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Background. Homologous recombination repair (HRR) is the main mechanism of repair of DNA double-strand breaks. Its deficiency (HRD) is a common feature of epithelial ovarian cancers (EOCs). BRCA1/2 mutations and/or other aberrations in genes of HRR are well known causes of HRD and genomic instability. Poly ADP-ribose polymerase inhibitors (PARPi) have revolutionized the management of BRCA mutant EOCs and demonstrated activity in HRD tumor cells. Determining HRD status can provide informations on the magnitude of benefit for PARPi therapy. Myriad MyChoice CDx is a next generation sequencing- based in vitro diagnostic test that assesses the Genomic Instability Score (GIS) which is an algorithmic measurement of loss of heterozygosity, telomeric allelic imbalance, and large-scale state transitions using DNA isolated from formalin-fixed paraffin embedded tumor tissue specimens. However Myriad MyChoice CDx, is a centrally performed and costly assay, with no reimbursement scheduled, at least in Italy. Methods. In this report, we described our experience in performing the HRD Focus AmoyDx (Amoy Diagnostics Ltd, Xiamen, Fujian, China) on the same samples of EOCs evaluated with Myriad MyChoiceCDx assay. Results. The overall percent agreement between AmoyDx and Myriad was 87.8% (65 of 74 tumors tested). All the 36 AmoyDx negative cases were confirmed to be negative by Myriad (negative predictive value, 100%). Conclusions. The concordance of the results with the gold standard Myriad MyChoice CDx assay suggest the feasibility and reliability of HRD testing in diagnostic laboratories with high-throughput NGS platforms and qualified personnel.

Objectives To evaluate the role of cervical re-injection of indocyanine green (ICG) to increase the detection rate of sentinel lymph node (SLN) in patients with endometrial cancer (EC) who underwent robotic-assisted surgical staging. Methods We retrospectively identified consecutive EC patients undergoing robotic-assisted staging with SLN biopsy at our Institution between June 2016 and April 2020. Patients were excluded if they had open abdominal surgical approach, neoadjuvant chemotherapy, and advanced stage [International Federation of Gynecology and Obstetrics (FIGO) stage III-IV] at diagnosis. According to our SLN protocol, in case of either unilateral or no SLN detection, we performed an ipsilateral or bilateral cervical re-injection of ICG. Results In total, 251 patients meeting inclusion criteria were included in the analysis. At first injection, bilateral detection was achieved in 184 (73.3%), unilateral detection in 57 (22.7%), and no detection in 10 (4.0%) patients. Cervical re-injection was performed in 51 of 67 patients with failed bilateral mapping. After cervical re-injection, bilateral detection rate increased to 94.5% (222/235), while unilateral and no detection were 5.1% (12/235) and 0.4% (1/235), respectively. Conclusions Our results suggest that cervical re-injection of ICG, in case of failed bilateral mapping of SLN, brings about a significant improvement in SLN detection rates, therefore reducing the number of side-specific required lymphadenectomies.

Objectives Wnt pathway mutations are a hallmark of endometrioid and clear cell subtypes of epithelial ovarian carcinoma (EOC). However, no drugs targeting the Wnt pathway in EOC are FDA-approved. Dickkopf-related protein 1 (DKK1), a modulator of the Wnt pathway, has emerged as a promising therapeutic target. We aimed to examine the role of DKK1 and the effects of a monoclonal antibody against DKK1 (DKN-01) in vivo and in a murine model of ovarian cancer. Methods We examined in vitro the role of DKK1 and the effects of DKK1 inhibition in EOC cell lines. We then studied in vivo the role of DKN-01 and DKK1 overexpression on tumor burden and anti-tumor immune cell populations using the ID8 syngeneic mouse model. Results DKN-01 did not phenotypically alter ES2 cells in vitro; however, DKK1 inhibition promoted Wnt signaling. Tumor burden and immune populations were unchanged in ID8 challenged mice treated with mDKN01. Mice challenged with ID8 cells overexpressing DKK1 had tumor burden similar to controls (p = 0.175). However, the overexpression of DKK1 decreased CD45+ leukocyte infiltration into the peritoneum (p = 0.008) and omentum (p = 0.032), reducing both natural killer (NK) and CD8 T cells, and reducing interferon-gamma (IFNγ) expression on activated CD8 T cells. Conclusions Our results suggest that DKK1 inhibition does not affect tumor growth in the ID8 ovarian cancer model. DKK1 overexpression alters anti-tumor immune populations within the tumor microenvironment. Thus, our findings confirm DKK1 as a new therapeutic target in EOC and suggest that DKK1 inhibition may function best in a combinatorial, immune-modulatory therapy.

