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Supplementary Figure 4. A) Two male eight-week-old NSG mice were injected in two ventral subcutaneous sites with 1x106 scrambled shRNA (scrCo) transduced and wild-type (wt) DU145 cells, respectively. Tumor volumes were calculated regularly and weights at the time of sacrifice as well as growth curves are shown. Time in days is shown on the x-axis and error bars indicate standard error of the mean (SEM). B) Three male eight-week-old NSG mice were injected in four ventral subcutaneous sites with 1x105 sorted CSC and non CSC subpopulations of shHIF1A as well as sorted control (scrCo) DU145 cells, respectively. Tumor volumes were calculated regularly and the growth curves of the CSC and non CSC populations from shHIF1A and scrCo DU145 cell lines are shown. Time in days is shown on the x-axis and error bars indicate SEM. C) Photograph of the xenografts arising from the four cell subpopulations in a representative NSG mouse at the time of sacrifice.

Supplementary Figure 2. A) Analysis of basal prostate cancer stem cells in 5 different primary TRAMP tumors. We identified a distinct Lin-/CD49f+/Sca-1+ subpopulation in 19 (82,61%) of the 23 analyzed TRAMP mice. B) Quantified and normalized flow cytometry results of hypoxic CSC and non-CSC subpopulations of DU145 control (scrCo) and DU145 HIF1-α knockdown (shHIF1A) cells labeled with 7-AAD and Annexin V.

Supplementary Figure 1. A) We identified a Lin-/CD49f+/Sca-1+ basal prostate stem cell population in wild-type C57BL/6 mice (left). HIF-1α mRNA was upregulated in sorted basal murine prostate stem cells (n=5, right). B) Treatment with increasing concentration of HIF-1α inhibitor leads to a dose dependent HIF-1α protein downregulation in hypoxic TRAMP-C1 cells. HIF-1α protein levels were assessed by immunoprecipitation and western blotting. IgG heavy chain (HC) is shown as a loading control for immunoprecipitation. Quantified and normalized protein levels are shown. C) HIF-1α shRNA expression in the DU145 cell line (shHIF1A) leads to reduced mRNA levels of HIF-1α and reduced expression of its target gene REDD1. Scrambled shRNA (scrCo) served as control. D) Knockdown of HIF-1α mRNA in the TRAMP-C1 murine cancer cell line. Scrambled shRNA (scrCo) served as control.

Supplementary Tables 1-3. Supplementary Table 1. Antibodies used for FACS (pTR=primary TRAMP; TR=TRAMP-C1; DU=DU145) Supplementary Table 2. Characteristics of analyzed TRAMP mice. Supplementary Table 3. Target Forward primer Reverse primer

Tumor-initiating subpopulations of cancer cells, also known as cancer stem cells (CSC), were recently identified and characterized in prostate cancer. A well-characterized murine model of prostate cancer was used to investigate the regulation of hypoxia-inducible factor 1α (HIF1A/HIF1α) in CSCs and a basal stem cell subpopulation (Lin−/Sca-1+/CD49f+) was identified, in primary prostate tumors of mice, with elevated HIF1α expression. To further analyze the consequences of hypoxic upregulation on stem cell proliferation and HIF1α signaling, CSC subpopulations from murine TRAMP-C1 cells (Sca-1+/CD49f+) as well as from a human prostate cancer cell line (CD44+/CD49f+) were isolated and characterized. HIF1α levels and HIF target gene expression were elevated in hypoxic CSC-like cells, and upregulation of AKT occurred through a mechanism involving an mTOR/S6K/IRS-1 feedback loop. Interestingly, resistance of prostate CSCs to selective mTOR inhibitors was observed because of HIF1α upregulation. Thus, prostate CSCs show a hypoxic deactivation of a feedback inhibition of AKT signaling through IRS-1. In light of these results, we propose that deregulation of the PI3K/AKT/mTOR pathway through HIF1α is critical for CSC quiescence and maintenance by attenuating CSC metabolism and growth via mTOR and promoting survival by AKT signaling. We also propose that prostate CSCs can exhibit primary drug resistance to selective mTOR inhibitors.Implications: This work contributes to a deeper understanding of hypoxic regulatory mechanisms in CSCs and will help devise novel therapies against prostate cancer. Mol Cancer Res; 13(3); 556–64. ©2014 AACR.

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Purpose: Although prognostic and predictive factors in ovarian cancer have been extensively studied for decades, only few have been identified and introduced to clinical practice. Here, we evaluate hVps37A (HCRP1) as a possible novel predictive marker for ovarian cancer. hVps37A was originally described as a member of the membrane-trafficking ESCRT-I complex mediating the internalization and degradation of ubiquitinated membrane receptors.Experimental Design: We analyzed an ovarian cancer tissue microarray for HCRP1, EGFR, and HER2 expression. We used a tetracycline inducible ovarian cancer cell culture model to show the effects of hVps37A knockdown in vitro and in vivo. In addition, we studied the effects of epidermal growth factor receptor (EGFR) inhibitors cetuximab and lapatinib on ovarian cancer cells under conditions of hVps37A knockdown.Results: We find that hVps37A is significantly downregulated in ovarian cancer and modifies the prognostic value of EGFR and HER2 expression. In addition, hVps37A downregulation in ovarian cancer cells leads to cytoplasmic pEGFR retention and hyperactivation of downstream pathways and is associated with enhanced xenograft growth in nude mice and invasion of the collagen matrix. Furthermore, due to subsequent sustained Akt- and MAPK-pathway activation, hVps37A-deficient cells become irresponsive to inhibition by the therapeutic antibody cetuximab.Conclusion: We propose that hVps37A status could become a novel prognostic and therapeutic marker for EGFR or HER2 driven tumors. Clin Cancer Res; 17(24); 7816–27. ©2011 AACR.

