Abstract Background Intraductal carcinoma (IDC) and cribriform architecture (CA) represent unfavorable subpathologies in localized prostate cancer. We recently showed that IDC shares a clonal ...ancestry with the adjacent glandular adenocarcinoma. Objective We investigated for the co-occurrence of “aggression” factors, genomic instability and hypoxia, and performed gene expression profiling of these tumors. Design, setting, and participants A total of 1325 men were treated for localized prostate cancer from four academic institutions (University Health Network, CHU de Québec-Université Laval, Memorial Sloan Kettering Cancer Center MSKCC, and Erasmus Medical Center). Pathological specimens were centrally reviewed. Gene copy number and expression, and intraprostatic oxygenation were assessed. Outcome measurements and statistical analysis IDC/CA was separately assessed for biochemical relapse risk in the Canadian and MSKCC cohorts. Both cohorts were pooled for analyses on metastasis. Results and limitation Presence of IDC/CA independently predicted for increased risks of biochemical relapse (HRCanadian 2.17, p < 0.001; HRMSKCC 2.32, p = 0.0035) and metastasis (HRpooled 3.31, p < 0.001). IDC/CA+ cancers were associated with an increased percentage of genome alteration (PGA median 7.2 vs 3.0, p < 0.001), and hypoxia (64.0% vs 45.5%, p = 0.17). Combinatorial genomic–pathological indices offered the strongest discrimination for metastasis (C-index 0.805 clinical + IDC/CA + PGA vs 0.786 clinical + IDC/CA vs 0.761 clinical). Profiling of mRNA abundance revealed that long noncoding RNA, SChLAP1 , was the only gene expressed at >3-fold higher ( p < 0.0001) in IDC/CA+ than in IDC/CA– tumors, independently corroborated by increased SChLAP1 RNA in situ hybridization signal. Optimal treatment intensification for IDC/CA+ prostate cancer requires prospective testing. Conclusions The poor outcome associated with IDC and CA subpathologies is associated with a constellation of genomic instability, SChLAP1 expression, and hypoxia. We posit a novel concept in IDC/CA+ prostate cancer, “ nimbosus ” (gathering of stormy clouds, Latin), which manifests as increased metastatic capacity and lethality. Patient summary A constellation of unfavorable molecular characteristics co-occur with intraductal and cribriform subpathologies in prostate cancer. Modern imaging for surveillance and treatment intensification trials should be considered in this adverse subgroup.
The identification of biomarker signatures in omics molecular profiling is usually performed to predict outcomes in a precision medicine context, such as patient disease susceptibility, diagnosis, ...prognosis, and treatment response. To identify these signatures, we have developed a biomarker discovery tool, called BioDiscML. From a collection of samples and their associated characteristics, i.e., the biomarkers (e.g., gene expression, protein levels, clinico-pathological data), BioDiscML exploits various feature selection procedures to produce signatures associated to machine learning models that will predict efficiently a specified outcome. To this purpose, BioDiscML uses a large variety of machine learning algorithms to select the best combination of biomarkers for predicting categorical or continuous outcomes from highly unbalanced datasets. The software has been implemented to automate all machine learning steps, including data pre-processing, feature selection, model selection, and performance evaluation. BioDiscML is delivered as a stand-alone program and is available for download at https://github.com/mickaelleclercq/BioDiscML.
Toll-like receptors (TLRs) have an important role in the activation of both innate and adaptive immunity in response to pathogens and danger signals. These receptors are expressed in immune cells and ...in some epithelia. They are expressed in the epithelium of the urinary bladder, where they actively participate in the fight against infection by uropathogens. TLR expression is decreased (although still evident) in bladder tumours, especially in non-muscle-invasive tumours. Intravesical immunotherapy with BCG to prevent recurrence of these tumours has been shown to involve the participation of three different TLRs (TLR2, TLR4, and TLR9). However, alternative therapies are needed as BCG fails in some patients and can sometimes cause severe adverse effects that are difficult to tolerate. In recent years, TLR2, TLR4, TLR7, and TLR9 agonists have been tested in vitro and in vivo for their ability to activate an antitumour immune response against bladder cancer. Promising results from these studies have led to the testing of TLR7 and TLR9 agonists in clinical trials.
