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

<b>OBJECTIVE</b>: 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. <b>PATIENTS AND METHODS</b>: Angiogenesis was assessed in 105 patients with either prostate cancer (79) or benign prostatic hyperplasia (BPH, 26) and these data correlated with levels of COX-2 expression in the same dataset. The mean microvessel density (MVD) was analysed as a marker of angiogenesis, using the endothelial antigen CD34 stained by immunohistochemistry. <b>RESULTS</b>: There was no difference in MVD in progressive tumour stages compared with BPH. There was a negative correlation between MVD and COX-2 expression, but the effect of increased COX-2 expression on MVD was not marked. <b>CONCLUSION</b>: These data suggest that COX-2 drives tumour spread in prostate cancer by means other than the promotion of angiogenesis

Gene amplifications associated with the development of hormone- resistant prostate cancer

Purpose: Hormone resistance remains a significant clinical problem in prostate cancer with few therapeutic options. Research into mechanisms of hormone resistance is essential. Experimental Design: We analyzed 38 paired (prehormone/posthormone resistance) prostate cancer samples using the Vysis GenoSensor. Archival microdissected tumor DNA was extracted, amplified, labeled, and hybridized to Amplione I DNA microarrays containing 57 oncogenes. Results: Genetic instability increased during progression from hormone-sensitive to hormone-resistant cancer (P = 0.008). Amplification frequencies of 15 genes (TERC, MYBL3, HRAS, PI3KCA, JUNB, LAMC2, RAF1, MYC, GARP, SAS, FGFR1, PGY1, MYCL1, MYB, FGR) increased by greater than 10% during hormone escape. Receptor tyrosine kinases were amplified in 73% of cases; this was unrelated to development of hormone resistance. However, downstream receptor tyrosine kinase signaling pathways showed increased amplification rates in resistant tumors for the mitogen-activated protein kinase (FGR/Src-2, HRAS, and RAF1; P = 0.005) and phosphatidylinositol 3'-kinase pathways (FGR/ Src-2, PI3K, and Akt; P = 0.046). Transcription factors regulated by these pathways were also more frequently amplified after escape (MYC family: 21% before versus 63% after, P = 0.027; MYB family: 26 % before versus 53 % after, P = 0.18). Conclusions: Development of clinical hormone escape is linked to phosphatidylinositol 3'-kinase and mitogen-activated protein kinase pathways. These pathways may function independently of the androgen receptor or via androgen receptor activation by phosphorylation, providing novel therapeutic targets

Chromosomal aberrations in transitional cell carcinoma that are predictive of disease outcome are independent of polyploidy

Objective To determine whether aneusomy for chromosomes 7, 9 and 17 (reported to predict recurrence in up to 65% of patients with superficial transitional cell bladder cancer and thus providing the opportunity for early and effective treatment) reflects specific genetic events on these chromosomes or merely wider unspecific genetic damage to the cell, e.g. that increased copy numbers for 7 and 17 reflect tumour polyploidy. Materials and methods The study comprised 25 primary tumours; 6 mu m thick sections from formalin-fixed and paraffin-embedded tumours were analysed. Chromosome copy numbers were determined by fluorescence in situ hybridization (FISH) using pericentromeric probes for chromosomes 7, 8, 9, 10, 11 and 17. A minimum of 200 nuclei per tumour area were scored by two independent observers. Results Eight of the 25 tumours examined (32%) showed no evidence of chromosomal abnormalities as detected by FISH for any chromosomes analysed. Twelve tumours (48%) showed abnormalities for one or two chromosomes, five tumours (20%) showed abnormalities for multiple chromosomes and one tumour showed abnormalities for all chromosomes analysed, suggestive of polyploidy. Conclusions Chromosomal abnormalities predictive of recurrence occur largely in the absence of other gross chromosomal lesions. In a small proportion of cases other chromosomes are affected, but this is almost always distinct from tumour polyploidy

