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Abstract:

This introductory webinar provides a foundational overview of molecular pathology specifically tailored for surgical pathologists. The session begins by reviewing the central dogma of molecular biology, detailing the structures of DNA and RNA, the processes of transcription and splicing (including alternative splicing), and translation via codons and reading frames. The presentation then categorizes and explains various types of genomic alterations, including large-scale changes like copy number variations (CNVs) and gene fusions, highlighting the utility of RNA sequencing for their detection. It also provides a detailed breakdown of point mutations (missense, nonsense, synonymous) and indel variants, explaining their functional consequences such as frameshifts and splice site disruptions, using MET exon 14 skipping as a key clinical example. Finally, the talk covers the standardized nomenclature for interpreting molecular reports and introduces crucial clinical concepts such as Variant Allele Frequency (VAF), Tumor Mutation Burden (TMB), and the basics of DNA repair deficiencies, including Mismatch Repair (MMR)/Microsatellite Instability (MSI) and Homologous Recombination Deficiency (HRD).

An Introduction to Molecular Pathology for Surgical Pathologists

• 00:00:18 Webinar Goal: To provide a foundational understanding of molecular pathology for surgical pathologists, enabling them to better understand and integrate molecular results into their diagnostic reports.
• 00:03:34 Molecular Biology Fundamentals: A review of the central dogma (DNA → RNA → Protein), covering the structures of DNA/RNA, gene components (exons and introns), transcription, and splicing, the process where non-coding introns are removed to create mature mRNA.
• 00:15:55 Reading Frames: Explanation of how a DNA sequence has multiple possible reading frames for translation. A frameshift—caused by an insertion or deletion of bases not divisible by three—alters the entire downstream protein sequence, usually resulting in a loss of function.
• 00:18:54 Copy Number Variations (CNVs): An overview of large-scale genomic changes involving gains (amplifications) or losses of DNA segments, with HER2 amplification cited as a key clinical example.
• 00:20:18 Gene Fusions and Detection: Gene fusions are created by chromosomal breaks and the rejoining of two separate genes. Key Takeaway: RNA sequencing is the most effective method for detecting fusions when the partner genes are unknown because it targets the much shorter, spliced mRNA transcript, bypassing the vast intronic regions where breakpoints typically occur.
• 00:28:02 Types of Point Mutations: A detailed breakdown of variants affecting one or a few base pairs:
  • Missense: A base change that results in a different amino acid.
  • Nonsense: A change that creates a premature stop codon, truncating the protein.
  • Synonymous (Silent): A change that does not alter the corresponding amino acid.
• 00:41:49 Splice Site Variants: These are mutations occurring at the exon-intron boundary that disrupt normal splicing. The MET exon 14 skipping mutation is used as a prime example of an actionable splice variant that leads to oncogenic activation.
• 00:51:59 Understanding Variant Nomenclature: A practical guide to interpreting molecular reports, explaining each component of a variant name: the gene symbol (TP53), transcript number (e.g., NM_...), coding DNA change (c.2369C>T), and the resulting protein change (p.Thr790Met).
• 01:07:53 Variant Allele Frequency (VAF): VAF is the percentage of sequencing reads that contain a specific variant. It is a crucial metric for estimating tumor purity, assessing tumor heterogeneity, and helping to distinguish between germline (~50% VAF) and somatic mutations.
• 01:11:11 Tumor Mutation Burden (TMB): TMB is defined as the total number of somatic mutations per megabase of DNA. It serves as a biomarker for predicting response to immunotherapy, but its value can vary significantly depending on the sequencing panel used for testing.
• 01:13:25 DNA Repair Deficiencies: An introduction to two critical DNA repair systems whose failure can drive cancer:
  • Mismatch Repair (MMR): Deficiency leads to Microsatellite Instability (MSI).
  • Homologous Recombination (HR): Deficiency (HRD) is associated with mutations in genes like BRCA1 and BRCA2.