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Purpose: Given discrepancies between preclinical and clinical observations of mammalian target of rapamycin (mTOR) inhibition in prostate cancer, we sought to determine the pharmacodynamic effects of the mTOR/TORC1 inhibitor rapamycin in men with intermediate- to high-risk prostate cancer undergoing radical prostatectomy.Experimental Design: Rapamycin was given at 3 or 6 mg orally for 14 days before radical prostatectomy in men with multifocal Gleason sum ≥7 prostate cancer; 10 untreated control subjects were included. The primary outcome was inhibition of phosphorylation of ribosomal S6 in posttreatment radical prostatectomy versus pretreatment biopsy tumor tissue, evaluated using a Simon two-stage design for pharmacodynamic efficacy.Results: Thirty-two subjects were accrued: 20 at 3 mg, 2 at 6 mg, and 10 controls. No dose-limiting toxicities were observed at 3 mg; however, two of two men enrolled at 6 mg experienced dose-limiting toxicities including thrombocytopenia and fever with grade 3 stomatitis. Adverse events observed at 3 mg included stomatitis, rash, ileus, and neutropenia. Pharmacodynamic studies showed tumor S6 phosphorylation inhibition in 50% of 10 evaluable rapamycin-treated men with sufficient paired tissue [median 58% inhibition (P = 0.049) versus 2% inhibition in controls (P = 0.75)] with no significant effect on AKT activity. We observed no change in Ki-67 or caspase-3 cleavage but noted a reduction in cytoplasmic p27 staining with increased nuclear localization with rapamycin treatment. Prostate tissue rapamycin concentrations were 3- to 4-fold higher than blood.Conclusions: At 3 mg daily, rapamycin successfully and safely inhibited prostate cancer S6 phosphorylation and achieved relatively high prostate tissue concentrations. No effect on AKT phosphorylation, tumor proliferation, or apoptosis was observed. Clin Cancer Res; 16(11); 3057–66. ©2010 AACR.

Supplementary Data from A Pharmacodynamic Study of Rapamycin in Men with Intermediate- to High-Risk Localized Prostate Cancer

Spatial transcriptomics (ST) is a powerful new approach to characterize the cellular and molecular architecture of the tumor microenvironment. Previous single-cell RNA-sequencing (scRNA-seq) studies of pancreatic ductal adenocarcinoma (PDAC) have revealed a complex immunosuppressive environment characterized by numerous cancer associated fibroblasts (CAFs) subtypes that contributes to poor outcomes. Nonetheless, the evolutionary processes yielding that microenvironment remain unknown. Pancreatic intraepithelial neoplasia (PanIN) is a premalignant lesion with potential to develop into PDAC, but the formalin-fixed and paraffin-embedded (FFPE) specimens required for PanIN diagnosis preclude scRNA-seq profiling. We developed a new experimental pipeline for FFPE ST analysis of PanINs that preserves clinical specimens for diagnosis. We further developed novel multi-omics analysis methods for threefold integration of imaging, ST, and scRNA-seq data to analyze the premalignant microenvironment. The integration of ST and imaging enables automated cell type annotation of ST spots at a single-cell resolution, enabling spot selection and deconvolution for unique cellular components of the tumor microenvironment (TME). Overall, this approach demonstrates that PanINs are surrounded by the same subtypes of CAFs present in invasive PDACs, and that the PanIN lesions are predominantly of the classical PDAC subtype. Moreover, this new experimental and computational protocol for ST analysis suggests a biological model in which CAF-PanIN interactions promote inflammatory signaling in neoplastic cells which transitions to proliferative signaling as PanINs progress to PDAC.SummaryPancreatic intraepithelial neoplasia (PanINs) are pre-malignant lesions that progress into pancreatic ductal adenocarcinoma (PDAC). Recent advances in single-cell technologies have allowed for detailed insights into the molecular and cellular processes of PDAC. However, human PanINs are stored as formalin-fixed and paraffin-embedded (FFPE) specimens limiting similar profiling of human carcinogenesis. Here, we describe a new analysis protocol that enables spatial transcriptomics (ST) analysis of PanINs while preserving the FFPE blocks required for clinical assessment. The matched H&E imaging for the ST data enables novel machine learning approaches to automate cell type annotations at a single-cell resolution and isolate neoplastic regions on the tissue. Transcriptional profiles of these annotated cells enable further refinement of imaging-based cellular annotations, showing that PanINs are predominatly of the classical subtype and surrounded by PDAC cancer associated fibroblast (CAF) subtypes. Applying transfer learning to integrate ST PanIN data with PDAC scRNA-seq data enables the analysis of cellular and molecular progression from PanINs to PDAC. This analysis identified a transition between inflammatory signaling induced by CAFs and proliferative signaling in PanIN cells as they become invasive cancers. Altogether, this integration of imaging, ST, and scRNA-seq data provides an experimental and computational approach for the analysis of cancer development and progression.

