Alessandro D. Santin

Preclinical Activity of Datopotamab Deruxtecan (Dato-DXd), an Antibody–Drug Conjugate Targeting TROP2, in Poorly Differentiated Endometrial Carcinomas

Abstract Datopotamab deruxtecan (Dato-DXd) is a novel antibody–drug conjugate (ADC) targeting trophoblast antigen-2 (TROP2), a cell surface glycoprotein highly expressed in many epithelial tumors, to deliver DXd, a potent topoisomerase I inhibitor. We evaluated TROP2 expression in primary endometrial cancer cell lines and the activity of Dato-DXd against endometrial cancer cell lines with different TROP2 expression in vitro and in vivo. TROP2 expression was assessed in nine primary tumor cell lines by flow cytometry. Cell viability after exposure to Dato-DXd was evaluated using flow cytometry–based assays to calculate the IC50. Bystander effect assay assessed the viability of TROP2-negative cells when cocultured with high TROP2-expressing cells. Fluorescent anti–phosphorylated histone H2AX antibody was used to demonstrate double-strand DNA breaks. Antibody-dependent cell cytotoxicity was tested in vitro using 4-hour chromium release assays. In vivo activity of Dato-DXd was evaluated against TROP2-positive endometrial cancer xenografts. A total of 78% (seven of nine) of the primary endometrial cancer cell lines expressed TROP2. Endometrial cancer cell lines expressing TROP2 were significantly more sensitive to Dato-DXd compared with control ADC. Dato-DXd–exposed, TROP2-positive endometrial cancer demonstrated increased double-strand DNA breaks compared with non-binding conjugate exposure. Dato-DXd mediated antibody-dependent cell cytotoxicity against TROP2-positive cell lines and induced significant bystander killing of TROP2-negative tumors when admixed with TROP2-positive tumors. In vivo, injection of Dato-DXd was well tolerated and demonstrated impressive tumor growth inhibition against chemotherapy-resistant poorly differentiated endometrial cancer xenografts (P < 0.0001). In conclusion, Dato-DXd is a novel ADC with remarkable preclinical activity against poorly differentiated endometrial cancer cell lines overexpressing TROP2. Clinical trials with Dato-DXd in patients with recurrent endometrial cancer are warranted. Significance: Targeted treatment of aggressive forms of endometrial cancer using the biomarker TROP2 is a significant opportunity for the development of treatments when patients are resistant to other lines of treatment. Here, we present data showing preclinical evidence of effectiveness of this biomarker-targeted therapy in endometrial cancer.

Preclinical evaluation of avutometinib and defactinib in high‐grade endometrioid endometrial cancer

AbstractBackgroundHigh‐grade endometrial cancers (EAC) are aggressive tumors with a high risk of progression after treatment. As EAC may harbor mutations in the RAS/MAPK pathways, we evaluated the preclinical in vitro and in vivo efficacy of avutometinib, a RAF/MEK clamp, in combination with the focal adhesion kinase (FAK) inhibitors defactinib or VS‐4718, against multiple primary EAC cell lines and xenografts.MethodsWhole‐exome sequencing (WES) was used to evaluate the genetic landscape of five primary EAC cell lines. The in vitro activity of avutometinib and defactinib as single agents and in combination was evaluated using cell viability, cell cycle, and cytotoxicity assays. Mechanistic studies were performed using Western blot assays while in vivo experiments were completed in UTE10 engrafted mice treated with either vehicle, avutometinib, VS‐4718, or their combination through oral gavage.ResultsWES results demonstrated multiple EAC cell lines to harbor genetic derangements in the RAS/MAPK pathway including KRAS/PTEN/PIK3CA/BRAF/ARID1A, potentially sensitizing to FAK and RAF/MEK inhibition. Five out of five of the EAC cell lines demonstrated in vitro sensitivity to FAK and/or RAF/MEK inhibition. By Western blot assays, exposure of EAC cell lines to defactinib, avutometinib, and their combination demonstrated decreased phosphorylated FAK (p‐FAK) as well as decreased p‐MEK and p‐ERK. In vivo the combination of avutometinib/VS‐4718 demonstrated superior tumor growth inhibition compared to single‐agent treatment and controls starting at Day 9 (p < 0.02 and p < 0.04) in UTE10 xenografts.ConclusionsAvutometinib, defactinib, and to a larger extent their combinations, demonstrated promising in vitro and in vivo activity against EAC cell lines and xenografts. These preclinical data support the potential clinical evaluation of this combination in high‐grade EAC patients.

Antibody–Drug Conjugates (ADC) in HER2/neu-Positive Gynecologic Tumors

Antibody–drug conjugates (ADCs) are a new class of targeted anti-cancer therapies that combine a monoclonal tumor-surface-receptor-targeting antibody with a highly cytotoxic molecule payload bonded through specifically designed cleavable or non-cleavable chemical linkers. One such tumor surface receptor is human epidermal growth factor 2 (HER2), which is of interest for the treatment of many gynecologic tumors. ADCs enable the targeted delivery of a variety of cytotoxic therapies to tumor cells while minimizing delivery to healthy tissues. This review summarizes the existing literature about HER2-targeting ADC therapies approved for use in gynecologic malignancies, relevant preclinical studies, strategies to address ADC resistance, and ongoing clinical trials.