Supplementary Data from Natural Immunity Enhances the Activity of a DR5 Agonistic Antibody and Carboplatin in the Treatment of Ovarian Cancer

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Supervised clustering of primary HGSOC (n=421) from the TCGA data using 59/126 immune related genes

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Purpose: Most high-grade serous ovarian cancer (HGSOC) patients develop recurrent disease after first-line treatment, frequently with fatal outcome. This work aims at studying the molecular biology of both primary and recurrent HGSOC.Experimental Design: Gene expression profiles of matched primary and recurrent fresh-frozen tumor tissues from 66 HGSOC patients were obtained by RNA sequencing. Clustering analyses and pairwise comparison of the profiles between matched samples and subsequent functional alignment were used for the identification of molecular characteristics of HGSOC.Results: Both primary and recurrent HGSOC samples presented predominant gene expression differences in their microenvironment, determined by a panel of genes covering all major pathways of immune activation together with a number of genes involved in the remodeling of extracellular matrix and adipose tissues. Stratifying tumor tissues into immune active and silent groups, we further discovered that although some recurrent tumors shared the same immune status as their primary counterparts, others switched the immune status, either from silent to active or active to silent. Interestingly, genes belonging to the B7-CD28 immune checkpoint family, known for their major role as negative regulators of the immune response, were overexpressed in the immune active tumors. Searching for potential tumor antigens, CEACAM21, a member of the carcinoembryonic antigen family, was found to be significantly overexpressed in immune active tissues in comparison with the silent ones.Conclusions: The results illustrate the complexity of the tumor microenvironment in HGSOC and reveal the molecular relationship between primary and recurrent tumors, which have multiple therapeutic implications. Clin Cancer Res; 23(24); 7621–32. ©2017 AACR.

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Functional assignment of the 142 genes with the highest expression difference in the two clusters revealed by unsupervised clustering of recurrent samples

The tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) induces apoptosis specifically in cancer cells with little effect on normal cells. We have previously shown that TRAIL signaling is altered in most ovarian cancer patients and that resistance to TRAIL contributes to ovarian cancer progression. In this study, we investigated whether resistance to TRAIL may be overcome by a monoclonal TRAILR2 (DR5) agonistic antibody (AD5-10). We found that the joint presence of AD5-10 with TRAIL and natural killer (NK) cells expressing TRAIL resensitizes ovarian cancer cells to apoptosis in vitro and in vivo, respectively. The combination of AD5-10 with carboplatin exerts a more than additive effect in vitro, which may at least partially be explained by the fact that carboplatin triggers DR5 expression on ovarian cancer cells. Moreover, AD5-10 restores the sensitivity of platin-resistant ovarian cancer to carboplatin in vivo. In addition, we found that TRAIL expression and NK cells are abundant in the tumor microenvironment and that depletion of NK cells abolishes the antitumor activity of AD5-10. This indicates that NK-mediated immunosurveillance against ovarian cancer might be mediated by TRAIL and that apoptosis induced by AD5-10 requires the presence of NK cells. In conclusion, this study indicates a key role and strong antitumorigenic effect of DR5 and highlights a novel link between NK-mediated immunosurveillance and activation of DR5-mediated apoptosis in ovarian cancer. Mol Cancer Ther; 9(4); 1007–18. ©2010 AACR.

Supplementary figure legends and the title and notes of supplementary tables

Objective Aim of the study was to investigate the expression of transforming growth factor-β1 (TGF-β1), a key regulator of the extracellular matrix composition, in the uterosacral ligaments (USLs) of women with pelvic organ prolapse (POP) compared with controls. We hypothesized that the expression pattern of TGF-β1 differs between postmenopausal women with or without POP. Methods Under ethical approval, USL samples were obtained from postmenopausal women undergoing vaginal hysterectomy for stage two or greater pelvic organ prolapse (cases, n = 70) and from postmenopausal women without pelvic organ prolapse undergoing vaginal hysterectomy for benign indications (controls, n = 30). Immunohistochemical staining was performed from paraffin embedded tissue using anti-TGF-β1 antibodies. The expression of TGF-β1 was evaluated by the pathologist, who was blinded to all clinical data. Results The expression of TGF-s1 was similar in patients with symptomatic POP (89 % positive) and in controls (90 % positive) without any signs of prolapse (p = 0.091). Age-adjusted analysis did not significantly alter these results. Regarding POP-Q stages, TGF-s1 was significantly more frequently expressed in severe prolapse cases compared to moderate/mild cases (POP-Q stage IV versus POP-Q stage II and III; p = 0.001). No significant association could be detected between TGF-s1 expression and age, BMI and parity in cases with POP (p > 0.05). As published previously, advanced patients’ age as well as early menopausal age remained independent risk factors associated with POP in multiple logistic regression analysis (p = 0.001; p = 0.02). Conclusion Although our study detected POP-Q stage related alterations in USL composition and TGF-s1 expression, there was no significant difference in the expression of TGF-β1 in cases with or without prolapse.

Introduction/Background In a recently published randomized, placebo-controlled phase II trial, we showed that topical imiquimod (IMQ) therapy is an efficacious and feasible treatment for selected patients with high-grade squamous intraepithelial lesion (HSIL). Aim of the present study was to investigate the non-inferiority of a 16-week topical IMQ therapy compared to large loop excision of the transformation zone (LLETZ) in patients diagnosed with cervical intraepithelial neoplasia (CIN) 2/3 (HSIL). Methodology Phase III randomized, controlled, multicenter, open, non-inferiority trial performed by Austrian Gynecologic Oncology group (AGO-Austria). Patients with histologically proven CIN 2 (older than 30 years) and CIN3 and satisfactory colposcopy were randomized (1:1) to topical IMQ treatment or LLETZ. Successful treatment was defined as negative HPV high-risk test (Roche cobas) result 6 months after start of the treatment. Secondary endpoints were long-term success of treatment and histological outcome. Results In total 93 patients were randomized and received the allocated treatment. The trial was stopped early due to poor accrual. CIN 2 and 3 at inclusion was present in 17 and 76 patients. In the IMQ group negative HPV test at 6 months after treatment start was observed in 66.7% of patients compared to 69.1% in LLETZ group (ITT analysis, rate difference 2.4%, 95% CI: -16.9 to -21.0). Negative HPV test at 12 months after treatment start was observed in 56.9% (IMQ) compared to 52.4% (LLETZ) of patients. In the IMQ group histologic regression 6 months after treatment was observed in 72.7% of patients. Complete resection was observed in 84.4% in the LLETZ group. Conclusion IMQ and LLETZ treatment seem to result in similar rates of negative HPV test results. We observed major difficulties in recruiting participants in the present academic RCT. Disclosure The present academic trial was partly funded by the Anniversary Fund of the Oesterreichische Nationalbank (OeNB). Stephan Polterauer, Alexander Reinthaller and Christoph Grimm have received honoraria for consultation from AstraZeneca, Celgene, MSD, PharmaMar, Roche,Roche Diagnostics, Meda Pharma, Tesaro, Vifor Pharma, outside the submitted work. Andreas Gleiss, Melanie Fraunschiel, Regina Berger, Gerhard Bogner, Olaf Reich, Gerda Trutnovsky, Stephan Aberle, Reinhard Horvat, Julia Ganhoer-Schimboeck, and Andreas Widschwendter have nothing to disclose.

