14 Critical Biomarkers in Your NGS Report

In your NGS report, what does an EGFR mutation mean?

Seeing “EGFR mutation” on a molecular (NGS) report means the tumor carries a change in the EGFR gene, and that this change may be one of the drivers behind the cancer's growth. In many cancers — most often non-small cell lung cancer (NSCLC) — this finding can open the door to targeted therapies. But the gene name alone doesn't clarify anything: whether your specific EGFR result is truly actionable depends on the exact variant, the type of cancer, and the stage of treatment you are in. Clarifying exactly that is the purpose of a molecular second opinion.

1Which EGFR mutations are actionable?

Not every EGFR mutation behaves the same way, and that is precisely the point. The classic “activating” mutations — exon 19 deletions and the L858R point mutation — are guideline-supported predictors of response to EGFR tyrosine kinase inhibitors (TKIs) in NSCLC. Exon 20 insertions are a different story: they generally do not respond to these classic TKIs and instead may require specific agents. Other EGFR alterations can fall into a gray zone — variants of uncertain significance (VUS) whose meaning is not yet clear. A second opinion clarifies which group your result belongs to, because it is the exact variant — not the word “EGFR” alone — that determines what is appropriate.

2Does an EGFR result mean the same thing in every cancer?

No. How actionable it is depends on the tumor type. An activating EGFR mutation in NSCLC sits on a well-mapped targeted-therapy pathway, while the same gene alteration in a different cancer may carry a different meaning — or no established meaning at all. The result should not be taken at face value from the gene name; it must be read in the context of your specific cancer and treatment line.

3Why should you get a second opinion on an EGFR result?

In practice, a molecular second opinion does four things with an EGFR finding. It pins down the exact variant (among exon 19 deletion, L858R, exon 20 insertion, a rare alteration, or a VUS). Using the AMP/ASCO/CAP framework, it re-grades how actionable that variant is for your tumor type and treatment line. It maps out the resistance you may encounter later (for example, T790M, C797S, or MET-driven escape) and what should be tested for at progression. It also checks your eligibility for molecular- and clinical-trial matching. All of this is brought together in a single written, physician-signed report for the oncologist treating you.

4What if my report says exon 20 insertion?

EGFR exon 20 insertion is the third most common group of EGFR alterations after exon 19 deletion and L858R, and it behaves differently from both. The structural reason matters: the inserted amino acids lock the kinase in an active conformation without enlarging the drug-binding pocket, so classic EGFR TKIs generally show limited activity — and reading only the phrase “EGFR mutation” on the report can lead to the wrong first-line choice. Exon 20 insertions are also not a single entity: they vary in position and length, and at least one well-characterized variant — A763_Y764insFQEA — can behave like a classic activating mutation and respond to standard EGFR TKIs, which is why the exact variant designation, not the label, must be the basis for decisions. The second pitfall is in detection: because of their variability in length and position, hotspot PCR assays and narrowly designed panels can miss these insertions, so a “no EGFR mutation” result from such a test does not prove an insertion is absent. Once an exon 20 insertion is confirmed, the treatment path diverges from the classic-mutation path; agents developed for this context — such as the EGFR-MET bispecific antibody amivantamab — are used together with platinum-based chemotherapy, and clinical trial options are frequently on the table. Approvals in this space have both been granted and withdrawn; the current status should be confirmed with the treating team.

5What happens when a TKI stops working, and what should be tested at that point?

Resistance to EGFR-targeted therapy is expected, not exceptional, and it emerges through three distinct routes, each requiring a different response. The first is on-target resistance: an additional mutation in EGFR itself that blocks drug binding — classically T790M after first- and second-generation TKIs, and tertiary changes such as C797S and L718Q or G724S after osimertinib. There is a detail here that reports can easily miss: when two resistance mutations co-occur, whether they sit on the same allele (cis) or on opposite alleles (trans) determines whether any combination-TKI strategy is biologically meaningful. The second is off-target (bypass) resistance: the tumor escapes through another pathway — MET amplification is the best characterized, alongside HER2 amplification and alterations in RAS/RAF, PIK3CA, and cell-cycle genes — and this changes the strategy, not just the EGFR drug. The third is histologic transformation, most often to small cell carcinoma; no plasma test can detect this — only a tissue biopsy can — and it changes treatment entirely. The practical takeaway is that the baseline report does not describe the tumor at progression. Molecular re-evaluation at progression is necessary; plasma ctDNA is a reasonable first step because it is fast and captures tumor heterogeneity, but a negative plasma result should prompt a tissue rebiopsy rather than closing the question. How the result is used is a decision for the treating oncologist.

In my NGS report, what does a KRAS mutation mean?

A KRAS mutation on your NGS (next-generation sequencing) report means the tumor carries a change in the KRAS gene — one of the most common drivers in cancer, seen especially often in non-small cell lung cancer (NSCLC) and colorectal cancer. The single most important detail is which exact variant you have. A KRAS G12C mutation may make you eligible for a guideline-recognized class of targeted inhibitors, while many other KRAS variants, such as G12D or G12V, do not yet have an approved targeted drug and are generally managed with standard therapy or a clinical trial. Whether any KRAS finding is actionable depends on your tumor type and treatment line, and the decision always belongs to the oncologist treating you. Think of this page not as a diagnosis, but as decision support to help you ask sharper questions.

1Is a KRAS mutation good or bad news, and is it treatable?