Integrated mutational landscape analysis of endometrial stromal sarcoma

Endometrial stromal sarcoma (ESS) is a rare uterine malignancy with limited treatment options. We performed integrated whole-genome, whole-exome, and transcriptome sequencing on 80 ESS tumors, comprising 32 low-grade (LG) and 48 high-grade (HG) tumors, to characterize their genetic landscape. The overall mutation burden was modest, with no significant difference between grades; however, we identified six hypermutated cases (7.5%) harboring POLE or mismatch repair mutations, genomic features predictive of immunotherapy response. We identified focal RAD54B amplifications in 15 tumors (18.8%), leading to elevated RAD54B expression and significantly shorter survival. This establishes RAD54B as an oncogenic driver in ESS. Known tumor suppressors (PTEN, TP53) were frequently mutated in HG-ESS but rare in LG-ESS, highlighting distinct grade-specific drivers of malignancy. HG-ESS exhibited widespread chromosomal gains, frequent loss of cell-cycle regulators (RB1, CDKN2A), and numerous private gene fusions arising from complex DNA rearrangements. In contrast, LG-ESS were defined by canonical fusions (e.g., JAZF1–SUZ12 ) and co-occurring deletions in metabolic regulator genes (TSC2, STK11). Finally, in an activating NRAS-mutant (p.Q61R) HG-ESS xenograft, the combination of MEK and FAK inhibition dramatically suppressed tumor growth and prolonged survival, highlighting a promising targeted treatment strategy. Overall, our comprehensive analysis defines the molecular basis of ESS and provides a strong preclinical rationale for precision therapies in this aggressive cancer.

Long-Term Follow-Up and Overall Survival in NRG258, a Randomized Phase III Trial of Chemoradiation Versus Chemotherapy for Locally Advanced Endometrial Carcinoma

This randomized phase III trial aimed to determine whether treatment with cisplatin and volume-directed radiation followed by carboplatin and paclitaxel for four cycles (chemoradiotherapy [C-RT]) increased recurrence-free survival (RFS) and overall survival (OS) when compared with carboplatin and paclitaxel for six cycles (chemotherapy [CT]) in locally advanced endometrial cancer (UC). Previously reported results showed that C-RT did not improve RFS compared with CT. Here we report the final OS analysis. Patients with International Federation of Gynecology and Obstetrics (FIGO) 2009 stage III-IVA UC or stage I/II serous or clear cell UC and positive cytology were enrolled. The primary objective was RFS. Secondary objectives were OS, toxicity, and quality of life. Cumulative probabilities of OS were estimated using the Kaplan-Meier method. Subgroup analyses of treatment effect for FIGO stage, age, race, gross residual disease, histology, lymph-vascular space invasion, and body mass index were performed. In total, 813 patients were randomly assigned (407 C-RT and 406 CT). The median follow-up was 112 months. Median OS was not achieved in either arm. The stratified hazard ratio for death comparing C-RT versus CT was 1.05 (95% CI, 0.82 to 1.34, log-rank two-sided P value = .72). None of the factors analyzed predicted OS benefit from C-RT. Although C-RT reduced the rate of local recurrence compared with CT, it did not increase OS or RFS in stage III/IVA UC.

Efficacy and Safety of Sacituzumab Govitecan in Patients With Advanced Solid Tumors (TROPiCS-03): Analysis in Patients With Advanced Endometrial Cancer

PURPOSE Patients with advanced endometrial cancer (EC) who progress on or after platinum-based therapy and immunotherapy have poor prognosis. We report efficacy and safety of sacituzumab govitecan (SG), a trophoblast cell-surface antigen 2 (Trop-2)–directed antibody-drug conjugate, in patients with advanced EC. METHODS TROPiCS-03 (ClinicalTrials.gov identifier: NCT03964727 ) is a multicohort, open-label, phase II basket study in patients with metastatic solid tumors. Eligible patients in the EC cohort received SG 10 mg/kg once on days 1 and 8 every 3 weeks. Primary end point was objective response rate (ORR) by investigator's assessment per RECIST v1.1. Secondary end points included clinical benefit rate (CBR; complete and partial response, and stable disease ≥6 months), duration of response (DOR), and progression-free survival (PFS) per investigator assessment, overall survival, and safety. Trop-2 expression of archival or baseline tumor specimens was analyzed by immunohistochemistry. RESULTS At data extraction date, 41 patients were enrolled. Median follow-up was 5.8 months (range, 0.7-19.3); median previous therapies was three (range, 1-6); and 85% of patients received previous chemotherapy and immunotherapy. ORR was 22% (95% CI, 11 to 38); CBR was 32% (95% CI, 18 to 48). Median DOR was 8.8 months (95% CI, 2.8 to not estimable); median PFS was 4.8 months (95% CI, 2.8 to 9.8). Trop-2 exploratory analysis was conducted retrospectively for 39 patients. Tumor Trop-2 protein was highly expressed in EC, showing limited correlation with efficacy. Grade ≥3 treatment-related adverse events (TRAEs) occurred in 73% of patients. Study drug discontinuation due to TRAEs was 5%. Two deaths occurred, deemed unrelated to SG. CONCLUSION Findings from TROPiCS-03 showed encouraging efficacy of SG with a manageable toxicity profile in a heavily pretreated population with advanced EC. Safety findings were consistent with the known SG safety profile.

