERT, and PI3K–AKT–mTOR pathway abnormalities.

3. Foundational Genomic and Transcriptomic Study

Landa, I., Ibrahimpasic, T., Boucai, L., Sinha, R., Knauf, J. A., Shah, R. H., Dogan, S., Ricarte-Filho, J. C., Krishnamoorthy, G. P., Xu, B., Schultz, N., Berger, M. F., Sander, C., Taylor, B. S., Ghossein, R., Ganly, I., & Fagin, J. A. (2016). Genomic and transcriptomic hallmarks of poorly differentiated and anaplastic thyroid cancers. Journal of Clinical Investigation, 126(3), 1052–1066. https://doi.org/10.1172/JCI85271

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Brief: A foundational study describing BRAF- and RAS-associated tumor groups, genomic complexity, dedifferentiation, and the accumulation of TP53, TERT, and other alterations during progression to ATC.

4. Integrated DNA and RNA Analysis

Yoo, S. K., Song, Y. S., Lee, E. K., Hwang, J., Kim, H. H., Jung, G., Kim, Y. A., Kim, S. J., Cho, S. W., Won, J. K., Chung, E. J., Shin, J. Y., Lee, K. E., Kim, J. I., Park, Y. J., & Seo, J. S. (2019). Integrative analysis of genomic and transcriptomic characteristics associated with progression of aggressive thyroid cancer. Nature Communications, 10, Article 2764. https://doi.org/10.1038/s41467-019-10680-5

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Brief: Integrates DNA and RNA findings to explain molecular progression, BRAF- and RAS-associated biology, TP53/CDKN2A loss, TERT alterations, PI3K pathway changes, and immune-marker expression.

5. Genomic and Evolutionary Landscape of ATC

Zeng, P. Y. F., Prokopec, S. D., Lai, S. Y., Pinto, N., Chan-Seng-Yue, M. A., Clifton-Bligh, R., Williams, M. D., Howlett, C. J., Plantinga, P., Cecchini, M. J., Lam, A. K., Siddiqui, I., Wang, J., Sun, R. X., Watson, J. D., Korah, R., Carling, T., Agrawal, N., Cipriani, N., . . . Nichols, A. C. (2024). The genomic and evolutionary landscapes of anaplastic thyroid carcinoma. Cell Reports, 43(3), Article 113826. https://doi.org/10.1016/j.celrep.2024.113826

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Brief: Provides a modern analysis of ATC evolution, clonal architecture, intratumoral heterogeneity, molecular subgroups, and progression from differentiated thyroid carcinoma.

6. Large Genomic Study of Advanced Thyroid Cancer

Pozdeyev, N., Gay, L. M., Sokol, E. S., Hartmaier, R. J., Deaver, K. E., Davis, S., French, J. D., Borre, P. V., LaBarbera, D. V., Tan, A. C., Schweppe, R. E., Fishbein, L., Ross, J. S., Haugen, B. R., & Bowles, D. W. (2018). Genetic analysis of 779 advanced differentiated and anaplastic thyroid cancers. Clinical Cancer Research, 24(13), 3059–3068. https://doi.org/10.1158/1078-0432.CCR-18-0373

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Brief: Defines the frequency of clinically relevant alterations in a large advanced-thyroid-cancer cohort, including BRAF, RAS, TP53, TERT, and uncommon actionable gene fusions.

7. Plasma-Based Genomic Profiling

Tarasova, V. D., Tsai, J., Masannat, J., Hernandez Prera, J. C., Hallanger Johnson, J., Veloski, C., Agosto Salgado, S., McIver, B., Drusbosky, L. M., & Chung, C. H. (2024). Characterization of the thyroid cancer genomic landscape by plasma-based circulating tumor DNA next-generation sequencing. Thyroid, 34(2), 197–205. https://doi.org/10.1089/thy.2023.0204

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Brief: Examines plasma circulating tumor DNA profiling in thyroid cancer and demonstrates the detection of BRAF, RAS, RET, ALK, NTRK, and other alterations through blood-based testing.

