Prospective verification of OvaPrint and multi-omic profiling for precise adnexal mass classification

Background: Patients with adnexal masses face both overtreatment, when benign disease is misclassified, and undertreatment, when cancers are not managed by gynecologic oncologists. CA125, the most widely used biomarker, lacks specificity. High-grade serous ovarian cancer (HGSOC), the most aggressive subtype of epithelial ovarian cancer (EOC), requires timely specialist management. OvaPrint, a cfDNA methylation liquid biopsy developed to improve adnexal mass evaluation, is currently optimized for HGSOC, with expansion underway. In a prospective multi-site study, we sought to clinically verify OvaPrint, compare it with CA125 and integrate it with 5-base sequencing, next generation proteomics and spatial transcriptomics analysis. Methods: Plasma was collected from six US sites preoperatively and analyzed with OvaPrint. Eligibility included adnexal mass on imaging without evidence of metastatic disease and planned surgery. Clinical data, including CA125 values, were abstracted from medical records. Diagnosis from surgical pathology reports served as the clinical ground truth. Of 121 cases (7 HGSOC, 16 other EOCs, 5 other cancers, 93 benign), 110 were evaluable (104 had CA125). Endpoints were sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV). Samples were also analyzed using the latest Illumina's technology including: Illumina's Protein Preparation (IPP) platform detecting 9500 proteins (80 plasma samples); 5-base sequencing solution for simultaneous whole-genome DNA methylation and variant detection (58 cfDNA samples); spatial transcriptomics for description of the tumor microenvironment (TME) and histologic context (23 fresh frozen tissue samples). Results: OvaPrint achieved 83.3% sensitivity for HGSOC. Specificity was 96.6% for benign cases and 97.1% for non-HGSOC tumors. The HGSOC-specific PPV and NPV was 62.5% and 99%, respectively. CA125 showed 100% sensitivity for HGSOC but had poor specificity (62.2%, 13% PPV). IPP, 5-base sequencing and spatial transcriptomics data are being evaluated to complement OvaPrint through an integrated multi-omic approach for the discrimination of malignant and benign adnexal masses. Conclusions: Accurate tools for EOC diagnosis are urgently needed. This prospective, multi-site study demonstrates OvaPrint's exceptional performance for the discernment of HGSOC. The high HGSOC-specific NPV and PPV of OvaPrint means a positive result is a near-certain indicator of HGSOC, supporting timely and appropriate care. OvaPrint is being expanded to other EOCs and is undergoing CLIA validation. Additional integration of other molecular endpoints could further enhance discrimination of adnexal masses. These findings establish it as the first liquid biopsy for HGSOC with potential to transform diagnostic practice and outcomes.