The establishment of PARP inhibitors in the treatment of epithelial ovarian carcinoma (EOC) has prompt BRCA assessment at the time of diagnosis. We described our five years of experience of tumor BRCA testing, as part of a multidisciplinary workflow for the management of EOC patients. We used a BRCA next-generation sequencing (NGS) test for profiling formalin-fixed, paraffin-embedded (FFPE) EOCs of 762 consecutive patients, with a success rate of 99.7% and a median turnaround time of 12 days. We found 178 (23.4%) cases with pathogenic/likely pathogenic (P/LP) mutations, 74 (9.7%) cases with variants of uncertain significance and 508 (66.8%) wild type tumors. Among 174 patients without P/LP mutations and investigated with multiple-ligation probe-amplification analysis on peripheral blood, two (1.1%) were positive for large rearrangements. Patients with P/LP alterations and/or with positive family history were referred to genetic counselling. Comparing tumor and blood NGS test results of 256 patients, we obtained a tumor test negative predictive value of 100% and we defined 76% of P/LP alterations as germline and 24% as somatic variants. The proposed workflow may successfully identify EOC patients with BRCA1/2 alteration, guiding both therapeutic and risk assessment clinical decisions.

ObjectiveTo compare the risk class attribution withmolecular classification unknownto those withmolecular classification known,according to the European Society of Gynaecological Oncology/European Society for Radiotherapy and Oncology/European Society of Pathology (ESGO/ESTRO/ESP) 2020 guidelines on endometrial cancer, with a focus on risk group migration. Additionally, to evaluate the capability of a novel molecular analysis algorithm to reduce the number of required tests.MethodsWe conducted a retrospective study including all consecutive patients with endometrial cancer undergoing surgery and comprehensive molecular analyses between April 2019 and December 2021. Molecular analyses including immunohistochemistry for p53 and mismatch repair (MMR) proteins, and DNA sequencing for POLE exonuclease domain were performed to classify tumors as POLE-mutated (POLE), MMR-deficient (MMR-d), p53 abnormal (p53abn), or non-specific molecular profile (NSMP). The two risk classifications of the ESGO/ESTRO/ESP 2020 guidelines were compared to estimate the proportion of patients in which the molecular analysis was able to change the risk class attribution. We developed a novel algorithm where the molecular analyses are reserved only for patients in whom incorporation of the molecular classification could change the risk class attribution.ResultsA total of 278 patients were included. Molecular analyses were successful for all cases, identifying the four subgroups: 27 (9.7%) POLE, 77 (27.7%) MMR-d, 49 (17.6%) p53abn, and 125 (45.0%) NSMP. Comparison of risk class attribution between the two classification systems demonstrated discordance in the risk class assignment in 19 (6.8%, 95% CI 4.2% to 10.5%) cases. The application of our novel algorithm would have led to a reduction in the number of POLE sequencing tests by 67% (95% CI 61% to 73%) and a decrease of p53 immunohistochemistry by 27% (95% CI 22% to 33%), as compared with the application of molecular classification to all patients.ConclusionMolecular categorization of endometrial cancer allows the reallocation of a considerable proportion of patients in a different risk class. Furthermore, the application of our algorithm enables a reduction in the number of required tests without affecting the risk classification.

Antibody-drug conjugates (ADCs) are complex engineered therapeutics consisting of monoclonal antibodies, directed toward tumor-associated antigens, to which highly potent cytotoxic agents are attached using chemical linkers. This targeted drug delivery strategy couples the precision of the antibody targeting moiety with the cytocidal activity of the payload, which is generally too toxic on its own to be systemically administered. In this manner, ADCs confer a means to reduce off-target toxicities in patients by limiting the exposure of normal tissues to the payload, thus broadening the potential therapeutic window compared with traditional chemotherapy. The pace of ADC development is accelerating, with the number of investigational agents in human trials having more than tripled over the past 5 years, underscoring the enthusiasm for this transformative approach to cancer treatment. Here, we review the key structural elements of ADC design (antibody, linker, and payload), highlighting critical aspects and technological advances that have affected the clinical effectiveness of this class of biopharmaceuticals. The ADC field continues to evolve, including ongoing efforts aimed at improving target selection, developing payloads with varied mechanisms of action and increased potency, designing innovative bioconjugation strategies, as well as maximizing efficacy and tolerability in patients. An overview of the current clinical trial landscape is provided, with emphasis on the clinical experience of the four ADCs to have received regulatory approval to date, as well as additional promising candidates currently in late-stage clinical development in both solid tumor and hematological malignancies.

