Integrated mutational landscape analysis of uterine leiomyosarcomas.

Uterine leiomyosarcomas (uLMS) are aggressive tumors arising from the smooth muscle layer of the uterus. We analyzed 83 uLMS sample genetics, including 56 from Yale and 27 from The Cancer Genome Atlas (TCGA). Among them, a total of 55 Yale samples including two patient-derived xenografts (PDXs) and 27 TCGA samples have whole-exome sequencing (WES) data; 10 Yale and 27 TCGA samples have RNA-sequencing (RNA-Seq) data; and 11 Yale and 10 TCGA samples have whole-genome sequencing (WGS) data. We found recurrent somatic mutations in TP53, MED12, and PTEN genes. Top somatic mutated genes included TP53, ATRX, PTEN, and MEN1 genes. Somatic copy number variation (CNV) analysis identified 8 copy-number gains, including 5p15.33 (TERT), 8q24.21 (C-MYC), and 17p11.2 (MYOCD, MAP2K4) amplifications and 29 copy-number losses. Fusions involving tumor suppressors or oncogenes were deetected, with most fusions disrupting RB1, TP53, and ATRX/DAXX, and one fusion (ACTG2-ALK) being potentially targetable. WGS results demonstrated that 76% (16 of 21) of the samples harbored chromoplexy and/or chromothripsis. Clinically actionable mutational signatures of homologous-recombination DNA-repair deficiency (HRD) and microsatellite instability (MSI) were identified in 25% (12 of 48) and 2% (1 of 48) of fresh frozen uLMS, respectively. Finally, we found olaparib (PARPi; P = 0.002), GS-626510 (C-MYC/BETi; P < 0.000001 and P = 0.0005), and copanlisib (PIK3CAi; P = 0.0001) monotherapy to significantly inhibit uLMS-PDXs harboring derangements in C-MYC and PTEN/PIK3CA/AKT genes (LEY11) and/or HRD signatures (LEY16) compared to vehicle-treated mice. These findings define the genetic landscape of uLMS and suggest that a subset of uLMS may benefit from existing PARP-, PIK3CA-, and C-MYC/BET-targeted drugs.

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

BACKGROUND: Microsatellite 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). METHODS: Patients 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). RESULTS: Twenty-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. CONCLUSIONS: This 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.

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

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/m2 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.

Declining use of inguinofemoral lymphadenectomy in the treatment of clinically negative, pathologic node positive vulvar cancer.

PURPOSE: The management of vulvar cancer with clinically negative groin lymph nodes but with positive sentinel lymph node biopsy (SLNB) is controversial, with options including inguinofemoral lymphadenectomy (IFL) and/or adjuvant chemotherapy and radiotherapy. We used the National Cancer Database (NCDB) to examine trends in the management of clinically node negative, pathologically node positive (cN-/pN+) patients. METHODS: The NCDB was used to identify cN-/pN+ vulvar cancer patients. Demographic and clinical data were compared with chi-squared and Wilcoxon rank-sum tests. OS was analyzed with the Kaplan-Meier method and log-rank test. Cox proportional hazards regression was used to determine factors associated with OS. RESULTS: A total of 885 cN-/pN+ vulvar cancer patients were identified between 2012 and 2016, during which the rate of SLNB alone increased from 3.6% to 11.7%, while the rate of IFL +/- SLNB decreased from 89.7% to 78.1% (p < 0.05). Radiation was used in 68.5% and 64.6% of the SLNB-alone and IFL +/- SLNB cohorts, respectively, with chemoradiation in 37.1% and 33.6%, respectively. OS was not different between patients who received SLNB-alone vs. IFL +/- SLNB (p = 0.644). Receipt of chemotherapy and radiation was associated with improved OS (p < 0.001). CONCLUSIONS: Among cN-/pN+ vulvar cancer patients in the NCDB, the practice of performing IFL decreased over time as SLNB-alone became more common and the majority received radiation +/- chemotherapy. There was no difference in OS between SLNB-alone vs. IFL +/- SLNB. Patients treated with adjuvant chemoradiation had improved survival. Whether the favorable outcomes in the SLNB-alone cohort may be attributed to radiotherapy dose escalation or use of chemotherapy warrants further study.

Synergistic activity of neratinib in combination with olaparib in uterine serous carcinoma overexpressing HER2/neu.

