The Journal of Molecular Diagnostics

Optimization of Pre-Analytical Handling to Maintain DNA Integrity in Diagnostic Papanicolaou Tests

Cell-free DNA (cfDNA) of ovarian carcinoma origin can be detected in samples from the gynecologic tract. This study aims to evaluate how pre-analytical handling affects DNA profile and integrity in Papanicolaou (Pap) tests, to optimize their potential for detection of ovarian cancers (OCs). Analysis of archived Pap tests from patients with OC, kept at room temperature for 48 hours and stored at -80°C, was complemented by in vitro experiments. Temperature-associated effects on DNA fragmentation were evaluated in samples stored at 4°C, -20°C, or -80°C. Time-dependent DNA degradation at room temperature was evaluated in comparison to storage at 4°C. Results were validated in prospectively collected Pap tests. The DNA integrity was assessed by fragment analysis. Accumulation of short DNA fragments was observed in archived Pap tests from patients with OC. In vitro, fragments of 100 to 350 bp increased 11.5-fold within 48 hours at room temperature compared with 1.7-fold when stored at 4°C. Consistent with the in vitro findings, prospectively collected samples showed reduced fragmentation when stored at 4°C compared with room temperature (P = 0.007). Long-term storage at 4°C had a significant negative effect on DNA stability (P = 0.013), whereas freezing slowed down fragmentation. Immediate storage at 4°C after sampling markedly reduces DNA degradation, suggesting a simple way to optimize pre-analytical handling and decrease unwanted fragmentation for cfDNA analysis in Pap tests.

Performance Evaluation of the Aptima hrHPV Nucleic Acid Amplification and Papanicolaou Co-Testing in Cervical Cancer Screening

Cervical cancer screening is evolving, with guidelines increasingly favoring high-risk human papillomavirus (hrHPV) nucleic acid amplification tests (NAATs) as a primary method. Although hrHPV NAATs offer high sensitivity for HPV-driven cervical cancers, Papanicolaou (Pap) cytology can detect additional gynecologic malignancies, including HPV-independent ones. This study evaluated the Hologic Aptima hrHPV NAAT and Pap co-testing using 61,089 samples from patients aged ≥21 years screened between 2018 and 2023. The cohort was predominantly White (78.4%), with 16.5% Black, 4.1% Asian, and 38.9% Hispanic subjects. Overall percentage agreement (PA) between hrHPV NAAT and Pap was 87.9%, with high negative PA (93.6%) and lower positive PA (52.3%). Excluding atypical squamous/glandular cell abnormalities improved overall and positive PA to 93.0% and 78.3%, respectively. The Aptima hrHPV NAAT showed high sensitivity for high-grade squamous intraepithelial lesions (98.3%) and squamous cell carcinoma (90.0%) but lower sensitivity for low-grade lesions (73.3%), adenocarcinoma (13.3%), and other malignancies (28.6%). Notably, 29 high-grade lesions or malignant cases had abnormal Pap but were hrHPV negative; 24 (82.8%) were histologically confirmed, including endometrial, cervical, ovarian, and fallopian tube carcinomas (mostly non-HPV-related). These findings emphasize cytology's role in detecting malignancies potentially missed by hrHPV testing alone and support co-testing in diverse populations. The Aptima hrHPV NAAT is not approved by the US Food and Drug Administration for primary HPV screening, reinforcing this need.

A Novel Droplet Digital PCR Assay for BRCA1 and RAD51C Methylation

Homologous recombination deficiency (HRD) affects 50% of ovarian cancers and influences poly (ADP-ribose) polymerase inhibitor efficacy. Although one-third of HRD tumors harbor a deleterious BRCA1/2 mutation, these mutations are not the sole cause of HRD. Promoter methylation of BRCA1 and RAD51C contributes to 19% and 2% of cases, respectively. A cohort of 224 patients with ovarian cancer tested for HRD validated the droplet digital PCR (ddPCR) technique. DNA was extracted from formalin-fixed, paraffin-embedded tissue, and the Genomic Instability Index (GII) was assessed using the SOPHiA DDM HRD Solution. BRCA1 and RAD51C promoter methylation was analyzed by ddPCR following enzymatic conversion, with a 10% threshold for methylation classification. Homologous recombination status was assessed in 194 patients. A positive GII (>0) was identified in 77 cases (39.7%), with 24 (31.2%) harboring a deleterious BRCA1/2 mutation, and 2 (2.6%) a RAD51C mutation. Methylation analysis revealed BRCA1 promoter methylation in 32 patients (41.6%) and RAD51C promoter methylation in 1 patient (1.3%), clarifying HRD status in 28 additional cases (36.3%). The study confirms HRD extends beyond BRCA1/2 mutations, with promoter methylation playing a significant role in HRD detection. ddPCR effectively identified methylation-driven HRD, explaining deficiency in 36.4% additional patients with a positive GII score. These findings highlight the importance of incorporating methylation analysis into HRD testing to improve patient stratification for poly (ADP-ribose) polymerase inhibitor therapy.