A KRAS mutation is not automatically good or bad news; what it means depends almost entirely on which variant and which cancer you have. The most therapeutically important variant is KRAS G12C. In advanced NSCLC, a guideline-recognized class of oral KRAS G12C inhibitors is available, typically after first-line therapy, and this is reflected in the NCCN and ESMO guidelines. In colorectal cancer, the same G12C variant is much rarer, and here the guideline-supported approach combines a KRAS G12C inhibitor with an anti-EGFR antibody, because the inhibitor alone appears less effective in colorectal tumors. For many other variants — such as G12D, G12V, G12A, or G13D — there is currently no approved targeted therapy in most tumor types, so treatment usually relies on standard chemotherapy, immunotherapy, or a clinical trial. A second opinion is most useful here for confirming the exact variant, assessing the tumor-type context, and checking whether a suitable trial is available.

2I have KRAS G12C. Does that mean I'm eligible for targeted therapy?

Possibly, but it depends on your tumor type and treatment line, which is why confirmation matters. In advanced NSCLC, a KRAS G12C mutation typically opens the door to a guideline-recognized class of KRAS G12C inhibitors, usually considered after a first-line therapy such as immunotherapy or chemotherapy, rather than at the very start. In colorectal cancer, a KRAS G12C inhibitor is used not alone but together with an anti-EGFR antibody, again typically after earlier treatment lines. Eligibility also depends on the result being confirmed on a validated test, your overall health status, your prior treatments, and other findings on your report. Because timing and the right drug pairing matter greatly, a common reason patients seek a second opinion is to make sure the correct treatment line and the correct combination are being considered. This is general guideline-level information, not a prescription; the oncologist treating you decides whether and when a targeted option is right for you.

3My report shows TP53, STK11, or KEAP1 alongside KRAS. Why does this matter?

When KRAS appears together with TP53, STK11, or KEAP1, these co-occurring mutations can change how physicians view immunotherapy — which is exactly why they are worth raising in a second opinion. In NSCLC, tumors carrying STK11 or KEAP1 alongside KRAS are often described as immunologically “cold” and, in published series, tend to derive less benefit from immune checkpoint inhibitors and carry a less favorable prognosis. A TP53 co-mutation has its own, context-dependent effects. Importantly, these patterns are signals, not on/off switches: on their own they do not activate or rule out a treatment, and guidelines still individualize the decision. This is exactly the kind of nuance a second opinion is built to handle — reading KRAS not in isolation but together with the full co-mutation picture, and checking whether immunotherapy, chemotherapy, a combination, or a trial best fits your tumor. The oncologist treating you remains the decision-maker.

What does an ALK fusion in your NGS report mean, and when does a second opinion help?

An ALK rearrangement (also called an ALK fusion) on your NGS (next-generation sequencing) report means your tumor — most often non-small cell lung cancer (NSCLC) — carries an ALK gene fusion that can drive its growth. This is generally one of the most actionable findings in lung cancer: in advanced NSCLC it is matched, at guideline category 1 level, with a drug class called ALK tyrosine kinase inhibitors (ALK TKIs). An ALK fusion is not the same thing as an ALK point mutation, and how it was detected genuinely matters, because some DNA-only panels can miss fusions that an RNA test would catch. Actionability again depends on your tumor type and treatment line, and the decision always belongs to the oncologist treating you.

1What does an ALK rearrangement or ALK fusion actually mean?

It generally means your tumor carries a structural rearrangement in which part of the ALK gene has fused to another gene, producing an abnormal protein that can drive cancer growth. This is most common in NSCLC, where ALK fusions occur in roughly 3 to 7 percent of cases, and the most common partner gene is EML4 (you may see a label such as EML4-ALK on your report). The key distinction is this: an ALK fusion or rearrangement is not the same thing as an ALK point mutation. It is the fusion that is matched with ALK-targeted therapy; ALK point mutations more often emerge as a resistance change that develops during treatment. In advanced NSCLC, an ALK fusion is generally treated as a strong, guideline-level (category 1) actionable driver and is matched with the ALK tyrosine kinase inhibitor class. ALK fusions can also appear in some other cancers (for example, in certain lymphomas and rarer tumors), but the evidence and degree of actionability vary by tumor type, so the same finding does not carry the same weight everywhere.

2My ALK results don't agree (FISH, IHC, and NGS) — or could the panel have missed it?

Discordant ALK results are a recognized issue, and yes, a DNA-only NGS panel can miss some genuine ALK fusions — which is why methodology matters. ALK status is commonly tested three ways: FISH (looks for the rearrangement in DNA), IHC (looks for the ALK protein), and NGS (sequencing). These do not always agree, and published case series describe tumors that read positive by one method and negative by another. An important practical point: in real-world datasets, RNA-based NGS detects meaningfully more ALK fusions than DNA-only NGS — roughly 15 to 18 percent more — because some fusion breakpoints and partners are difficult to capture from DNA. So if your clinical picture strongly points to an ALK-driven tumor (for example, a never- or light-smoking patient with lung adenocarcinoma) but a DNA panel comes back negative, it is reasonable to ask your oncologist whether orthogonal confirmation — RNA fusion testing, IHC, or FISH — is warranted before concluding ALK is absent. The reverse also matters: a positive result obtained by only one method, or an unusual fusion partner, may deserve closer scrutiny before being treated as fully actionable. This is exactly the kind of testing-methodology question a second opinion can help resolve.

3If I'm ALK-positive, what are my options, and what happens when the drug stops working?