INTRODUCTION: Uterine serous carcinoma (USC) is an aggressive variant of endometrial cancer with a poor prognosis. Approximately 30% of USC overexpress HER2/neu, a recognized target for trastuzumab in advanced/recurrent HER2/neu-positive USC. We evaluated the efficacy of the pan-c-erb inhibitor neratinib and the poly (ADP-ribose)-polymerase (PARP) inhibitor olaparib as single agents and in combination against USC cell lines and xenografts. METHODS: In-vitro cell-viability assays with olaparib, neratinib, and olaparib/neratinib were assessed using flow-cytometry based assays against a panel of USC cell lines with high and low HER2/neu expression. Homologous recombination deficiency (HRD) signatures were evaluated as described by Alexandrov et al. (Nature;2020;578:94-101) while downstream signaling affected by neratinib/olaparib exposure was assessed with immunoblotting. Efficacy of single- versus dual-agent inhibition was evaluated in-vivo using two USC-xenografts with 3+ HER2/neu expression. RESULTS: Neratinib was more potent than olaparib in suppression of in-vitro growth of HER2/neu 3+ cell lines (ARK1: p = 0.0047; ARK2: p = 0.0428) while no difference was noted against HER2/neu 1+ tumors (ARK4). Importantly, the combination of olaparib with neratinib synergistically improved tumor suppression compared to either single-agent in vitro. USC cells exposed to olaparib upregulated HER2/neu expression, while neratinib treatment increased PARP activity (ARK1: p < 0.0001; ARK2: p < 0.0001). Single-agent neratinib transiently inhibited in vivo growth of USC xenografts harboring HER2/neu gene amplification (ARK1: p < 0.05; ARK2: p < 0.05). In contrast, the combination of the two inhibitors caused a stronger and durable growth inhibition in both USC xenografts (ARK1: p < 0.05; ARK2: p < 0.05). CONCLUSION: The combination of olaparib and neratinib is active and synergistic against primary HER2/neu + USC. This combination may represent a novel therapeutic option for USC patients with HER2/neu+, homologous recombination-proficient tumors resistant to chemotherapy.

Homologous recombination deficiency (HRD) signature-3 in ovarian and uterine carcinosarcomas correlates with preclinical sensitivity to Olaparib, a poly (adenosine diphosphate [ADP]- ribose) polymerase (PARP) inhibitor.

OBJECTIVES: Carcinosarcoma (CS) of the ovary and uterus are highly aggressive malignancies associated with poor survival. Poly(ADP-ribose)-polymerase inhibitors (PARPi) are targeted agents impairing DNA repair via homologous-recombination-deficiency (HRD) mechanisms. We used whole-exome-sequencing (WES) data from a cohort of fresh tumor samples of ovarian (OCS) and uterine carcinosarcoma (UCS), primary cell lines and xenografts to investigate the role for olaparib in CSs. METHODS: WES data from 73 CS samples (48 UCS and 25 OCS) were analyzed for HRD signatures. Olaparib activity was evaluated using cell-viability, cell-cycle, apoptosis and cytotoxicity assays against primary CS cell lines. Olaparib antitumor activity was tested in vivo against HRD CS xenografts. RESULTS: Signature-3 (i.e. HRD-related signature) was identified in 60% of OCS (15 of 25) vs 25% of UCS (12 of 48) (p = 0.005). CS cell lines harboring Signature-3/HRD (3 OCS/1 UCS) were significantly more sensitive to olaparib when compared to HRP cell lines (5 UCS/1 OCS) [mean IC50 ± SEM = 2.94 µM ± 0.07 vs mean ± SEM = 23.3 µM ± 0.09, (p = 0.02), respectively]. PARPi suppressed CS cell growth through cell cycle arrest in the G2/M phase and caused more apoptosis in HRD vs HRP primary tumors (p < 0.0001). In vivo, olaparib significantly impaired HRD CS xenografts tumor growth (p = 0.0008) and increased overall animal survival (p < 0.0001). CONCLUSIONS: OCS and UCS cell lines harboring HRD signature-3 were significantly more sensitive to olaparib in vitro and in vivo when compared to HRP CS. Clinical studies with PARPi in CS patients with a dominant signature 3 (HRD-related) are warranted.

Mutational landscape of primary, metastatic, and recurrent ovarian cancer reveals c-MYC gains as potential target for BET inhibitors.