Distinct Mechanisms of Mismatch-Repair Deficiency Delineate Two Modes of Response to Anti–PD-1 Immunotherapy in Endometrial Carcinoma

Abstract Mismatch repair–deficient (MMRd) cancers have varied responses to immune-checkpoint blockade (ICB). We conducted a phase II clinical trial of the PD-1 inhibitor pembrolizumab in 24 patients with MMRd endometrial cancer (NCT02899793). Patients with mutational MMRd tumors (6 patients) had higher response rates and longer survival than those with epigenetic MMRd tumors (18 patients). Mutation burden was higher in tumors with mutational MMRd compared with epigenetic MMRd; however, within each category of MMRd, mutation burden was not correlated with ICB response. Pretreatment JAK1 mutations were not associated with primary resistance to pembrolizumab. Longitudinal single-cell RNA-seq of circulating immune cells revealed contrasting modes of antitumor immunity for mutational versus epigenetic MMRd cancers. Whereas effector CD8+ T cells correlated with regression of mutational MMRd tumors, activated CD16+ NK cells were associated with ICB-responsive epigenetic MMRd tumors. These data highlight the interplay between tumor-intrinsic and tumor-extrinsic factors that influence ICB response. Significance: The molecular mechanism of MMRd is associated with response to anti–PD-1 immunotherapy in endometrial carcinoma. Tumors with epigenetic MMRd or mutational MMRd are correlated with NK cell or CD8+ T cell–driven immunity, respectively. Classifying tumors by the mechanism of MMRd may inform clinical decision-making regarding cancer immunotherapy. This article is highlighted in the In This Issue feature, p. 247

Immune checkpoint inhibitors for recurrent endometrial cancer

Endometrial cancer (EC) is the most common gynecologic malignancy. Outcomes for patients with advanced and/or recurrent disease have been modest with the use of chemotherapy. The approval of immune checkpoint inhibitors targeting PD-1 has recently revolutionized human cancer treatment. Recent trials with immune checkpoint inhibitors used alone or in combination with other agents, have demonstrated remarkable efficacy in the treatment of the all-comers EC patient population. In this article, we review major clinical trials on PD-1/PD-L1 inhibitors in advanced and recurrent EC and discuss the response rates of these agents in the context of their genomic background. Immune checkpoint inhibitors have significantly changed our approach to the treatment of advanced/recurrent EC. Single agent anti-PD-1 regimens are highly effective in MMRd/MSI-H patients, but their clinical efficacy remains modest in MMR proficient/TMB low EC patients. Combination regimens that can decrease the tumor microenvironments immunosuppression and increase tumor immunogenicity represent a viable treatment option to broaden the activity of immune checkpoint inhibitors in advanced/recurrent EC patients. An increased understanding of the biomarkers of response and the molecular mechanisms of resistance to immune checkpoint inhibitors remains key for the next advancement of the field.

A phase 2 evaluation of pembrolizumab for recurrent Lynch‐like versus sporadic endometrial cancers with microsatellite instability

BackgroundMicrosatellite instability–high (MSI‐H)/mismatch repair deficiency (dMMR) is a biomarker for responses to immune checkpoint inhibitors (ICIs). Whether mechanisms underlying microsatellite instability alter responses to ICIs is unclear. This article reports data from a prospective phase 2 pilot study of pembrolizumab in patients with recurrent MSI‐H endometrial cancer (EC) analyzed by whole exome sequencing (WES) and potential mechanisms of primary/secondary ICI resistance (NCT02899793).MethodsPatients with measurable MSI‐H/dMMR EC confirmed by polymerase chain reaction/immunohistochemistry were evaluated by WES and received 200 mg of pembrolizumab every 3 weeks for ≤2 years. The primary end point was the objective response rate (ORR). Secondary end points included progression‐free survival (PFS) and overall survival (OS).ResultsTwenty‐five patients (24 evaluable) were treated. Six patients (25%) harbored Lynch/Lynch‐like tumors, whereas 18 (75%) had sporadic EC. The tumor mutation burden was higher in Lynch‐like tumors (median, 2939 mutations/megabase [Mut/Mb]; interquartile range [IQR], 867‐5108 Mut/Mb) than sporadic tumors (median, 604 Mut/Mb; IQR, 411‐798 Mut/Mb; P = .0076). The ORR was 100% in Lynch/Lynch‐like patients but only 44% in sporadic patients (P = .024). The 3‐year PFS and OS proportions were 100% versus 30% (P = .017) and 100% versus 43% (P = .043), respectively.ConclusionsThis study suggests prognostic significance of Lynch‐like cancers versus sporadic MSI‐H/dMMR ECs for ORR, PFS, and OS when patients are treated with pembrolizumab. Larger confirmatory studies in ECs and other MSI‐H/dMMR tumors are necessary. Defective antigen processing/presentation and deranged induction in interferon responses serve as mechanisms of resistance in sporadic MSI‐H ECs. Oligoprogression in MSI‐H/dMMR patients appears salvageable with surgical resection and/or local treatment and the continuation of pembrolizumab off study. Clinical studies evaluating separate MSI‐H/dMMR EC subtypes treated with ICIs are warranted.

