Ardeshir Hakam

Expression of insulin-like growth factor-1 receptor in human colorectal cancer

The activation of the insulinlike growth factor 1/IGF-1 receptor system (IGF1/IGF1-R) has recently emerged as critical event in transformation and tumorigenicity of several murine and human tumors. Expression of IGF1 and of IGF1-R has been demonstrated in normal and neoplastic intestinal cell lines of rats and humans. However, the modulation of IGF1-R expression during the progression from normal colonic mucosa to adenoma, to carcinoma, and to metastasis, has not been evaluated. In this retrospective study, we investigated the expression of IGF1-R in 12 colonic adenomas (AD), 36 primary colorectal adenocarcinomas (CA), and in 27 corresponding metastases (MT). Normal colonic mucosa (N) was adjacent to the CA in 34 cases. Formalin-fixed, paraffin-embedded tissues of each case were immunostained using the avidin-biotin-peroxidase method. We used an anti-IGF1-R rabbit polyclonal antibody (Santa Cruz Biotechnology, CA; dilution 1:100). Positive staining was quantitated by CAS-200. Moderate to strong cytoplasmic immunostaining was observed in 34 of 36 CA (96%), and in 25 of 27 MT (93%). In all of the positive MTs, the intensity of the staining was always strong. In 10 of 12 ADs (83%), only a faint cytoplasmic stain was identified. Normal mucosa when present was negative. Strong IGF1-R positivity correlated with higher grade and higher-stage tumors (P < .01). These data suggest a role of IGF1-R expression during the progression of colorectal adenoma to carcinoma. An increased number of IGF1-R receptors may favor the metastasis of colorectal cancer.

MicroRNAs and their target messenger RNAs associated with endometrial carcinogenesis.

OBJECTIVE Recent advances in gene expression technology have provided insights into global messenger RNA (mRNA) expression changes associated with endometrial cancer development. However, the post-transcriptional events that may also have phenotypic consequences remain to be completely delineated. MicroRNAs (miRNAs) are small non-coding RNA transcripts, that influence cell function via modulation of post-transcriptional activity of multiple target mRNA genes. Although recent reports suggest that miRNAs may influence human cancer development, their role in endometrial carcinogenesis remains to be described. METHODS We measured expression of 335 unique human miRNAs in 61 fresh-frozen endometrial specimens, including 37 endometrial cancers, 20 normal endometrium, and 4 complex atypical hyperplasia samples. In parallel, expression of 22,000 mRNA genes was analyzed using the Affymetrix Human U133A GeneChips in 29 of the endometrial samples, including 20 endometrial carcinomas and 9 normal endometrial samples. Differentially expressed mRNAs, miRNAs, and predicted miRNA-mRNA targets were integrated and evaluated for representation of relevant functional biologic pathways. RESULTS Thirteen miRNAs (p<0.02) and 90 mRNAs (FDR; 0%) were identified to be associated with endometrial cancer development. Twenty-six of the 90 (29%) differentially expressed mRNAs are Sangar-database predicted mRNA targets of the 13 miRNAs. Pathway analysis demonstrates significant involvement of these 26 mRNA genes in processes including cell death, growth, proliferation, and carcinogenesis. CONCLUSION We have identified miRNAs and mRNAs associated with endometrial cancer development. Further, our strategy of integrating miRNA/mRNA data may also aid in the identification of important biologic pathways and additional unique genes that have importance in endometrial pathogenesis.

MicroRNAs and their target messenger RNAs associated with ovarian cancer response to chemotherapy

OBJECTIVE Few successful therapeutic options exist for patients with recurrent ovarian cancer (OVCA). This is due in part to an incomplete understanding of the molecular determinants of chemotherapy-response. Recently, it has been shown that microRNAs (miRNAs) influence messenger-RNA (mRNA) post-transcriptional control and can contribute to human carcinogenesis. The objective of the current study was to explore the role of miRNAs, and their predicted mRNA targets, in OVCA in-vitro response to chemotherapy. METHODS The expression of 335 unique miRNAs was measured in 16 OVCA cell lines. In parallel, the sensitivity of these cell lines to 6 commonly used chemotherapeutic agents (cisplatin, doxorubicin, topotecan, paclitaxel, docetaxel, and gemcitabine) was evaluated by in-vitro cell proliferation assay. MiRNAs associated with cell line drug response were identified by linear regression analysis, and their predicted mRNA targets subject to functional biologic pathway analyses. RESULTS Twenty-seven miRNAs were found to be associated with response to the one or more of the 6 salvage chemotherapies tested (p<0.05). Predicted targets of these miRNAs included 52 mRNAs, previously reported to be associated with chemo-responsiveness, and which are also involved in functional biologic pathways that influence cancer cell cytotoxicity, carcinogenesis, cell mitosis, p53 signaling, and tumor cell growth and invasion. CONCLUSION We have identified miRNAs and their predicted target mRNAs associated with ovarian cancer cell response to chemotherapeutic agents. Our strategy of integrating miRNA and mRNA data may aid in the characterization of important molecular pathways associated with OVCA chemo-response.