Ovarian cancer remains the most lethal gynecologic malignancy. We analyzed the mutational landscape of 64 primary, 41 metastatic, and 17 recurrent fresh-frozen tumors from 77 patients along with matched normal DNA, by whole-exome sequencing (WES). We also sequenced 13 pairs of synchronous bilateral ovarian cancer (SBOC) to evaluate the evolutionary history. Lastly, to search for therapeutic targets, we evaluated the activity of the Bromodomain and Extra-Terminal motif (BET) inhibitor GS-626510 on primary tumors and xenografts harboring c-MYC amplifications. In line with previous studies, the large majority of germline and somatic mutations were found in BRCA1/2 (21%) and TP53 (86%) genes, respectively. Among mutations in known cancer driver genes, 77% were transmitted from primary tumors to metastatic tumors, and 80% from primary to recurrent tumors, indicating that driver mutations are commonly retained during ovarian cancer evolution. Importantly, the number, mutation spectra, and signatures in matched primary-metastatic tumors were extremely similar, suggesting transcoelomic metastases as an early dissemination process using preexisting metastatic ability rather than an evolution model. Similarly, comparison of SBOC showed extensive sharing of somatic mutations, unequivocally indicating a common ancestry in all cases. Among the 17 patients with matched tumors, four patients gained PIK3CA amplifications and two patients gained c-MYC amplifications in the recurrent tumors, with no loss of amplification or gain of deletions. Primary cell lines and xenografts derived from chemotherapy-resistant tumors demonstrated sensitivity to JQ1 and GS-626510 (P = 0.01), suggesting that oral BET inhibitors represent a class of personalized therapeutics in patients harboring recurrent/chemotherapy-resistant disease.

Whole-exome sequencing of cervical carcinomas identifies activating ERBB2 and PIK3CA mutations as targets for combination therapy.

The prognosis of advanced/recurrent cervical cancer patients remains poor. We analyzed 54 fresh-frozen and 15 primary cervical cancer cell lines, along with matched-normal DNA, by whole-exome sequencing (WES), most of which harboring Human-Papillomavirus-type-16/18. We found recurrent somatic missense mutations in 22 genes (including PIK3CA, ERBB2, and GNAS) and a widespread APOBEC cytidine deaminase mutagenesis pattern (TCW motif) in both adenocarcinoma (ACC) and squamous cell carcinomas (SCCs). Somatic copy number variants (CNVs) identified 12 copy number gains and 40 losses, occurring more often than expected by chance, with the most frequent events in pathways similar to those found from analysis of single nucleotide variants (SNVs), including the ERBB2/PI3K/AKT/mTOR, apoptosis, chromatin remodeling, and cell cycle. To validate specific SNVs as targets, we took advantage of primary cervical tumor cell lines and xenografts to preclinically evaluate the activity of pan-HER (afatinib and neratinib) and PIK3CA (copanlisib) inhibitors, alone and in combination, against tumors harboring alterations in the ERBB2/PI3K/AKT/mTOR pathway (71%). Tumors harboring ERBB2 (5.8%) domain mutations were significantly more sensitive to single agents afatinib or neratinib when compared to wild-type tumors in preclinical in vitro and in vivo models (P = 0.001). In contrast, pan-HER and PIK3CA inhibitors demonstrated limited in vitro activity and were only transiently effective in controlling in vivo growth of PIK3CA-mutated cervical cancer xenografts. Importantly, combinations of copanlisib and neratinib were highly synergistic, inducing long-lasting regression of tumors harboring alterations in the ERBB2/PI3K/AKT/mTOR pathway. These findings define the genetic landscape of cervical cancer, suggesting that a large subset of cervical tumors might benefit from existing ERBB2/PIK3CA/AKT/mTOR-targeted drugs.

Trastuzumab tolerability in the treatment of advanced (stage III-IV) or recurrent uterine serous carcinomas that overexpress HER2/neu.

