Yves Allory

Molecular Characterization of Neuroendocrine Prostate Cancer and Identification of New Drug Targets

UNLABELLED Neuroendocrine prostate cancer (NEPC) is an aggressive subtype of prostate cancer that most commonly evolves from preexisting prostate adenocarcinoma (PCA). Using Next Generation RNA-sequencing and oligonucleotide arrays, we profiled 7 NEPC, 30 PCA, and 5 benign prostate tissue (BEN), and validated findings on tumors from a large cohort of patients (37 NEPC, 169 PCA, 22 BEN) using IHC and FISH. We discovered significant overexpression and gene amplification of AURKA and MYCN in 40% of NEPC and 5% of PCA, respectively, and evidence that that they cooperate to induce a neuroendocrine phenotype in prostate cells. There was dramatic and enhanced sensitivity of NEPC (and MYCN overexpressing PCA) to Aurora kinase inhibitor therapy both in vitro and in vivo, with complete suppression of neuroendocrine marker expression following treatment. We propose that alterations in Aurora kinase A and N-myc are involved in the development of NEPC, and future clinical trials will help determine from the efficacy of Aurora kinase inhibitor therapy. SIGNIFICANCE We report on the largest in-depth molecular analysis of NEPC and provide new insight into molecular events involved in the progression of prostate cancer.

Localization and phosphorylation of HP1 proteins during the cell cycle in mammalian cells

Abstract.Mammalian heterochromatin proteins 1 (HP1α, HP1β, and HP1γ) are nonhistone proteins that interact in vitro with a set of proteins that play a role in chromatin silencing, transcription, and chromatin remodeling. Using antibodies specific for each HP1 isoform, we showed that they segregate in distinct nuclear domains of human HeLa cells. By contrast, in mouse 3T3 interphase cells, HP1α and HP1β are strictly colocalized. In mitotic HeLa cells, all of HP1α and a fraction of HP1β and HP1γ remain associated with chromosomes. Immunostaining of spread HeLa chromosomes showed that HP1α is mainly localized on centromeres as shown previously for HP1β, while HP1γ is distributed on discrete sites on the arms of chromosomes. Biochemical analysis showed that HP1α and HP1γ are phosphorylated throughout the cell cycle, although more extensively in mitosis than in interphase, while HP1β apparently remains unphosphorylated. Therefore, despite their extensive sequence conservation, mammalian HP1 isoforms differ widely in their nuclear localization, mitotic distribution and cell cycle-related phosphorylation. Thus, subtle differences in primary sequence and in posttranslational modifications may promote their targeting at different chromatin sites, generating pleiotropic effects.

Regional copy number–independent deregulation of transcription in cancer

Genetic and epigenetic alterations have been identified that lead to transcriptional deregulation in cancers. Genetic mechanisms may affect single genes or regions containing several neighboring genes, as has been shown for DNA copy number changes. It was recently reported that epigenetic suppression of gene expression can also extend to a whole region; this is known as long-range epigenetic silencing. Various techniques are available for identifying regional genetic alterations, but no large-scale analysis has yet been carried out to obtain an overview of regional epigenetic alterations. We carried out an exhaustive search for regions susceptible to such mechanisms using a combination of transcriptome correlation map analysis and array CGH data for a series of bladder carcinomas. We validated one candidate region experimentally, demonstrating histone methylation leading to the loss of expression of neighboring genes without DNA methylation.

Gene Expression Signatures Predict Outcome in Non–Muscle-Invasive Bladder Carcinoma: A Multicenter Validation Study

Purpose: Clinically useful molecular markers predicting the clinical course of patients diagnosed with non–muscle-invasive bladder cancer are needed to improve treatment outcome. Here, we validated four previously reported gene expression signatures for molecular diagnosis of disease stage and carcinoma in situ (CIS) and for predicting disease recurrence and progression. Experimental Design: We analyzed tumors from 404 patients diagnosed with bladder cancer in hospitals in Denmark, Sweden, England, Spain, and France using custom microarrays. Molecular classifications were compared with pathologic diagnosis and clinical outcome. Results: Classification of disease stage using a 52-gene classifier was found to be highly significantly correlated with pathologic stage (P < 0.001). Furthermore, the classifier added information regarding disease progression of Ta or T1 tumors (P < 0.001). The molecular 88-gene progression classifier was highly significantly correlated with progression-free survival (P < 0.001) and cancer-specific survival (P = 0.001). Multivariate Cox regression analysis showed the progression classifier to be an independently significant variable associated with disease progression after adjustment for age, sex, stage, grade, and treatment (hazard ratio, 2.3; P = 0.007). The diagnosis of CIS using a 68-gene classifier showed a highly significant correlation with histopathologic CIS diagnosis (odds ratio, 5.8; P < 0.001) in multivariate logistic regression analysis. Conclusion: This multicenter validation study confirms in an independent series the clinical utility of molecular classifiers to predict the outcome of patients initially diagnosed with non–muscle-invasive bladder cancer. This information may be useful to better guide patient treatment.

