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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.

BackgroundHundreds of ongoing clinical trials combine radiation therapy, mostly delivered as stereotactic body radiotherapy (SBRT), with immune checkpoint blockade. However, our understanding of the effect of radiotherapy on the intratumoral immune balance is inadequate, hindering the optimal design of trials that combine radiation therapy with immunotherapy. Our objective was to characterize the intratumoral immune balance of the malignant prostate after SBRT in patients.MethodsSixteen patients with high-risk, non-metastatic prostate cancer at comparable Gleason Grade disease underwent radical prostatectomy with (n = 9) or without (n = 7) neoadjuvant SBRT delivered in three fractions of 8 Gy over 5 days completed 2 weeks before surgery. Freshly resected prostate specimens were processed to obtain single-cell suspensions, and immune-phenotyped for major lymphoid and myeloid cell subsets by staining with two separate 14-antibody panels and multicolor flow cytometry analysis.ResultsMalignant prostates 2 weeks after SBRT had an immune infiltrate dominated by myeloid cells, whereas malignant prostates without preoperative treatment were more lymphoid-biased (myeloid CD45+ cells 48.4 ± 19.7% vs. 25.4 ± 7.0%; adjusted p-value = 0.11; and CD45+ lymphocytes 51.6 ± 19.7% vs. 74.5 ± 7.0%; p = 0.11; CD3+ T cells 35.2 ± 23.8% vs. 60.9 ± 9.7%; p = 0.12; mean ± SD).ConclusionSBRT drives a significant lymphoid to myeloid shift in the prostate-tumor immune infiltrate. This may be of interest when combining SBRT with immunotherapies, particularly in prostate cancer.

Prostate cancer is a heterogeneous cancer with widely varying levels of morbidity and mortality. Approaches to prostate cancer screening, diagnosis, surveillance, treatment and management differ around the world. To identify the highest priority research needs across the prostate cancer biomedical research domain, Movember conducted a landscape analysis with the aim of maximizing the effect of future research investment through global collaborative efforts and partnerships. A global Landscape Analysis Committee (LAC) was established to act as an independent group of experts across urology, medical oncology, radiation oncology, radiology, pathology, translational research, health economics and patient advocacy. Men with prostate cancer and thought leaders from a variety of disciplines provided a range of key insights through a range of interviews. Insights were prioritized against predetermined criteria to understand the areas of greatest unmet need. From these efforts, 17 research needs in prostate cancer were agreed on and prioritized, and 3 received the maximum prioritization score by the LAC: first, to establish more sensitive and specific tests to improve disease screening and diagnosis; second, to develop indicators to better stratify low-risk prostate cancer for determining which men should go on active surveillance; and third, to integrate companion diagnostics into randomized clinical trials to enable prediction of treatment response. On the basis of the findings from the landscape analysis, Movember will now have an increased focus on addressing the specific research needs that have been identified, with particular investment in research efforts that reduce disease progression and lead to improved therapies for advanced prostate cancer., The Movember global Landscape Analysis Committee (LAC) was established to act as an independent group of experts across urology, medical oncology, radiation oncology, radiology, pathology, translational research, health economics and patient advocacy to identify the highest priority research needs across the prostate cancer biomedical research domain. Findings from the landscape analysis illustrate the research priorities in prostate cancer and will enable Movember to focus on specific needs, with particular investment in research to reduce disease progression and improve therapies for advanced prostate cancer.

Melanoma of the skin is the sixth most common type of cancer in Europe and accounts for 3.4% of all diagnosed cancers. More alarming is the degree of recurrence that occurs with approximately 20% of patients lethally relapsing following treatment. Malignant melanoma is a highly aggressive skin cancer and metastases rapidly extend to the regional lymph nodes (stage 3) and to distal organs (stage 4). Targeted oncotherapy is one of the standard treatment for progressive stage 4 melanoma, and BRAF inhibitors (e.g. vemurafenib, dabrafenib) combined with MEK inhibitor (e.g. trametinib) can effectively counter BRAFV600E-mutated melanomas. Compared to conventional chemotherapy, targeted BRAFV600E inhibition achieves a significantly higher response rate. After a period of cancer control, however, most responsive patients develop resistance to the therapy and lethal progression. The many underlying factors potentially causing resistance to BRAF inhibitors have been extensively studied. Nevertheless, the remaining unsolved clinical questions necessitate alternative research approaches to address the molecular mechanisms underlying metastatic and treatment-resistant melanoma. In broader terms, proteomics can address clinical questions far beyond the reach of genomics, by measuring, i.e. the relative abundance of protein products, post-translational modifications (PTMs), protein localisation, turnover, protein interactions and protein function. More specifically, proteomic analysis of body fluids and tissues in a given medical and clinical setting can aid in the identification of cancer biomarkers and novel therapeutic targets. Achieving this goal requires the development of a robust and reproducible clinical proteomic platform that encompasses automated biobanking of patient samples, tissue sectioning and histological examination, efficient protein extraction, enzymatic digestion, mass spectrometry-based quantitative protein analysis by label-free or labelling technologies and/or enrichment of peptides with specific PTMs. By combining data from, e.g. phosphoproteomics and acetylomics, the protein expression profiles of different melanoma stages can provide a solid framework for understanding the biology and progression of the disease. When complemented by proteogenomics, customised protein sequence databases generated from patient-specific genomic and transcriptomic data aid in interpreting clinical proteomic biomarker data to provide a deeper and more comprehensive molecular characterisation of cellular functions underlying disease progression. In parallel to a streamlined, patient-centric, clinical proteomic pipeline, mass spectrometry-based imaging can aid in interrogating the spatial distribution of drugs and drug metabolites within tissues at single-cell resolution. These developments are an important advancement in studying drug action and efficacy in vivo and will aid in the development of more effective and safer strategies for the treatment of melanoma. A collaborative effort of gargantuan proportions between academia and healthcare professionals has led to the initiation, establishment and development of a cutting-edge cancer research centre with a specialisation in melanoma and lung cancer. The primary research focus of the European Cancer Moonshot Lund Center is to understand the impact that drugs have on cancer at an individualised and personalised level. Simultaneously, the centre increases awareness of the relentless battle against cancer and attracts global interest in the exceptional research performed at the centre.

Abstact Background Many men diagnosed with prostate cancer are active surveillance (AS) candidates. However, AS may be associated with increased risk of disease progression and metastasis due to delayed therapy. Genomic classifiers, e.g., Decipher, may allow better risk-stratify newly diagnosed prostate cancers for AS. Methods Decipher was initially assessed in a prospective cohort of prostatectomies to explore the correlation with clinically meaningful biologic characteristics and then assessed in diagnostic biopsies from a retrospective multicenter cohort of 266 men with National Comprehensive Cancer Network (NCCN) very low/low and favorable-intermediate risk prostate cancer. Decipher and Cancer of the Prostate Risk Assessment (CAPRA) were compared as predictors of adverse pathology (AP) for which there is universal agreement that patients with long life-expectancy are not suitable candidates for AS (primary pattern 4 or 5, advanced local stage [pT3b or greater] or lymph node involvement). Results Decipher from prostatectomies was significantly associated with adverse pathologic features (p-values

The MM500 study is an initiative to map the protein levels in malignant melanoma tumor samples, focused on in‐depth histopathology coupled to proteome characterization. The protein levels and localization were determined for a broad spectrum of diverse, surgically isolated melanoma tumors originating from multiple body locations. More than 15,500 proteoforms were identified by mass spectrometry, from which chromosomal and subcellular localization was annotated within both primary and metastatic melanoma. The data generated by global proteomic experiments covered 72% of the proteins identified in the recently reported high stringency blueprint of the human proteome. This study contributes to the NIH Cancer Moonshot initiative combining detailed histopathological presentation with the molecular characterization for 505 melanoma tumor samples, localized in 26 organs from 232 patients., The MM500 study is an initiative to map the protein levels in malignant melanoma tumor samples, focused on in‐depth histopathology coupled to proteome characterization. The protein levels and localization were determined for a broad spectrum of diverse melanoma tumors originating from multiple body locations. More than 15,500 proteoforms were identified by mass spectrometry, from which chromosomal and sub‐cellular localization was annotated within both primary and metastatic melanoma.

