Atypical Spitz tumors in patients younger than 18 years.

BACKGROUND: Diagnosis and proper management of atypical Spitz tumors in pediatric age are still controversial. OBJECTIVE: We sought to investigate the clinicopathological and molecular features of atypical Spitz tumors in patients aged 18 years or younger. METHODS: We performed a retrospective clinicopathological and fluorescence in situ hybridization study on 50 pediatric atypical Spitz tumors. RESULTS: Parameters that were significantly correlated with a diagnosis of atypical Spitz tumors over Spitz nevus included asymmetry, level IV/V, lack of maturation, solid growth, nuclear pleomorphism, high nuclear-cytoplasmic ratio, atypical and deep mitoses, and more than 6 mitoses/mm(2). In the atypical Spitz tumors group, a significantly higher mitotic rate was observed in prepuberal age (P = .04). The 4-probe fluorescence in situ hybridization melanoma assay did not discriminate atypical Spitz tumors from Spitz nevi. Heterozygous 9p21 loss was found in 3 of 37 cases and homozygous 9p21 loss in 2 of 37 cases. Only 1 child experienced a fatal outcome, showing genetic abnormalities by melanoma fluorescence in situ hybridization probe and a heterozygous 9p21 deletion. LIMITATIONS: The limited number of adverse outcomes did not allow the prognostic analysis of single morphologic features. CONCLUSION: Pediatric atypical Spitz tumors are associated with minimal lethal potential. Atypical Spitz tumors require complete excision and careful follow-up while our data do not support any clinical benefit for the sentinel lymph node biopsy procedure and completion lymphadenectomy.

Fluorescence in-situ hybridization analysis for melanoma diagnosis.

Melanocytic proliferation constitutes a heterogeneous group of lesions with remarkable differences in their biology and clinical outcome. Thus, accurate histological diagnosis of these cases is mandatory to establish the most appropriate surgical treatment and follow-up. Although histological examination alone is usually sufficient to identify melanomas among the greater number of nevi, the definition of the benign or malignant nature of a subset of melanocytic tumours, exhibiting atypical features, is a challenging task. Novel techniques that may assist in the histopathological diagnosis in difficult cases have been extensively researched over recent years. Fluorescence in-situ hybridization (FISH), performed with a panel of four probes, including three locus-specific identifier (RREB1, MYB, and CCND1) genes, seems to represent a sensitive and specific molecular tool for the diagnosis of non-ambiguous melanocytic lesions. Some studies have agreed that FISH may be an ancillary diagnostic instrument, but cannot replace light microscopy, to distinguish benign nevi from malignant melanomas in daily practice. However, in the context of ambiguous melanocytic tumours, results are still controversial, and additional and substantial work is needed to develop reliable probes that may identify, with high sensitivity, specific subsets of ambiguous melanocytic lesions, including spitzoid proliferation.

MAML2 rearrangement in Warthin's tumour: a fluorescent in situ hybridisation study of metaplastic variants.

BACKGROUND: Warthin's tumour (WT) is a common benign lesion of the major salivary glands. The nature of WT remains controversial, with particular regard to the presence of clonal chromosomal abnormalities, including the t(11;19) translocation involving the CRTC1 and MAML2 genes, that have been identified in both WT and mucoepidermoid carcinoma. In this study, we focused our attention on metaplastic WT variants, and we conducted a fluorescent in situ hybridisation (FISH) analysis for the presence of MAML2 gene rearrangement. METHODS: Dual-colour FISH analysis was performed on paraffin-embedded sections of eight WTs showing metaplastic changes (five with squamous metaplasia, two with mucinous metaplasia and one with both) using a MAML2 break-apart probe. RESULTS: Presence of split signals indicative of gene rearrangement was identified in a subset of cells in areas of squamous metaplasia in two samples of WT. No rearrangement was observed in the oncocytic epithelium, in lymphocytes and in areas of mucinous metaplasia. CONCLUSIONS: The presence of a small subpopulation of cells carrying MAML2 rearrangement in areas of squamous metaplasia within WT could predispose these lesions to malignant transformation in mucoepidermoid carcinoma and could represent a molecular link between the two entities.