AimsAnalysis of microsatellite instability (MSI) is strongly recommended in endometrial cancer (EC) and colorectal cancer to screen for Lynch syndrome, to predict prognosis and to determine optimal treatment and follow-up. In a large monoinstitutional series of ECs, we evaluated the reliability and accuracy of Idylla assay, a rapid, fully automated system to detect MSI, and we compared its performance with two routine reference methods.MethodsWe evaluated MSI status in 174 formalin-fixed, paraffin-embedded EC tissue samples using immunohistochemistry (IHC) for mismatch repair (MMR) proteins and Idylla assay. Samples with discordant or equivocal results were analysed with a third technique, the Promega MSI kit.ResultsIdylla MSI assay and IHC were highly concordant (overall agreement: 154/170=90.59%, 95% CI 85.26% to 94.12%). However, in four samples, MMR-IHC staining was equivocal; moreover, 16 cases showed discordant results, that is, MMR deficient using IHC and microsatellite stable using Idylla. These 20 samples were reanalysed using the MSI-Promega kit, which showed the same results of Idylla assay in 18/20 cases (overall agreement: 90%, 95% CI 69.90% to 97.21%).ConclusionsOur results suggest that IHC is an efficient method to determine MMR status in ECs. However, the Idylla MSI assay is a rapid and reliable tool to define MSI status, and it could represent a valuable alternative to conventional MSI-PCR methods.

Ovarian transposition aims to minimize ovarian exposure and damage during pelvic radiotherapy. One or both ovaries are separated from the uterus and mobilized away from the area where the radiation will be administered. A review of the available literature was conducted to evaluate the efficacy and safety of ovarian transposition among pre-menopausal women diagnosed with cervical cancer and eligible for pelvic radiotherapy. Outcomes evaluated were ovarian function preservation and complication rates. We also searched for information on pregnancy/live birth rates after ovarian transposition. Our search yielded a total of 635 manuscripts, of which 33 were considered eligible. A total of 28 full texts were selected for the current review, including 1377 patients who underwent ovarian transposition. The median or mean follow-up ranged between 7 and 87 months. Ovarian function preservation after ovarian transposition and pelvic radiotherapy, with or without chemotherapy, was 61.7% (431/699 patients), ranging from 16.6% to 100%. A total of 12 studies reported on 117 complications, accounting for 8.5%. Ovarian metastases were described in 5 (0.4%). Data about fertility preservation after ovarian transposition are scarce and definitive conclusions cannot be drawn. Based on the available data, ovarian transposition could be performed on young patients with tumors smaller than 4 cm, and it should be avoided in those with bulky tumors. A risk/benefit assessment should be carefully evaluated by a multidisciplinary team, and the decision regarding ovarian transposition should be always guided by the values and informed preferences of the patient.

Prognosis for women with epithelial ovarian cancer remains poor. One new molecular target in epithelial ovarian cancer is folate receptor alpha (FRα). This commentary discusses the characteristics that contribute to its attractiveness as a candidate for therapeutic intervention.

To evaluate oncologic outcomes of early stage cervical cancer patients who underwent robotic radical hysterectomy (RRH) in a referral center, a retrospective analysis was performed. From January 2010 to December 2018, medical records of stage IA2–IIA1 cervical cancer patients, who underwent radical hysterectomy at our institute, were retrospectively reviewed. We focused our analysis on those who underwent RRH. A total of 198 patients were included in the final analysis. Median follow up was 52 months. At last follow-up, 188 (94.9%) women were disease-free, 9 (4.5%) had died, and 1 (0.5%) was alive with recurrent disease. At 4.5 years, PFS was 93.1% (SE ± 2.1) and OS was 95.1% (SE ± 1.8). Stratified by tumor size, PFS for tumor