Validation and Performance of Quantitative BRCA1 and RAD51C Promoter Hypermethylation Testing in Breast and Ovarian Cancers

Poly (adenosine diphosphate-ribose) polymerase (PARP) inhibitors represent a significant advancement in the treatment of epithelial ovarian cancer, triple-negative breast cancer, pancreatic cancer, and castrate-resistant prostate cancer, and they are poised to improve treatment in an increasing number of other cancer types. PARP inhibitor efficacy as monotherapy has been primarily observed in tumors with deleterious genetic variants in genes involved in the homologous recombination repair pathway. Tumors without these variants have also been shown to respond; notably, those with hypermethylation at all alleles of the BRCA1 or RAD51C promoter can respond to PARP inhibitors. These epigenetic biomarkers therefore represent a patient population that may also benefit from this targeted therapy. However, no robust test has been conducted to identify these biomarkers in routine clinical specimens that is amenable to implementation for decentralized testing. This study describes the analytical and clinical validation of a BRCA1 and RAD51C promoter methylation test that can be run with a single-day library preparation workflow for sequencing on any next-generation sequencing platform. The results show that this test can accurately quantitate the level of promoter methylation at the BRCA1 and RAD51C genes using formalin-fixed, paraffin-embedded samples, even when the extracted DNA is extremely degraded or the input amount is limited. This test increases the precision of diagnostic tests aimed at identifying patients who are likely and unlikely to respond to PARP inhibitor therapy.

Improving Specificity for Ovarian Cancer Screening Using a Novel Extracellular Vesicle–Based Blood Test

The low incidence of ovarian cancer (OC) dictates that any screening strategy needs to be both highly sensitive and highly specific. This study explored the utility of detecting multiple colocalized proteins or glycosylation epitopes on single tumor-associated extracellular vesicles from blood. The novel Mercy Halo Ovarian Cancer Test (OC Test) uses immunoaffinity capture of tumor-associated extracellular vesicles, followed by proximity-ligation real-time quantitative PCR to detect combinations of up to three biomarkers to maximize specificity, and measures multiple combinations to maximize sensitivity. A high-grade serous carcinoma (HGSC) case-control training set of EDTA plasma samples from 397 women was used to lock down the test design, the data interpretation algorithm, and the cutoff between cancer and noncancer. Performance was verified and compared with cancer antigen 125 in an independent blinded case-control set of serum samples from 390 women (132 controls, 66 HGSC, 83 non-HGSC OC, and 109 benign). In the verification study, the OC Test showed a specificity of 97.0% (128/132; 95% CI, 92.4%-99.6%), a HGSC sensitivity of 97.0% (64/66; 95% CI, 87.8%-99.2%), and an area under the curve of 0.97 (95% CI, 0.93-0.99) and detected 73.5% (61/83; 95% CI, 62.7%-82.6%) of the non-HGSC OC cases. This test exhibited fewer false positives in subjects with benign ovarian tumors, nonovarian cancers, and inflammatory conditions when compared with cancer antigen 125. The combined sensitivity and specificity of this new test suggests that it may have potential in OC screening.

