Tumour NGS Panels

Tumour next generation sequencing (NGS) panels read the DNA, and sometimes the RNA, of many genes in a tumour at the same time. They find mutations, insertions and deletions, copy number changes, gene fusions, and signatures such as tumour mutation burden and microsatellite instability. One test replaces a series of single gene tests and saves tissue.

Panels range from small hotspot panels (20 to 50 genes) to full genomic profiling (300 to 500 genes or more), and whole exome or whole genome sequencing. RNA-based panels are better at finding fusions and splice variants. Tumour only testing is common, and paired tumour and normal sequencing separates germline from somatic variants.

DNA is extracted from formalin fixed, paraffin embedded tumour (at least 10 to 50 ng, with at least 20% tumour cells for most assays), libraries are prepared, and sequencing is done. Bioinformatic analysis, variant classification and expert review take place before the report. The turnaround is 7 to 21 days.

Most advanced cancers carry at least one variant that affects treatment choice, and a matched treatment is possible in a minority, from about 10 to 15% in many tumour types to over 50% in lung adenocarcinoma. Panels also identify patients for treatment studies, and hereditary cancer risk.

Indications.

• Advanced non-small cell lung cancer, and metastatic colorectal, melanoma, breast, ovarian, prostate and biliary cancers, where several drugs depend on molecular results.
• Cancers of unknown primary, rare tumours and sarcomas, for diagnostic and treatment information.
• Cancers that have progressed on treatment, and no established option is left.
• Haematological cancers (acute myeloid leukaemia, myelodysplastic syndromes, lymphoma), with disease specific panels.
• Tumours with suspected hereditary syndromes, for tumour based clues.
• Assessment of eligibility for tumour agnostic treatments (NTRK fusion, high tumour mutation burden, mismatch repair deficiency).

Not indicated for early cancers where results will not affect treatment, or when the patient is too unwell to receive treatment.

Specimen requirements.

• Formalin fixed, paraffin embedded tumour (biopsy, resection or cell block), fixed in 10% neutral buffered formalin for 6 to 72 hours, with minimal delay to fixation.
• Pathologist review of tumour percentage, and macrodissection to enrich tumour.
• Fresh frozen tissue or plasma as alternatives.
• A matched blood sample for paired normal sequencing, when ordered.
• Avoidance of acid decalcification. EDTA is used for bone.
• Clinical details: tumour type, stage, previous treatment and family history.

Normal appearances.

A negative panel for actionable variants shows the genes that were tested, their coverage, and no pathogenic variants. A report states the quality of the sample (tumour content, depth of coverage) and the limits of detection.

Interpretation.

• Variants are classed by their effect (pathogenic, likely pathogenic, variant of uncertain significance, likely benign, benign), and by levels of evidence for clinical action (for example, variants with approved drugs, variants with strong evidence in other tumours, variants with early evidence, and variants of no clinical use).
• Actionable variants: EGFR, ALK, ROS1, RET, MET, BRAF, KRAS G12C, HER2, NTRK, PIK3CA, BRCA1 and BRCA2, FGFR2, IDH1 and others, each linked to a drug in the relevant tumour type.
• Tumour mutation burden: the number of mutations per megabase (a threshold of 10 or more per megabase is used as high in some settings), which suggests benefit from immune checkpoint treatment in some tumours.
• Microsatellite instability and homologous recombination deficiency scores are reported by some panels.
• Copy number gains (HER2, MET, MYC amplification) and losses, and fusions, are listed.
• Germline like variants (allele frequency near 50%) in genes such as BRCA1, BRCA2, TP53, MLH1 and MSH2 are flagged, and confirmation on a germline sample is advised.
• Co-occurring variants (for example, TP53 and STK11 or KEAP1 in lung cancer) alter prognosis and sometimes the treatment response.
• Variants of uncertain significance are not acted on, and they are reviewed when the evidence changes.

Critical values and findings needing immediate action.

• A matched, approved therapy for an advanced cancer: urgent referral to the oncology team to start treatment.
• A variant suggesting hereditary cancer (BRCA1 or BRCA2, TP53, mismatch repair genes): referral for genetic counselling and testing of relatives.
• Failure of the test because of low tumour content or poor DNA quality: repeat on a new sample, or plasma testing, without waiting.
• Actionable findings in a patient with symptomatic or life threatening disease: communication to the team on the same day.

Limitations and artefacts.

• Low tumour content and low quality DNA (old blocks, heavy fixation, decalcification) give false negatives and poor coverage.
• Formalin artefacts cause low level C to T changes, and bioinformatic filters are needed to avoid false positive calls.
• DNA-only panels can miss fusions with breakpoints in uncovered regions, so RNA-based tests are used for fusions.
• Panels cover selected genes, and variants outside the panel are not found. Differences in content and quality between panels affect results.
• Interpretation of variants of uncertain significance and of resistance changes is difficult, and databases are updated.
• Tumour mutation burden values differ between assays, and thresholds are validated only for some tumours.
• Tumour heterogeneity and changes under treatment mean one biopsy can under represent the disease.
• Incidental germline findings need consent, counselling and confirmation.
• Cost, access, turnaround and the lack of matched treatments limit benefit.

Special groups.

• Patients with small biopsies and cytology material: a single combined panel saves tissue.
• Young patients and those with unusual cancers: broader panels and RNA testing find rare drivers.
• Patients with hereditary risk (family history, early onset): germline testing alongside tumour testing.
• Children with cancer: specialised paediatric panels and fusion testing.
• Patients with haematological cancers: panels for specific diseases inform diagnosis, risk and treatment.
• Regions: sequencing is limited to a few reference centres, and samples are shipped long distances. Tests are often paid for by the patient. A focused panel covering the genes with approved and affordable treatments for common local cancers is a practical choice, and results are discussed with a molecular tumour board where one exists.

Follow up tests.

• Immunohistochemistry for PD-L1, mismatch repair proteins, HER2 and other proteins.
• In situ hybridisation for confirmation of amplifications and fusions.
• Germline testing of the blood sample for suspected hereditary variants.
• Plasma sequencing at progression.
• Staging and response imaging.
• Review at a molecular tumour board.
• Enrolment in a treatment study, where available.
• Specialist genetic counselling for patients and families.