If you have an ALK fusion in advanced NSCLC, the guideline-preferred first-line approach is generally a drug from the ALK tyrosine kinase inhibitor (ALK TKI) class, not chemotherapy or immunotherapy alone. Several generations of ALK TKIs are available, and current guidelines tend to favor newer-generation ALK inhibitors as initial therapy in many patients because they tend to work better and control brain metastases more effectively; the specific agent and sequencing are decisions your oncologist makes for your situation. Over time the cancer can develop resistance, and this is where NGS becomes useful a second time. At progression, retesting the tumor tissue or a blood-based (liquid biopsy) NGS test can sometimes reveal the mechanism — for example, G1202R, an acquired ALK point mutation that is among the resistance changes seen more often after second-generation ALK inhibitors. Identifying this mechanism can help your oncologist choose the next ALK inhibitor, because different resistance mutations respond differently to different drugs. The honest bottom line is that not every ALK report is equally actionable, the sequencing of treatments is individualized, and none of this replaces the assessment of the oncologist treating you.

In my NGS report, what does a BRAF V600E mutation mean?

A BRAF V600E mutation means your tumor carries a specific change at codon 600 of the BRAF gene that keeps an important growth-signaling pathway (the MAPK pathway) switched on. It is the most common and best-understood BRAF variant, and in many cancers it is a recognized, guideline-level target for BRAF-plus-MEK inhibitor therapy (a drug class). Two things determine whether it is actionable for you: your exact tumor type and your exact BRAF variant. V600E (called Class I) behaves very differently from non-V600 (Class II/III) BRAF mutations, where classic BRAF inhibitors can be ineffective or can even open the pathway instead of blocking it. In the large majority of cases, BRAF V600E is a somatic (tumor-specific only) change, not an inherited one. This page explains what the result means and where a second reading of your report can change the plan. This content is decision support, not a diagnosis, and the final decision belongs to the oncologist treating you.

1Is BRAF V600E targetable, and does that depend on my cancer type?

Yes, BRAF V600E is one of the most actionable findings on an NGS report, but how it is treated depends heavily on your tumor type and treatment line. In melanoma, BRAF V600E-positive disease is a classic indication for combined BRAF-plus-MEK inhibition (a drug class), and the same combination is recognized at guideline level in BRAF V600E non-small cell lung cancer and BRAF V600E anaplastic thyroid cancer. There is also a tissue-agnostic pathway: in 2022, the US FDA granted accelerated approval, based on basket trials, to a BRAF-plus-MEK inhibitor combination for a range of BRAF V600E solid tumors that had progressed on prior therapy and had no satisfactory alternative. Colorectal cancer is the key exception. Here, a BRAF inhibitor alone works poorly because the tumor reactivates the pathway through EGFR; that is why the established, guideline-level first-line standard for BRAF V600E metastatic colorectal cancer combines a BRAF inhibitor with an anti-EGFR antibody and fluorouracil-based chemotherapy. In other words, the same mutation can mean a single targeted combination in one cancer and a different, multi-drug strategy in another. Verify the tumor-type-specific recommendation against current NCCN and ESMO guidelines and the OncoKB BRAF page, and remember that actionability also depends on whether you are in the first line or a later treatment line.

2My report says BRAF but not V600E (a non-V600 / Class II/III mutation). Is this the same thing?

No, and this distinction is one of the strongest reasons for a second reading of a BRAF result. Not all BRAF mutations behave like V600E. Scientists divide them into three classes. Class I is V600E and its close relatives (such as V600K); these switch the pathway on by themselves, independent of upstream RAS signaling. Class II comprises non-V600 mutations that signal as active BRAF dimers. Class III has little or no kinase activity of its own and depends on upstream RAS signaling. The treatment implications differ markedly. Classic BRAF inhibitors designed for V600E can be ineffective against many non-V600 variants, and in some cases BRAF inhibitors can paradoxically open the MAPK pathway rather than block it. For Class II/III BRAF mutations, there is currently no widely approved targeted standard comparable to V600E, and management is far less well defined. If your report simply says “BRAF mutation,” or names a non-V600 alteration such as a fusion, an in-frame insertion or deletion, or a codon other than 600, it is worth confirming the exact variant and class before assuming a V600E-style treatment applies.

3Does BRAF V600E mean my cancer is hereditary or more aggressive, and what is the prognosis?

In the large majority of cases, BRAF V600E is a somatic mutation — meaning it arose in the tumor itself, is not inherited, generally is not passed from parent to child, and a tumor-only NGS result does not mean your relatives carry it. Whether it points to more aggressive disease depends on the cancer type and is not a verdict on your individual result. In some settings, such as papillary thyroid cancer and BRAF V600E metastatic colorectal cancer, the literature associates this mutation with, on average, a more aggressive biology, but this is a statistical pattern across groups, not a prediction for any one individual, and the data carry some debate. Equally important, the presence of BRAF V600E also opens targeted options that did not exist a decade ago. Prognosis is shaped by stage, tumor type, overall health, and treatment — not by a single word “BRAF” on a report. If hereditary risk is a concern for your family, that is a separate question, best addressed through dedicated germline testing and genetic counseling rather than inference from a tumor NGS panel.

What does HER2 (ERBB2) mean in my NGS report?