OBJECTIVE: Due to previously reported trastuzumab safety concerns and the scant data available in endometrial cancer patients, we sought to assess the safety, tolerability and toxicity profile of trastuzumab in patients with advanced/recurrent uterine serous carcinoma (USC) that overexpress HER2/neu in our multicenter randomized phase II trial. METHODS: Patients were randomized 1:1 to receive carboplatin/paclitaxel (C/P) for 6 cycles ± trastuzumab (T) with the experimental arm continuing to receive single agent trastuzumab maintenance treatment until disease progression/toxicity. Progression-free-survival was the primary endpoint; overall-survival and toxicity were secondary endpoints. Adverse events (AEs) were compared between treatment arms. RESULTS: There were 28 patients in the C/P arm and 32 patients in the experimental (C/P + T) arm. Fifty-eight patients (97%) experienced 977 treatment-related AEs of which 875 (89.6%) were low-grade (grade 1-2) and 102 (10.4%) were high-grade (grade 3-5). The mean ± standard deviation of AEs per patient was 15.5 ± 16.3 in the C/P arm and 17.0 ± 16.0 in the C/P + T arm. Gastrointestinal AEs were the most common in both arms (n = 155, 15.7%) of which 94.2% were low-grade (n = 146). Importantly, no significant difference between treatment arms was detected in any system-organ class of AE including cardiac AE. Five (17%) of 29 patients who received prolonged trastuzumab maintenance therapy had no sign of cumulative toxicity after an average (range) of 5.1 (4.2-6.3) years. CONCLUSIONS: Trastuzumab appears to be safe and has a manageable toxicity profile both when used in combination with chemotherapy and when used for single agent maintenance in patients with HER2/neu positive USC. This safety profile is reassuring given the proven efficacy of trastuzumab in advanced/recurrent HER2/neu positive USC.

DHES0815A, a novel antibody-drug conjugate targeting HER2/neu, is highly active against uterine serous carcinomas in vitro and in vivo.

OBJECTIVE: Uterine serous carcinoma (USC) is an aggressive histologic variant of endometrial cancer which portends a poor prognosis. DHES0815A is a novel antibody-drug-conjugate (ADC) which binds specifically to HER2 overexpressing tumors at a distinct epitope from that bound by trastuzumab and pertuzumab after which it delivers the toxic payload, PBD-MA, a DNA mono-alkylating agent. The objective of this study was to evaluate the preclinical activity of DHES0815A against primary USC cell lines and xenografts. METHODS: Twelve primary USC cell lines were assessed by immunohistochemistry (IHC) for HER2 protein expression and for C-erbB2 gene amplification using fluorescent in situ hybridization (FISH) analysis. Cell viability and bystander killing in USC cell lines after exposure to DHES0815A, the non-targeted ADC, and the unconjugated antibody (i.e. MHES0488A) were evaluated using flow cytometry-based-assays. In vivo activity of DHES0815A was tested against HER2/neu overexpressing USC xenografts. RESULTS: High HER2/neu protein expression was seen in 25% (3/12) of the primary USC cell lines. USC cell lines overexpressing HER2/neu were significantly more sensitive to DHES0815A when compared to the non-targeted control ADC (p < 0.001). DHES0815A did not induce significant bystander killing of HER2/neu negative tumors when admixed with HER2/neu positive tumors. DHES0815A caused growth-inhibition and increased survival in USC HER2/neu overexpressing xenografts when compared to controls (p < 0.01). CONCLUSIONS: DHES0815A is both highly selective and toxic to USC tumors overexpressing HER2/neu both in vitro and in vivo. HER2-directed ADCs, alone or in combination with other HER2/neu targeted agents may represent a novel treatment option for patients with tumors harboring HER2/neu overexpression refractory to trastuzumab and traditional chemotherapy.

Robotic Low Anterior Resection and Partial Bladder Resection for Management of Locoregional Endometrial Cancer Recurrence.

