Arginine-based PNA microarrays for APOE genotyping.

Four modified PNAs containing one chiral monomer bearing two arginine-derived side chains, with the correct configuration for specific and stable DNA binding, were synthesized, complementary to two DNA tracts in the APOE gene containing SNPs related to the insurgence of Alzheimer's disease. PNA binding performances were first tested in solution against complementary and mismatched oligonucleotides by measuring melting temperatures, and showed high specificity in SNP recognition. In order to set up a new diagnostic platform for APOE genotyping, PNA microarrays were then developed with the synthesized modified PNAs. PNA probe deposition protocols on microarrays were optimized in order to minimize cross-contamination due to carry over. The microarrays obtained by arginine-based PNA deposition were incubated with complementary and mismatched oligonucleotides, showing excellent mismatch recognition on the microarray platform. The specificity of the microarrays was finally tested with oligonucleotide mixtures simulating the real genotype profiles. Six different hybridisation patterns related to six different genotypes in the APOE gene were found to be clearly distinct in microarray experiments, demonstrating the potential of this approach for highly specific genetic analysis.

Distinct molecular patterns based on proximal and distal sporadic colorectal cancer: arguments for different mechanisms in the tumorigenesis.

BACKGROUND AND AIMS: Colorectal cancer (CRC) ranks as the fourth most frequently diagnosed cancer worldwide. CRCs that arise proximally or distally to the splenic flexure show differences in epidemiologic incidence, morphology, and molecular alterations, suggesting the existence of two categories of CRC based on the site of origin. The aim of the present work is to investigate the histological and molecular differences between CRCs located proximally and distally to the splenic flexure, and their potential involvement in tumor prognosis and therapeutic strategies. METHODS: We evaluated 120 patients affected by sporadic CRC for clinicopathologic features, microsatellite instability (MSI), loss of heterozygosity (LOH) of chromosomes 18q, 8p, and 4p; they were also investigated for hMlh1, hMsh2, Fhit, p27, and Cox-2 immunostaining. RESULTS: The mucinous histotype was more frequent in the proximal than in the distal CRCs (p<0.004). The frequency of MSI phenotype was higher in proximal than in distal tumors (p<0.001); moreover, reduced or absent hMlh1, Fhit, p27 immunohistochemical expressions were more frequent in proximal than in distal tumors (p<0.001 and 0.01 for p27). In contrast, the frequency of LOH in 18q was higher in distal than in proximal tumors (p=0.002). No significant differences were observed between proximal and distal tumors in the frequency of LOH in 8p and altered expression of hMsh2 and p53 protein. CONCLUSION: These different features may reflect different genetic pathways of carcinogenesis and support the hypothesis of a different mechanism of cancer development between the proximal and the distal colon, with potential implications in the therapeutic approach.

Loss of p27 expression and microsatellite instability in sporadic colorectal cancer.

BACKGROUND: The role of the loss of p27 protein expression in the oncogenesis of colorectal cancer is still in debate. In this study, we prospectively examined the immunohistochemical expression of p27 in 108 consecutive colorectal cancers, and we analysed the relationship with the results, the clinicopathological data, microsatellite instability (MSI) and other genetic alterations of tumours. METHODS: Unselected patients (108) who underwent curative colorectal resection for sporadic colorectal cancer in a three-year period were evaluated for MSI using 6 microsatellite markers, and for the presence of p27, p53, Fhit, Mlh1 and Msh2 proteins by means of immunostaining. The relationships between these markers were analysed. p27 protein expression was examined for association with disease recurrences and survival. RESULTS: Lack of p27 expression was noted in 33 out of 108 (30.5%) colorectal cancer cases (P<0.05). This altered expression was significantly higher in proximal cancers (P<0.05), mucinous tumours (P<0.001), poorly differentiated histology (P<0.01), cancers with MSI (P<0.05), tumours with altered expression of Mlh1 (P<0.01), of Msh2 (P<0.05), and of Fhit (P<0.01). Overall survival was better in the patient group with altered level of phenotypic p27 expression, although the difference does not reach statistical significance (P=0.069). The analysis performed only for patients with tumour at stage II showed significantly better survival when the tumour exhibited altered p27 expression (P<0.02). CONCLUSIONS: The results of the present study support the hypothesis that altered expression of p27 may be part of the genetic pathway involving MSI, which is responsible for the development of some colorectal cancers.

Abnormal Fhit protein expression and high frequency of microsatellite instability in sporadic colorectal cancer.

The role of Fhit protein in the oncogenesis of colorectal cancer is still in debate. Recent studies have revealed that reduced Fhit protein expression is associated with a deficiency of the mismatch repair protein. One hundred and twenty unselected patients who underwent curative resection for sporadic colorectal cancer in a three-year period were evaluated for microsatellite instability (MSI) using six microsatellite markers, and for the presence of Fhit and mismatch repair (MMR) proteins (Mlh1 and Msh2) by means of immunostaining. The relations between these markers were analysed. Reduced or absent Fhit expression was noted in 18 out of 118 patients. This altered expression was significantly higher in right-sided cancer (P = 0.005), mucinous tumours (P = 0.005) and in poorly differentiated histological types (P = 0.0001). MSI was found in 22 out of 109 patients, more so in right-sided cancer (P = 0.0001), poorly differentiated histology (P = 0.0001), and mucinous tumours (P = 0.0001). No association was found with TNM stage. MSI was present in 66.7% of tumours with altered Fhit expression and in only 10% of tumours with preserved or intermediate Fhit expression (P = 0.0001). Of the tumours with reduced or absent Fhit expression, 72.2% had loss of nuclear Mlh1 or Msh2 expression compared with only 14% of the preserved or intermediate Fhit expression tumours (P = 0.0001). These results support the hypothesis that deficiency in a MMR gene could be a cause of the high frequency of alterations in Fhit expression, and they permit the suggestion that FHIT gene alteration may be part of the genetic pathway involving MSI through which some colorectal cancers arise.