George Theodoropoulos

Circulating MicroRNA in inflammatory bowel disease

BACKGROUND MicroRNAs (miRNAs) consist of a group of small noncoding RNAs that partially regulate gene expression. We investigated the expression patterns of commonly deregulated miRNAs in Crohns disease (CD) and ulcerative colitis (UC) in peripheral blood samples of inflammatory bowel disease patients. PATIENTS AND METHODS This study consisted of 128 CD and 88 UC patients, as well as 162 healthy controls. The expression patterns of the miRNA species were quantitatively assayed using reverse transcription and real-time RT-PCR. Stem-loop complementary DNAs (cDNAs) were synthesized using looped reverse transcription primers specific for each miRNA. RESULTS MiR-16, miR-23a, miR-29a, miR-106a, miR-107, miR-126, miR-191, miR-199a-5p, miR-200c, miR-362-3p and miR-532-3p were expressed at significantly higher levels in the blood from patients with CD compared with the healthy controls. No significant differences were observed when the CD patients were classified according to disease location and phenotype. In the UC cases three miRNAs (miR-16, miR-21, miR-28-5p, miR-151-5p, miR-155 and miR-199a-5p) were significantly increased compared to healthy controls. miR-155 was the most highly expressed of the UC-associated miRNA in blood samples. CONCLUSIONS Our results suggest that several miRNAs could distinguish CD from UC by real-time PCR. This further highlights the putative role of miRNAs as contributors to IBD pathogenesis. They may help develop new non-invasive biomarkers to distinguish UC and CD.

COMMON POLYMORPHISMS IN THE VASCULAR ENDOTHELIAL GROWTH FACTOR GENE AND COLORECTAL CANCER DEVELOPMENT, PROGNOSIS, AND SURVIVAL

Angiogenesis plays an important role in growth, progression, and metastasis of tumors. The most important regulator of angiogenesis is vascular endothelial growth factor (VEGF). VEGF expression has been associated with advance stage and poor survival of several cancers. In the present study we evaluated the association of functional polymorphisms in the VEGF gene with colorectal cancer development, prognosis, and survival. Three hundred twelve consecutive patients with surgically treated colorectal adenocarcinoma were enrolled in the present study. The genomic DNA was extracted from paraffin‐embedded tissue and five VEGF (−2578C>A, −1154G>A, −634G>C, −460T>C, and +936C>T) gene polymorphisms were determined using a polymerase chain reaction‐restriction fragment length polymorphism assay. VEGF −2578C>A, −1154G>A, −634G>C, −460T>C, and +936C>T genotype and allele frequencies were similar among patients and controls. There was a trend showing carriers of the −2578A and +936T alleles more frequent among patients with CRC, but these differences did not reach statistical significance. Furthermore, no correlation was found between all these variants and tumor characteristics like size, histological grading, positive regional lymph node metastases or tumor stage. However, the −2578AA, −634CC, and +936TT genotypes found to be related with a significantly lower overall survival in our study. In conclusion, VEGF gene polymorphisms were found to be an independent prognostic marker for Greek CRC patients.

Evaluation of hypoxia‐inducible factor 1α overexpression as a predictor of tumour recurrence and progression in superficial urothelial bladder carcinoma

To investigate the possible role of hypoxia‐inducible factor 1α (HIF‐1α, a transcription factor important in regulating O2 homeostasis and physiological responses to oxygen deprivation) in the recurrence and progression of superficial urothelial bladder cancer, and to examine its expression in relation to proliferation status, apoptotic activity and intratumoral angiogenesis.

Prognostic significance of the co-expression of p53 and c-erbB-2 proteins in breast cancer

The immunohistochemical expression of p53 and c‐erbB‐2 gene proteins was examined in a series of 130 breast adenocarcinomas. This study intended to investigate whether the frequency of the altered expression of the tumour suppressor gene p53 and the overexpression of the oncogene c‐erbB‐2 in breast cancer tissue cells correlated with other variables known to affect the biological behaviour of these tumours and the overall survival of the patients (median follow‐up time: 6 years). The expression of p53 protein and c‐erbB‐2 gene product was evaluated immunohistochemically. Expression of p53 protein was detected in 30 (23 per cent) of the neoplasms examined, while 26 (20 per cent) out of the 130 cases demonstrated positive c‐erbB‐2 immunoreactivity. There was a statistically significant association between p53 protein expression and primary tumour size, lymph node involvement, and oestrogen receptor positivity. The incidence of c‐erbB‐2 positivity was significantly correlated with high tumour grade, axillary node invasion, large tumour size, and the absence of steroid receptors. p53 immuno‐expression was clearly associated with c‐erbB‐2 protein overexpression. Concomitant p53 and c‐erbB‐2 positive immunolabelling, which emerged in 14 out of the 130 cases (10·7 per cent), was clearly associated with high grade, large size, positive nodal status, ductal infiltrating (NOS) histological type, and low values of progesterone receptors. Overall survival of patients was not significantly related to the immunoreactivity of either p53 or c‐erbB‐2 considered separately, whereas there was a clearly significant trend to worse overall prognosis in cancers with double p53/c‐erbB‐2 positive phenotype. The simultaneous immunodetection of p53/c‐erbB‐2 appears to have greater negative prognostic relevance than their separate expression.

