Rono Mukherjee

Observer variation in immunohistochemical analysis of protein expression, time for a change?

Aim : Immunohistochemical analysis of protein expression is central to most clinical translational studies and defines patient treatment or selection criteria for novel drugs. Interobserver variation is rarely analysed despite recognition that this is a key area of potential inaccuracy. Therefore our aim was to examine observer variation and suggest the revision of current standards.

Expression levels of the JAK/STAT pathway in the transition from hormone-sensitive to hormone-refractory prostate cancer

The main cause of prostate cancer-related mortality is the development of hormone-refractory disease. Circulating serum levels of IL-6 are raised in hormone-refractory prostate cancer patients and evidence from cell line studies suggests that the IL-6R/JAK/STAT3 pathway may be involved in development of this disease. In the current study we investigate if expression levels of these family members are implicated in the development of hormone-refractory prostate cancer. Immunohistochemistry using IL-6R, JAK1, STAT3, pSTAT3Tyr705 and pSTAT3Ser727 antibodies was performed on 50 matched hormone-sensitive and hormone-refractory tumours pairs. An increase in expression of cytoplasmic IL-6 receptor, with the development of hormone-refractory prostate cancer was associated with reduced time to relapse (P=0.0074) while an increase in expression of cytoplasmic pSTAT3Tyr705 was associated with reduced patient survival (P=0.0003). In addition, those patients with high expression of cytoplasmic pSTAT3Tyr705 in their hormone-refractory tumours had significantly shorter time to death from biochemical relapse and overall survival in comparison to those patients with low expression of cytoplasmic pSTAT3Tyr705 (P=0.002 and P=0.0027, respectively). Activation of STAT3, via phosphorylation is associated with reduced patient survival, suggesting that activation of the IL-6R/JAK/STAT3 pathway is involved with development of hormone-refractory prostate cancer.

Phosphorylation of the androgen receptor is associated with reduced survival in hormone-refractory prostate cancer patients

Cell line studies demonstrate that the PI3K/Akt pathway is upregulated in hormone-refractory prostate cancer (HRPC) and can result in phosphorylation of the androgen receptor (AR). The current study therefore aims to establish if this has relevance to the development of clinical HRPC. Immunohistochemistry was employed to investigate the expression and phosphorylation status of Akt and AR in matched hormone-sensitive and -refractory prostate cancer tumours from 68 patients. In the hormone-refractory tissue, only phosphorylated AR (pAR) was associated with shorter time to death from relapse (P=0.003). However, when an increase in expression in the transition from hormone-sensitive to -refractory prostate cancer was investigated, an increase in expression of PI3K was associated with decreased time to biochemical relapse (P=0.014), and an increase in expression of pAkt473 and pAR210 were associated with decreased disease-specific survival (P=0.0019 and 0.0015, respectively). Protein expression of pAkt473 and pAR210 also strongly correlated (P<0.001, c.c.=0.711) in the hormone-refractory prostate tumours. These results provide evidence using clinical specimens, that upregulation of the PI3K/Akt pathway is associated with phosphorylation of the AR during development of HRPC, suggesting that this pathway could be a potential therapeutic target.

The role of c‐Jun and c‐Fos expression in androgen‐independent prostate cancer

Molecular mechanisms underlying the development of androgen‐insensitive prostate cancer (AIPC) are poorly understood. However, there is growing evidence that different molecular profiles may result in the development of AIPC. Cell line studies demonstrate that c‐Jun and c‐Fos, via formation of the transcription factor activated protein 1 (AP‐1), activate androgen‐regulated genes independent of androgens and that c‐Jun alone acts as an androgen receptor co‐factor. The aim of this study was to investigate whether increased levels of c‐Jun and phosphorylated c‐Jun are associated with the development of AIPC using clinical material. Material from a cohort of 51 patients with paired tumours, obtained before and after the development of AIPC, and with full clinical biochemical follow‐up, was retrieved from the archives. Tumour c‐Jun, activated c‐Jun, c‐Fos, and pan protein kinase C (PKC) protein expression were analysed by immunohistochemistry and protein expression was scored by two independent observers using a weighted histoscore. No evidence was found to suggest that c‐Jun acting as an androgen receptor co‐factor influences the development of AIPC. However, it was observed that patients with high expression levels of phosphorylated c‐Jun had a significantly shorter survival from relapse compared with patients with low phosphorylated c‐Jun protein expression (p = 0.023), suggesting that increased AP‐1 levels may promote AIPC tumour growth. Whilst PKC did not appear to activate c‐Jun in vivo, increased PKC expression in AIPC tumours was also associated with decreased patient survival from time of relapse (p = 0.014). In conclusion, the data support the hypothesis that activation of c‐Jun plays a role in the development of AIPC via AP‐1 formation in some patients. However, PKC appears to promote the development of AIPC independently of c‐Jun activation via an as yet unexplained mechanism. Copyright

Amplification of the androgen receptor may not explain the development of androgen-independent prostate cancer

Objective To examine the role of androgen receptor (AR) gene amplification and aneusomy of the X chromosome in the development of antiandrogen‐resistant prostate cancer.