8. Emerging Comprehensive Genomic Profiling of ATC

Trivedi, V., Noronha, V., Bal, M., Chandrani, P., Poojary, D., Saldanha, E., Choughule, A., Pange, P., Gupta, V., Menon, N., Patil, V., Shah, M., Chaturvedi, P., Prabhash, K., & Dutt, A. (2025). Comprehensive genomic profiling of anaplastic thyroid cancer identifies alterations in THRA, a potential modifier of cellular plasticity. JCO Global Oncology, 11, e2400610. https://doi.org/10.1200/GO-24-00610

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Brief: Reports emerging THRA alterations that may influence tumor-cell plasticity; however, THRA is not yet an established therapeutic target in routine ATC treatment.

9. Diagnostic Evaluation in Core-Biopsy Specimens

Riascos, M. C., & Barletta, J. A. (2025). Diagnostic evaluation of anaplastic thyroid carcinoma in core biopsy specimens: Morphologic, immunohistochemical, molecular, and therapeutic considerations. Endocrine Pathology, 36(1), Article 32.

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Brief: Addresses ATC diagnosis in core-biopsy specimens, including morphology, immunohistochemistry, differential diagnosis, tissue adequacy, and preservation of material for molecular analysis.

10. VE1 BRAF V600E Immunohistochemistry

Na, J. I., Kim, J. H., Kim, H. J., Kim, H. K., Moon, K. S., Lee, J. S., Lee, J. H., Lee, K. H., & Park, J. T. (2015). VE1 immunohistochemical detection of the BRAF V600E mutation in thyroid carcinoma: A review of its usefulness and limitations. Virchows Archiv, 467(2), 155–168.

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Brief: Explains how VE1 immunohistochemistry detects BRAF V600E, its concordance with molecular testing, and the technical or interpretive limitations that can lead to inaccurate results.

11. BRAF Immunohistochemistry Versus Genomic Testing

Onaga, R., Enokida, T., Sakashita, S., Tanaka, N., Hoshi, Y., Kishida, T., Kuboki, R., Fujisawa, T., Okano, S., Nishino, H., Ito, M., Ishii, G., Ishikawa, S., & Tahara, M. (2025). Concordance of BRAF V600E mutation between immunohistochemistry and genomic testing for thyroid cancer. International Journal of Clinical Oncology, 30(6), 1143–1151. https://doi.org/10.1007/s10147-025-02760-y

Access: PubMed

Brief: Evaluates agreement between rapid BRAF V600E immunohistochemistry and genomic testing, including practical problems associated with limited tissue and inadequate tumor content.

12. Tissue- and Liquid-Based NGS in ATC

Yeh, C.-N., Lin, S.-F., Wu, C.-L., Liou, M.-J., Chen, I.-W., Chen, C.-P., Chang, C.-F., Wang, Q.-A., & Wu, C.-E. (2025). Genomic landscape and comparative analysis of tissue and liquid-based NGS in Taiwanese anaplastic thyroid carcinoma. npj Precision Oncology, 9, Article 16. https://doi.org/10.1038/s41698-025-00802-2

Access: PubMed | Full text

Brief: Directly compares tissue and plasma NGS in ATC and demonstrates why a negative liquid-biopsy result does not reliably exclude an alteration that may be detectable in tumor tissue.

13. Liquid-Biopsy Systematic Review

Zeyghami, W., Hansen, M.-L. U., Jakobsen, K. K., Groenhøj, C., Feldt-Rasmussen, U., von Buchwald, C., & Hahn, C. H. (2023). Liquid biopsies in thyroid cancers: A systematic review and meta-analysis. Endocrine-Related Cancer, 30(12), e230002. https://doi.org/10.1530/ERC-23-0002

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Brief: Summarizes the diagnostic performance and limitations of circulating tumor material and supports liquid biopsy as a complementary test rather than a replacement for tumor-tissue analysis.

