Your NGS Report Is In: What Does It Leave Unsaid?

No targetable mutation was found on the tumor's DNA panel; the report looked practically "empty." Had we relied on that result alone, the patient would have been planned for standard chemotherapy only.

However, RNA-based fusion analysis of the same sample revealed an FGFR2 fusion. In biliary tract cancer, this is the direct target of FDA-approved targeted drugs (FGFR inhibitors).

Why was it not seen on DNA? A significant proportion of FGFR2 fusions cannot be captured by a DNA panel because the breakpoint falls in a region not covered by the panel's probes; RNA, by reading the mature transcript, shows the fusion directly.

The report said "BRAF mutation." The first reflex might be to think of the BRAF inhibitors known in melanoma or colorectal cancer. But the mutation here was not the classic BRAF V600E; it was a Class III (non-V600) BRAF mutation (BRAF p.G464E), a subtype with low kinase activity that signals through RAS-dependent dimers (CRAF).

For this reason, the classic (Class I) BRAF inhibitors (vemurafenib, dabrafenib, encorafenib) designed to target V600E monomers are ineffective on their own against this mutation and may paradoxically activate the MAPK pathway.

Low-grade serous ovarian cancer is relatively resistant to chemotherapy; the expected benefit from a classic cytotoxic approach is limited. The report contained a dominant KRAS mutation.

In this subtype, the standard drug-mutation match would, based on the KRAS driver, highlight a MEK/RAF-axis targeted combination. But this combination was neither accessible for this patient nor, even if it were, anything but unnecessary and toxic at this point.

Brief rationale: in a setting where chemotherapy does not work and a toxic or inaccessible targeted combination is also unnecessary, the tumor's own biology (hormone receptor positivity) pointed to the least toxic and most feasible path. So not only "what to do" but also "what not to do" was clearly defined.

The patient carried a KRAS G12R mutation; a variant that currently has no allele-specific approved drug. At first glance it may look "untargetable."

The molecular interpretation turned the phrase "no target" into the insight "the strongest opportunity is actually immunotherapy."

Let us state it up front: the standard report (Guardant), based on a CHEK2 alteration, recommended a PARP inhibitor (olaparib, talazoparib) for this patient. We rejected that recommendation. This case shows how we diverged at three separate decision points within a single report:

• 1We closed off the false-positive PARP path. The VAF of the alteration was only 0.6%; far below the tumor fraction (16.2%), i.e., a deeply subclonal signal; CHEK2-related clonal hematopoiesis (CHIP) variants in the same patient were confounding the signal; and there is no CHEK2-based PARP-i approval in urothelial cancer. Result: an unnecessary and unfounded targeted therapy was avoided.
• 2We placed an isolated number into a holistic biology. The standard report left a high TMB value (28.47 mut/Mb) as just an independent data line. We synthesized it together with ARID1A loss + TERT promoter mutation + urothelial histology into a coherent immunotherapy-favoring biology; with the caveat that "blood-TMB is not a tissue-approved analyte; tissue confirmation is advised."
• 3The most valuable part: we prevented an error. A clinician seeing the FGFR1 amplification in the report might reflexively consider erdafitinib. We warned explicitly: erdafitinib is approved only for FGFR3 mutations or FGFR2/3 fusions; FGFR1 amplification is not eligible (and the amplification here was low level, copy number ~2.3). Avoiding giving the right drug to the wrong target.

The standard NGS report recommended an older, more generic MEK-axis combination against KRAS (avutometinib + defactinib).

The principle is what matters: in precision oncology, evidence changes within months; the recommendation must align with the most current phase 3 literature at the moment the report is written. Standard vendor reports often do not update at this pace.

There was a discordance here: while DNA sequencing showed no mutation or loss in SMARCA4, immunohistochemistry (IHC) revealed SMARCA4 protein loss.

This is a classic example of not falling into the "if it is not in the report, it does not exist" fallacy.

The report contained several alterations at once: an AR amplification, biallelic PTEN loss, an FGL1-NRG1 fusion and a borderline-high blood-TMB. The standard report recommended capivasertib for PTEN loss and zenocutuzumab for the NRG1 fusion side by side, without any prioritization.

