Valentina Agnese

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.

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.

Targeting apoptosis in solid tumors: the role of bortezomib from preclinical to clinical evidence

The ubiquitin–proteasome pathway is the main proteolytic system present in the nucleus and cytoplasm of all eukaryotic cells. Apoptosis activation induced by ubiquitin–proteasome pathway inhibition makes the proteasome a new target of anticancer therapy. Bortezomib is the first proteasome inhibitor to be approved by the US FDA; in 2003 as a third line and in 2005 as a second line therapy for the treatment of multiple myeloma only. This review focuses on the use of bortezomib, not only in its therapeutic role but also, more specifically, in its biologic role and discusses the most recent applications of the drug in solid tumors, both at a preclinical and clinical level.

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.

Is BRCA1-5083del19, identified in breast cancer patients of Sicilian origin, a Calabrian founder mutation?

Various studies have been published in Italy regarding the different BRCA1 mutations, but only the BRCA1-5083del19 mutation is recurrent and specific to individuals of Italian descent with a founder effect on the Calabrian population. In our previous study, BRCA1-5083del19 mutation carriers were found in four index cases of 106 Sicilian patients selected for familial and/or hereditary breast/ovarian cancers. The high frequency rate of this mutation identified in the Sicilian population led us to perform haplotype analysis in all family carriers. Five highly polymorphic microsatellite markers were used (D17S1320, D17S932, D17S1323, D17S1326, D17S1325) to establish whether or not all these families had a common ancestor. This analysis showed that all mutation carriers of these families had a common allele. None of the non-carriers of the mutation or of the 50 healthy Sicilian controls showed this haplotype. This allelotype analysis highlighted the presence of a common allele (ancestor), thus suggesting the presence of a founder effect in the Sicilian population. Our results are in contrast with other studies but only the allelotype analysis of all the BRCA1-5083del19 mutation carriers of two neighboring regions of the south of Italy (Calabria and Sicily) will make it possible to identify the real ancestor of this mutation.

Assessment of “grading” with Ki-67 and c-kit immunohistochemical expressions may be a helpful tool in management of patients with flat epithelial atypia (FEA) and columnar cell lesions (CCLs) on core breast biopsy

It is essential to reach a better understanding of “flat epithelial atypia/columnar cell lesions” (FEA/CCLs) in breast core biopsies. Our aim was to explore their biological nature, in order to predict the likelihood of an upgrade to carcinoma. “Cytological grading” has been specially focused, in view of its possible utility in the choice of management. One hundred thirty of a total of 900 cases core needle (CN)/vacuum‐assisted biopsies (VABs), with diagnoses of “hyperplasia” and “atypia” were retrospectively re‐evaluated. Pathological findings of further excision biopsies (FEBs) performed in 40/75 patients with follow‐up were compared with the previous diagnoses. In all cases, both Ki‐67 and c‐kit immunoreactivities were explored and compared with both normal breast tissues and subsequently documented cancers, with special reference to the hyperplastic FEA/CCLs, with “mild” atypia (FEA/CCHAm). Sixteen cases were re‐diagnosed as “usual ductal hyperplasia” (UDH), 60 as “columnar cell hyperplasia” (CCH), and 54 as FEA/CCHA, 30 of which FEA/CCHAm and 24 FEA/CCHAh (with high atypia). Significantly, the Ki‐67 index proved to be on the increase and c‐kit expression on the decrease in FEA/CCHA lesions, mainly in the FEA/CCHAh group and in the subsequently observed cancers, compared with either benign tissues or the FEA/CCH cases. It was also significant that most of the carcinomas were found in FEBs within the FEA/CCHAh group. In this study cytological grading, together with Ki‐67 and c‐kit indices, proved to be helpful in FEA/CCLs evaluation. With regard to FEA/CCHAm lesions, an adequate surveillance appears to be a more appropriate management tool than FEB, as a result of their biological nature and behavior. J. Cell. Physiol. 221: 343–349, 2009.

The Genetics of Breast Cancer

Breast cancer (BC) is a complex and heterogeneous disease caused by interaction of both genetic and nongenetic risk factors. The biological diversity of sporadic BCs consists in the development of several BC subtypes, which are systematically different from one another and which present specific genetic and phenotypic features. Recently, with the advent of cDNA microarrays it has been possible to associate a distinctive “molecular portrait” to a single BC subtype and, consequently, improve BC taxonomy. From a clinical point of view, the gene expression profiles could supply the classic pathological experiment with the aim to select patients with a better prognosis and that could have a benefit from a specific chemotherapy treatment. Recently a new role in BC progression has been identified in a group of small, noncoding RNAs, the MicroRNA (miRNA), which regulate gene expression. Of particular interest is the identification of different miRNA expression levels according to the five molecular BC subtypes identified by experiments on microarray gene expression. About 3–8% of all breast cancers are hereditary, and it is estimated that the main contributors to this type of cancer are mutations in autosomal dominant genes segregating with the disease. Nowadays, BRCA1 and BRCA2 are considered as the two main BC susceptibility genes, although the involvement of additional low penetrance genes implicated in particular syndromes should not be overlooked. Epidemiologic studies reported that women who are BRCA1 mutation carriers have a 45–60% cumulative risk to develop BC before age 35–40, and the average cumulative risk in BRCA1-mutation carriers by age 70 years is 65% whereas the corresponding risk to develop this neoplasm for BRCA2 mutation carriers is estimated to be 25–40% and 45%, respectively. Oncogenetic counseling requires a “multidisciplinary approach,” involving geneticists, oncologists, and psychologists and is offered by many diagnostic clinical genetic services. Nowadays, risk evaluation regarding BC patients is performed with the use of specific mathematical models, such as BRCAPro, the Couch and the Myriad models.

Ductal lavage: A way of carefully tracing the breast-secreting duct

Background: Breast cancer is the most commonly diagnosed neoplasia in women after nonmelanoma skin tumors. Unfortunately, present-day diagnostic methods are unable to identify the presence of a cancer until it has been developing for several years. Currently, ductal lavage seems to represent a new method of reaching an early diagnosis of breast cancer. Materials & methods: This study analyzed 30 patients with ages ranging from 40 to 55 years; and in 26 of these patients, we were able to obtain a sufficient quantity of material for cytological and biomolecular analysis. Results & conclusion: We propose an easy, reproducible method that makes it possible to obtain a detailed map of the nipple, in order to re-identify the duct orifice and take a series of repeated samples from it over a period of time. This procedure is a promising screening and translational research tool since it provides the quantity and quality of ductal fluid required for subsequent cytological and biomolecular analyses.