Purpose This study aimed to evaluate the feasibility and safety of prostate stereotactic body radiation therapy (SBRT) neoadjuvant to radical prostatectomy (RP) in a phase 1 trial. The primary endpoint was treatment completion rate without severe acute surgical complications. Secondary endpoints included patient-reported quality of life and physician-reported toxicities. Methods and Materials Patients with nonmetastatic high-risk or locally advanced prostate cancer received 24 Gy in 3 fractions to the prostate and seminal vesicles over 5 days, completed 2 weeks before RP. Patients with pN1 disease were treated after multidisciplinary discussion and shared decision making. Patient-reported quality of life (International Prostate Symptom Score and Expanded Prostate Cancer Index Composite 26-item version questionnaires) and physician-reported toxicity (Common Terminology Criteria for Adverse Events, version 4.03) were assessed before SBRT, immediately before surgery, and at 3-month intervals for 1 year. Results Twelve patients were enrolled, and 11 completed treatment (1 patient had advanced disease on prostate-specific membrane antigen positron emission tomography after enrollment but before treatment). There were no significant surgical complications. After RP, 2 patients underwent additional radiation therapy to nodes with androgen suppression for pN1 disease. Median follow-up after completion of treatment was 20.1 months, with 9 of 11 patients having a follow-up period of >12 months. Two patients had biochemical recurrence (prostate-specific antigen ≥0.05) within the first 12 months, with an additional 2 patients found to have biochemical recurrence after the 12-month period. The highest Common Terminology Criteria for Adverse Events genitourinary grades were 0, 1, 2, and 3 (n = 1, 4, 4, and 2, respectively), and the highest gastrointestinal grades were 0, 1, and 2 (n = 9, 1, and 1, respectively). At 12 months, incontinence was the only grade ≥2 toxicity. One and 2 of 9 patients had grade 2 and 3 incontinence, respectively. On the Expanded Prostate Cancer Index Composite (26-item version), the mean/median changes in scores from baseline to 12 months were –32.8/–31.1 for urinary incontinence, –1.6/–6.2 for urinary irritative/obstructive, –2.1/0 for bowel, –34.4/–37.5 for sexual function, and –10.6/–2.5 for hormonal. The mean/median change in International Prostate Symptom Score from baseline to 12 months was 0.5/0.5. Conclusions RP after neoadjuvant SBRT appears to be feasible and safe at the dose tested. The severity of urinary incontinence may be higher than RP alone.

An amendment to this paper has been published and can be accessed via the original article.

Background Multiplex immunohistochemistry (mIHC) permits the labeling of six or more distinct cell types within a single histologic tissue section. The classification of each cell type requires detection of uniquely colored chromogens localized to cells expressing biomarkers of interest. The most comprehensive and reproducible method to evaluate such slides is to employ digital pathology and image analysis pipelines to whole-slide images (WSIs). Our suite of deep learning tools quantitatively evaluates the expression of six biomarkers in mIHC WSIs. These methods address the current lack of readily available methods to evaluate more than four biomarkers and circumvent the need for specialized instrumentation to spectrally separate different colors. The use case application for our methods is a study that investigates tumor immune interactions in pancreatic ductal adenocarcinoma (PDAC) with a customized mIHC panel. Methods Six different colored chromogens were utilized to label T-cells (CD3, CD4, CD8), B-cells (CD20), macrophages (CD16), and tumor cells (K17) in formalin-fixed paraffin-embedded (FFPE) PDAC tissue sections. We leveraged pathologist annotations to develop complementary deep learning-based methods: (1) ColorAE is a deep autoencoder which segments stained objects based on color; (2) U-Net is a convolutional neural network (CNN) trained to segment cells based on color, texture and shape; and (3) ensemble methods that employ both ColorAE and U-Net, collectively referred to as ColorAE:U-Net. We assessed the performance of our methods using: structural similarity and DICE score to evaluate segmentation results of ColorAE against traditional color deconvolution; F1 score, sensitivity, positive predictive value, and DICE score to evaluate the predictions from ColorAE, U-Net, and ColorAE:U-Net ensemble methods against pathologist-generated ground truth. We then used prediction results for spatial analysis (nearest neighbor). Results We observed that (1) the performance of ColorAE is comparable to traditional color deconvolution for single-stain IHC images (note: traditional color deconvolution cannot be used for mIHC); (2) ColorAE and U-Net are complementary methods that detect six different classes of cells with comparable performance; (3) combinations of ColorAE and U-Net in ensemble methods outperform ColorAE and U-Net alone; and (4) ColorAE:U-Net ensemble methods can be employed for detailed analysis of the tumor microenvironment (TME). Summary We developed a suite of scalable deep learning methods to analyze 6 distinctly labeled cell populations in mIHC WSIs. We evaluated our methods and found that they reliably detected and classified cells in the PDAC tumor microenvironment. We also utilized the ColorAE:U-Net ensemble method to analyze 3 mIHC WSIs with nearest neighbor spatial analysis. We demonstrate a proof of concept that these methods can be employed to quantitatively describe the spatial distribution of immune cells within the tumor microenvironment. These complementary deep learning methods are readily deployable for use in clinical research studies.

Background Metastatic prostate cancer (PC) is highly lethal. The ability to identify primary tumors capable of dissemination is an unmet need in the quest to understand lethal biology and improve patient outcomes. Previous studies have linked chromosomal instability (CIN), which generates aneuploidy following chromosomal missegregation during mitosis, to PC progression. Evidence of CIN includes broad copy number alterations (CNAs) spanning > 300 base pairs of DNA, which may also be measured via RNA expression signatures associated with CNA frequency. Signatures of CIN in metastatic PC, however, have not been interrogated or well defined. We examined a published 70-gene CIN signature (CIN70) in untreated and castration-resistant prostate cancer (CRPC) cohorts from The Cancer Genome Atlas (TCGA) and previously published reports. We also performed transcriptome and CNA analysis in a unique cohort of untreated primary tumors collected from diagnostic prostate needle biopsies (PNBX) of localized (M0) and metastatic (M1) cases to determine if CIN was linked to clinical stage and outcome. Methods PNBX were collected from 99 patients treated in the VA Greater Los Angeles (GLA-VA) Healthcare System between 2000 and 2016. Total RNA was extracted from high-grade cancer areas in PNBX cores, followed by RNA sequencing and/or copy number analysis using OncoScan. Multivariate logistic regression analyses permitted calculation of odds ratios for CIN status (high versus low) in an expanded GLA-VA PNBX cohort (n = 121). Results The CIN70 signature was significantly enriched in primary tumors and CRPC metastases from M1 PC cases. An intersection of gene signatures comprised of differentially expressed genes (DEGs) generated through comparison of M1 versus M0 PNBX and primary CRPC tumors versus metastases revealed a 157-gene “metastasis” signature that was further distilled to 7-genes (PC-CIN) regulating centrosomes, chromosomal segregation, and mitotic spindle assembly. High PC-CIN scores correlated with CRPC, PC-death and all-cause mortality in the expanded GLA-VA PNBX cohort. Interestingly, approximately 1/3 of M1 PNBX cases exhibited low CIN, illuminating differential pathways of lethal PC progression. Conclusions Measuring CIN in PNBX by transcriptome profiling is feasible, and the PC-CIN signature may identify patients with a high risk of lethal progression at the time of diagnosis.

Background Immune cell infiltrates (ICI) of tumors are scored by pathologists around tumor glands. To obtain a better understanding of the immune infiltrate, individual immune cell types, their activation states and location relative to tumor cells need to be determined. This process requires precise identification of the tumor area and enumeration of immune cell subtypes separately in the stroma and inside tumor nests. Such measurements can be accomplished by a multiplex format using immunohistochemistry (IHC). Method We developed a pipeline that combines immunohistochemistry (IHC) and digital image analysis. One slide was stained with pan-cytokeratin and CD45 and the other slide with CD8, CD4 and CD68. The tumor mask generated through pan-cytokeratin staining was transferred from one slide to the other using affine image co-registration. Bland-Altman plots and Pearson correlation were used to investigate differences between densities and counts of immune cell underneath the transferred versus manually annotated tumor masks. One-way ANOVA was used to compare the mask transfer error for tissues with solid and glandular tumor architecture. Results The overlap between manual and transferred tumor masks ranged from 20%–90% across all cases. The error of transferring the mask was 2- to 4-fold greater in tumor regions with glandular compared to solid growth pattern (p