In an NGS report, HER2 (also written ERBB2) can refer to one of three different things, and they are not interchangeable: gene amplification (extra copies of the gene), an activating mutation (a change in the gene sequence, most often an exon 20 insertion in lung cancer), or protein overexpression measured by a separate stain. Which one your report shows matters greatly, because for most anti-HER2 therapies the established, guideline-level threshold is generally a protein-level or a specific DNA-level result confirmed with the right test (IHC and/or FISH), not RNA overexpression alone. Whether any given HER2 finding is actionable depends heavily on your tumor type and treatment line. Some HER2 findings open a clear, established treatment pathway; others are not actionable today. The decision belongs to the oncologist directing your treatment.

1What is the difference between HER2 amplification, an ERBB2 mutation, and HER2 overexpression?

These are three separate biological findings measured in different ways, and your NGS report may show one without the others. HER2 amplification means the tumor carries extra copies of the ERBB2 gene (a copy-number gain), and an NGS panel derives this from DNA. An ERBB2 (HER2) mutation is a change in the gene's sequence; in lung cancer the most common is an exon 20 in-frame insertion that can switch on the receptor independent of copy number. HER2 protein overexpression is the amount of HER2 protein on the cell surface, and DNA sequencing never measures this at all; it is read by immunohistochemistry (IHC) and graded as 0, 1+, 2+, or 3+. Gene amplification is the main driver of protein overexpression, and the two usually track together, though not always, which is why a confirmatory protein or FISH test is often required. An important caveat: if your report flags HER2 based on RNA expression alone, this is generally not by itself the approved actionability threshold, and should be confirmed with IHC and/or FISH. Guideline frameworks such as NCCN and ESMO define HER2 positivity by tumor type using these protein and in situ hybridization criteria.

2Is the HER2 (ERBB2) finding in my report actionable, and which cancers is anti-HER2 therapy used in?

It can be, but actionability depends on your tumor type, your treatment line, and which HER2 finding you have; a HER2 result is therefore never automatically a treatment plan. As a class, anti-HER2 therapy — including antibody-drug conjugates that deliver chemotherapy directly to HER2-positive cells — is most solidly established in HER2-positive breast and gastric/gastroesophageal cancers, and has long been part of guideline-based care. Its role is expanding to other tumor types as well: activating ERBB2 mutations in NSCLC are a recognized target, and HER2 amplification or overexpression is becoming increasingly important in colorectal and biliary tract cancers. A notable development is the 2024 tumor-agnostic accelerated approval of a HER2-directed antibody-drug conjugate for previously treated, unresectable or metastatic HER2-positive (IHC 3+) solid tumors without a satisfactory alternative, which underscores that the actionable threshold is generally a protein-level (IHC) result rather than RNA alone. Even so, eligibility, treatment line, and the exact testing required are defined by NCCN's and ESMO's tumor-type-specific guidelines, and some HER2 findings — for example, a low-level copy-number gain or an RNA-only signal — may not be actionable at all.

3My HER2 results don't agree (NGS says amplified but IHC/FISH says negative): what should I do?

Discordant HER2 results are a recognized situation, and the right next step is usually to resolve them with the correct confirmatory test, not to assume one result is simply wrong. While NGS measures the gene (DNA copy number or sequence), IHC directly measures the HER2 protein and FISH directly measures gene copies in the tissue; because they look at different things, they can diverge in a minority of cases. The clinically important point is this: approval and guideline thresholds are tied to specific tests — for most indications, HER2 status is defined by IHC, with FISH used to resolve equivocal protein results, as specified by NCCN and ESMO. So an NGS amplification result that conflicts with a negative IHC, or a HER2 flag based on RNA expression, generally needs to be reconciled by ordering or reviewing the guideline-standard test (IHC and/or FISH) on sufficient tumor tissue for your tumor type. A second opinion here is essentially about making sure the correct confirmatory test was actually performed and interpreted against the criteria for your tumor type, because that is what determines whether anti-HER2 therapy is on the table. This is decision support to help you ask the right questions; the testing and treatment decision belongs to the oncologist and pathologist directing your care.

In an NGS report, what do MSI-H / dMMR and TMB mean, and when should a second opinion be sought

If your tumor NGS (next-generation sequencing) report says MSI-H (high microsatellite instability), dMMR (mismatch repair deficiency), or TMB-high (high tumor mutational burden), the core message is the same: these are guideline-level biomarkers that point toward immunotherapy with immune checkpoint inhibitors. MSI-H/dMMR is one of the few markers that can guide treatment across many cancer types regardless of where the tumor started, and it was the basis for the first tumor-agnostic immunotherapy approval. Whether this translates into a treatment for your specific situation still depends on your tumor type, treatment line, and other findings, and that decision belongs to the oncologist directing your care. This page explains in plain language what these results mean and when a careful second look is worth requesting.

1What does MSI-H or dMMR mean, and is it good or bad?

MSI-H and dMMR describe the same underlying problem in the tumor: the cell's DNA "proofreading" system (mismatch repair) is broken, so the tumor accumulates many small DNA errors, especially in repetitive regions called microsatellites. dMMR usually refers to loss of one or more repair proteins (MLH1, MSH2, MSH6, PMS2) seen on immunohistochemistry, while MSI-H refers to the resulting instability measured by PCR or NGS; in most tumors the two findings agree. From a treatment standpoint this is generally a favorable finding, because tumors with this defect tend to respond to immune checkpoint inhibitors. The evidence was strong enough that in 2017 the US FDA granted the first tumor-agnostic (tissue/site-independent) accelerated approval of a checkpoint inhibitor for MSI-H/dMMR solid tumors, and a second class of checkpoint inhibitor agent later received a tumor-agnostic approval for dMMR solid tumors as well. An honest caveat: a "favorable biomarker" does not mean every MSI-H tumor will respond, and the strength of the recommendation still depends on your cancer type and where you are in your treatment course, which is why guidelines (NCCN, ESMO) treat it as one input feeding into a decision rather than an automatic prescription. MSI-H/dMMR also carries a second meaning beyond treatment: it can be a clue pointing to Lynch syndrome, an inherited condition, discussed below.