14. FDA Companion Diagnostic for BRAF V600E

U.S. Food and Drug Administration. (2024, January 26). Oncomine Dx Target Test—P160045/S025.

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Brief: Describes an FDA-authorized tumor-tissue test for identifying BRAF V600E in ATC and selecting eligible patients for dabrafenib plus trametinib; the device approval date was September 29, 2023.

15. TAFINLAR Prescribing Information

Novartis Pharmaceuticals Corporation. (2026). Tafinlar (dabrafenib) capsules for oral use and tablets for oral suspension [Prescribing information]. DailyMed.

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Brief: Provides the ATC indication, requirement for BRAF confirmation, dosage guidance, adverse reactions, monitoring recommendations, safety warnings, and drug-interaction information for dabrafenib.

16. Initial Dabrafenib and Trametinib Trial

Subbiah, V., Kreitman, R. J., Wainberg, Z. A., Cho, J. Y., Schellens, J. H. M., Soria, J.-C., Wen, P. Y., Zielinski, C., Cabanillas, M. E., Urbanowitz, G., Mookerjee, B., Wang, D., Rangwala, F., & Keam, B. (2018). Dabrafenib and trametinib treatment in patients with locally advanced or metastatic BRAF V600-mutant anaplastic thyroid cancer. Journal of Clinical Oncology, 36(1), 7–13. https://doi.org/10.1200/JCO.2017.73.6785

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Brief: Provides the original prospective evidence that combined BRAF and MEK inhibition can produce substantial and sometimes rapid tumor responses in BRAF V600-mutant ATC.

17. Updated ROAR Trial Analysis

Subbiah, V., Kreitman, R. J., Wainberg, Z. A., Cho, J. Y., Schellens, J. H. M., Soria, J.-C., Wen, P. Y., Zielinski, C. C., Cabanillas, M. E., Boran, A., Ilankumaran, P., Burgess, P., Romero Salas, T., & Keam, B. (2022). Dabrafenib plus trametinib in patients with BRAF V600E-mutant anaplastic thyroid cancer: Updated analysis from the phase II ROAR basket study. Annals of Oncology, 33(4), 406–415. https://doi.org/10.1016/j.annonc.2021.12.014

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Brief: Provides updated prospective data on objective response, response duration, progression-free survival, overall survival, and treatment-related toxicity.

18. Neoadjuvant BRAF and MEK Inhibition

Wang, J. R., Zafereo, M. E., Dadu, R., Ferrarotto, R., Busaidy, N. L., Lu, C., Ahmed, S., Gule-Monroe, M. K., Williams, M. D., Sturgis, E. M., Goepfert, R. P., Gross, N. D., Lai, S. Y., Gunn, G. B., Phan, J., Rosenthal, D. I., Fuller, C. D., Morrison, W. H., Iyer, P., & Cabanillas, M. E. (2019). Complete surgical resection following neoadjuvant dabrafenib plus trametinib in BRAF V600E-mutated anaplastic thyroid carcinoma. Thyroid, 29(8), 1036–1043. https://doi.org/10.1089/thy.2019.0133

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Brief: Describes a six-patient series in which neoadjuvant dabrafenib plus trametinib reduced initially unresectable or difficult-to-resect disease sufficiently to permit subsequent surgery.

19. Surgery After BRAF-Directed Therapy

Zhao, X., Wang, J. R., Dadu, R., Busaidy, N. L., Xu, L., Learned, K. O., Chasen, N. N., Vu, T., Maniakas, A., Eguia, A. A., Diersing, J., Gross, N. D., Goepfert, R., Lai, S. Y., Hofmann, M. C., Ferrarotto, R., Lu, C., Gunn, G. B., Spiotto, M. T., . . . Zafereo, M. E. (2023). Surgery after BRAF-directed therapy is associated with improved survival in BRAF V600E-mutant anaplastic thyroid cancer: A single-center retrospective cohort study. Thyroid, 33(4), 484–491. https://doi.org/10.1089/thy.2022.0504

Access: PubMed | Full text

Brief: Reports an association between surgery after BRAF-directed treatment and improved survival, but the findings remain vulnerable to patient-selection bias and other retrospective limitations.