Evaluation of treatment effects in patients with endometrial cancer and POLE mutations: An individual patient data meta‐analysis

BACKGROUNDEndometrial cancers (ECs) with somatic mutations in DNA polymerase epsilon (POLE) are characterized by unfavorable pathological features, which prompt adjuvant treatment. Paradoxically, women with POLE‐mutated EC have outstanding clinical outcomes, and this raises concerns of overtreatment. The authors investigated whether favorable outcomes were independent of treatment.METHODSA PubMed search for POLE and endometrial was restricted to articles published between March 1, 2012, and March 1, 2018, that provided individual patient data (IPD), adjuvant treatment, and survival. Following the Preferred Reporting Items for Systematic Review and Meta‐Analysis (PRISMA) reporting guidelines for IPD, the authors used univariate and multivariate one‐stage meta‐analyses with mixed effects Cox models (random effects for study cohorts) to infer the associations of treatment, traditional prognostic factors, and outcome, which was defined as the time from first diagnosis to any adverse event (progression/recurrence or death from EC).RESULTSThree hundred fifty‐nine women with POLE‐mutated EC were identified; 294 (82%) had pathogenic mutations. Worse outcomes were demonstrated in patients with nonpathogenic POLE mutations (hazard ratio, 3.42; 95% confidence interval, 1.47‐7.58; log‐rank P < .01). Except for stage (P < .01), traditional prognosticators were not associated with progression/recurrence or death from disease. Adverse events were rare (11 progressions/recurrences and 3 disease‐specific deaths). Salvage rates in patients who experienced recurrence were high and sustained, with 8 of 11 alive without evidence of disease (range, 5.5‐14.2 years). Adjuvant treatment was not associated with outcome.CONCLUSIONSClinical outcomes for ECs with pathogenic POLE mutations are not associated with most traditional risk parameters, and patients do not appear to benefit from adjuvant therapy. The observed low rates of recurrence/progression and the high and sustained salvage rates raise the possibility of safely de‐escalating treatment for these patients.LAY SUMMARY Ten percent of all endometrial cancers have mutations in the DNA repair gene DNA polymerase epsilon (POLE). Women who have endometrial cancers with true POLE mutations experience almost no recurrences or deaths from their cancer even when their tumors appear to have very unfavorable characteristics. Additional therapy (radiation and chemotherapy) does not appear to improve outcomes for women with POLE‐mutated endometrial cancer, and this supports the move to less therapy and less associated toxicity. Diligent classification of endometrial cancers by molecular features provides valuable information to inform prognosis and to direct treatment/no treatment.

Stage III uterine serous carcinoma: modern trends in multimodality treatment

To examine outcomes in a modern treatment era for stage III uterine serous carcinoma (USC). Fifty women were retrospectively identified as 2009 International Federation of Gynecology and Obstetrics stage III USC patients who received radiotherapy (RT) at our institution between 1/2003-5/2018. The patients were divided into 2 cohorts: 20 in the early era (2003-2010) and 30 in the modern era (2011-2018). Patient characteristics were compared using χ² tests for categorical variables and t-tests for continuous variables. Recurrence free survival (RFS) and overall survival (OS) were analyzed with Kaplan-Meier estimates, the log-rank test, and Cox proportional hazards. The modern era differed from the early era in the increased use of volume-directed external beam RT (EBRT) as opposed to vaginal brachytherapy (VB) alone (33.3% vs 5.0%, p=0.048), minimally invasive surgery (56.7% vs. 25%, p=0.027), sentinel node sampling (26.7% vs. 0%, p=0.012), computed tomography imaging in the perioperative period (63.3% vs. 30%, p=0.044), and human epidermal growth factor receptor 2/ Modern era treatment was associated with improved RFS and OS in patients with stage III USC. Regional nodal recurrences were significantly reduced in patients who received EBRT.

Sacituzumab govitecan, an antibody‐drug conjugate targeting trophoblast cell‐surface antigen 2, shows cytotoxic activity against poorly differentiated endometrial adenocarcinomas in vitro and in vivo

