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Background: The need to better understand the molecular underpinnings of the heterogeneous outcomes of patients with prostate cancer is a pressing global problem and a key research priority for Movember. To address this, the Movember Global Action Plan 1 Unique tissue microarray (GAP1-UTMA) project constructed a set of unique and richly annotated tissue microarrays (TMA) from prostate cancer samples obtained from multiple institutions across several global locations. Methods: Three separate TMA sets were built that differ by purpose and disease state. Results: The intended use of TMA1 (Primary Matched LN) is to validate biomarkers that help determine which clinically localized prostate cancers with associated lymph node metastasis have a high risk of progression to lethal castration-resistant metastatic disease, and to compare molecular properties of high-risk index lesions within the prostate to regional lymph node metastases resected at the time of prostatectomy. TMA2 (Pre vs. Post ADT) was designed to address questions regarding risk of castration-resistant prostate cancer (CRPC) and response to suppression of the androgen receptor/androgen axis, and characterization of the castration-resistant phenotype. TMA3 (CRPC Met Heterogeneity)'s intended use is to assess the heterogeneity of molecular markers across different anatomic sites in lethal prostate cancer metastases. Conclusions: The GAP1-UTMA project has succeeded in combining a large set of tissue specimens from 501 patients with prostate cancer with rich clinical annotation. Impact: This resource is now available to the prostate cancer community as a tool for biomarker validation to address important unanswered clinical questions around disease progression and response to treatment.

Fluoroquinolones are a commonly used prophylaxis in transrectal ultrasound-guided prostate biopsy (TRUS-Bx), even though fluoroquinolone-resistant Escherichia coli has been associated with infectious complications after TRUS-Bx. The present study describes fluoroquinolone resistance mechanisms and antimicrobial susceptibility among intestinal E. coli, isolated from TRUS-Bx patients in a prospective study showing very few infectious prostate biopsy adverse events. This Multi-IMPROD sub-study included a total of 336 patients who received either ciprofloxacin, levofloxacin, or fosfomycin as prophylaxis before TRUS-Bx. E. coli could be cultured from 278 fecal swab samples, and 27 (9.7%) of these showed resistance to ciprofloxacin, and 14 (5.0%) were susceptible with increased exposure (I). Chromosomal and transferable fluoroquinolone resistance mechanisms were found among ciprofloxacin non-susceptible isolates, but both qnr genes and single gyrA mutations were found also among the ciprofloxacin-susceptible E. coli population. Low-level fluoroquinolone resistance is commonly associated with ESBL production in Enterobacterales. However, ESBL and qnr genes were not associated in our material, 14 isolates were ESBL producers and only 14.3% of them had the qnr gene, although 85.7% of the ESBL producers were ciprofloxacin non-susceptible. In the Multi-IMPROD substudy, only two mild urinary tract infections were reported, indicating that the antimicrobial susceptibility or resistance pattern of E. coli does not correlate with the onset of post-biopsy adverse events. We conclude that in our clinical settings, ciprofloxacin and levofloxacin prophylaxis is effective, and no severe post-biopsy infections were detected despite the intestinal colonization of genotypically and phenotypically fluoroquinolone-resistant E. coli.

Background: Accurate detection of clinically significant prostate cancer (csPCa), Gleason Grade Group ≥ 2, remains a challenge. Prostate MRI radiomics and blood kallikreins have been proposed as tools to improve the performance of biparametric MRI (bpMRI). Purpose: To develop and validate radiomics and kallikrein models for the detection of csPCa. Study Type: Retrospective. Population: A total of 543 men with a clinical suspicion of csPCa, 411 (76%, 411/543) had kallikreins available and 360 (88%, 360/411) did not take 5-alpha-reductase inhibitors. Two data splits into training, validation (split 1: single center, n = 72; split 2: random 50% of pooled datasets from all four centers), and testing (split 1: 4 centers, n = 288; split 2: remaining 50%) were evaluated. Field strength/Sequence: A 3 T/1.5 T, TSE T2-weighted imaging, 3x SE DWI. Assessment: In total, 20,363 radiomic features calculated from manually delineated whole gland (WG) and bpMRI suspicion lesion masks were evaluated in addition to clinical parameters, prostate-specific antigen, four kallikreins, MRI-based qualitative (PI-RADSv2.1/IMPROD bpMRI Likert) scores. Statistical Tests: For the detection of csPCa, area under receiver operating curve (AUC) was calculated using the DeLong's method. A multivariate analysis was conducted to determine the predictive power of combining variables. The values of P-value < 0.05 were considered significant. Results: The highest prediction performance was achieved by IMPROD bpMRI Likert and PI-RADSv2.1 score with AUC = 0.85 and 0.85 in split 1, 0.85 and 0.83 in split 2, respectively. bpMRI WG and/or kallikreins demonstrated AUCs ranging from 0.62 to 0.73 in split 1 and from 0.68 to 0.76 in split 2. AUC of bpMRI lesion-derived radiomics model was not statistically different to IMPROD bpMRI Likert score (split 1: AUC = 0.83, P-value = 0.306; split 2: AUC = 0.83, P-value = 0.488). Data Conclusion: The use of radiomics and kallikreins failed to outperform PI-RADSv2.1/IMPROD bpMRI Likert and their combination did not lead to further performance gains. Level of Evidence: 1. Technical Efficacy: Stage 2. publishedVersion

Due to its dynamic nature, the evolution of cancer cell-extracellular matrix (ECM) crosstalk, critically affecting metastasis and treatment resistance, remains elusive. Our results show that platinum-chemotherapy itself enhances resistance by progressively changing the cancer cell-intrinsic adhesion signaling and cell-surrounding ECM. Examining ovarian high-grade serous carcinoma (HGSC) transcriptome and histology, we describe the fibrotic ECM heterogeneity at primary tumors and distinct metastatic sites, prior and after chemotherapy. Using cell models from systematic ECM screen to collagen-based 2D and 3D cultures, we demonstrate that both specific ECM substrates and stiffness increase resistance to platinum-mediated, apoptosis-inducing DNA damage via FAK and β1 integrin-pMLC-YAP signaling. Among such substrates around metastatic HGSCs, COL6 was upregulated by chemotherapy and enhanced the resistance of relapse, but not treatment-naïve, HGSC organoids. These results identify matrix adhesion as an adaptive response, driving HGSC aggressiveness via co-evolving ECM composition and sensing, suggesting stromal and tumor strategies for ECM pathway targeting., Platinum chemotherapy is standard of care in ovarian cancers but treatment resistance commonly develops. Here, the authors show that the extracellular microenvironment is modulated following chemotherapy and the changes in matrix proteins and stiffness alter the cell death response of tumour cells.

High-grade serous ovarian cancer (HGSC) is the most prevalent and lethal ovarian cancer type. While HGSC usually responds well to primary treatment, most cases eventually relapse. Functional precision medicine - tailoring individualized treatments based on functional in vitro assays on patient-derived cells - has been recently employed in cancer clinical trials. Cancer organoids - three-dimensional, self-organizing, self-renewing cell cultures that recapitulate original tissue structure and function - have been applied as cellular models in these trials. However, in case of HGSC, organoid derivation has proven time consuming and inefficient, hindering their application in functional precision medicine due to a short time window, in which therapy for each patient needs to be selected. To address this problem, we aimed to establish whether drug vulnerabilities at HGSC relapse could be predicted using organoids derived from the primary disease cells. We derived sequential organoid models from material sampled during primary treatment and at relapse. Then, for organoid pairs (primary-relapse), we performed large-scale drug response profiling of a library of 370 compounds (approved drugs or drugs in clinical development), in 384-well microplate format, alone or in combination with a standard HGSC chemotherapeutic agent carboplatin. First, we found that HGSC organoid responses to standard chemotherapeutics retrospectively correlated to observed clinical treatment outcomes. But further, for each patient we identified compounds with pronounced cytotoxicity both in the primary and in the relapsed model, amounting to 66% of all hits (7% were primary-specific and 27% relapse-specific). We then focused on identifying patient-specific hits rather than compounds displaying general toxicity in all patient models. Based on a potential clinical applicability, for three patients we selected compounds for validation in organoid outgrowth assay, with prolonged (>1 month) drug-free period post-treatment. In two patients, AZD4573, a selective CDK9 inhibitor in clinical development for hematological malignancies, at nanomolar concentrations caused eradication of organoids when combined with carboplatin. Organoids from the third patient were vulnerable to nitazoxanide, an approved anti-helminthic agent and an inhibitor of mitochondrial oxidative phosphorylation. Importantly, the selected final hits were identified solely based on screening in organoid models from primary disease. In summary, we here demonstrate that HGSC organoids derived from primary disease material predict a majority of patient-specific drug vulnerabilities of organoids derived from the relapsed HGSC lesions. This indicates that patient stratification in functional precision medicine for treatment of HGSC relapse could be prospectively performed at the primary disease stage. Citation Format: Wojciech Senkowski, Laura Gall-Mas, Matias M. Falco, Yilin Li, Kari Lavikka, Mette C. Kriegbaum, Jaana Oikkonen, Daria Bulanova, Elin J. Pietras, Karolin Voßgröne, Yan-Jun Chen, Erdogan P. Erkan, Mia K. Høg, Ida M. Larsen, Tarja Lamminen, Katja Kaipio, Jutta Huvila, Anni Virtanen, Lars H. Engelholm, Pernille Christiansen, Eric Santoni Rugiu, Kaisa Huhtinen, Olli Carpén, Johanna Hynninen, Sampsa Hautaniemi, Anna Vähärautio, Krister Wennerberg. A platform utilizing high-grade serous ovarian cancer organoids for prospective patient stratification in functional precision medicine. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5779.

