Solid Tumor Biomarkers and Companion Diagnostics

Somatic testing

Solid tumor testing looks for somatic changes - mutations acquired by the tumor, not inherited - that predict response to specific drugs. A test that FDA approves alongside a drug, as the required way to select patients, is a companion diagnostic (CDx) ¹.

Lung cancer

Non-small cell lung cancer, especially adenocarcinoma, is tested broadly, usually by an NGS panel:

• EGFR: exon 19 deletions and exon 21 L858R predict response to EGFR tyrosine kinase inhibitors (osimertinib, erlotinib, gefitinib, afatinib); the cobas EGFR Mutation Test v2 is one companion diagnostic ¹. T790M is the common acquired resistance mutation to first- and second-generation EGFR TKIs; osimertinib was developed to overcome it.
• ALK rearrangements (most often EML4::ALK) select ALK inhibitors such as crizotinib, alectinib, and brigatinib; the Vysis ALK Break Apart FISH probe is one CDx ¹. A break-apart probe shows split red and green signals when the gene is rearranged.
• ROS1, RET, NTRK fusions, BRAF V600E, MET exon 14 skipping, and KRAS G12C each have targeted drugs.

Colorectal cancer

• KRAS and NRAS (exons 2, 3, and 4): activating mutations mean anti-EGFR antibodies (cetuximab, panitumumab) won’t work; they benefit only RAS wild-type tumors ². The therascreen KRAS kit is a CDx for panitumumab, cetuximab, and the KRAS G12C inhibitors sotorasib and adagrasib ¹.
• BRAF V600E: predicts poor response to anti-EGFR antibodies alone ², selects BRAF-targeted combinations, and in MSI-high tumors points to a sporadic rather than Lynch syndrome cancer ³.
• Mismatch repair / MSI: every new colorectal cancer is screened for mismatch repair deficiency by IHC or MSI testing ³. MSI-high/dMMR tumors respond to immune checkpoint inhibitors, and the result also screens for Lynch syndrome (next lesson).

MSI testing compares the lengths of short repeat markers (mononucleotide repeats such as BAT-25 and BAT-26) in tumor and normal DNA by PCR and capillary electrophoresis; new allele sizes in the tumor mean instability.

Breast cancer

HER2 (ERBB2) amplification selects HER2-targeted drugs (trastuzumab, pertuzumab, ado-trastuzumab emtansine), with FISH kits as CDx ¹. HER2 is assessed by IHC first, with in situ hybridization (ISH) for equivocal cases. A dual-probe ISH result is clearly positive (group 1) when the HER2/CEP17 ratio is ≥2.0 and the average HER2 copy number is ≥4.0 signals per cell; the less common discordant groups 2 to 4 need concurrent IHC review ⁴.

PIK3CA mutations and BRCA1/2 (somatic or germline) also select therapies.

Melanoma

BRAF V600E/K mutations select BRAF plus MEK inhibitors (dabrafenib plus trametinib, encorafenib plus binimetinib); the THxID BRAF kit is a CDx ¹.

Specimen issues

• FFPE tissue gives fragmented, cross-linked DNA; assays use short amplicons and must handle formalin artifacts.
• Tumor content: a pathologist marks tumor areas and estimates the percentage of tumor nuclei. A heterozygous mutation in a sample that’s 20% tumor appears at about 10% variant allele frequency; if the assay’s limit of detection is 5% VAF, samples below about 10% tumor risk false negatives. Macrodissection enriches tumor.
• Liquid biopsy: circulating tumor DNA (ctDNA) in plasma can be tested when tissue is unavailable or to find resistance mutations such as EGFR T790M. A negative ctDNA result doesn’t rule out a mutation, since some tumors shed little DNA.

Reporting somatic variants

The AMP/ASCO/CAP guideline sorts somatic variants into four tiers ⁵:

• Tier I: strong clinical significance (FDA-approved therapy or guideline-endorsed).
• Tier II: potential clinical significance.
• Tier III: unknown clinical significance.
• Tier IV: benign or likely benign.

Tumor-only sequencing can find germline variants (a BRCA2 variant near 50% VAF, for example), which should prompt germline testing and counseling.

What to take from this

Companion diagnostics tie a somatic biomarker to a drug. In lung cancer, EGFR exon 19 deletions and L858R predict TKI response and T790M causes resistance; ALK and other fusions select their own inhibitors. In colorectal cancer, RAS mutations rule out anti-EGFR antibodies, BRAF V600E predicts poor response and suggests sporadic MSI, and MMR/MSI screening selects immunotherapy and flags Lynch syndrome. HER2 ISH is positive at a ratio ≥2.0 with ≥4 copies. Tumor percentage limits sensitivity, ctDNA can’t rule out a mutation, and somatic variants are reported in four tiers.