Laser pressure catapulting (LPC): optimization LPC-system and genotyping of colorectal carcinomas.

Genotype analysis is becoming more and more useful in clinical practice, since specific mutations in tumors often correlate with prognosis and/or therapeutic response. Unfortunately, current molecular analytical techniques often require time-consuming and costly steps of analysis, thus making their routine clinical use difficult. Moreover, one of the most difficult problems arising during tumor research is that of their cell heterogeneity, which depends on their clear molecular heterogeneity. SSCP analysis discriminates by means of aberrant electrophoresis migration bands, mutated alleles which may represent as little as 15-20% of their total number. Nevertheless, in order to identify by sequencing the type of alteration revealed by this technique, only the mutated allele must be isolated. The advent of laser microdissection is a procedure which easily solves these problems of accuracy, costs, and time. The aims of this study were to perfect the system of laser pressure catapulting (LPC) laser microdissection for the assessment of the mutational status of p53 and k-ras genes in a consecutive series of 67 patients with colorectal carcinomas (CRC), in order to compare this technique with that involving hand-dissection and to demonstrate that since the LPC system guarantees more accurate biomolecular analyses, it should become part of clinical routine in this field. The LPC-system was perfected with the use of mineral oil and the LPC-membrane. To compare the techniques of hand- and LPC-microdissection, alcohol-fixed, paraffin-embedded tissue from 67 cases of CRC were both hand- and laser-microdissected. In either case, dissected samples were analyzed by SSCP/sequencing and direct sequencing for k-ras and p53 gene mutations. LPC-microdissection made it possible to pick up mutations by direct sequencing or SSCP/sequencing, whereas hand-microdissection mutations were identified only by means of SSCP followed by sequencing; direct sequencing did not reveal any mutation. In the 67 patients examined by either method, 36% (24/67) showed p53 mutations, 32 of which identified. Seventy-eight percent (25/32) were found in the conserved areas of the gene, while 12% (4/32) were in the L2 loop, 50% (16/32) were in the L3 loop, and 12% (4/32) in the LSH motif of the protein. Moreover, of the 67 cases examined, 40% (27/67) showed mutations in k-ras, with a total of 29 mutations identified. Of these, 14 (48%) were found in codon 12 and 15 (52%) in codon 13. The modifications which we brought to the LPC system led to a vast improvement of the technique, making it an ideal substitution for hand-microdissection and guaranteeing a considerable number of advantages regarding facility, accuracy, time, and cost. Furthermore, the data obtained from the mutational analyses performed confirm that the LPC system is more efficient and rapid than hand-microdissection for acquiring useful information regarding molecular profile and can therefore be used with success in clinical routine.

Heterogeneity within and between primary colorectal carcinomas and matched metastases as revealed by analysis of Ki-ras and p53 mutations.

Analysis of the genetic status of Ki-ras and p53 in primary colorectal carcinomas and matched colorectal liver metastasis from 30 patients reveals an overall heterogeneity both within and between the two tumoral tissues. Both genes were found mutated with a similar frequency in both tissues; however, identical mutations in primary tumor and matched metastasis were found less frequently in the case of the Ki-ras than the p53 gene. Only in three cases the same p53 and Ki-ras mutations found in the primary tumor were found also in the metastasis. In several metastatic specimens the DNA bearing a mutation detected also in the primary tumor appears significantly less abundant than the wild-type DNA. These data are discussed in the light of current models of primary tumor/metastasis relationships.

TP53 in gastric cancer: mutations in the l3 loop and LSH motif DNA-binding domains of TP53 predict poor outcome.