BACKGROUND: Previously, we showed that carbohydrate restriction with calorie restriction slowed tumor growth in xenograft mouse prostate cancer models. Herein, we examined the impact of carbohydrate restriction without calorie restriction on tumor development within the context of diet-induced obesity in the Hi-Myc transgenic mouse model of prostate cancer.METHODS: Mice were randomized at 5 weeks of age to ad libitum western diet (WD; 40% fat, 42% carbohydrate; n=39) or ad libitum no carbohydrate ketogenic diet (NCKD; 82% fat, 1% carbohydrate; n=44). At age 3 or 6 months, mice were killed, prostates weighed and prostate histology, proliferation, apoptosis and macrophage infiltration evaluated by hematoxylin and eosin, Ki67, TUNEL and F4/80 staining, respectively. Body composition was assessed by DEXA, serum cytokines measured using multiplex, and Akt/mTOR signaling assessed by Western.RESULTS: Caloric intake was higher in the NCKD group, resulting in elevated body weights at 6 months of age, relative to the WD group (45 g vs 38g; P=0.008). Despite elevated body weights, serum monocyte chemoattractant protein (MCP)-1 and interleukin (IL)-1α levels were lower in NCKD versus WD mice (P=0.046 and P=0.118, respectively), and macrophage infiltration was reduced in prostates of NCKD versus WD mice (P=0.028). Relative Akt phosphorylation and phospho-S6 ribosomal protein levels were reduced in prostates of NCKD versus WD mice. However, while mice randomized to NCKD had smaller prostates after adjustment for body weight at 3 and 6 months (P=0.004 and P=0.002, respectively), NCKD mice had higher rates of adenocarcinoma at 6 months compared to WD mice (100 vs 80%, P=0.04).CONCLUSIONS: Despite higher caloric intake and elevated body weights, carbohydrate restriction lowered serum MCP-1 levels, reduced prostate macrophage infiltration, reduced prostate weight, but failed to slow adenocarcinoma development. Together, these data suggest that although carbohydrate restriction within the context of obesity may reduce obesity-associated systemic inflammation and perhaps slow tumor growth, it is not sufficient to counteract obesity-associated tumor development.

Men with high‐grade prostate cancer (HGPC) are at greatest risk of disease progression. Clinical risk factors associated with castration‐resistant prostate cancer (CRPC), metastases, and prostate cancer‐specific mortality (PCSM) were identified in a contemporary HGPC cohort. Clinical data was collected from men diagnosed with Gleason sum (GS) ≥8 at the Greater Los Angeles Veterans Affairs (GLA‐VA) Healthcare System between 2000 and 2013. Multivariable competing risks regression analyses assessed progression to CRPC, metastases, and PCSM within three treatment strata. The cumulative incidence of disease progression was calculated at 2, 5, and 10‐year time points. Review of 2149 prostate cancer cases yielded 322 with HGPC. Median survival times for cancer‐specific and overall mortality were significantly shorter in men treated with primary androgen deprivation therapy (ADT) (P = 0.0002 and P

INTRODUCTION AND OBJECTIVES:Conventional histological analysis cannot differentiate diagnostic prostate needle biopsies (PNBX) from patients with localized disease (stage M0) versus those with de n...

INTRODUCTION AND OBJECTIVES:Prostate cancer (PC) is the most common non-cutaneous cancer in men and a leading cause of cancer death. In the US, black men are disproportionately affected with higher...

During the diagnostic workup of lung adenocarcinomas (LAC), pathologists evaluate distinct histological tumor growth patterns. The percentage of each pattern on multiple slides bears prognostic significance. To assist with the quantification of growth patterns, we constructed a pipeline equipped with a convolutional neural network (CNN) and soft-voting as the decision function to recognize solid, micropapillary, acinar, and cribriform growth patterns, and non-tumor areas. Slides of primary LAC were obtained from Cedars-Sinai Medical Center (CSMC), the Military Institute of Medicine in Warsaw and the TCGA portal. Several CNN models trained with 19,924 image tiles extracted from 78 slides (MIMW and CSMC) were evaluated on 128 test slides from the three sites by F1-score and accuracy using manual tumor annotations by pathologist. The best CNN yielded F1-scores of 0.91 (solid), 0.76 (micropapillary), 0.74 (acinar), 0.6 (cribriform), and 0.96 (non-tumor) respectively. The overall accuracy of distinguishing the five tissue classes was 89.24%. Slide-based accuracy in the CSMC set (88.5%) was significantly better (p

There is limited information regarding the biobanking of pleural and peritoneal fluids that might supplement storage of pulmonary and thoracic tissue biospecimens. Such fluids are sometimes collected for clinical analyses and may have uses that obviate or supplement tissue samples. There has been a growing interest in using liquid biopsies as they are less invasive and may be amenable to analyses that guide targeted therapies. Integrating cytology and biobanking approaches, we describe techniques that may be used for collecting and banking pleural and peritoneal fluids.

// Adriana C. Vidal 1 , Zinan Chen 2 , Lauren E. Howard 2 , Daniel M. Moreira 3 , Ramiro Castro-Santamaria 4 , Gerald L. Andriole 5 , Emanuela Taioli 6 , Jay H. Fowke 7 , Beatrice Knudsen 8 , Charles G. Drake 9 , J. Curtis Nickel 10 and Stephen J. Freedland 1 1 Department of Surgery, Division of Urology, Cedars Sinai Medical Center, Los Angeles, CA, USA 2 Department of Biostatistics and Bioinformatics, Duke University School of Medicine, Durham, NC, USA 3 Department of Urology, Mayo Clinic, Rochester, MN, USA 4 GlaxoSmithKline Inc., R&D, King of Prussia, PA, USA 5 Washington University School of Medicine in St. Louis, St. Louis, MO, USA 6 Institute for Translational Epidemiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA 7 Department of Medicine and Urologic Surgery, Vanderbilt University Medical Center, TN, USA 8 Department of Medicine, Cedars-Sinai Biobank, Cedars Sinai Medical Center, Los Angeles, CA, USA 9 Johns Hopkins Sidney Kimmel Comprehensive Cancer Center, and the Brady Urology Institute, John Hopkins University, Baltimore, MD, USA 10 Department of Urology, Queen’s University, Kingston, ON, Canada Correspondence to: Adriana C. Vidal, email: // Keywords : prostate biopsy, inflammation, race, prostate cancer, acute inflammation Received : May 27, 2016 Accepted : July 07, 2016 Published : July 18, 2016 Abstract Prostate cancer (PC) risk differs between races, and we previously showed prostate inflammation in benign prostate tissue was linked with a lower future PC risk. However, whether prostate tissue inflammation varies by race is unknown. We analyzed baseline acute and chronic prostate inflammation by race in REDUCE, a 4-year, multicenter, placebo-controlled study where all men had a negative prostate biopsy prior to enrollment. We included 7,982 men with standardized central pathology review to determine the presence or absence of chronic or acute inflammation in baseline prostate biopsy tissue. Logistic regression was used to compare prostate inflammation by race, adjusting for confounders. Of 7,982 men, 7,271 were white (91.1%), 180 (2.3%) black, 131 (1.6%) Asian, 319 (4.0%) Hispanic and 81 (1%) unknown. A total of 78% had chronic and 15% had acute inflammation. On multivariable analysis relative to white men, black men were less likely (OR = 0.65, 95%CI: 0.41-1.03, p = 0.07) and Asian men more likely to have acute inflammation (OR = 1.74, 95%CI: 1.14-2.65, p = 0.001). Hispanic men had similar levels of acute inflammation as white men. Chronic inflammation did not significantly differ across races. We identified racial differences in acute inflammation, particularly in Asian men, in benign prostate tissue that inversely mirrored population-level data on PC race disparity. As we showed in REDUCE that acute inflammation is linked with lower future PC risk, if validated in future studies, these data suggest racial differences in prostatic acute inflammation may contribute in part to race differences in PC risk, especially among Asian men.