2My report shows MSI-H but TMB is low (or the results don't agree). Should I have this re-confirmed?

Yes; an internally inconsistent result is one of the clearest reasons to request a second look before acting on it. MSI-H and high TMB usually occur together, because a broken repair system that produces microsatellite instability also tends to produce a generally high mutation count. If a report calls a tumor MSI-H while TMB is low, or the MSI signal is borderline, this discordance raises the possibility of a borderline or false-positive result, and confirmation is reasonable — for example, mismatch repair immunohistochemistry (MMR-IHC), PCR-based MSI testing, or central pathology review. Concordance between methods is generally high, but the rare discordant cases are exactly the ones where a wrong result would most change the plan. There is also a tumor-biology nuance your oncologist will weigh: TMB performs more consistently as an immunotherapy signal in cancers with known mutagenic exposure (for example, smoking- or UV-related tumors) and less consistently in others, so a TMB number should be read in the context of cancer type rather than as a verdict on its own. The practical takeaways: don't treat an ambiguous MSI-H/low-TMB combination as settled, and ask whether an orthogonal confirmatory test (such as MMR-IHC) is warranted before an immunotherapy decision is finalized.

3Does this come from blood or tissue, and does MSI-H mean I need to be tested for Lynch syndrome?

These are two separate but common questions, and both matter for a second opinion. The first concerns the sample type: MSI and TMB results derived from tumor tissue are the validated, guideline-anchored basis for immunotherapy decisions. A blood-based (liquid biopsy) TMB or MSI result depends heavily on how much tumor DNA has shed into the blood, so it can miss findings when circulating tumor fraction is low, and current practice generally treats blood TMB as a proxy used mainly when tissue is scarce, not a substitute for a tissue result. If an important decision rests solely on a blood-based TMB or MSI value, it is reasonable to ask whether tissue confirmation is feasible. The second concerns heredity: an MSI-H/dMMR tumor can be a signal for Lynch syndrome, an inherited cancer predisposition condition, which is why guidelines support considering referral for genetic counseling and germline testing, particularly with a younger age at diagnosis or a suggestive family history. The tumor finding alone does not confirm Lynch syndrome (especially in colorectal cancer, where additional steps such as MLH1 promoter methylation or BRAF testing help distinguish sporadic from inherited cases), but it is the trigger that starts this conversation, and it carries implications for you and your blood relatives. Your oncologist and a genetics unit can determine the right path.

What does a ROS1 fusion mean in my NGS report?

ROS1 is a driver alteration seen in roughly 1–2 percent of lung adenocarcinomas and, more rarely, in a few other tumor types; frequency estimates vary by series. The key point is this: what is actionable is a fusion (rearrangement) finding. Simply seeing "ROS1" mentioned in your report is not, by itself, enough; a point mutation or copy-number change reported in the ROS1 gene is generally not considered actionable today. Once a ROS1 fusion is confirmed, however, guidelines favor a targeted ROS1 tyrosine kinase inhibitor in advanced non-small cell lung cancer. The decision belongs to the oncologist directing your treatment; this page explains how the report is read and where a second look makes a difference.

1What are the treatment options once a ROS1 fusion is found?

When a confirmed ROS1 fusion is present in advanced non-small cell lung cancer, the guideline-preferred approach is generally a ROS1 inhibitor rather than chemotherapy or immunotherapy alone. This class includes agents such as crizotinib, entrectinib, and repotrectinib; ceritinib and lorlatinib have also been used at some centers. The distinction between them is not just efficacy but central nervous system (CNS) activity: in a patient with brain metastasis or at risk of brain recurrence, agents with better CNS penetration are preferred — a practical distinction that can change the first-line choice. Agent availability and approval status vary from country to country and change over time, so current approval and reimbursement status should be confirmed with the treating team. The second-opinion question here is usually not "should a ROS1 inhibitor be given?" but "which ROS1 inhibitor, in what sequence, for this patient?"

2Could the panel have missed ROS1?

Yes, and this is one of the clinically most important technical details. Fusions may not be reliably captured by DNA-based panels alone, because breakpoints fall in intronic regions and some partner genes fall outside panel coverage. RNA-based fusion testing, IHC (ROS1 protein staining), and FISH are orthogonal methods that complement one another. If the clinical picture strongly suggests a driver mutation — for example, a never- or light-smoker with adenocarcinoma histology whose results are negative for EGFR/ALK/KRAS — and the DNA panel is negative, it is worth asking whether RNA-based fusion testing or IHC should be ordered before concluding ROS1 is absent. The reverse also holds: a result positive only by IHC, or an unusual fusion partner, may warrant molecular-level confirmation before starting treatment. A report saying "negative" is not the same as saying "not tested"; one of the first things checked in a second opinion is the panel's method and coverage.

3What happens when the drug stops working?

