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// Eirwen M. Miller 2 , Nicole E. Patterson 1 , Jenna Marcus Zechmeister 2 , Michal Bejerano-Sagie 1 , Maria Delio 1 , Kunjan Patel 1 , Nivedita Ravi 1 , Wilber Quispe-Tintaya 1 , Alexander Maslov 1 , Nichelle Simmons 1 , Maria Castaldi 1 , Jan Vijg 1 , Rouzan G. Karabakhtsian 3 , John M. Greally 1 , Dennis Y.S. Kuo 2 and Cristina Montagna 1 1 Department of Genetics, Albert Einstein College of Medicine, Bronx, NY 10461, USA 2 Department of Obstetrics & Gynecology and Women’s Health, Division of Gynecologic Oncology, Montefiore Medical Center, Bronx, NY 10461, USA 3 Department of Pathology, Montefiore Medical Center, Bronx, NY 10461, USA Correspondence to: Cristina Montagna, email: cristina.montagna@einstein.yu.edu Keywords: target sequencing; next generation sequencing; endometrial carcinoma; tumor recurrence Received: July 29, 2017 Accepted: September 08, 2017 Published: October 26, 2017 ABSTRACT Next generation sequencing (NGS) technologies have revolutionized our approach to genomic research. The use of whole genome sequencing (WGS), whole exome sequencing (WES), transcriptome profiling, and targeted DNA sequencing has exponentially improved our understanding of the human genome and the genetic complexities underlying malignancy. Yet, WGS and WES clinical applications remain limited due to high costs and the large volume of data generated. When utilized to address biological questions in basic science studies, targeted sequencing panels have proven extremely valuable due to reduced costs and higher sequencing depth. However, the routine application of targeted sequencing to the clinical setting is limited to a few cancer subtypes. Some highly aggressive tumor types, like type 2 endometrial cancer (EC), could greatly benefit from routine genomic analysis using targeted sequencing. To explore the potential utility of a mid size panel (~150 genes) in the clinical setting, we developed and validated a custom panel against WGS, WES, and another commercially available targeted panel. Our results indicate that a mid size custom designed panel is as efficient as WGS and WES in mapping variants of biological and clinical relevance, rendering higher coverage, at a lower cost, with fewer variants of uncertain significance. Because of the much higher sequencing depth that could be achieved, our results demonstrate that targeted sequencing outperformed WGS and WES in the mapping of pathogenic variants in a breast cancer case, as well as a case of mixed serous and high-grade endometrioid EC, the most aggressive EC subtype.

Background: Resident experience and opinions regarding robotic surgical training as part of the formal obstetrics and gynecology curriculum has not been reported. Objective: To evaluate residents’ experience with the newly introduced Robotic Olympics and a robotic surgical trainings curriculum in general, especially in correlation with future career goals. Methods: All residents of the Obstetrics and Gynecology Residency Program at the Montefiore Medical Center, who participated in the Robotic Olympics 2014, a team-based simulation competition, completed a de-identified pre- and post-Olympics survey. Results: For the participating 31 residents, the mean number of bedside-assistant robotic and console cases was 8 (0-50) and 4 (0-30), respectively. Both were positively associated with postgraduate level. The majority of residents (89%) reported that they were best trained in open surgery. Only 52% anticipated using robotic surgery in their future practice. Anticipated use of the robot and interest in robotic training were correlated with surgical subspecialty career goals. 100% of residents aspiring a career in gynecologic oncology and none interested in maternofetal medicine anticipated future use of robotic surgery. However, all residents desired the Robotic Olympics to be integral part of resident education. Conclusions: The majority of residents welcomed the addition of the Robotic Olympics to the robotic-surgical curriculum. However, the residents’ interest in robotic surgical training in general was disparate and correlated with the anticipated use of the robot in the residents’ future career. This data suggests the need for directed robotic surgical training for residents interested in surgical sub-specialties to focus resources early on.

Objective: The objective of this study is to assess the outcome of port-a-cath (PAC) maintenance every three months in patients with gynecologic malignancies with the goal of standardizing a safe and appropriate interval that would maintain patency and minimize side effects. Methods: We performed a retrospective medical record review and evaluation of all patients with a Bard? PAC who were noted to have no evidence of disease (NED) during the years 2003 to 2010. The interval between accessions and any complications related to the presence of the PAC were recorded. Relevant complications included skin infections, bacteremia, thrombosis, and occlusions. Statistical analysis was done using the Fisher’s exact test. Results: A total of 201 patients had PAC placed and 43 patients underwent PAC accessions to maintain patency. The total number of accessions was 150 with a median number per patient of 2.0 (range 1 - 10). The mean time between flushes was 112 days (SD = 57). When comparing women in maintenance who had flushes within 90 days versus those who had flushes over 90 days apart, there was no difference in infection or occlusion rates between these groups (p = 0.515). In the Conclusion: Infections and occlusions are rare in women with gynecologic malignancies undergoing maintenance of their PAC. Longer intervals between PAC flushes do not appear to affect the outcome in our patients. Our ongoing data and follow-up confirm that extending the interval of PAC accession to every 3 months, rather than monthly, is safe, effective and convenient in the patient population with gynecologic malignancies.