Heat shock protein 70 and HLA-DR molecules tissue expression. Prognostic implications in colorectal cancer.

PURPOSE: The expression of 70,000-Da heat shock protein (HSP 70) and HLA-DR molecules on cancer cells influences immunologic mechanisms that may be of some prognostic significance. The purpose of this study was to examine the relationship among immunohistochemical HSP 70, HLA-DR expression, and clinicopathologic tumor variables, as well as patient survival in a series of 128 colorectal carcinomas. METHOD: A three-step immunoperoxidase staining technique was undertaken for detection of both markers. RESULTS: Of the examined carcinomas 77.3 percent were HSP 70-positive and 74.2 percent were HLA-DR-positive. Increased HSP 70-positive expression correlated significantly with low differentiation (P<0.05), showed a tendency to characterize advanced stages of disease, and was clearly associated with worse overall survival (P<0.05). The highest rate of HLA-DR positivity was demonstrated in early stages and was significantly associated with more favorable prognosis (P<0.001). HSP 70-positive/HLA-DR-negative patients had worse overall survival compared with the rest (P<0.001). CONCLUSIONS: The resulting opposite effects on prognosis of examined markers seem to be related to different pathophysiologic functional roles on tumor immunology.

Survivin -31G/C promoter polymorphism and sporadic colorectal cancer

IntroductionSurvivin is an apoptotic inhibitor, plays an important role in cell cycle regulation, and may be involved in the development and progression of cancer. A common polymorphism at the survivin gene promoter (-31 G/C) has been shown to influence survivin expression and the risk for cancer.AimThe aim of the present study was to investigate whether this polymorphism could be involved in the sporadic colorectal cancer (CRC) development, prognosis, and survival.Materials and methodsThe -31G/C polymorphism of survivin promoter was analyzed by polymerase chain reaction (PCR) restriction fragment length polymorphism method in biopsies from 312 patients with sporadic CRC and 362 healthy individuals. Survivin messenger RNA (mRNA) expression in CRC tissues was detected by quantitative reverse transcriptase PCR.Results and discussionThe genotype frequencies for -31GG, -31GC, and -31CC were 21.79%, 41.99%, and 36.22% in CRC patients and 33.98%, 45.03%, and 20.99% in healthy subjects, respectively. The frequencies of the survivin -31C allele and CC genotype were significantly higher in CRC patients than in healthy subjects (p < 0.0001). Homozygotes for the -31CC survivin genotype, expressed 1.6-fold higher mRNA levels of survivin compared to cases with the -31GG and -31GC genotypes.ConclusionThe -31CC genotype of survivin promoter is associated with CRC and may be a risk factor for CRC.

Adjuvant chemotherapy in rectal cancer: Defining subgroups who may benefit after neoadjuvant chemoradiation and resection: A pooled analysis of 3,313 patients.

Recent literature suggests that the benefit of adjuvant chemotherapy (aCT) for rectal cancer patients might depend on the response to neoadjuvant chemoradiation (CRT). Aim was to evaluate whether the effect of aCT in rectal cancer is modified by response to CRT and to identify which patients benefit from aCT after CRT, by means of a pooled analysis of individual patient data from 13 datasets. Patients were categorized into three groups: pCR (ypT0N0), ypT1‐2 tumour and ypT3‐4 tumour. Hazard ratios (HR) for the effect of aCT were derived from multivariable Cox regression analyses. Primary outcome measure was recurrence‐free survival (RFS). One thousand seven hundred and twenty three (1723) (52%) of 3,313 included patients received aCT. Eight hundred and ninety eight (898) patients had a pCR, 966 had a ypT1‐2 tumour and 1,302 had a ypT3‐4 tumour. For 122 patients response, category was missing and 25 patients had ypT0N+. Median follow‐up for all patients was 51 (0–219) months. HR for RFS with 95% CI for patients treated with aCT were 1.25(0.68–2.29), 0.58(0.37–0.89) and 0.83(0.66–1.10) for patients with pCR, ypT1‐2 and ypT3‐4 tumours, respectively. The effect of aCT in rectal cancer patients treated with CRT differs between subgroups. Patients with a pCR after CRT may not benefit from aCT, whereas patients with residual tumour had superior outcomes when aCT was administered. The test for interaction did not reach statistical significance, but the results support further investigation of a more individualized approach to administer aCT after CRT and surgery based on pathologic staging.

