Rosa Maria Tomasino

Expression pattern of receptor activator of NFκB (RANK) in a series of primary solid tumors and related bone metastases.

Receptor activator of NFκB ligand (RANKL), RANK, and osteoprotegerin (OPG) represent the key regulators of bone metabolism both in normal and pathological conditions, including bone metastases. To our knowledge, no previous studies investigated and compared RANK expression in primary tumors and in bone metastases from the same patient. We retrospectively examined RANK expression by immunohistochemistry in 74 bone metastases tissues from solid tumors, mostly breast, colorectal, renal, lung, and prostate cancer. For 40 cases, tissue from the corresponding primary tumor was also analyzed. Sixty‐six (89%) of the 74 bone metastases were RANK‐positive and, among these, 40 (59.5%) showed more than 50% of positive tumor cells. The median percentage of RANK‐positive cells was 60% in primary tumors and metastases, without any statistically significant difference between the two groups (P = 0.194). The same percentage was obtained by considering only cases with availability of samples both from primary and metastasis. Our study shows that RANK is expressed by solid tumors, with high concordance between bone metastasis and corresponding primary tumor. These data highlight the central role of RANK/RANKL/OPG pathway as potential therapeutic target not only in bone metastasis management, but also in the adjuvant setting. J. Cell. Physiol. 226: 780–784, 2011.

Local reactions to tick bites.

A retrospective histological and immunohistochemical study has been carried out in 25 cases of tick bites recorded in our Departments. The samples that included an attached tick showed a cement cone anchoring the mouthparts to the skin and a blood-soaked, spongiform appearance of the superficial dermis, with a mild neutrophilic and eosinophilic infiltration. The vessels displayed a loose multilayered endothelial proliferation, with plump endothelia, permeated with erythrocytes. A few of them were severed, allowing copious blood extravasation. The established lesions included the following: erythema chronicum migrans-like cases, foreign body granulomas-sometimes containing remnants of the mouthparts-cutaneous lymphoid hyperplasia, either of the T-cell or the B-cell type, and tick-bite alopecia. In both the T-cell and B-cell pseudolymphomas, several vessels showed concentric endothelial and perithelial proliferation similar to that seen in the acute lesions. In the tick-bite alopecia, a lymphocytic infiltrate attacked the permanent portion of the hair follicles, whose reaction was a noticeable hyperplasia of the fibrous sheaths, although only a minority of the hairs was destroyed. The observed alterations are specific in the acute lesions and in the alopecia, where they directly arise as a result of the interactions between the hosts tissues and the antihemostatic, anti-inflammatory, and immunomodulatory chemicals contained in the tick saliva. In the other lesions, the changes seem less characteristic, although the fragments of mouthparts and the special vascular changes provide a clue to their etiology.

Prognostic significance of proliferative activity, DNA-ploidy, p53 and Ki-ras point mutations in colorectal liver metastases

Abstract. Paired colorectal liver metastases (CLM) and normal tissue samples from a consecutive series of 36 patients were studied prospectively. MIB‐1 expression was studied by immunohistochemistry on paraffin‐embedded sections. DNA ploidy and S‐phase fraction (SPF) measurements were performed by flow cytometry on frozen tissues. Mutations within the p53 (exons 5‐8) and c‐Ki‐ras (codons 12 and 13) genes were detected by PCR single‐strand conformation polymorphism analysis followed by sequencing. A high correlation was observed between the MIB‐1 LI and SPF value (rho=0·81; P<0·01). Moreover, p53 gene mutations were associated with either high MIB‐1 LI and high SPF. In univariate analysis, SPF and MIB‐1 levels were related to risk of death. The association between overall survival and DNA‐ploidy or p53 mutations did not reach statistical significance, but a slightly better survival was observed for patients either with DNA‐diploid tumours or without mutations (P=0·05 and P=0.06, respectively). SPF was shown by multivariate Cox model analysis to be an independent prognostic variable and thus it might be a useful prognostic factor in patients with CLM.

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.

Reelin expression in human prostate cancer: a marker of tumor aggressiveness based on correlation with grade

Reelin is a glycoprotein that plays a critical role in the regulation of neuronal migration during brain development and, since reelin has a role in the control of cell migration, it might represents an important factor in cancer pathology. In this study, 66 surgical specimens of prostate cancer were analyzed for reelin expression by immunohistochemical method. The reelin expression was correlated with Gleason score and individual Gleason patterns. Reelin expression was found in 39% prostate cancers. Stromal tissues, normal epithelial cells and prostate intraepithelial neoplasia (PIN) of any grade around and distant from cancer were always negative for reelin. Reelin was found in malignant prostatic epithelial glands of 50% cases Gleason score 10, 52% Gleason score 9, 56% Gleason score 8, 18% Gleason score 7, while no sample of prostate cancers with Gleason score 6 showed reelin expression (P=0,005). As reelin staining is frequently found in high Gleason score prostate cancers, we explored whether reelin expression is influenced by single Gleason patterns. While Gleason 3 pattern did not show reelin immunoreactivity, reelin expression was found in 35% Gleason 4 patterns and 45% Gleason 5 patterns (P<0.001). Our results demonstrated for the first time that reelin is expressed in prostate cancer and not in benign prostate tissue and its expression occurs in higher Gleason score and correlates significantly with increasing of single Gleason patterns. This suggests reelin may behave as a specific histological marker and may represent a useful biomarker to predict aggressive phenotypic behavior of prostatic cancer cells.

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.

Extramammary Paget Disease of the Axilla Associated With Comedo-like Apocrine Carcinoma In Situ.

Extramammary Paget disease of the axilla with underlying apocrine carcinoma has been reported only in six cases until now. This report deals with a seventh case characterized by the unique finding of comedo-like features evocative of large cell ductal breast carcinoma within an otherwise typical in situ apocrine carcinoma. This is characterized by spiral-shaped foci of epithelial proliferation with decapitation secretion and central masses of necrotic debris. A possible connection between the solid neoplasm and the overlying Paget disease is illustrated by a few apocrine-follicular units colonized by both the Paget cells and the structured adenocarcinoma. Here, although they display the same immunohistologic pattern of glandular differentiation, the two populations seem to be cytologically different and do not show signs of gradual transition to one another. Thus, they give the impression of parallel but distinct processes, which is consistent with the hypothesis of proliferative induction of a preexisting intraepidermal scattered population from the underlying adnexal carcinoma. The observed resemblance between apocrine carcinoma and comedo carcinoma of the breast, with its ontogenetic and phylogenetic implications, links not only the two neoplasms and the corresponding glands of origin but also mammary and extramammary Paget disease. This reinforces the unifying conception of Paget disease.