BackgroundWith the approval of the poly (ADP-ribose) polymerase (PARP) inhibitor olaparib for newly diagnosed, breast cancer gene (BRCA)1/2 mutated, ovarian cancer women, the assessment of BRCA1/2 tumour status will be shortly required at the time of diagnosis.AimTo investigate the feasibility of next-generation sequencing (NGS)-based BRCA tumour test on cytological specimens from ovarian cancer ascites.MethodsWe evaluated the BRCA1/2 status on neoplastic ascites and corresponding tumour tissue of 11 patients with ovarian cancer, using the NGS ‘Oncomine BRCA Research Assay’.ResultsThe NGS-based BRCA test on cytological samples had a success rate of 100%, with 11 of 11 concordant BRCA1/2 results between ascites and tumour tissues analyses, including two wild type samples and nine cases harbouring somatic or germline variants.ConclusionBRCA test may be performed on ovarian cancer ascites, reproducing BRCA1/2 tumour status and representing a useful tool for clinical decision-making.

Introduction/Background To estimate the incidence of abdominal wall metastases (AWM) and their impact on overall survival in patients with advanced low-grade serous ovarian cancer (LGSOC). Methodology Patients with advanced LGSOC (FIGO stage III–IV) undergoing primary and/or secondary cytoreductive surgery at our Institution between August 2001 and August 2016 were retrospectively identified. Incidence of AWM was evaluated by review of electronic medical records. Overall survival was estimated using the Kaplan-Meier method. Results Total of 60 women who met inclusion criteria were identified. Of them, 30 (50%) patients had AWM, including 12 (20%) at primary surgery, 18 (30%) at relapse, or both 8 (13.3%). Among patients with evidence of AWM at primary surgery, 11 (91.7%) had prior surgery (10 laparoscopic, 1 laparotomy). Median follow-up was 87 (IQR 51, 127) years. Among the 60 patients, 24 (40%) died of disease [17/30 (56.7%) vs. 8/30 (26.7%) in the groups with and without AWM, respectively]. The median overall survival in the group of patients with AWM was lower than in the group without AWM (80 months; 95% CI 80–103 vs. 89 months; 95% CI 73–104, respectively, Log rank test, P=0.085), although not statistically significant. Conclusion Incidence of AWM in advanced LGSOC is high (50% in the present series) compared to high-grade serous epithelial ovarian cancer (3–5%) (Cormio et al, 2003, Gynecologic Oncology) and is associated with decreased overall survival. Disclosure Nothing to disclose.

The PARP inhibitor olaparib has been approved in the maintenance setting of platinum-sensitive epithelial ovarian cancer patients with germline or somatic BRCA1/2 mutation. Therefore, the availability of a tumor BRCA test has become a clinical need. We report the results of the clinical implementation of a tumor BRCA test within the frame of an institutional workflow for the management of patients with nonmucinous and nonborderline epithelial ovarian cancer. In total, 223 patients with epithelial ovarian cancer were prospectively analyzed. BRCA1/2 status was evaluated on formalin-fixed, paraffin-embedded tumor specimens using next-generation sequencing technology. The tumor BRCA test had a success rate of 99.1% (221 of 223 successfully analyzed cases) and a median turnaround time of 17 calendar days. Among the 221 cases, BRCA1 or BRCA2 pathogenic/likely pathogenic mutations were found in 62 (28.1%) cases and variants of uncertain significance in 25 (11.3%) cases. The concordance rate between tumor BRCA test results and germline BRCA1/2 status was 87%, with five cases harboring pathogenic/likely pathogenic somatic-only mutations. The next-generation, sequencing-based tumor BRCA test showed a high success rate and a turnaround time compatible with clinical purposes. The tumor BRCA test could be implemented in a molecular diagnostic setting and it may guide the clinical management of patients with epithelial ovarian cancer.

Objectives To evaluate the impact of cervical re-injection on the detection rate of fluorescence-guided sentinel lymph node (SLN) mapping in endometrial cancer (EC) patients undergoing robotic-assisted surgical staging. Methods From April, 1 2017 to December, 31 2018 patients undergoing robotic-assisted surgery for apparently early-stage EC at our Institution were prospectively treated with SLN mapping using indocyanine green (ICG) accordingly to the Memorial Sloan Kettering Cancer Center (MSKCC) surgical algorithm. As per MSKCC algorithm, four mL (1.25 mg/mL) of ICG were injected into the cervical submucosa and stroma, at the 3 and 9 o’clock positions (1 mL each). In case of either no detection or unilateral detection, cervical re-injection was performed followings the same steps as previously described. Overall (successful mapping of at least one hemipelvis) and bilateral detection were evaluated pre- and post-re-injection. Results Of the 107 patients undergoing robotic-assisted surgical staging for EC during the study period, 7 cases with no detection or unilateral detection who did not underwent re-injection were excluded. Among the remaining 100 patients, after a single injection the overall detection rate was 98% (95% CI, 92.2–99.6%) with a 69% (95% CI, 58.8–77.7%) of bilateral detection rate. After re-injection, overall and bilateral detection rate were 100% (95% CI, 95.3–100%) and 91% (95% CI, 8.32–95.5%), respectively. Conclusions In the case of no detection or unilateral sentinel lymph node detection, cervical re-injection of ICG can increase overall and bilateral detection rate, thus decreasing the number of patients requiring a complete bilateral or side-specific lymphadenectomy.