Purpose: Most high-grade serous ovarian cancer (HGSOC) patients develop recurrent disease after first-line treatment, frequently with fatal outcome. This work aims at studying the molecular biology of both primary and recurrent HGSOC. Experimental Design: Gene expression profiles of matched primary and recurrent fresh-frozen tumor tissues from 66 HGSOC patients were obtained by RNA sequencing. Clustering analyses and pairwise comparison of the profiles between matched samples and subsequent functional alignment were used for the identification of molecular characteristics of HGSOC. Results: Both primary and recurrent HGSOC samples presented predominant gene expression differences in their microenvironment, determined by a panel of genes covering all major pathways of immune activation together with a number of genes involved in the remodeling of extracellular matrix and adipose tissues. Stratifying tumor tissues into immune active and silent groups, we further discovered that although some recurrent tumors shared the same immune status as their primary counterparts, others switched the immune status, either from silent to active or active to silent. Interestingly, genes belonging to the B7-CD28 immune checkpoint family, known for their major role as negative regulators of the immune response, were overexpressed in the immune active tumors. Searching for potential tumor antigens, CEACAM21, a member of the carcinoembryonic antigen family, was found to be significantly overexpressed in immune active tissues in comparison with the silent ones. Conclusions: The results illustrate the complexity of the tumor microenvironment in HGSOC and reveal the molecular relationship between primary and recurrent tumors, which have multiple therapeutic implications. Clin Cancer Res; 23(24); 7621–32. ©2017 AACR.

High grade serous ovarian cancer (HGSOC) is the most frequent type of ovarian cancer. Most patients have primary response to platinum-based chemotherapy but frequently relapse, which leads to patient death. A lack of well documented and characterized patient-derived HGSOC cell lines is so far a major barrier to define tumor specific therapeutic targets and to study the molecular mechanisms underlying disease progression. We established 34 patient-derived HGSOC cell lines and characterized them at cellular and molecular level. Particularly, we demonstrated that a cancer-testis antigen PRAME and Estrogen Receptor could serve as therapeutic targets. Notably, data from the cell lines did not demonstrate acquired resistance due to tumor recurrence that matched with clinical observations. Finally, we presented that all HGSOC had no or very low CDKN1A (p21) expression due to loss of wild-type TP53, suggesting that loss of cell cycle control is the determinant for tumorigenesis and progression. In conclusion, patient-derived cell lines reveal that PRAME is a potential tumor specific therapeutic target in HGSOC and counteracting the down-regulation of p21 caused by loss of wild-type TP53 might be the key to impede disease progression. ispartof: CANCER LETTERS vol:459 pages:1-12 ispartof: location:Ireland status: published

Summary: High-accuracy next-generation DNA sequencing promises a paradigm shift in early cancer detection by enabling the identification of mutant cancer molecules in minimally invasive body fluid samples. We demonstrate 80% sensitivity for ovarian cancer detection using ultra-accurate Duplex Sequencing to identify TP53 mutations in uterine lavage. However, in addition to tumor DNA, we also detect low-frequency TP53 mutations in nearly all lavages from women with and without cancer. These mutations increase with age and share the selection traits of clonal TP53 mutations commonly found in human tumors. We show that low-frequency TP53 mutations exist in multiple healthy tissues, from newborn to centenarian, and progressively increase in abundance and pathogenicity with older age across tissue types. Our results illustrate that subclonal cancer evolutionary processes are a ubiquitous part of normal human aging, and great care must be taken to distinguish tumor-derived from age-associated mutations in high-sensitivity clinical cancer diagnostics. : Salk et al. demonstrate that ultra-sensitive DNA sequencing to identify TP53 mutations among cells shed into uterine fluid shows potential for minimally invasive ovarian cancer detection. Yet they also reveal ubiquitous age-related accumulations of cancer-like TP53 mutations in the normal tissues of healthy women. This highlights an important challenge of using tumor driver mutations for cancer screening. Keywords: Duplex Sequencing, next-generation sequencing, TP53, clonal evolution, early detection, somatic mutations, high-grade serous ovarian cancer, uterine lavage, aging, gynecologic oncology

High accuracy next-generation DNA sequencing promises a paradigm shift in early cancer detection by enabling the identification of mutant cancer molecules in minimally-invasive body fluid samples. We demonstrate 80% sensitivity for ovarian cancer detection using ultra-accurate Duplex Sequencing to identify TP53 mutations in uterine lavage. However, in addition to tumor DNA, we also detect low frequency TP53 mutations in nearly all lavages from women with and without cancer. These mutations increase with age and share the selection traits of clonal TP53 mutations commonly found in human tumors. We show that low frequency TP53 mutations exist in multiple healthy tissues, from newborn to centenarian, and progressively increase in abundance and pathogenicity with older age across tissue types. Our results illustrate that subclonal cancer evolutionary processes are a ubiquitous part of normal human aging and great care must be taken to distinguish tumor-derived, from age-associated mutations in high sensitivity clinical cancer diagnostics.