Within the prostate tumor microenvironment (TME) there are complex multi-faceted and dynamic communication occurring between cancer cells and immune cells. Macrophages are key cells which infiltrate ...and surround tumor cells and are recognized to significantly contribute to tumor resistance and metastases. Our understanding of their function in the TME is commonly based on in vitro and in vivo models, with limited research to confirm these model observations in human prostates. Macrophage infiltration was evaluated within the TME of human prostates after 72 h culture of fresh biopsies samples in the presence of control or enzalutamide. In addition to immunohistochemistry, an optimized protocol for multi-parametric evaluation of cellular surface markers was developed using flow cytometry. Flow cytometry parameters were compared to clinicopathological features. Immunohistochemistry staining for 19 patients with paired samples suggested enzalutamide increased the expression of CD163 relative to CD68 staining. Techniques to validate these results using flow cytometry of dissociated biopsies after 72 h of culture are described. In a second cohort of patients with Gleason grade group ≥ 3 prostate cancer, global macrophage expression of CD163 was unchanged with enzalutamide treatment. However, exploratory analyses of our results using multi-parametric flow cytometry for multiple immunosuppressive macrophage markers suggest subgroup changes as well as novel associations between circulating biomarkers like the neutrophil to lymphocyte ratio (NLR) and immune cell phenotype composition in the prostate TME. Further, we observed an association between B7-H3 expressing tumor-associated macrophages and the presence of intraductal carcinoma. The use of flow cytometry to evaluate ex vivo cultured prostate biopsies fills an important gap in our ability to understand the immune cell composition of the prostate TME. Our results highlight novel associations for further investigation.
The gut microbiota modulates response to hormonal treatments in prostate cancer (PCa) patients, but whether it influences PCa progression remains unknown. Here, we show a reduction in fecal ...microbiota alpha-diversity correlating with increase tumour burden in two distinct groups of hormonotherapy naïve PCa patients and three murine PCa models. Fecal microbiota transplantation (FMT) from patients with high PCa volume is sufficient to stimulate the growth of mouse PCa revealing the existence of a gut microbiome-cancer crosstalk. Analysis of gut microbial-related pathways in mice with aggressive PCa identifies three enzymes responsible for the metabolism of long-chain fatty acids (LCFA). Supplementation with LCFA omega-3 MAG-EPA is sufficient to reduce PCa growth in mice and cancer up-grading in pre-prostatectomy PCa patients correlating with a reduction of gut Ruminococcaceae in both and fecal butyrate levels in PCa patients. This suggests that the beneficial effect of omega-3 rich diet is mediated in part by modulating the crosstalk between gut microbes and their metabolites in men with PCa.
Introduction The localization, density but mostly the phenotype of tumor infiltrating lymphocytes (TIL) provide important information on the initial interaction between the host immune system and the ...tumor. Our objective was to assess the prognostic significance of T (CD3 + ), T regulatory (T reg ) (FoxP3 + ) and T memory (T mem ) (CD45RO + ) infiltrating lymphocytes and of genes associated with TIL in prostate cancer (PCa). Methods Immunohistochemistry (IHC) was used to assess the infiltration of CD3 + , FoxP3 + and CD45RO + cells in the tumor area, tumor margin and adjacent normal-like epithelium of a series of 98 PCa samples with long clinical follow-up. Expression of a panel of 31 TIL-associated genes was analyzed by Taqman Low-Density Array (TLDA) technology in another series of 50 tumors with long clinical follow-up. Kaplan-Meier and Cox proportional hazards regression analyses were performed to determine association of these markers with biochemical recurrence (BCR), need for definitive androgen deprivation therapy (ADT) or lethal PCa. Results TIL subtypes were present at different densities in the tumor, tumor margin and adjacent normal-like epithelium, but their density and phenotype in the tumor area were the most predictive of clinical outcomes. In multivariate analyses, a high density of T reg (high FoxP3 + /CD3 + cell ratio) predicted a higher risk for need of definitive ADT (HR=7.69, p=0.001) and lethal PCa (HR=4.37, p=0.04). Conversely, a high density of T mem (high CD45RO + /CD3 + cell ratio) predicted a reduced risk of lethal PCa (HR=0.06, p=0.04). TLDA analyses showed that a high expression of FoxP3 was associated with a higher risk of lethal PCa (HR=5.26, p=0.02). Expression of CTLA-4, PD-1, TIM-3 and LAG-3 were correlated with that of FoxP3. Amongst these, only a high expression of TIM-3 was associated with a significant higher risk for definitive ADT in univariate Cox regression analysis (HR=3.11, p=0.01). Conclusion These results show that the proportion of T reg and T mem found within the tumor area is a strong and independent predictor of late systemic progression of PCa. Our results also suggest that inhibition of TIM-3 might be a potential approach to counter the immunosuppressive functions of T reg in order to improve the anti-tumor immune response against PCa.