Endometrial cancer is the most common gynecologic malignancy in developed countries. The antibody–drug conjugate (ADC) sacituzumab govitecan (SG) targets trophoblast cell‐surface antigen‐2 (Trop‐2) – a cell‐surface glycoprotein highly expressed in many epithelial tumors – and delivers the active metabolite of irinotecan SN‐38 to Trop‐2‐positive tumor cells. We evaluated Trop‐2 expression in endometrial endometrioid carcinoma (EC) tissues and the activity of SG against primary poorly differentiated EC cell lines and xenografts. Trop‐2 expression was assessed in 143 formalin‐fixed–paraffin‐embedded tumors and seven primary tumor cell lines by immunohistochemistry and flow cytometry, respectively. Cell viability of primary tumor cell lines was assessed following exposure to SG, or control antibodies. Antibody‐dependent cell cytotoxicity (ADCC) against Trop‐2‐positive and Trop‐2‐negative EC cell lines was measured in vitro using 4‐h chromium release assays. A Trop‐2‐positive EC xenograft model was used to determine the in vivo activity of SG. Moderate‐to‐strong staining was detected in 84% (120/143) of EC samples, whereas 43% (3/7) of the primary EC cell lines tested overexpressed Trop‐2. EC cell lines overexpressing Trop‐2 were significantly more sensitive to SG compared to control ADC (P = 0.014 and P = 0.005). Both SG and the unconjugated parental antibody hRS7 mediated high ADCC against Trop‐2‐positive cell lines. Moreover, SG induced significant bystander killing of Trop‐2‐negative tumors cocultured with Trop‐2‐positive tumors. In the xenograft model, intravenous administration of SG twice weekly for three weeks was well tolerated and demonstrated impressive tumor growth inhibition against poorly differentiated, chemotherapy‐resistant EC xenografts (P = 0.011). In summary, SG is a novel ADC with remarkable preclinical activity against poorly differentiated EC cell lines overexpressing Trop‐2. These findings warrant future clinical trials.

Randomised phase II trial of weekly ixabepilone ± biweekly bevacizumab for platinum-resistant or refractory ovarian/fallopian tube/primary peritoneal cancer

Abstract Background This multi-center RP2 study assessed activity/safety of ixabepilone + bevacizumab compared to ixabepilone in platinum-resistant/refractory ovarian/fallopian tube/primary peritoneal cancer. Additional objectives were to examine the role of prior bevacizumab and taxanes, and explore class III-ß-tubulin (TUBB3) as a predictive biomarker. Methods Participants were randomised to receive ixabepilone 20 mg/m 2 days 1, 8, 15 with (IXA + BEV) or without (IXA) bevacizumab 10 mg/kg days 1, 15 every 28 days. Patients were stratified by prior BEV. The primary endpoint was PFS. OS, safety, and ORR served as secondary endpoints. Results Among 76 evaluable patients who received IXA + BEV ( n  = 39) compared to IXA ( n  = 37), the ORR was 33% ( n  = 13) versus 8% ( n  = 3)( P  = 0.004), durable at 6 months in 37% ( n  = 14) and 3% ( n  = 1) ( P  < 0.001). BEV significantly improved PFS (median:5.5 vs 2.2 months, HR = 0.33, 95%CI 0.19–0.55, P  < 0.001) and OS (median:10.0 vs 6.0 months, HR = 0.52, 95%CI 0.31–0.87, P  = 0.006). Both regimens were well-tolerated. TUBB3 expression did not predict response. Subgroup analyses revealed minimal effect of prior BEV or taxane resistant/refractory status on response to IXA + BEV. Conclusions IXA + BEV is a well-tolerated, effective combination for platinum/taxane-resistant ovarian cancer that extends PFS and likely OS relative to IXA monotherapy. Prior receipt of BEV should not preclude the use of IXA + BEV. TUBB3 is not a predictive biomarker. Clinical trial registration NCT3093155.

Preclinical Efficacy of the Estrogen Receptor Degrader Fulvestrant in Combination with RAF/MEK Clamp Avutometinib and FAK Inhibitor in a Low-Grade Serous Ovarian Cancer Animal Model with Acquired Resistance to Chemotherapy and Aromatase Inhibitor

Low-grade-serous ovarian carcinomas (LGSOC) are rare tumors characterized by a high recurrence rate and limited treatment options. Most LGSOC are estrogen receptor (ER)-positive and demonstrate alterations in the RAS/MAPK pathway. Avutometinib is a dual RAF/MEK clamp, whereas defactinib and VS-4718 are focal adhesion kinase (FAK) inhibitors. Fulvestrant is an ER antagonist/degrader. We assessed the preclinical efficacy of fulvestrant, avutometinib + VS-4718 (FAKi), and the triple combination in a chemotherapy/aromatase inhibitor-resistant LGSOC patient-derived tumor xenograft (PDX) model. Tissue obtained from a LGSOC patient wild-type for KRAS/NRAS/BRAF mutations in progression after chemotherapy/anastrozole was transplanted into female CB17/lcrHsd-Prkdc/SCID mice (PDX-OVA(K)250). The animals were treated with either saline/control, fulvestrant, avutometinib/FAKi, or the triple combination of avutometinib/FAKi/fulvestrant. Avutometinib and FAKi were given five-days on and two-days off through oral gavage. Fulvestrant was administered subcutaneously weekly. Mechanistic studies were performed ex vivo using Western blot assays. Animals treated with the triple combination demonstrated stronger tumor growth inhibition compared to all the other experimental groups including control/saline (p < 0.001), single-agent fulvestrant (p = 0.04 from day eight and onwards), and avutometinib/FAKi (p = 0.02 from day 18). Median survival for mice treated with saline/control was 29 days while mice in all other experimental groups were alive at day 60 (p < 0.0001). Treatment was well tolerated across all experimental treatments. By Western blot, exposure of OVA(K)250 to the triple combination demonstrated a decrease in phosphorylated MEK (p-MEK) and p-ERK levels. The addition of fulvestrant to avutometinib/FAKi is well tolerated in vivo and enhances the antitumor activity of avutometinib/FAKi in a LGSOC-PDX model with acquired resistance to chemotherapy/aromatase inhibitors. These results support the clinical evaluation of avutometinib/defactinib in combination with fulvestrant or an aromatase inhibitor in patients with recurrent LGSOC.