// Katharina Auer 1 , Anna Bachmayr-Heyda 1 , Nyamdelger Sukhbaatar 1 , Stefanie Aust 1 , Klaus G. Schmetterer 2 , Samuel M. Meier 3 , Christopher Gerner 3 , Christoph Grimm 1 , Reinhard Horvat 4 , Dietmar Pils 1 1 Department of Obstetrics and Gynecology, Medical University of Vienna and Comprehensive Cancer Center, Vienna, Austria 2 Department for Laboratory Medicine, Medical University of Vienna, Vienna, Austria 3 Department of Analytical Chemistry, University of Vienna, Vienna, Austria 4 Department of Pathology, Medical University of Vienna, Vienna, Austria Correspondence to: Dietmar Pils, email: dietmar.pils@univie.ac.at Keywords: epithelial ovarian cancer, peritoneal tumor spread, flow cytometry, PD-1, next generation sequencing Received: May 05, 2016 Accepted: July 14, 2016 Published: August 03, 2016 ABSTRACT The immune system plays a critical role in cancer progression and overall survival. Still, it is unclear if differences in the immune response are associated with different patterns of tumor spread apparent in high grade serous ovarian cancer patients and previously described by us. In this study we aimed to assess the role of the immune system in miliary (widespread, millet-sized lesions) and non-miliary (bigger, exophytically growing implants) tumor spread. To achieve this we comprehensively analyzed tumor tissues, blood, and ascites from 41 patients using immunofluorescence, flow cytometry, RNA sequencing, multiplexed immunoassays, and immunohistochemistry. Results showed that inflammation markers were systemically higher in miliary. In contrast, in non-miliary lymphocyte and monocyte/macrophage infiltration into the ascites was higher as well as the levels of PD-1 expression in tumor associated cytotoxic T-lymphocytes and PD-L1 expression in tumor cells. Furthermore, in ascites of miliary patients more epithelial tumor cells were present compared to non-miliary, possibly due to the active down-regulation of anti-tumor responses by B-cells and regulatory T-cells. Summarizing, adaptive immune responses prevailed in patients with non-miliary spread, whereas in patients with miliary spread a higher involvement of the innate immune system was apparent while adaptive responses were counteracted by immune suppressive cells and factors.

Purpose Type II ovarian cancer (OC) and endometrial cancer (EC) are generally diagnosed at an advanced stage, translating into a poor survival rate. There is increasing evidence that Müllerian duct cancers may exfoliate cells. We have established an approach for lavage of the uterine cavity to detect shed cancer cells. Patients and Methods Lavage of the uterine cavity was used to obtain samples from 65 patients, including 30 with OC, five with EC, three with other malignancies, and 27 with benign lesions involving gynecologic organs. These samples, as well as corresponding tumor tissue, were examined for the presence of somatic mutations using massively parallel sequencing (next-generation sequencing) and, in a subset, singleplex analysis. Results The lavage technique could be applied successfully, and sufficient amounts of DNA were obtained in all patients. Mutations, mainly in TP53, were identified in 18 (60%) of 30 lavage samples of patients with OC using next-generation sequencing. Singleplex analysis of mutations previously determined in corresponding tumor tissue led to further identification of six patients. Taken together, in 24 (80%) of 30 patients with OC, specific mutations could be identified. This also included one patient with occult OC. All five analyzed lavage specimens from patients with EC harbored mutations. Eight (29.6%) of 27 patients with benign lesions tested positive for mutations, six (75%) as a result of mutations in the KRAS gene. Conclusion This study proved that tumor cells from ovarian neoplasms are shed and can be collected via lavage of the uterine cavity. Detection of OC and EC and even clinically occult OC was achieved, making it a potential tool of significant promise for early diagnosis.

Abnormalities of connective tissue structure or its repair mechanism may predispose women to pelvic organ prolapse (POP). We hypothesized that the expression of tenascin-X in the uterosacral ligament of postmenopausal women with symptomatic POP is increased compared with postmenopausal women without POP. Furthermore, we identified clinical risk factors associated with POP in our study population. We conducted a retrospective case-control study in which 33 postmenopausal women with symptomatic POP ≥ pelvic organ prolapse quantification system (POP-Q) stage II were matched with 33 postmenopausal women without POP. Studied tissue specimens were taken from hysterectomy specimens, and tenascin-X expression was investigated by immunohistochemistry. The immunohistochemical profile of the uterosacral connective tissue of cases and controls was compared. Tenascin-X was expressed in 94% of POP cases and in 91% of controls. Our study failed to show any statistically significant differences in tenascin-X expression between women with and without POP (p = 0.64). However, tenascin-X was significantly more expressed in cases with severe prolapse (POP-Q stage IV) compared with moderate prolapse stages (POP-Q stage II and III) (p = 0.001). Advanced patient age as well as early menopausal age remained independent risk factors associated with POP in multiple logistic regression analysis (p = 0.001). No difference could be demonstrated between tenascin-X expression in patients with or without POP. Tenascin-X does not seem to play a major role in the pathogenesis of POP in postmenopausal women.