// Tamara L. Lotan 1, 2 , Alba Torres 1 , Miao Zhang 3 , Jeffrey J. Tosoian 4 , Liana B. Guedes 1 , Helen Fedor 1 , Jessica Hicks 1 , Charles M. Ewing 4 , Sarah D. Isaacs 4 , Dorhyun Johng 4 , Angelo M. De Marzo 1, 2, 4 , William B. Isaacs 2, 4 1 Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD, USA 2 Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, MD, USA 3 Department of Pathology the University of Texas MD Anderson Cancer Center, Houston, TX, USA 4 Brady Urological Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USA Correspondence to: Tamara L. Lotan, email: tlotan1@jhmi.edu Keywords: prostatic adenocarcinoma, HOXB13 G84E, familial prostate cancer, PTEN, ERG Received: December 20, 2016 Accepted: January 21, 2017 Published: February 08, 2017 ABSTRACT A recurrent germline mutation (G84E) in the HOXB13 gene is associated with early onset and family history-positive prostate cancer in patients of European descent, occurring in up to 5% of prostate cancer families. To date, the molecular features of prostate tumors occurring in HOXB13 G84E carriers have not been studied in a large cohort of patients. We identified 101 heterozygous carriers of G84E who underwent radical prostatectomy for prostate cancer between 1985 and 2011 and matched these men by race, age and tumor grade to 99 HOXB13 wild-type controls. Immunostaining for HOXB13, PTEN, ERG, p53 and SPINK1 as well as RNA in situ hybridization for ETV1/4/5 were performed using genetically validated assays. Tumors from G84E carriers generally expressed HOXB13 protein at a level comparable to benign and wild-type glands. ETS gene expression (either ERG or ETV1/4/5 ) was seen in 36% (36/101) of tumors from G84E carriers compared to 68% (65/96) of the controls ( p < 0.0001). PTEN was lost in 11% (11/101) of G84E carriers compared to 25% (25/99) of the controls ( p = 0.014). PTEN loss was enriched among ERG-positive compared to ERG-negative tumors in both groups of patients. Nuclear accumulation of the p53 protein, indicative of underlying TP53 missense mutations, was uncommon in both groups, occurring in 1% (1/101) of the G84E carriers versus 2% (2/92) of the controls ( p = NS). Taken together, these data suggest that genes other than ERG and PTEN may drive carcinogenesis/progression in the majority of men with germline HOXB13 mutations.

Context.— Phosphatase and tensin homolog (PTEN) is a promising prognostic and potentially predictive biomarker in prostate cancer. Objective.— To assess the effects of preanalytic variables on an analytically validated and fully automated PTEN immunohistochemistry assay. Design.— PTEN immunohistochemistry was performed on Ventana immunostaining systems. In benign prostate tissues, immunostaining intensity across variable conditions was assessed by digital image analysis. In prostate tumor tissues, immunostaining was scored visually. Results.— Delay of fixation for 4 hours or longer at room temperature or 48 hours or longer at 4°C and duration of formalin fixation did not significantly alter immunostaining intensity. Intensity of staining was highest in 10% formalin compared with other fixatives. Tumor tissues with PTEN loss processed using protocols from 11 academic institutions were all evaluable and scored identically. PTEN immunostaining of needle biopsies where tissue blocks had been stored for less than 10 years was more frequently scored as nonevaluable compared with blocks that had been stored for 10 years or longer. This effect was less evident for radical prostatectomy specimens, where low rates of nonevaluable staining were seen for 23 years or more of storage. Storage of unstained slides for 5 years at room temperature prior to immunostaining resulted in equivalent scoring compared with freshly cut slides. Machine-to-machine variability assessed across 3 Ventana platforms and 2 institutions was negligible in 12 tumors, and platform-to-platform variability was also minor comparing Ventana and Leica instruments across 77 tumors (κ = 0.926). Conclusions.— Automated PTEN immunostaining is robust to most preanalytic variables in the prostate and may be performed on prostate tumor tissues subjected to a wide range of preanalytic conditions. These data may help guide assay development if PTEN becomes a key predictive biomarker.