The role of HER1-HER4 and EGFRvIII in hormone-refractory prostate cancer

<b>Purpose</b>: The role of the type I receptor tyrosine kinase (HER) family in progression of prostate cancer is controversial. Breast cancer studies show that these receptors should be investigated as a family. The current study investigates expression of HER1-HER4 and EGFRvIII in matched hormone-sensitive and hormone-refractory prostate tumors. <b>Experimental Design</b>: Immunohistochemical analysis was used to investigate protein expression of HER1-HER4, EGFRvIII, and phosphorylated Akt (pAkt) in matched hormone-sensitive and hormone-refractory prostate tumors. <b>Results</b>: Surprisingly, high HER2 membrane expression in hormone-sensitive tumors was associated with an increased time to biochemical relapse (<i>P</i> = 0.0003), and this translated into longer overall survival (<i>P</i> = 0.0021). Consistent with other studies, HER4 membrane expression in hormone-sensitive tumors was associated with longer time to biochemical relapse (<i>P</i> = 0.042), and EGFRvIII membrane expression was associated with shorter time to biochemical relapse (<i>P</i> = 0.015). An increase in pAkt expression was associated with reduced survival (<i>P</i> = 0.0098). Multivariate analysis showed that HER2 was an independent positive predictive marker of time to relapse in hormone-sensitive prostate tumors (<i>P</i> = 0.014). In contrast, high HER2 expression in hormone-refractory tumors was associated with decreased time to death from biochemical relapse (<i>P</i> = 0.039), and EGFRvIII nuclear expression was associated with decreased time to death from biochemical relapse and decreased overall survival (<i>P</i> = 0.02 and <i>P</i> = 0.005). <b>Conclusion</b>: These results suggest that the HER family may have multiple roles in prostate cancer, and that expression of the proteins alone is insufficient to predict the biological response that they may elicit

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. Patients and methods Twenty patients with prostate cancer resistant to androgen-deprivation therapy were selected for study. The records of patients with tumours before and after antiandrogen therapy, and with a full clinical follow-up, were retrieved. AR gene amplification and X chromosome copy number were assessed by fluorescence in situ hybridization using a labelled probe at locus Xq11-13 for the AR gene and a labelled a-satellite probe for the X chromosome. At least 20 nuclei were scored over three tumour areas by two independent observers. Results Aneusomy of the X chromosome was reported respectively in seven (35%) and 11 (55%) tumours before and after hormone relapse, the AR gene copy number was increased in seven (35%) and 13 (65%), respectively, and AR gene amplification was detected in one (5%) and three (15%), respectively. Neither increased AR copy number nor AR amplification in primary tumours precluded a biological response to androgen-deprivation therapy. Conclusion The rate of AR gene amplification is too low to be solely responsible for the development of antiandrogen-resistant prostate cancer. Also, the presence of amplified AR and cells aneusomic for the X chromosome in primary tumours that respond to androgen-deprivation therapy suggests that an increase in AR gene copy number does not prevent a tumour from responding to this therapy. Therefore other mechanisms which could cause hormone-refractory prostate cancer must be investigated before it is understood why so many patients relapse with this disease

The CAG trinucleotide repeat length in the androgen receptor does not predict the early onset of prostate cancer

Objective To relate the repeat length of the androgen-receptor CAG trinucleotide to the age of onset of prostate cancer, stage and grade of disease. Patients and methods After obtaining ethical approval, 265 patients with locally confined or locally advanced/metastatic prostate cancer were identified and evaluated for age at diagnosis (less than 65 years and greater than 75 years). DNA was extracted from peripheral blood lymphocytes and 1 mug aliquots subjected to polymerase chain reaction using fluorescently labelled primers. Samples were then run on an ABI 377 gene scan analysis gel with an internal molecular weight marker. The length of the CAG repeat was determined by comparing the gene scan product size to samples where the CAG repeat length had been quantified using direct sequencing. The Kruskal-Wallis, Mann-Whitney and Wilcoxon two sample tests were used to analyse the data. Results The mean (range) length of the CAG repeat in the androgen receptor was 22.2 (10-31) in the younger and 22.5 (16-32) in the older group, and was not statistically different. There was no significant association between the CAG repeat length and the age of onset of prostate cancer (P = 0.568) or with stage (P = 0.577) and grade (P = 0.891) of prostate cancer. Conclusion These results suggest that there is no correlation between the androgen receptor CAG repeat length and the age of onset, stage and grade of prostate cancer, confirming recent doubts from other similar studies of a suggested correlation between shorter androgen receptor CAG repeat and early onset and aggressiveness of prostate cancer

An increase in N-Ras expression is associated with development of hormone refractory prostate cancer in a subset of patients