HER2/neu as a Signaling and Therapeutic Marker in Uterine Serous Carcinoma

Research into aggressive gynecologic cancers such as uterine serous carcinoma (USC) has recently evolved from chemotherapy to the development of drugs targeting specific biomarkers differentially expressed/active in tumor cells. One such target is HER2/neu, which plays an important role in the coordination of cell growth and differentiation. Importantly, when overexpressed and/or amplified in tumor cells, the downstream tyrosine kinase of HER2/neu becomes constitutively activated, causing dysregulated gene transcription. In breast cancer patients, HER2/neu has been successfully utilized for many years as a target for multiple monoclonal antibodies and more recently antibody–drug conjugates (ADCs). Use in gynecologic malignancies has been slower, however, due to recently identified unique characteristics of HER2/neu protein expression and gene amplification in biologically aggressive tumors such as USC including its major heterogeneity and lack of apical staining when compared to breast cancer. Accordingly, the use of optimal testing algorithms for HER2/neu status in patients with USC may have important implications for the development of novel, effective, and targeted treatment modalities against this lethal variant of endometrial cancer. In this review, we discuss HER2/neu gene expression in USC, evaluate the efficacy of HER2/neu-directed therapies in both preclinical and clinical settings, and discuss possible mechanisms of resistance to HER2/neu targeting agents.

Preclinical Activity of Datopotamab Deruxtecan, an Antibody–Drug Conjugate Targeting Trophoblast Cell-Surface Antigen 2, in Uterine Serous Carcinoma

Abstract Uterine serous carcinoma (USC) is a rare subset of endometrial cancer with a poor prognosis and high recurrence rate. Datopotamab deruxtecan (Dato-DXd) is a novel antibody–drug conjugate (ADC). The objective of this study was to evaluate the preclinical activity of Dato-DXd in USC in vitro against primary USC cell lines with various trophoblast cell-surface antigen 2 (TROP2) expression and in vivo in TROP2-overexpressing cell line–derived mice xenografts. USC primary tumor cell lines were treated with Dato-DXd and a control ADC (CTL ADC) to evaluate cell viability following exposure. Antibody-dependent cell-mediated cytotoxicity against TROP2-overexpressing and -nonexpressing cell lines was evaluated using a 4-hour chromium release assay. USC xenografts in mice were treated with Dato-DXd, CTL ADC, datopotamab, and vehicle to assess the in vivo effects via retro-orbital Dato-DXd administration. We found USC cell lines with TROP2 overexpression to be significantly more sensitive to killing induced by Dato-DXd compared with CTL ADC in vitro (e.g., IC50: 0.11 µmol/L vs. 30.07 µmol/L, P = 0.0074 and 0.11 µmol/L vs. 48.95 µmol/L, P = 0.0127, respectively). Dato-DXd induced antibody-dependent cell-mediated cytotoxicity in the presence of peripheral blood lymphocytes from healthy donors. TROP2-nonexpressing cell lines demonstrated minimal killing by Dato-DXd; however, when admixed with TROP2-overexpressing cells, a significant bystander effect was appreciated. In vivo, mice xenografts overexpressing TROP2 treated with Dato-DXd demonstrated tumor growth suppression and longer overall survival compared with CTL ADC–treated xenografts. These data demonstrate Dato-DXd to be highly active against TROP2-overexpressing USC in vitro and in vivo. Our preclinical activity results warrant future clinical trials for patients with advanced or recurrent USC. Significance: Targeted treatment of USC using the biomarker TROP2 represents a significant opportunity for further treatment options for patients already resistant to other lines of treatment. In this study, we present data showing preclinical evidence of effectiveness of this biomarker-targeted therapy in USC.

Integrated mutational landscape analysis of poorly differentiated high-grade neuroendocrine carcinoma of the uterine cervix

High-grade neuroendocrine cervical cancers (NETc) are exceedingly rare, highly aggressive tumors. We analyzed 64 NETc tumor samples by whole-exome sequencing (WES). Human papillomavirus DNA was detected in 65.6% (42/64) of the tumors. Recurrent mutations were identified in PIK3CA, KMT2D/MLL2, K-RAS, ARID1A, NOTCH2, and RPL10. The top mutated genes included RB1, ARID1A, PTEN, KMT2D / MLL2, and WDFY3, a gene not yet implicated in NETc. Somatic CNV analysis identified two copy number gains (3q27.1 and 19q13.12) and five copy number losses (1p36.21/5q31.3/6p22.2/9q21.11/11p15.5). Also, gene fusions affecting the ACLY-CRHR1 and PVT1-MYC genes were identified in one of the eight samples subjected to RNA sequencing. To resolve evolutionary history, multiregion WES in NETc admixed with adenocarcinoma cells was performed (i.e., mixed-NETc). Phylogenetic analysis of mixed-NETc demonstrated that adenocarcinoma and neuroendocrine elements derive from a common precursor with mutations typical of adenocarcinomas. Over one-third (22/64) of NETc demonstrated a mutator phenotype of C > T at CpG consistent with deficiencies in MBD4 , a member of the base excision repair (BER) pathway. Mutations in the PI3K/AMPK pathways were identified in 49/64 samples. We used two patient-derived-xenografts (PDX) (i.e., NET19 and NET21) to evaluate the activity of pan-HER (afatinib), PIK3CA (copanlisib), and ATR (elimusertib) inhibitors, alone and in combination. PDXs harboring alterations in the ERBB2/PI3K/AKT/mTOR/ATR pathway were sensitive to afatinib, copanlisib, and elimusertib ( P < 0.001 vs. controls). However, combinations of copanlisib/afatinib and copanlisib/elimusertib were significantly more effective in controlling NETc tumor growth. These findings define the genetic landscape of NETc and suggest that a large subset of these highly lethal malignancies might benefit from existing targeted therapies.