Recent commercialization of methods for in situ hybridization using Z-pair probe/branched DNA amplification has led to increasing adoption of this technology for interrogating RNA expression in formalin-fixed, paraffin-embedded (FFPE) tissues. Current practice for FFPE block storage is to maintain them at room temperature, often for many years.To examine the effects of block storage time on FFPE tissues using a number of RNA in situ probes with the Advanced Cellular Diagnostic's RNAscope assay.We report marked reductions in signals after 5 years and significant reductions often after 1 year. Furthermore, storing unstained slides cut from recent cases (1 year old) at -20°C can preserve hybridization signals significantly better than storing the blocks at room temperature and cutting the slides fresh when needed.We submit that the standard practice of storing FFPE tissue blocks at room temperature should be reevaluated to better preserve RNA for in situ hybridization.

The aim of this study was to characterize patterns of local progression following resection for pancreatic intraductal papillary mucinous neoplasms (IPMN) using targeted next-generation sequencing (NGS).Progression of neoplastic disease in the remnant pancreas following resection of IPMN may include development of a new IPMN or ductal adenocarcinoma (PDAC). However, it is not clear whether this progression represents recurrence of the same neoplasm or an independent second neoplasm.Targeted-NGS on genes commonly mutated in IPMN and PDAC was performed on tumors from (1) 13 patients who developed disease progression in the remnant pancreas following resection of IPMN; and (2) 10 patients who underwent a resection for PDAC and had a concomitant IPMN. Mutations in the tumors were compared in order to determine the relationship between neoplasms. In parallel, clinical and pathological characteristics of 260 patients who underwent resection of noninvasive IPMN were reviewed to identify risk factors associated with local progression.We identified 3 mechanisms underlying local progression in the remnant pancreas: (1) residual microscopic disease at the resection margin, (2) intraparenchymal spread of neoplastic cells, leading to an anatomically separate but genetically related recurrence, and (3) multifocal disease with genetically distinct lesions. Analysis of the 260 patients with noninvasive IPMNs showed that family history of pancreatic cancer (P = 0.027) and high-grade dysplasia (HGD) (P = 0.003) were independent risk factors for the development of an IPMN with HGD or an invasive carcinoma in the remnant pancreas.Using NGS, we identify distinct mechanisms for development of metachronous or synchronous neoplasms in patients with IPMN. Patients with a primary IPMN with HGD or with positive family history are at an increased risk to develop subsequent high-risk neoplasms in the remnant pancreas.

PURPOSE: Prostate cancer is clinically and molecularly heterogeneous. We determined the prognosis of men with ERG-ETS fusions and SPINK1 over expression. MATERIALS AND METHODS: Men were identified with intermediate or high risk localized prostate cancer treated with radical prostatectomy and no therapy before metastasis. A case-cohort design sampled a cohort (262) enriched with metastasis from the entire cohort and a cohort (213) enriched with metastasis from patients with biochemical recurrence. We analyzed transcriptomic profiles and subtyped tumors as m-ERG(+), m-ETS(+), m-SPINK1(+) or Triple Negative (m-ERG(−)/m-ETS(−)/m-SPINK1(−)), and multivariable logistic regression analyses, Kaplan-Meier and multivariable Cox models were used to evaluate subtypes as predictors of clinical outcomes. RESULTS: Overall 36%, 13%, 11% and 40% of prostate cancer was classified as m-ERG(+), m-ETS(+), m-SPINK1(+) and Triple Negative, respectively. Univariable analysis demonstrated that m-SPINK1(+) tumors were more common in African-American men (OR 5, 95% CI 1.6–16) but less commonly associated with positive surgical margins (OR 0.16, 95% CI 0.03–0.69) compared to the m-ERG(+) group. Compared to the Triple Negative group, m-SPINK1(+) showed similar associations with race and surgical margins in univariable and multivariable analyses across the entire cohort. Survival analyses did not show significant differences among m-ERG(+), m-ETS(+) and Triple Negative cases. m-SPINK1(+) independently predicted prostate cancer specific mortality after metastasis (HR 2.48, 95% CI 0.96–6.4) and biochemical recurrence (HR 3, 95% CI 1.1–8). CONCLUSIONS: SPINK1 over expression is associated with prostate cancer specific mortality in at risk men with biochemical and clinical recurrence after prostatectomy. ERG-ETS alterations are not prognostic for outcome.