Coexpression of IGF-1R and c-Src proteins in human pancreatic ductal adenocarcinoma

Aberrant c-Src protein kinase activation has been identified as one of the molecular alterations involved in human pancreatic carcinogenesis. It has been postulated that c-Src may induce transformation by causing the overexpression of the insulinlike growth factor-1 receptor (IGF-1R) in pancreatic tumor cell lines. To further study the interaction between c-Src and IGF-1R proteins in human pancreatic cancer, we examined their coexpression in 47 human pancreatic ductal adenocarcinomas (PDA). Formalin-fixed, paraffin-embedded sections from 47 cases of PDA were stained using the immunohistochemical avidin–biotin–peroxidase method. We used an anti-human IGF-1R mouse monoclonal antibody (dilution 1:100 with antigen retrieval), and an anti-c-Src mouse monoclonal antibody (dilution 1:100 with antigen retrieval). The stains were semiquantitatively evaluated using the Allred score system, assessing intensity of stain and percentage of positive tumor cells. High cytoplasmic c-Src expression (Allred score 7–8) was seen in 33/47 (70%) tumors. In only 4 cases was c-Src either negative or low (Allred score 3). Strong and diffuse membranous IGF-1R stain (Allred score 7–8) was identified in 30/47 (64%) tumors. IGF-1R staining was low (Alled score 2–4) in 2 cases and negative in 1. Interestingly, in 40/47 (85%) cases c-Src and IGF-1R stains had similar scores. An inverse staining pattern was detected in only 6/47 (13%) tumors. Normal pancreatic ducts as well as areas of chronic pancreatitis were negative for IGF-1R. In conclusion, our data support the role of IGF-1R and c-Src in human pancreatic carcinogenesis; the coexpression of both these molecules may play an important role in transformation of pancreatic ductal cells.

Expression of the Antiapoptotic Protein Survivin in Colon Cancer

BACKGROUND The antiapoptotic protein survivin has been demonstrated to play an important role in colorectal carcinogenesis. However it is unclear whether the upregulation of survivin is maintained through progressive stages of disease, or if other apoptosis-related genes are coexpressed and/or repressed. We sought to evaluate survivin expression in colonic neoplasia and identify relationships with additional regulators of apoptosis. PATIENTS AND METHODS Tissue samples from 168 patients with primary colorectal cancer were profiled using the GeneChip Human Genome U133 Plus 2.0 Array (Affymetrix, Santa Clara, CA) and evaluated for survivin expression. Immunohistochemical staining for survivin and a panel of apoptosis-associated proteins were used in 86 patients with tissue microarray (TMA) blocks; scoring was by stain intensity and percentage of positive cells (range, 0-9). RESULTS Survivin mRNA was upregulated (1.8-fold increase) in primary colon cancers- irrespective of American Joint Committee on Cancer (AJCC) stage- and metastases compared with normal colonic tissue (P < .0001). Survivin staining was positive in 93% of adenocarcinomas (median immunohistochemistry [IHC] score: 2 [range, 1-6]), 100% of adenomas (1 [range,1-2]), and 43% of normal colonic mucosa (1, [range 1-2]) (P = .006). Survivin expression increased with worsening tumor grade (P < .05). In colon cancers, survivin expression positively correlated with the coexpression of PUMA (P < .001), TACE (P = .003), and MCL1 (P = .01), and trended toward an inverse correlation with BAX (P = .058). CONCLUSIONS Survivin expression increases during the normal mucosa-adenoma-carcinoma sequence and is maintained throughout progression of disease, which strengthens its appeal as a therapeutic target. Furthermore, we have demonstrated co-overexpression of several other apoptosis-related genes, which may in turn serve as additional and potentially synergistic therapeutic targets.