ROS1 inhibitors provide durable control in most patients, but the development of resistance is an expected process and it can arise in two different forms. The first is resistance mutations within the kinase domain that block the drug's binding; the best known is the solvent-front mutation G2032R, which confers resistance to some first-generation agents while later-generation inhibitors have been designed to retain activity against this variant. The second is activation of bypass pathways independent of ROS1, or histologic transformation, both of which call for a different strategy. In practice, this means that before simply "moving to the next drug" at progression, discussing a renewed molecular workup (tissue rebiopsy and/or plasma ctDNA) to understand the resistance mechanism is worthwhile. If progression is limited to the brain while disease elsewhere remains controlled, continuing the current therapy with local treatment rather than switching drugs can also be an option. The treating oncologist evaluates all of these distinctions.

In my NGS report, MET: exon 14 skipping and amplification are not the same thing

MET can appear in a report in two entirely different forms, and their treatment implications are not the same. The first is the MET exon 14 skipping alteration: a classic driver mutation in which a change in the gene sequence causes a portion of the protein to be skipped, reported in roughly 3–4 percent of lung adenocarcinomas. The second is MET amplification: the presence of extra copies of the gene. Amplification is not as firmly established an indication as exon 14 skipping; the threshold values, measurement method, and clinical context (for example, resistance after an EGFR inhibitor) substantially change what the result means. When you see "MET" in your report, the first question to ask is which of the two it is.

1Is MET exon 14 skipping actionable?

A confirmed MET exon 14 skipping alteration in advanced non-small cell lung cancer is a guideline-recognized biomarker for targeted therapy; selective MET inhibitors such as capmatinib and tepotinib are used for this indication. Two clinically important points apply. The first is patient profile: this alteration can occur in older patients and those with a smoking history, more so than other driver mutations — meaning it should be sought even in a patient who does not fit the "classic driver mutation" profile. The second is toxicity: manageable but frequent side effects such as edema and nausea are expected with MET inhibitors, and dose management determines the sustainability of treatment. This is decision support; the indication, treatment line, and choice of agent are determined by the treating oncologist.

2My report shows MET amplification. Is this a target?

This is a valid finding but one that requires careful reading. With MET amplification, the critical question is not "present or absent" but how much and how it was measured: low-level copy-number gains and high-level focal amplification do not mean the same thing, and chromosome 7 polysomy can mimic true focal amplification. NGS panels report copy number using different algorithms and different thresholds, and a gene-to-centromere ratio reported by FISH does not directly substitute for the copy number reported by a panel. In addition, amplification can be underreported when tumor purity is low. Clinically, the most established context for MET amplification is as a resistance mechanism that emerges after EGFR inhibitor therapy in EGFR-mutant lung cancer, a scenario in which combination strategies and clinical trials come into play. It is not correct to assume, on its own, that a pre-treatment MET amplification automatically has a treatment counterpart.

3Why is the MET result so prone to misreading?

For three reasons. First, the alterations that cause exon 14 skipping are not a single "hotspot": numerous different point mutations, small deletions, and insertions in the splice region can produce the same outcome, so narrowly designed, DNA-only panels can miss non-canonical variants, and RNA-based confirmation has value here. Second, not every alteration reported as a "MET mutation" is exon 14 skipping; other mutations in the kinase domain, or VUS findings, do not carry the same clinical meaning. Third, protein-level MET overexpression (IHC), gene amplification, and exon 14 skipping are separate concepts and cannot be used interchangeably. Clarifying which of these is being reported in a given report is the first task performed for MET in a second opinion.

In my NGS report, RET: fusion, mutation, or hereditary?

The first distinction when reading a RET result is this: is a fusion being reported, or a point mutation? RET fusions occur in a range of tumors, most notably lung adenocarcinoma and papillary thyroid cancer, and are an established target for selective RET inhibitors. RET point mutations, on the other hand, are primarily associated with the biology of medullary thyroid cancer, and here an additional dimension comes into play: some of these mutations are hereditary (MEN2 syndromes). In other words, RET is one of the few findings that can have consequences for both treatment and family, and these two questions must be addressed separately.

1A RET fusion was found. What comes next?

Selective RET inhibitors — selpercatinib and pralsetinib — are agents developed specifically for RET fusion-positive tumors and are guideline-listed for RET fusion-positive advanced lung cancer and RET-altered thyroid cancers; a tumor-agnostic approval pathway has also been defined for solid tumors carrying a RET fusion (current approval and reimbursement status varies by country and should be verified). The practical distinction here is between selective RET inhibitors and multikinase inhibitors that inhibit RET more broadly alongside a wider side-effect profile (for example, cabozantinib, vandetanib); selective agents generally offer a more targeted effect and a more manageable toxicity profile. Treatment sequencing is determined by disease type and prior therapies.

2I have a RET mutation; is genetic counseling needed?

A RET mutation reported in the context of medullary thyroid cancer is a clear trigger for germline (hereditary) testing. A subset of medullary thyroid cancer cases are part of MEN2A or MEN2B syndromes, and this diagnosis raises the need to screen not only the patient but also first-degree relatives, and in some cases to consider preventive interventions. The critical technical point is this: an NGS panel run on the tumor cannot definitively distinguish whether a variant is hereditary or acquired; that requires a separate germline test from blood and genetic counseling. Seeing a RET mutation in a tumor report and reading it only as a treatment target often means missing the most important finding in the report.

3Why is fusion testing method-dependent?

Like other fusions, RET fusions are a type of alteration that DNA-based panels are structurally challenged to detect: breakpoints can be scattered across large intronic regions, and partner gene diversity is high. RNA-based sequencing is generally more sensitive for fusion detection; FISH, and in some cases IHC, provide orthogonal confirmation. Clinically, this means that in a never-smoker lung adenocarcinoma patient whose driver mutation testing comes back negative, before closing the case as "no driver found," one should ask which method the panel used to search for fusions. In a second opinion, this is often the single question that changes the plan.