Somatic Genomic and Transcriptomic Characterization of Primary Ovarian Serous Borderline Tumors and Low-Grade Serous Carcinomas

Low-grade serous carcinoma (LGSC) may develop from serous borderline tumor (SBT) tissue, where the micropapillary type (mSBT) presents the highest risk for progression. The sensitivity of LGSC to standard chemotherapy is limited, so alternative therapeutic approaches, including targeted treatment, are needed. However, knowledge about the molecular landscape of LGSC and mSBT is limited. A sample set of 137 pathologically well-defined cases (LGSC, 97; mSBT, 40) was analyzed using capture DNA next-generation sequencing (727 genes) and RNA next-generation sequencing (147 genes) to show the landscape of somatic mutations, gene fusions, expression pattern, and prognostic and predictive relevance. Class 4/5 mutations in the main driver genes (KRAS, BRAF, NRAS, ERBB2, USP9X) were detected in 48% (14/29) of mSBT cases and 63% (47/75) of LGSC cases. The USP9X mutation was detected in only 17% of LGSC cases. RNA next-generation sequencing revealed gene fusions in 6 of 64 LGSC cases (9%) and 2 of 33 mSBT cases (9%), and a heterogeneous expression profile across LGSC and mSBT. No molecular characteristics were associated with greater survival. The somatic genomic and transcriptomic profiles of 35 mSBT and 85 LGSC cases are compared for the first time. Candidate oncogenic gene fusions involving BRAF, FGFR2, or NF1 as a fusion partner were identified. Molecular testing of LGSC may be used in clinical practice to reveal therapeutically significant targets.

Comparing the Diagnostic Performance of Quantitative PCR, Digital Droplet PCR, and Next-Generation Sequencing Liquid Biopsies for Human Papillomavirus–Associated Cancers

Human papillomavirus (HPV)-associated cancers, including oropharyngeal squamous cell carcinoma (HPV + OPSCC), cervical cancer, and squamous cell carcinoma of the anus (HPV + SCCA), release circulating tumor HPV DNA (ctHPVDNA) into the blood. The diagnostic performance of ctHPVDNA detection depends on the approaches used and the individual assay metrics. A comparison of these approaches has not been systematically performed to inform expected performance, which in turn affects clinical interpretation. A meta-analysis was performed using Ovid MEDLINE, Embase, and Web of Science Core Collection databases to assess the diagnostic accuracy of ctHPVDNA detection across cancer anatomic sites, detection platforms, and blood components. The population included patients with HPV + OPSCC, HPV-associated cervical cancer, and HPV + SCCA with pretreatment samples analyzed by quantitative PCR (qPCR), digital droplet PCR (ddPCR), or next-generation sequencing (NGS). Thirty-six studies involving 2986 patients met the inclusion criteria. The sensitivity, specificity, and quality of each study were assessed and pooled for each analysis. The sensitivity of ctHPVDNA detection was greatest with NGS, followed by ddPCR and then qPCR when pooling all studies, whereas specificity was similar (sensitivity: ddPCR > qPCR, P  ddPCR, P = 0.014). ctHPVDNA from OPSCC was more easily detected compared with cervical cancer and SCCA, overall (P = 0.044). In conclusion, detection platform, anatomic site of the cancer, and blood component used affects ctHPVDNA detection and must be considered when interpreting results. Plasma NGS-based testing may be the most sensitive approach for ctHPVDNA overall.

Comparative Analysis of Gene Expression Analysis Methods for RNA in Situ Hybridization Images

Gene expression analysis is pivotal in cancer research and clinical practice. Although traditional methods lack spatial context, RNA in situ hybridization (RNA-ISH) is a powerful technique that retains spatial tissue information. Here, RNAscope score, RT-droplet digital PCR, and automated QuantISH and QuPath were used for quantifying RNA-ISH expression values from formalin-fixed, paraffin-embedded samples. The methods were compared using high-grade serous ovarian carcinoma samples, focusing on CCNE1, WFDC2, and PPIB genes. The findings demonstrate good concordance between automated methods and RNAscope, with RT-droplet digital PCR showing less concordance. Additionally, QuantISH exhibits robust performance, even for low-expressed genes like CCNE1, showcasing its modular design and enhancing accessibility as a viable alternative for gene expression analysis.

Preanalytical Histology Review Improves POLE Mutation Detection in Endometrial Carcinomas

