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The recommended treatment for patients with high-risk non–muscle-invasive bladder cancer (HR-NMIBC) is tumor resection followed by adjuvant Bacillus Calmette-Guérin (BCG) bladder instillations. However, only 50% of patients benefit from this therapy. If progression to advanced disease occurs, then patients must undergo a radical cystectomy with risks of substantial morbidity and poor clinical outcome. Identifying tumors unlikely to respond to BCG can translate into alternative treatments, such as early radical cystectomy, targeted therapies, or immunotherapies. Here, we conducted molecular profiling of 132 patients with BCG-naive HR-NMIBC and 44 patients with recurrences after BCG (34 matched), which uncovered three distinct BCG response subtypes (BRS1, 2 and BRS3). Patients with BRS3 tumors had a reduced recurrence-free and progression-free survival compared with BRS1/2. BRS3 tumors expressed high epithelial-to-mesenchymal transition and basal markers and had an immunosuppressive profile, which was confirmed with spatial proteomics. Tumors that recurred after BCG were enriched for BRS3. BRS stratification was validated in a second cohort of 151 BCG-naive patients with HR-NMIBC, and the molecular subtypes outperformed guideline-recommended risk stratification based on clinicopathological variables. For clinical application, we confirmed that a commercially approved assay was able to predict BRS3 tumors with an area under the curve of 0.87. These BCG response subtypes will allow for improved identification of patients with HR-NMIBC at the highest risk of progression and have the potential to be used to select more appropriate treatments for patients unlikely to respond to BCG.

Extensive intratumoral heterogeneity (ITH) is believed to contribute to therapeutic failure and tumor recurrence, as treatment-resistant cell clones can survive and expand. However, little is known about ITH in triple-negative breast cancer (TNBC) because of the limited number of single-cell sequencing studies on TNBC. In this study, we explored ITH in TNBC by evaluating gene expression-derived and imaging-derived multi-region differences within the same tumor. We obtained tissue specimens from 10 TNBC patients and conducted RNA sequencing analysis of 2–4 regions per tumor. We developed a novel analysis framework to dissect and characterize different types of variability: between-patients (inter-tumoral heterogeneity), between-patients across regions (inter-tumoral and region heterogeneity), and within-patient, between-regions (regional intratumoral heterogeneity). We performed a Bayesian changepoint analysis to assess and classify regional variability as low (convergent) versus high (divergent) within each patient feature (TNBC and PAM50 subtypes, immune, stroma, tumor counts and tumor infiltrating lymphocytes). Gene expression signatures were categorized into three types of variability: between-patients (108 genes), between-patients across regions (183 genes), and within-patients, between-regions (778 genes). Based on the between-patient gene signature, we identified two distinct patient clusters that differed in menopausal status. Significant intratumoral divergence was observed for PAM50 classification, tumor cell counts, and tumor-infiltrating T cell abundance. Other features examined showed a representation of both divergent and convergent results. Lymph node stage was significantly associated with divergent tumors. Our results show extensive intertumoral heterogeneity and regional ITH in gene expression and image-derived features in TNBC. Our findings also raise concerns regarding gene expression based TNBC subtyping. Future studies are warranted to elucidate the role of regional heterogeneity in TNBC as a driver of treatment resistance.

Objective To describe early experience of replacing PSA with Stockholm3 for detection of prostate cancer in primary care. Design and methods Longitudinal observations, comparing outcome measures before and after the implementation of Stockholm3. Setting Stavanger region in Norway with about 370,000 inhabitants, 304 general practitioners (GPs) in 97 primary care clinics, and one hospital. Intervention GPs were instructed to use Stockholm3 instead of PSA as standard procedure for diagnosis of prostate cancer. Main outcome measures Proportion of GP clinics that had ordered a Stockholm3 test. Number of men referred to needle biopsy. Distribution of clinically significant prostate cancer (csPC) (Gleason Score ≥7) and clinically non-significant prostate cancer (cnsPC) (Gleason Score 6), in needle biopsies. Estimation of direct healthcare costs. Results Stockholm3 was rapidly implemented as 91% (88/97) of the clinics started to use the test within 14 weeks. After including 4784 tested men, the percentage who would have been referred for prostate needle biopsy was 29.0% (1387/4784) if based on PSA level ≥3ng/ml, and 20.8% (995/4784) if based on Stockholm3 Risk Score (p

