Luigi Cavazzini

E2F1-Regulated MicroRNAs Impair TGFβ-Dependent Cell-Cycle Arrest and Apoptosis in Gastric Cancer

Deregulation of E2F1 activity and resistance to TGFbeta are hallmarks of gastric cancer. MicroRNAs (miRNAs) are small noncoding RNAs frequently misregulated in human malignancies. Here we provide evidence that the miR-106b-25 cluster, upregulated in a subset of human gastric tumors, is activated by E2F1 in parallel with its host gene, Mcm7. In turn, miR-106b and miR-93 regulate E2F1 expression, establishing a miRNA-directed negative feedback loop. Furthermore, upregulation of these miRNAs impairs the TGFbeta tumor suppressor pathway, interfering with the expression of CDKN1A (p21(Waf1/Cip1)) and BCL2L11 (Bim). Together, these results suggest that the miR-106b-25 cluster is involved in E2F1 posttranscriptional regulation and may play a key role in the development of TGFbeta resistance in gastric cancer.

Immunohistochemical Test for MLH1 and MSH2 Expression Predicts Clinical Outcome in Stage II and III Colorectal Cancer Patients

PURPOSE To evaluate the prognostic significance of DNA mismatch repair (MMR) status in a large series of stage II and III colorectal cancer patients. The relationship among MMR status, adjuvant chemotherapy, and clinical outcome was also investigated. PATIENTS AND METHODS The study included 718 patients with colorectal adenocarcinoma (393 stage II and 325 stage III) who underwent curative surgical resection. MMR status was determined by immunohistochemical analysis of MLH1 and MSH2 expression. Microsatellite instability (MSI) was assessed in 363 patients using mononucleotide and dinucleotide markers. RESULTS One hundred fourteen (15.9%) carcinomas showed abnormal MMR protein (MMRP) expression (96 MLH1 negative and 18 MSH2 negative) and were classified as MMRP negative, whereas 604 tumors demonstrated normal MLH1/MSH2 immunoreactivity (MMRP positive). MLH1/MSH2 expression was closely related to MSI status (P < .001) and several clinicopathologic features. Patients with MMRP-negative carcinomas demonstrated a marked reduction in the risk of cancer-related death with respect to patients with MMRP-positive tumors (hazard ratio, 0.2579; 95% CI, 0.1289 to 0.5159). A better clinical outcome for patients with MMRP-negative tumors was observed in both stage II (P = .0006) and stage III (P = .0052) disease. In stage III disease, the survival advantage conferred by MMRP-negative tumors was more evident among patients treated with surgery alone than among patients who received adjuvant chemotherapy. A nonsignificant trend for survival benefit from adjuvant chemotherapy was observed among patients with MMRP-positive carcinomas but not among those with MMRP-negative carcinomas. CONCLUSION Immunohistochemical testing for MLH1/MSH2 expression provides useful prognostic information for the management of stage II and III colorectal cancer patients.

Expression of P2X7 receptor increases in vivo tumor growth

The P2X7 receptor is an ATP-gated ion channel known for its cytotoxic activity. However, recent evidence suggests a role for P2X7 in cell proliferation. Here, we found that P2X7 exhibits significant growth-promoting effects in vivo. Human embryonic kidney cells expressing P2X7 exhibited a more tumorigenic and anaplastic phenotype than control cells in vivo, and the growth rate and size of these tumors were significantly reduced by intratumoral injection of the P2X7 inhibitor-oxidized ATP. The accelerated growth of P2X7-expressing tumors was characterized by increased proliferation, reduced apoptosis, and a high level of activated transcription factor NFATc1. These tumors also showed a more developed vascular network than control tumors and secreted elevated amounts of VEGF. The growth and neoangiogenesis of P2X7-expressing tumors was blocked by intratumoral injection of the VEGF-blocking antibody Avastin (bevacizumab), pharmacologic P2X7 blockade, or P2X7 silencing in vivo. Immunohistochemistry revealed strong P2X7 positivity in several human cancers. Together, our findings provide direct evidence that P2X7 promotes tumor growth in vivo.

Elevated Expression of A3 Adenosine Receptors in Human Colorectal Cancer Is Reflected in Peripheral Blood Cells