Purpose/Objectives: Her2-neu positive breast cancers have a good overall prognosis with the advent of Her2-directed therapies such as trastuzumab. However, despite the increased efficacy with Her-2-directed therapies 20-30% of patients still have local and/or distant failure despite being Her-2 amplified on pathology. The etiology of this local failure remains unknown. Recent evidence has suggested that there may be immune factors that contribute to the progression of breast cancer and the response to therapy so we undertook a study to examine the relationship between immune-based markers and traditional pathologic and clinical markers of outcome. Materials/Methods: Paraffin-embedded sections were generation and clinical records were reviewed for 88 patients, age ≥ 18 years, with pathologically-proven Her2-neu+ breast cancer who were treated at a single institution from 01/2001 to 12/2013. Single-color immunohistochemical staining was performed for CK5/6, CK14 and EGFR and scored by a breast pathologist. Adjacent sections were also then stained for CD45, CD4, CD8 and CD68 using a multi-color immunohistochemical approach. Slides were scanned using the Vectra Automated Quantitative Pathology Imaging System and analyzed using an in-house algorithm to quantitate the number of immune cells within the tumor, tumor margin and within 2 mm outside the tumor. We then compared the level of CK5/6, CK14, EGFR with the number of immune cells. The number of different immune cells were also analyzed with respect to other clinical parameters including age, tumor size, nodal status, hormone receptor status, time to progression, progression-free survival and overall survival. Results: At a median follow-up of 66.5 months, 20 (22.7%) patients had progressed. We found that the number of CD45+ leukocytes at the margin correlates with the expression of CK5/6 (p = 0.015) which predicted for local failure. Further, we found that the ratio of CD8 to CD4 cells within the tumor and margin highly correlates with the expression of the hormone receptors (p = 0.01). Conclusions: Our preliminary results suggest that immune markers may be important predictors of a basal-like phenotype as defined by CK5/6 expression in Her2+ breast cancers which itself correlated with significantly higher local failure. Further higher CD8 to CD4 ratios were highly correlated with hormone receptor expression, particularly PR expression suggesting that in the Her2+ population the more favorable prognosis for the "triple-positive" subtype of Her2+ cancers may be in part due to a more favorable immune microenvironment. Citation Format: Shiao SL, Gertych A, Ma Z, Zhang X, Burnison CM, Mirhadi AJ, Giuliano A, Knudsen BS, Chung A. Quantitative analysis of T cell and macrophage immune markers in Her2-positive breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-07-37.

339 Background: Stereotactic Body Radiotherapy (SBRT) delivers high dose per fraction radiotherapy to targets with high precision. Such hypofractionated RT appears to act as an immune adjuvant, altering the tumor infiltrating immune landscape and enriching it for lymphocytes as numerous preclinical investigations would suggest. Based on this hypothesis, hundreds of ongoing trials listed in clinicaltrials.gov currently test the combination of RT (largely SBRT) with various immunotherapies. However, studies directly measuring the representation of infiltrating immune cells after SBRT in patients are few and far between and none exist in the context of prostate cancer. We therefore sought to interrogate the tumor-immune interface after prostate SBRT using fresh tissue in patients. Methods: Fresh prostate tissue from patients (N=10) enrolled in a clinical trial of prostate SBRT (three fractions of 8 Gy directed to the prostate and seminal vesicles) in the neoadjuvant setting two weeks prior to radical prostatectomy was subjected to multicolor flow cytometry and compared to that of Gleason Grade and T stage matched controls who did not undergo neoadjuvant therapy. Results: With a threshold of significance level of 0.05 for unadjusted p-values, using two-sided two-sample t-test, myeloid cells and particularly CD14+/hiCD16+DR+ intermediate monocytes/macrophages were enriched, while lymphocytes, including T cells and CD56+16− NK cells were decreased in SBRT-treated prostates as compared to unirradiated controls. Conclusions: The immune infiltrates in prostates two weeks after SBRT demonstrates a significant lymphoid to myeloid shift consistent with a tumor microenvironment after SBRT that is likely immunosuppressive beyond what can be targeted through the PD-1/L1 or CTLA-4 axis alone. This may have implications for the design of immunotherapy trials, especially in prostate cancer, that test SBRT in combination with immunotherapies.

Prostate cancer is the most common and second most deadly form of cancer in men in the United States. The classification of prostate cancers based on Gleason grading using histological images is important in risk assessment and treatment planning for patients. Here, we demonstrate a new region-based convolutional neural network framework for multi-task prediction using an epithelial network head and a grading network head. Compared with a single-task model, our multi-task model can provide complementary contextual information, which contributes to better performance. Our model is achieved a state-of-the-art performance in epithelial cells detection and Gleason grading tasks simultaneously. Using fivefold cross-validation, our model is achieved an epithelial cells detection accuracy of 99.07% with an average area under the curve of 0.998. As for Gleason grading, our model is obtained a mean intersection over union of 79.56% and an overall pixel accuracy of 89.40%.

Robust delineation of tissue components in hematoxylin and eosin (H&E) stained slides is a critical step in quantifying tissue morphology. Fully convolutional neural networks (FCN) are ideally suited for automatic and efficient segmentation of tissue components in H&E slides. However, their performance relies on the network architecture, quality and depth of training. Here we introduce a set of 802 image tiles of colon biopsies from 2 subjects with inflammatory bowel disease (IBD) annotated for glandular epithelium (EP), gland lumen together with goblet cells (LG), and stroma (ST). We either trained the FCN-8s de-novo on our images (DN-FCN-8s) or pre-trained on the ImageNet dataset and fine-tuned on our images (FT-FCN-8s). For comparison, we used the U-Net trained de-novo. The training involved 700/802 images, leaving 102 images as a testing set. Ultimately, each model was validated in an independent digital biopsy slide. We also determined how the number of images used for training affects the performance of the model and observed a plateau in trainability at 700 images. In the testing set, U-Net and FT-FCN-8s achieved accuracies of 92.30% and 92.26% respectively. In the independent biopsy slide, U-Net demonstrated a segmentation accuracy of 88.64%, with F1-scores of 0.74 (EP), 0.92 (LG), and 0.93 (ST). The performance of the FT-FCN-8s was slightly worse, but the model required fewer images to reach a high classification performance. Our data demonstrate that all 3 FCNs are appropriate for segmentation of glands in biopsies from patients with IBD and open the door for quantification of IBD associated pathologies.

Aims To test the hypothesis that the hypoxia marker carbonic anhydrase IX (CAIX), and the cholangiocytic/progenitor markers cytokeratin (CK) 19 and epithelial cell adhesion molecule (EpCAM), may be expressed in areas of hypoxia in hepatocellular carcinoma (HCC) after transarterial chemoembolization (TACE). Methods and results Immunohistochemistry for CAIX, CK19 and EpCAM (BerEP4) was performed in 57 HCCs, including 40 residual/recurrent tumours adjacent to the TACE treatment site and 17 untreated tumours from the same 40 patients. CAIX was exxpressed in 19 of 40 residual/recurrent HCCs and in two of 17 untreated HCCs. The rate of CAIX immunoreactivity in the treated tumours was significantly higher than that in the non-treated tumours (47.5% versus 11.8%, P = 0.015). CK19 and EpCAM were expressed in six of 19 and in seven of 19 CAIX-positive TACE-treated HCCs, respectively, but were not expressed in CAIX-negative tumours or untreated tumours. There were significant associations between CK19 and CAIX immunoreactivity, and between EpCAM and CAIX immunoreactivity (P = 0.007 and P = 0.003, respectively). Double staining of CAIX and CK19 showed co-localization of both proteins in five of six cases. Three of seven EpCAM-positive tumours were also positive for CK19. Conclusions Hypoxia may trigger the expression of proteins that are normally associated with cholangiocytic/progenitor cell differentiation, suggesting that TACE paradoxically causes an aggressive tumour phenotype.

Prostate cancer is a biologically heterogeneous disease with variable molecular alterations underlying cancer initiation and progression. Despite recent advances in understanding prostate cancer heterogeneity, better methods for classification of prostate cancer are still needed to improve prognostic accuracy and therapeutic outcomes. In this study, we computationally assembled a large virtual cohort (n = 1,321) of human prostate cancer transcriptome profiles from 38 distinct cohorts and, using pathway activation signatures of known relevance to prostate cancer, developed a novel classification system consisting of three distinct subtypes (named PCS1–3). We validated this subtyping scheme in 10 independent patient cohorts and 19 laboratory models of prostate cancer, including cell lines and genetically engineered mouse models. Analysis of subtype-specific gene expression patterns in independent datasets derived from luminal and basal cell models provides evidence that PCS1 and PCS2 tumors reflect luminal subtypes, while PCS3 represents a basal subtype. We show that PCS1 tumors progress more rapidly to metastatic disease in comparison with PCS2 or PCS3, including PSC1 tumors of low Gleason grade. To apply this finding clinically, we developed a 37-gene panel that accurately assigns individual tumors to one of the three PCS subtypes. This panel was also applied to circulating tumor cells (CTC) and provided evidence that PCS1 CTCs may reflect enzalutamide resistance. In summary, PCS subtyping may improve accuracy in predicting the likelihood of clinical progression and permit treatment stratification at early and late disease stages. Cancer Res; 76(17); 4948–58. ©2016 AACR.

Active surveillance (AS) is an important management option for men with low-risk, clinically localized prostate cancer. The clinical parameters for patient selection and definition of progression for AS protocols are evolving as data from several large cohorts matures. Vital to this process is the critical role pathologic parameters play in identifying appropriate candidates for AS. These findings need to be reproducible and consistently reported by surgical pathologists. This report highlights the importance of accurate pathology reporting as a critical component of these protocols.