Preanalytical tissue assessment is an important step in cancer molecular testing; however, its impact on molecular test results has not been systematically evaluated. This study describes a quality-improvement project in which routine histology review was implemented at a US molecular diagnostics laboratory. The effects of implementation on laboratory compliance and the analytical performance of a targeted POLE assay were measured as changes in tumor cellularity documentation, tumor sample enrichment (in samples with <40% tumor cellularity), POLE mutation rate, tumor signal intensity, and repeat-testing rate. Endometrial carcinoma samples (N = 1752) and tested for POLE mutations using a multiplex PCR assay. POLE mutation rates were 6.3% and 5.0% before and after intervention, respectively (P = 0.25), with the mutations most commonly detected being p.Pro286Arg (47%) and p.Val411Leu (21%). Documentation of tumor cellularity increased from 29% to 100%, and the rate of tumor enrichment increased from 1.4% to 31.5% (both, P < 0.0001). Mutation signal intensity increased from 0.32 to 0.58, and the repeat-testing rate decreased from 8.8% to 2.3% (P = 0.004 and <0.0001, respectively). Systematic preanalytical histology review was associated with improved analytical performance of a targeted POLE assay, accompanied by compliance in tumor cellularity documentation, increased tumor enrichment, and decreased repeated testing, supporting preanalytical assessment in improving somatic mutation detection in pathology specimens with low tumor content.

Clinical Performance of the Full Genotyping Agena MassARRAY HPV Assay Using SurePath Screening Samples within the VALGENT4 Framework

The clinical performance evaluation of the novel MassARRAY human papillomavirus (MA-HPV) assay was performed using Danish SurePath cervical cancer screening samples under the fourth Validation of HPV Genotyping Tests (VALGENT) framework. The MA-HPV assay is a mass array-based assay that individually detects 14 oncogenic HPV genotypes and five nononcogenic types. The MA-HPV assay was validated using the VALGENT4 panel, which constitutes 997 consecutive samples from a screening population in addition to 297 disease-enriched samples with abnormal cytology findings. The clinical accuracy of the MA-HPV assay for sensitivity and specificity was assessed relative to that of the general primer 5+/6+ PCR enzyme immunoassay (GP-EIA), by a noninferiority test. The type-specific concordance of the MA-HPV assay was assessed as well. The relative sensitivity of the MA-HPV assay for cervical intraepithelial neoplasia ≥2 or ≥3 was 1.02 (95% CI, 0.98-1.05) and 1.01 (95% CI, 0.99-1.04), respectively. The sensitivity of the MA-HPV was noninferior to that of the GP-EIA (P = 0.0001), whereas the specificity of the MA-HPV was inferior (0.89; 95% CI, 0.85-0.91; P > 0.99). The MA-HPV assay is a clinical sensitive assay with a lower clinical specificity compared with the GP-EIA. The assay in its current form seems more suited to play a role where specificity is of lesser importance but where high sensitivity is paramount.

Evaluation of Pre-Analytical Variables for Human Papillomavirus Primary Screening from Self-Collected Vaginal Swabs

Human papillomavirus (HPV) primary screening is an effective approach to assessing cervical cancer risk. Self-collected vaginal swabs can expand testing access, but the data defining analytical performance criteria necessary for adoption of self-collected specimens are limited, especially for those occurring outside the clinic, where the swab remains dry during transport. Here, we evaluated the performance of self-collected vaginal swabs for HPV detection using the Cobas 6800. There was insignificant variability between swabs self-collected by the same individual (n = 15 participants collecting 5 swabs per participant), measured by amplification of HPV and human β-globin control DNA. Comparison of self-collected vaginal swab and provider-collected cervical samples (n = 144 pairs) proved highly concordant for HPV detection (total agreement = 90.3%; positive percentage agreement = 84.2%). There was no relationship between the number of dry storage days and amplification of HPV (n = 68; range, 4 to 41 days). Exposure of self-collected dry swabs to extreme summer and winter temperatures did not affect testing outcomes. A second internal control (RNase P) demonstrated that lack of amplification for β-globin from self-collected specimens was consistent with poor, but not absent, cellularity. These data suggest that self-collected vaginal samples enable accurate clinical HPV testing, and that extended ambient dry storage or exposure to extreme temperatures does not influence HPV detection. Furthermore, lack of β-globin amplification in HPV-negative samples accurately identified participants who required recollection.

Robust Assessment of Homologous Recombination Deficiency Genomic Instability by OncoScan Microarrays

Genomic instability scars are markers for detecting homologous recombination deficiency (HRD) status in patients with ovarian cancer and predicting the response to poly (ADP-ribose) polymerase inhibitor treatment. Currently, only a few reliable and validated assays are available, with the Myriad myChoice CDx being the most commonly used commercial assay for genomic instability scar score determination. Given the need for a more straightforward, accessible, and reliable method for detecting genomic instability scars methods, in this work, we describe the feasibility of using the microarray OncoScan copy number variant assay and open-source software packages to quantify genomic instability scores, and the development of an open-access online platform for genomic instability score calculation. The laboratory-developed test accurately classified homologous recombination-proficient and recombination-deficient samples based on genomic instability scores derived from the OncoScan copy number variant assay. Internally evaluated genomic instability scores demonstrated a 92% overall agreement and a higher sample success rate compared with externally analyzed genomic instability scar scores. The availability of HRD determination has doubled the number of patients eligible for poly (ADP-ribose) polymerase therapy. The assay can be conveniently performed on individual samples, and the open-access online platform facilitates HRD determination without the need for specialized bioinformatics support.