OBJECTIVE: To demonstrate a robotic tumor debulking for management of locoregional endometrial cancer recurrence. DESIGN: Case report. SETTING: Tertiary referral center in New Haven, CT. INTERVENTIONS: A 70-year-old patient with a history of stage IB endometrioid endometrial cancer presented with rectal bleeding 3 years after the completion of treatment. A mass involving the distal sigmoid colon/upper rectum and bilateral distal periureteral masses were visualized on imaging. There was no distant metastatic disease. Colonoscopic biopsies were consistent with endometrial cancer recurrence. Because the patient was symptomatic with rectal bleeding and had no distant metastasis, it was recommended that she undergo surgical resection for management of this locoregional recurrence. The patient was placed in reverse Trendelenburg position with a rightward tilt to mobilize the splenic flexure. Once the cephalad aspect of the descending colon mobilization was completed, the patient was placed in Trendelenburg lithotomy position to expose the pelvis. A robot was docked at this point and the pelvic avascular spaces were delineated. A medial-to-lateral approach was used in mobilization of the sigmoid colon mesentery. The left ureter was identified and the sigmoid branches of inferior mesenteric artery were sealed. The descending/sigmoid colon junction was stapled. After complete mobilization of the sigmoid colon, the tumor-free upper rectum was delineated and stapled. Attention was then turned to the distal peri-ureteral masses. The 2-cm mass on the right, which was densely adherent to the distal right ureter, was completely resected after extensive ureterolysis. The resection of the 4-cm mass on the left which involved both the distal left ureter and the bladder dome required an intentional cystotomy and a partial cystectomy to attain negative margins (Supplemental Figure 1). The procedure was continued with the bowel anastomosis. The anvil was introduced through the vagina and was placed into the proximal limb through an antimesenteric incision. An end-to-end tension-free anastomosis was performed and adequate vascularization was confirmed with intravenous indocyanine green. CONCLUSION: Robotic low anterior resection and partial bladder resection were performed without any complications with negative margins. Robotic tumor debulking should be considered in appropriate patients when managing locoregional recurrence of endometrial cancer [1,2].

Derangements in HUWE1/c-MYC pathway confer sensitivity to the BET bromodomain inhibitor GS-626510 in uterine cervical carcinoma.

OBJECTIVE: Whole-exome-sequencing (WES) studies reported c-MYC gene-amplification and HUWE1 gene deletion/mutations in a significant number of cervical-cancer-patients (CC) suggesting HUWE1/c-MYC pathway as potential therapeutic target. We investigated HUWE1/c-MYC expression in fresh-frozen-CC and the activity of the novel BET inhibitor GS-626510 (Gilead-Science-Inc) against primary WES CC-cultures and CC-xenografts. METHODS: HUWE1 and c-MYC expression were evaluated by qRT-PCR in 23 CC including 12 fresh-frozen-tumor-tissues and 11 primary-cell-lines. c-Myc expression was also evaluated by Western-Blot (WB) and fluorescence-in-situ-hybridization (FISH) in all 11 fully sequenced primary-CC-cell-lines. Primary tumors were evaluated for sensitivity to GS-626510 in-vitro using proliferation and viability-assays. siRNA experiments were used to evaluate the effect of HUWE1 silencing on primary-CC-cell-line growth and sensitivity to GS-626510. Finally, the in-vivo activity of GS-626510 was studied in CC-CVX8-mouse-xenografts. RESULTS: Fresh-frozen-CC and primary-CC-cell-lines overexpressed c-MYC when compared to normal tissues (p = .01). FISH demonstrated amplification of c-MYC in 9/11 (82%) of the primary-CC-cell-lines. Cell-lines with derangements in HUWE1/c-MYC pathway were highly sensitive to GS-626510, with a dose-response decrease in cell proliferation and viability. siRNA silencing of HUWE1 significantly increased c-MYC expression and CC cell-proliferation and enhanced the in-vitro sensitivity to GS-626510. Twice-daily oral doses of GS-626510 were well tolerated in-vivo and highly effective in decreasing tumor-growth (p = .004) and increasing survival (p = .004) of CC-CVX8 xenografts. CONCLUSIONS: Downregulation/inactivation of HUWE1 may increase c-MYC expression and proliferation in primary-CC-cell-lines. GS-626510 may represent a novel, potentially highly effective therapeutic agent against CC overexpressing c-MYC and/or harboring HUWE1 mutations. Clinical studies with BET inhibitor in CC-patients harboring radiation/chemotherapy-resistant disease are warranted.

In vitro and in vivo activity of sacituzumab govitecan, an antibody-drug conjugate targeting trophoblast cell-surface antigen 2 (Trop-2) in uterine serous carcinoma.