Purpose The proliferation marker Ki-67 has been used as a prognostic marker to separate low- and high-risk breast cancer subtypes and guide treatment decisions for adjuvant chemotherapy. The association of Ki-67 with response to tamoxifen therapy is unclear. High-throughput automated scoring of Ki-67 might enable standardization of quantification and definition of clinical cut-off values. We hypothesized that digital image analysis (DIA) of Ki-67 can be used to evaluate proliferation in breast cancer tumors, and that Ki-67 may be associated with tamoxifen resistance in early-stage breast cancer. Patients and methods Here, we apply DIA technology from Visiopharm using a custom designed algorithm for quantifying the expression of Ki-67, in a case-control study nested in the Danish Breast Cancer Group clinical database, consisting of stages I, II, or III breast cancer patients of 35-69 years of age, diagnosed during 1985-2001, in the Jutland peninsula, Denmark. We assessed DIA-Ki-67 score on tissue microarrays (TMAs) from breast cancer patients in a case-control study including 541 ER-positive and 300 ER-negative recurrent cases and their non-recurrent controls, matched on ER-status, cancer stage, menopausal status, year of diagnosis, and county of residence. We used logistic regression to estimate odds ratios and associated 95% confidence intervals to determine the association of Ki-67 expression with recurrence risk, adjusting for matching factors, chemotherapy, type of surgery, receipt of radiation therapy, age category, and comorbidity. Results Ki-67 was not associated with increased risk of recurrence in tamoxifen-treated patients (ORadj =0.72, 95% CI 0.54, 0.96) or ER-negative patients (ORadj =0.85, 95% CI 0.54, 1.34). Conclusion Our findings suggest that Ki-67 digital image analysis in TMAs is not associated with increased risk of recurrence among tamoxifen-treated ER-positive breast cancer or ER-negative breast cancer patients. Overall, our findings do not support an increased risk of recurrence associated with Ki-67 expression.

Implementation of high-risk human papilloma virus (HPV) screening and the increasing proportion of HPV vaccinated women in the screening program will reduce the percentage of HPV positive women with oncogenic potential. In search of more specific markers to identify women with high risk of cancer development, we used RNA sequencing to compare the transcriptomic immune-profile of 13 lesions with cervical intraepithelial neoplasia grade 3 (CIN3) or adenocarcinoma in situ (AIS) and 14 normal biopsies from women with detected HPV infections. In CIN3/AIS lesions as compared to normal tissue, 27 differential expressed genes were identified. Transcriptomic analysis revealed significantly higher expression of a number of genes related to proliferation, (CDKN2A, MELK, CDK1, MKI67, CCNB2, BUB1, FOXM1, CDKN3), but significantly lower expression of genes related to a favorable immune response (NCAM1, ARG1, CD160, IL18, CX3CL1). Compared to the RNA sequencing results, good correlation was achieved with relative quantitative PCR analysis for NCAM1 and CDKN2A. Quantification of NCAM1 positive cells with immunohistochemistry showed epithelial reduction of NCAM1 in CIN3/AIS lesions. In conclusion, NCAM1 and CDKN2A are two promising candidates to distinguish whether women are at high risk of developing cervical cancer and in need of frequent follow-up.

The purpose of this study was to establish a gene signature that may predict CIN3 regression and that may aid in selecting patients who may safely refrain from conization. Oncomine mRNA data including 398 immune-related genes from 21 lesions with confirmed regression and 28 with persistent CIN3 were compared. L1000 mRNA data from a cervical cancer cohort was available for validation (n = 239). Transcriptomic analyses identified TDO2 (p = 0.004), CCL5 (p &lt, 0.001), CCL3 (p = 0.04), CD38 (p = 0.02), and PRF1 (p = 0.005) as upregulated, and LCK downregulated (p = 0.01) in CIN3 regression as compared to persistent CIN3 lesions. From these, a gene signature predicting CIN3 regression with a sensitivity of 91% (AUC = 0.85) was established. Transcriptomic analyses revealed proliferation as significantly linked to persistent CIN3. Within the cancer cohort, high regression signature score associated with immune activation by Gene Set enrichment Analyses (GSEA) and immune cell infiltration by histopathological evaluation (p &lt, 0.001). Low signature score was associated with poor survival (p = 0.007) and large tumors (p = 0.01). In conclusion, the proposed six-gene signature predicts CIN regression and favorable cervical cancer prognosis and points to common drivers in precursors and cervical cancer lesions.

Epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 3 (HER3) have been investigated as triple-negative breast cancer (TNBC) biomarkers. Reduced EGFR levels can be compensated by increases in HER3; thus, assaying EGFR and HER3 together may improve prognostic value. In a multi-institutional cohort of 510 TNBC patients, we analyzed the impact of HER3, EGFR, or combined HER3-EGFR protein expression in pre-treatment samples on breast cancer-specific and distant metastasis-free survival (BCSS and DMFS, respectively). A subset of 60 TNBC samples were RNA-sequenced using massive parallel sequencing. The combined HER3-EGFR score outperformed individual HER3 and EGFR scores, with high HER3-EGFR score independently predicting worse BCSS (Hazard Ratio [HR] = 2.30, p = 0.006) and DMFS (HR = 1.78, p = 0.041, respectively). TNBCs with high HER3-EGFR scores exhibited significantly suppressed ATM signaling and differential expression of a network predicted to be controlled by low TXN activity, resulting in activation of EGFR, PARP1, and caspases and inhibition of p53 and NFκB. Nuclear PARP1 protein levels were higher in HER3-EGFR-high TNBCs based on immunohistochemistry (p = 0.036). Assessing HER3 and EGFR protein expression in combination may identify which adjuvant chemotherapy-treated TNBC patients have a higher risk of treatment resistance and may benefit from a dual HER3-EGFR inhibitor and a PARP1 inhibitor.