Chemotherapy resistance is a critical contributor to cancer mortality and thus an urgent unmet challenge in oncology. To characterize chemotherapy resistance processes in high-grade serous ovarian cancer, we prospectively collected tissue samples before and after chemotherapy and analyzed their transcriptomic profiles at a single-cell resolution. After removing patient-specific signals by a novel analysis approach, PRIMUS, we found a consistent increase in stress-associated cell state during chemotherapy, which was validated by RNA in situ hybridization and bulk RNA sequencing. The stress-associated state exists before chemotherapy, is subclonally enriched during the treatment, and associates with poor progression-free survival. Co-occurrence with an inflammatory cancer–associated fibroblast subtype in tumors implies that chemotherapy is associated with stress response in both cancer cells and stroma, driving a paracrine feed-forward loop. In summary, we have found a resistant state that integrates stromal signaling and subclonal evolution and offers targets to overcome chemotherapy resistance.

PurposeAlthough prostate cancer is the most common cancer in men in Western countries, there is significant variability in geographical incidence. This might result from genetic factors, discrepancies in screening policies or differences in lifestyle. Gut microbiota has been recently associated with cancer progression, but its role in prostate cancer is unclear.MethodsIn a prospective multicenter clinical trial (NCT02241122), the gut microbiota profiles of 181 men with a clinical suspicion of prostate cancer were assessed utilizing 16S rRNA gene sequencing. Sequences were assigned to operational taxonomic units, and differential abundance analysis, α- and β-diversities, and predictive functional (PICRUSt) analyses were performed. Additionally, plasma steroid hormone levels were correlated with the predicted microbiota functions.ResultsSeveral differences in the gut microbiota between the subjects with and without prostate cancer were noted. Prevotella 9, members of the Erysipelotrichaceae family and Escherichia-Shigella were higher, and Jonquetella, Moryella, Anaeroglobus, Corynebacterium and CAG-352 were lower in the cancer group. Predictive functional analyses revealed higher 5-α-reductase, copper absorption, and retinal metabolism in the prostate cancer associated microbiome. Plasma testosterone associated negatively with the microbial 5-α-reductase activity (p=0.030). In a subgroup of men taking 5-α-reductase inhibitors (n=17), plasma estrone (p=0.027) and estradiol (p=0.059) levels were lower in men with predicted elevation of the microbial 5-α-reductase function.ConclusionsGut microbiota of the prostate cancer patients differed significantly compared to benign subjects. Microbial 5-α-reductase, copper absorption and retinol metabolism are potential mechanisms of action. These findings could explain the observed association of lifestyle, geography, and prostate cancer incidence.

Background Multiparametric MRI of the prostate has been shown to improve the risk stratification of men with an elevated prostate-specific antigen (PSA). However, long acquisition time, high cost, and inter-center/reader variability of a routine prostate multiparametric MRI limit its wider adoption. Purpose To develop and validate nomograms based on unique rapid biparametric MRI (bpMRI) qualitative and quantitative derived variables for prediction of clinically significant cancer (SPCa). Study type Retrospective analyses of single (IMPROD, NCT01864135) and multiinstitution trials (MULTI-IMPROD, NCT02241122). Population 161 and 338 prospectively enrolled men who completed the IMPROD and MULTI-IMPROD trials, respectively. Field strength/sequence IMPROD bpMRI: 3T/1.5T, T2 -weighted imaging, three separate diffusion-weighted imaging (DWI) acquisitions: 1) b-values 0, 100, 200, 300, 500 s/mm2 ; 2) b values 0, 1500 s/mm2 ; 3) values 0, 2000 s/mm2 . Assessment The primary endpoint of the combined trial analysis was the diagnostic accuracy of the combination of IMPROD bpMRI and clinical variables for detection of SPCa. Statistical tests Logistic regression models were developed using IMPROD trial data and validated using MULTI-IMPROD trial data. The model's performance was expressed as the area under the curve (AUC) values for the detection of SPCa, defined as ISUP Gleason Grade Group ≥2. Results A model incorporating clinical variables had an AUC (95% confidence interval) of 0.83 (0.77-0.89) and 0.80 (0.75-0.85) in the development and validation cohorts, respectively. The corresponding values for a model using IMPROD bpMRI findings were 0.93 (0.89-0.97), and 0.88 (0.84-0.92), respectively. Further addition of the quantitative DWI-based score did not improve AUC values (P Data conclusion A prediction model using qualitative IMPROD bpMRI findings demonstrated high accuracy for predicting SPCa in men with an elevated PSA. Online risk calculator: http://petiv.utu.fi/multiimprod/ Level of Evidence: 1 Technical Efficacy Stage: 2 J. Magn. Reson. Imaging 2020;51:1556-1567.

Many patients with muscle-invasive bladder cancer (BC) are either ineligible for or do not benefit from cisplatin-based chemotherapy, and there is an unmet need to estimate individuals' drug sensitivities. We investigated the suitability of conditionally reprogrammed (CR) cells for the characterization of BC properties and their feasibility for personalized drug sensitivity screening. The CR cultures were established from six BC tumors with varying histology and stage. Four cultures were successfully propagated for genomic, transcriptomic, and protein expression profiling and compared to the parental tumors. Two out of four CR cultures (urothelial carcinoma and small cell neuroendocrine carcinoma [SmCC]) corresponded well to their parental tumors and underwent drug sensitivity screening to identify novel drugs for the respective tumors. Both cultures were sensitive to standard BC chemotherapy agents (eg cisplatin and gemcitabine) and to conventional drugs such as taxanes and inhibitors of topoisomerase and proteasome. The SmCC cells were also sensitive to statins (eg, atorvastatin and pitavastatin). In summary, after confirming their representativeness and origin, we conclude that CR cells are a feasible platform for personalized drug sensitivity testing and might thus add to the approaches used to personalize BC treatment strategies. PATIENT SUMMARY: We investigated the conditional reprogramming method for generating patient-derived bladder cancer cell cultures and studied their feasibility for planning personalized treatment strategies.