Background Infection with high-risk human papillomaviruses (hrHPVs) is a frequent cause of cervical intraepithelial neoplasias and carcinomas. The recently developed p16/Ki67 dual stain of cytologic preparations possesses superior specificity over current HPV testing for detecting moderate- and high-grade dysplasias and can potentially be applied in routine cytology screening. Image analysis can possibly improve the efficiency of evaluating Papanicolaou (Pap) smears, if dual-stained nuclei are accurately localized and reliably distinguished from the background of other cells. Methods Here, we describe a technique comprising color deconvolution, radial symmetry detector, and a superpixel-based segmentation for computerized delineation of nuclei in Pap smears stained with p16/Ki67. Results The performance of the method was determined by the precision and recall in 99 images (n = 19,323 cells) and reached 0.952 and 0.958, respectively. The accuracy of delineation, assessed by the Jaccard index (n = 1,080 cells), was 0.794. In single cells the precision and recall was higher than in clumps (P = 0.005). Conclusions In summary, the new technique delineates large and small nuclei irrespectively of coloration with a significantly better performance than a method solely involving the radial symmetry detector. Therefore, it is suited to automatically define nuclear areas for quantification of nuclear biomarkers in smears. © 2014 International Clinical Cytometry Society

The complexity of survival mechanisms in cancer cells from patients remains poorly understood. To obtain a comprehensive picture of tumor cell survival in prostate cancer metastases, we interrogated 5 survival proteins that operate within 3 survival pathways in a cohort of 185 lethal metastatic prostate metastases obtained from 44 patients. The expression levels of BCL-2, BCL-XL, MCL-1, cytoplasmic Survivin, nuclear Survivin, and Stathmin were measured by immunohistochemistry in a tissue microarray. Simultaneous expression of 3 or more proteins occured in 81% of lethal prostate cancer metastasis and BCL-2, cytoplasmic Survivin and MCL-1 were co-expressed in 71% of metastatic sites. An unsupervised cluster analysis separated bone and soft tissue metastases according to patterns of survival protein expression. BCL-2, cytoplasmic Survivin and MCL-1 had significantly higher expression in bone metastases (p < 10−5), while nuclear Survivin was significantly higher in soft tissue metastases (p = 3×10−14). BCL-XL overexpression in soft tissue metastases almost reached significance (p =0.09), while stathmin expression did not (p=0.28). In addition, the expression of MCL-1 was significantly higher in AR-positive tumors. Neuroendocrine differentiation was not associated with specific survival pathways. These studies demonstrate for the first time that bone and soft tissue metastases from the same patient differ significantly in expression of a panel of survival proteins and that with regards to survival protein expression, expression is associated with metastatic site and not patient. Altogether this study suggests that optimal therapeutic inhibition may require combinations of drugs that target both bone as well as soft tissue-specific survival pathways.

Author(s): Liu, Sandy; Cadaneanu, Radu M; Zhang, Baohui; Huo, Lihong; Lai, Kevin; Li, Xinmin; Galet, Colette; Grogan, Tristan R; Elashoff, David; Freedland, Stephen J; Rettig, Matthew; Aronson, William J; Knudsen, Beatrice S; Lewis, Michael S; Garraway, Isla P | Abstract: BackgroundBenign human prostate tubule-initiating cells (TIC) and aggressive prostate cancer display common traits, including tolerance of low androgen levels, resistance to apoptosis, and microenvironment interactions that drive epithelial budding and outgrowth. TIC can be distinguished from epithelial and stromal cells that comprise prostate tissue via cell sorting based upon Epcam, CD44, and CD49f antigenic profiles. Fetal prostate epithelial cells (FC) possess a similar antigenic profile to adult TIC and are capable of inducing tubule formation. To identify the TIC niche in human prostate tissue, differential keratin (KRT) expression was evaluated.ResultsGene expression data generated from Affymetrix Gene Chip human U133 Plus 2.0 array of sorted adult and fetal epithelial cells revealed KRT13 to be significantly enriched in FC and TIC compared to basal cells (BC) and luminal cells (LC) (pl0.001). Enriched KRT13 expression was confirmed by RT-PCR and cytospin immunostaining. Immunohistochemical analysis of KRT13 expression revealed rare KRT13+ epithelia throughout prostatic ducts/acini in adult tissue specimens and differentiated tubules in 24-week recombinant grafts, In contrast, abundant KRT13 expression was observed in developing ducts/acini in fetal prostate and cord-like structures composing 8-week recombinant grafts. Immunostaining of a prostate tissue microarray revealed KRT13+ tumor foci in approximately 9% of cases, and this subset displayed significantly shorter time to recurrence (p = 0.031), metastases (p = 0.032), and decreased overall survival (p = 0.004). Diagnostic prostate needle biopsies (PNBX) from untreated patients with concurrent bone metastases (clinical stage M1) displayed KRT13+ tumor foci, as did bone metastatic foci.ConclusionsThe expression profile of KRT13 in benign fetal and adult prostate tissue and in recombinant grafts, as well as the frequency of KRT13 expression in primary and metastatic prostate cancer indicates that it may be a marker of a stem/progenitor-like cell state that is co-opted in aggressive tumor cells. KRT13 is enriched in benign stem-like cells that display androgen-resistance, apoptosis-resistance, and branching morphogenesis properties. Collectively our data demonstrate that KRT13 expression is associated with poor prognosis at multiple stages of disease progression and may represent an important biomarker of adverse outcome in patients with prostate cancer.

The bitter taste receptor TAS2R38 is a G protein coupled receptor (GPCR) that has been found in many extra-oral locations like the gastrointestinal (GI) system, respiratory system, and brain, though its function at these locations is only beginning to be understood. To probe the receptor's potential metabolic role, immunohistochemistry of human ileum tissues was performed, which showed that the receptor was co-localized with glucagon-like peptide 1 (GLP-1) in L-cells. In a previous study, we had modeled the structure of this receptor for its many taste-variant haplotypes (Tan etal. 2011), including the taster haplotype PAV. The structure of this haplotype was then used in a virtual ligand screening pipeline using a collection of ∼2.5 million purchasable molecules from the ZINC database. Three compounds (Z7, Z3, Z1) were purchased from the top hits and tested along with PTU (known TAS2R38 agonist) in invitro and invivo assays. The dose-response study of the effect of PTU and Z7 on GLP-1 release using wild-type and TAS2R38 knockout HuTu-80cells showed that the receptor TAS2R38 plays a major role in GLP-1 release due to these molecules. Invivo studies of PTU and the three compounds showed that they each increase GLP-1 release. PTU was also chemical linked to cellulose to slow its absorption and when tested invivo, it showed an enhanced and prolonged GLP-1 release. These results suggest that the GI lumen location of TAS2R38 on the L-cell makes it a relatively safe drug target as systemic absorption is not needed for a TAS2R38 agonist drug to effect GLP-1 release.

Molecular and genomic analysis of microscopic quantities of tumor from formalin-fixed, paraffin-embedded biopsy specimens has many unique challenges. Herein, we evaluated the feasibility of obtaining transcriptome-wide RNA expression to measure prognostic classifiers in diagnostic prostate needle core biopsy specimens. One-hundred fifty-eight samples from diagnostic needle core biopsy specimens (BX) and radical prostatectomies (RPs) were collected from 33 patients at three hospitals; each patient provided up to six tumor and benign samples. Genome-wide transcriptomic profiles were generated using Affymetrix Human Exon arrays for comparison of gene expression alterations and prognostic signatures between the BX and RP samples. A sufficient amount of RNA (>100 ng) was obtained from all RP specimens (n = 77) and from 72 of 81 of BX specimens. Of transcriptomic features detected in RP, 95% were detectable in BX tissues and demonstrated a high correlation (r=0.96). Likewise, an expression signature pattern validated on RPs (Decipher prognostic test) showed correlation between BX and RP (r=0.70). Of matched BX and RP pairs, 25% showed discordant molecular subtypes. Genome-wide exon arrays yielded data of comparable quality from biopsy and RP tissues. The high concordance of tumor-associated gene expression changes between BX and RP samples provides evidence for the adequate performance of the assay platform with samples from prostate needle biopsy specimens with limited tumor volume.

