Simona Corsale

Prognostic significance of p16INK4a alterations and 9p21 loss of heterozigosity 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.

TP53 and p16INK4A, but not H-KI-Ras, are involved in tumorigenesis and progression of pleomorphic adenomas.

The putative role of TP53 and p16INK4A tumor suppressor genes and Ras oncogenes in the development and progression of salivary gland neoplasias was studied in 28 cases of pleomorphic adenomas (PA), 4 cases of cystic adenocarcinomas, and 1 case of carcinoma ex‐PA. Genetic and epigenetic alterations in the above genes were analyzed by Polymerase Chain Reaction/Single Strand Conformational Polymorphism (PCR/SSCP) and sequencing and by Methylation Specific‐PCR (MS‐PCR). Mutations in TP53 were found in 14% (4/28) of PAs and in 60% (3/5) of carcinomas. Mutations in H‐Ras and K‐Ras were identified in 4% (1/28) and 7% (2/28) of PAs, respectively. Only 20% (1/5) of carcinomas screened displayed mutations in K‐Ras. p16INK4A promoter hypermethylation was found in 14% (4/28) of PAs and 100% (5/5) carcinomas. All genetic and epigenetic alterations were detected exclusively in the epithelial and transitional tumor components, and were absent in the mesenchymal parts. Our analysis suggests that TP53 mutations and p16INK4A promoter methylation, but not alterations in the H‐Ras and K‐Ras genes, might be involved in the malignant progression of PA into carcinoma.

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.

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. J. Cell. Physiol. 191: 237–246, 2002.

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.

DNA aneuploidy and high proliferative activity but not K-ras-2 mutations as independent predictors of clinical outcome in operable gastric carcinoma

The prognostic value of DNA ploidy, S‐phase fraction (SPF) and K‐ras‐2 mutations in gastric carcinoma (GC) has not yet been clearly defined. The aim of this study was to clarify the association between biomolecular variables, tumor characteristics, and clinical outcome in GC patients.

Pharmacogenomics in Colorectal Carcinomas: Future Perspectives in Personalized Therapy

The recent introduction of new drugs such as capecitabine, irinotecan, and oxaliplatinum has greatly improved the clinical outcome of patients with advanced/metastatic colorectal cancer. Nevertheless, some patients may suffer from the adverse drug reactions which will probably be the main cause of chemotherapy failure. The goal of pharmacogenomics is to find correlations between therapeutic responses to drugs and the genetic profiles of patients; the different responses to a particular drug are due, in fact, not only to the specific clinico‐pathological features of the patient or to environmental factors, but also to the ethnic origins and the particular individuals genetic profile. Genes which codify for the metabolism enzymes, receptor proteins, or protein targets of chemotherapy agents often present various genetic polymorphisms. The main aim of this review is to provide an overview of the known polymorphisms present in the genes which codify for factors (thymidylate synthase dihydropyrimidine dehydrogenase, uridine diphosphate (UDP)‐glucuronosyl‐transferase 1A1, enzymes implicated in DNA repair) involved in the action mechanisms of the drugs now utilized in chemotherapeutic treatment of colorectal carcinoma, such as fluoropyrimidines, irinotecan, and platinum agents.

TP53 Mutations and S-Phase Fraction but not DNA-Ploidy Are Independent Prognostic Indicators in Laryngeal Squamous Cell Carcinoma

To prospectively evaluate the prognostic significance of TP53, H‐, K‐, and N‐Ras mutations, DNA‐ploidy and S‐phase fraction (SPF) in patients affected by locally advanced laryngeal squamous cell carcinoma (LSCC). Eight‐one patients (median follow‐up was 71 months) who underwent resective surgery for primary operable locally advanced LSCC were analyzed. Tumor DNA was screened for mutational analysis by PCR/SSCP and sequencing. DNA‐ploidy and SPF were performed by flow cytometric analyses. Thirty‐six patients (44%) had, at least, a mutation in the TP53 gene. Of them, 22% (8/36) had double mutations and 3% (1/36) had triple mutations. In total, 46 TP53 mutations were observed. The majority (41%) of these occur in exon 5 (19/46), while the mutations in exons 6, 7, and 8 were represented in 14, 7, and 6 patients, respectively (31%, 15%, and 16%). Five LSCC patients (6%) showed a mutation in H‐Ras gene. Sixty‐three percent of the cases (51/81) were DNA aneuploidy, 14% of these (7/51) were multiclonal. Thirty‐nine patients (48%) had an high SPF value. At Univariate analysis, the DNA aneuploidy, high SPF (>15.1%), TP53 mutations and, in particular, the mutations that occur in exons 5 and 8 were significantly related to quicker disease relapse and short OS. At Multivariate analysis, the major significant predictors for both disease relapse and death were high SPF and any TP53 mutations. While histological grade G3 was an independent factor only for relapse. In conclusions, any TP53 mutations and high SPF are important biological indicators to predict the outcome of LSCC patients.