Bakkum‐Gamez, Jamie N.

INTRODUCTION: To compare two published risk stratification criteria (Milwaukee Model vs. Mayo Criteria) to predict lymphatic dissemination (LD) in endometrioid endometrial cancer (EC). METHODS: Patients who underwent surgery for EC between 1/1/2004-9/30/2013 and had endometrioid histology without stage IV disease were included and classified as either low-risk or at-risk for LD using two independent risk stratification systems. LD was defined as positive nodes at time of surgery or lymph node recurrence within 2 years of surgery after negative lymph node assessment (LNA) or when LNA was not performed. False positive (FP) and false negative (FN) rates for each risk model were calculated. RESULTS: Among 1103 patients, 81 (7.3%) had LD (72 positive LN and 9 LN recurrences). Most patients (90.2%) had stage I disease. Integration of the Milwaukee Model was associated with a low at-risk rate for LD (38.1%) but a relatively high FN rate (13.6%, 95% CI 7.0-23.0). Conversely, the traditional Mayo Criteria using a tumor diameter (TD) cutoff of ≤2 cm (vs >2 cm) had a higher at-risk rate for LD (69.5%) but a FN rate of 0.0% (95% CI, 0-4.5). Modifying Mayo Criteria using a TD cutoff of ≤3 cm (vs >3 cm) identified fewer women at-risk than using ≤2 cm cutoff (56.8% vs. 69.5%), but had a higher FN rate of 3.7% (95% CI, 0.8-10.4). CONCLUSIONS: The Milwaukee Model had the lowest at-risk rate of LD but an unacceptable FN rate. Modifying the Mayo Criteria by increasing the TD cutoff from the traditional ≤2 cm to ≤3 cm would spare an estimated 12.7% of patients LNA, but the accompanying FN rate is unacceptably high. The traditional Mayo Criteria for low-risk EC remains the most accurate in determining which patients LNA can be omitted.

INTRODUCTION: Leiomyomas are benign uterine smooth muscle tumors. After menopause, most women have a reduction in size and number of leiomyomas. Conversely, uterine sarcomas present as a rapidly enlarging pelvic mass in postmenopausal women. The distinction of uterine leiomyoma from sarcoma is difficult and definitive diagnosis requires pathologic examination. METHODS: A 74 year-old multiparous female with a history of ductal carcinoma in situ (DCIS) of the breast presented with significant postmenopausal bleeding, requiring blood transfusion, and a rapidly enlarging pelvic mass. Abdominal imaging including ultrasound and computerized tomography confirmed a 15 cm uterine mass. The patient underwent a total abdominal hysterectomy and bilateral salpingo-oophorectomy with concern for uterine sarcoma. Grossly, the uterus contained a 15.5 cm well demarcated intramural mass. Microscopically, the tumor was a spindle cell neoplasm with anastomosing fascicles interrupted by thick-walled blood vessels or fibrous regions. The cells exhibited uniformly mild to moderate cytologic atypia. Immunohistochemically, the tumor strongly and diffusely expressed desmin and h-caldesmon and exhibited patchy, strong expression of CD10. RNA-seq, confirmed by RT-PCR and Sanger sequencing, revealed a gene fusion involving KAT6B and KANSL1. The fusion joins exon 3 of KAT6B to exons 11 and 12 of KANSL1. The final diagnosis was a benign uterine leiomyoma. The patient remains without evidence of disease six months post-operatively. CONCLUSION: This is the first case, to our knowledge, that has identified the KAT6B-KANSL1 fusion in a uterine leiomyoma. Further efforts to classify the pathologic characteristics and prevalence of this fusion may contribute to understanding of tumorigenesis of uterine smooth muscle tumors.