20. Pembrolizumab Added to Dabrafenib and Trametinib

Hamidi, S., Iyer, P. C., Dadu, R., Gule-Monroe, M. K., Maniakas, A., Zafereo, M. E., Wang, J. R., Busaidy, N. L., & Cabanillas, M. E. (2024). Checkpoint inhibition in addition to dabrafenib/trametinib for BRAF V600E-mutated anaplastic thyroid carcinoma. Thyroid, 34(3), 336–346. https://doi.org/10.1089/thy.2023.0573

Access: PubMed

Brief: Evaluates pembrolizumab added to BRAF/MEK inhibition, including survival outcomes and immune-related adverse events; prospective validation remains necessary.

21. PD-L1 Blockade Plus Matched Targeted Therapy

Cabanillas, M. E., Dadu, R., Ferrarotto, R., Gule-Monroe, M., Liu, S., Fellman, B., Williams, M. D., Zafereo, M., Wang, J. R., Lu, C., Ning, M., McKinley, B. A., Woodman, S. E., Duose, D., Gunn, G. B., & Busaidy, N. L. (2024). Anti-programmed death ligand 1 plus targeted therapy in anaplastic thyroid carcinoma: A nonrandomized clinical trial. JAMA Oncology, 10(12), 1672–1680. https://doi.org/10.1001/jamaoncol.2024.4729

Access: PubMed | Full text

Brief: Evaluates molecularly matched targeted therapy combined with PD-L1 inhibition in ATC; because the trial was nonrandomized, it should not be interpreted as a definitive randomized comparison.

22. Spartalizumab PD-1 Trial

Capdevila, J., Wirth, L. J., Ernst, T., Ponce Aix, S., Lin, C.-C., Ramlau, R., Butler, M. O., Delord, J.-P., Gelderblom, H., Ascierto, P. A., Fasolo, A., Führer, D., Hütter-Krönke, M. L., Forde, P. M., Wrona, A., Santoro, A., Sadow, P. M., Szpakowski, S., Wu, H., . . . Taylor, M. (2020). PD-1 blockade in anaplastic thyroid carcinoma. Journal of Clinical Oncology, 38(23), 2620–2627. https://doi.org/10.1200/JCO.19.02727

Access: PubMed | Full text

Brief: Evaluates spartalizumab in ATC and explores associations between PD-L1 expression, immune biomarkers, and treatment response.

23. NTRK Fusion-Positive Thyroid Cancer

Waguespack, S. G., Drilon, A., Lin, J. J., Brose, M. S., McDermott, R., Almubarak, M., Bauman, J., Casanova, M., Krishnamurthy, A., Kummar, S., Leyvraz, S., Oh, D.-Y., Park, K., Sohal, D., Sherman, E., Norenberg, R., Silvertown, J. D., Brega, N., Hong, D. S., & Cabanillas, M. E. (2022). Efficacy and safety of larotrectinib in patients with TRK fusion-positive thyroid carcinoma. European Journal of Endocrinology, 186(6), 631–643. https://doi.org/10.1530/EJE-21-1259

Access: PubMed

Brief: Supports NTRK fusion testing and larotrectinib treatment in TRK fusion-positive thyroid malignancies; the study includes several thyroid-cancer histologies and is not limited to ATC.