PURPOSE: Although the discovery of prions was rewarded with a Nobel Prize, their existence was only attributed to a limited number of diseases. Recent evidence suggests that their role has been underestimated and several other proteins carry prion-like properties, like ß-amyloid, and most recently p53. High-grade serous ovarian cancers (HGSOC) harbor TP53 mutations in about 96% of cases. These mutations promote p53 aggregation, which might be responsible for complete abrogation of tumor suppressor functions, resulting in dominant-negative activity and oncogenic gain-of-function. Here, we describe the use of an ELISA-based technology for p53 prion detection in fresh-frozen tumor tissue and their clinical relevance in ovarian cancer. EXPERIMENTAL PROCEDURES: Fresh-frozen tumor tissue specimens of 81 HGSOC patients, who previously had been included in the EU-funded OVCAD study, were analyzed. For each of these patients at least 5-year follow-up data were available. For the detection of p53 prions the Seprion-ELISA, previously designed for the detection of BSE and scrapie was adapted and optimized. To investigate the impact of p53 aggregation on clinical outcomes (overall survival and progression-free survival), we performed a complete-case analysis. In a subset of patients the Ki67 proliferation index and a homologous recombination deficiency (HRD) score, based on the Myriad score, were available. Correlations were analyzed using ANOVA and t-test. RESULTS: In 39 of 46 (84.8%) patients with missense mutated cancers a p53 prion specific signal was observed. The aggregation propensity varied considerably within samples carrying the same mutations. Multivariable Cox regression models, with respect to other prognostic factors significantly associated with overall survival in patients with late-stage serous epithelial ovarian cancer (age, FIGO stage and presence of a residual tumor), show superiority of the group with extensive p53 aggregation in overall survival and in progression-free survival. No and moderate p53 aggregation are associated with a worse overall survival in contrast to high p53 aggregation (P values 0.025 and 0.011). Similar results in these groups are assessed for progression-free survival (P values 0.030 and 0.008). Interestingly, the group with extensive p53 aggregation was associated with a non-significant trend toward higher HR deficiency. Furthermore, this group had a significantly higher Ki67 index compared to patients with moderate p53 aggregation (P value 0.033). CONCLUSIONS: This study reports the first specific and quantitative screening for p53 prions in patient material. We were able to demonstrate that the p53-Seprion-ELISA is a robust and highly sensitive detection tool for p53 prions. Our data show that the aggregation propensity is not only depending on the TP53 mutation and that other cofactors may be involved. Moreover, we show that p53 aggregation is an independent prognostic marker for survival. The higher Ki67 proliferation index and the trend towards higher genomic instability in patients with extensive p53 aggregation suggest that these tumors have an increased likelihood of response to platinum-containing therapy. To conclude, we demonstrated the high potential of p53 aggregation as a biomarker for patients' survival, suggesting that classification of patients based on the amount of aggregated p53 could allow therapy decisions. Citation Format: Nicole Heinzl, Elisabeth Maritschnegg, Katarzyna Koziel, Stuart Wilson, Georg Heinze, Christine Wallisch, Reinhard Horvat, Jalid Sehouli, Ioana Braicu, Ignace Vergote, Els van Nieuwenhuysen, Sven Mahner, Eva Obermayr, Eva Schuster, Barbara Holzer, Nicole Concin, Robert Zeillinger. IDENTIFICATION OF P53 PRIONS AS AN INDEPENDENT PROGNOSTIC MARKER FOR SURVIVAL IN HIGH-GRADE SEROUS OVARIAN CANCER [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr AP15.

Aim of this study was to investigate the histologic outcome of cervical intraepithelial neoplasia (CIN) during observational management. Consecutive women with histologically verified CIN and observational management were included. Histologic findings of initial and follow-up visits were collected and persistence, progression and regression rates at end of observational period were assessed. Uni- and multivariate analyses were performed. A systematic review of the literature and meta-analysis was performed. In 783 women CIN I, II, and III was diagnosed by colposcopically guided biopsy in 42.5%, 26.6% and 30.9%, respectively. Younger patients had higher rates of regression (p 40 years, regression rates were 44.7%, 33.7%, 30.9%, 27.3%, and 24.9%, respectively. Pooled analysis of published data showed similar results. Multivariable analysis showed that with each five years of age, the odds for regression reduced by 21% (p

Cancer cell lines are good in vitro models to study molecular mechanisms underlying chemoresistance and cancer recurrence. Recent works have demonstrated that most of the available ovarian cancer cell lines are most unlikely high grade serous (HGSOC), the major type of epithelial ovarian cancer. We aimed at establishing well characterized HGSOC cell lines, which can be used as optimal models for ovarian cancer research. We successfully established seven cell lines from HGSOC and provided the major genomic alterations and the transcriptomic landscapes of them. They exhibited different gene expression patterns in the key pathways involved in cancer resistance. Each cell line harbored a unique TP53 mutation as their corresponding tumors and expressed cytokeratins 8/18/19 and EpCAM. Two matched lines were established from the same patient, one at diagnosis and being sensitive to carboplatin and the other during chemotherapy and being resistant. Two cell lines presented respective BRCA1 and BRCA2 mutations. To conclude, we have established seven cell lines and well characterized them at genomic and transcriptomic levels. They are optimal models to investigate the molecular mechanisms underlying the progression, chemo resistance and recurrence of HGSOC. publisher: Elsevier articletitle: Molecular characterization of 7 new established cell lines from high grade serous ovarian cancer journaltitle: Cancer Letters articlelink: http://dx.doi.org/10.1016/j.canlet.2015.03.040 content_type: article copyright: Copyright © 2015 The Authors. Published by Elsevier Ireland Ltd. ispartof: Cancer Letters vol:362 issue:2 pages:218-28 ispartof: location:Ireland status: published

Tumor-initiating subpopulations of cancer cells, also known as cancer stem cells (CSC), were recently identified and characterized in prostate cancer. A well-characterized murine model of prostate cancer was used to investigate the regulation of hypoxia-inducible factor 1α (HIF1A/HIF1α) in CSCs and a basal stem cell subpopulation (Lin−/Sca-1+/CD49f+) was identified, in primary prostate tumors of mice, with elevated HIF1α expression. To further analyze the consequences of hypoxic upregulation on stem cell proliferation and HIF1α signaling, CSC subpopulations from murine TRAMP-C1 cells (Sca-1+/CD49f+) as well as from a human prostate cancer cell line (CD44+/CD49f+) were isolated and characterized. HIF1α levels and HIF target gene expression were elevated in hypoxic CSC-like cells, and upregulation of AKT occurred through a mechanism involving an mTOR/S6K/IRS-1 feedback loop. Interestingly, resistance of prostate CSCs to selective mTOR inhibitors was observed because of HIF1α upregulation. Thus, prostate CSCs show a hypoxic deactivation of a feedback inhibition of AKT signaling through IRS-1. In light of these results, we propose that deregulation of the PI3K/AKT/mTOR pathway through HIF1α is critical for CSC quiescence and maintenance by attenuating CSC metabolism and growth via mTOR and promoting survival by AKT signaling. We also propose that prostate CSCs can exhibit primary drug resistance to selective mTOR inhibitors. Implications: This work contributes to a deeper understanding of hypoxic regulatory mechanisms in CSCs and will help devise novel therapies against prostate cancer. Mol Cancer Res; 13(3); 556–64. ©2014 AACR.