Background European treatment guidelines for pTa and pT1 urinary bladder urothelial carcinoma depend highly on stage and WHO-grade. Both the WHO73 and the WHO04 grading systems show some intra- and interobserver variability. The current pilot study investigates which histopathological features are especially sensitive for this undesired lack of reproducibility and the influence on prognostic value. Methods Thirty-eight cases of primary non-muscle invasive urothelial carcinomas, including thirteen cases with stage progression, were reviewed by three pathologists. Thirteen microscopic features were extracted from pathology textbooks and evaluated separately. Reproducibility was measured using Gwet’s agreement coefficients. Prognostic ability regarding progression was estimated by the area under curve (AUC) of the receiver operating characteristics (ROC) function. Results The best reproducible features (Gwet’s agreement coefficient above 0.60) were papillary architecture, nuclear polarity, cellular maturation, nuclear enlargement and giant nuclei. Nucleoli was the strongest prognostic feature, and the only feature with an AUC above 0.70 for both grading systems, but reproducibility was not among the strongest. Nuclear polarity also had prognostic value with an AUC of 0.70 and 0.67 for the WHO73 and WHO04, respectively. The other features did not have significant prognostic value. Conclusions The reproducibility of the histopathological features of the different WHO grading systems varied considerably. Of all the features evaluated, only nuclear polarity was both prognostic and significantly reproducible. Further validation studies are needed on these features to improve grading of urothelial carcinomas.

Tamoxifen is an important targeted endocrine therapy in breast cancer. However, side effects and early discontinuation of tamoxifen remains a barrier for obtaining the improved outcome benefits of long-term tamoxifen treatment. Biomarkers predictive of tamoxifen side effects remain unidentified. The objective of this prospective population-based study was to investigate the value of tamoxifen metabolite concentrations as biomarkers for side effects. A second objective was to assess the validity of discontinuation rates obtained through pharmacy records with the use of tamoxifen drug monitoring. Longitudinal serum samples, patient-reported outcome measures and pharmacy records from 220 breast cancer patients were obtained over a 6-year period. Serum concentrations of tamoxifen metabolites were measured by LC–MS/MS. Associations between metabolite concentrations and side effects were analyzed by logistic regression and cross table analyses. To determine the validity of pharmacy records we compared longitudinal tamoxifen concentrations to discontinuation rates obtained through the Norwegian Prescription database (NorPD). Multivariable Cox regression models were performed to identify predictors of discontinuation. At the 2nd year of follow-up, a significant association between vaginal dryness and high concentrations of tamoxifen, Z-4′-OHtam and tam-NoX was identified. NorPD showed a tamoxifen-discontinuation rate of 17.9% at 5 years and drug monitoring demonstrated similar rates. Nausea, vaginal dryness and chemotherapy-naive status were significant risk factors for tamoxifen discontinuation. This real-world data study suggests that measurements of tamoxifen metabolite concentrations may be predictive of vaginal dryness in breast cancer patients and verifies NorPD as a reliable source of adherence data.

Simple Summary Obesity increases the risk of postmenopausal, hormone receptor-positive breast cancer and has been linked to a higher risk of recurrence and mortality. During obesity, adipose tissue can become dysfunctional, resulting in chronic low-grade inflammation. Crown-like structures in breast adipose tissue (CLS-B), composed of macrophages surrounding dead or dying adipocytes in a crown-like pattern, are a new histologic marker of local inflammation. In this review, we aim to evaluate the early evidence of CLS-B in breast cancer. There is consistent evidence that CLS-B are more frequently detected among obese compared to non-obese breast cancer patients. Additionally, several studies have found that CLS-B presence is associated with metabolic and inflammatory factors that contribute to breast cancer development and progression. However, more studies are needed to understand the potential clinical utility of CLS-B as a marker of breast cancer risk or prognosis. Abstract Obesity is an established risk factor for postmenopausal breast cancer and has been linked to worse breast cancer prognosis, most clearly for hormone receptor-positive breast cancers. The underlying mechanisms of the obesity–breast cancer association are not fully understood, but growing evidence points to the breast adipose tissue microenvironment playing an important role. Obesity-induced adipose tissue dysfunction can result in a chronic state of low-grade inflammation. Crown-like structures of the breast (CLS-B) were recently identified as a histologic marker of local inflammation. In this review, we evaluate the early evidence of CLS-B in breast cancer. Data from preclinical and clinical studies show that these inflammatory lesions within the breast are associated with local NF-κB activation, increased aromatase activity, and elevation of pro-inflammatory mediators (TNFα, IL-1β, IL-6, and COX-2-derived PGE2)—factors involved in multiple pathways of breast cancer development and progression. There is also substantial evidence from epidemiologic studies that CLS-B are associated with greater adiposity among breast cancer patients. However, there is insufficient evidence that CLS-B impact breast cancer risk or prognosis. Comparisons across studies of prognosis were complicated by differences in CLS-B evaluation and deficiencies in study design, which future studies should take into consideration. Breast adipose tissue inflammation provides a plausible explanation for the obesity–breast cancer association, but further study is needed to establish its role and whether markers such as CLS-B are clinically useful.