Randomized Phase II Trial of Imiquimod with or without 9-Valent HPV Vaccine versus Observation in Patients with High-grade Pre-neoplastic Cervical Lesions (NCT02864147)

Abstract Purpose: We report the results of a randomized phase II trial of imiquimod, a topical immune-response modulator versus imiquimod plus a 9-valent human papillomavirus (HPV) vaccine (9vHPV) versus clinical surveillance in cervical intraepithelial neoplasia (CIN2/3) patients. Patients and Methods: We randomly allocated 133 patients with untreated CIN2/3 in equal proportions to a 4-month treatment with self-applied vaginal suppositories containing imiquimod (Arm B) or imiquimod plus a 9vHPV (Arm C) versus clinical surveillance (Arm A). The main outcome was efficacy, defined as histologic regression to CIN1 or less. Secondary outcomes were HPV clearance and tolerability. Exploratory objectives included the comparison of cervical CD4/CD8 T-cell infiltration at baseline, mid-study, and posttreatment by flow cytometry among study arms. Results: Of the 114 evaluable patients 77% and 23% harbored CIN2 and CIN3, respectively. Regression to CIN1 or less was observed in 95% of patients in the imiquimod group (Arm B) compared with 79% in the control/surveillance (Arm A); P = 0.043 and 84% in the imiquimod+9vHPV group (Arm C; P = 0.384 vs. Arm A). Neither of the treatment-arm differences from Arm A reached the prespecified α = 0.025 significance level. No significant differences were noted in the secondary outcome of rate of HPV clearance. The number of tissue-resident memory CD4/CD8 T cells in cytobrush samples demonstrated a >5-fold increase in Arm B/imiquimod when compared with Arm A/surveillance (P < 0.01). In contrast, there was no significant difference in T-cell responses among participants in Arm C when compared with Arm A. Imiquimod treatment was well tolerated. Conclusions: Although imiquimod induced a higher regression to CIN1 or less and significant increases in CD4/CD8 T cells infiltrating the cervix, it did not meet its prespecified statistical outcome for efficacy. A higher regression rate than expected was observed in the surveillance arm of this prospective trial. Future clinical trials with imiquimod targeting CIN3 patients are warranted.

Phase II evaluation of nivolumab in the treatment of persistent or recurrent cervical cancer (NCT02257528/NRG-GY002)

Patients with persistent/recurrent cervical cancer following platinum-based chemotherapy have limited therapeutic options. The Gynecologic-Oncology-Group conducted a phase II trial to assess efficacy and tolerability of nivolumab, an immune checkpoint inhibitor, in persistent/recurrent cervical carcinoma. Key eligibility criteria included persistent/recurrent cervical cancer, failure of prior systemic therapy and ECOG PS 0-1. Nivolumab 3 mg/kg was given IV every 2 wk. until disease progression or intolerable toxicity. Response was assessed every 8 wk. for 6 months and every 12 wk. thereafter. The primary endpoints were objective response as assessed by RECIST 1.1. The study used a 2-stage group sequential design. PD-L1 expression was evaluated in tumor specimens by immunohistochemistry (IHC) using a combined-positive-score (CPS) cutoff of ≥1%. Of 26 enrolled patients with persistent/recurrent cervical cancer, 25 were evaluable for response/toxicity with a median age of 45. 36% had ECOG PS of 1, and 100% had received one prior systemic chemotherapy regimen. PD-L1 expression (≥1%) was identified in 77.3% of tumor samples. As of 03/05/19, all patients were off study treatment; median follow-up for survival status was 32 months (range, 2-41.5). There were 21 (84%) patients with a treatment-related adverse event (TRAE) and most were grades 1-2. Six (24%) patients had grade 3 TRAEs with 1 discontinuing nivolumab due to hepatic toxicity. No grade 5 TRAEs occurred, and 2 patients had grade 4 TRAEs. One confirmed partial response (4%; 90% CI, 0.4%-22.9%), duration of response 3.8 months. Thirty-six percent of patients had stable disease (SD) (9/25; 90% CI, 20.2%-54.4%); the median duration of SD was 5.7 months (range, 3.5-12.7). Estimated PFS and OS at 6 months were 16% and 78.4%, respectively. Single agent nivolumab exhibited low antitumor activity and an acceptable safety profile in patients with persistent/recurrent cervical cancer previously treated with platinum-based chemotherapy.