Purpose: Adenosine is a ubiquitous nucleoside that accumulates at high levels in hypoxic regions of solid tumors, and A3 adenosine receptors have been recently demonstrated to play a pivotal role in the adenosine-mediated inhibition of tumor cell proliferation. In the present work, we addressed the question of the putative relevance of A3 subtypes in colorectal adenocarcinomas. Experimental Design: Seventy-three paired samples of tumor and surrounding peritumoral normal mucosa at a distance of 2 and 10 cm from the tumor and blood samples obtained from a cohort of 30 patients with colorectal cancer were investigated to determine the presence of A3 receptors by means of binding, immunocytochemistry, and real-time reverse transcription-polymerase chain reaction studies. Results: As measured by receptor binding assays, the density of A3 receptor was higher in colon carcinomas as compared with normal mucosa originating from the same individuals (P < 0.05). Overexpression of A3 receptors at the protein level was confirmed by immunohistochemical studies, whereas no changes in A3 mRNA accumulation in tumors as compared with the corresponding normal tissue were revealed. The overexpression of A3 receptors in tumors was reflected in peripheral blood cells, where the density was approximately 3-fold higher compared with healthy subjects (P < 0.01). In a cohort of 10 patients studied longitudinally, expression of A3 receptors in circulating blood cells returned to normal after surgical resection for colorectal cancer. Conclusions: This study provides the first evidence that A3 receptor plays a role in colon tumorigenesis and, more importantly, can potentially be used as a diagnostic marker or a therapeutic target for colon cancer.

Genetic progression in microsatellite instability high (MSI-H) colon cancers correlates with clinico-pathological parameters: A study of the TGFβRII, BAX, HMSH3, HMSH6, IGFIIR and BLM genes

Colon carcinomas with microsatellite mutator phenotype exhibit specific genetic and clinico‐pathological features. This report describes the analysis of 63 “microsatellite instability‐high” (MSI‐H) tumors for the presence of mutations in microsatellites located in the coding regions (CDRs) of 6 genes: TGFβRII, BAX, hMSH3, hMSH6, IGFIIR, and BLM. The following frequencies of mutations were detected: TGFβRII (70%), BAX (54%), hMSH3 (36.5%), IGFIIR (22%), hMSH6 (17.5%), and BLM (16%). The overall picture revealed combinations of mutations suggestive of a progressive order of accumulation, with mutations of TGFβRII and BAX first, followed by frameshifts in hMSH3, hMSH6, IGFIIR, and BLM. Correlations with 12 clinico‐pathological parameters revealed that tumors with frameshifts in 1 or 2 CDRs were significantly better differentiated than tumors with frameshifts in more than 2 CDRs. We also found that mutations in the hMSH3 gene were significantly associated with decreased wall invasiveness and aneuploidy, and frameshifts in the BLM gene were significantly associated with the mucinous histotype. A trend toward an association between hMSH3 and IGFIIR with the medullary and conventional adenocarcinoma histotypes, respectively, was seen. Our results strengthen the concept that mutations in target genes have a role in the tumorigenic process of MSI‐H tumors, and indicate that frameshifts in microsatellites located in CDRs occur in a limited number of combinations that could determine distinct clinico‐pathological traits. Int. J. Cancer 89:230–235, 2000.

BRAF V600E mutation analysis increases diagnostic accuracy for papillary thyroid carcinoma in fine needle aspiration biopsies

OBJECTIVE Papillary thyroid carcinoma (PTC) represents the majority of differentiated thyroid cancers, presenting the V600E activating BRAF mutation in 29-83% of cases. The aim of our study is to analyze the influence of BRAF mutation analysis on the diagnostic accuracy of fine-needle aspiration biopsy (FNAB) in patients with suspected PTC. DESIGN AND METHODS Thyroid cytoaspirates from 469 nodules (size: 1.1+/-0.8 cm) with ultrasonographic features suspicious of malignant lesion, performed in 374 patients, were submitted to cytological evaluation and to biomolecular analysis, carried out after somatic DNA isolation, specific PCR amplification, and subsequent automated direct sequencing. All PCR fragments were also processed by specific enzyme restriction analysis. RESULTS BRAF V600E mutation was found in 48 samples, 41 of which were also cytologically diagnosed as PTC, with histologic confirmation after thyroidectomy. Total thyroidectomy was perfomed also in seven patients with negative cytology but positive BRAF mutation, with histological confirmation of PTC in all. Among the 429 BRAF-negative samples, 407 had negative cytology for PTC, while 22 were diagnosed as suspected PTC and underwent total thyroidectomy with histological diagnosis of PTC in 17 and benign lesion in five. The prevalence of BRAF V600E mutation among histologically diagnosed PTC patients was 64%. Biomolecular analysis significantly increased cytology sensitivity for PTC from 77.3 to 86.7% (P<0.01). CONCLUSIONS These data indicate that BRAF V600E mutation analysis can significantly improve FNAB diagnostic accuracy. However, biomolecular analysis is complementary to cytology, which should always be performed.