Extensive utilization of organoids from high-grade serous ovarian carcinoma (HGSOC), the most common and lethal ovarian cancer, has been hampered by low success rates of long-term culture and scarcity of fresh tumor material. Here we present the development of a novel method for efficient generation, expansion and use of HGSOC organoids from cryopreserved tumor material. First, we assessed commonly used organoid media components and found that supplements such as FGF-2, R-Spondin1, Wnt or Noggin had negative impact on the HGSOC organoid derivation. But further, we found that supplementation with FGF-4, which has not been used in cancer organoid culture before, is beneficial for HGSOC organoid growth. Through extensive testing of various supplements and their combinations, we designed two novel HGSOC organoid media formulations - Medium 1 (M1) and Medium 2 (M2). Using M1 and M2 enabled generation and long-term expansion of living HGSOC organoid biobank with markedly improved success rate than in previous reports (55% vs. 23-38%). The organoids were established from cryopreserved tumor material, demonstrating the feasibility of using frozen tissue biobanks for HGSOC organoid derivation. Overall, we generated a collection of 18 expandable HGSOC organoid lines from 11 patients, encompassing samples from different tissue sites and disease progression stages. We validated the organoids using whole-genome sequencing, immunohistochemistry and single-cell RNA sequencing and demonstrated that they are genetically and phenotypically representative of original patient samples over long-term culture. Based on available patient consents, we deposited 3 organoid lines in a publicly accessible biobank. Finally, we investigated whether organoid drug responses correlate to those observed earlier in the clinic in the corresponding patients. Organoid-based drug-response profiling of clinically used HGSOC drug collection was performed in 384-well microplate format. To explore whether growth conditions impact correlation between organoid drug responses and clinical response, we compared the organoid drug responses in the nutrient-rich M1/M2 growth media to the ones observed in human plasma-like medium (HPLM), supplemented with relevant niche factors from M1/M2. Organoid drug responses correlated with clinical treatment outcomes, but only for organoids maintained in HPLM (Spearman r = 0.987, p=0.007 in HPLM vs 0.607, p=0.167 in growth medium, n=7), highlighting the importance of culture conditions in organoid-based functional assays. Taken together, we introduce a resource for efficient development and use of HGSOC organoids from cryopreserved material in ovarian cancer research. Citation Format: Wojciech Senkowski, Laura Gall Mas, Matias M. Falco, Yilin Li, Kari Lavikka, Mette C. Kriegbaum, Jaana Oikkonen, Daria Bulanova, Elin J. Pietras, Karolin Voßgröne, Erdogan P. Erkan, Terese K. Høj, Mia K. Høg, Tarja Lamminen, Katja Kaipio, Anni Virtanen, Lars H. Engelholm, Pernille Christiansen, Eric Santoni-Rugiu, Kaisa Huhtinen, Olli Carpén, Johanna Hynninen, Sampsa Hautaniemi, Anna Vähärautio, Krister Wennerberg. Efficient establishment and utilization of a high-grade serous ovarian cancer organoid biobank [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3069.

Chemotherapy resistance is the greatest contributor to cancer mortality and the most urgent unmet challenge in oncology. In order to reveal transcriptomics changes due to platinum-based chemotherapy we analyzed single-cell RNA-seq data from fresh tissue samples taken at the time of diagnosis and after neoadjuvant chemotherapy from 11 high-grade serous ovarian cancer (HGSOC) patients. With a novel clustering method accounting for patient-specific variability and technical confounders, we identified 12 clusters. Of these, a stress-related transcriptional profile was enriched during chemotherapy and associated significantly to poor progression-free survival (PFS) and disease-free interval (DFI) in deconvoluted bulk RNA-seq data analysis of treatment-naive samples in TCGA cohort. Pan-cancer cell line analysis suggests that patients with high stress-related transcriptional profile may benefit from MEK1/2 inhibitors instead of platinum. Further, high proportion of stromal components and high interaction score between tumor and stromal suggest the tumor cells with high-stress profile actively interact with and potentially recruit stromal cells to their microenvironment already prior to chemotherapy, potentially facilitating protection from chemotherapeutic treatments. In summary, we have identified a stress-related transcriptional profile, which is present at the time of diagnosis, enriched during platinum treatment, independent predictor for poor PFS and DFI, and, based on in vitro data, targetable with MEK1/2 inhibitors.Translational relevanceWe discovered a stress-related transcriptional profile that is significantly enriched in fresh tissue samples after chemotherapy and is significantly associated with poor progression-free survival in an independent patient cohort. The survival association is independent of age, tumor purity or BRCAness. Therefore, this chemotherapy resistance associated profile is intrinsic and could thus be targeted already in treatment-naive patients. The translation potential of the stress-related transcriptomics profile was further supported by pan-cancer cell line analysis that showed that cell lines with high stress-related transcriptional profile are not affected by platinum, corroborating our results, whereas they were more sensitive to MEK1/2 inhibitors. Taken together, the stress-related transcriptional profile, quantifiable with a set of 35 marker genes, provides a basis for improved prediction of platinum response as well as novel avenues to treat this patient group more effectively.

Prostate Magnetic Resonance Imaging (MRI) is increasingly being used in men with a clinical suspicion of prostate cancer (PCa). Performing prostate MRI without the use of an intravenous contrast (IV) agent in men with a clinical suspicion of PCa can lead to reduced MRI scan time. Enabling a large array of different medical providers (from mid-level to specialized radiologists) to evaluate and potentially report prostate MRI in men with a clinical suspicion of PCa with a high accuracy could be one way to enable wide adoption of prostate MRI in men with a clinical suspicion of PCa. The aim of this pictorial review is to provide an insight into acquisition, quality control and reporting of prostate MRI performed without IV contrast agent in men with a clinical suspicion of PCa, aimed specifically at radiologists starting reporting prostate MRI, urologists, urology/radiology residents and mid-level medical providers without experience in reporting prostate MRI. Free public access ( http://petiv.utu.fi/improd/ and http://petiv.utu.fi/multiimprod/ ) to complete datasets of 161 and 338 men is provided. The imaging datasets are accompanied by clinical, laboratory and histopathological findings. Several topics are simplified in order to provide a solid base for the development of skills needed for an unsupervised review and potential reporting of prostate MRI in men with a clinical suspicion of PCa. The current review represents the first step towards enabling a large array of different medical providers to review and report accurately prostate MRI performed without IV contrast agent in men with a clinical suspicion of PCa.

Background Multiparametric prostate magnetic resonance imaging (mpMRI) can be considered the gold standard in prostate magnetic resonance imaging (MRI). Biparametric prostate MRI (bpMRI) is faster and could be a feasible alternative to mpMRI. Objective To determine the negative predictive value (NPV) of Improved Prostate Cancer Diagnosis (IMPROD) bpMRI as a whole and in clinical subgroups in primary diagnostics of clinically significant prostate cancer (CSPCa). Design, setting, and participants This is a pooled data analysis of four prospective, registered clinical trials investigating prebiopsy IMPROD bpMRI. Men with a clinical suspicion of prostate cancer (PCa) were included. Intervention Prebiopsy IMPROD bpMRI was performed, and an IMPROD bpMRI Likert scoring system was used. If suspicious lesions (IMPROD bpMRI Likert score 3–5) were visible, targeted biopsies in addition to systematic biopsies were taken. Outcome measurements and statistical analysis Performance measures of IMPROD bpMRI in CSPCa diagnostics were evaluated. NPV was also evaluated in clinical subgroups. Gleason grade ≥3 + 4 in any biopsy core taken was defined as CSPCa. Results and limitations A total of 639 men were included in the analysis. The mean age was 64 yr, mean prostate-specific antigen level was 8.9 ng/ml, and CSPCa prevalence was 48%. NPVs of IMPROD bpMRI Likert scores 3–5 and 4–5 for CSPCa were 0.932 and 0.909, respectively, and the corresponding positive predictive values were 0.589 and 0.720. Only nine of 132 (7%) men with IMPROD bpMRI Likert score 1–2 had CSPCa and none with Gleason score >7. Thus, 132 of 639 (21%) study patients could have avoided biopsies without missing a single Gleason >7 cancer in the study biopsies. In the subgroup analysis, no clear outlier was present. The limitation is uncertainty of the true CSPCa prevalence. Conclusions IMPROD bpMRI demonstrated a high NPV to rule out CSPCa. IMPROD bpMRI Likert score 1–2 excludes Gleason >7 PCa in the study biopsies. Patient summary We investigated the feasibility of prostate magnetic resonance imaging (MRI) with the Improved Prostate Cancer Diagnosis (IMPROD) biparametric MRI (bpMRI) protocol in excluding significant prostate cancer. In this study, highly aggressive prostate cancer was excluded using the publicly available IMPROD bpMRI protocol ( http://petiv.utu.fi/multiimprod/ ).

A major challenge in analyzing cancer patient transcriptomes is that the tumors are inherently heterogeneous and evolving. We analyzed 214 bulk RNA samples of a longitudinal, prospective ovarian cancer cohort and found that the sample composition changes systematically due to chemotherapy and between the anatomical sites, preventing direct comparison of treatment-naive and treated samples. To overcome this, we developed PRISM, a latent statistical framework to simultaneously extract the sample composition and cell type specific whole-transcriptome profiles adapted to each individual sample. Our results indicate that the PRISM-derived composition-free transcriptomic profiles and signatures derived from them predict the patient response better than the composite raw bulk data. We validated our findings in independent ovarian cancer and melanoma cohorts, and verified that PRISM accurately estimates the composition and cell type specific expression through whole-genome sequencing and RNA in situ hybridization experiments. PRISM is freely available with full source code and documentation.