receptors, competitively blocking muscarinic receptors and highly selective towards urinary bladder receptors. In anticipation of the clinical trial: “multicenter, open-label, randomized, comparative clinical study of efficiency and safety of medicinal product Imidafenacin, 0,2 mg per day and Tolterodinum, 4 mg per dayfor treatment of overactive bladder”, was conducted preclinical study. METHODS: The immunotoxic properties study included tests to evaluate humoral, cell-mediated, and non-specific immune responses conducted in mature male and female Balb/C mice. In all these tests, Imidafenacin was administered intragastrically at doses 0.083 mg/kg (TD) and 0.83 mg/kg (10TD) over a period of 28 days. The effects of the medicinal product on humoral immune response was evaluated by the antibody titre following an injection of sheep erythrocyte suspension. Study animals were administered the sheep erythrocyte suspension intraperitoneally to achieve immunization. In 7 days after immunization, the antibody titre in the serum was measured. Cell-mediated immune response was evaluated using the delayed-type hypersensitivity reaction to the hapten dinitrochlorobenzene (DNCB). Experimental animals were immunized by subcutaneous injection of 2 % DNCB solution in DMSO between the shoulder blades. Five days later, immunized mice were administered 0.1 % DNCB solution under the aponeurosis of a hind leg. In 24 hours after the challenging injection, the animals were euthanized and the DTH reaction index calculated by the difference between the weight of the experimental leg and that of the control leg. RESULTS: Statistical analyses of obtained data revealed no effect of multiple dose intragastric administration of imidafeniacin 0.083 mg/kg (TD) and 0.83 mg/kg (10TD) on the anti-sheep erythrocyte antibody titre in sexually mature male and female mice. The delayed-type hypersensitivity reaction test demonstrated that the investigational medicinal product administered over a 28-day period intragastrically at 0.083 mg/kg had no effect on the development of cell-mediated immune response in mice of either gender. Administration of Uritos at 0.83 mg/kg increased with statistical significance the cell-mediated immune response by 40 % in males and 45 % in female animals. The phagocytic activity test carried out on murine peritoneal macrophages demonstrated that the intragastric course of the TD and 10TD doses of Uritos increased, with statistical significance and in a dose-dependent manner, the non-specific immune response in male animals by 25 % and 43 %, respectively, and in female mice by 26 % and 40 %, respectively, as compared with animals given the vehicle. CONCLUSIONS: Imidafenacin administered at the 0.083 mg/kg (TD) or 0.83 mg/kg (10TD) dose demonstrated no immunotoxic properties

The World Health Organization recommends subclassification of lung cancer according to the percentages of histologic subtypes within a tumor. The manual quantification of lung tumor composition is very time consuming, but it can potentially be aided by a machine learning application. We have updated our previously developed methodology to segment and distinguish solid and micropapillary lung tumor subtypes. Binary tumor masks delineated by machine learning were defined by the mean area of binary objects and by the number of objects found in an image frame. These two features distinguished solid (\(n=31\)) and micropapillary (\(n=61\)) histologic subtypes with excellent performance (\(p

Expression of E-cadherin is used to monitor the epithelial phenotype, and its loss is suggestive of epithelial-mesenchymal transition (EMT). EMT triggers tumor metastasis. Exit from EMT is marked by increased E-cadherin expression and is considered necessary for tumor growth at sites of metastasis; however, the mechanisms associated with exit from EMT are poorly understood. Herein are analyzed 185 prostate cancer metastases, with significantly higher E-cadherin expression in bone than in lymph node and soft tissue metastases. To determine the molecular mechanisms of regulation of E-cadherin expression, three stable isogenic cell lines from DU145 were derived that differ in structure, migration, and colony formation on soft agar and Matrigel. When injected into mouse tibia, the epithelial subline grows most aggressively, whereas the mesenchymal subline does not grow. In cultured cells, ZEB1 and Src family kinases decrease E-cadherin expression. In contrast, in tibial xenografts, E-cadherin RNA levels increase eight- to 10-fold despite persistent ZEB1 expression, and in all ZEB1-positive metastases (10 of 120), ZEB1 and E-cadherin proteins were co-expressed. These data suggest that transcriptional regulation of E-cadherin differs in cultured cells versus xenografts, which more faithfully reflect E-cadherin regulation in cancers in human beings. Furthermore, the aggressive nature of xenografts positive for E-cadherin and the frequency of metastases positive for E-cadherin suggest that high E-cadherin expression in metastatic prostate cancer is associated with aggressive tumor growth.

Background: Prostate needle biopsies (PNBX) that are obtained during the initial diagnostic workup are often the only source of tumor tissue that is available to determine the severity of prostate cancer (PC). Interestingly, tumor grades and pathologic features in PNBXs of men with lethal metastatic disease (M1 stage) are indistinguishable from those of high-grade nonmetastatic tumors of patients who do not progress after treatment (M0 stage). We hypothesize that the morphology of tumor nuclei can be used as a source of biomarker development to distinguish M1 tumors from high-grade localized M0 tumors. Methods: Our study consists of a cohort of 85 high-grade M0 and 78 M1 cases, within a biorepository of 2150 patients at the Greater Los Angeles Veterans Affairs hospital. PNBX slides were digitized at 40X and pathologists annotated all cancer foci. These annotated regions were divided into smaller image tiles (dimensions) and fed into our digital image analysis pipeline for nuclear segmentation and feature extraction. We applied two distinct feature extraction methods to capture morphologic information within tumor nuclei. The 64 Handcrafted (HC) features describe nuclear properties such as shape, area, and chromatin conformation. The 62 Autoencoder (AE) features are abstract descriptors generated by a deep learning algorithm, which learns to redraw the nuclei. We built 2 machine learning models using AE or/and HC features for classification of M1 versus M0 cases. We divided our patients into 3 groups: training, testing and validation. A 7-fold cross-validation was performed in the training and testing sets and the area under the curve of the receiver operating characteristic (ROC) was calculated in the validation set. Results: Model 1 utilizes processed features derived from patient-level distributions and generalized linear model. The estimated AUC for predicting M1 stage is 0.79 and 0.74 for HC and AE features, respectively. Model 2 uses the average of each features obtained from dominant group of nuclei within individual tiles and neural network models. Nuclei groups were assigned by unsupervised clustering method. For this model, the AUC of predicting M1 stage is 0.77 and 0.75 for HC and AE features, respectively. Conclusion: By applying two distinct feature extraction methods and two approaches to summarize features, we obtain similar prediction accuracies. These results demonstrate that quantitative nuclear features contain information to classify M1 and M0 cases, which cannot be classified based on tumor grade or pathologic features. Citation Format: Fangjin Huang, Nathan Ing, Eric N. Miller, Hootan Salemi, Michael S. Lewis, Isla P. Garraway, Arkadiusz Gertych, Beatrice S. Knudsen. Nuclear morphology predicts prostate cancer metastasis at diagnosis [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 3042.

71 Background: The Decipher 22-feature genomic classifier (GC) has been validated to predict metastasis and prostate cancer specific mortality in needle biopsy (Bx) tissue of men with intermediate and high-risk prostate cancer. We validate GC in diagnostic Bx specimens for the prediction of high-grade/stage (HGS) disease at radical prostatectomy (RP) in men with low and favorable-intermediate (fav-int) NCCN risk group disease. Methods: We identified 176 men diagnosed with low or fav-int NCCN risk group disease who had available Bx GC scores and pathological information after RP from Cedars-Sinai, University of Calgary, Cleveland Clinic, MD Anderson, and Johns Hopkins. The GC score was calculated based on a locked random forest model. Scores range from 0-1 with cut points for GC low, intermediate and high risk groups as 0.6, respectively. The primary endpoint was HGS (Gleason group 3-5 or pT3b or lymph node invasion (LNI)). Univariable (UVA) and multivariable (MVA) logistic regression models were used to evaluate GC and CAPRA. Results: Median age of the cohort was 62 years, 87% and 13% had Bx grade group (GG) 1 or 2 disease. 76% and 24% were NCCN low and fav-int risk, respectively. CAPRA classified 70% as low (0-2) and 30% as average risk (3-5). GC classified 80% low, 16% intermediate and 4% high genomic risk. After RP, 41% had RP GG 1, 46% GG 2 and 13% had GG 3-5 disease. pT3b or positive lymph nodes were observed in 7 men (4%), overall 27 (15%) of men had HGS at RP. In the UVA and MVA, GC was the only significant predictor of HGS with odds ratio (OR) of 1.38 and 1.34 per 10% unit increase, before and after adjusting for CAPRA (p=0.011, 0.027). A low risk score (GC