Objectives: Dysbiosis is thought to promote endothelial dysfunction and chronic inflammation, resulting in many chronic human diseases including cancer. This pilot study sought to characterize the cervicovaginal, endometrial and anorectal micro- and mycobiota of post-menopausal women with endometrioid endometrial adenocarcinoma (EAC), uterine serous carcinoma (USC), and controls without cancer. Methods: Post-menopausal patients undergoing hysterectomy for endometrial cancer or non-cancerous disease were enrolled after informed consent. Women were excluded if they reported history of prior cancer, recent urinary or gynecologic infection, antibiotic or probiotic use. Microbial swabs were obtained from the cervicovaginal, the anorectal, and the endometrial cavity after removal of the uterus. DNA was extracted and PCR amplified using barcoded 16SV4 (bacterial) and ITS1 (fungal) primers. Next generation libraries were prepared and sequenced on an Illumina MiSeq 2x300. Sequence reads were processed using our internally developed bioinformatics pipeline and publically available software. Results: Next-generation sequencing of bacterial and fungal DNA revealed substantial differences of cervicovaginal, endometrial and anorectal microbiota in patients with USC (n= 8) compared to those with EAC (n= 13) or benign disease (n= 6). Uterine fungal reads were much higher in patients with USC than EAC (average of 3237 reads versus 938 reads respectively, p = 0.05 Wilcox test). Candida species were reduced, but not eliminated in cervicovaginal samples, and there was a dominance of Malassezia restrica in USC and EAC. A non-significant predominance of Porphyromonas species was noted in USC specimens compared to EAC specimens (p = 0.063). Conclusions: Alpha-diversity of 16S bacterial rRNA and fungal species of the cervicovaginal microbiome was significantly associated with endometrial cancer in our study. USC had a fundamentally different microbiome from control samples and demonstrated a predominance of Porphyromonas species. Further sampling and analysis is needed to confirm these results and to identify rare pathogens. Characterization of microbiota in women with endometrial cancer may help identify risk factors for gynecologic malignancy, and drive investigation regarding preventative and therapeutic intervention. Citation Format: Gregory M. Gressel, Marina Frimer, Christine P. Zolnik, Mykhaylo Usyk, Devin T. Miller, Eirwen M. Miller, D. Y.S. Kuo, Robert D. Burk. Characterization of the cervicovaginal, endometrial and anorectal microbiota and mycobiota of post-menopausal women with endometrioid and serous endometrial cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5143.

Purpose/Objectives: Integration of human papillomavirus (HPV) DNA into the human genome occurs in a fraction of viral infections and is a near ubiquitous factor in cervical tumorigenesis. While use of next-generation sequencing (NGS) methods has significantly advanced our understanding of HPV DNA integration and its consequences, short read approaches may yield false positives or misinterpretation of associated genomic structural variants. With the goal of resolving genomic rearrangements, we applied both HPV16 hybridization capture-NGS and long read Nanopore technology to the well-studied CaSki cervical carcinoma cell line that has many sites of HPV16 DNA integration. Methods: CaSki genomic DNA was sheared, ligated to Illumina adaptors, hybridized to HPV16 capture probes (Roche Nimblegen SeqCap EZ System), and sequenced by Illumina HiSeq 2500, paired end, 300 bp mode. Reads were aligned to a custom human (GRCh37/hg19) plus HPV16 reference genome using BWA mem, and junction fragments were computationally identified using Delly and SplazerS. For long read analysis, isolated CaSki DNA was sheared to 20 kb. Genomic libraries were prepared and sequenced on an Oxford Nanopore MinION Mk1b device (ONT) using standard 48 hour scripts. Base calling and fastq file extraction were performed with Albacore v2.0. Reads were aligned using Ngmlr, and structural variants called using Sniffles. Results: For Illumina analysis, 34 million demultiplexed read pairs were obtained with average, HPV genome coverage depth of 2.8*106. 47 previously identified, HPV-human junctions were verified along with 193 novel junctions. Use of PCR has validated 4 of 6 novel junctions tested to date. Multiple junctions were noted to be clustered at some chromosomal locations, with 30 (13.5%) called less than 1kb apart on the human genomic side. For long read analysis, ~150K reads were obtained. Mean read length was 16K with genome and HPV16 coverage of 0.60X and 220X, respectively. Integrations were detected on chromosomes 2, 5, 7, 10, 19, 20, and X, and all consisted of >2 tandem viral genomes. Discussion: Our molecular approaches generated unprecedented long-range analyses of HPV DNA integration sites in the CaSki cervical carcinoma cell line. HPV DNA was often present in tandem arrays. We interpret the clusters of nearby HPV-human junctions as potential evidence of frequent, ongoing, subclonal, genomic rearrangements at these sites. Combining deep-sequencing and long-range methodologies allowed important insights, and we are currently analyzing clinical cancer samples using them. Citation Format: Anne Van Arsdale, Jack Lenz, Nicole Patterson, Elaine Maggi, Dennis Y. Kuo, Cristina Montagna. Elucidation of integrated HPV DNA structure in a cervical carcinoma cell line by combined high resolution and long range sequencing analyses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-334.