DEVELOPMENT OF CUSTOMIZED TESTS FOR THE EARLY DIAGNOSIS OF RELAPSES OF GYNECOLOGICAL TUMORS USING LIQUID BIOPSY

Global statistics regarding endometrial, cervical, and ovarian cancers show that gynecological tumors often tend to relapse, especially those initially diagnosed at advanced stages. Recurrent gynecological tumors are a significant concern and are generally associated with a poor prognosis. This underscores the importance of early detection and effective management of recurrences. The current biochemical tumor markers used in clinical practice, such as CA125, do not provide sufficient sensitivity and specificity for the early detection of gynecological cancer relapses. This limitation highlights the need for more accurate and reliable diagnostic tools. In addition, the absence of universally shared guidelines for the management and follow-up of gynecological tumor survivors contributes to the challenges of early relapse detection. Consistent and evidence-based guidelines are essential for improving patient care and outcomes. Considering these challenges, the development of our project aimed to create a rapid, patient-specific somatic mutation detection system for early relapse detection in gynecological cancer survivors. Tumors often release DNA into the bloodstream, and this circulating tumor DNA can contain mutations specific to the cancer. By analyzing this ctDNA, we aimed to detect the presence of mutations associated with the tumor by using liquid biopsy that can provide valuable information about the presence of cancer without the need for a traditional tissue biopsy. These mutations serve as unique markers for each patient’s cancer, and we can use them to discriminate ctDNA from the total cfDNA extracted from plasma samples during follow-up after surgical treatment. We have enrolled 63 patients who received a final diagnosis of endometrial, cervical, and ovarian cancers and we collected biological samples from each of them. Whole Exome Sequencing and bioinformatic analysis were performed for 36 patients for the tumor DNA samples. Our detection system employed two distinct molecular biology approaches: 1) Allele-Specific Real-Time PCR method: in this case, we also used an allele-specific restriction enzyme that specifically cleaved wildtype DNA to increase the sensitivity of allele-specific real-time PCR amplification. We tested by this method the somatic mutations in the gDNA and tDNA. For cfDNA extracted from plasma samples during follow-up, two patients were tested, and both were negative at the end of the follow-up in agreement with the clinical examinations and biochemical marker levels. 2) Digital PCR custom assay: using the digital PCR method, we tested one mutation in the KRAS gene that was detected in two endometrial patients. For the first patient, we predicted the presence of few tumor cells that carried this mutation during the follow-up although the biochemical and clinical investigations were negative. For the second patient, we detected this mutation after 6 months of the surgical operation, but she was negative for this mutation at the end of the follow-up in agreement with the clinical investigations. We also characterized the genetic profile at the level of somatic mutations of 36 patients enrolled in the study. In particular, we identified the most altered signaling pathways, tumor clonality, and drug-genome interactions for each patient. Moreover, 7 out of 36 tDNA samples had been found to have potentially actionable somatic mutations, described in the new Actionability in Precision Oncology product released by the COSMIC database, which can be targeted using specific drugs, strengthening the link between the genetic profile of the tumor and clinical oncology.