24. Selpercatinib in RET-Altered Thyroid Cancer

Wirth, L. J., Brose, M. S., Subbiah, V., Worden, F., Solomon, B., Robinson, B., Hadoux, J., Tomasini, P., Weiler, D., Deschler-Baier, B., Tan, D. S. W., Maeda, P., Lin, Y., Singh, R., Bayt, T., Drilon, A., & Cassier, P. A. (2024). Durability of response with selpercatinib in patients with RET-activated thyroid cancer: Long-term safety and efficacy from LIBRETTO-001. Journal of Clinical Oncology, 42(27), 3187–3195. https://doi.org/10.1200/JCO.23.02503

Access: PubMed | Full text

Brief: Provides long-term evidence for selective RET inhibition in RET-mutant medullary thyroid cancer and RET fusion-positive thyroid carcinoma; the overall outcomes should not be presented as ATC-specific.

25. FDA Approval Summary for Selpercatinib

Bradford, D., Larkins, E., Mushti, S. L., Rodriguez, L., Skinner, A. M., Helms, W. S., Price, L. S. L., Fourie Zirkelbach, J., Li, Y., Liu, J., Charlab, R., Reyes Turcu, F., Liang, D., Ghosh, S., Roscoe, D., Philip, R., Zack-Taylor, A., Tang, S., Kluetz, P. G., . . . Singh, H. (2021). FDA approval summary: Selpercatinib for the treatment of lung and thyroid cancers with RET gene mutations or fusions. Clinical Cancer Research, 27(8), 2130–2135. https://doi.org/10.1158/1078-0432.CCR-20-3558

Access: PubMed

Brief: Summarizes the regulatory evidence supporting selpercatinib and clarifies the clinically important distinction between RET mutations and RET gene fusions.

26. Primary and Metastatic Molecular Heterogeneity

Ricarte-Filho, J. C., Ryder, M., Chitale, D. A., Rivera, M., Heguy, A., Ladanyi, M., Janakiraman, M., Solit, D., Knauf, J. A., Tuttle, R. M., Ghossein, R. A., & Fagin, J. A. (2009). Mutational profile of advanced primary and metastatic radioactive iodine-refractory thyroid cancers reveals distinct pathogenetic roles for BRAF, PIK3CA, and AKT1. Cancer Research, 69(11), 4885–4893. https://doi.org/10.1158/0008-5472.CAN-09-0727

Access: PubMed | Full text

Brief: Demonstrates molecular differences between primary and metastatic tumor sites and supports the principle that a single biopsy may not capture every clinically relevant tumor clone; the cohort was not limited to ATC.

27. BRAF Biology Review

Scheffel, R. S., Dora, J. M., & Maia, A. L. (2022). BRAF mutations in thyroid cancer. Current Opinion in Oncology, 34(1), 9–18. https://doi.org/10.1097/CCO.0000000000000797

Access: PubMed

Brief: Explains BRAF signaling, the biological effects of the V600E mutation, therapeutic targeting, and mechanisms relevant to BRAF-inhibitor treatment.

28. Molecular Targeted-Therapy Review

Cabanillas, M. E., Zafereo, M., Gunn, G. B., & Ferrarotto, R. (2016). Anaplastic thyroid carcinoma: Treatment in the age of molecular targeted therapy. Journal of Oncology Practice, 12(6), 511–518. https://doi.org/10.1200/JOP.2016.012013

Access: PubMed

Brief: Describes the transition from conventional ATC treatment toward molecularly targeted and multidisciplinary treatment strategies. This is the corrected citation for the previously mismatched PMID.

29. Real-World Outcomes With Modern Systemic Therapy

Luong, A., Poei, D., Chow, L., Li, M., Nie, Q., Angell, T., Tang, L., Hsu, R., & Thomas, J. (2025). Impact of modern systemic therapies on survival in patients with anaplastic thyroid cancer: A single-center retrospective cohort review. Drugs – Real World Outcomes, 12(2), 295–300. https://doi.org/10.1007/s40801-025-00493-y

Access: PubMed | Full text

Brief: Describes recent real-world survival outcomes associated with targeted and immune-based treatments, but its small, single-center, retrospective design prevents firm causal conclusions.