Ovarian cancer (OC) is caused by genetic aberrations in networks that control growth and survival. Importantly, aberrant cancer metabolism interacts with oncogenic signaling providing additional drug targets. Tumors overexpress the lipogenic enzyme fatty acid synthase (FASN) and are inhibited by FASN blockers, whereas normal cells are FASN-negative and FASN-inhibitor-resistant. Here, we demonstrate that this holds true when ovarian/oviductal cells reside in their autochthonous tissues, whereas in culture they express FASN and are FASN-inhibitor-sensitive. Upon subculture, nonmalignant cells cease growth, express senescence-associated β-galactosidase, lose FASN and become FASN-inhibitor-resistant. Immortalized ovarian/oviductal epithelial cell lines—although resisting senescence—reveal distinct growth activities, which correlate with FASN levels and FASN drug sensitivities. Accordingly, ectopic FASN stimulates growth in these cells. Moreover, FASN levels and lipogenic activities affect cellular lipid composition as demonstrated by thin-layer chromatography. Correlation between proliferation and FASN levels was finally evaluated in cancer cells such as HOC-7, which contain subclones with variable differentiation/senescence and corresponding FASN expression/FASN drug sensitivity. Interestingly, senescent phenotypes can be induced in parental HOC-7 by differentiating agents. In OC cells, FASN drugs induce cell cycle blockade in S and/or G2/M and stimulate apoptosis, whereas in normal cells they only cause cell cycle deceleration without apoptosis. Thus, normal cells, although growth-inhibited, may survive and recover from FASN blockade, whereas malignant cells get extinguished. FASN expression and FASN drug sensitivity are directly linked to cell growth and correlate with transformation/differentiation/senescence only indirectly. FASN is therefore a metabolic marker of cell proliferation rather than a marker of malignancy and is a useful target for future drug development.

Summary Background Even if vulvar cancer is not common, over one hundred women are affected in Austria per year. There is strong evidence that basaloid and warty variants are associated with types of human papillomavirus (HPV). Methods The aim of this study is to analyze the types of HPV in vulvar cancer in Austria. This cross-sectional period-prevalence international collaborative study on archival specimens was performed in cooperation with the Institut Catalan di Oncologia in Barcelona, Spain. A total of 177 consecutive samples of Austrian women were analyzed to detect the presence of various HPV types using the SPF10 PCR/DEIA/LiPA25 system. Furthermore, the expression of the tumor suppressor protein p16INK4a was analyzed by immunohistochemistry (CINtec histology kit, ROCHE). A tumor was considered HPV-driven if an overexpression of p16INK4a was detected. Results In all, 41 cases of vulvar cancer tested positive for HPV DNA (23%) and 32 (18%) were p16 positive. Patients with warty and basaloid squamous cell cancer were significantly younger than those with keratinizing squamous cell cancer (63.3 years vs. 71.0 years, p = 0.021). In addition, 77.4% of all cases suffering from warty or basaloid squamous cell cancer tested positive for HPV, compared to 9.5% of the keratinizing squamous cell cancer cases (p < 0.001). The most commonly detected HPV strain was type 16, followed by 31 and 33. Conclusion Infection with HPV type 16 appears to be strongly correlated to the development of warty or basaloid squamous cell cancer. Vaccination against HPV can be expected to prevent this type of vulvar cancer.

High-grade serous ovarian cancer (HGSOC) is characterized by a TP53 mutation rate of up to 96.7% and associated with a more aggressive tumor biology. The origin of HGSOC is thought to arise either from fallopian tube secretory cells or the ovarian surface epithelium/inclusion cysts, the former with more evidence. Peritoneal tumor spread is heterogeneous, either excessive in the peritoneum (with miliary appearance) or more confined to the ovaries with only few (bigger and exophytically growing) peritoneal implants. Using RNA sequencing and DNA digital droplet polymerase chain reaction (PCR), we identified two different functional TP53 mutations in one HGSOC patient: one exclusively in the ovarian tumor mass and the other exclusively in ascites tumor cells, peritoneal tumor masses, and a lymph node metastasis. In blood, both mutations could be detected, the one from the peritoneal tumors with much higher frequency, presumably because of the higher tumor load. We conclude that this mutually exclusive distribution of two different TP53 mutations in different tumor tissues indicates the development of two independent carcinomas in the peritoneal cavity, probably one originating from a precancerous lesion in the fallopian tube and the other from the ovaries. In addition, in the patient's ascites CD45 and EpCAM, double-positive cells were found—proliferating but testing negative for the above-mentioned TP53 mutations. This mutually exclusive distribution of two TP53 mutations is probably further evidence that HGSOC can originate either from the fallopian tube or (more seldom) the ovaries, the former more prone for excessive peritoneal tumor spread.

