Comprehensive ctDNA analysis to identify genome-instability and actionable mutation landscape in gynecologic cancers.
Authors: Bharde Atul , Bhonde Mandar , Bose Chirantan , Raut Nirmal Vivek , Kothavade Hrishita , D'Souza Alain , Kad Trupti , Jadhav Bhagwat , Prajapati Sangeeta , Jadhav Vikas Balasaheb Leelavati , Joshi Sujit Krishnarao , Hariramani Kanchan , Uttarwar Mohan , Khandare Jayant , Shafi Gowhar 
Published: June 2023

e17503

Background: Gynaecology malignancies are the second most prevalent cancers in women worldwide with rapid progression, high relapse, and mortality. Although HRR pathway inhibitors can greatly benefit clinical outcomes, patients without HRR pathway mutations may remain unbenefited while being profiled for tissue DNA or with a small target gene panel. Compelling data suggest that plasma genotyping shows a wide diversity of cancer mutations along genome instability indicators compared to tumor tissue. Here, we analyzed the mutational landscape of ctDNA and tissue DNA (tDNA) obtained from gynecologic cancer patients. We demonstrate the clinical utility of a comprehensive gene panel to identify multiple actionable mutations and genome instability indicators from ctDNA samples. Methods: We retrospectively analyzed the mutational landscape of 22 ovarian and endometrial cancer patients using target-hybridization enrichment of ctDNA and tDNA. Libraries prepared using a custom-designed comprehensive gene panel OncoIndx, targeting 600 cancer-relevant exons including MSI and HRR pathway genes, were sequenced on the Illumina NGS platform in pair-end mode. Variant calling was performed with an in-house developed bioinformatics pipeline. Results: NGS analysis revealed a total of 127 mutations consisting SNVs (45.6 %), truncations (40.94 %), amplifications (4.72 %), splice variants (4.72 %), and frameshift (3.94 %). Genome-wide mutational analysis showed a slightly higher mean TMB (15.7) for endometrial tumors compared to ovarian samples (11.85). The mean HRD score (45 %) was high for 90 % of patients and corroborated well with the HRR pathway gene mutation. 30 % of patients without HRR gene mutations including BRCA 1/2 had a high average HRD score (43 %) (LOH/TAI/LST) and may qualify for PARP inhibitors (PARPi). Alterations in Ras isoforms (H, K, and N) were the most frequent (36 %) followed by TP53 and PI3K (32 %). At the pathway level, DDR gene-variants were most frequently followed by alterations in proliferative signaling genes. ctDNA showed a wider mutational landscape, including clinically significant mutations, compared to tDNA. Additionally, ctDNA analysis revealed the presence of novel variants of SUFU and KIT, which remain unexplored in the context of cancer stem cells and relapse. Conclusions: Comprehensive mutational landscape showed the presence of genome-wide instability and the presence of mutations in the HRR pathway. Our results suggest that patients with high HRD scores and Ras pathway instability may benefit from Ras pathway inhibitors-PARPi combination. Mutational landscapes obtained from ctDNA were significantly wider and revealed multiple actionable targets compared to tDNA suggesting ctDNA as a viable and informative alternative for genotyping of gynecological cancers.