The aim of this study was to clarify whether specific p53 mutations may have biological relevance in terms of disease relapse or death in gastric carcinomas (GC). Resected specimens from a consecutive series of 62 patients with GC undergoing potentially curative surgery were prospectively studied. The mutational status of exons 5-8 of the p53 gene was investigated in 62 cases using the PCR-SSCP and sequencing. Presence of microsatellite instability (MSI) was evaluated in 56 cases by analyzing loci highly sensitive of MSI. Twenty mutations of p53 were detected in 17 of the 62 cases analyzed (27%). Ten mutations (50%) occurred in highly conserved domains. According to the p53 specific functional domains: 4/20 mutations (20%) were in the L3 loop and 3/20 (15%) in LSH motif. Eight of the 56 GC resulted MSI-H, 5 (9%) MSI-L, and 43 (77%) MSI stable (MSS). None of the 8 (14%) MSI-H GC showed p53 mutations. p53 mutations were associated with intestinal histotype. Moreover, specific mutations in functional domain (L3 and LSH), together with advanced TNM stage, node involvement, depth of invasion, diffuse histotype, proved to be significantly related to quicker relapse and to shorter overall survival. Specific mutations in p53 functional domains, rather than any mutations in this gene, may be biologically more significant in terms of patients outcome, indicating that these mutations might have biological relevance to identify subgroups of patients at higher risk of relapse or death who might benefit from a more aggressive therapeutic approach.

STAT proteins: from normal control of cellular events to tumorigenesis.

Signal transducers and activators of transcription (STAT) proteins comprise a family of transcription factors latent in the cytoplasm that participate in normal cellular events, such as differentiation, proliferation, cell survival, apoptosis, and angiogenesis following cytokine, growth factor, and hormone signaling. STATs are activated by tyrosine phosphorylation, which is normally a transient and tightly regulates process. Nevertheless, several constitutively activated STATs have been observed in a wide number of human cancer cell lines and primary tumors, including blood malignancies and solid neoplasias. STATs can be divided into two groups according to their specific functions. One is made up of STAT2, STAT4, and STAT6, which are activated by a small number of cytokines and play a distinct role in the development of T-cells and in IFNgamma signaling. The other group includes STAT1, STAT3, and STAT5, activated in different tissues by means of a series of ligands and involved in IFN signaling, development of the mammary gland, response to GH, and embriogenesis. This latter group of STATS plays an important role in controlling cell-cycle progression and apoptosis and thus contributes to oncogenesis. Although an increased expression of STAT1 has been observed in many human neoplasias, this molecule can be considered a potential tumor suppressor, since it plays an important role in growth arrest and in promoting apoptosis. On the other hand, STAT3 and 5 are considered as oncogenes, since they bring about the activation of cyclin D1, c-Myc, and bcl-xl expression, and are involved in promoting cell-cycle progression, cellular transformation, and in preventing apoptosis.

Nm23-H1 expression does not predict clinical survival in colorectal cancer patients.

The gene Nm23, which encodes for a nucleoside diphosphate kinase, has been defined as a metastasis-suppressor gene because of the inverse correlation between its expression and the metastatic capacity of the tumor cells. For colorectal cancer, however, the findings are equivocal. The aim of our study was to assess, in 160 patients undergoing surgery for colorectal cancer (CRC), the expression of the Nm23-H1 protein and to evaluate its possible associations with traditional clinicopathologic variables, with DNA-ploidy and proliferative activity (S-phase fraction, SPF), and with disease-free and overall survival of patients. Nm23-H1 expressions were evaluated on paraffin-embedded tissue by immunohistochemistry; DNA-ploidy and SPF on frozen tissue by flow-cytometric analysis. The median follow-up time in our study group was 71 months (range 34-115 months). No association was observed between Nm23-H1 protein expression and clinicopathological variables, S-phase fraction and DNA-ploidy. Furthermore, no significant differences were observed in the survival of patients with either moderate or strong Nm23-H1 expression. The major significant predictors for both disease relapse and death were advanced Dukes' stage, DNA aneuploid tumors and high SPF, while lymphohematic invasion was the only independent factor for relapse and non-curative resection for death. Our results indicate that Nm23-H1 activity is tissue-specific and that in CRCs the expression of the protein is not associated with tumor progression and patient prognosis, although further studies are required in order to throw more light on the possible clinical significance of the overexpression of the protein Nm23-H1 in such tumors.

p53 mutations in L3-loop zinc-binding domain, DNA-ploidy, and S phase fraction are independent prognostic indicators in colorectal cancer: a prospective study with a five-year follow-up.