BackgroundMagnetic resonance imaging (MRI) combined with targeted biopsy (TB) is increasingly used in men with clinically suspected prostate cancer (PCa), but the long acquisition times, high costs, and inter-center/reader variability of routine multiparametric prostate MRI limit its wider adoption.Methods and findingsThe aim was to validate a previously developed unique MRI acquisition and reporting protocol, IMPROD biparametric MRI (bpMRI) (NCT01864135), in men with a clinical suspicion of PCa in a multi-institutional trial (NCT02241122). IMPROD bpMRI has average acquisition time of 15 minutes (no endorectal coil, no intravenous contrast use) and consists of T2-weighted imaging and 3 separate diffusion-weighed imaging acquisitions. Between February 1, 2015, and March 31, 2017, 364 men with a clinical suspicion of PCa were enrolled at 4 institutions in Finland. Men with an equivocal to high suspicion (IMPROD bpMRI Likert score 3-5) of PCa had 2 TBs of up to 2 lesions followed by a systematic biopsy (SB). Men with a low to very low suspicion (IMPROD bpMRI Likert score 1-2) had only SB. All data and protocols are freely available. The primary outcome of the trial was diagnostic accuracy-including overall accuracy, sensitivity, specificity, negative predictive value (NPV), and positive predictive value-of IMPROD bpMRI for clinically significant PCa (SPCa), which was defined as a Gleason score ≥ 3 + 4 (Gleason grade group 2 or higher). In total, 338 (338/364, 93%) prospectively enrolled men completed the trial. The accuracy and NPV of IMPROD bpMRI for SPCa were 70% (113/161) and 95% (71/75) (95% CI 87%-98%), respectively. Restricting the biopsy to men with equivocal to highly suspicious IMPROD bpMRI findings would have resulted in a 22% (75/338) reduction in the number of men undergoing biopsy while missing 4 (3%, 4/146) men with SPCa. The main limitation is uncertainty about the true PCa prevalence in the study cohort, since some of the men may have PCa despite having negative biopsy findings.ConclusionsIMPROD bpMRI demonstrated a high NPV for SPCa in men with a clinical suspicion of PCa in this prospective multi-institutional clinical trial.Trial registrationClinicalTrials.gov NCT02241122.

Background Biparametric magnetic resonance imaging (bpMRI) combined with prostate-specific antigen density (PSAd) may be an effective strategy for selecting men for prostate biopsy. It has been shown that performing biopsy only for men with bpMRI Likert scores of 4–5 or PSAd ≥0.15 ng/ml/cm3 is the most efficient strategy. Objective To externally validate previously published biopsy strategies using two prospective bpMRI trial cohorts. Design, setting, and participants After IMPROD bpMRI, 499 men had systematic transrectal prostate biopsies and men with IMPROD bpMRI Likert scores of 3–5 had an additional two to four targeted biopsies. Outcome measurements and statistical analysis Various IMPROD bpMRI Likert score and PSAd thresholds were assessed using detection rates for significant prostate cancer (sPCa; Gleason score ≥3 + 4), predictive values, and proportion of biopsies avoided. Net benefits and decision curve analyses (DCA) were compared with the aim of finding an optimal strategy for sPCa detection. Combined biopsies were used for reference. Results and limitations The negative predictive value (NPV) for sPCa in IMPROD bpMRI Likert 3–5 and 4–5 score groups was 93% and 92%, respectively, while the corresponding positive predictive value (PPV) was 57% and 72%, respectively. In DCA, the optimal combination was IMPROD bpMRI Likert score 4–5 or Likert 3 with PSAd ≥0.20 ng/ml/cm3, which had NPV of 93% and PPV of 67%. Using this combination, 35% of the study patients would have avoided biopsies and 13 sPCas (6%, 13/229, of all sPCas diagnosed) would have been missed. Conclusions IMPROD bpMRI demonstrated a good NPV for sPCa. PSAd improved the NPV mainly among men with equivocal suspicion on IMPROD bpMRI. However, the additional value of PSAd was marginal: the NPV and PPV for IMPROD bpMRI Likert 4–5 score group were 92% and 72%, respectively, while the corresponding values for the best combination strategy were 93% and 67%. Patient summary We investigated a rapid prostate magnetic resonance imaging protocol (IMPROD bpMRI) combined with prostate-specific antigen (PSA) density for detection of significant prostate cancer. Our results show that IMPROD bpMRI is a good diagnostic tool, but the additional value provided by PSA density is marginal.

The functional properties of actin-regulating formin proteins are diverse and in many cases cell-type specific. FHOD1, a formin expressed predominantly in cells of mesenchymal lineage, bundles actin filaments and participates in maintenance of cell shape, migration and cellular protrusions. FHOD1 participates in cancer associated epithelial to mesenchymal transition (EMT) in oral squamous cell carcinoma and breast cancer. The role of FHOD1 in melanomas has not been characterized. Here, we show that FHOD1 expression is typically strong in cutaneous melanomas and cultured melanoma cells while the expression is low or absent in benign nevi. By using shRNA to knockdown FHOD1 in melanoma cells, we discovered that FHOD1 depleted cells are larger, rounder and have smaller focal adhesions and inferior migratory capacity as compared to control cells. Importantly, we found FHOD1 depleted cells to have reduced colony-forming capacity and attenuated tumor growth in vivo, a finding best explained by the reduced proliferation rate caused by cell cycle arrest. Unexpectedly, FHOD1 depletion did not prevent invasive growth at the tumor margins. These results suggest that FHOD1 participates in key cellular processes that are dysregulated in malignancy, but may not be essential for melanoma cell invasion.

Extracellular matrix (ECM) interactions contribute to cancer metastasis and chemotherapy resistance by regulating invasive cancer growth and apoptosis evasion. However, the key ECM pathways activated during cancer evolution, including tumor metastasis and tissue responses to chemotherapy, remain to be systematically identified. Using a unique longitudinal high-grade serous ovarian carcinoma (HGSOC) cohort to understand the ECM deposition and remodeling in response to disease development and chemotherapeutics, we conducted ECM proteome, i.e., matrisome, analysis on HGSOC tissues of distinct anatomic sites from treatment-naïve and post-chemotherapy primary samples. Together with an unbiased functional ECM screen for platinum-treatment responses, we report how the solid tumor ECM evolves with disease progression and how specific stromal ECM proteins, including fibronectin, promote ovarian cancer cell motility, invasion, and resistance to platinum treatment. Importantly, we observed these specific stromal ECM proteins to be expressed already at HGSOC omental microlesions with remarkable desmoplastic reaction, as shown by immunohistochemistry of longitudinally collected patient cohort samples. Further, by using clinical The Cancer Genome Atlas (TCGA) microarray data we found that at transcriptional level the overexpression of a specific ECM protein, with platinum-induced cancer cell spreading and migratory response, also associated with poor survival in the treatment-naïve TCGA patient data, and was specifically upregulated after chemotherapy in our longitudinal cohort. In ovarian cancer cell cultures and in patient-derived 3D HGSOC organoid cultures, this specific ECM-rich microenvironment associated with an epithelial-to-mesenchymal transition transcriptional markers coupled to a stem-like morphology and properties with increased resistance. These results underline the importance of cell-matrix communication in invasion and chemotherapy response, revealing certain ECM proteins as important promoters of aggressiveness and chemotherapy resistance in the metastatic HGSOC lesions. Citation Format: Elina A. Pietilä, Jordi Gonzales-Molina, Lidia Moyano-Galceran, Laura Lehtinen, Pauliina Turunen, Tomas Santos Martins, Ville Rantanen, Antti Häkkinen, Sanaz Jamalzadeh, Kaiyang Zhang, Tarja Lamminen, Katja Kaipio, Johanna Hynninen, Sakari Hietanen, Seija Grénman, Rainer Lehtonen, Sampsa Hautaniemi, Olli Carpén, Kaisa Lehti. Extracellular matrix proteins increase invasive growth and chemotherapy resistance of ovarian cancer cells [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A64.