OBJECTIVE: Uterine serous carcinoma (USC) is an aggressive variant of endometrial cancer with poor prognosis. Sacituzumab govitecan (SG) is a novel antibody-drug-conjugate (ADC) targeting trophoblast cell-surface antigen 2 (Trop-2), a transmembrane-calcium-signal-transducer, to deliver SN-38, the active metabolite of irinotecan. The objective of this study was to evaluate the expression of Trop-2 in USC and the preclinical activity of SG against primary USC cell-lines and xenografts. METHODS: We used immunohistochemistry (IHC) and flow-cytometry-based assays to evaluate Trop-2 expression and cell-viability in USC tissue and primary tumor-cell-lines after exposure to SG, non-targeting control ADC, and naked antibody hRS7-IgG. Antibody-dependent-cell-cytotoxicity (ADCC) against Trop-2+ and Trop-2- USC cell-lines was evaluated in vitro using 4-hr-Chromium-release-assays. In vivo activity of SG was tested against Trop-2+ USC xenografts by intravenous administration of SG, control ADC, and hRS7. RESULTS: Trop-2 expression by IHC was detected in 95.1% of USC samples (99/104). Primary tumor cell-lines overexpressing Trop-2 were significantly more sensitive to SG when compared to control ADC (p <0.05). Both SG and hRS7 mediated ADCC in Trop2+ USC cell-lines while no cytotoxicity was detected against Trop-2- cells. SG induced significant bystander killing of Trop-2- tumors when admixed with Trop-2+ tumors. SG caused growth-inhibition and increased survival in SG treated mice harboring Trop-2+ xenografts when compared to controls (p <0.05). CONCLUSIONS: SG is remarkably active against USC overexpressing Trop-2 in vitro and in vivo. Our results combined with SG clinical responses recently reported against multiple chemotherapy resistant human tumors further support clinical development of SG in USC patients with advanced/recurrent disease.

Robotic Colostomy Takedown in a Patient with Extensive Ventral Hernias and Adhesive Disease.

STUDY OBJECTIVE: To demonstrate a surgical video wherein a robot-assisted colostomy takedown was performed with anastomosis of the descending colon to the rectum after reduction of ventral hernias and extensive lysis of adhesions. DESIGN: Case report and a step-by-step video demonstration of a robot-assisted colostomy takedown and end-to-side anastomosis. SETTING: Tertiary referral center in New Haven, Connecticut. A 64-year-old female was diagnosed with stage IIIA endometrioid endometrial adenocarcinoma in 2015 when she underwent an optimal cytoreductive surgery. In addition, she required resection of the sigmoid colon and a descending end colostomy with Hartmann's pouch, mainly secondary to extensive diverticulitis. After adjuvant chemoradiation, she remained disease free and desired colostomy reversal. Body mass index at the time was 32 kg/m2. Computed tomography of her abdomen and pelvis did not show any evidence of recurrence but was notable for a large ventral hernia and a parastomal hernia. She then underwent a colonoscopy, which was negative for any pathologic condition, except for some narrowing of the distal rectum above the level of the levator ani. INTERVENTIONS: Enterolysis was extensive and took approximately 2 hours. The splenic flexure of the colon had to be mobilized to provide an adequate proximal limb to the anastomosis site. An anvil was then introduced into the distal descending colon through the colostomy site. A robotic stapler was used to seal the colostomy site and detach it from the anterior abdominal wall. Unfortunately, the 28-mm EEA sizer (Covidien, Dublin, Ireland) perforated through the distal rectum, caudal to the stricture site. A substantial length of the distal rectum had to be sacrificed secondary to the perforation, which mandated further mobilization of the splenic flexure. The rectum was then reapproximated with a 3-0 barbed suture in 2 layers. This provided us with approximately 6- to 8-cm distal rectum. An end-to-side anastomosis of the descending colon to the distal rectum was performed. Anastomotic integrity was confirmed using the bubble test. Because of the lower colorectal anastomosis, a protective diverting loop ileostomy was performed. The patient had an uneventful postoperative course. A hypaque enema performed 3 months after the colostomy takedown showed no evidence of anastomotic leak or stricture. The ileostomy was then reversed without any complications. CONCLUSION: Robot-assisted colostomy takedown and anastomosis of the descending colon to rectum were successfully performed. Although there is a paucity of literature examining this technique within gynecologic surgery, the literature on general surgery has supported laparoscopic Hartmann's reversal and has demonstrated improved rates of postoperative complications and incisional hernia and reduced duration of hospitalization [1]. Minimally invasive technique is a feasible alternative to laparotomy for gynecologic oncology patients who undergo colostomy, as long as the patients are recurrence free.