Papillary renal cell carcinoma

A series of 43 papillary renal cell carcinomas (PRCCs) were analyzed to investigate the prognostic value of the morphological subtyping (type 1/type 2) proposed by Delahunt and Eble [6]. Twenty-six cases were type 1 (small cuboid cells arranged in single or double layers), 13 cases were type 2 (voluminous eosinophilic cells with irregular pseudostratification pattern), and four cases with oncocytic cells (large eosinophilic cells with round regular nuclei) were distinct from type 2 and grouped apart. All type-1 and oncocytoid-type PRCCs were staged pT1 or pT2, whereas 8/13 type-2 PRCCs were staged pT3 or pT4. Follow-up information (range, 3–113 months; median, 43 months) showed 12 deaths from disease: 2 in the type-1 group,10 in the type-2 group, 0 in the oncocytoid-type group. The Kaplan-Meier analysis showed that pejorative outcome was associated (P<0.001) with high stage (pT3/pT4), high nuclear grade (3/4), morphological type 2, absence of foam cells, and abundant fibrous stroma. The multivariate analysis showed that stage and morphological type were independently associated with survival (P<0.05). These results support the clinical interest of morphological subtyping of PRCCs in the prognosis evaluation of the patients. The four oncocytoid-type PRCCs had a favorable outcome, but additional data are required to evaluate this type of neoplasm.

Inflammation in benign prostatic hyperplasia: A 282 patients' immunohistochemical analysis

Prostatic inflammation could be a key component in prostate enlargement and benign prostatic hyperplasia (BPH) progression. Our aim was to characterize inflammatory cells infiltrate within BPH tissue and to correlate inflammation and clinical data.

Prostate Cancer Antigen 3 Score Accurately Predicts Tumour Volume and Might Help in Selecting Prostate Cancer Patients for Active Surveillance

BACKGROUND The optimal selection of prostate cancer (PCa) patients for active surveillance (AS) is currently being debated. OBJECTIVE To assess the impact of urinary prostate cancer antigen 3 (PCA3) score as an AS criterion instead of and in addition to the current criteria. DESIGN, SETTING, AND PARTICIPANTS We prospectively studied 106 consecutive low-risk PCa patients (prostate-specific antigen [PSA] ≤10 ng/ml, clinical stage T1c-T2a, and biopsy Gleason score 6) who underwent a PCA3 urine test before radical prostatectomy (RP). MEASUREMENTS Performance of AS criteria (biopsy criteria, PCA3 score, PSA density, and magnetic resonance imaging [MRI] findings) was tested in predicting four prognostic pathologic findings in RP specimens: (1) pT3-4 disease; (2) overall unfavourable disease (OUD) defined by pT3-4 disease and/or pathologic primary Gleason pattern 4; (3) tumour volume <0.5 cm(3); and (4) insignificant PCa. RESULTS AND LIMITATIONS The PCA3 score was strongly correlated with the tumour volume in a linear regression analysis (p<0.001, r=0.409). The risk of having a cancer ≥0.5 cm(3) and a significant PCa was increased three-fold in men with a PCA3 score of ≥25 compared with men with a PCA3 score of <25 with most AS biopsy criteria used. There was a trend towards higher PCA3 scores in patients with unfavourable and non-organ-confined disease and Gleason >6 cancers. In a multivariate analysis taking into account each AS criterion, a high PCA3 score (≥25) was an important predictive factor for tumour volume ≥0.5 cm(3) (odds ratio [OR]: 5.4; p=0.010) and significant PCa (OR: 12.7; p=0.003). Biopsy criteria and MRI findings were significantly associated with OUD (OR: 3.9 and 5.0, respectively; p=0.030 and p=0.025, respectively). CONCLUSIONS PCA3 score may be a useful marker to improve the selection for AS in addition to the current AS criteria. With a predictive cut-off of 25, PCA3 score is strongly indicative for tumour volume and insignificant PCa.

Abrogation of De novo Lipogenesis by Stearoyl-CoA Desaturase 1 Inhibition Interferes with Oncogenic Signaling and Blocks Prostate Cancer Progression in Mice