Background The antitumor efficacy of PARP inhibitors (PARPi) for breast cancer patients harboring germline BRCA1/2 (gBRCA1/2) mutations is well established. While PARPi monotherapy was ineffective in patients with metastatic triple negative breast cancer (TNBC) wild type for BRCA1/2, we hypothesized that PARPi may be effective in primary TNBCs without previous chemotherapy exposure. Patients and methods In the phase II PETREMAC trial, patients with primary TNBC >2 cm received olaparib for up to 10 weeks before chemotherapy. Tumor biopsies collected before and after olaparib underwent targeted DNA sequencing (360 genes) and BRCA1 methylation analyses. In addition, BRCAness (multiplex ligation-dependent probe amplification), PAM50 gene expression, RAD51 foci, tumor-infiltrating lymphocytes (TILs) and PD-L1 analyses were performed on pretreatment samples. Results The median pretreatment tumor diameter was 60 mm (range 25-112 mm). Eighteen out of 32 patients obtained an objective response (OR) to olaparib (56.3%). Somatic or germline mutations affecting homologous recombination (HR) were observed in 10/18 responders [OR 55.6%, 95% confidence interval (CI) 33.7-75.4] contrasting 1/14 non-responders (OR 7.1%; CI 1.3-31.5, P = 0.008). Among tumors without HR mutations, 6/8 responders versus 3/13 non-responders revealed BRCA1 hypermethylation (P = 0.03). Thus, 16/18 responders (88.9%, CI 67.2-96.9), in contrast to 4/14 non-responders (28.6%, CI 11.7-54.7, P = 0.0008), carried HR mutations and/or BRCA1 methylation. Excluding one gPALB2 and four gBRCA1/2 mutation carriers, 12/14 responders (85.7%, CI 60.1-96.0) versus 3/13 non-responders (23.1%, CI 8.2-50.3, P = 0.002) carried somatic HR mutations and/or BRCA1 methylation. In contrast to BRCAness signature or basal-like subtype, low RAD51 scores, high TIL or high PD-L1 expression all correlated to olaparib response. Conclusion Olaparib yielded a high clinical response rate in treatment-naïve TNBCs revealing HR deficiency, beyond germline HR mutations. Trial registration ClinicalTrials.gov identifier: NCT02624973. 2020 The Author(s). Published by Elsevier Ltd on behalf of European Society for Medical Oncology. This is an open access article under theCC BY license(http://creativecommons.org/licenses/by/4.0/).

Concentrations of active tamoxifen metabolites may be predictive of tamoxifen efficacy, and several therapeutic thresholds have been reported. This study of 406 ER+ breast cancer patients replicated an association between a previously reported active tamoxifen threshold (Z‐4OHtam ≤ 3.26 nm) and adverse BC outcome. The association was identified in early‐stage premenopausal patients using tamoxifen alone without sequential aromatase inhibitor use., Low steady‐state levels of active tamoxifen metabolites have been associated with inferior treatment outcomes. In this retrospective analysis of 406 estrogen receptor‐positive breast cancer (BC) patients receiving adjuvant tamoxifen as initial treatment, we have associated our previously reported thresholds for the two active metabolites, Z‐endoxifen and Z‐4‐hydroxy‐tamoxifen (Z‐4OHtam), with treatment outcomes in an independent cohort of BC patients. Among all patients, metabolite levels did not affect survival. However, in the premenopausal subgroup receiving tamoxifen alone (n = 191) we confirmed an inferior BC ‐specific survival in patients with the previously described serum concentration threshold of Z‐4OHtam ≤ 3.26 nm (HR = 2.37, 95% CI = 1.02–5.48, P = 0.039). The ‘dose–response’ survival trend in patients categorized to ordinal concentration cut‐points of Z‐4OHtamoxifen (≤ 3.26, 3.27–8.13, > 8.13 nm) was also replicated (P‐trend log‐rank = 0.048). Z‐endoxifen was not associated with outcome. This is the first study to confirm the association between a published active tamoxifen metabolite threshold and BC outcome in an independent patient cohort. Premenopausal patients receiving 5‐year of tamoxifen alone may benefit from therapeutic drug monitoring to ensure tamoxifen effectiveness.