Abstract Introduction. VSV-IFNβ-NIS is an oncolytic vesicular stomatitis virus (VSV; Rhabdovirus family) with rapid replication kinetics and potent antitumor activity. VSV-IFNβ-NIS encodes the human interferon beta (IFNβ) gene as a STING agonist and the human sodium iodide symporter (NIS) as a reporter gene for tracking the pharmacokinetics (PK) of virus replication in infected tumors. VSV replicates selectively in cancer cells and has promising preclinical antitumor activity across a broad spectrum of cancer types. We report here the safety and correlative data from an FIH trial of intravenous (IV) administration of VSV-IFNβ-NIS in patients with stage IV or recurrent endometrial cancer (EC). Methods. There are two ongoing IV FIH trials using VSV-IFN-NIS, in patients with EC (NCT03120624) and one in patients with hematological malignancies (NCT03017820). In EC, it is a classical 3+3 phase I trial, starting at 5x109 TCID50 through 5x1011 TCID50, given as a single IV dose. The primary objective is safety and tolerability; secondary objectives include monitoring the PK of viral replication through SPECT/CT imaging with NIS gene, viremia, virus shedding, preliminary efficacy, changes in the immune profile of peripheral blood leukocytes, and immunohistochemistry for immune cell infiltrates in tumors. Results. Nine patients have received IV VSV-IFNβ-NIS to date; three with EC and six with hematologic malignancies. The highest dose administered to date is 1.7x1010 TCID50 and dose escalation is ongoing. No DLTs have been observed. Patients experienced the expected infusion related AEs including rigors, chills, nausea, fever, hypotension, and hot flashes. Multiple cytokines increased at 4h post infusion of virus, but most returned to baseline levels by 24h. Viremia was detectable in all patients at the end of infusion, and to varying levels at 30 mins, 1, 2, 4, 24, 48h or 72 hours post virus infusion. No persistent viremia was observed. No infectious virus was recovered in buccal swabs or urine and neutralizing anti-VSV antibodies were present by day 29. Extensive immune phenotyping for T cells, NK, MSDC, myeloid cells performed on peripheral blood cells collected at baseline and at day 3, 8, 15 and 29 post virus infusion showed a trend towards increased PD-1 expression on CD8+ cells. Early IHC data suggests an increase in CD3+ and CD8+ cells in tumor biopsies at day 29 and 3 months in patients with EC treated at the first dose level. Elispot assays for shared EC antigens are pending. Conclusions. IV administration of VSV-IFNβ-NIS up to doses of 1.7x1010 is safe and well tolerated. There is evidence of T cell activation with increased PD-1 expression in CD8+ T cells in the peripheral blood and increased in CD3+ and CD8+ cells in tumor biopsies. Updated results for the EC study will be reported. Citation Format: Jamie Bakkum-Gamez, Matthew S. Block, Nanda Packiriswamy, Bethany A. Brunton, Upreti Deepak, Jonathan M. Mitchell, Lukkana Suksanpaisan, Pamela Atherton, Amylou Dueck, Stephen J. Russell, Martha Q. Lacy, Kah-Whye Peng. First in human (FIH) dose escalation studies of intravenous administration of VSV-IFNβ-NIS (Voyager-V1™) in Stage IV or recurrent endometrial cancer [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 CT072.

Abstract Objectives: To investigate the antitumor potential of the widely used anticholesterol drug Atorvastatin (ATV) alone, in combination with Carboplatin (CBPT) and with the glycolytic inhibitor PFK158 (PFKi) in ovarian cancer (OC). Methods: MTT assay and the Chou-Talalay methodology were used to test anticancer activity in vitro; half maximal inhibitory concentration (IC50) and combination index (CI) were calculated. Cellular changes were studied with transmission electron microscopy (TEM). ATV was combined with PFKi as we previously showed that PFKi, in addition to glycolysis, inhibits lipid metabolism by targeting PLA2G3. PLA2G3 expression was studied by western blot given that downregulation in OVCAR8 using two shRNAs (sh33, sh35) induced autophagy, a prodeath condition in OC, and was associated with chemosensitization compared to nontargeted controls (NTC) (unpublished data). A random sample of 10 patient-derived OC xenografts (PDX) was studied by TEM. Results: OVCAR8 exhibited a dose dependent proliferation inhibition when treated with ATV (IC50 8μM). PFKi and CBPT IC50 were 4μM and 78μM. PFKi treatment combined with 4μM and 8μM of ATV resulted in ATV sensitizing cells to PFKi by shifting PFKi IC50 down to 2.7μM and 0.4μM, respectively. When ATV was combined in constant ratio with CBPT, CIs were >1.1 indicating antagonism. Combination of ATV 4μM with PFKi 2μM resulted in greater decrease in PLA2G3 and p62 and greater increase in cleaved PARP compared to PFKi alone suggesting higher levels of autophagy and apoptosis. ATV and combination treatment resulted in increased caveolin-1 levels, previously shown to be associated with apoptotic cell death. TEM analysis of ATV and PFKi treated cells showed greater number of lipid rich multilamellar bodies (MLB) compared to controls. PLA2G3 knockdowns exhibited higher MBL formation, lower p62, and when treated with statin and with PFKi, cleaved PARP was significantly higher compared to NTCs. TEM showed that PDXs clustered in low and high MLB groups suggesting a distinct novel phenotype. Conclusions: ATV could enhance PFKi antitumor activity by jointly targeting PLA2G3 leading to metabolic reprogramming of OC. Importantly, ATV antagonized CBPT. Additional studies are in progress to explore the mechanistic pathway of ATV and characterize the significance of MLB in OC as a prodeath phenotype and biomarker of response. Citation Format: Eleftheria Kalogera, Debarshi Roy, Jamie N. Bakkum-Gamez, William A. Cliby, Sean C. Dowdy, Viji Shridhar. Atorvastatin antagonizes carboplatin, synergizes with the glycolytic inhibitor PFK-158, and induces lipid laden multilamellar bodies in ovarian cancer [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 5821.