Development, Validation, and Implementation of an Augmented Multiwell, Multitarget Quantitative PCR for the Analysis of Human Papillomavirus Genotyping through Software Automation, Data Science, and Artificial Intelligence

The value of human papillomavirus (HPV) testing for cervical cancer screening is well established; however, its use as a primary screening option or as a reflex test after atypical cytology results is now gaining wider acceptance. The importance of full genotyping and viral load determination has been demonstrated to enhance the clinical understanding of the viral infection progression during follow-up or after treatment, thereby providing clinicians with supplementary tools for optimized patient management. We developed a new analysis method for the RIATOL quantitative PCR assay, and validated and implemented it in the laboratory of clinical molecular pathology at Algemeen Medisch Laboratorium (AML), under national accreditation and following the International Organization for Standardization guidelines. This study presents the successful validation of a high-throughput, multitarget HPV analysis method, with enhanced accuracy on both qualitative and quantitative end results. This is achieved by software standardization and automation of PCR curve analysis and interpretation, using data science and artificial intelligence. Moreover, the user-centric functionality of the platform was demonstrated to enhance both staff training and routine analysis workflows, thereby saving time and laboratory personnel resources. Overall, the integration of the FastFinder plugin semi-automatic analysis algorithm with the RIATOL real-time quantitative PCR assay proved to be a remarkable advancement in high-throughput HPV quantification, with demonstrated capability to provide highly accurate clinical-grade results and to reduce manual variability and analysis time.

Equivalent Clinical Accuracy of Human Papillomavirus DNA Testing Using Cobas 4800 and 6800 Human Papillomavirus Systems in Paired Urine and Cervical Samples

The use of urine for cervical cancer screening is gaining international attention, although more data on the relative clinical accuracy of validated human papillomavirus (HPV) DNA tests on urine versus cervical samples are needed. This study primarily seeks to evaluate the clinical performance of Roche cobas 4800 and 6800 HPV Systems in first-void urine, collected at home, compared with clinician-collected cervical samples. Paired first-void urine (index test) and cervical samples (comparator test) from 499 females enrolled at five Belgian colposcopy clinics were analyzed with cobas HPV Systems. Colposcopy and histology of biopsies were used as reference test (trial registration number: NCT03064087). Sample processing protocols and clinical thresholds proposed by the manufacturer for cervical samples were also applied for first-void urine. In the total study population, HPV testing on first-void urine was similarly sensitive [ratio

MLH1 Promotor Hypermethylation in Colorectal and Endometrial Carcinomas from Patients with Lynch Syndrome

Screening for Lynch syndrome (LS) in colorectal cancer (CRC) and endometrial cancer patients generally involves immunohistochemical staining of the mismatch repair (MMR) proteins. In case of MLH1 protein loss, MLH1 promotor hypermethylation (MLH1-PM) testing is performed to indirectly distinguish the constitutional MLH1 variants from somatic epimutations. Recently, multiple studies have reported that MLH1-PM and pathogenic constitutional MMR variants are not mutually exclusive. This study describes 6 new and 86 previously reported MLH1-PM CRCs or endometrial cancers in LS patients. Of these, methylation of the MLH1 gene promotor C region was reported in 30 MLH1, 6 MSH2, 6 MSH6, and 3 PMS2 variant carriers at a median age at diagnosis of 48.5 years [interquartile range (IQR), 39-56.75 years], 39 years (IQR, 29-51 years), 58 years (IQR, 53.5-67 years), and 68 years (IQR, 65.6-68.5 years), respectively. For 31 MLH1-PM CRCs in LS patients from the literature, only the B region of the MLH1 gene promotor was tested, whereas for 13 cases in the literature the tested region was not specified. Collectively, these data indicate that a diagnosis of LS should not be excluded when MLH1-PM is detected. Clinicians should carefully consider whether follow-up genetic MMR gene testing should be offered, with age <60 to 70 years and/or a positive family history among other factors being suggestive for a potential constitutional MMR gene defect.