Tumor heterogeneity in high-grade serous ovarian cancer (HGSOC) stems from highly variable copy number changes that lack shared actionable drug targets and contribute to inevitable recurrence and death. To unveil the vast heterogeneity in HGSOC, and understand the molecular changes that occur during chemotherapy, we analyzed single-cell transcriptomes of 49 dissociated HGSOC tumor or ascites specimens, along with six normal postmenopausal fimbria/tuba specimens as controls. This set contains 13 pairs of samples from patients who underwent neoadjuvant chemotherapy, taken before treatment (primary), and after three cycles of standard carboplatin-paclitaxel chemotherapy (interval). Clustering single-cell transcriptomes based on acknowledged cell type markers revealed epithelial cancer cells, as well as diverse stromal and immune cell types. The cell type proportions displayed high variability across HGSOC tumors, intriguingly with lowest tumor purity in patients with short platinum-free interval. To identify the impact of chemotherapy on transcriptional programs, we extracted shared transcriptomic differences in the cancer cells of seven primary-interval pairs. Regardless of the clinical response, three cycles of chemotherapy induced an oxidative stress response program that inhibits apoptosis, while inducing genes involved in MHC II antigen presentation to T helper cells. Next, to study how cell states change during chemotherapy, we ordered the cells in pseudotime based on their transcriptomic similarity, revealing a mixture of primary and interval cells in all but two primary-interval pairs. Furthermore, based on inferred copy number analysis, we estimate that genomic differences contribute variably, between 20%, to the overall transcriptomic differences between the primary and interval pairs. These results suggest that pre- and post-chemotherapy states are not completely separate and that genomic evolution has a highly variable effect on the state differences, providing support for an intrinsic chemoresistance model with genomic and nongenomic components. To further the understanding of intrinsic resistance, we next focused on patients’ clinical response mechanisms in HGSOC cancer cells. Genes encoding well-known serum markers of HGSOC, WFDC2 and MUC16, were expressed at higher levels in primary specimens of patients with incomplete response, whereas patients with complete response displayed higher expression of genes related to chromatin repression/heterochromatin. Finally, we focused on tumor resectability to understand the transcriptomic patterns underlying HGSOC metastasis. Already prior to chemotherapy, patients with residual disease showed induced FOXO3-driven regulon linked to quiescence, stress resistance, and stemness, whereas patients with no residual disease express increased level of E2F1/DP1-driven regulon linked to BRCAness, high proliferation, and high metabolic rate. Citation Format: Erdogan Pekcan Erkan, Jun Dai, Kaiyang Zhang, Katja Kaipio, Tarja Lamminen, Naziha Mansuri, Lasse Suominen, Amjad Alkodsi, Kaisa Huhtinen, Sakari Hietanen, Johanna Hynninen, Seija Grénman, Olli Carpén, Sampsa Hautaniemi, Anna Vähärautio. Singling out tumor heterogeneity and chemoresistance in high-grade serous ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A40.

Objectives To determine, in a prospective, multicentre setting, the prevalence of fluoroquinolone-resistant (FQ-R) and extended-spectrum β-lactamase (ESBL)-producing Escherichia coli (E. coli) strains in men undergoing transrectal ultrasonography-guided prostate biopsy (TRUS-Bx) in Finland; and to survey the associated risk factors for having the previously mentioned strains. Patients and methods This is a substudy of the trial investigating the role of magnetic resonance imaging (MRI) in prostate cancer diagnosis (Improved Prostate Cancer Diagnosis - Combination of Magnetic Resonance Imaging Targeted Biopsies and Biomarkers Multi-institutional Study [multi-IMPROD], NCT02241122). In all, 359 patients from four study centres were recruited to this prospective study. After having signed the informed consent form, these men with suspicion of prostate cancer completed a detailed questionnaire on their medical, smoking, and travelling history, as well as their recent use of antibiotics. After the bi-parametric MRI scan, TRUS-Bx was taken and a rectal swab sample was collected and cultured for determining the antimicrobial susceptibility profile of E. coli strains. The potential risk factors for having FQ-R or third-generation cephalosporin-resistant (3GC-R) E. coli strains were analysed using univariate and multivariate logistic regression analysis. Results The percentage of FQ-R and 3GC-R E. coli strains amongst the study population was 13% and 8%, respectively. Amongst patients having E. coli strains, the rate of FQ-R and 3GC-R strains was 14% and 8%, respectively. Of the 3GC-R E. coli strains, 62% proved to be ESBL-producers and 88% were also FQ-R. In multivariate analysis, international travel during the preceding year significantly increased the risk of having a FQ-R E. coli strain (odds ratio [OR] 3.592, P = 0.001) and, unexpectedly, use of antibiotics during the previous year significantly decreased this risk (OR 0.442, P = 0.035). No significant risk factors for having 3GC-R E. coli were identified. Conclusion The occurrence of intestinal FQ-R and/or 3GC-R (potentially ESBL-producing) E. coli strains in men undergoing TRUS-Bx in Finland is notable. The finding is consistent with the global increase in antimicrobial resistance. International travel appears to be an indisputable risk factor for having intestinal FQ-R E. coli strains. The contemporary antimicrobial resistance situation should be taken into account in the care of post-TRUS-Bx infections.

Background Transrectal ultrasound-guided prostate biopsy (TRUS-Bx) is typically considered a safe procedure. However, infectious complications have been increasing. Objective To determine the contemporary rate of biopsy-related infectious and noninfectious complications after TRUS-Bx, and identify potential risk factors associated with the complications. Design, setting, and participants This was a prospective multicenter study and a substudy of a trial investigating the role of magnetic resonance imaging (MRI) in prostate cancer diagnosis (multi-IMPROD, NCT02241122). Intervention TRUS-Bx was performed for all patients included in the study. Ciprofloxacin, levofloxacin, or fosfomycin was administered for antibiotic prophylaxis. Outcome measurements and statistical analysis On inclusion, patients completed a detailed questionnaire and underwent MRI scanning. Antibiotic prophylaxis was prospectively recorded. After collection of a rectal swab, TRUS-Bx (total of 14–18 biopsy cores) was performed and. The rectal swabs were cultured and the antimicrobial susceptibility profile of Escherichia coli strains was analyzed. Biopsy complications leading to a visit to a health care unit were recorded and potential risk factors for complications were analyzed. Results and limitations Twelve of the 294 patients (4.1%) had a biopsy-related complication, of which two (0.7%) were infectious and managed in the outpatient setting. Some 11% of the patients had an E. coli strain resistant to the prophylactic antibiotic administered. Conclusions The risk of an infectious or noninfectious complication after TRUS-Bx is very low, although the FQ resistance rate in the study population was significant. Accordingly, the present TRUS-Bx procedure and antibiotic prophylaxis are efficient in guarding against biopsy complications, but regional resistance rates may affect the generalizability of the results. Patient summary We examined the rate of complications after prostate biopsies in 294 patients. The risk of having a biopsy-related complication was low (4.1%). The rate of infectious complications was reasonably low (0.7%) although antibiotic resistance to the prophylactic antibiotic regimen was significant (11%).

High-grade serous ovarian cancer (HGSOC) is the most common cause of gynecologic-cancer-associated death. Although HGSOC is initially extremely sensitive to platinum-based chemotherapy, relapse and progression to chemotherapy resistance are frequently seen, resulting in the low 5-year survival rate of this disease. Therefore, we aim to establish a feasible methodology that combines genome editing and single-cell RNA-seq to high-throughput screen new potential targets that could be utilized alone or combined with platinum for HGSOC treatment. Firstly, we constructed several inducible Cas9-expressed HGSOC cell lines, including both platinum-sensitive and -resistant cells. Secondly, we transduced these cell lines with a pooled sgRNA lentivirus consisting of 105 non-target scrambled sgRNAs and sgRNAs against 188 genes (10 sgRNAs per gene) that are currently targets of FDA/EMA approved drugs or drugs undergoing clinical trials in ovarian cancer treatment. Our analysis of multiple HGSOC dissociated tumors by single-cell RNA-seq has revealed intertumoral heterogeneity in expression of these drug targets in vivo. The majority of these 188 genes, approximately two thirds, are expressed in low level in most epithelial ovarian cancer (EOC) cells. Only 4 genes (AKT1, IL6, C1QA, INHBA) show relatively high expression, and only in a small subset of EOC cells. Also, the remaining drug target genes that are expressed show intertumoral heterogeneity, e.g., high expression of PARP-1 in tumors that are potentially deficient in homologous recombination repair. To elucidate the functionality of these drug targets, we will next analyze the transcriptomic effects of specific gene CRISPR/Cas9 knockouts in these HGSOC cell lines by droplet-based single-cell RNA-seq. From the data, we will compare the differences between platinum-sensitive and -resistant cell lines after the gene knockouts to identify the correlations between specific genetic perturbations and platinum resistance. As a future perspective, we will continue optimizing our platform for screening other potential targets. Overall, this study provides a comprehensive and unbiased approach for potential HGSOC target screening. Note: This abstract was not presented at the conference. Citation Format: Jun Dai, Erdogan Pekcan Erkan, Kaiyang Zhang, Katja Kaipio, Tarja Lamminen, Kaisa Huhtinen, Johanna Hynninen, Seija Grenman, Olli Mikael Carpen, Sampsa Hautaniemi, Anna Vähärautio. High-throughput screening of new potential targets for high-grade serous ovarian cancer treatment. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B40.