Upregulation of MAPK pathway is associated with survival in castrate-resistant prostate cancer

Background:Recent evidence has implicated the MAP kinase (MAPK) pathway with the development of castrate-resistant prostate cancer (CRPC). We have previously reported gene amplification of critical members of this pathway with the development of castrate-resistant disease. In addition, we have shown that rising Raf-1 expression, with the development of CRPC, influences time to biochemical relapse. We therefore sought to further analyse the role of both Raf-1 and its downstream target MAPK in the molecular pathogenesis of CRPC.Methods:Protein expression of Raf-1 and MAPK, including their activation status, was analysed using immunohistochemistry in a database of 65 paired tumour specimens obtained before and after the development of CRPC and correlated with other members of the pathway.Results:Patients whose nuclear expression of MAPK rose with the development of CRPC had a significantly shorter median time to death following biochemical relapse (1.40 vs 3.00 years, P=0.0255) as well as reduced disease-specific survival when compared with those whose expression fell or remained unchanged (1.16 vs 2.62 years, P=0.0005). Significant correlations were observed between protein expression of Raf-1 and MAPK with the type 1 receptor tyrosine kinases, Her2 and epidermal growth factor receptor, as well as the transcription factor AP-1 in CRPC tumours.Conclusion:We conclude that the Her2/Raf-1/MAPK/AP-1 axis may promote the development of CRPC, leading to early relapse, and reduced disease-specific survival. In addition, members of the pathway may act as novel therapeutic and/or diagnostic targets for prostate cancer.

The relationship between angiogenesis and cyclooxygenase‐2 expression in prostate cancer

To test the hypothesis that angiogenesis in prostate cancer is associated with tumour invasion and metastasis, and that this is mediated through increased cyclooxygenase‐2 (COX‐2) expression.

Bad expression influences time to androgen escape in prostate cancer

To assess the role of selected downstream Bcl‐2 family members (Bad, Bax, Bcl‐2 and Bcl‐xL) in the development of androgen‐independent prostate cancer (AIPC), as androgen‐deprivation therapy is the treatment of choice in advanced prostate cancer, yet patients generally relapse and progress to an AI state within 18–24 months.

A rare case of a multi-focal corpora cavernosal abscess

Review of the current literature suggests that an abscess originating in the corpus cavernosum is rare, with a paucity of reported cases in the literature. Upon reviewing the evidence, there have been reported cases of cavernosal abscess following a local nidus for infection including trauma or local injection, however few have reported a spontaneous abscess formation within the penile corporal cavernosal region. Here, we present the case of a 49-year old previously fit and healthy gentleman, with a spontaneous bilateral penile cavernosal abscess. Magnetic resonance (MR) imaging confirmed the diagnosis of an extensive abscess within the corpora cavernosa bilaterally. Subsequent incision and drainage of the abscess was performed followed by a 5 week course of antibiotics. Microscopy & culture demonstrated Streptococcus Anginosus in both blood cultures and wound pus swabs. Of note, lengthy critical care input was also required for inotropic support secondary to significant sepsis.

Abstract 4711: The role ERK1/2 and Androgen Receptor phosphorylation at serine 81 in the progression from hormone naive to castrate resistant prostate cancer

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Prostate cancer incidence in the UK is approximately 35,500 cases per year and the second most common cause of male cancer-specific death. The high mortality rate associated with prostate cancer is attributed to the development of castrate resistance prostate cancer (CRPC). Following the development of CRPC there are few therapeutic options with the mean survival period being only 18-24 months. Cell line studies implement ERK1/2 activation in the transition from hormone naive prostate cancer (HNPC) to CRPC. Recent evidence suggests that ERK kinase activation induces phosphorylation of AR at Serine 81 (AR81) to stimulate prostate cancer cell growth and induce PSA expression (Shigemura K et al Prostate 2009, 69(9) 949-955). This data suggests that the ERK kinase pathway may provide a novel therapeutic target for treatment of CRPC The aim of the current study was to investigate if the relationship between the AR and ERK1/2 is upheld in clinical prostate cancer specimens. A cohort of 55 patients with matched HNPC and CRPC tissue was established with full clinical follow-up. Immunohistochemistry was employed to assess expression of AR, AR81, ERK1/2 and phosphorylated ERK1/2 (pERK). All 55 patients in the cohort were diagnosed with HNPC and subsequently progressed to CRPC. The median age at diagnosis was 70(66-73), median Gleason sum at diagnosis was 8(6-9) and median time to biochemical relapse was 2.3 years. Mean follow-up was 7.5 (4.3-11.7) years, 4 patients were alive at last follow-up, 35 patients died of their disease and 16 patients died of other causes. In the transition of HNPC to CRPC 8% of patients exhibited an increase in AR81 expression and 20% exhibited an increase in ERK1/2 expression. PSA at diagnosis (p=0.034) and high pERK expression in HNPC (p=0.03) were associated with shorter time to biochemical relapse. A negative correlation between AR and ERK1/2 expression was observed in HNPC (R2=-0.383, p=0.004), but no association with AR81 and ERK1/2 or pERK was observed in this stage of disease. Gleason sum (p=0.009), presence of metastasis at diagnosis (p=0.0002), presence of metastasis at relapse (p=0.003) and high ERK1/2 expression in CRPC (p=0.003) were associated with shorter disease specific survival. In CRPC no correlations were observed between AR or AR81 and ERK1/2 or pERK. AR81 expression did however positively correlate with proliferation index as assessed by Ki67 expression (R2=0.339, p=0.025). Combined expression of AR81 and ERK1/2 resulted in a significantly shorter disease specific survival. Whereas, expression of these protein alone (4.2 years vs. 5.4 years) or not at all (7.2 years) resulted in a longer disease specific survival p=0.0006. In conclusion, although no correlations with AR81 and ERK1/2 or pERK were observed in the current study, high expression of AR81 and ERK1/2 combined had a synergistic effect of decreased disease specific survival. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4711.