ABPath Molecular Genetic Pathology 3 — Questions and Answers
Question 1: A 15% variant allele fraction is reported for a somatic mutation in a tumor with 40% tumor cellularity. This VAF is most consistent with:
- A heterozygous mutation in the tumor cells (Correct answer)
- A germline homozygous variant
- A sequencing error
- Complete loss of heterozygosity
Correct answer: A heterozygous mutation in the tumor cells
With ~40% tumor content, a heterozygous somatic mutation is expected around ~20% VAF, so 15% fits a heterozygous clonal event.
Question 2: Which control is essential to confirm that a negative PCR result is not due to amplification failure?
- An internal amplification (endogenous) control (Correct answer)
- A no-template control only
- A positive control only
- A reagent blank only
Correct answer: An internal amplification (endogenous) control
An internal/endogenous control confirms amplifiable DNA is present, ruling out inhibition or failed extraction behind a true-negative call.
Question 3: FISH break-apart probes are most useful for detecting:
- Gene rearrangements with variable partners such as ALK (Correct answer)
- Single-nucleotide variants
- Small insertions and deletions
- Promoter methylation
Correct answer: Gene rearrangements with variable partners such as ALK
Break-apart FISH detects rearrangement of a gene regardless of the fusion partner by separating flanking probes.
Question 4: Which is the most common cause of a false-positive low-level variant call in FFPE-derived DNA?
- Formalin-induced cytosine deamination artifacts (Correct answer)
- Bacterial contamination
- Excess magnesium
- Primer dimers
Correct answer: Formalin-induced cytosine deamination artifacts
Formalin fixation causes cytosine deamination producing C>T/G>A artifacts that mimic low-VAF variants.
Question 5: In next-generation sequencing, 'read depth' at a position primarily determines:
- Sensitivity for detecting low-frequency variants (Correct answer)
- The length of each read
- The GC content
- The base-calling chemistry
Correct answer: Sensitivity for detecting low-frequency variants
Higher coverage improves confidence and sensitivity for calling low variant allele fraction mutations.
Question 6: A KRAS mutation in metastatic colorectal cancer predicts:
- Resistance to anti-EGFR monoclonal antibody therapy (Correct answer)
- Sensitivity to imatinib
- Sensitivity to trastuzumab
- Response to tamoxifen
Correct answer: Resistance to anti-EGFR monoclonal antibody therapy
Activating KRAS mutations downstream of EGFR confer resistance to cetuximab and panitumumab.
Question 7: Multiplex ligation-dependent probe amplification (MLPA) is best suited to detect:
- Copy number changes such as exon-level deletions or duplications (Correct answer)
- Point mutations
- Methylation of CpG islands only
- RNA fusion transcripts
Correct answer: Copy number changes such as exon-level deletions or duplications
MLPA quantifies relative copy number and readily detects single- or multi-exon deletions and duplications.
ABPath Molecular Genetic Pathology 3
A 15% variant allele fraction is reported for a somatic mutation in a tumor with 40% tumor cellularity.
This VAF is most consistent with: