Principles of Molecular Diagnostics in Cancer
📌 The initial diagnosis relies on histology, but molecular testing is essential for an integrated diagnosis (histology + WHO grade + molecular data) to refine treatment pathways.
🔬 Pathologists must assess tissue quality, specifically looking at tumor cellularity, the percentage of tumor tissue, and avoiding areas with necrosis before molecular testing.
🧬 DNA comprises three billion code letters made of four nucleotides (A, G, C, T); genes (about 20,000) have crucial functions, and pathological changes like base substitutions or frameshifts (due to insertions/deletions) can drive cancer.

Immunohistochemistry (IHC) as a Screening Tool
🌟 IHC uses antibodies to stain proteins (appearing brown) on cell surfaces, quickly indicating the presence or absence of specific molecular markers, such as the mutant p53 protein.
📉 Absence of a protein, like ATRX, can indicate a mutation, as seen in certain brain tumors, illustrating how IHC helps categorize disease types (e.g., astrocytoma vs. oligodendroglioma).
🎯 IHC is quick, cheap, and useful for screening targetable mutations like BRAF V600E, which guides treatment via mech inhibitors, but may require validation with other techniques due to potential ambiguity.
✂️ When tissue samples are small (a few millimeters), test selection must be strategic; sequencing might be preferred over extensive IHC panels to conserve precious material.

DNA Methylation Profiling
🧬 Methylation involves adding methyl groups, typically at CpG islands within promoter regions of genes, which generally switches gene expression off when present.
🧠 The Heidelberg Brain Tumor Classifier utilizes methylation arrays to analyze 850,000 CpG sites to assign a diagnosis score (0 to 1) for challenging cases, revolutionizing neuropathology diagnostics.
📊 Methylation data visualization via t-SNE plots shows tumor types clustering together, facilitating diagnosis and aiding in class discovery (identifying new tumor subtypes).
⬆️ Methylation profiling can change initial histological diagnoses or up/down-grade tumors in about 12% of cases, significantly impacting the prescribed treatment intensity.

Copy Number Interpretation
🔍 Copy number changes refer to gains (amplifications) or losses (deletions) of chromosome arms or specific genes (oncogenes), crucial for characterization and prognosis.
🟢 Loss of 1p and 19q is diagnostic for oligodendroglioma, identified via characteristic patterns on copy number plots, where deviations from the zero line indicate changes.
📌 Deletion of the CDKN2AB gene is linked to a worse prognosis in certain tumors, highlighting copy number analysis' value in risk stratification.
🧩 Copy number plots can also screen for fusions (e.g., BRAF fusion associated with pylocytic astrocytoma), though copy-neutral fusions require sequencing for detection.

Fusions, Translocations, and Sequencing
🔗 Fusions/translocations occur when a break in one or two chromosomes causes two non-adjacent genes to join, often resulting in the over-activation of one gene, driving cancer proliferation.
🧪 Intronic regions, previously considered "junk DNA," are critical as break points for fusion formation, often occurring at consistent locations (e.g., in ALK fusions).
💡 Targeted fusions like ETV6-NTRK3 are pathognomonic in some cancers (e.g., 96% in secretory carcinoma) and are targetable with specific drugs, leading to dramatic positive outcomes in poor-prognosis cases.
🔬 Paired-end sequencing confirms fusions by comparing sequence reads to a reference genome; if a fusion exists, one end of the read aligns at one location while the other aligns elsewhere.
🧬 Whole genome sequencing detects both mutations and fusions, whereas whole exome sequencing only covers exonic regions, missing gene fusions.

Test Selection and Future Directions
🧐 Test selection must be guided by the known disease characteristics, expected targetable mutations, and the amount of tissue available to maximize information yield sparingly.
💸 Cost is a major factor; IHC is cheap, but sequencing can be expensive, requiring a balance, especially considering patient funding for advanced treatments.
✂️ CRISPR-Cas9 technology represents future research by using "molecular scissors" to cut out mutations, helping understand their function and guiding future drug target discovery.
🤝 Engagement with molecular pathology is crucial for oncologists and pathologists to achieve the most accurate diagnosis, ensuring patients receive the right treatment pathway and optimizing clinical trial entry.

Key Points & Insights
➡️ Histology is not replaced; it must always be correlated with molecular findings to form an integrated diagnosis, which is increasingly incorporated into new WHO tumor classifications.
➡️ DNA methylation profiling is providing revolutionary diagnostic support, evidenced by changes in tumor grading and diagnosis in about 12% of cases reviewed.
➡️ When investigating fusions, remember that IHC and copy number analysis are screening tools; sequencing provides necessary validation for specific, targetable alterations like ALK or NTRK fusions.
➡️ Pathologists must balance the need for information against tissue consumption, preferring techniques that yield the most diagnostic utility from limited biopsy material.

📸 Video summarized with SummaryTube.com on Nov 03, 2025, 13:19 UTC