DNA Repair and Response to Therapy: Exploring their Relation in Epithelial Ovarian Cancer Models

Epithelial ovarian cancer (EOC) is often characterized by defects in DNA repair pathways, which partially explain its sensitivity to platinum compounds and to poly (ADP-ribose) polymerase inhibitors (PARPi). However, EOC almost invariably relapses with a drug resistant, incurable disease. Biomarkers able to predict the response to therapy could ameliorate the management of EOC patients. The studies herein reported aimed to elucidate the role of different DNA repair pathways as possible determinants of cisplatin (DDP) and olaparib (a PARPi) response in a collection of patient-derived xenografts (PDXs) characterized for their response to both drugs. We analysed the expression of 35 different DNA repair genes involved in DDP and olaparib response, and found CDK12, XPF, PALB2, USP28, ARTEMIS, ARID1A and MDR1 associated with DDP response. In particular, CDK12 expression was significantly higher in DDP resistant PDXs and associated with higher recurrence rate in EOC patients with low residual tumour. We analysed the basal level of RAD51 foci in proliferating cells of formalin-fixed paraffin-embedded PDX tumours, as possible readout of homologous recombination pathway, and found that the percentage of RAD51 foci-positive cells predicted olaparib, but not DDP response. As DDP adducts can be repaired by multiple pathways including nucleotide excision repair (NER) and base excision repair (BER), we studied POLB, ERCC1, XPF and ERCC1/XPF complex as read out of NER and BER activity, but no correlation with DDP response was found. These findings support the role of CDK12 and RAD51 foci expressed in untreated tumours in the response to DDP and olaparib, respectively, in our EOC PDX models and warrant the setup of DNA repair functional assays able to capture the complexity of factors determining DDP and PARPi response, that could be easily translated in the clinical practice to prioritize treatment choice in EOC patients

PO-473 Quantification of ERCC1-XPF complexes in ovarian cancer xenografts with different sensitivity to cisplatin

Introduction Epithelial ovarian cancer is the most lethal gynaecological cancer due to the development of resistance to a platinum based therapy. As DNA repair capacity is a key determinant for the cellular response to platinum (DDP) agents, DNA repair functional assays are required to study its relevance in DDP resistance. We set up a proximity ligation assay (PLA) to study the activity of nucleotide excision repair (NER) in patient derived ovarian carcinoma xenografts (PDXs) sensitive (S) and resistant (R) to DDP. Material and methods Patient derived xenografts from fresh ovarian carcinomas were recently established in our laboratory. DDP antitumour activity was evaluated in most of the PDXs. Mice were sacrificed when tumours reached 1,5–2 gr. Tumours were fixed in formalin and paraffin embedded (FFPE). PLA was performed on tumour slides, using DuolinkII reagents (Sigma-Aldrich) and following the manufacturer instructions. PLA detects the presence of the protein complexes ERCC1-XPF, that are quantified as foci per nucleus and represent a biomarker of NER activity. Images were acquired by Olympus Virtual Slider (Olympus) and analysed with ImageJ software. Statistical analysis was performed with GraphPad Prism7. Results and discussions Our xenobank comprises PDXs with different response to DDP: MNHOC266 and MNHOC230 are very sensitive to the drug, while MNHOC315 is resistant. We also obtained three sublines resistant to DDP (MNHOC124R, MNHOC124LPR and MNHOC239R) starting from sensitive PDXs (MNHOC124S, MNHOC124LPS and MNHOC239S), after several in vivo drug treatments. Statistically significant higher level of ERCC1-XPF foci could be observed in MNHOC124R and MNHOC124LPR as compared to their sensitive counterparts. No differences were observed between MNHOC239S and R PDXs, even if the number of ERCC1-XPF foci in MNHOC239S were statistically higher than the ones observed in MNHOC124S and in MNHOC124LPS. MNHOC266 and MNHOC230 showed levels of foci comparable to those of MNHOC124S and MNHOC124LPS. mRNA and protein levels of the different isoforms of ERCC1 and of XPF were not different among the PDXs studied. Conclusion PLA for the detection of ERCC1-XPF complexes was set up in FFPE xenograft tumour slides. These preliminary results highlight a possible link between DDP resistance and higher NER activity that need to be confirmed in a wider panel of PDXs. In addition, these data confirm the importance to develop functional assays to directly evaluate the activity of different DNA repair pathways to predict DDP activity

Platinum sensitivity and DNA repair in a recently established panel of patient-derived ovarian carcinoma xenografts

A xenobank of patient-derived (PDX) ovarian tumor samples has been established consisting of tumors with different sensitivity to cisplatin (DDP), from very responsive to resistant. As the DNA repair pathway is an important driver in tumor response to DDP, we analyzed the mRNA expression of 20 genes involved in the nucleotide excision repair, fanconi anemia, homologous recombination, base excision repair, mismatch repair and translesion repair pathways and the methylation patterns of some of these genes. We also investigated the correlation with the response to platinum-based therapy. The mRNA levels of the selected genes were evaluated by Real Time-PCR (RT-PCR) with ad hoc validated primers and gene promoter methylation by pyrosequencing. All the DNA repair genes were variably expressed in all 42 PDX samples analyzed, with no particular histotype-specific pattern of expression. In high-grade serous/endometrioid PDXs, the CDK12 mRNA expression levels positively correlated with the expression of TP53BP1, PALB2, XPF and POLB. High-grade serous/endometrioid PDXs with TP53 mutations had significantly higher levels of POLQ, FANCD2, RAD51 and POLB than high-grade TP53 wild type PDXs. The mRNA levels of CDK12, PALB2 and XPF inversely associated with the in vivo DDP antitumor activity; higher CDK12 mRNA levels were associated with a higher recurrence rate in ovarian patients with low residual tumor. These data support the important role of CDK12 in the response to a platinum based therapy in ovarian patients