p53 gene alterations are among the most common events observed in colorectal cancer,and are accompanied frequently by DNA aneuploidy and high proliferative activity. The prognostic significance of such mutations remains controversial. We prospectively evaluated the prognostic significance of p53 mutations, DNA-ploidy, and S phase fraction (SPF) in a consecutive series of 160 colorectal cancer patients (median follow-up 71 months). Tumor DNA was screened for p53 mutations by PCR/single-strand conformational polymorphism/sequencing. DNA-ploidy and SPF were assessed by DNA flow cytometry. p53 mutations were detected in 68 of 160 (42.5%) cases. In 56% (38 of 68) of these, p53 mutations were found in conserved areas of the gene and in 44% (30 of 68 cases) outside the conserved regions. Eighteen of the 68 cases (26%) had mutations in the L3 loop, 11 of 68 (16%) in the L1 loop-sheet-alpha helix motif, and 39 of 68 (58%) outside L3 and loop-sheet-alpha helix. Seventy-five percent of the cases (120 of 160) showed DNA aneuploidy, whereas 18% of these (22 of 120) were multiclonal. The major independent predictors for both disease relapse and death were advanced Dukes' stage, p53 mutations affecting L3 loop, DNA-aneuploid tumors, and high SPF (>18.5%). Our results show that mutations in L3 functional domain, more than any mutations, are important biological indicators to predict the outcome of patients indicating that these mutations have biological relevance in terms of colorectal cancer disease course.

Prognostic significance of p16INK4a alterations and 9p21 loss of heterozygosity in locally advanced laryngeal squamous cell carcinoma.

The p16INK4a gene, localized within chromosome 9p21, has been identified as a cyclin-dependent kinase inhibitor and may negatively regulate the cell cycle acting as a tumor suppressor. Genetic alterations involving the 9p21 region are common in human cancers. A consecutive series of 64 untreated patients (median of follow up 53 months) undergoing surgical resection for locally advanced laryngeal squamous-cell carcinomas (LSCCs) has been studied prospectively. Our purpose was to investigate p16 alterations (9p21 allelic loss, hypermethylation and point mutations) and their possible association with clinico-pathological data and flow cytometric variables (DNA-ploidy and S-phase fraction (SPF)), and to determine the possible prognostic role of this gene in these tumors. PCR-based techniques were used for investigating 9p21 loss of heterozygosity (LOH) and methylation promoter status of the p16 gene. p16 mutations were detected by PCR-SSCP (single strand conformation polymorphism) and sequencing. 9p21 LOH was detected in 16/62 (26%) informative tumors, point mutations in 5% (3/64) and hypermethylation in 9% (6/64) of the cases. p16 alterations were significantly associated with high SPF and DNA-aneuploidy. By univariate analysis, poor histologic differentiation, stage IV, DNA-aneuploidy and p16 point mutations proved to be significantly related to quicker relapse, whereas these same factors, and in addition high SPF, 9p21 LOH and any p16 alterations were significantly related to shorter overall survival. By Cox proportional hazards analysis only histologic grade (G3) and p16 point mutations were independently related to both disease relapse and death. Our study has identified p16 point mutations as important biomolecular indicators in LSCCs.

Have p53 gene mutations and protein expression a different biological significance in colorectal cancer?

p53 alterations are considered the most common genetic events in many types of neoplasms, including colorectal carcinoma (CRC). These alterations include mutations of the gene and/or overexpression of the protein. The aim of our study was to assess whether in 160 patients undergoing resective surgery for primary operable CRC there was an association between p53 mutations and protein overexpression and between these and other biological variables, such as cell DNA content (DNA-ploidy) and S-phase fraction (SPF), and the traditional clinicopathological variables. p53 mutations, identified by PCR-SSCP-sequencing analysis, were found in 68/160 patients (43%) and positive staining for p53 protein, detected with the monoclonal antibody DO-7, was present in 48% (77/160) of the cases, with agreement of 57% (91/160). In particular, a significant association was found between increased p53 expression and genetic alterations localized in the conserved regions of the gene or in the L3 DNA-binding domain and the specific type of mutation. Furthermore, both overexpression of p53 and mutations in the conserved areas of the gene were found more frequently in distal than in proximal CRCs, suggesting that they might be "biologically different diseases." Although p53 mutations in conserved areas were associated with flow cytometric variables, overexpression of p53 and mutations in its L3 domain were only related respectively to DNA-aneuploidy and high SPF. These data may reflect the complex involvement of p53 in the different pathways regulating cell-cycle progression. In conclusion, the combination of the mutational status and immunohistochemistry of p53, and flow cytometric data may provide an important insight into the biological features of CRCs.