Immunohistochemical Pattern of MLH1/MSH2 Expression Is Related to Clinical and Pathological Features in Colorectal Adenocarcinomas with Microsatellite Instability

Detection of colorectal carcinomas with high-frequency microsatellite instability (MSI-H) is clinically important for several reasons. Recent studies suggested that immunohistochemical analysis of MLH1 and MSH2 expression is a rapid and accurate method for identifying large bowel tumors of the MSI-H phenotype. In this study, we evaluated by immunohistochemistry MLH1 and MSH2 protein expression in 132 MSI-H, 23 MSI-L (low-frequency MSI), and 150 microsatellite stable (MSS) colorectal adenocarcinomas. Loss of MLH1 or MSH2 expression was detected in 120 (90.9%) MSI-H carcinomas, whereas all MSI-L and MSS tumors showed normal expression of both proteins. Lack of MLH1 nuclear staining was observed much more often than absence of MSH2 nuclear staining (106 and 14 cases, respectively). Among MSI-H carcinomas, MLH1/MSH2 pattern of expression was significantly related to several clinical and pathological variables. In particular, MSI-H MLH1/MSH2-positive carcinomas were more often located in the distal colon, were more frequently classified as ordinary adenocarcinomas, and were more likely to be well or moderately differentiated, p53 positive, and <7 cm in diameter than were MLH1-negative and MSH2-negative carcinomas. In addition, MLH1-negative carcinomas were less common among patients with hereditary nonpolyposis colorectal cancer (HNPCC) or suspected HNPCC and in the group of patients aged <50 years. Patients with MLH1-negative carcinomas more frequently died of disease than did patients with MLH1/MSH2-positive and MSH2-negative MSI-H tumors, but the difference was not statistically significant. The results of the present investigation strongly indicate that immunohistochemical analysis of MLH1 and MSH2 expression is a practical and reliable method for the routine detection of the vast majority of MSI-H large bowel adenocarcinomas. Our data also point out that MSI-H MLH1/MSH2-positive colorectal carcinomas are characterized by distinctive pathological features.

Differentiated Thyroid Cancers 11–20 mm in Diameter Have Clinical and Histopathologic Characteristics Suggesting Higher Aggressiveness than Those ≤10 mm

OBJECTIVE To compare characteristics and outcomes of differentiated thyroid cancers < or =10 mm with those 11-20 mm in diameter. DESIGN Retrospective chart review of 426 patients with thyroid carcinoma < or =20 mm diagnosed and treated between 1990 and 2004 in one university clinic. MAIN OUTCOMES Lymph node metastases were more frequent at diagnosis in 11-20 mm than in < or =10 mm cancers (p < 0.001). The prevalence of distant metastases did not differ between < or =10 mm and 11-20 mm cancers. One hundred and thirty-three patients (73%) with tumors 11-20 mm were disease free 2 years after 131I treatment, and no recurrence has been observed over 2-14 years of follow-up. Forty-one patients (22%) with cancers 11-20 mm (N1 or M1) required 2-4 years to become disease free. Neck lymph node recurrence was observed in nine patients (4.9%) 4 months to 14 years after surgery and (131)I therapy. Four patients (1.6%) with cancers < or =10 mm in diameter had cancer recurrence (p = 0.05 compared to the 11-20 mm cancers). Based on the presence of distant metastases at diagnosis and recurrence of disease during follow-up, cancers 11-20 mm in diameter seemed more aggressive than those < or =10 mm (p < 0.05). CONCLUSION Cancers 11-20 mm seem more aggressive than those < or =10 mm.

Colonic mucosa adjacent to adenomas and hyperplastic polyps—a morphological and histochemical study

The morphological and histochemical features of colonic mucosa adjacent to 142 adenomas and 31 hyperplastic polyps were studied. Three predominant patterns were identified: (1) normal mucosa, showing normal histological architecture and secretion of sulphomucins; (2) N+ type, histologically normal mucosa with predominance of sialomucins; (3) transitional mucosa; hyperplastic mucosa secreting sialomucins. Hyperplastic changes were observed in the immediate neighbourhood or at the base of adenomas and were more frequent and extensive near large adenomas than around smaller lesions. Sialomucins were often predominant in the mucosa adjacent to large adenomas, but N+ type mucosa was also seen near minute adenomas and hyperplastic polyps and remote from polypoid lesions. Moreover, both hyperplastic and secretory changes were more frequent in the left colon than in the right. These findings seem to suggest that mucosal hyperplasia more likely represents a local change, parallel with or secondary to tumour development rather than a pre‐adenomatous lesion. Secretory modifications are widespread and may result from the action of various factors among which carcinogens cannot be excluded.

Histological Features of Non-A, Non-B Hepatitis in Hemodialysis Patients

In 12 hemodialysis (HD) patients with persistently raised serum alanine amino-transferase concentrations, 6 of which with anti-HCV antibodies, a liver biopsy was performed. The histological examination showed chronic persistent hepatitis or less significant changes in 11 patients and mild chronic active hepatitis in only 1. Non-A, non-B hepatitis seems to demonstrate in HD patients a low tendency to induce an active and progressive liver disease.