Increased CNV-Region Deletions in Mild Cognitive Impairment (MCI) and Alzheimer\u27s Disease (AD) Subjects in the ADNI Sample

We investigated the genome-wide distribution of CNVs in the Alzheimer\u27s disease (AD) Neuroimaging Initia- tive (ADNI) sample (146 with AD, 313 with Mild Cognitive Impairment (MCI), and 181 controls). Comparison of single CNVs between cases (MCI and AD) and controls shows overrepresentation of large hetero- zygous deletions in cases (p-value b 0.0001). The analysis of CNV-Regions identifies 44 copy number variable loci of heterozygous deletions, with more CNV-Regions among affected than controls (p = 0.005). Seven of the 44 CNV-Regions are nominally significant for association with cognitive impairment. We validated and con- firmed our main findings with genome re-sequencing of selected patients and controls. The functional pathway analysis of the genes putatively affected by deletions of CNV-Regions reveals enrichment of genes implicated in axonal guidance, cell–cell adhesion, neuronal morphogenesis and differentiation. Our findings support the role of CNVs in AD, and suggest an association between large deletions and the development of cognitive impairment

High Risk of Secondary Infections Following Thrombotic Complications in Patients With COVID-19

Background. This study’s primary aim was to evaluate the impact of thrombotic complications on the development of secondary infections. The secondary aim was to compare the etiology of secondary infections in patients with and without thrombotic complications. Methods. This was a cohort study (NCT04318366) of coronavirus disease 2019 (COVID-19) patients hospitalized at IRCCS San Raffaele Hospital between February 25 and June 30, 2020. Incidence rates (IRs) were calculated by univariable Poisson regression as the number of cases per 1000 person-days of follow-up (PDFU) with 95% confidence intervals. The cumulative incidence functions of secondary infections according to thrombotic complications were compared with Gray’s method accounting for competing risk of death. A multivariable Fine-Gray model was applied to assess factors associated with risk of secondary infections. Results. Overall, 109/904 patients had 176 secondary infections (IR, 10.0; 95% CI, 8.8–11.5; per 1000-PDFU). The IRs of secondary infections among patients with or without thrombotic complications were 15.0 (95% CI, 10.7–21.0) and 9.3 (95% CI, 7.9–11.0) per 1000-PDFU, respectively (P = .017). At multivariable analysis, thrombotic complications were associated with the development of secondary infections (subdistribution hazard ratio, 1.788; 95% CI, 1.018–3.140; P = .043). The etiology of secondary infections was similar in patients with and without thrombotic complications. Conclusions. In patients with COVID-19, thrombotic complications were associated with a high risk of secondary infections

Combinations of ATR, Chk1 and Wee1 Inhibitors with Olaparib Are Active in Olaparib Resistant Brca1 Proficient and Deficient Murine Ovarian Cells

Background. Poly(ADP-ribose) polymerases inhibitor (PARPi) have shown clinical efficacy in ovarian carcinoma, especially in those harboring defects in homologous recombination (HR) repair, including BRCA1 and BRCA2 mutated tumors. There is increasing evidence however that PARPi resistance is common and develops through multiple mechanisms. Methods. ID8 F3 (HR proficient) and ID8 Brca1-/- (HR deficient) murine ovarian cells resistant to olaparib, a PARPi, were generated through stepwise drug concentrations in vitro. Both sensitive and resistant cells lines were pharmacologically characterized and the molecular mechanisms underlying olaparib resistance. Results. In ID8, cells with a HR proficient background, olaparib resistance was mainly caused by overexpression of multidrug resistance 1 gene (MDR1), while multiple heterogeneous co-existing mechanisms were found in ID8 Brca1-/- HR-deficient cells resistant to olaparib, including overexpression of MDR1, a decrease in PARP1 protein level and partial reactivation of HR repair. Importantly, combinations of ATR, Chk1 and Wee1 inhibitors with olaparib were synergistic in sensitive and resistant sublines, regardless of the HR cell status. Conclusion. Olaparib-resistant cell lines were generated and displayed multiple mechanisms of resistance, which will be instrumental in selecting new possible therapeutic options for PARPi-resistant ovarian tumors

Combinations of ATR, Chk1 and Wee1 Inhibitors with Olaparib Are Active in Olaparib Resistant <i>Brca1</i> Proficient and Deficient Murine Ovarian Cells

Background. Poly(ADP-ribose) polymerases inhibitor (PARPi) have shown clinical efficacy in ovarian carcinoma, especially in those harboring defects in homologous recombination (HR) repair, including BRCA1 and BRCA2 mutated tumors. There is increasing evidence however that PARPi resistance is common and develops through multiple mechanisms. Methods. ID8 F3 (HR proficient) and ID8 Brca1-/- (HR deficient) murine ovarian cells resistant to olaparib, a PARPi, were generated through stepwise drug concentrations in vitro. Both sensitive and resistant cells lines were pharmacologically characterized and the molecular mechanisms underlying olaparib resistance. Results. In ID8, cells with a HR proficient background, olaparib resistance was mainly caused by overexpression of multidrug resistance 1 gene (MDR1), while multiple heterogeneous co-existing mechanisms were found in ID8 Brca1-/- HR-deficient cells resistant to olaparib, including overexpression of MDR1, a decrease in PARP1 protein level and partial reactivation of HR repair. Importantly, combinations of ATR, Chk1 and Wee1 inhibitors with olaparib were synergistic in sensitive and resistant sublines, regardless of the HR cell status. Conclusion. Olaparib-resistant cell lines were generated and displayed multiple mechanisms of resistance, which will be instrumental in selecting new possible therapeutic options for PARPi-resistant ovarian tumors