Objective: The global rise of HPV(+) oropharyngeal squamous cell carcinoma (OPSCC) has generated considerable interest underlying its etiology and management. Relative to HPV(-) OPSCC, HPV(+) patients are younger and more responsive to chemoradiation, though non-responders within this sub-class of OPSCC indicate divergent molecular pathophysiology, and continue to challenge clinicians. Rather than an adaptive approach to what many consider distinct disease processes, treatment remain the same for all OPSCC. Improved molecular stratification would greatly enhance the clinician's ability to precisely tailor treatment without jeopardizing outcomes. Here, two HPV(+) OPSCC cohorts delineated based on treatment response are compared via a hybrid Data Dependent Acquisition/Data Independent Acquisition (DDA/DIA) approach via mass spectrometry, to detect low-abundance proteins and highlight pathways not detected through genomic platforms alone. Methods: Fourteen HPV(+) OPSCC patients who underwent definitive chemoradiation were stratified as clinical responders (n=6) or nonresponders (n=8). Responders were defined as having no recurrence for at least three years after treatment. Formalin-fixed paraffin tissue (FFPE) slides of tissue at diagnosis were confirmed to be p16(+) OPSCC by an independent pathologist, microdissected to achieve >80% tumor purity, and subjected to heat and pressure cycling via barocycler to enhance protein yield and identification. Equal mass of protein was digested and run on a Sciex TripleTOF mass spectrometer. Peptides identified with Results: Collectively, 1440 proteins were identified and quantified. Of those, 212 were found to be significantly different between responders and nonresponders (FDR Conclusions: We demonstrate the feasibility of a modified barocycler approach to obtain robust proteomic data from FFPE tissues at a comparable rate to fresh frozen tissues. We further identify via a hybrid mass spectrometry approach targets of insulin receptor signaling that may be a driver of pathogenesis at the proteomic level. Such targets may stratify HPV(+) OPSCC patients at diagnosis and help tailor the intensity of therapy to mitigate toxicities based on recurrence risk. Citation Format: Aaron Robinson, Sarah J. Parker, Shiao L. Stephen, Beatrice S. Knudsen, Zachary S. Zumsteg, Jennifer E. Van Eyk, Allen S. Ho. Proteomic stratification of HPV(+) oropharyngeal squamous cell carcinoma identifies targets in insulin receptor signaling within nonresponders [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr 41.

The inappropriate expression of the c-MET cell surface receptor in many human solid tumors necessitates the development of companion diagnostics to identify those patients who could benefit from c-MET targeted therapies. Tumor tissues are formalin-fixed and paraffin embedded (FFPE) for histopathological evaluation, making the development of an antibody against c-MET that accurately and reproducibly detects the protein in FFPE samples an urgent need. We have developed a monoclonal antibody, designated MET4, from a panel of MET-avid monoclonal antibodies, based on its specific staining pattern in FFPE preparations of normal human prostate tissues. The accuracy of MET4 immunohistochemistry (MET4-IHC) was assessed by comparing MET4-IHC in FFPE cell pellets with immunoblotting analysis. The technical reproducibility of MET4-IHC possessed a percentage coefficient of variability (%CV) of 6.25% in intra-assay and inter-assay testing. Comparison with other commercial c-MET antibody detection reagents demonstrated equal specificity and increased sensitivity for c-MET detection in prostate tissues. In two cohorts of ovarian cancers and gliomas, MET4 reacted with ovarian cancers of all histological subtypes (strong staining in 25%) and with 63% of gliomas. In addition, MET4 bound c-Met on the surfaces of cultured human cancer cells and tumor xenografts. In summary, the MET4 monoclonal antibody accurately and reproducibly measures c-MET expression by IHC in FFPE tissues and can be used for molecular imaging in-vivo. These properties encourage further development of MET4 as a multipurpose molecular diagnostics reagent to help to guide appropriate selection of patients being considered for treatment with c-MET-antagonistic drugs.

Purpose: We examined the feasibility of using gene expression changes in human prostate epithelium as a measure of response to a dietary intervention. Materials and Methods: Eight men with newly diagnosed prostate cancer were randomized to a low-fat/low-glycemic load intervention arm (200). Prostate tissue was collected before randomization and ∼6 weeks later, at the time of radical prostatectomy. Epithelium was acquired by laser capture microdissection, and transcript abundance levels were measured by cDNA microarray hybridization and confirmed by quantitative reverse transcription-PCR. Results: Men in the intervention arm consumed 39% less total energy (P = 0.004) and the difference in weight change between intervention and control arms was −6.1 kg (P = 0.02). In the intervention arm, 23 (0.46%) of 5,711 cDNAs with measurable expression were significantly altered (P < 0.05; false discovery rate, ≤10%). In the control arm, there were no significant changes in transcript expression, even when using a false discovery rate as high as 50%. Conclusions: A 6-week, low-fat/low-glycemic load diet was associated with significant gene expression changes in human prostate epithelium. These results show the feasibility of using prostate tissues collected at diagnosis and at surgery to study the effects of dietary manipulation on prostate tissue, which may give insight into the molecular mechanisms underlying the associations of diet and obesity with the development or progression of prostate cancer. (Cancer Epidemiol Biomarkers Prev 2007;16(10):2150–4)

Androgen deprivation therapy (ADT) remains the primary treatment for advanced prostate cancer. The efficacy of ADT has not been rigorously evaluated by demonstrating suppression of prostatic androgen activity at the target tissue and molecular level. We determined the efficacy and consistency of medical castration in suppressing prostatic androgen levels and androgen-regulated gene expression. Androgen levels and androgen-regulated gene expression (by microarray profiling, quantitative reverse transcription-PCR, and immunohistochemistry) were measured in prostate samples from a clinical trial of short-term castration (1 month) using the gonadotropin-releasing hormone antagonist, Acyline, versus placebo in healthy men. To assess the effects of long-term ADT, gene expression measurements were evaluated at baseline and after 3, 6, and 9 months of neoadjuvant ADT in prostatectomy samples from men with localized prostate cancer. Medical castration reduced tissue androgens by 75% and reduced the expression of several androgen-regulated genes (NDRG1, FKBP5, and TMPRSS2). However, many androgen-responsive genes, including the androgen receptor (AR) and prostate-specific antigen (PSA), were not suppressed after short-term castration or after 9 months of neoadjuvant ADT. Significant heterogeneity in PSA and AR protein expression was observed in prostate cancer samples at each time point of ADT. Medical castration based on serum testosterone levels cannot be equated with androgen ablation in the prostate microenvironment. Standard androgen deprivation does not consistently suppress androgen-dependent gene expression. Suboptimal suppression of tumoral androgen activity may lead to adaptive cellular changes allowing prostate cancer cell survival in a low androgen environment. Optimal clinical efficacy will require testing of novel approaches targeting complete suppression of systemic and intracrine contributions to the prostatic androgen microenvironment. [Cancer Res 2007;67(10):5033–41]

Men who enter active surveillance because their biopsy exhibits only Gleason grade 3 (G3) frequently have higher grade tumor missed by biopsy. Thus, biomarkers are needed that, when measured on G3 tissue, can predict the presence of higher grade tumor in the whole prostate. We evaluated whether PTEN loss, chromosome 8q gain (MYC) and/or 8p loss (LPL) measured only on G3 cores is associated with un-sampled G4 tumor. A tissue microarray was constructed of prostatectomy tissue from patients whose prostates exhibited only Gleason score 3+3, only 3+4, or only 4+3 tumor (n=50 per group). Cores sampled only from areas of G3 were evaluated for PTEN loss by immunohistochemistry, and PTEN deletion, LPL/8p loss, and MYC/8q gain by fluorescence in situ hybridization (FISH). Biomarker results were compared between Gleason score 6 vs. 7 tumors using conditional logistic regression. PTEN protein loss, odds ratio=4.99, p=.033, MYC/8q gain, odds ratio=5.36, p=.010, and LPL/8p loss, odds ratio=3.96, p=.003 were significantly more common in G3 cores derived from Gleason 7 vs. Gleason 6 tumors. PTEN gene deletion was not statistically significant. Associations were stronger comparing Gleason 4+3 vs. 6 than for Gleason 3+4 vs. 6. MYC/8q gain, LPL/8p loss, and PTEN protein loss measured in G3 tissue microarray cores strongly differentiate whether the core comes from a Gleason 6 or Gleason 7 tumor. If validated to predict upgrading from G3 biopsy to prostatectomy these biomarkers could reduce the likelihood of enrolling high risk men and facilitate safe patient selection for active surveillance.