Background:The Wnt/β-catenin pathway is linked to tumorigenesis in a variety of tumors and promotes T cell exclusion and resistance to checkpoint inhibitors. We sought to determine whether a small molecule inhibitor of this pathway, WNT974, would impair tumor growth, affect gene expression patterns, and improve the immune response in human and murine ovarian cancer models.Methods:Human ovarian cancer cells were treated with WNT974 in vitro. RNAseq libraries were constructed and differences in gene expression patterns between responders and nonresponders were compared to The Cancer Genome Atlas (TCGA). Mice with subcutaneous or intraperitoneal ID8 ovarian cancer tumors were treated with WNT974, paclitaxel, combination, or control. Tumor growth and survival were measured. Flow cytometry and β-TCR repertoire analysis were used to determine the immune response.Results:Gene expression profiling revealed distinct signatures in responders and nonresponders, which strongly correlated with T cell infiltration patterns in the TCGA analysis of ovarian cancer. WNT974 inhibited tumor growth, prevented ascites formation, and prolonged survival in mouse models. WNT974 increased the ratio of CD8+T cells to T regulatory cells (Tregs) in tumors and enhanced the effector functions of infiltrating CD4+and CD8+T cells. Treatment also decreased the expression of inhibitory receptors on CD8+T cells. Combining WNT974 with paclitaxel further reduced tumor growth, prolonged survival, and expanded the T cell repertoire.Conclusions:These findings suggest that inhibiting the Wnt/β-catenin pathway may have a potent immunomodulatory effect in the treatment of ovarian cancer, particularly when combined with paclitaxel.

5572 Background: The PARPi Olaparib has been approved in maintenance setting of recurrent platinum-sensitive and BRCA mutated, ovarian cancer (OC) patients. However, according to the striking results of SOLO 1 trial, it could be shortly proposed even to newly diagnosed BRCA mutated OC women. We report the results of the implementation of tumor BRCA test in diagnostic setting within the frame of institutional workflow for the management of OC patients. Methods: 223 women with OC were consecutively referred over 25 months to tumor BRCA test. The test was requested by gynecologic oncologist at the diagnosis of non-mucinous and non-borderline OC, upon discussion on the implications of the test result and written consent collected for each patient. Formalin-fixed paraffin embedded (FFPE) specimens of OC were tested using the automatized “Oncomine BRCA Research Assay” Next Generation Sequencing (NGS) panel. The tumor BRCA test was also performed on 5 archetypal cytological samples from ascites. Results: All the cases were considered adequate for the NGS analysis according to the tumor cell content (more than 10%) and the DNA yield extracted (more than 10 ng). The tumor BRCA test had a successful rate of 99.1% . The median Turn-Around Time (TAT) was 17 calendar days, from 33 days of the first trimester to 14 days of the last trimester of this analysis. Overall BRCA1 or BRCA2 pathogenic (P)/likely pathogenic (LP) mutations were found in 62 (28.1%) cases and variants of uncertain significance (VUS) in 25 (11.3%) cases, including 3 cases with a BRCA1 P variant and a concurrent BRCA2 VUS alteration. In detail, 47 P/LP variants and 16 VUS were identified in BRCA1 whereas 15 P/LP mutations and 9 VUS occurred in BRCA2. Complete concordance in tumor BRCA test results were seen between ascites cytological samples and matched tumors. Conclusions: The tumor BRCA test could be implemented in routine diagnostic setting, at diagnosis of non-mucinous and non-borderline OC. The test could be performed on FFPE specimens, had an high successful rate and a TAT compatible with clinical needs. The promising data on cytological samples will be confirmed in larger series.