Immunohistochemical expression of C-myc oncogene, heat shock protein 70 and HLA-DR molecules in malignant cutaneous melanoma

The clinical course of malignant melanomas is frequently unpredictable, although a number of prognostically useful variables can be identified. There is a need for additional markers of prognostic value. In a series of 60 malignant cutaneous melanomas, we analysed the immunohistochemical expression of c-myc proto-oncogene, heat shock protein 70 (HSP70) and HLA-DR molecules in order to investigate their prognostic significance. C-myc, HSP70 and HLA-DR were expressed in 43.3%, 56.6% and 38.3% of all melanoma cases, respectively. Advanced Clark levels (Clark III–V) were significantly associated with c-myc expression rate (P<0.05), HSP70 detection (P<0.01) and HLA-DR positivity (P<0.01). Increased Breslow thickness (>1.5 mm) was related to HLA-DR expression (P<0.05). High mitotic rate was closely associated with c-myc positivity (P<0.05), while HSP70 and HLA-DR expression separately correlated to clinical stage of the disease (P<0.05). The evaluation of these variables may be of immunological and prognostic significance. They were found to be associated with melanocyte subpopulations of the vertical growth phase which are arguably characterized by an increased invasive potential.

Association between mutations in the CARD15/NOD2 gene and colorectal cancer in a Greek population

Epidemiological observations suggest that cancer arises from chronically inflamed tissues. Inflammatory bowel disease (IBD) is a typical example since patients with longstanding IBD are at increased risk for development of colorectal cancer (CRC). Therefore, genetic factors predisposing to or implicated in the chronic inflammatory process in IBD may simultaneously predispose to CRC. Recently CARD15/NOD2 has been associated with IBD, which further strengthens the notion that the inflammatory response plays a crucial role in this disease. Several mutations have been identified in the CARD15/NOD2 gene, which appear with significantly higher frequency in patients with IBD. In this report, we have examined the frequency of the 3 major mutations R702W, G908R and 3020insC of the CARD2/NOD2 gene in a series of 104 consecutive Greek patients with sporadic colorectal cancer and 100 healthy individuals. The frequency of all the mutations was significantly elevated compared to the control population (R702W, OR=5.00, p=0.023; G908R, OR=2.78, p=0.025; 3020insC, OR=2.44, p=0.017). Patients with advanced stage tumors were more frequently carriers of at least 1 variant in the CARD15/NOD2 gene (p=0.009). Our results suggest that CARD2/NOD2 may be a genetic factor that predispose to sporadic colorectal cancer.

Assessment of JC polyoma virus in colon neoplasms.

PURPOSEResearch data have recently emphasized an intriguing association of JC polyoma virus with colon carcinogenesis. Tumorigenicity of JC virus is attributed to the T-antigen of its Mad-1 variant. Controversy arose when another research group did not confirm this association. The purpose of this study was to detect JC virus in a series of colon neoplasms from Greek patients.METHODSA nested polymerase chain reaction assay was used to detect JC virus in 80 cancerous, 25 adenomatous specimens of large bowel, and 20 colonoscopic biopsy samples from normal patients without colorectal neoplasia. Quantitation of JC virus DNA was performed by real-time polymerase chain reaction.RESULTSJC polyoma virus nucleotide sequence was detected in 61 percent of colon adenocarcinomas and in 60 percent of adenomas, at a viral load of 9 × 103 to 20 × 103 copies/µg DNA. Adjacent normal mucosa in 35 positive colon adenocarcinoma specimens, and normal mucosa from six patients of the control group, had low viral loads (50–450 copies/µg DNA).CONCLUSIONSJC polyoma virus genome is present in colon neoplasms. JC virus detection in adenomas at comparable viral loads to malignant tumors suggests its implication at early steps of colonic carcinogenesis. Taking into consideration other published data, infection of colonic epithelium with JC virus might be a prime candidate for a role in chromosomal and genomic instability.