30. Survival in the Modern Treatment Era

Maniakas, A., Dadu, R., Busaidy, N. L., Wang, J. R., Ferrarotto, R., Lu, C., Williams, M. D., Gunn, G. B., Hofmann, M. C., Cote, G., Sperling, J., Gross, N. D., Sturgis, E. M., Goepfert, R. P., Lai, S. Y., Cabanillas, M. E., & Zafereo, M. (2020). Evaluation of overall survival in patients with anaplastic thyroid carcinoma, 2000–2019. JAMA Oncology, 6(9), 1397–1404. https://doi.org/10.1001/jamaoncol.2020.3362

Access: PubMed | Full text

Brief: Examines changes in ATC survival across treatment eras and highlights molecular testing, targeted therapy, immunotherapy, neoadjuvant treatment, surgery, and multidisciplinary care.

31. Clinicopathology, Genetics, and Advanced-Therapy Review

Molinaro, E., Romei, C., Biagini, A., Sabini, E., Agate, L., Mazzeo, S., Materazzi, G., Sellari-Franceschini, S., Ribechini, A., Torregrossa, L., Basolo, F., Vitti, P., & Elisei, R. (2017). Anaplastic thyroid carcinoma: From clinicopathology to genetics and advanced therapies. Nature Reviews Endocrinology, 13(11), 644–660. https://doi.org/10.1038/nrendo.2017.76

Access: PubMed

Brief: Provides a comprehensive overview of ATC pathology, dedifferentiation, tumor progression, molecular genetics, clinical behavior, and the biological rationale for precision-treatment approaches.

32. STRN–ALK Fusion in Aggressive Thyroid Cancer

Kelly, L. M., Barila, G., Liu, P., Evdokimova, V. N., Trivedi, S., Panebianco, F., Gandhi, M., Carty, S. E., Hodak, S. P., Luo, J., Dacic, S., Yu, Y. P., Nikiforova, M. N., Ferris, R. L., Altschuler, D. L., & Nikiforov, Y. E. (2014). Identification of the transforming STRN-ALK fusion as a potential therapeutic target in the aggressive forms of thyroid cancer. Proceedings of the National Academy of Sciences of the United States of America, 111(11), 4233–4238. https://doi.org/10.1073/pnas.1321937111

Access: PubMed

Brief: Identifies the transforming STRN–ALK fusion and supports fusion-sensitive, RNA-based testing as a method for detecting rare but potentially actionable molecular drivers.

33. Evidence-Based Integrative Oncology

Greenlee, H., DuPont-Reyes, M. J., Balneaves, L. G., Carlson, L. E., Cohen, M. R., Deng, G., Johnson, J. A., Mumber, M., Seely, D., Zick, S. M., Boyce, L. M., & Tripathy, D. (2017). Clinical practice guidelines on the evidence-based use of integrative therapies during and after breast cancer treatment. CA: A Cancer Journal for Clinicians, 67(3), 194–232. https://doi.org/10.3322/caac.21397

Access: PubMed | Full text

Brief: Supports evaluating integrative interventions according to defined outcomes such as anxiety, fatigue, sleep, quality of life, and treatment-related symptoms; it is not ATC-specific and does not show that integrative therapies directly treat ATC mutations or control the tumor.

34. Drug, Food, and Supplement Interaction Safety

Lohr, L. K., Blake, K. T., Chan, C. M., Sturm, S., & Walsh, G. T. (2023). Managing drug interactions with oral anticancer treatments. Journal of the Advanced Practitioner in Oncology, 14(5), 419–438. https://doi.org/10.6004/jadpro.2023.14.5.7

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Brief: Reviews clinically important drug–drug, drug–food, and drug–supplement interactions involving oral anticancer treatments and supports careful review of herbs and complementary products during targeted therapy.