• Biallelic PTEN loss → capivasertib (moved to the front): a genuinely actionable target with phase 3-level evidence in prostate cancer (AKT pathway inhibition) and a direct match to the tumor type.
• FGL1-NRG1 fusion → zenocutuzumab (not prioritized): its approved indications are NRG1-fusion lung, pancreatic and biliary tract cancers; there is no approval in prostate. Reliable confirmation also requires tissue-based RNA, and this case was a liquid biopsy.
• AR amplification: this was a marker of resistance rather than a targetable opportunity (it implies cross-resistance when switching hormonal agents).
• Blood-TMB: it was borderline and liquid-based; a conditional signal that would require tissue confirmation before any single immunotherapy decision.

Here the difference was not finding a new target; it was performing the prioritization the standard report did not. Choosing the right target matters, and so does not promoting an appealing but inappropriate option; that too is part of this guidance.

The standard report pointed this patient toward two targeted therapies and immunotherapy together: a PARP inhibitor for CHEK2, an inhibitor for MET amplification, and immunotherapy given the high PD-L1 (22C3, CPS 60). But it did not prioritize.

• CHEK2 → PARP inhibitor (we rejected it): a high VAF does not make a variant a PARP target. CHEK2 p.I157T was a low-penetrance, founder-type missense variant and, at 42.6% VAF (~66% adjusted), in the heterozygous germline range; not a biallelic loss acquired by the tumor. PARP requires genuine HRD. We referred the high VAF to germline confirmation and genetic counseling.
• MET amplification → targeted inhibitor (we put it in context): MET inhibitors (capmatinib/tepotinib) are approved in NSCLC and off-label in urothelial; they should be considered only within an appropriate clinical trial, as a later-line option.

We also maintained the "do not give the right drug to the wrong target" discipline: erdafitinib was not appropriate because FGFR3 was wild-type; and because of RB1 loss (together with dual TP53 loss), cautious surveillance was advised for neuroendocrine/small-cell transformation at progression; not a treatment change, but a monitoring signal.

There was a discordance in the same report: while RNA sequencing showed HER2 (ERBB2) and ERBB3 overexpression, the DNA panel showed no HER2 amplification. RNA said "could be a target," while DNA said "no amplification."

• If IHC 3+ / FISH+: an effective treatment door opens, such as trastuzumab deruxtecan (tumor-agnostic) or zanidatamab; otherwise it could have gone unnoticed.
• If not: an unnecessary and ineffective anti-HER2 therapy based on the RNA signal alone is avoided.

Cross-platform mismatch (RNA vs DNA vs protein) is not "confusion"; managed correctly, it is a decision opportunity.

The vendor's report listed some expression values on RNA-Seq (high TOP2A, low MGMT); but provided them only as Research Use Only (RUO) data and made no treatment recommendation. For the clinician these lines could have remained "numbers that do not tell me what to do."

• High TOP2A: topoisomerase II-alpha is the direct target of anthracyclines (doxorubicin). High expression also supported, at the molecular level, the first-line doxorubicin-based regimen that is already NCCN Category 1.
• Low MGMT: raises a hypothesis of sensitivity to alkylating agents (dacarbazine / temozolomide); an option that could be considered at the next line.

In clinical practice this is exactly the difference: turning a data line marked "no recommendation" in a vendor report into an actionable treatment rationale for the patient.

The first-line treatment is clear: a RET-specific inhibitor (selpercatinib), NCCN Category 1. But the real contribution was the warning that anticipated what would happen later, before treatment had even started.

• Histologic transformation (SCLC) surveillance: biallelic TP53 inactivation and RET-driven genomic instability create a substrate for phenotypic transformation to SCLC. If rapid progression or neuroendocrine features appear at progression, re-biopsy; if transformation is confirmed, treatment shifts toward carboplatin + etoposide ± atezolizumab (IMpower133).
• Alternative (bypass) pathway warning: ERBB3 (HER3) and MET overexpression on RNA-Seq pointed to a bypass-resistance route via PI3K-AKT under a RET inhibitor. Investigation by re-biopsy at progression was defined in advance.
• IO position: even if PD-L1 looks favorable, single-agent immunotherapy is not recommended in the first line when a RET fusion is confirmed (RET-fusion NSCLC is "immune-cold"); the RET inhibitor takes priority.

The difference here is proactive: while treatment is just beginning, mapping out for the physician in advance how resistance and transformation might emerge and what to do at that moment.

The report returned an MSI status of "borderline/equivocal." A clinician could have read this as a possible MSI-H signal, i.e., an immunotherapy opportunity.

The difference was, instead of jumping to a single "promising" line, checking biological consistency and protecting the patient from being steered toward an immunotherapy that would probably not work.