Prostate cancer (PC) is the most frequently diagnosed cancer in North American men. Pathologists are in critical need of accurate biomarkers to characterize PC, particularly to confirm the presence ...of intraductal carcinoma of the prostate (IDC-P), an aggressive histopathological variant for which therapeutic options are now available. Our aim was to identify IDC-P with Raman micro-spectroscopy (RμS) and machine learning technology following a protocol suitable for routine clinical histopathology laboratories.
We used RμS to differentiate IDC-P from PC, as well as PC and IDC-P from benign tissue on formalin-fixed paraffin-embedded first-line radical prostatectomy specimens (embedded in tissue microarrays TMAs) from 483 patients treated in 3 Canadian institutions between 1993 and 2013. The main measures were the presence or absence of IDC-P and of PC, regardless of the clinical outcomes. The median age at radical prostatectomy was 62 years. Most of the specimens from the first cohort (Centre hospitalier de l'Université de Montréal) were of Gleason score 3 + 3 = 6 (51%) while most of the specimens from the 2 other cohorts (University Health Network and Centre hospitalier universitaire de Québec-Université Laval) were of Gleason score 3 + 4 = 7 (51% and 52%, respectively). Most of the 483 patients were pT2 stage (44%-69%), and pT3a (22%-49%) was more frequent than pT3b (9%-12%). To investigate the prostate tissue of each patient, 2 consecutive sections of each TMA block were cut. The first section was transferred onto a glass slide to perform immunohistochemistry with H&E counterstaining for cell identification. The second section was placed on an aluminum slide, dewaxed, and then used to acquire an average of 7 Raman spectra per specimen (between 4 and 24 Raman spectra, 4 acquisitions/TMA core). Raman spectra of each cell type were then analyzed to retrieve tissue-specific molecular information and to generate classification models using machine learning technology. Models were trained and cross-validated using data from 1 institution. Accuracy, sensitivity, and specificity were 87% ± 5%, 86% ± 6%, and 89% ± 8%, respectively, to differentiate PC from benign tissue, and 95% ± 2%, 96% ± 4%, and 94% ± 2%, respectively, to differentiate IDC-P from PC. The trained models were then tested on Raman spectra from 2 independent institutions, reaching accuracies, sensitivities, and specificities of 84% and 86%, 84% and 87%, and 81% and 82%, respectively, to diagnose PC, and of 85% and 91%, 85% and 88%, and 86% and 93%, respectively, for the identification of IDC-P. IDC-P could further be differentiated from high-grade prostatic intraepithelial neoplasia (HGPIN), a pre-malignant intraductal proliferation that can be mistaken as IDC-P, with accuracies, sensitivities, and specificities > 95% in both training and testing cohorts. As we used stringent criteria to diagnose IDC-P, the main limitation of our study is the exclusion of borderline, difficult-to-classify lesions from our datasets.
In this study, we developed classification models for the analysis of RμS data to differentiate IDC-P, PC, and benign tissue, including HGPIN. RμS could be a next-generation histopathological technique used to reinforce the identification of high-risk PC patients and lead to more precise diagnosis of IDC-P.
Using a modified RNA-sequencing (RNA-seq) approach, we discovered a new family of unusually short RNAs mapping to ribosomal RNA 5.8S, which we named dodecaRNAs (doRNAs), according to the number of ...core nucleotides (12 nt) their members contain. Using a new quantitative detection method that we developed, we confirmed our RNA-seq data and determined that the minimal core doRNA sequence and its 13-nt variant C-doRNA (doRNA with a 5′ Cytosine) are the two most abundant doRNAs, which, together, may outnumber microRNAs. The C-doRNA/doRNA ratio is stable within species but differed between species. doRNA and C-doRNA are mainly cytoplasmic and interact with heterogeneous nuclear ribonucleoproteins (hnRNP) A0, A1 and A2B1, but not Argonaute 2. Reporter gene activity assays suggest that C-doRNA may function as a regulator of Annexin II receptor (AXIIR) expression. doRNAs are differentially expressed in prostate cancer cells/tissues and may control cell migration. These findings suggest that unusually short RNAs may be more abundant and important than previously thought.
