What is the preferred tissue specimen type for homologous recombination deficiency (HRD) testing in ovarian cancer, and what alternative sample types are acceptable?

Preferred Tissue Specimen Type for HRD Testing in Ovarian Cancer

Surgical tissue specimens from primary or interval cytoreductive surgery are the preferred sample type for HRD testing in ovarian cancer, with a minimum tumor cell content of ≥30% and tumor area ≥0.5 cm² to optimize test success rates.

Primary Recommendation: Surgical Specimens

The evidence strongly supports using surgical tissue as the gold standard for HRD testing. In a large real-world analysis of 2,654 samples, surgical specimens achieved success rates up to 98% when optimal tissue parameters were met 1. A multivariable analysis demonstrated that surgical specimens were significantly more likely to yield conclusive HRD test results compared to biopsies, cytology, or other tissue sources 2.

Critical Tissue Requirements

For optimal HRD testing performance, pathologists should select samples meeting these specifications 1:

• Tumor cell content: ≥30% (significantly affects success probability, p < 0.0001)
• Tumor area: ≥0.5 cm² based on hematoxylin and eosin staining
• Histology consideration: High-grade serous ovarian cancer samples perform best

These parameters are essential because HRD assays measure genomic instability patterns that require adequate tumor DNA for reliable analysis. Samples with abundant inflammatory infiltrates or low tumor cellularity are at higher risk of yielding inconclusive results.

Alternative Sample Types

Cytological Samples

Cytological specimens can be used as an acceptable alternative when surgical tissue is unavailable, though with some caveats. A study of 110 matched cytological and histological samples from treatment-naïve high-grade serous carcinoma patients demonstrated 3:

• 100% concordance for BRCA1/2 mutation detection
• 89.7% overall concordance for homologous recombination repair-associated gene mutations
• 90.9% concordance for HRD status classification
• Moderate correlation for genomic instability metrics (Pearson coefficient 0.48)

This evidence supports cytological samples as viable alternatives, particularly for BRCA mutation testing, though genomic instability scoring may be less reliable than with surgical specimens.

Biopsy Specimens

Laparoscopic biopsies obtained before initiation of neoadjuvant chemotherapy are preferable to post-chemotherapy samples. The timing is critical because:

• Chemotherapy exposure is a significant risk factor for inconclusive HRD testing on multivariable analysis 2
• Interval cytoreductive surgery specimens (post-chemotherapy) have higher rates of inconclusive results compared to primary surgery specimens 2
• Pre-treatment biopsies preserve the genomic landscape needed for accurate HRD assessment

Practical Implementation Algorithm

Step 1: Prioritize surgical specimens from primary cytoreductive surgery whenever possible

Step 2: If neoadjuvant chemotherapy is planned, obtain laparoscopic biopsy before chemotherapy initiation

Step 3: Ensure pathologist reviews sample adequacy:

• Confirm ≥30% tumor cell content
• Verify ≥0.5 cm² tumor area
• Assess for excessive necrosis or inflammatory infiltrate

Step 4: If surgical tissue unavailable, cytological samples are acceptable, particularly for BRCA1/2 mutation detection

Step 5: Avoid post-chemotherapy specimens when possible due to increased failure rates

Critical Pitfalls to Avoid

Chemotherapy-exposed tissue: The single most important modifiable risk factor for test failure. In real-world practice, 12% of HRD tests yielded inconclusive results, with chemotherapy exposure being independently associated with failure 2. Plan tissue acquisition before treatment initiation.

Inadequate tumor cellularity: Samples with <30% tumor content have significantly reduced success rates 1. The pathologist must carefully select representative tumor areas and quantify inflammatory cells and necrosis, as HRD-positive cancers often have abundant immune infiltrates.

Wrong histologic subtype: HRD testing has limited utility in non-high-grade serous histologies. No clear cell or endometrioid ovarian cancers tested HRD-positive in one cohort 2, and HRD detection rates were significantly lower in non-serous subtypes (12% versus 49%, p < 0.001) 4.

Delayed or improper fixation: Inappropriate tissue handling modifies sample quality and impacts molecular test results 5. Pathology departments must maintain quality standards (ISO 15189 or equivalent) for pre-analytical steps.

Pathologist's Essential Role

The ESMO guidelines emphasize that pathological evaluation is essential for HRD testing success 5. A pathologist with gynecological pathology experience should:

• Confirm diagnosis (particularly distinguishing high-grade serous from endometrioid with SET features)
• Assess sample adequacy before testing
• Select optimal tumor areas
• Quantify tumor cells, inflammatory cells, and necrosis
• Provide integrated pathology-molecular reports

This integrated approach achieved 90.3% successful testing rates in a cohort of 2,654 real-world samples 1, demonstrating the critical importance of proper specimen selection and handling.

Tumor Fraction Considerations

Recent evidence indicates that tumor fraction (TF) strongly influences HRD performance 6. Optimal sensitivity requires:

• TF ≥0.4 for hybrid-capture panel-based assays
• TF ≥0.2 for shallow whole-genome sequencing approaches

Lower tumor fractions increase the risk of false-negative results, making specimen selection even more critical in samples with low cellularity or high stromal/inflammatory components.