Protein expression of H, K and N-Ras was assessed in hormone sensitive and hormone refractory prostate tumour pairs from 61 patients by immunohistochemistry. Expression of H-Ras and K- Ras was not associated with any known clinical parameters. In contrast an increase in N-Ras membrane expression in the transition from hormone sensitive to hormone refractory prostate cancer was associated with shorter time to relapse (p=0.01) and shorter disease specific survival (p=0.008). In addition, patients with an increase in N-Ras membrane expression had lower levels of PSA at relapse (p=0.02) and expression correlated with phosphorylated MAP kinase (p=0.010) and proliferation index (Ki67, p=0.02). These results suggest that in a subgroup patients N-Ras expression is associated with development of hormone refractory prostate cancer via activation of the MAP kinase cascade

Ras/Raf-1/MAPK pathway mediates response to tamoxifen but not chemotherapy in breast cancer patients

<b>Purpose</b>: The expression and activation of the Ras/Raf-1/mitogen-activated protein kinase (MAPK) pathway plays an important role in the development and progression of cancer, and may influence response to treatments such as tamoxifen and chemotherapy. In this study we investigated whether the expression and activation of the key components of this pathway influenced clinical outcome, to test the hypothesis that activation of the MAPK pathway drives resistance to tamoxifen and chemotherapy in women with breast cancer. <b>Experimental Design</b>: Breast tumors from patients at the Glasgow Royal Infirmary and others treated within the BR9601 trial were analyzed for expression of the three Ras isoforms, total Raf-1, active and inactive forms of Raf-1 [pRaf(ser338) and pRaf(ser259), respectively], MAPK, and phospho-MAPK using an immunohistochemical approach. Analyses were done with respect to disease free-survival and overall survival. <b>Results</b>: Expression and activation of the Ras pathway was associated with loss of benefit from treatment with tamoxifen but not chemotherapy. Overexpression of pRaf(ser338) was associated with shortened disease-free and overall survival time in univariate analyses. Multivariate analysis suggested pRaf(ser338) was independent of known prognostic markers in predicting outcome following tamoxifen treatment (<i>P</i>=0.03). <b>Conclusion</b>: This study suggests that activation of the Ras pathway predicts for poor outcome on tamoxifen but not chemotherapy, and identifies pRaf(ser338) as a potential marker of resistance to estrogen receptor–targeted therapy. In addition, it suggests that expression of pRaf(ser338) could identify patients for whom tamoxifen alone is insufficient adjuvant systemic therapy, but for whom the addition of chemotherapy may be of benefit

Gene amplification and overexpression of HER2 in renal cell carcinoma

Objective To determine the frequency of HER2 genetic abnormalities in renal cell carcinoma (RCC) and hence assess the potential suitability of Herceptin(TM) immunotherapy. Patients and methods Tumours from 2 7 patients with RCC were assessed: all patients had undergone nephrectomy. Benign renal tissue from the nephrectomy specimens was studied in seven patients. Gene amplification was assessed using fluorescent in- situ hybridization, and protein over-expression using immunohistochemistry. Results Twenty-four patients had clear cell renal carcinoma, two had papillary renal carcinoma and one a sarcomatoid variant carcinoma. There was no HER2 amplification in the tumours or the benign renal tissue. Polysomy 17 was detected in 11 of 27 tumours (41%) and increased HER2 copy number in seven (26%). Both polysomy 17 and increased HER2 copy number were absent in the benign renal tissue. Three tumours (11%) and six of the seven benign renal tissue samples over-expressed the HER2 protein. Conclusions HER2 amplification is absent and protein over-expression uncommon in RCC. This casts doubt on the suitability of Herceptin(TM) in the treatment of RCC

Updated recommendations for HER2 testing in the UK

This paper serves to update previously published guidance on rationale and methodology for HER2 laboratory testing following the recommendation for the use of HER2 targeted treatment in the management of advanced breast cancer in the UK. Emphasis is placed on the standardisation of methodology and assessment and strategies to achieve high quality performance. A two phase testing algorithm based on first line immunocytochemistry evaluation and second line fluorescence in situ hybridisation assessment of borderline cases is recommended. To ensure maintenance of expertise, an annual caseload volume of at least 250 cases is recommended for laboratories providing a testing service