Numerical and/or structural centrosome amplification (CA) is a hallmark of cancers that is often associated with the aberrant tumor karyotypes and poor clinical outcomes. Mechanistically, CA compromises mitotic fidelity and leads to chromosome instability (CIN), which underlies tumor initiation and progression. Recent technological advances in microscopy and image analysis platforms have enabled better-than-ever detection and quantification of centrosomal aberrancies in cancer. Numerous studies have thenceforth correlated the presence and the degree of CA with indicators of poor prognosis such as higher tumor grade and ability to recur and metastasize. We have pioneered a novel semi-automated pipeline that integrates immunofluorescence confocal microscopy with digital image analysis to yield a quantitative centrosome amplification score (CAS), which is a summation of the severity and frequency of structural and numerical centrosome aberrations in tumor samples. Recent studies in breast cancer show that CA increases across the disease progression continuum, while normal breast tissue exhibited the lowest CA, followed by cancer-adjacent apparently normal, ductal carcinoma in situ and invasive tumors, which showed the highest CA. This finding strengthens the notion that CA could be evolutionarily favored and can promote tumor progression and metastasis. In this review, we discuss the prevalence, extent, and severity of CA in various solid cancer types, the utility of quantifying amplified centrosomes as an independent prognostic marker. We also highlight the clinical feasibility of a CA-based risk score for predicting recurrence, metastasis, and overall prognosis in patients with solid cancers.

In pathology labs worldwide, we see an increasing number of tissue samples that need to be assessed without the same increase in the number of pathologists. Computational pathology, where digital scans of histological samples called whole-slide images (WSI) are processed by computational tools, can be of help for the pathologists and is gaining research interests. Most research effort has been given to classify slides as being cancerous or not, localization of cancerous regions, and to the “big-four” in cancer: breast, lung, prostate, and bowel. Urothelial carcinoma, the most common form of bladder cancer, is expensive to follow up due to a high risk of recurrence, and grading systems have a high degree of inter- and intra-observer variability. The tissue samples of urothelial carcinoma contain a mixture of damaged tissue, blood, stroma, muscle, and urothelium, where it is mainly muscle and urothelium that is diagnostically relevant. A coarse segmentation of these tissue types would be useful to i) guide pathologists to the diagnostic relevant areas of the WSI, and ii) use as input in a computer-aided diagnostic (CAD) system. However, little work has been done on segmenting tissue types in WSIs, and on computational pathology for urothelial carcinoma in particular. In this work, we are using convolutional neural networks (CNN) for multiscale tile-wise classification and coarse segmentation, including both context and detail, by using three magnification levels: 25x, 100x, and 400x. 28 models were trained on weakly labeled data from 32 WSIs, where the best model got an F1-score of 96.5% across six classes. The multiscale models were consistently better than the single-scale models, demonstrating the benefit of combining multiple scales. No tissue-class ground-truth for complete WSIs exist, but the best models were used to segment seven unseen WSIs where the results were manually inspected by a pathologist and are considered as very promising.

The Covid-19 pandemic represents an unsolved problem which has caused numerous fatalities At the present time, there is no vaccination available or curative therapy However, recent reports suggest that SARS-CoV2 acts upon its functional receptor ACE2 inducing a variety of deleterious effects such as inflammation, endothelial dysfunction, microangiopathy, myocarditis, thrombosis and myocardial infarction The details of the SARS-CoV2-ACE2 interaction are poorly understood and most of the hypothesis related to this are based on the extrapolation of previous research focusing on ACE2 as a receptor for SARS-CoV Considering the similarities between SARS-CoV2 and SARS-CoV we have conducted a physiopathological analysis focusing on the key pathogenic role of ACE2 as a functional receptor for SARS-CoV2 In this context, we have identified several potential therapeutic targets which should be further evaluated in patients with Covid-19 It is likely that an efficient therapy for Covid-19 will be revealed by research investigating the binding of viral spike S protein to ACE2, and the immunological response determined by SARS-CoV2-ACE2 interaction, including the anti-viral role of plasmacytoid dendritic cells and anti-inflammatory reprogramming of macrophages © Eugen B Petcu, Christopher Andry, Eric J Burks, Stephen Hamlet, Emiel AM Janssen, Kjell H Kjellevold, Antonio de Las Morenas, Nancy S Miller, Rodica I Miroiu, Iulian Nusem, Aurel Popa-Wagner

Protein structural alterations, including misfolding and aggregation, are a hallmark of several diseases, including cancer. However, the possible clinical application of protein conformational analysis using infrared spectroscopy to detect cancer-associated structural changes in proteins has not been established yet. The present study investigates the applicability of Fourier transform infrared spectroscopy in distinguishing the sera of healthy individuals and breast cancer patients. The cancer-associated alterations in the protein structure were analyzed by fitting the amide I (1600–1700 cm−1) band of experimental curves, as well as by comparing the ratio of the absorbance values at the amide II and amide III bands, assigning those as the infrared spectral signatures. The snapshot of the breast cancer-associated alteration in circulating DNA and RNA was also evaluated by extending the spectral fitting protocol to the complex region of carbohydrates and nucleic acids, 1140–1000 cm−1. The sensitivity and specificity of these signatures, representing the ratio of the α-helix and β-pleated sheet in proteins, were both 90%. Likewise, the ratio of amides II and amide III (I1556/I1295) had a sensitivity and specificity of 100% and 80%, respectively. Thus, infrared spectroscopy can serve as a powerful tool to understand the protein structural alterations besides distinguishing breast cancer and healthy serum samples.