Abstract Background: Endometrial cancer (EC) is the most common gynecologic malignancy in developed countries and the incidence and mortality of EC continue to rise in the US. When detected early, EC is highly curable, suggesting that early detection strategies could reduce EC mortality. Women who develop EC often have a prolonged history of abnormal bleeding, offering a window for identifying many at-risk women prior to the development of advanced cancer. To evaluate risk prediction, novel sampling and early detection strategies in women at increased risk of EC, we established a prospective cohort of women with abnormal bleeding that is followed for endometrial hyperplasia and cancer endpoints. Methods: We consecutively enrolled women ≥45 years old presenting to the Mayo Clinic for evaluation of abnormal uterine bleeding (AUB), postmenopausal bleeding (PMB), thickened endometrial stripe (ES) on imaging, or Lynch Syndrome (LS). From all women, an intravaginal tampon and an endometrial brushing via Tao brush were collected before endometrial biopsy. Epidemiological EC risk factor data, clinical data, and endometrial biopsy histology results were abstracted from medical records. Absolute risk of endometrial hyperplasia and EC were calculated overall and in strata based on clinical symptoms and risk factors. Results: Among the first 1,000 women enrolled into the study, 712 (71%) were overweight or obese, 534 (53%) were postmenopausal, and 214 (22%) were current hormone replacement therapy (HRT) users. Endometrial biopsy and, if indicated, subsequent hysterectomy, detected 39 ECs, 16 atypical hyperplasias, and 28 hyperplasias without atypia at baseline evaluation. Among 534 postmenopausal women, 453 (85%) reported any type of bleeding, with spotting (39%) and light bleeding (27%) being more common than heavy bleeding (14%). Among perimenopausal women with AUB (n=452), the absolute risk of EC at baseline was 0.9% and among women with PMB, the absolute risk of EC at baseline was 7.5%. Among women with PMB, EC risk was modified by HRT use (risk of 2.6% in women on HRT and 8.2% in women not on HRT, respectively), obesity (risk of 5.3%, 8.1% and 9.5% for women with normal, overweight, and obese BMI, respectively), and type of bleeding (risk of 9.7%, 4.9%, and 8.2% in women with spotting, light or heavy bleeding, respectively). Discussion: While risk of endometrial cancer in perimenopausal women with AUB is low, approximately 8% of women presenting for clinical work up of PMB have underlying EC. Clinical presentation and risk factors modify the absolute risk of EC and may inform clinical management. Additional recruitment and prospective follow-up is underway to improve estimates of short- and long-term EC risk in women with PMB. With its collection of tampon and Tao brush samples, the study will provide a unique resource to evaluate molecular markers of EC and EC risk in elevated-risk women. Citation Format: Nicolas A. Wentzensen, Megan Clarke, Viji Shridhar, Maureen Lemens, Matthew Hopkins, Lisa Ahlberg, Shannon Laughlin-Tommaso, Karl Podratz, Mark Sherman, Jamie Bakkum-Gamez. A prospective study of endometrial cancer detection in women presenting for evaluation of abnormal peri- and postmenopausal bleeding [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 2203.

Objective We describe a novel way to calculate estimated blood loss (EBL) using an intraoperative pictographic tool in gynecologic surgery. Methods A pictographic tool to estimate sponge saturation was developed to calculate EBL during surgery. A prospective cohort of women 18 years or older undergoing benign vaginal hysterectomy with planned adnexal surgery at Mayo Clinic were consented for use of the pictographic tool. Demographic, preoperative, intraoperative, and postoperative data were abstracted. Estimated blood loss was compared among surgeons, anesthesia providers, and the pictographic tool and then correlated with change in hemoglobin. Results Eighty-one patients met inclusion with mean age of 45.3 ± 8.7 years. Successful vaginal hysterectomy was achieved in all patients with successful completion of planned adnexectomy in 69 (85.2%). Mean EBL among surgeons, anesthesia providers, and pictographic estimates, respectively, was as follows: 199.4 ± 81.9 mL, 195.5 ± 152.2 mL, and 288.5 ± 186.6 mL, with concordance correlation coefficients for surgeons and anesthesia providers versus pictographic tool of 0.40 (95% confidence interval, 0.29–0.51) and 0.68 (95% confidence interval, 0.57–0.79), respectively. The mean postoperative change in hemoglobin was −1.8 g/dL; there were no postoperative transfusions. Change in hemoglobin was more correlated with blood loss estimates from surgeons ( r = −0.31, P = 0.008) and anesthesia providers ( r = −0.37, P = 0.003) than the pictographic tool ( r = −0.19, P = 0.11). Conclusions Use of a pictographic tool to objectively estimate blood loss demonstrated significant overestimations compared with both anesthesia providers’ and surgeons’ estimates because the pictographic tool was less correlated with postoperative change in hemoglobin than anesthesia provider and surgeon estimates.