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.

Perineural pattern of aggregation of cellular blue nevus: probable histoarchitectural reminiscence of histogenesis.

A striking feature of cellular blue nevus consists in the presence, in its histologic picture, of numerous hypertrophic nerves and nerve-like figures, positive for histochemical and immunohistochemical methods for nerve fibers and myelin sheaths. These findings, first described in Masson's original article and repeatedly highlighted in the past for their possible histogenetic significance, are currently considered as merely coincidental. However, the thin conventional histologic sections, catching only short tracts of the nerves, preclude a correct observation of their route and do not allow us to verify if there is an architectural relationship between them and the nevus as a whole. With this aim, we observed a few specimens of cellular blue nevus on digitally overlapped images of contiguous 25-microm-thick sections, processed with Winkelmann's technique of silver impregnation for nerve fibers, which supplied an overall, 3-dimensional view of the lesions and the nerves running through them. In these images, the lobular form of the nevus could be seen gathering around a branching hypertrophic nerve, whose stem stretched vertically from the depth to the most superficial tract of the lesion. The nevus cell aggregates invested the stem and the limbs individually, and these followed the curvilinear contour of the nevus lobules. Our images represent evidence of a preferential perineural aggregation of cellular blue nevus, at least in its lobular form. This indicates that the numerous nerves and the neuroid figures, observed in detail-but within a limited perspective- in the conventional sections, are not merely coincidental and they could indeed be a sign of neural differentiation and/or a clue to the possible neural origin of the nevus.

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 host's 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.

Correlation between GP-170 expression, prognosis, and chemoresistance of superficial bladder carcinoma.

PURPOSE: To study GP-170 in superficial bladder cancer at initial diagnosis and at recurrence and to evaluate if intravesical chemoprophylaxis modifies the expression of GP-170 in tumor recurrences. MATERIALS AND METHODS: GP-170 was retrospectively assessed in 160 patients affected by primary superficial transitional cell carcinoma of the bladder and followed for up to 10 years. Eighty-four patients (52.5%) recurred after transurethral resection (TUR). Adjuvant intravesical chemotherapy after TUR was adopted in 52 patients. The correlations between GP-170 and G-grade, T-category, risk of recurrence and of progression, and adoption of adjuvant intravesical chemotherapy were investigated. The correlations between variations in grade and stage at recurrence and modifications in GP-170 expression were also studied. RESULTS: No significant correlation between GP-170 expression and G-grade and T-category was found. A significant correlation was detected between GP-170 expression and recurrence ( P=0.0383). It showed a biphasic pattern, i.e., tumors that did not express GP-170 had a higher recurrence rate, but high GP-170 levels were also associated with an increasing risk of recurrence. Intravesical chemotherapy did not induce significative variations in GP-170 expression. No correlation was found between progression and GP-170. CONCLUSION: GP-170 seems to be an independent prognostic factor for recurrence in superficial bladder tumors. A negative GP-170 pattern and high levels of GP-170 are associated with an increasing risk of recurrence but have no impact upon progression. In our experience, GP-170 is neither induced nor modified by intravesical chemotherapy, although it might represent a factor of chemoresistance when strongly expressed.

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.

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 p16(INK4A) 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. p16(INK4A) 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 p16(INK4A) promoter methylation, but not alterations in the H-Ras and K-Ras genes, might be involved in the malignant progression of PA into carcinoma.

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.

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.

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.