Different miRNA profiling protocols and technologies introduce differences in the resulting quantitative expression profiles. These include differences in the presence (and measurability) of certain miRNAs. We present and examine a method based on quantile normalization, Adjusted Quantile Normalization (AQuN), to combine miRNA expression data from multiple studies in breast cancer into a single joint dataset for integrative analysis. By pooling multiple datasets, we obtain increased statistical power, surfacing patterns that do not emerge as statistically significant when separately analyzing these datasets. To merge several datasets, as we do here, one needs to overcome both technical and batch differences between these datasets. We compare several approaches for merging and jointly analyzing miRNA datasets. We investigate the statistical confidence for known results and highlight potential new findings that resulted from the joint analysis using AQuN. In particular, we detect several miRNAs to be differentially expressed in estrogen receptor (ER) positive versus ER negative samples. In addition, we identify new potential biomarkers and therapeutic targets for both clinical groups. As a specific example, using the AQuN-derived dataset we detect hsa-miR-193b-5p to have a statistically significant over-expression in the ER positive group, a phenomenon that was not previously reported. Furthermore, as demonstrated by functional assays in breast cancer cell lines, overexpression of hsa-miR-193b-5p in breast cancer cell lines resulted in decreased cell viability in addition to inducing apoptosis. Together, these observations suggest a novel functional role for this miRNA in breast cancer. Packages implementing AQuN are provided for Python and Matlab: https://github.com/YakhiniGroup/PyAQN., Author summary This work demonstrates a practical approach to the joint-analysis of multiple miRNA expression profiling datasets acquired with different measurement technologies. The use of different platforms in miRNA profiling can lead to major differences in results. In particular, some miRNA species are less amenable to detection and quantification by certain platforms or designs. Our approach, termed AQuN, combines quantile normalization with special attention to missing entities, to normalize miRNA expression across datasets, technologies, designs and platforms. As we show, our proposed approach uncovers patterns of interest that would not have emerged as statistically significant when analyzing the datasets individually or with other standard-practice normalization methods. Amongst our findings, we noted a previously undocumented miRNA that is significantly over-expressed in samples from estrogen-receptor positive breast cancer patients as compared to samples from estrogen-receptor negative patients. We further investigated this miRNA, hsa-miR-193b-5p, and experimentally show, in cell lines, that its expression level impacts the viability of tumor cells. AQuN is available to the community in the form of Python and Matlab packages. The joint-processed data is also made available for further investigation.

Background and rationale: Side effects of adjuvant treatment with tamoxifen (tam) may impair Quality of Life (QoL) and have been suggested as an independent variable for discontinuation of tam1. There are large inter-patient variabilities in prevalence and severance of side effects among tam users. Therefore, there is a need for biological markers that can predict side effects. A potential biological predictor is the serum concentrations of tam and/or its metabolites. In this prospective observational study we have analyzed serum concentrations of tam and 9 metabolites over 3 years. Patients Reported Outcome Measures (PROM) were obtained to elucidate possible associations between side effects, adherence and tam metabolism. Methods: Breast cancer patients using adjuvant tam (20mg/d) were recruited through the Prospective Breast Cancer Biobank project between 2011 and 2016. Inclusion criteria were ER positive status, ≥ 6 months tam use, tumor size of ≥0.1 cm and being able to read and write Norwegian. Concentration levels of tam and metabolites in serum were analyzed by LC-MS/MS2 and adherence data were collected through the Norwegian prescription database. PROM-data comprised of validated questioners reporting side effects of endocrine treatment and QoL. Statistical analyses comprised non-parametric tests, logistic regression, chi square tests and the Benjamin-Hochberg procedure to correct for multiple testing. Results: Associations between metabolite concentrations and side effects were run as a cross sectional analysis (N=149) and separate analysis of each year of follow-up with 85, 77 and 65 patients at the 1st, 2nd and 3rd year respectively. We found that 78 % of patients reported side effects, 66 % reported mood swings, 21 % reported severe hot flushes and 71 % reported decreased libido. When analyzing years separately, we found that on the 2nd year patients experiencing vaginal dryness had significantly higher levels of tamoxifen (P=0.032, after correction for multiple testing and adjustment for clinical relevant variables) compared to patients not experiencing vaginal dryness. Also, on year 2 the patients in the lower quartile of Z-endoxifen (≤17.9 nM) had significantly lower libido (p=0.015) compared to patients with Z-endoxifen levels >17.9 nM after adjustment for clinical relevant variables and correction for multiple testing. Analyses regarding adherence are not complete and more results will be presented in the poster. Discussion: Our data indicates that high serum concentrations of tam and low concentrations of Z-endoxifen are associated with vaginal dryness and sexual dysfunction. Patients reporting “very low libido” had the highest levels of tam, suggesting that slow metabolic conversion and accumulation of tam may contribute to sexual dysfunction. Our results were only significant in the second year of follow-up, possibly because patients wait to resume sexual activity after diagnosis, chemo and surgery. After receiving advice (i.e. lubricants), the symptoms are often reduced in the subsequent follow-up (3rd year). In conclusion, our results indicate that monitoring tam serum concentrations may be used to predict side effects. 1 Owusu C. et al. JCO. 2008 2 Helland T. et al. BCR. 2017 Citation Format: Helland T, Haugstøyl ME, Hagen KB, Kvaløy JT, Lunde S, Lode K, Lind RA, Gripsrud BH, Bifulco E, Gebreslase NS, Jonassen J, Hustad SS, Aas T, Lende TH, Lien EA, Janssen EA, Mellgren G, Søiland H. Serum concentrations of tamoxifen and Z-endoxifen may predict sexual dysfunction in the 2nd year of adjuvant endocrine treatment [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-14-08.