Efficacy and Safety of Avutometinib ± Defactinib in Recurrent Low-Grade Serous Ovarian Cancer: Primary Analysis of ENGOT-OV60/GOG-3052/RAMP 201

PURPOSE This study evaluated the efficacy and safety of avutometinib (rapidly accelerated fibrosarcoma/mitogen-activated extracellular signal-regulated kinase [MEK] clamp) alone or in combination with defactinib (focal adhesion kinase inhibitor) in patients with recurrent low-grade serous ovarian cancer (LGSOC). METHODS In this phase II, open-label study, patients with recurrent, measurable LGSOC after ≥1 line of platinum chemotherapy were stratified by tumor Kirsten rat sarcoma virus homolog ( KRAS ) mutation status and randomly assigned to oral avutometinib 4.0 mg two times per week monotherapy or avutometinib 3.2 mg two times per week in combination with oral defactinib 200 mg two times per day. The combination was selected as the go-forward regimen for expansion. The primary end point was objective response rate (ORR) by blinded independent central review. RESULTS A total of 115 patients received the go-forward combination regimen. Patients had a median of 3 (range, 1-9) prior lines of therapy, including hormonal (86%), bevacizumab (51%), and MEK inhibitor (22%). Confirmed ORR was 31% (95% CI, 23% to 41%) with a median duration of response of 31.1 months (95% CI, 14.8 to 31.1). ORR was 44% in KRAS- mutant and 17% in KRAS wild-type cohorts. The median progression-free survival was 12.9 months (95% CI, 10.9 to 20.2) overall and 22.0 months (95% CI, 11.1 to 36.6) and 12.8 months (95% CI, 7.4 to 18.4) in KRAS- mutant and wild-type cohorts, respectively. The most frequent grade ≥3 treatment-related adverse events (AEs) were elevated creatine phosphokinase (24%), diarrhea (8%), and anemia (5%). Ten percent of patients discontinued because of AEs. CONCLUSION The efficacy and safety profile of avutometinib in combination with defactinib support this combination as a potential standard of care for recurrent LGSOC. A randomized phase 3 study of avutometinib and defactinib versus investigator's choice of therapy for women with recurrent LGSOC is currently enrolling (RAMP301; ClinicalTrials.gov identifier: NCT06072781 ).

A Study of Avutometinib (VS-6766) + Defactinib (VS-6063) in Recurrent Low-Grade Serous Ovarian Cancer

This study will assess the safety and efficacy of avutometinib (VS-6766) in combination with defactinib versus Investigator's choice of treatments (ICT) in subjects with recurrent LGSOC who have progressed on a prior platinum-based therapy.

Nivolumab-ipilimumab and Chemoradiation for Cervical Cancer

A total of 112 patients with locally advanced cervical cancer will be randomized 1:1 to standard therapy with cisplatin-based chemoradiation or nivolumab-ipilimumab induction followed by cisplatin-based chemoradiation. The primary outcome will be 3-year disease-free survival.

Zimberelimab Combined With Concurrent Radiotherapy and Chemotherapy for Locally Advanced Cervical Cancer

This is a prospective, single arm, phase II clinical study on the treatment of locally advanced cervical cancer (Ⅱ B to Ⅳ a) with Zimberelimab combined with concurrent radiotherapy and chemotherapy.

A Study of Avutometinib (VS-6766) v. Avutometinib (VS-6766) + Defactinib in Recurrent Low-Grade Serous Ovarian Cancer With and Without a KRAS Mutation

This study will assess the safety and efficacy of avutometinib (VS-6766) monotherapy and in combination with defactinib in subjects with recurrent Low-Grade Serous Ovarian Cancer (LGSOC)

Evaluation of Weekly Ixabepilone With or Without Biweekly Bevacizumab

This is a randomized, two-arm, open-label Phase II multicenter study designed to examine the effects of adding bevacizumab to ixabepilone for the treatment of patients who have recurrent or persistent platinum-resistant/refractory epithelial (non-mucinous) ovarian, fallopian tube, or primary peritoneal cancer. Its primary objective is to assess whether adding bevacizumab to ixabepilone improves progression-free survival in its target population. Study participants will be stratified by (a) study site and (b) previous receipt of bevacizumab prior to randomization.

Pembrolizumab in Ultramutated and Hypermutated Endometrial Cancer

Primary Objectives: To assess the antitumor activity (proportion of objective response by RECIST 1.1 criteria) of pembrolizumab with objective tumor response in patients with persistent, recurrent or metastatic endometrial cancer harboring an ultra-mutated or hyper-mutated (MMR gene-defective) phenotype identified by next generation sequencing (NGS) and comprehensive genomic profiling (CGP). To determine the nature and degree of toxicity of pembrolizumab as assessed by CTCAE in patients with persistent, recurrent or metastatic endometrial carcinoma. Secondary Objective(s): To estimate the duration of progression-free survival (PFS) and overall survival (OS).

Nivolumab in Treating Patients With Persistent, Recurrent, or Metastatic Cervical Cancer

This phase II trial studies the side effects and how well nivolumab works in treating patients with cervical cancer that has grown, come back, or spread to other places in the body. Monoclonal antibodies, such as nivolumab, may block tumor growth in different ways by targeting certain cells.