Gynaecological carcinomas (ovarian, endometrial, cervical, vaginal, vulvar and breast) are among the ones leading to the highest cancer-related deaths worldwide. The presence of germline pathogenic... Show moreGynaecological carcinomas (ovarian, endometrial, cervical, vaginal, vulvar and breast) are among the ones leading to the highest cancer-related deaths worldwide. The presence of germline pathogenic variants in the breast cancer susceptibility genes BRCA1 and BRCA2 is associated with an increased risk for breast and ovarian cancer as well as other cancers. The BRCA proteins play a crucial role in the homologous recombination (HR) pathway, which is the only DNA damage repair pathway that can repair DNA double-strand breaks error-free. The repair of DNA damage by HR is crucial to maintain genomic integrity. Patients whose tumors are HR-Deficient (HRD) are particularly sensitive to platinum-based chemotherapy and poly-ADP ribose polymerase inhibitors (PARPi). BRCA1/2 deficiency, but also (epi)genetic defects in other HR-related genes or other unknown factors can lead to HRD. However, BRCA1/2 deficiency, and not HRD, is still the leading prerequisite to be eligible for PARPi therapy. In this thesis, two functional RAD51-based HRD tests (RECAP and RAD51-FFPE test), were developed and validated using gynaecological carcinomas. These functional HRD tests were benchmarked with BRCA1/2 deficiency and with other DNA-based HRD tests measuring genomic scars and mutational signatures. In addition, the prevalence of functional HRD was explored among a variety of gynaecological carcinomas. Show less
PurposeBRCA-deficient breast cancers (BC) are highly sensitive to platinum-based chemotherapy and PARP inhibitors due to their deficiency in the homologous recombination (HR) pathway. However, HR... Show morePurposeBRCA-deficient breast cancers (BC) are highly sensitive to platinum-based chemotherapy and PARP inhibitors due to their deficiency in the homologous recombination (HR) pathway. However, HR deficiency (HRD) extends beyond BRCA-associated BC, highlighting the need for a sensitive method to enrich for HRD tumors in an alternative way. A promising approach is the use of functional HRD tests which evaluate the HR capability of tumor cells by measuring RAD51 protein accumulation at DNA damage sites. This study aims to evaluate the performance of a functional RAD51-based HRD test for the identification of HRD BC.MethodsThe functional HR status of 63 diagnostic formalin-fixed paraffin-embedded (FFPE) BC samples was determined by applying the RAD51-FFPE test. Samples were screened for the presence of (epi)genetic defects in HR and matching tumor samples were analyzed with the RECAP test, which requires ex vivo irradiated fresh tumor tissue on the premise that the HRD status as determined by the RECAP test faithfully represented the functional HR status.ResultsThe RAD51-FFPE test identified 23 (37%) of the tumors as HRD, including three tumors with pathogenic variants in BRCA1/2. The RAD51-FFPE test showed a sensitivity of 88% and a specificity of 76% in determining the HR-class as defined by the RECAP test.ConclusionGiven its high sensitivity and compatibility with FFPE samples, the RAD51-FFPE test holds great potential to enrich for HRD tumors, including those associated with BRCA-deficiency. This potential extends to situations where DNA-based testing may be challenging or not easily accessible in routine clinical practice. This is particularly important considering the potential implications for treatment decisions and patient stratification. Show less
Introduction Carcinomas with defects in the homologous recombination (HR) pathway are sensitive to PARP inhibitors (PARPi). A robust method to identify HR-deficient (HRD) carcinomas is therefore of... Show moreIntroduction Carcinomas with defects in the homologous recombination (HR) pathway are sensitive to PARP inhibitors (PARPi). A robust method to identify HR-deficient (HRD) carcinomas is therefore of utmost clinical importance. Currently, available DNA-based HRD tests either scan HR-related genes such as BRCA1 and BRCA2 for the presence of pathogenic variants or identify HRD-related genomic scars or mutational signatures by using whole-exome or whole-genome sequencing data. As an alternative to DNA-based HRD tests, functional HRD tests have been developed that assess the actual ability of tumors to accumulate RAD51 protein at DNA double-strand breaks as a proxy for HR proficiency. Areas covered This review presents an overview of currently available HRD tests and discusses the pros and cons of the different methodologies including their sensitivity for the identification of HRD tumors, their concordance with other HRD tests, and their capacity to predict therapy response. Expert opinion With the increasing use of PARPi in the treatment of several cancers, there is an urgent need to implement HRD testing in routine clinical practice. To this end, calibration of HRD thresholds and clinical validation of both DNA-based and RAD51-based HRD tests should have top-priority in the coming years. Show less