Background: About 60-80% of ductal carcinoma in situ (DCIS) cases are high-grade (HG) DCIS with an elevated risk of local recurrence (LR) even after a lumpectomy. Patients are often under or over treated due to the lack of accurate recurrence risk prediction models. Current prognostic models such as OncotypeDX and Van Nuys Prognostic Index (VNPI) lack consistency and are limited to a specific subset of patients. Here in this study, we show that the extent of centrosome amplification (CA) in a DCIS lesion can predict the risk of LR after lumpectomy. CA refers to presence of supernumerary or large centrosomes and is a characteristic of pre-invasive lesions, and breast tumors, and promotes erroneous mitoses and chromosomal instability. Methods: We have pioneered a semi-automated pipeline that integrates immunofluorescence confocal microscopy with digital image analysis and yields a quantitative Centrosomal Amplification Score (CAS) for each patients' tumor sample by evaluating severity and frequency of centrosomal aberrations therein. To this end, we first immunofluorescently stained centrosomes in formalin fixed paraffin embedded resection samples from DCIS patients (discovery cohort n=133 and a validation cohort n=119) using an antibody against γ-tubulin, and co-stained nuclei with DAPI. Next, we imaged the slides and processed the raw 3D image data using IMARIS Biplane 8.2 3D volume rendering software. Finally, we calculated centrosome numbers and volume in ˜250 cells from each patient sample. Using a mathematical algorithm, we generated a composite CAS score for each patient sample by integrating the numerical (CASi) and structural (CASm) aberrations. Results: We found that DCIS patients with recurrence exhibited higher CAS. Intriguingly, higher CAS was also associated with greater risk of developing ipsilateral breast events [Hazard ratio (HR) =7.58 for discovery cohort and HR=5.8 for validation cohort, p Conclusion: Our data compellingly show that CAS quantifies the risk of recurrence in DCIS patients with the highest concordance and provides a novel and innovative tool to tailor their treatment based on their risk profile. Citation Format: Mittal K, Kaur J, Wei G, Toss MS, Osan RM, Janssen EA, Søiland H, Rakha EA, Rida PC, Aneja R. A quantitative centrosomal amplification score (CAS) predicts local recurrence in ductal carcinoma in situ [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-18-02.

Background Previously, we have shown that miR-18a and miR-18b gene expression strongly correlates with high proliferation, oestrogen receptor -negativity (ER−), cytokeratin 5/6 positivity and basal-like features of breast cancer. Methods We investigated the expression and localization of miR-18a and -18b in formalin fixed paraffin embedded (FFPE) tissue from lymph node negative breast cancers (n = 40), by chromogenic in situ hybridization (CISH). The expression level and in situ localization of miR-18a and -18b was assessed with respect to the presence of tumour infiltrating lymphocytes (TILs) and immunohistochemical markers for ER, CD4, CD8, CD20, CD68, CD138, PAX5 and actin. Furthermore, in two independent breast cancer cohorts (94 and 377 patients) the correlation between miR-18a and -18b expression and the relative quantification of 22 immune cell types obtained from the CIBERSORT tool was assessed. Results CISH demonstrated distinct and specific cytoplasmic staining for both miR-18a and miR-18b, particularly in the intratumoural stroma and the stroma surrounding the tumour margin. Staining by immunohistochemistry revealed some degree of overlap of miR-18a and -18b with CD68 (monocytes/macrophages), CD138 (plasma cells) and the presence of high percentages of TILs. CIBERSORT analysis showed a strong correlation between M1-macrophages and CD4+ memory activated T-cells with mir-18a and -18b. Conclusions Our study demonstrates that miR-18a and miR-18b expression is associated with ER- breast tumours that display a high degree of inflammation. This expression is potentially associated specifically with macrophages. These results suggest that miR-18a and miR-18b may play a role in the systemic immunological response in ER− tumours.

