Thread by @pathcej_md

🧬#MolecularPath “Selecting the Right Tool for the Job: Practical Considerations for the Application of Molecular Diagnostics Methods in SurgPath” with @LJTafeMD and Dr. Larissa Furtado

A few takeaways (just scratching the surface of this excellent talk!) ⬇️🧵#MolecularPath_CEJ

First up: Pre-analytical Considerations

Purposes of molecular oncology testing:

🔵 Diagnosis
🔵 Therapy response/management
🔵 Monitoring

1/

Considerations for molecular oncology testing:

🔵 Availability of specimens
🔵 Testing options: targets 🎯, analytical sensitivity, TAT ⏱, cost
🔵 Clinical scenario
🔵 NCCN guidelines

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🧰 Molecular pathology toolkit is based on target (NOT a comprehensive list!!):

📌 Chromosomes ➡️ SNP array/array CGH
📌 Nucleic acid variants ➡️ Sanger sequencing, NGS
📌 Epigenetics ➡️ hypermethylation analysis
📌 mRNA ➡️ expression array, RT-PCR

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What can be tested?

🔵 FNA, FFPE, fresh frozen tissue >> bony mets (use mild decal like EDTA)
🔵 Testing multiple areas in a tumor is usually unnecessary, but if multiple primaries, test all

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🔬🧬 “Pathologists are the gatekeepers of specimen quality and adequacy”

Consider two types of heterogeneity in specimens for molecular testing:
📌 Tissue heterogeneity ⬇️
📌 Tumor heterogeneity: multiple clones of tumor (important in recurrent disease/mets)

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🔬 Tissue heterogeneity: a section of tumor is not 100% tumor - there are also inflammatory and stromal cells

🔵 Important because of minimum tumor cellularity needed to perform testing (established during assay validation)

Helpful to have a pathologist score H&E… ⬇️

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…though that’s not without its problems (interobserver variability, inaccuracy): nature.com/articles/modpa…

To improve estimation of tumor %:
📌 Use tumor nuclei ("tumor dilution")
📌 Limit # of pathologists performing
📌 Training sets/feedback for individual pathologists

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Next: Analytical Considerations

🎯 Targeted testing (e.g. IHC, FISH) vs comprehensive (molecular) testing

Obv not all assays are equal in terms of what info they will or won’t give you, as well as other test characteristics, so choosing the right test is important! ⬇️

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E.g. DNA vs RNA seq in fusion detection ⬇️

📌 DNA sequencing: Since most gene fusion breakpoints occur in introns (highly variable) ➡️ DNA sequencing will include many probes targeting intron sequences (but this can get $$$)

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📌 RNA sequencing: performed on spliced mRNA, 1️⃣ either specific fusions, or 2️⃣ using partner-agnostic libraries (i.e. knowledge of fusion partner not required) ➡️ multiplexing with novel fusion detection

RNA seq used more often clinically than DNA seq

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⚠️ Critical to understand the characteristics, limitations of different fusion detection methods (esp if sending out to a reference lab!) ➡️ assist in interpretation and reporting of results

Check details of test designs before ordering d/t differences across laboratories!

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Another example, multiple methods for BRAF mutational testing:

🔵 Allele-specific PCR (⬆️ analytical sensitivity, but can't detect variable mutations which may be clinically relevant)
🔵 Sanger seq (can detect small indels, but not as sensitive)
⬇️

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🔵 NGS (higher analytical sensitivity, can detect multiple anomalies simultaneously)
⬇️
nature.com/articles/s4138… (would have been missed with FISH, allele-specific PCR)

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And of course, all molecular test results must be put into context with diagnosis, histology, and clinical course

Best accomplished in conjunction with colleagues from multiple disciplines 🤝

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Post- analytical considerations: essential elements of a report 📄

📌 Patient/clinical info
📌 Test background/methodology, results & interpretation, limitations
📌 References and recommendations
pubmed.ncbi.nlm.nih.gov/27993330/

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⬆️ is just scratching the surface 🎥 Highly recommend watching the recording on @Pathologists #CAP21 if you didn’t catch this excellent session live!!

Thank you Dr. @LJTafeMD and Dr. Furtado!

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