The purpose is to present the Austrian data of an observational, cross-sectional, multicentre study conducted in 13 European countries. This study was undertaken to assess human papillomavirus (HPV) type distribution in high-grade cervical lesions. Two hundred and ninety cases of high-grade cervical intraepithelial neoplasia (CIN2, CIN3) or adenocarcinoma in situ of the cervix from 2004 to 2007 were analysed by histopathological review and polymerase chain reaction for HPV. Two hundred and nine cases were “histologically eligible” and of those 206 were HPV+. Median age was 34 years (19–76 years). CIN3 was the most frequent diagnosis (64.6 %), followed by CIN2/3 (19.4 %) and CIN2 (13.6 %). Adenocarcinoma in situ was only detected together with a squamous lesion in 2.4 % cases. Majority of women (75.2 %) were infected with a single HPV type, 98.1 % of them with high-risk HPV types: HPV 16 (56.1 %), HPV 33 (12.3 %), HPV 31 (11.0 %), HPV 18 (3.9 %), HPV 35 (3.9 %), HPV 51 (3.2 %) and HPV 52 (2.6 %), HPV 58 (1.9 %) and HPV 45 (0.6 %). The low-risk HPV 6 was found in 1.3 % cases. Forty-six lesions (22.3 %) contained multiple infections, 71.1 % of those were HPV 16 or 18 and 91.1 % were HPV 16, 18, 31, 33 or 45 positive. These Austrian data clearly demonstrate that HPV 16 is the predominant type in high-grade CIN, the immediate precursor lesions of cervical cancer. More than 60 % of the lesions were associated with HPV 16 and 18 and more than 85 % with HPV 16, 18, 31, 33 or 45. The prevalence of adenocarcinoma in situ is underestimated, since its detection is difficult with the current cytology screening.

Tumor-infiltrating immune cells and their prognostic value have been analyzed in various malignancies. Although tissue microarray (TMA) has been used in some of these studies, it is still questionable whether this technique can represent the results of infiltrating CD8+ cells obtained from whole-tissue sections (WTS). The aim of this study was to assess and compare the density of tumor-infiltrating CD8+ cells in ovarian cancer using TMA and WTS. CD8+ lymphocytes were immunohistochemically stained on WTS and TMA cores from 37 ovarian cancer patients and quantified using the image analysis software HistoQuest. Four different areas were selected on the WTS, namely (i) tumor; (ii) stroma; (iii) mixed; and (iv) dense, whereby dense represented areas of most abundant CD8+ cells. On the TMA, (i) the whole TMA cores and (ii) areas containing only epithelial tumor tissue were analyzed. The Pearson correlation and principal component analysis was used to estimate the correlation of results from different techniques. CD8+ lymphocytes showed highly correlated measurements between tumor, mixed, and dense areas. Moderate correlations were found between each of these 3 measurements and stroma. CD8+ cell counts from WTS showed moderate correlation with TMA cell counts. Consistently, principal component analysis showed 3 clusters (i) tumor, dense, mixed; (ii) stroma; and (iii) TMA areas. Taken together, when the prognostic impact of tumor-infiltrating CD8+ cells in ovarian cancer is investigated with TMA technique, a moderate correlation with WTS results has to be considered.

Background: Expression of p95HER2 (p95), a truncated form of HER2 also known as p110 or M611-CTF, is a possible trastuzumab resistance mechanism and has been associated with poor prognosis in trastuzumab-treated HER2-positive metastatic breast cancer (MBC). Previously we reported on optimal clinical cutoffs for quantitative p95 (Clin Cancer Res, 16:4226, 2010) and quantitative HER2 protein expression (H2T) by HERmark® (Cancer, 116:5168, 2010) that defined patient subsets with different progression-free survival (PFS). These cutoffs were confirmed in an independent trastuzumab-treated MBC cohort (ASCO 2011, #586). Here, using individual patient data, we performed an analysis on the combined data set of 243 cases from the discovery and validation cohorts to derive optimal cutoffs for quantitative p95 and H2T. Methods: Both quantitative H2T (HERmark, Monogram Biosciences) and p95 assays employed the VeraTag® method to quantify protein expression in formalin-fixed, paraffin-embedded tumor samples from two cohorts of 101 and 142 cases of trastuzumab-treated MBC with 7.4 and 9.2 months median PFS, respectively. All analyses were stratified by hormone receptor status, tumor grade (3 vs. 1+2) and cohort. H2T measurements were compared to pre-specified cutoffs for HERmark negative (H2T17.8 RF/mm2), derived from the 95th percentile of centrally determined HER2-negatives, respectively, within a reference database of 1,090 breast cancer patient samples. Results: Patients classified as HERmark-positive had longer PFS than those classified as HERmark-negative (HR = 0.52; p = 0.0006; medians 10.0 and 5.9 months). The previously determined optimal H2T cutoff of 13.8 RF/mm2 in the center of the HERmark-equivocal zone, gave a similar result (HR = 0.54; p = 0.0005). This was close to the optimal cutoff of 12.75 RF/mm2 (HR = 0.48; p < 0.0001, unadjusted) for the combined data set. The PFS for the small group of patients in the HERmark-equivocal zone (n = 20) was more similar to the HERmark-negatives (equivocal vs. negative: HR=0.98; p = 0.9) than the HERmark-positives (positive vs. equivocal: HR=0.57; p = 0.057). The pre-specified p95 cutoff at 2.8 RF/mm2 separated the 174 HERmark-positive cases into two groups of longer (p95 Conclusions: HERmark positive and negative categories, defined by analytical comparison with centrally determined HER2 status, were confirmed to have significantly different PFS in trastuzumab-treated MBC patients. The optimal H2T clinical cutoff for this combined analysis was centered in the HERmark analytical equivocal zone. An optimal p95 clinical cutoff of 2.7 RF/mm2 derived from this combined analysis was nearly identical to the previously established cutoff of 2.8 RF/mm2. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-10-31.