In 2005, the International Society of Urological Pathology (ISUP) introduced several modifications to the original Gleason system that were intended to enhance the prognostic power of Gleason score (GS). The objective of this study was to clinically validate the 2005 ISUP Gleason grading system for its ability to detect metastasis. We queried our institutional RP database for men with NCCN clinically localized intermediate to high-risk disease undergoing radical prostatectomy (RP) between 1992 and 2010 with no additional treatment until the time of metastatic progression. A case-cohort design was utilized. A total of 333 available RP samples were re-reviewed and GS was reassigned per the 2005 ISUP Gleason system. Cumulative incidence of metastasis was 0%, 8.4%, 24.5% and 44.4% among specimens that were downgraded, unchanged, had one point GS increase and two point GS increase, respectively. The hazard ratio for metastasis raised in GS 8 and 9 compared to GS 7 from 2.77 and 5.91 to 3.49 and 9.31, respectively. The survival c-index of GS increased from 0.70 to 0.80 when samples were re-graded at 5 years post RP. The c-index of the reassigned GS was higher than the original GS (0.77 vs 0.64) for predicting PCSM at 10 years post RP. The regraded GS improved the prediction of metastasis and PCSM. This validates the updated Gleason grading system using an unambiguous clinical endpoint and highlights the need for reassignment of Gleason grading according to 2005 ISUP system when considering comparisons of novel biomarkers to clinicopathological variables in archival cohorts.

Prostate cancer often manifests as morphologically distinct tumour foci and is frequently found adjacent to presumed precursor lesions such as high-grade prostatic intraepithelial neoplasia (HGPIN). While there is some evidence to suggest that these lesions can be related and exist on a pathological and morphological continuum, the precise clonal and temporal relationships between precursor lesions and invasive cancers within individual tumours remain undefined. Here, we used molecular genetic, cytogenetic, and histological analyses to delineate clonal, temporal, and spatial relationships between HGPIN and cancer lesions with distinct morphological and molecular features. First, while confirming the previous finding that a substantial fraction of HGPIN lesions associated with ERG-positive cancers share rearrangements and overexpression of ERG, we found that a significant subset of such HGPIN glands exhibit only partial positivity for ERG. This suggests that such ERG-positive HGPIN cells either rapidly invade to form adenocarcinoma or represent cancer cells that have partially invaded the ductal and acinar space in a retrograde manner. To clarify these possibilities, we used ERG expression status and TMPRSS2-ERG genomic breakpoints as markers of clonality, and PTEN deletion status to track temporal evolution of clonally related lesions. We confirmed that morphologically distinct HGPIN and nearby invasive cancer lesions are clonally related. Further, we found that a significant fraction of ERG-positive, PTEN-negative HGPIN and intraductal carcinoma (IDC-P) lesions are most likely clonally derived from adjacent PTEN-negative adenocarcinomas, indicating that such PTEN-negative HGPIN and IDC-P lesions arise from, rather than give rise to, the nearby invasive adenocarcinoma. These data suggest that invasive adenocarcinoma can morphologically mimic HGPIN through retrograde colonization of benign glands with cancer cells. Similar clonal relationships were also seen for intraductal carcinoma adjacent to invasive adenocarcinoma. These findings represent a potentially undervalued indicator of pre-existing invasive prostate cancer and have significant implications for prostate cancer diagnosis and risk stratification.

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.

significantly enhanced DPYSL3 gene expression in the mRNA and protein levels at day 3 after transfection in 22RV1, DU145 and PC-3 cells. CONCLUSIONS: Our data reveal that isoform-1 of tumor suppressor gene DPYSL3 expression is significantly reduced in malignant tissues and is responsive to up-regulation by a saRNA-based approach. Further animal based in vivo testing of the saRNAs is being undertaking by our group to evaluate their clinical significance as a novel gene therapy strategy.