Objective: High-grade serous ovarian cancer (HGSOC) remains the most lethal gynecologic malignancy. More than 50% of high-grade serous ovarian cancer (HGSOC) patients die due to recurrent disease within 5 years. Recurrent tumor clones are selected by chemotherapy treatment from heterogeneous subpopulations of cancer cells, and differ phenotypically. The objective of current study is to develop a high-throughput chemical biology approach to analyze phenotypic heterogeneity in HGSOC following progression of the disease. Methods: We utilize the Drug Sensitivity and Resistance Testing (DSRT) platform to assess the effects of 528 approved and investigational anti-cancer agents on primary HGSOC isolates. Samples are collected from ascites and/or solid high-grade ovarian tumors from the patients at different disease stages. DSRT profiling of the samples integrates viability and toxicity readouts with high-content imaging in order to assess the drug responses of phenotypically different cell subpopulations of epithelial cancer cells and stromal component. Results: Drug sensitivity profiling of ten primary and six relapse patient samples revealed heterogeneous drug sensitivity profiles characterized by common responsiveness to HDAC inhibitors, CDK inhibitors, and PI3K/mTOR inhibitors (mean drug sensitivity score (DSS) > 10). Relapse HGSOC samples demonstrated general trend towards resistance to a broad spectrum of chemotherapeutics and targeted drugs. Imaging-based DSRT profiling revealed that in both primary and relapse ascites-derived cancer cells the response to platinum drugs and DNA damaging agents associated with upregulation of transcriptional regulators of stemness, including BMI1, SOX2 and NANOG. The ascites-derived CK18-expressing epithelial cells collected from the same patient a) before chemotherapy, b) after 6 cycles of Carboplatin treatment and c) at the relapse demonstrated an increasing sensitivity to aldehyde dehydrogenase inhibitor disulfiram. These preliminary findings suggest that increase in a subpopulation of cancer cells with stem-like characteristics may accompany recurrence in HGSOC. Conclusions: Compound sensitivity testing combined with high-content imaging appears an informative tool to explore therapeutic vulnerabilities in chemoresistant subpopulations of ovarian cancer cells, and improves the accuracy of drug sensitivity profiling. Chemical biology screening of prospectively collected primary HGSOC samples can also help identifying phenotypic changes associated with chemoresistance development. Citation Format: Daria R. Bulanova, Katja Kaipio, Tarja Lamminen, Aleksandr Ianevski, Tero Aittokallio, Olli Carpen, Krister Wennerberg. Chemical biology approach to phenotypic intra-tumor heterogeneity in high-grade serous 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 875.

Hydroxysteroid (17β) dehydrogenase 7 (HSD17B7) has been shown to catalyze the conversion of both estrone to estradiol (17-ketosteroid reductase activity) and zymosterone to zymosterol (3-ketosteroid reductase activity involved in cholesterol biosynthesis) in vitro. To define the metabolic role of the enzyme in vivo, we generated knockout mice deficient in the enzyme activity (HSD17B7KO). The data showed that the lack of HSD17B7 results in a blockage in the de novo cholesterol biosynthesis in mouse embryos in vivo, and HSD17BKO embryos die at embryonic day (E) 10.5. Analysis of neural structures revealed a defect in the development of hemispheres of the front brain with an increased apoptosis in the neuronal tissues. Morphological defects in the cardiovascular system were also observed from E9.5 onward. Mesodermal, endodermal, and hematopoietic cells were all detected by the histological analysis of the visceral yolk sac, whereas no organized vessels were observed in the knockout yolk sac. Immunohistological staining for platelet endothelial cell adhesion molecule-1 indicated that the complexity of the vasculature also was reduced in the HSD17B7KO embryos, particularly in the head capillary plexus and branchial arches. At E8.5–9.5, the heart development and the looping of the heart appeared to be normal in the HSD17B7KO embryos. However, at E10.5 the heart was dilated, and the thickness of the cardiac muscle and pericardium in the HSD17B7KO embryos was markedly reduced, and immunohistochemical staining for GATA-4 revealed that HSD17B7KO embryos had a reduced number of myocardial cells. The septum of the atrium was also defected in the knockout mice.

When present in excess amounts during fetal life, androgens can impair female development by inducing masculinization. On way to modify fetal steroid concentration is by altering the expression of hydroxysteroid (17beta) dehydrogenases (HSD17Bs). Human HSD17B1 converts weak estrogen estrone to estradiol, and with lower catalytic efficiency, weak androgen androstenedione to testosterone. We have recently shown that over-expression of human HSD17B1 in transgenic mice results in masculinized phenotype in female mice. In the present study, we further show that in addition to the Müllerian ducts, HSD17B1TG females have internal structures resembling Wolffian ducts, and enlarged Skene paraurethral gland, also called the female prostate. HSD17B1 expression has been found in fetal human ovary, thus, it is possible that HSD17B1 contributes to maintain the normal steroid hormone concentration during development. Thereby, abnormal increase in the fetal expression of HSD17B1 could contribute to the development of hormonal imbalances, and so result in female masculinization.

Hydroxysteroid (17beta) dehydrogenases (HSD17Bs) belong to the short-chain dehydrogenase/reductase family consisting of a diverse pool of enzymes with oxidoreductase activity. HSD17B enzymes catalyze the conversion between 17-keto and 17-hydroxy steroids, either activating or inactivating sex steroids. Previous studies have demonstrated a role for human HSD17B1 enzyme in estradiol (E2) biosynthesis both in gonads and extragonadal steroid target tissues and various estrogen-dependent diseases. In the present study, five transgenic (TG) mouse lines universally overexpressing human HSD17B1 were generated and characterized at fetal and adult ages, especially to study the enzyme function in vivo. Activity measurements in vivo indicated that in addition to activating estrone to E2, the enzyme is able to significantly reduce androstenedione to testosterone, and TG females presented increased testosterone concentration preceding birth. As a consequence, TG females suffered from several phenotypic features typical to enhanced fetal androgen exposure. Furthermore, the ovaries developed androgen-dependent ovarian benign serous cystadenomas at adulthood. Androgen dependency of the phenotypes was confirmed by rescuing them by antiandrogen treatment, or by transplanting wild-type ovaries to the TG females. In conclusion, the data evidently show that, in addition to activating estrone to E2, human HSD17B1 enhances androgen action in vivo. Thus, the relative amounts of androgenic and estrogenic substrates available partially determine the physiological function of the enzyme in vivo. The novel function observed for human HSD17B1 is likely to open new possibilities also for the use of HSD17B1-inhibitors as drugs against androgen-related dysfunctions in females.