The Mainz Classification of Renal Cell Tumors.

BACKGROUND: Tumors arising from the renal tubular epithelium have variable characteristics and have been subject to a variety of histologic classifications. METHODS: The authors describe the distinct clinical, pathologic, phenotypic, and genotypic features of different types of renal tumors. RESULTS: The Mainz classification is now widely accepted because characteristic genetic alterations have been demonstrated in each tumor type. CONCLUSIONS: The increasing emphasis on utilizing genetic characteristics of specific tumors is reflected by the more widespread use of the Mainz classification for renal cell tumors.

Quantitative DNA methylation analysis of candidate genes in cervical cancer.

Aberrant DNA methylation has been observed in cervical cancer; however, most studies have used non-quantitative approaches to measure DNA methylation. The objective of this study was to quantify methylation within a select panel of genes previously identified as targets for epigenetic silencing in cervical cancer and to identify genes with elevated methylation that can distinguish cancer from normal cervical tissues. We identified 49 women with invasive squamous cell cancer of the cervix and 22 women with normal cytology specimens. Bisulfite-modified genomic DNA was amplified and quantitative pyrosequencing completed for 10 genes (APC, CCNA, CDH1, CDH13, WIF1, TIMP3, DAPK1, RARB, FHIT, and SLIT2). A Methylation Index was calculated as the mean percent methylation across all CpG sites analyzed per gene (~4-9 CpG site) per sequence. A binary cut-point was defined at >15% methylation. Sensitivity, specificity and area under ROC curve (AUC) of methylation in individual genes or a panel was examined. The median methylation index was significantly higher in cases compared to controls in 8 genes, whereas there was no difference in median methylation for 2 genes. Compared to HPV and age, the combination of DNA methylation level of DAPK1, SLIT2, WIF1 and RARB with HPV and age significantly improved the AUC from 0.79 to 0.99 (95% CI: 0.97–1.00, p-value = 0.003). Pyrosequencing analysis confirmed that several genes are common targets for aberrant methylation in cervical cancer and DNA methylation level of four genes appears to increase specificity to identify cancer compared to HPV detection alone. Alterations in DNA methylation of specific genes in cervical cancers, such as DAPK1, RARB, WIF1, and SLIT2, may also occur early in cervical carcinogenesis and should be evaluated.

miR-21, miR-221 and miR-222 expression and prostate cancer recurrence among obese and non-obese cases.

Recent evidence shows that certain microRNAs (miRNAs) play a role in both obesity and prostate cancer recurrence, but the association between the expression of these miRNAs and obesity in prostate cancer recurrence is unknown. In this study, we examined the effect of the interaction between obesity and miR-21, miR-221 or miR-222 expression on prostate cancer recurrence among 28 recurrent and 37 non-recurrent prostate cancer cases. miRNA expression was determined using quantitative real-time polymerase chain reaction. Cox proportional hazard models adjusting for age at diagnosis, clinical stage and Gleason score were used to estimate hazard ratios (HR) and 95% confidence intervals (95% CI) for recurrence free survival. A significantly (P=0.014) higher proportion of recurrent cases (78.6%) than non-recurrent cases (48.6%) had a low expression of miR-21 and the difference was more prominent in obese than non-obese patients. Multivariate analysis showed that the expression of miR-21 was an independent risk factor for recurrence in obese (HR=6.15, 95% CI=1.04-36.48, P=0.045), but not in non-obese (HR=1.28, 95% CI=0.30-5.49, P=0.74) cases. A significant association with recurrence was not observed for the expression of miR-221 and miR-222. In summary, our findings show that miR-21 is associated with prostate cancer recurrence after radical prostatectomy and suggest that the differential expression of miR-21 is more prominent in obese than in non-obese cases. Future larger studies are warranted to confirm these initial findings and to elucidate the mechanisms involved.