Taxanes are widely employed chemotherapies for patients with metastatic prostate and breast cancer. Here, we show that loss of Diaphanous-related formin-3 (DIAPH3), frequently associated with metastatic breast and prostate cancers, correlates with increased sensitivity to taxanes. DIAPH3 interacted with microtubules (MT) and its loss altered several parameters of MT dynamics as well as decreased polarized force generation, contractility and response to substrate stiffness. Silencing of DIAPH3 increased the cytotoxic response to taxanes in prostate and breast cancer cell lines. Analysis of drug activity for tubulin-targeted agents in the NCI-60 cell line panel revealed a uniform positive correlation between reduced DIAPH3 expression and drug sensitivity. Low DIAPH3 expression correlated with improved relapse-free survival in breast cancer patients treated with chemotherapeutic regimens containing taxanes. Our results suggest that inhibition of MT stability arising from DIAPH3 downregulation enhances susceptibility to MT poisons and that the DIAPH3 network potentially reports taxane sensitivity in human tumors.

Preclinical urothelial carcinoma models suggest activity of dasatinib, an oral SRC-family kinase (SFK) inhibitor. We sought to determine the feasibility and biologic activity of neoadjuvant dasatinib (Neo-D) in patients with muscle-invasive urothelial carcinoma of the bladder (miUCB) preceding radical cystectomy (RC).A prospective multisite phase II trial was conducted. Key eligibility criteria included: resectable miUCB (T2-T4a, N0, M0), and Eastern Cooperative Oncology Group performance status 0 to 1. Patients received oral Neo-D 100mg once daily for 28±7 days followed by RC 8 to 24 hours after the last dose. The primary end point was feasibility, defined as≥60% of patients with miUCB completing therapy without treatment-related dose-limiting toxicity (DLT). Pre- and posttreatment tumor immunohistochemistry of phosphorylated SFK (pSFK), Ki-67, and cleaved caspase (Cas)-3 results were analyzed by paired t test.The study completed full accrual with enrollment of 25 patients of whom 23 were evaluable for feasibility. The study achieved its primary end point with 15 patients (65%) completing therapy without treatment-related DLTs. DLTs included: fatigue (n = 2), pulmonary embolism, abdominal pain, supraventricular tachycardia, enteric fistula, hematuria, and dyspnea (n = 1 each). At RC, 5 patients (23%) hadpT2 disease. Analysis of pre- and posttreatment tumors demonstrated significantly decreased pSFK (P = 0.003) but no overall significant changes in Ki-67 or Cas3. In total, 4 cases demonstrated a nonsignificant decrease in Ki-67, of which 3 cases also demonstrated a decrease in pSFK and 2 cases had marginal increase in Cas3.Neo-D in miUCB patients was feasible and safe. Overall, significant inhibition of pSFK was observed without overall reduction of cellular proliferation or increase of apoptosis, although biologic anti-tumor activity may exist in a small subset of patients. These results highlight the potential utility of the neoadjuvant trial paradigm and suggest that clinical benefit of single-agent SFK inhibition in unselected patients with miUCB is unlikely.

Polyps of the gastrointestinal tract are very common lesions and most frequently sporadic in nature. Some polyp subtypes are associated with rare hereditary polyposis syndromes, including juvenile polyposis syndrome, Peutz-Jeghers syndrome, and Cowden syndrome. However, many sporadic benign lesions of the gastrointestinal tract can mimic some of these syndromic hamartomatous polyps. The role of the surgical pathologist is to raise the possibility of a hereditary condition in case of suggestive polyp histology and to look for clinical information to support the suspected diagnosis. In this review, the clinical presentation and the pathology associated with these rare hamartomatous polyposis syndromes are discussed in an attempt to provide pathologists clues in suggesting one such syndrome on the basis of histologic findings and clinical context. Identification of affected individuals is important because of the increased gastrointestinal and other malignancies. Recently, new adenomatous polyposis syndromes have been discovered, expanding the genetic causes of patient diagnosed with multiple colonic adenomas. By being aware of the clinical phenotype and the tumor spectrum associated with gastrointestinal polyposis syndromes, surgical pathologists can play a critical role in recommending genetic counseling when suspicious of such a diagnosis. This may lead to the identification of a genetic cause and appropriate surveillance of affected family members to screen for associated malignancies.

Purpose: Met, an oncogene product and receptor tyrosine kinase, is a keystone molecule for malignant progression in solid human tumors. We are developing Met-directed imaging and therapeutic agents, including anti-Met monoclonal antibodies (MetSeekTM). In this study, we compared two antibodies, Met5 and Met3, for nuclear imaging of human and canine Met-expressing tumor xenografts in nude mice. Experimental Design: Xenografts representing cancers of three different human tissue origins and metastatic canine prostate cancer were raised s.c. in host athymic nude mice. Animals were injected i.v. with I-125-Met5 or I-125-Met3, posterior total body gamma camera images were acquired for several days postinjection, and quantitative region-of-interest activity analysis was done. Results: PC-3, SK-LMS-1/HGF, and CNE-2 xenografts imaged with I-125-Met5 were compared with PC-3, SK-LMS-1/HGF, and DU145 xenografts imaged with I-125-Met3. Nuclear imaging contrast was qualitatively similar for I-125-Met5 and I-125-Met3 in PC-3 and SK-LMS-1/HGF host mice. However, by region-of-interest analysis, the set of human tumors imaged with I-125-Met3 exhibited a pattern of rapid initial tumor uptake followed by a continuous decline in activity, whereas the set of human tumors imaged with I-125-Met5 showed slow initial uptake, peak tumor-associated activity at 1 day postinjection, and persistence of activity in xenografts for at least 5 days. GN4 canine prostate cancer xenografts were readily imaged with I-125-Met5. Conclusions: We conclude that radioiodinated Met3 and Met5 offer qualitatively similar nuclear images in xenograft-bearing mice, but quantitative considerations indicate that Met5 might be more useful for radioimmunotherapy. Moreover, canine prostate cancer seems to be a suitable model for second-stage preclinical evaluation of Met5.

Background: Immunohistochemical studies use antibodies to stain tissues with the goal of quantifying protein expression. However, protein expression is often heterogeneous resulting in variable degrees and patterns of staining. This problem is particularly acute in prostate cancer, where tumors are infiltrative and heterogeneous in nature. In this article, we introduce analytic approaches that explicitly consider both the frequency and intensity of tissue staining. Methods: Compositional data analysis is a technique used to analyze vectors of unit-sum proportions, such as those obtained from soil sample studies or species abundance surveys. We summarized specimen staining patterns by the proportion of cells staining at mild, moderate, and intense levels and used compositional data analysis to summarize and compare the resulting staining profiles. Results: In a study of Syndecan-1 staining patterns among 44 localized prostate cancer cases with Gleason score 7 disease, compositional data analysis did not detect a statistically significant difference between the staining patterns in recurrent (n = 22) versus nonrecurrent (n = 22) patients. Results indicated only modest increases in the proportion of cells staining at a moderate intensity in the recurrent group. In contrast, an analysis that compared quantitative scores across groups indicated a (borderline) significant increase in staining in the recurrent group (P = 0.05, t test). Conclusions: Compositional data analysis offers a novel analytic approach for immunohistochemical studies, providing greater insight into differences in staining patterns between groups, but possibly lower statistical power than existing, score-based methods. When appropriate, we recommend conducting a compositional data analysis in addition to a standard score-based analysis.

Objectives To determine the significance of syndecan-1 expression, a cell-surface heparan sulfate proteoglycan in localized and metastatic prostate cancer. Methods We performed a retrospective analysis of 76 men with Gleason sum 6 or 7 prostate cancer treated by radical prostatectomy and a separate cohort of 75 men with metastatic prostate cancer. Syndecan-1 immunoreactivity was measured in primary prostate specimens or in samples from metastatic sites and correlated with patient outcome. Results Syndecan-1 was expressed in normal basal and secretory epithelial cells, 26% of radical prostatectomy specimens, and 35% of metastatic disease. No association was found between syndecan-1 positivity and prostate-specific antigen recurrence in the collective cohort of Gleason sum 6 and 7 cancers. However, when stratified by Gleason sum, syndecan-1 immunoreactivity (immunoreactivity score 150 or greater) was associated with a greater recurrence rate in Gleason sum 7 cancers. Expression of syndecan-1 was significantly greater in soft tissue than in bone metastasis (P = 0.048, Fisher's exact test). Conclusions Consistent with a possible biochemical role for syndecan-1 in prostate cancer progression and metastasis, syndecan-1 expression correlated with serologic recurrence in Gleason sum 7 prostate cancer and was highly expressed in soft-tissue metastases.