Hydroxysteroid (17beta) dehydrogenase 2 (HSD17B2) has been shown to inactivate both estrogens and androgens and activate 20alpha-hydroxyprogesterone to progesterone. In the present study, we generated transgenic (TG) mice ubiquitously expressing human HSD17B2. The TG mice produced showed growth retardation and delayed eye opening at the postnatal age. Disrupted spermatogenesis was evident in the presence of normal serum and intratesticular testosterone, progesterone, and normal circulating LH concentrations. A proper androgen action in the target tissues was confirmed by normal histological appearance of the prostate and epididymis. Furthermore, quantitative RT-PCR analysis indicated only a slight decrease in androgen-dependent gene expression in the prostate. The disrupted spermatogenesis was not associated with increased germ cell apoptosis as analyzed by caspase-3 activation. However, it resulted in infertility in the HSD17B2 TG males after the age of 3 months, and at the age of 6 months the seminiferous tubules showed a Sertoli cell-only phenotype. The data indicate that the growth retardation and disrupted spermatogenesis are not due to a lack of proper estrogen or androgen action. Interestingly, the testicular phenotype and some of the other phenotypic changes described are typically observed in mice with reduced action of retinoic acid signaling. This, together with the rescue of the testis phenotype by a synthetic retinoic acid receptor agonist (4-[(E)-2-(5, 6, 7, 8-tetrahydro-5, 5, 8, 8-tetramethyl-2-naphthalenyl)-1-propenyl] benzoic acid), suggests a role for HSD17B2 in the action of retinoids, in addition to its oxidative HSD17B activity on sex steroids.

FSH is a pituitary gonadotropin that along with LH plays a key role in the regulation of gonadal function. The gonadotropic hormones are composed of two subunits, the common alpha subunit and the hormone-specific beta subunit, which determines the binding to specific receptors and induction of biological response. Unlike the LHbeta gene, shown in earlier studies to harbour several amino acid-altering polymorphisms and mutations, information about the eventual sequence variation of the human FSHbeta subunit is not available. In this study, we made sequence analysis and comparison of polymorphisms found in FSHbeta in two Caucasian populations, the Finns and the Danes. It was found that FSHbeta subunit is highly conserved in these populations. Compared with the published sequences, only three silent polymorphisms were detected in the coding regions of the gene, and the promoter sequence was completely identical with the reported sequence. Two of the polymorphisms found were novel, one in the Finnish and one in the Danish population. The results of the sequence analysis show that the human FSHbeta gene is highly conserved and amino acid changing mutations are apparently extremely rare, at least in the samples collected randomly from control populations. This may be due to the crucial role of normal FSH function in the regulation of fertility.

Upon screening for polymorphisms in the human luteinizing hormone beta-subunit (LH beta) gene, we discovered a novel mutation in the LH beta signal peptide with functional consequences for signal transduction in mouse Leydig tumour cells (mLTC-1). This G(52)A point mutation in exon 2 of the LH beta gene, detected in heterozygous form in several normal DNA samples, caused an Ala(-3)Thr amino acid substitution. Recombinant forms of wild-type (WT) and Ala(-3)Thr variant (V) LH were produced in human embryonic kidney (HEK) 293 cells and purified. The immunoreactivities of the recombinant LH were determined by immunofluorometric assays and in-vitro bioactivities in mLTC-1 cells were assessed by using cAMP, progesterone and inositol trisphosphate (IP(3)), and activation of mitogen-activated protein kinase (MAPK) as end-points. Whereas both LH forms stimulated progesterone production and MAPK in similar fashion, WT-LH was more potent in stimulating cAMP, and V-LH was more potent in stimulating IP(3) generation. Both LH forms bound to LH receptors with similar affinities. No evidence was found for influence of the signal peptide mutation on efficacy of alpha- and beta-subunit dimerization. Sequencing of the recombinant V-LH beta protein also revealed that the mutation did not interfere with signal peptide cleavage. In summary, the present findings indicate that the Ala(-3)Thr mutation in the LH beta-subunit signal peptide has functional consequences, in the form of dissociation of stimulatory potency for different signal transduction pathways in vitro.

Resistance to chemotherapy poses a major clinical problem in the treatment of ovarian cancer. The efficacy of platinum- based chemotherapy relies on defective DNA repair mechanisms, and increased expression of homologous recombination (HR)/DNA repair genes in primary tumor samples is associated with improved chemoresponse. However, the effects of neo-adjuvant chemotherapy (NACT) on the expression DNA repair genes in cancer cells are poorly understood. In addition, high-grade serous ovarian cancers (HGSOCs) display significant inter- and intratumoral heterogeneity, and the identification of mechanisms of drug resistance has been hampered by the lack of data at the single cell resolution. We collected 34 primary and 20 interval tumor samples from 15 patients treated for HGSOC at the Turku University Hospital, Finland. Tumor tissue total RNA was sequenced with Illumina HiSeq. For this study, we selected 363 genes selected due to their validated role in DNA repair pathways and compared their expression profiles between primary and interval samples. The treatment naïve primary tumor samples highly expressed genes related to HR, such as BRCA2, RAD54, PARPBP, FANCD2, and POLQ. Whereas after NACT the interval samples highly expressed genes regulating cell cycle, inflammatory response, and the drug efflux pump ABCB1. Moreover, higher expression of BRCA2 or POLQ in the interval samples correlated with poor primary therapy outcome (p To reveal the transcriptomics landscapes of treatment naïve and NACT treated HGSOCs at the single cell level, we performed single cell mRNA sequencing of primary and interval samples using the Fluidigm C1 Platform. Unsupervised clustering revealed eight distinct cell subpopulations, of which two clusters were annotated HGSOC cells based on enriched expression of cancer specific genes. In global gene expression analysis of 98 HGSOC cells, the most differentially expressed genes in primary HGSOC cells were heat shock proteins reflecting active protein synthesis. By contrast, the cancer cells from interval samples highly expressed genes regulating the cell cycle and immune response. Of the DNA repair pathway genes, the interval samples expressed lower levels of HES1, a transcription factor that is known to promote cancer progression and stemness. The herein elucidated DNA repair pathway transcriptomics landscape at single-cell and whole tumor level provides novel targets for overcoming chemoresistance in HGSOC. Citation Format: Anniina Farkkila, Kaiyang Zhang, Katja Kaipio, Tarja Lamminen, Rainer Lehtonen, Johanna Hynninen, Seija Grénman, Olli Carpén, Sampsa Hautaniemi, Anna Vähärautio. Single-cell transcriptomics reveals differential DNA repair signatures after chemotherapy in high-grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1428. doi:10.1158/1538-7445.AM2017-1428

Leber hereditary optic neuropathy (LHON) is a maternally inherited eye disease most commonly caused by mitochondrial DNA (mtDNA) point mutation at position 11778, 3460, or 14484. Approximately 14% of families show heteroplasmy for the pathogenic mutations but little is known about the mutational burden in different tissues of these heteroplasmic individuals. Consequently, estimating the risks of visual loss is difficult. This study presents quantitative mutation analyses of tissues representing all embryonal layers in two families heteroplasmic for the 11778 mutation. These analyses show that a high amount of mutated mtDNA in leukocytes is correlated with a high proportion of mutated mtDNA in other tissues.

Local estrogen production plays a key role in proliferative endometrial disorders, such as endometrial hyperplasia and cancer. Hydroxysteroid (17beta) dehydrogenase 1 (HSD17B1) is an enzyme that catalyzes with high efficiency the conversion of weakly active estrone into highly potent estradiol. Here we report that female transgenic mice expressing human HSD17B1 invariably develop endometrial hyperplasia in adulthood. These mice also fail to ovulate and have enhanced peripheral conversion of estrone into estradiol in a variety of target tissues, including the uterus. As in humans, endometrial hyperplasia in HSD17B1 transgenic female mice was reversible on ovulation induction, which triggers a rise in circulating progesterone levels, and in response to exogenous progestins. Strikingly, a treatment with an HSD17B1 inhibitor failed to restore ovulation yet completely reversed the hyperplastic morphology of epithelial cells in the glandular compartment, although less so in the luminal epithelium. The data indicate that human HSD17B1 expression enhances endometrial estrogen production, and consequently, estrogen-dependent proliferation. Therefore, HSD17B1 is a promising new therapeutic target in the management of estrogen-dependent endometrial diseases.

Summary Hydroxysteroid (17β) dehydrogenase 1 (HSD17B1) catalyzes the reaction between the low active 17-ketosteroids and the highly active 17beta-hydroxysteroids. In the present study, we have generated transgenic (TG) mice expressing human (h) HSD17B1 under mouse mammary tumor virus (MMTV) promoter (MMTV-hHSD17B1TG mice). The MMTV-hHSD17B1TG mice were used to characterize HSD17B1 enzyme activity and properties of HSD17B1 inhibitor in vivo. Expression of the transgene was detected by enzyme activity and RT-PCR analysis. Increased HSD17B1 activity in the TG mice was detected in vivo by applying estrone as a substrate via an intravenous injection. The developed enzyme activity measurement was then applied to analyze the efficacy of HSD17B1 inhibitor in vivo. The results indicated that the MMTV-hHSD17B1TG mouse model is a valuable novel tool to test human HSD17B1 inhibition by various compounds in vivo. With the potent hHSD17B1 inhibitor compound tested, at highest an 85% and 33% inhibition of the enzyme activity in males and in females, respectively, was observed.