Robotic Tumor Debulking off External Iliac Vessels for the Management of Recurrent Ovarian Cancer.

OBJECTIVE: To show a surgical video in which an isolated mass was resected off the external iliac vessels for the management of recurrent ovarian cancer. DESIGN: Case report. SETTING: Tertiary referral center in New Haven, Connecticut. INTERVENTIONS: This is a step-by-step demonstration of a robotic tumor debulking in a patient with isolated recurrence of epithelial ovarian cancer [1-3]. The patient is a 70-year-old woman with Lynch syndrome who received a diagnosis for stage IIC high-grade serous ovarian adenocarcinoma and underwent complete debulking in 1996. She had most recently been on pembrolizumab for microsatellite instability-high tumor until February 2019, when she received a diagnosis for isolated hypermetabolic mass in close proximity to the external iliac vessels and right iliac fossa. The patient was placed in dorsal low lithotomy Trendelenburg position, and 15° leftward tilt of the table was obtained to expose the right pelvic sidewall and iliac fossa. To optimally target the surgical field of interest, all robotic trocars were placed in a straight line starting from 5 cm above symphysis pubis on the left side to left subcostal line between the midline vertical and the left midclavicular lines, as per the manufacturer's port placement guidelines (Fig. 1). CONCLUSION: Robotic resection of the tumor nodule off the external iliac vessels was successfully performed with adequate range of motion provided by the arms and without any complications. Trocar placement should be tailored to the site of surgical interest. Robotic-assisted laparoscopy should be considered as a valid alternative to the traditional open approach, when managing solitary masses in patients with recurrent ovarian cancer.

Robotic type II B posterior exenteration for recurrent vaginal cancer.

OBJECTIVE: To demonstrate a surgical video, wherein a robotic-assisted posterior exenteration was performed for management of recurrent vaginal cancer. METHODS: We present a case of a 55 year-old female with a history of stage II squamous cell vaginal carcinoma. Patient recurred two years after completion of her primary chemoradiation at the posterior upper vagina. Pelvic MRI showed an approximately 4 cm tumoral nodule, without invasion into rectum or to bilateral parametria. PET-CT ruled out any metastatic disease. She was explained of the palliative systemic treatment versus potentially curative pelvic exenteration, as her options. After extensive counseling, she opted for the surgical option. Given her extensive comorbidities, including poorly controlled diabetes, COPD, obesity and heavy smoking, decision was made to attempt the procedure with a robotic approach (Behbehani et al.; Kammar et al. [1,2]). The technical steps of posterior Type IIB exenteration have been detailed in the video with an emphasis on anatomic landmarks by utilizing visual illustrations (Cibula [3]). The surgical margins were deemed to be negative with frozen section evaluation. Intravenous indocyanine green injection confirmed adequate blood supply to the end colostomy site. Patency of bilateral ureters was confirmed at the end of the procedure. RESULTS: Robotic-assisted Type IIB posterior pelvic exenteration was successfully completed without any intra-operative complications. Patient was discharged home on post-operative day 8. She has been dispositioned to surveillance. CONCLUSIONS: Robotic approach to highly morbid pelvic exenteration procedures should be considered in selected patients with recurrent gynecologic malignancies, who present without evidence of distant metastatic disease.

PARP-1 activity (PAR) determines the sensitivity of cervical cancer to olaparib.

OBJECTIVES: Cervical cancer (CC) remains a major health problem worldwide. Poly (adenosine diphosphate [ADP]-ribose) polymerase (PARP) inhibitors (PARPi) have emerged as a promising class of chemotherapeutics in ovarian cancer. We explored the preclinical in vitro and in vivo activity of olaparib against multiple primary whole exome sequenced (WES) CC cells lines and xenografts. METHODS: Olaparib cell-cycle, apoptosis, homologous-recombination-deficiency (HRD), PARP trapping and cytotoxicity activity was evaluated against 9 primary CC cell lines in vitro. PARP and PAR expression were analyzed by Western blot assays. Finally, olaparib in vivo antitumor activity was tested against CC xenografts. RESULTS: While none of the cell lines demonstrated HRD, three out of 9 (33.3%) primary CC cell lines showed strong PARylation activity and demonstrated high sensitivity to olaparib in vitro treatment (cutoff IC50 values < 2 µM, p = 0.0012). Olaparib suppressed CC cell growth through cell cycle arrest in the G2/M phase and caused apoptosis (p < 0.0001). Olaparib activity in CC involved both PARP enzyme inhibition and trapping. In vivo, olaparib significantly impaired CC xenografts tumor growth (p = 0.0017) and increased overall animal survival (p = 0.008). CONCLUSIONS: A subset of CC primary cell lines is highly responsive to olaparib treatment in vitro and in vivo. High level of PARylation correlated with olaparib preclinical activity and may represent a useful biomarker for the identification of CC patients benefitting the most from PARPi.

