You have your NGS report in hand — but what about what it doesn't tell you?

Interpreting multi-platform testing (DNA + RNA) together opened the door to a survival-extending targeted therapy option that would otherwise have been missed entirely. Our recommendation is clear: DNA alone is insufficient in bile duct cancers; RNA fusion analysis should be considered mandatory.

We emphasized that classic (Class I) BRAF inhibitors should be avoided in this profile; the rational target is the MEK inhibition axis. We also drew attention to relevant early-phase trial pathways for the accompanying MTAP deletion and ARID1A/KDM6A losses.

Because of the ER and PR expression detected on the RNA layer of the NGS (hormone receptor positivity), we recommended hormonal suppression (endocrine therapy), which is much better tolerated; this choice is supported by the fact that low-grade serous ovarian cancer is often hormone-sensitive. Single-agent MEK inhibitor was also noted as an option.

Immunotherapy was not recommended due to low TMB, MSS status, and PD-L1 negativity.

KRAS was accompanied by TP53, and STK11/KEAP1 were absent; this combination defines the molecular subgroup with the best immunotherapy response in KRAS-mutant lung cancer. As a priority, we recommended a first-line chemo-immunotherapy combination, requested that PD-L1 testing be completed urgently, and drew attention to a next-generation pan-RAS inhibitor trial in case of progression.

The CHEK2 alteration's VAF was only 0.6%, far below the tumor fraction (16.2%) — that is, a deeply subclonal signal; the same patient had CHEK2-associated clonal hematopoiesis (CHIP) variants confounding the signal; and there is no CHEK2-based PARP inhibitor approval in urothelial cancer. Outcome: an unnecessary, unsupported targeted therapy was avoided.

The standard report left a high TMB value (28.47 mut/Mb) without an associated treatment. We synthesized this, together with ARID1A loss + TERT promoter mutation + urothelial histology, into a biology consistent with — and favoring — immunotherapy, and converted it into a numeric IO-suitability score; we added the caveat that "blood-based TMB is not an analyte validated against tissue; tissue confirmation is recommended."

A clinician seeing FGFR1 amplification on the report might reflexively consider erdafitinib. We explicitly cautioned against this: erdafitinib is approved only for FGFR3 mutation or FGFR2/3 fusion; FGFR1 amplification is not eligible (and here the amplification was low-level, with a copy number of ~2.3). Avoiding giving the right drug to the wrong target is the highest-value type of contribution from a safety standpoint.

Based on very recently reported positive Phase 3 data for this variant, we prioritized the pan-RAS inhibitor daraxonrasib, which directly targets RAS. In previously treated metastatic pancreatic cancer, this agent showed a significant overall survival advantage over standard chemotherapy (RASolute 302 Phase 3, KRAS G12 mutant population). The drug is still under regulatory review and is accessible through an appropriate clinical trial.

Rather than glossing over the discrepancy, we explained its mechanism: a protein disappearing without a detectable DNA alteration usually points to epigenetic silencing (promoter methylation) or other mechanisms a panel cannot capture. To resolve the discrepancy, we recommended an additional methylation test. The correct treatment decision can only be made after this confirmation.

PTEN biallelic loss → capivasertib (moved to front): a genuinely actionable target that matches the tumor type directly and has Phase 3-level evidence in prostate cancer (AKT pathway inhibition).

AR amplification was more a marker of resistance than a targetable opportunity. Blood-based TMB was borderline and liquid-derived — a conditional signal requiring tissue confirmation before any standalone immunotherapy decision.

Given this profile and treatment history, due to the high PD-L1 we prioritized immunotherapy (pembrolizumab). High PD-L1 together with the accompanying ARID1A loss was a strong checkpoint-sensitivity signal; it was the most robust, evidence-based option at this line.

A high VAF does not make a variant a PARP target. CHEK2 p.I157T was a low-penetrance, founder-type missense variant, and at a purity-corrected VAF of ~66% it sat in the heterozygous germline range rather than representing biallelic loss. A PARP inhibitor requires genuine HRD. We placed the high VAF in the context of germline confirmation and genetic counseling and referred the patient to clinical genetics.

MET inhibitors (capmatinib/tepotinib) are approved in NSCLC but are off-label in urothelial cancer; they should be considered only within an appropriate clinical trial, as a later-line option. Also, since FGFR3 was wild-type, erdafitinib was not applicable; given the combined RB1 + TP53 loss, we recommended careful monitoring for neuroendocrine transformation.

For anti-HER2 therapy (e.g., ADCs such as trastuzumab deruxtecan), the actionable and approved threshold is not RNA overexpression but the protein level — that is, IHC, supported by FISH when needed. RNA overexpression alone is not a validated biomarker; the only correct way to resolve the discrepancy was to request HER2 IHC/FISH.

TOP2A high: the direct target of anthracyclines (doxorubicin); this molecularly supported the already NCCN Category 1 first-line doxorubicin-based regimen.MGMT low: a hypothesis of sensitivity to alkylating agents (dacarbazine/temozolomide); could be considered at a later line.CCNE1 high: the Cyclin E1 / CDK2 axis; a rationale for an appropriate CDK2 inhibitor trial.

This RNA expression data is RUO; there is no validated companion diagnostic test. We did not present these as "proven predictive biomarkers"; we explicitly labeled them as hypothesis-generating biological rationale that prioritizes treatment.

Monitoring for histologic transformation (SCLC): biallelic TP53 inactivation and RET-driven instability create the groundwork for phenotypic transformation to SCLC. At progression, re-biopsy in case of unexpectedly rapid progression/neuroendocrine features; if transformation is confirmed, carboplatin + etoposide ± atezolizumab (IMpower133).Bypass pathway warning: ERBB3 (HER3) and MET overexpression on RNA-Seq pointed to bypass resistance via PI3K-AKT; an appropriate next-generation agent/trial plan was defined in advance.

Although PD-L1 may look technically eligible, single-agent immunotherapy is not recommended first-line in the presence of a confirmed RET fusion (RET fusion-positive NSCLC is an "immune-cold" phenotype); the RET inhibitor takes priority.

We evaluated this signal not in isolation, but together with TMB. TMB was low (5.3 mut/Mb); dMMR/MSI-H is strongly associated with high TMB, so against a low TMB the most likely meaning of an indeterminate MSI result is MSS/pMMR, and it should first be reconfirmed. We nonetheless requested MMR-IHC. The accompanying MUTYH being in the germline range supported a non-hypermutated profile and was flagged for germline counseling.