What tests are done on tissue samples for optimal treatment of different types of cancers, including breast, lung, ovarian, colon, and hematological malignancies?

Tissue Testing for Optimal Cancer Treatment

For optimal cancer treatment, tissue samples should undergo comprehensive molecular profiling including immunohistochemistry, next-generation sequencing (NGS), and specific biomarker testing tailored to each cancer type, with core needle biopsies (minimum 3-5 cores using 14-gauge or larger needles) being the preferred sampling method across most malignancies.

General Principles of Tissue Acquisition

Optimal Biopsy Technique

• Core needle biopsy is the gold standard for obtaining diagnostic tissue, requiring at minimum three 14-gauge cores from various tumor locations to ensure adequate material for both diagnosis and molecular testing 1
• For bone sarcomas, collect 5-7 core biopsies using 8-, 11-, or 14-gauge needles, or obtain 2 cm³ open biopsy specimens divided into multiple 0.2 cm³ pieces 1
• Each core biopsy yields approximately 10 mm³ (~10 mg) of tissue, with larger gauge needles (12G, 14G, 16G) providing higher tissue volumes 1
• Samples should contain 30-40% neoplastic nuclei with less than 20% necrosis for adequate molecular analysis 1

Critical Handling Requirements

• Time from tissue acquisition to fixation must be minimized (cold ischemia time <60 minutes) 1
• Fix samples in 10% neutral buffered formalin for 6-72 hours 1
• Slice specimens at 5-mm intervals and immerse in sufficient formalin volume for adequate penetration 1
• Document cold ischemia time, fixative type, and timing on all accession slips 1
• Unstained slides cut more than 6 weeks before analysis should not be used 1

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Cancer-Specific Testing Protocols

Non-Small Cell Lung Cancer (NSCLC)

Essential Molecular Testing:

• EGFR mutation analysis is mandatory for all nonsquamous NSCLC and should be considered in squamous histology for never-smokers 1
• ALK rearrangement testing (by immunohistochemistry or FISH) is required 1
• ROS1 translocation testing should be implemented in routine procedures 1
• PD-L1 immunohistochemistry for immunotherapy selection 1

Testing Methodology:

• NGS-based multiplex panels are preferred as they detect point mutations, rearrangements, copy number variations, and insertions/deletions simultaneously 1
• Acceptable assay sensitivity: 1-5% mutant DNA detection 1
• High-depth NGS (≥200x depth) can detect mutations at 1-10% frequency depending on error rates 1
• Plasma-based testing (liquid biopsy) is acceptable when tissue is inadequate, though tissue remains preferable 1

Sample Requirements:

• 70-85% of tested samples are core needle biopsies, fine-needle aspirations, or fluid specimens 1
• Cytologic samples with abundant malignant cells are acceptable alternatives 1
• Failure rates of 5-30% occur primarily due to inadequate cellularity 1

Breast Cancer

Mandatory Biomarker Testing:

• Estrogen receptor (ER) by immunohistochemistry - positive if ≥1% tumor nuclei stain 1
• Progesterone receptor (PgR) by immunohistochemistry - positive if ≥1% tumor nuclei stain 1
• HER2 status by immunohistochemistry and/or FISH 1
• Tumor grade assessment from pretreatment core biopsies (essential before neoadjuvant therapy as post-treatment grading is unreliable) 1

Special Reporting for ER:

• Samples with 1-10% ER positivity should be reported as "ER Low Positive" with explanatory comment 1
• This distinction is critical as limited data exists on treatment response in this subgroup 1

Additional Considerations:

• Ki67 proliferation index is optional but useful for research 1
• Gene expression assays (mRNA-based) provide additional prognostic information 1
• Cold ischemia time significantly impacts ER/PR assessment accuracy 1

Bone Sarcomas (Osteosarcoma and Ewing Sarcoma)

Comprehensive Sampling Strategy:

• At diagnosis: Collect 5-7 core biopsies (0.2 cm³ sections each) using 8-11-14 gauge needles 1
• Consider metastatic site biopsy if oligometastases are detectable at presentation 1
• At relapse: Obtain fresh tissue even if diagnosis seems certain, as relapsed tissue paired with diagnostic samples is highly valuable 1

Sample Processing Priorities:

1. FFPE for standard pathology and neoadjuvant chemotherapy response assessment 1
2. Snap-frozen samples stored at -80°C for genomic analysis 1
3. Fresh tissue in RNA-preserving medium 1
4. Fresh tissue in culture-compatible medium for functional studies 1

Blood Sample Collection:

• Obtain at diagnosis, before/after surgery, and at follow-up 1
• Use EDTA tubes for circulating tumor DNA (ctDNA), circulating tumor cells (CTCs), PBMCs, and plasma 1
• PAXgene tubes for RNA analysis 1
• Cell-free Streck tubes for ctDNA analysis 1

Colorectal Cancer

Essential Molecular Testing:

• RAS mutation status (KRAS and NRAS) - mandatory before anti-EGFR therapy
• BRAF V600E mutation - prognostic and predictive marker
• Microsatellite instability (MSI) or mismatch repair (MMR) protein testing - predicts immunotherapy response
• HER2 amplification in RAS wild-type tumors

Ovarian Cancer

Standard Testing:

• BRCA1/2 mutation testing (germline and somatic) - predicts PARP inhibitor response
• Homologous recombination deficiency (HRD) testing - broader predictor of platinum sensitivity
• Mismatch repair/microsatellite instability - for immunotherapy eligibility

Hematological Malignancies

Specialized Requirements:

• Fresh tissue in RPMI medium is essential for lymphoma diagnosis and flow cytometry 2
• Coordinate with pathology department regarding collection medium choice 2
• Flow cytometry requires viable cells, precluding formalin fixation 1
• Cytogenetic analysis and molecular testing for specific translocations based on suspected subtype

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Quality Control and Workflow Optimization

Multidisciplinary Approach

• Establish standard operating procedures (SOPs) adaptable to local resources with continuous quality control 1
• Multidisciplinary tumor boards are essential for individualized diagnostic and therapeutic planning 1
• Reflex testing initiated by pathologists reduces turnaround time significantly 1

Rapid On-Site Evaluation (ROSE)

• ROSE improves diagnostic yield from 71.4% to 78.0% (OR 2.35) for bronchoscopy procedures 2
• Reduces needle passes required (3.4 vs 6.1, p<0.001) and non-diagnostic specimens (0.9% vs 4.4%, p=0.018) 2
• Provides immediate feedback on sample adequacy with 91% diagnostic accuracy 2
• Perform ≥4 needle passes to maximize diagnostic efficacy in malignancies (92.1% final yield) 2

Sample Prioritization

• Prioritize tissue for molecular testing over lower-priority analyses during initial workup 1
• Obtain unstained sections sufficient for all necessary molecular markers during initial diagnostic workup 1
• Store initial core biopsies for ≥10 years, especially critical when pathologic complete response occurs 1

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Emerging Technologies and Liquid Biopsy

Liquid Biopsy Applications

• Plasma-based testing is acceptable when tissue rebiopsy is not feasible (feasible in only 74.6% of patients, with 20% yielding inadequate tissue) 1
• Liquid biopsy components include ctDNA, CTCs, circulating RNA, exosomes, and tumor platelets 3, 4
• Ultra-sensitive techniques like digital droplet PCR (<0.1% sensitivity) and CAPP-Seq (0.02% sensitivity) enable detection of minimal residual disease 1
• Non-invasive, rapid, and enables serial monitoring during treatment 3, 4

Limitations and Considerations

• Tissue biopsy remains the gold standard due to higher standardization and accuracy 3
• Liquid biopsy cannot assess tumor microenvironment or perform comprehensive immunohistochemistry 1
• Best used complementary to tissue biopsy, particularly for monitoring and detecting resistance mechanisms 5

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Critical Pitfalls to Avoid

• Never use fine-needle aspiration alone when core biopsy is feasible - insufficient tissue for comprehensive molecular testing 1
• Do not delay fixation - prolonged cold ischemia compromises biomarker accuracy, particularly ER/PR 1
• Avoid testing only primary sites - consider metastatic site sampling as molecular discordance occurs between sites 1
• Do not assume cytology is inadequate - properly processed cytologic samples (cytoblocks) can be sufficient for molecular analysis 6
• Never skip germline testing when indicated - particularly important in ovarian cancer and bone sarcomas for family counseling and treatment selection 1