The sensitivity to PARP inhibitors (PARPi) is related to tumor-specific defects in homologous recombination (HR) and extends beyond BRCA1/2-related deficiencies. A robust method to identify HR... Show moreThe sensitivity to PARP inhibitors (PARPi) is related to tumor-specific defects in homologous recombination (HR) and extends beyond BRCA1/2-related deficiencies. A robust method to identify HR-deficient (HRD) carcinomas is therefore of utmost clinical importance. In this study, we evaluated the use of a functional test (the RECAP test) for the identification of HRD ovarian carcinomas. Forty-nine epithelial ovarian carcinomas (EOC) were analyzed by the RECAP test. Thirty-nine of these tumors were of the high-grade serous (HGSOC) histologic subtype. Ten out of these 39 HGSOC specimens showed HRD (26%), whereas ovarian carcinomas of other histologic subtypes (n = 10) were all HR-proficient (HRP). Eight out of 9 sequenced HRD tumors showed pathogenic BRCA1/2 variants or BRCA1 promoter hypermethylation. This study shows that the RECAP test is a reliable and rapid test to identify functional deficiencies in HR and a good alternative to DNA-based HRD tests.Recent studies have shown that the efficacy of PARP inhibitors in epithelial ovarian carcinoma (EOC) is related to tumor-specific defects in homologous recombination (HR) and extends beyond BRCA1/2 deficient EOC. A robust method with which to identify HR-deficient (HRD) carcinomas is therefore of utmost clinical importance. In this study, we investigated the proficiency of a functional HR assay based on the detection of RAD51 foci, the REcombination CAPacity (RECAP) test, in identifying HRD tumors in a cohort of prospectively collected epithelial ovarian carcinomas (EOCs). Of the 39 high-grade serous ovarian carcinomas (HGSOC), the RECAP test detected 26% (10/39) to be HRD, whereas ovarian carcinomas of other histologic subtypes (n = 10) were all HR-proficient (HRP). Of the HRD tumors that could be sequenced, 8/9 showed pathogenic BRCA1/2 variants or BRCA1 promoter hypermethylation, indicating that the RECAP test reliably identifies HRD, including but not limited to tumors related to BRCA1/2 deficiency. Furthermore, we found a trend towards better overall survival (OS) of HGSOC patients with RECAP-identified HRD tumors compared to patients with HRP tumors. This study shows that the RECAP test is an attractive alternative to DNA-based HRD tests, and further development of a clinical grade RECAP test is clearly warranted. Show less
Kopper, O.; Witte, C.J. de; Lohmussaar, K.; Valle-Inclan, J.E.; Hami, N.; Kester, L.; ... ; Clevers, H. 2019
Ovarian cancer (OC) is a heterogeneous disease usually diagnosed at a late stage. Experimental in vitro models that faithfully capture the hallmarks and tumor heterogeneity of OC are limited and... Show moreOvarian cancer (OC) is a heterogeneous disease usually diagnosed at a late stage. Experimental in vitro models that faithfully capture the hallmarks and tumor heterogeneity of OC are limited and hard to establish. We present a protocol that enables efficient derivation and long-term expansion of OC organoids. Utilizing this protocol, we have established 56 organoid lines from 32 patients, representing all main subtypes of OC. OC organoids recapitulate histological and genomic features of the pertinent lesion from which they were derived, illustrating intra-and interpatient heterogeneity, and can be genetically modified. We show that OC organoids can be used for drug-screening assays and capture different tumor subtype responses to the gold standard platinum-based chemotherapy, including acquisition of chemoresistance in recurrent disease. Finally, OC organoids can be xenografted, enabling in vivo drug-sensitivity assays. Taken together, this demonstrates their potential application for research and personalized medicine. Show less
Jonge, M.M. de; Auguste, A.; Wijk, L.M. van; Schouten, P.C.; Meijers, M.; Haar, N.T. ter; ... ; Bosse, T. 2019