Prostate cancer (PCa) shows considerable variation in clinical outcomes between individuals with similar diseases. The initial host-tumor interaction as assessed by detailed analysis of tumor ...infiltrating immune cells within the primary tumor may dictate tumor evolution and late clinical outcomes. In this study, we assessed the association between clinical outcomes and dendritic cell (DC) or macrophage (MΦ) tumor infiltration as well as with expression of genes related to their functions.
Infiltration and localization of immature DC, mature DC, total MΦ and M2-type MΦ was analyzed by immunohistochemistry in 99 radical prostatectomy specimens from patients with 15.5 years median clinical follow-up using antibodies against CD209, CD83, CD68 and CD163, respectively. The density of positive cells for each marker in various tumor areas was determined. In addition, expression of immune genes associated with DC and MΦ was tested in a series of 50 radical prostatectomy specimens by Taqman Low-Density Array with similarly long follow-up. Gene expression was classified as low and high after unsupervised hierarchical clustering. Numbers and ratio of positive cells and levels of gene expression were correlated with endpoints such as biochemical recurrence (BCR), need for definitive androgen deprivation therapy (ADT) or lethal PCa using Cox regression analyses and/or Kaplan-Meier curves.
Positive immune cells were observed in tumor, tumor margin, and normal-like adjacent epithelium areas. CD209
and CD163
cells were more abundant at the tumor margin. Higher CD209
/CD83
cell density ratio at the tumor margin was associated with higher risk of ADT and lethal PCa while higher density of CD163
cells in the normal-like adjacent epithelium was associated with a higher risk of lethal PCa. A combination of 5 genes expressed at high levels correlated with a shorter survival without ADT and lethal PCa. Among these five genes, expression of
and
was correlated to each other and was associated with shorter survival without BCR and ADT/lethal PCa, respectively.
A higher level of infiltration of CD209
immature DC and CD163
M2-type MΦ in the peritumor area was associated with late adverse clinical outcomes.
The identification of patients with high-risk prostate cancer (PC) is a major challenge for clinicians, and the improvement of current prognostic parameters is an unmet clinical need. We and others ...have identified an association between the nuclear localization of NF-κB p65 and biochemical recurrence (BCR) in PC in small and/or single-centre cohorts of patients.
In this study, we accessed 2 different multi-centre tissue microarrays (TMAs) representing cohorts of patients (Test-TMA and Validation-TMA series) of the Canadian Prostate Cancer Biomarker Network (CPCBN) to validate the association between p65 nuclear frequency and PC outcomes. Immunohistochemical staining of p65 was performed on the Test-TMA and Validation-TMA series, which include PC tissues from patients treated by first-line radical prostatectomy (n = 250 and n = 1,262, respectively). Two independent observers evaluated the p65 nuclear frequency in digital images of cancer tissue and benign adjacent gland tissue. Kaplan-Meier curves coupled with a log-rank test and univariate and multivariate Cox regression models were used for statistical analyses of continuous values and dichotomized data (cutoff of 3%). Multivariate analysis of the Validation-TMA cohort showed that p65 nuclear frequency in cancer cells was an independent predictor of BCR using continuous (hazard ratio HR 1.02 95% CI 1.00-1.03, p = 0.004) and dichotomized data (HR 1.33 95% CI 1.09-1.62, p = 0.005). Using a cutoff of 3%, we found that this biomarker was also associated with the development of bone metastases (HR 1.82 95% CI 1.05-3.16, p = 0.033) and PC-specific mortality (HR 2.63 95% CI 1.30-5.31, p = 0.004), independent of clinical parameters. BCR-free survival, bone-metastasis-free survival, and PC-specific survival were shorter for patients with higher p65 nuclear frequency (p < 0.005). As the small cores on TMAs are a limitation of the study, a backward validation of whole PC tissue section will be necessary for the implementation of p65 nuclear frequency as a PC biomarker in the clinical workflow.
We report the first study using the pan-Canadian multi-centre cohorts of CPCBN and validate the association between increased frequency of nuclear p65 frequency and a risk of disease progression.
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