In my NGS report, NTRK: only fusions count

NTRK1, NTRK2, and NTRK3 fusions are one of the few findings that can guide treatment regardless of where the tumor originated; larotrectinib and entrectinib are agents that have received tumor-agnostic (tissue-agnostic) approval in this context. However, the most common error in this area is not technical but one of interpretation: not every NTRK finding mentioned in a report is a fusion. Point mutations and copy number alterations in NTRK genes are common in some cancers and do not constitute an indication for TRK inhibitors. NTRK fusions are also very rare; they are reported in under 1 percent of common adult cancers, while in certain rare tumors such as secretory carcinoma and infantile fibrosarcoma they are almost defining.

1My report says NTRK. Am I a candidate for targeted therapy?

This depends entirely on the type of finding. If there is a confirmed NTRK fusion (for example with a partner such as ETV6–NTRK3), TRK inhibitors are a guideline-listed option in advanced disease when no other satisfactory option exists, and this is one of the rare situations defined independently of tumor type. By contrast, if the report states only something like "NTRK1 p.… variant" or "NTRK2 amplification," this is not actionable at the current level of evidence and should not create an expectation of a TRK inhibitor. This distinction matters greatly in practice: some cases referred for a second opinion as "NTRK positive" turn out, once the report is re-read, to involve not a fusion but a meaningless background variant.

2How is a fusion confirmed?

Three methods are used together, and none is flawless on its own. Pan-TRK immunohistochemistry is a fast screening tool but can give false positives in some tissues, and its sensitivity drops for some fusions, particularly certain cases involving NTRK3; a positive IHC therefore requires molecular confirmation. DNA-based NGS panels can miss fusions due to intronic breakpoints and partner gene diversity. RNA-based sequencing is generally the most reliable approach for fusion detection and is reasonable to request when clinical suspicion is high. When searching for a rare finding, the choice of method is as decisive as the result itself.

3What happens if resistance develops after a TRK inhibitor?

Responses achieved with TRK inhibitors are often rapid and pronounced, but not durable. Resistance frequently develops through secondary mutations in the kinase domain (for example, solvent-front mutations) or through activation of alternative pathways. Distinguishing between these two mechanisms matters, because next-generation TRK inhibitors have been developed specifically to address kinase domain mutations, whereas bypass resistance requires a different strategy. It is therefore reasonable to discuss repeating molecular assessment (tissue or plasma) at the time of progression. The decision on subsequent treatment rests with your treating oncologist.

In my NGS report, what do BRCA1 / BRCA2 and HRD mean?

A BRCA1 or BRCA2 finding opens two distinct questions at once in a report, and these must not be conflated. The first concerns treatment: BRCA alterations point to a defect in DNA repair, and this defect has been associated with sensitivity to PARP inhibitors and platinum-based therapies; there are guideline-defined uses in ovarian, breast, prostate, and pancreatic cancers. The second concerns inheritance: a significant proportion of BRCA variants are germline, meaning they can be carried in the family. A tumor panel cannot definitively distinguish between the two. HRD (homologous recombination deficiency), meanwhile, is a broader concept than BRCA and requires a separate interpretive discipline.

1Is the BRCA finding in the tumor report hereditary?

NGS performed on tumor tissue alone cannot reliably tell whether a BRCA variant is hereditary or acquired, because the tumor also contains any germline variant the patient already carries. Variant allele frequency and zygosity can offer some clues, but these are not definitive. The clinically correct step is to consider referral for a separate germline test from blood and genetic counseling whenever a pathogenic or likely pathogenic BRCA variant is reported in the tumor report. This distinction does not concern the family alone; in some contexts, the treatment indication itself is also defined according to the germline versus somatic distinction. This is one of the most commonly missed steps encountered in second opinions.

2What is the HRD score, and are the tests interchangeable?

HRD is not the result of a single gene but a measure of the damage signatures left in the tumor's genome; different commercial tests calculate it using different components (for example, loss of heterozygosity, telomeric allelic imbalance, large-scale transitions) and different thresholds. The practical consequence is this: an "HRD positive/negative" result from one test is not directly equivalent to the result from another test, and which test was used in which study matters when interpreting the indication. Furthermore, an HRD-negative result does not always mean no benefit will be seen from a PARP inhibitor; in some indications, use is defined independently of this biomarker. When reading an HRD result, which test was used, what the threshold was, and to which clinical context it was applied must all be considered together.

3How does resistance develop after a PARP inhibitor?

One of the best-characterized mechanisms is reversion mutations: the tumor develops a second alteration that restores the reading frame of the disrupted BRCA gene and partially regains DNA repair function; this has been associated with resistance to both PARP inhibitors and platinum. Alterations of this type are not present in the baseline report but can be detected in a new sample obtained at progression, particularly in plasma ctDNA. For a patient progressing on a PARP inhibitor, it is therefore worth discussing an updated molecular assessment rather than re-reading the old report. The choice of subsequent treatment is determined by the treating oncologist.

What does a PIK3CA mutation in my NGS report mean?

PIK3CA is one of the most frequently mutated genes in cancer, reported in roughly one-third of hormone receptor–positive, HER2-negative breast cancers (frequency estimates vary by series). Being common does not mean every PIK3CA finding has a treatment counterpart: actionability depends on both the tumor type and the variant itself. In breast cancer, defined targeted options exist for specific activating mutations, while in other tumor types the same variant today usually points not to a standard treatment but to clinical trial evaluation.