Medullary thyroid carcinomas (MTCs) are mostly aggressive but slowly growing malignant tumours that metastasize early to loco-regional lymph nodes. Desmoplastic stroma reaction is a strong risk factor associated with lymph node metastases. We evaluated immunohistochemically the expression of two hypoxia-associated proteins, carbonic anhydrase IX (CAIX) and hypoxia-induced factor 1α (HIF1α), and ki-67, intercellular matrix adhesion molecule E-cadherin and the stroma remodelling marker tenascin C in a series of 100 sporadic MTCs and corresponding lymph node metastases, if present. Moderate to strong expression of CAIX was seen in 53 cases, and of HIF1α in 51 cases, showing a strong correlation (p < 0.001; Spearman's coefficient of correlation, 0.59). Expression correlated with the degree of desmoplasia (p CAIX = 0.001 and pHIF1α = 0.001), with tenascin C expression (pCAIX = 0.001, pHIF1α = 0.038), with the ki-67 proliferation index (pCAIX = 0.001, pHIF1α = 0.001) and with the presence of lymph node metastases (pCAIX < 0.001 and pHIF1α = 0.007). The absence of membranous E-cadherin staining was significantly associated with the grade of desmoplasia, tenascin expression and lymph node metastases (p≤0.05) but not with ki67 proliferation index or expression of hypoxia-associated factors. Expression of hypoxia-associated proteins was in most cases identical between primary tumours and lymph node metastases. Two cases showed strong uniform expression of CAIX and HIF1α in the primary tumour as well as in the lymph node metastases, and sequencing revealed mutations in the coding regions of the Von-Hippel-Lindau gene (VHL). Our findings suggest that despite of the fact that MTCs have only slowly growth, tumour hypoxia plays an important role in the development of loco-regional metastases. Since traditional cytotoxic chemotherapy has only little effect on MTCs, targeting hypoxia-associated and -regulated proteins might be of benefit for patients. Copyright © 2011 Pathological Society of Great Britain and Ireland. Published by John Wiley and Sons, Ltd.

Highlights ► Comparative oncological trials could speed up the development of novel therapies. ► Modeling revealed that human and canine ErbB-1 and ErbB-2 molecules are highly conserved. ► The canine ErbB molecules harbour epitopes for cetuximab and trastuzumab antibodies and indeed transmitted growth inhibitory signals. ► This knowledge opens up perspectives for comparative immunotherapy trials with canine cancer patients., To facilitate comparative oncology trials we compared the biological and molecular homologies of canine (dog; Canis lupus familiaris) and human tumor-associated antigens ErbB-1 and -2. Further, we investigated whether they could serve as targets for anti-ErbB-1 (cetuximab) and anti-ErbB-2 antibodies (trastuzumab), which are highly relevant in human clinical oncology. Immunohistochemistry of canine mammary cancer showed ErbB-1 overexpression in 3/10 patients and ErbB-2 in 4/10. We report 91% amino acid homology for ErbB-1 and 92% for ErbB-2 between canine and human molecules. Modeling of canine on human ErbB-1 revealed that the cetuximab epitope only differs by 4 amino acids: Lys443 is replaced by Arg, Ser468 by Asn, Gly471 by Asp, and Asn473 by Lys in canines. The trastuzumab binding site is identical in human and canine ErbB-2 apart from a single amino acid change (Pro557 to Ser). Binding of cetuximab and trastuzumab to canine mammary carcinoma cells CF33, CF41, Sh1b and P114 was confirmed by flow cytometry. Both antibodies significantly inhibited canine tumor cell proliferation partly due to growth arrest in G0/G1 phase. We explain the lower efficiency on the tested canine than on human SKBR3 and A431 cells, by a 2-log lower expression level of the canine ErbB-1 and -2 molecules. Our results indicate significant homology of human and canine Erb-1 and -2 tumor associated antigens. The fact that the canine homologues express the cetuximab and trastuzumab epitopes may facilitate antibody-based immunotherapy in dogs. Importantly, the striking similarities of ErbB-1 and -2 molecules open up avenues towards comparative strategies for targeted drug development.

Patients receiving tumour necrosis factor alpha (TNF-α) inhibitors are at increased risk of exacerbation of (myco-)bacterial and some viral infections. However, information on anogenital human papillomavirus (HPV) infection in these patients is sparse or conflicting. In this study 222 patients with psoriasis or inflammatory bowel disease (IBD), who received either anti-TNF-α inhibitors or alternatives (purine-, folic acid analogues, phototherapy, fumaric ester, mesalazine) continuously for at least 6 months, were evaluated for the presence of anogenital HPV-induced lesions, mucosal HPV DNA, and serological status of mucosal low-risk HPV6 and high-risk HPV16/HPV18. Hallmarks of anogenital HPV infection were more frequently detected in patients with psoriasis than in those with IBD. HPV-induced lesions, viral DNA, and seroprevalence were not elevated in participants with psoriasis or IBD, who received TNF-α inhibitors for a mean duration of 31.4 months (range 6-96 months) compared with recipients of alternative or no treatment. TNF-α blockade for a mean period of 31.4 months does not increase detectable anogenital HPV infection or disease.

Purpose: Although prognostic and predictive factors in ovarian cancer have been extensively studied for decades, only few have been identified and introduced to clinical practice. Here, we evaluate hVps37A (HCRP1) as a possible novel predictive marker for ovarian cancer. hVps37A was originally described as a member of the membrane-trafficking ESCRT-I complex mediating the internalization and degradation of ubiquitinated membrane receptors. Experimental Design: We analyzed an ovarian cancer tissue microarray for HCRP1, EGFR, and HER2 expression. We used a tetracycline inducible ovarian cancer cell culture model to show the effects of hVps37A knockdown in vitro and in vivo. In addition, we studied the effects of epidermal growth factor receptor (EGFR) inhibitors cetuximab and lapatinib on ovarian cancer cells under conditions of hVps37A knockdown. Results: We find that hVps37A is significantly downregulated in ovarian cancer and modifies the prognostic value of EGFR and HER2 expression. In addition, hVps37A downregulation in ovarian cancer cells leads to cytoplasmic pEGFR retention and hyperactivation of downstream pathways and is associated with enhanced xenograft growth in nude mice and invasion of the collagen matrix. Furthermore, due to subsequent sustained Akt- and MAPK-pathway activation, hVps37A-deficient cells become irresponsive to inhibition by the therapeutic antibody cetuximab. Conclusion: We propose that hVps37A status could become a novel prognostic and therapeutic marker for EGFR or HER2 driven tumors. Clin Cancer Res; 17(24); 7816–27. ©2011 AACR.