In 2005, the International Society of Urological Pathology (ISUP) introduced several modifications to the original Gleason system that were intended to enhance the prognostic power of Gleason score (GS). The objective of this study was to clinically validate the 2005 ISUP Gleason grading system for its ability to detect metastasis. We queried our institutional RP database for men with NCCN clinically localized intermediate to high-risk disease undergoing radical prostatectomy (RP) between 1992 and 2010 with no additional treatment until the time of metastatic progression. A case-cohort design was utilized. A total of 333 available RP samples were re-reviewed and GS was reassigned per the 2005 ISUP Gleason system. Cumulative incidence of metastasis was 0%, 8.4%, 24.5% and 44.4% among specimens that were downgraded, unchanged, had one point GS increase and two point GS increase, respectively. The hazard ratio for metastasis raised in GS 8 and 9 compared to GS 7 from 2.77 and 5.91 to 3.49 and 9.31, respectively. The survival c-index of GS increased from 0.70 to 0.80 when samples were re-graded at 5 years post RP. The c-index of the reassigned GS was higher than the original GS (0.77 vs 0.64) for predicting PCSM at 10 years post RP. The regraded GS improved the prediction of metastasis and PCSM. This validates the updated Gleason grading system using an unambiguous clinical endpoint and highlights the need for reassignment of Gleason grading according to 2005 ISUP system when considering comparisons of novel biomarkers to clinicopathological variables in archival cohorts. PLOS ONE | DOI:10.1371/journal.pone.0146189 January 5, 2016 1 / 13 OPEN ACCESS Citation: Faraj SF, Bezerra SM, Yousefi K, Fedor H, Glavaris S, Han M, et al. (2016) Clinical Validation of the 2005 ISUP Gleason Grading System in a Cohort of Intermediate and High Risk Men Undergoing Radical Prostatectomy. PLoS ONE 11(1): e0146189. doi:10.1371/journal.pone.0146189 Editor: Xin Gao, Sun Yat-sen University, CHINA Received: October 18, 2015 Accepted: December 14, 2015 Published: January 5, 2016 Copyright: © 2016 Faraj et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: Kasra Yousefi and Elai Davicione are employees of GenomeDx Biosciences Inc. The remaining authors have no conflict of interest to declare. The funder provided support in the form of salaries for authors [KY and ED], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.

Background: Prostate cancers composed exclusively of Gleason pattern 3 have an excellent prognosis,while those containing Gleason pattern 4 or higher are more aggressive. Gleason pattern 4 is composed of either fused glands, poorly formed glands or cribriform structures with cribriform structures being most readily recognized and shown to be of independent prognostic significance (PMIDS: 24145642;25189638). However, the biological properties of these cribriform structures have not been studied. There is a spectrum of nuclear features in cribriform glands with cells towards the perimeter containing enlarged nucleoli, and open chromatin as compared to cells towards the center. Since MYC overexpression drives nucleolar enlargement we examined the pattern of expression of MYC protein in these structures. Also, we examined p27 staining since it has been reported to be more intense in the nuclei in the center of these structures (PMID:9731735). Design: Cases with cribriform Gleason score 7 were used for IHC for MYC, p27 and Ki67 as well as double label immunofluorescence staining for p27 and MYC. Slides were scanned and regions were selected containing cribriform glands (N = 18 total regions) and analyzed using image analysis software by Aperio Inc. We quantified the level of nuclear staining for MYC and p27 in relation to the outside/perimeter regions versus the inner aspect of the glands. Chromogenic in-situ hybridization for MYC mRNA was also performed. Results: The increase in MYC staining in the perimeter aspects of the cribriform structures was highly significant as compared to the inner regions (P Conclusion: Two distinct populations of cancer cells comprise cribriform carcinomas of the prostate; the central aspect is composed of cells with relatively small nucleoli that are non-proliferating with high levels of nuclear p27, and an outer layer that is composed of metabolically more active cells with increased MYC, nucleolar size and proliferative fraction. These findings provide new biological insights into the unique structure of cribriform prostate cancer lesions and suggest a complex interplay between the two cell types. Future studies are needed to determine how and whether these findings relate to disease aggressiveness. Citation Format: Ibrahim Kulac, Jessica Hicks, Qizhi Zheng, Helen Fedor, Toby C. Cornish, Angelo M. De Marzo. Inverse relation of Myc and p27 in cribriform pattern prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2025. doi:10.1158/1538-7445.AM2015-2025