1What does a PIK3CA mutation mean in breast cancer?

In hormone receptor–positive, HER2-negative advanced breast cancer, a PIK3CA mutation is an established selection marker for therapies targeting the PI3K pathway; alpelisib is a PI3Kα inhibitor developed in this context, capivasertib is an AKT inhibitor used in patients with a PIK3CA/AKT1/PTEN alteration, and inavolisib is defined within a combination regimen in PIK3CA-mutant, endocrine-resistant disease. These are not interchangeable options: each has been studied in a different clinical scenario, with different combinations, and in patients with different prior treatment histories. Which agent is appropriate is determined by prior endocrine therapy, CDK4/6 inhibitor use, and overall clinical status. Approval and reimbursement status vary by country and should be verified.

2Are all PIK3CA mutations the same?

No, and this is one of the details most often overlooked in reports. PIK3CA has specific hotspot mutations defined in clinical trials; these classic variants in the helical and kinase domains are considered activating. Rarer variants or those of uncertain significance outside these do not carry the same level of evidence and cannot automatically be used as grounds for access to a drug. The co-occurrence of more than one activating PIK3CA mutation is also a distinct biological situation. One of the things done in a second opinion is checking exactly which trial population the specific variant reported corresponds to — that is, whether the patient actually falls within that evidence.

3Should I be tested from blood or tissue, and what should I know before starting treatment?

PIK3CA mutations can be tested for in both tumor tissue and plasma ctDNA; plasma is a practical starting point, but a negative plasma result does not prove the mutation's absence, and tissue testing is recommended in that case. On the treatment side, the most important thing to know in advance is toxicity: hyperglycemia is an expected side effect of agents targeting the PI3K/AKT pathway and must be managed; baseline glucose and HbA1c assessment is therefore required, along with a pre-planned monitoring strategy if diabetes or prediabetes is present. Preventive approaches have also been defined for side effects such as rash and stomatitis. This preparation directly affects the likelihood of treatment discontinuation and should be discussed before starting.

In my NGS report, FGFR: the type of alteration changes everything

The FGFR family (FGFR1–4) is commonly seen in reports, but actionability depends almost entirely on three questions: which gene, which type of alteration, and which tumor. FGFR2 fusions or rearrangements are an established target in intrahepatic cholangiocarcinoma; FGFR3 mutations and fusions open a defined treatment pathway in urothelial carcinoma. FGFR1 amplification, by contrast — despite being frequently reported in squamous lung cancer and breast cancer — has not to date become established as a reliable response marker. In other words, the phrase "FGFR-positive" carries no meaning on its own.

1Which FGFR finding actually opens up treatment?

Today there are two clearest contexts. First, FGFR2 fusion or rearrangement in previously treated advanced intrahepatic cholangiocarcinoma: in this group, FGFR inhibitors such as pemigatinib and futibatinib are options included in guidelines. Second, advanced urothelial carcinoma carrying an appropriate FGFR3 (or FGFR2) alteration: erdafitinib is a defined option in this context. FGFR findings outside these two contexts — particularly amplifications and non-canonical mutations — generally do not constitute a standard indication and more often point to clinical trial evaluation. Eligibility and approval status vary by country and should be verified with the treating team.

2Can an FGFR2 fusion be missed on a panel?

Yes. FGFR2 rearrangements are alterations with high partner diversity, and breakpoints can fall outside a panel's design coverage; in some cases, more complex structural rearrangements are found instead of a fusion. For this reason, in an intrahepatic cholangiocarcinoma case with high clinical suspicion, a negative result on a DNA-based panel should not be accepted as definitive without confirmation by RNA-based fusion testing or FISH. Likewise, a finding reported as an "FGFR2 mutation" does not carry the same clinical meaning as a fusion; exactly what has been reported needs to be clarified. This is one of the points where a second opinion most often adds value in cholangiocarcinoma.

3What should I know before starting an FGFR inhibitor?

This drug class has class-specific side effects that must be planned for in advance. Hyperphosphatemia is an almost expected finding and is managed with phosphate level monitoring, dietary adjustment, and phosphate-binder therapy when needed. Eye-related side effects — particularly the picture described as subretinal fluid accumulation — require regular ophthalmologic evaluation, and visual changes should be reported without delay. Nail and skin changes, along with dry mouth, are also common. In addition, secondary mutations arising in the kinase domain during treatment can lead to resistance; repeat molecular evaluation at progression is worth discussing. This monitoring plan should be clarified before starting treatment.

TP53: the most frequently found, least drug-selecting gene

TP53 is the gene most frequently encountered in reports, yet the one that changes treatment least directly. This contradiction is the point where a report is most easily misread: TP53 is not a predictive marker but a prognostic and contextual one. Its value emerges not on its own, but when read together with the rest of the tumor's molecular picture.

The right time for a second opinion

If you already have your NGS report and any of the following applies, contact us:

• The report contains an actionable finding, but the treatment plan does not reflect it.
• The report is "negative," but the clinical picture (age, smoking history, tumor type) suggests there may be more to find.
• Different laboratories have returned conflicting results.
• Disease progressed during treatment and the resistance mechanism was never investigated.

The NgsAdvisory team re-evaluates your report using the IYGAS™ methodology and prepares a structured second-opinion report you can share with your treating physician.