Increased de novo fatty acid (FA) synthesis is one hallmark of tumor cells, including prostate cancer. We present here our most recent results showing that lipid composition in human prostate cancer is characterized by an increased ratio of monounsaturated FA to saturated FA, compared with normal prostate, and evidence the overexpression of the lipogenic enzyme stearoyl-CoA desaturase 1 (SCD1) in human prostate cancer. As a new therapeutic strategy, we show that pharmacologic inhibition of SCD1 activity impairs lipid synthesis and results in decreased proliferation of both androgen-sensitive and androgen-resistant prostate cancer cells, abrogates the growth of prostate tumor xenografts in nude mice, and confers therapeutic benefit on animal survival. We show that these changes in lipid synthesis are translated into the inhibition of the AKT pathway and that the decrease in concentration of phosphatidylinositol-3,4,5-trisphosphate might at least partially mediate this effect. Inhibition of SCD1 also promotes the activation of AMP-activated kinase and glycogen synthase kinase 3α/β, the latter on being consistent with a decrease in β-catenin activity and mRNA levels of various β-catenin growth-promoting transcriptional targets. Furthermore, we show that SCD1 activity is required for cell transformation by Ras oncogene. Together, our data support for the first time the concept of targeting the lipogenic enzyme SCD1 as a new promising therapeutic approach to block oncogenesis and prostate cancer progression. Mol Cancer Ther; 9(6); 1740–54. ©2010 AACR.

Prostate Cancer Detection Rate in Patients with Repeated Extended 21-Sample Needle Biopsy

BACKGROUND Prevalence of prostate cancer (PCa) after a negative first extended prostate needle biopsy protocol is unknown. OBJECTIVE To evaluate the prevalence of significant PCa in patients who have had a negative first extended prostate biopsy protocol. DESIGN, SETTING, AND PARTICIPANTS Between March 2001 and May 2007, 2500 consecutive patients underwent an extended protocol of 21 biopsies. Of 953 patients who had a negative first extended prostate biopsy procedure, 231 patients underwent a second or more set of 21-core biopsies. Indications for repeated biopsies were persistently elevated prostate-specific antigen (PSA), PSA increase during the follow-up, or prior prostatic intraepithelial neoplasia (PIN), or atypical small acinar proliferation (ASAP). INTERVENTION All participants underwent at least two extended prostate needle biopsy protocols. MEASUREMENTS Clinical and pathologic factors (age, PSA, PSA doubling time, PIN, ASAP, digital rectal exam [DRE]) were analyzed for their ability to predict positive biopsy, and tumour parameters were assessed in patients undergoing radical prostatectomy. RESULTS AND LIMITATIONS Second, third, and fourth extended 21-sample biopsy procedures yielded a diagnosis of PCa in 18%, 17%, and 14% of patients respectively. Patients with prior PIN had 16% risk of prostate cancer; patients with ASAP had a 42% risk. The mean number of positive cores was 2.19. Prostate volume and PSA density were statistically significant predictors of positive biopsy (p<0.05). For the 43 patients who underwent radical prostatectomy, pathologic findings revealed mean Gleason score of 6.7 (6-8), pT2a-c in 72%, pT3a in16%, and pT4 in 7%. Mean cancer volume was 1.15 cc and 85.2% of tumours were clinically significant (tumour volume > 0.5 cc, Gleason > or = 7 and/or pT3). CONCLUSIONS Negative first extended biopsies should not reassure a patient of not having PCa. However, prostate cancers detected after two or more sets of extended procedures, appear to be localized (intracapsular disease) and well-differentiated prostate cancers, although they are still clinically significant.

EGFR as a potential therapeutic target for a subset of muscle-invasive bladder cancers presenting a basal-like phenotype

A subtype of aggressive human muscle-invasive bladder cancer expresses basal epithelial markers and is sensitive to EGFR inhibition in preclinical models. Bladder Cancer’s Basal Instincts Like most cancers, bladder tumors are much easier to treat when they do not invade deep into the tissue, accounting for the poor outcomes of patients with muscle-invasive bladder cancer. By performing genetic analysis on a large number of these tumors, Rebouissou et al. identified a specific subgroup of muscle-invasive bladder cancers expressing basal markers. Although these are aggressive tumors, the authors showed that they have a weak spot and are unusually dependent on the activity of a signaling pathway called epidermal growth factor receptor (EGFR). As a result, these tumors are sensitive to treatment with drugs that inhibit the EGFR pathway, which the authors confirmed in preclinical models. Muscle-invasive bladder carcinoma (MIBC) constitutes a heterogeneous group of tumors with a poor outcome. Molecular stratification of MIBC may identify clinically relevant tumor subgroups and help to provide effective targeted therapies. From seven series of large-scale transcriptomic data (383 tumors), we identified an MIBC subgroup accounting for 23.5% of MIBC, associated with shorter survival and displaying a basal-like phenotype, as shown by the expression of epithelial basal cell markers. Basal-like tumors presented an activation of the epidermal growth factor receptor (EGFR) pathway linked to frequent EGFR gains and activation of an EGFR autocrine loop. We used a 40-gene expression classifier derived from human tumors to identify human bladder cancer cell lines and a chemically induced mouse model of bladder cancer corresponding to human basal-like bladder cancer. We showed, in both models, that tumor cells were sensitive to anti-EGFR therapy. Our findings provide preclinical proof of concept that anti-EGFR therapy can be used to target a subset of particularly aggressive MIBC tumors expressing basal cell markers and provide diagnostic tools for identifying these tumors.