Considerable attention has been paid to the role of sex steroids during periods of major skeletal turnover, but the interaction of the gonadotropic hormones, which include LH, FSH, and human chorionic gonadotropin (hCG), within bone tissue have been overlooked. The question is pertinent due to the recent detection of extragonadal expression of gonadotropin receptors. Western blotting, immunolocalization, and RT-PCR supported the presence of osteoblast LH receptors. However, osteoblast cells failed to bind [125I]hCG and treatment with hCG failed to generate either cAMP or phosphorylated ERK 1/2. Bone mineral density (BMD) and bone histomorphometry were examined in the following models: 1) LH receptor null mutant (LuRKO) mice; 2) transgenic mice overexpressing hCG (hCG αβ+); and 3) ovariectomized (OVX) hCG αβ+ model. Male LuRKO mice showed a decrease in BMD after 5 months, apparently secondary to suppressed gonadal steroid production. Similarly, 9- to 10-wk-old female LuRKO mice exhibited decreases in histomorphometric parameters tested. The data indicate that loss of LH signaling results in a reduction in bone formation or an increase in bone resorption. By contrast, there were significant increases in BMD and histomorphometric indices for female, but not male, hCG αβ+ mice, indicating that chronic exposure to hCG results in bone formation or a decrease in bone resorption. However, OVX of the hCG αβ+ mice resulted in a significant reduction in BMD comparable to OVX WT controls. Although gonadotropin levels are tightly linked to sex steroid titers, it appears that their effects on the skeleton are indirect.

Mutations of the luteinizing hormone (LH) subunit genes are extremely rare. Only one polymorphic LHbeta gene variant makes an exception. In 1992, an immunologically anomalous form of LH was found in a healthy woman, and it was subsequently found to be caused by two point mutations leading to two amino acid substitutions in the LHbeta subunit. Of the two point mutations, Trp(8)Arg and Ile(15)Thr, the first one is mainly responsible for the altered immunoreactivity and the latter one introduces an extra glycosylation site into Asn(13) of the mutated LHbeta peptide. The frequency of this variant LHbeta allele differs widely between ethnic groups, being most common in aboriginal Australians (carrier frequency >50%; allelic frequency 28.3%) and totally lacking from Kotas of Southern India. Functional differences have been detected when wild-type LH and variant LH have been compared. Variant LH possesses increased in vitro bioactivity, whereas its half-life in circulation is shorter in comparison to wild-type LH. Also the regulation of the variant LHbeta gene differs due to additional changes in its promoter sequence. Correlations of occurrence of variant LH with various clinical conditions involving LH function suggest that it represents a biologically less active form of LH and may be related to borderline suppression of gonadal function, including subfertility. In this article, we will review the current information about the differences observed in structure and functions between the wild-type and variant LH, as well as their possible pathophysiological correlations.

A lowered efficiency of oxidative phosphorylation was recently found in a Leber hereditary optic neuropathy (LHON) proband carrying a mutation in the mtDNA gene for subunit 6 of the membrane-bound F0 segment of the F1F0-ATP synthase [9]. This phenotype was transferred to cytoplasmic hybrid cells together with the mutation, proving its functional significance. Increasing the respiratory rate in the mitochondria from this mutant raised the ATP/2e− ratio back to normal values. A different mutation in the same mtDNA gene has been found in patients with the NARP syndrome [10]. Although the ATP/2e− ratio is also decreased in this mutant, in this case an increase in the respiratory rate could not compensate for it. Whilst both mutations affect subunit 6 of the proton-translocating F0 segment, the LHON mutation induces a proton leak whereas the NARP mutation blocks proton translocation. Hence, the latter will have much more destructive metabolic consequences in agreement with the large clinical differences between the two diseases.

Ovarian cancer is the most lethal gynecologic malignancy in developed countries. The incidence is 1 in 70 women, and 5-year survival rate below 50 %. Although major achievements have been reached in understanding the molecular alterations underlying the disease, the discoveries need yet to be integrated into clinical practice. A major obstacle in this process may be the translation of in vitro findings into diagnostic tests and novel therapeutic approaches. Well-characterized and representative preclinical mouse models that recapitulate the heterogeneity of tumors in patients are crucial in narrowing the gap between experimental and clinical studies. The aim of this work was to test and validate various mouse xenograft models for their applicability in ovarian cancer research. We tested several xenograft mouse models using (1) established commercially available ovarian cancer cell lines, (2) primary cell lines originating from high grade serous ovarian cancers or ascitic fluid, and (3) primary or metastatic tumor tissue directly from operation. Cell growth, tumor take rate and histopathological correlation between mouse and human tumors were applied for selection of commercial cell lines in xenograft experiments into athymic nude mice. Intrabursal orthotopic inoculations were used to model early, and intraperitoneal inoculations to model late stage ovarian cancer. Translational aspects were studied by comparing the histopathology between mouse tumors and human ovarian cancer specimens and by testing the utility of serum biomarkers CA-125 and HE-4 in mouse models. The mouse xenograft models faithfully represented the morphology of human serous ovarian cancer and many of the tissue markers were similarly expressed in human and mouse tumors. Importantly, the serum biomarker analysis developed in this study allowed follow-up of the tumor burden ex vivo. Such biomarker will be useful in studying drug responses and other tumor modifying therapies. The primary cell lines were inoculated subcutaneously to immunodeficient NOD-SCID and NOG mice in order to enrich and characterize ovarian cancer initiating cells (OCICs). Due to the low rate of tumor formation of in vitro cultured OCICs, we have started to use patient derived tumor xenograft (PDTX) transplantations. In this model fresh surgical tissue, sectioned in app. 3 mm3 pieces are implanted into the flanks of immunodeficient NOG mice. The preliminary results show a take rate of 75 % with PDTXs. Histopathological comparisons of original human specimen to mouse tumors reveals that this model also recapitulates the human microenvironment in PDTX modelling. Also, the PDTX model appears to increase the probability for successfully establishing novel primary cell lines. In conclusion, mouse xenograft models offer translational and important validation methods for pre-clinical cancer drug development and diagnostics. Citation Format: Tarja Lamminen, Katja Kaipio, Piia Mikkonen, Pia Roering, Olli Carpén. Xenograft mouse models for ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr B43.

The mitochondrial complex I genes were sequenced in seven Leber hereditary optic neuroretinopathy (LHON) families without the ND4/11,778 and ND1/3460 mutations. Four replacement mutations restricted only to LHON families were found, one in the ND1 gene at nt 4025, and three in the ND5 gene at nt 12,811, 13,637, and 13,967. The mutations did not change evolutionarily conserved amino acids suggesting that they are not primary LHON mutations in these families. They may be considered as secondary LHON mutations serving as exacerbating factors in an appropriate genetic background. A complex III mutation, cyt b/15,257, has been suggested to be one of the primary mutations causing LHON. Its presence was determined for 23 Finnish LHON families, and it was detected in two families harboring the ND4/11,778 mutation. Similarly, complex IV mutation COI/7444 was screened in Finnish LHON families, and it was found in one family carrying the ND1/3460 mutation.

The broad research use of organoids from high-grade serous ovarian carcinoma (HGSC) has been hampered by low culture success rates and limited availability of fresh tumor material. Here we describe a method for generation and long-term expansion of HGSC organoids with efficacy markedly improved over previous reports (55% vs. 23-38%). We established organoids from cryopreserved material, demonstrating the feasibility of using viably biobanked tissue for HGSC organoid derivation. Genomic, histologic and single-cell transcriptomic analyses revealed that organoids recapitulated genetic and phenotypic features of original tumors. Organoid drug responses correlated with clinical treatment outcomes, although in culture conditions-dependent manner and only in organoids maintained in human plasma-like medium (HPLM). Organoids from consenting patients are available to the research community through a public biobank and organoid genomic data explorable through an interactive online tool. Taken together, this resource facilitates the application of HGSC organoids in basic and translational ovarian cancer research.