PI3K oncogenic mutations mediate resistance to afatinib in HER2/neu overexpressing gynecological cancers.

OBJECTIVE: Aberrant expression of HER2/neu and PIK3CA gene products secondary to amplification/mutations are common in high-grade-serous-endometrial (USC) and ovarian-cancers (HGSOC). Because scant information is currently available in the literature on the potential negative effect of PIK3CA mutations on the activity of afatinib, in this study we evaluate for the first time the role of oncogenic PIK3CA mutations as a potential mechanism of resistance to afatinib in HGSOC and USC overexpressing HER2/neu. METHODS: We used six whole-exome-sequenced primary HGSOC/USC cell-lines and three xenografts overexpressing HER2/neu and harboring mutated or wild-type PIK3CA/PIK3R1 genes to evaluate the role of PI3K-mutations as potential mechanism of resistance to afatinib, an FDA-approved pan-c-erb-inhibitor in clinical trials in USC. Primary-USC harboring wild-type-PIK3CA gene was transfected with plasmids encoding oncogenic PIK3CA-mutations (H1047R/E545K). The effect of afatinib on HER2/PI3K/AKT/mTOR pathway was evaluated by immunoblotting. RESULTS: We found PI3K wild-type cell-lines to be significantly more sensitive (lower IC50) than PI3K-mutated cell-lines p = 0.004). In vivo, xenografts of primary cell-line USC-ARK2, transfected with the PIK3CA-H1047R or E545K hotspot-mutations, exhibited significantly more rapid tumor growth when treated with afatinib, compared to mice harboring ARK2-tumors transfected with wild-type-PIK3CA (p = 0.041 and 0.001, respectively). By western-blot, afatinib effectively reduced total and phospho-HER2 proteins in all cell-lines. However, H1047R/E545K-PIK3CA-transfected-ARK2-cells demonstrated a greater compensatory increase in phosphorylated-AKT proteins after afatinib exposure when compared to controls ARK2. CONCLUSIONS: Oncogenic PI3K mutations may represent a major mechanism of resistance to afatinib. Combinations of c-erb with PIK3CA, AKT or mTOR inhibitors may be necessary to more efficiently block the PIK3CA/AKT/mTOR pathway.

Challenges of Robotic Gynecologic Surgery in Morbidly Obese Patients and How to Optimize Success.

PURPOSE OF REVIEW: Robotic surgery has been shown to have a significant benefit in obese gynecologic patients over open surgery. However, robotic surgery in these patients requires a thorough understanding of the physiologic adaptations caused by obesity, adequate preoperative optimization, specialized equipment and techniques, and careful attention to intra- and postoperative management in order to minimize complications. This article reviews the benefits of a minimally invasive approach in obese patients and provides a thorough guide to perioperative management of obese patients undergoing robotic gynecologic surgery. A useful set of tips and tricks to overcome many of the technical challenges in performing robotic surgery in the obese patients is included. RECENT FINDINGS: In the USA, obesity has risen to affect 39.8% of the population, which leads to increased incidence of mortality, hypertension, diabetes, heart disease, and stroke. Moreover, obese patients are at greater risk of perioperative complications during gynecologic surgery. With the use of laparoscopy, many of the perioperative risks of surgery in obese patients can be ameliorated. However, minimally invasive surgery in obese patients is technically challenging. Robotic-assisted laparoscopy addresses several of these challenges, allowing surgeons to offer minimally invasive approaches to patients with extreme BMIs while reducing perioperative risk. Obese patients undergoing gynecologic surgery receive a greater benefit than their non-obese counterparts from a laparoscopic approach, and current data support the safety and feasibility of robotic surgery in the obese population. Therefore, every effort to offer a minimally invasive surgery to obese patients should be made.