Clinical Accuracy of Alinity m HR HPV Assay on Self- versus Clinician-Taken Samples Using the VALHUDES Protocol

The VALHUDES protocol was established to evaluate clinical accuracy of human papillomavirus (HPV) assays to detect cervical precancer on first-void urine (FVU) and vaginal self-samples versus matched clinician-collected cervical samples (CCSs). Here we evaluated clinical performance of Alinity m HR HPV assay in a colposcopy referral population. Home-collected FVU (Colli-Pee FV 5020) 1 day before colposcopy (n = 492), at-clinic collected dry vaginal self-samples [multi-Collect Swab (mC; n = 493), followed by Evalyn Brush (EB; n = 233) or Qvintip (QT; n = 260)] and matched CCSs, were available for the study. Sensitivity to detect cervical intraepithelial neoplasia grade 2 or higher (CIN2

Comparison of the Clinical Accuracy of Xpert HPV Assay on Vaginal Self-Samples and Cervical Clinician-Taken Samples within the VALHUDES Framework

The accuracy of high-risk human papillomavirus testing with the Xpert HPV assay on vaginal self-samples was compared with clinician-taken samples within the VALidation of HUman papillomavirus assays and collection DEvices for Self-samples and urine samples (VALHUDES) framework. Five-hundred and twenty-three women were recruited in five Belgian colposcopy clinics, of whom 483 (median age, 40 years; interquartile range, 31 to 49 years) were included in the main analysis (226 collected with Evalyn Brush and 257 collected with Qvintip). Cervical samples were collected with Cervex-Brush. Colposcopy and histology outcomes were considered as the reference standard. The Xpert HPV assay had similar accuracy for cervical intraepithelial neoplasia ≥2 on self-collected versus clinician-collected samples [relative sensitivity, 0.96 (95% CI, 0.91-1.02); and relative specificity, 0.96 (95% CI, 0.89-1.04)]. The relative accuracy slightly differed by vaginal collection device [sensitivity ratios of 0.98 (95% CI, 0.90-1.06) and 0.94 (95% CI, 0.87-1.02) for Evalyn and Qvintip, respectively; specificity ratios of 1.06 (95% CI, 0.95-1.19) and 0.88 (95% CI, 0.80-0.98) for Evalyn and Qvintip, respectively]. No difference in cycle threshold values was observed between vaginal and cervical samples. In conclusion, the sensitivity of Xpert HPV assay for cervical intraepithelial neoplasia ≥2 on vaginal self-samples was similar to that of cervical specimens. The clinical specificity was lower than on clinician-collected samples when self-samples were taken with Qvintip.

Poly (ADP Ribose) Polymerase Inhibitors for Cancer

Homologous Recombination Deficiency as an Ovarian Cancer Biomarker in a Real-World Cohort

The diagnostic evaluation of homologous recombination deficiency (HRD) is central to define targeted therapy strategies for patients with ovarian carcinoma. We evaluated HRD in 514 ovarian carcinoma samples by next-generation sequencing of DNA libraries, including BRCA1/BRCA2 and 26,523 single-nucleotide polymorphisms using the standardized Myriad HRD assay, with the predefined cut point of ≥42 for a positive genomic instability score (GIS). All samples were measured in the central Myriad laboratory and in an academic molecular pathology laboratory. A positive GIS was detected in 196 (38.1%) of tumors, whereas 318 (61.9%) were GIS negative. Combining GIS and BRCA mutations, a total of 200 (38.9%) of the 514 tumors were HRD positive. A positive GIS was significantly associated with high-grade serous histology (P < 0.000001), grade 3 tumors (P = 0.001), and patient age <60 years (P = 0.0003). The concordance between both laboratories for the GIS status was 96.9% (P < 0.000001), with a sensitivity of 94.6% and a specificity of 98.4%. Concordance for HRD status was 97.1% (499 of 514 tumors). The percentage of HRD-positive tumors in our real-life cohort was similar to the proportion observed in the recently published PAOLA-1 trial, with high concordance between central and local laboratories. Our results support introduction of the standardized HRD assay in academic molecular pathology laboratories, thus broadening access to personalized oncology strategies for patients with ovarian cancer worldwide.