Approximately 20-40% of high-grade Cervical Intraepithelial Neoplasia (CIN) regresses spontaneously, but the natural prognosis of an individual lesion is unpredictable. Gain of the chromosomal 3q region, which contains the human telomerase RNA gene on 3q26, is found in CIN lesions and cervical carcinoma and shows correlation with disease grade. The aim of this study is to assess whether 3q26 gain as a single genetic marker can predict the natural prognosis of high-grade CIN, by performing a review of the literature and pilot study. A literature review was conducted. Additionally, we performed a pilot study in 19 patients with histologically confirmed high-grade CIN lesions who were followed for a mean of 115days, after which loop excision was performed. Fluorescent in situ hybridization analysis was performed on the initial diagnostic biopsies to determine gain of 3q26. Eight studies were included in the literature overview, with a total of 407 patients. Of these, only 22 patients had high-grade lesions. All studies found an association between 3q26 gain and disease prognosis. Positive predictive values (PPV) ranged from 50 to 93%, negative predictive values (NPV) ranged from 75 to 100%. Only five out of 155 patients (3.2%) without 3q26 gain showed disease persistence or progression. In our pilot study on3q26 gain inhigh-grade CIN, the PPV of 3q26 gain for disease persistence was 67%, the NPV 100%. All four patients without 3q26 gain showed disease regression. In conclusion, the absence of 3q26 gain in diagnostic biopsies may be applied to identify high-grade CIN lesions with a high probability of disease regression.

Background Tamoxifen (tam) is the main adjuvant endocrine treatment option in premenopausal breast cancer (BC) patients comprising luminal-like tumors. However, a significant proportion of tam-users will experience a relapse within 15 years of primary surgery. We postulate that some patients do not achieve the full clinical benefit of tam due to inter-individual differences in the metabolism of the drug and that the clinical relevance of this may be different between molecular subtypes of BC. Here, we have compared the prognostic value of threshold levels of active tam metabolites in PAM50 luminal (lum) A and B molecular subtypes. Material and Methods A number of 64 lum-like BC patients who were relapse-free 3 years after surgery, were retrospectively analyzed in the observational Oslo1 study. All patients received 20 mg tamoxifen daily for 5 years. Serum was obtained at the time of the 3 years follow-up. A sensitive and accurate LC-MS/MS method was developed and validated for the detection and quantification of tam and 9 metabolites in human serum. The median follow-up time from serum sampling to BC death or last follow-up was 13.9 years (0.6-16.5). Recurrence score and molecular subtype of the patients were determined on FFPE-tumor samples using the PAM50 classification algorithm. Results A linear trend was identified for the correlation between active metabolite Z-4OHtam and BCSS (p=0.021, HR=0.64, CI95=0.43–0.93). There was no linear association between the remaining metabolites and BC outcome. We further explored the possible association between survival and concentration thresholds for the active metabolites Z-4OHtam and Z-endoxifen and identified supervised cut off values representing low concentrations for Z-4OHtam (≤3.26 nM) and Z-endoxifen (≤9.00 nM). BC patients with low Z-4OHtam had a BCSS of 33.3% compared to 82.8% in patients with Z-4OHtam >3.26 nM (p Discussion Low levels (≤ 3.26 nM) of the active tam metabolite Z-4OHtam was associated with a poorer long-term outcome in tam-treated BC patients. However, when grouping patients according to the PAM50-based molecular subtype, this was only significant in patients belonging to the lum B subtype. Our results suggest that higher levels of active tam metabolites and thus better ER blockage are more important in the more aggressive lum B subtype. Citation Format: Helland T, Søiland H, Hustad S, Lash TL, Kvaløy JT, Renolen A, Borgen E, Bifulco E, Henne N, Lien EA, Mellgren G, Naume B, Janssen EA. Serum levels of the active tamoxifen metabolite Z-4OHtam is predictive of long-term survival in luminal B subtype of breast cancer patients [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-12-05.

In urothelial cell type non-muscle invasive urinary bladder carcinoma, TNM stage and WHO grade are widely used to classify patients into low and high‑risk groups for prognostic and therapeutic decision-making. However, stage and grade reproducibility and prediction accuracy are wanting. This may lead to suboptimal treatment. We evaluated whether proliferation features, nuclear area of the epithelial cancer cells and the composition of stromal and tumor infiltrating lymphocytes have independent prognostic value. In 183 primary non-muscle invasive bladder cancer patients with long follow-up (median for stage progression cohort: 119 months, range 5-173; median for tumor recurrence cohort: 82, range 3-165) proliferation features Ki67, PPH3 and Mitotic Activity Index (MAI), Mean Nuclear Area (MNA), lymphocyte subsets (CD8+, CD4+, CD25+) and plasma cells (CD138+) were assessed on consecutive sections. Post-resection instillation treatments (none, mitomycin, BCG) were strictly standardized during the intake period. Risk of recurrence was associated with expression of Ki67 (≤ 39 vs. > 39) and Multifocality (p = 0.01). Patients with low Ki67 had a higher recurrence rate than those with high Ki67. Lymphocyte composition did not predict recurrence. Stage progression was strongly associated with high values for MAI (>15) and CD25+ (>0.2%). In a multivariate analysis the combination of MAI and CD25+ was the single most prognostic feature (p