Significance of histologic pattern of carcinoma and sarcoma components on survival outcomes of uterine carcinosarcoma

BACKGROUND To examine the effect of the histology of carcinoma and sarcoma components on survival outcome of uterine carcinosarcoma. PATIENTS AND METHODS A multicenter retrospective study was conducted to examine uterine carcinosarcoma cases that underwent primary surgical staging. Archived slides were examined and histologic patterns were grouped based on carcinoma (low-grade versus high-grade) and sarcoma (homologous versus heterologous) components, correlating to clinico-pathological demographics and outcomes. RESULTS Among 1192 cases identified, 906 cases were evaluated for histologic patterns (carcinoma/sarcoma) with high-grade/homologous (40.8%) being the most common type followed by high-grade/heterologous (30.9%), low-grade/homologous (18.0%), and low-grade/heterologous (10.3%). On multivariate analysis, high-grade/heterologous (5-year rate, 34.0%, P = 0.024) and high-grade/homologous (45.8%, P = 0.017) but not low-grade/heterologous (50.6%, P = 0.089) were independently associated with decreased progression-free survival (PFS) compared with low-grade/homologous (60.3%). In addition, older age, residual disease at surgery, large tumor, sarcoma dominance, deep myometrial invasion, lymphovascular space invasion, and advanced-stage disease were independently associated with decreased PFS (all, P < 0.01). Both postoperative chemotherapy (5-year rates, 48.6% versus 39.0%, P < 0.001) and radiotherapy (50.1% versus 44.1%, P = 0.007) were significantly associated with improved PFS in univariate analysis. However, on multivariate analysis, only postoperative chemotherapy remained an independent predictor for improved PFS [hazard ratio (HR) 0.34, 95% confidence interval (CI) 0.27-0.43, P < 0.001]. On univariate analysis, significant treatment benefits for PFS were seen with ifosfamide for low-grade carcinoma (82.0% versus 49.8%, P = 0.001), platinum for high-grade carcinoma (46.9% versus 32.4%, P = 0.034) and homologous sarcoma (53.1% versus 38.2%, P = 0.017), and anthracycline for heterologous sarcoma (66.2% versus 39.3%, P = 0.005). Conversely, platinum, taxane, and anthracycline for low-grade carcinoma, and anthracycline for homologous sarcoma had no effect on PFS compared with non-chemotherapy group (all, P > 0.05). On multivariate analysis, ifosfamide for low-grade/homologous (HR 0.21, 95% CI 0.07-0.63, P = 0.005), platinum for high-grade/homologous (HR 0.36, 95% CI 0.22-0.60, P < 0.001), and anthracycline for high-grade/heterologous (HR 0.30, 95% CI 0.14-0.62, P = 0.001) remained independent predictors for improved PFS. Analyses of 1096 metastatic sites showed that carcinoma components tended to spread lymphatically, while sarcoma components tended to spread loco-regionally (P < 0.001). CONCLUSION Characterization of histologic pattern provides valuable information in the management of uterine carcinosarcoma.

BCL2 antagonist of cell death kinases, phosphatases, and ovarian cancer sensitivity to cisplatin

Objective The BCL2 family proteins are critical mediators of cellular apoptosis and, as such, have been implicated as determinants of cancer cell chemo-sensitivity. Recently, it has been demonstrated that the phosphorylation status of the BCL2 antagonist of cell death (BAD) protein may influence ovarian cancer (OVCA) cell sensitivity to cisplatin. Here, we sought to evaluate how kinase and phosphatase components of the BAD apoptosis pathway influence OVCA chemo-sensitivity. Methods Protein levels of cyclin-dependent kinase 1 (CDK1) and protein phosphatase 2C (PP2C) were measured by immunofluorescence in a series of 64 primary advanced-stage serous OVCA patient samples. In parallel, levels of cAMP-dependent protein kinase (PKA), AKT, and PP2C were quantified by Western blot analysis in paired mother/daughter platinum-sensitive/resistant OVCA cell lines (A2008/C13, A2780S/A2780CP, Chi/ChiR). BAD pathway kinase CDK1 was depleted using siRNA transfection, and the influence on BAD phosphorylation and cisplatin-induced apoptosis was evaluated. Results OVCA patient samples that demonstrated complete responses to primary platinum-based therapy demonstrated 4-fold higher CDK1 (p<0.0001) and 2-fold lower PP2C (p=0.14) protein levels than samples that demonstrated incomplete responses. Protein levels of PP2C were lower in the platinum-resistant versus that shown in the platinum-sensitive OVCA cell line sub-clones. Levels of PKA were higher in all platinum-resistant than in platinum-sensitive OVCA cell line sub-clones. Selective siRNA depletion of CDK1 increased sensitivity to cisplatin-induced apoptosis (p<0.002). Conclusion BAD pathway kinases and phosphatases, including CDK1 and PP2C, are associated with OVCA sensitivity to platinum and may represent therapeutic opportunities to enhance cytotoxic efficacy.