Main clues in the pathologic diagnosis of melanoma: is molecular genetics helping?

Although conventional histopathologic examination is still the undisputable mainstay for the diagnosis of melanocytic skin neoplasms, application of molecular testing has experienced tremendous growth and will continue to expand in the future as the need for more specific diagnoses and new targeted therapies evolve. Ancillary molecular methods, including comparative genomic hybridization and fluorescence in situ hybridization, have the potential to provide important new information to challenging cases, and will help improve diagnostic accuracy, particularly in cases in which morphology is not conclusive. Pathologists are increasingly involved in the prospective genotyping of melanoma, which leads to patient stratification in light of the novel personalized therapeutic approaches in the advanced setting.

Prognostic significance of the alterations of the G1-S checkpoint in localized leiomyosarcoma of the peripheral soft tissue.

BACKGROUND: The aim of this study was to investigate the expression of cell cycle regulators p53, p16, cyclin-D1, and retinoblastoma (Rb) gene protein in leiomyosarcoma of the peripheral soft in order to identify expression profiles potentially useful for clinical prognostic purposes. MATERIALS AND METHODS: A tissue microarray representing 70 localized leiomyosarcomas of the limbs and limb girdles was created with 3 representative cores from each tumor. Immunohistochemical staining was performed for p53, p16, cyclin-D1, and Rb using standard techniques. Staining was scored as either absent-low (<20% of neoplastic cells) or moderate-diffuse (≥20%). Outcome analysis was performed for local recurrence-free survival (LFS), metastatic disease-free survival (MDFS), and disease-specific survival (DSS). RESULTS: Kaplan-Meier analysis of survival revealed that no single alteration of the factors examined was associated with outcome, but tumors showing concomitant alteration of p16 and p53 were characterized by reduced MDFS and DSS (P = 0.01 and P < 0.001, respectively). In addition, patients who received adjuvant therapy consisting of radiotherapy alone or radiotherapy and chemotherapy had a better DSS than those receiving surgery alone or surgery and chemotherapy (P = 0.05). In multivariate analysis, altered p16/p53 remained the only parameter predictive of MDFS and DSS (P = 0.048, hazard ratio [HR] = 2.488, 95% confidence interval [95% CI] 1.006-5.116; P = 0.043, HR = 2.498, 95% CI 1.029-5.909, respectively). CONCLUSIONS: Accumulation of cell cycle alterations represents a prognostic indicator in localized soft tissue leiomyosarcoma, and in particular altered p16/p53 expression is associated with an unfavorable prognosis. This may help the clinical management of patients with leiomyosarcomas.

Immunohistochemical investigation of tumorigenic pathways in sinonasal intestinal-type adenocarcinoma. A tissue microarray analysis of 62 cases.

AIMS: Sinonasal intestinal-type adenocarcinoma (ITAC) is an uncommon neoplasm morphologically similar to colorectal adenocarcinoma, with a well-recognized association with occupational exposure to wood or leather dusts. Here, we analyse several gene products with pivotal roles in tumorigenesis, including p53, p16, deleted in colon cancer (DCC), retinoblastoma, adenomatous polyposis coli, ß-catenin, E-cadherin and CD10, and discuss their relation to clinical behaviour and to similar pathways in colorectal adenocarcinomas. METHODS AND RESULTS: Immunohistochemical analysis of 62 ITACs was conducted on a tissue microarray. Aberrant expression of p53 and p16 were the most commonly observed alterations (61.3% and 64.5% of cases, respectively). Analysis according to the histological subtype showed that p53 overexpression was less frequent in mucinous ITACs (35.3% versus 71.1%, P = 0.018), while loss of DCC and E-cadherin were observed more frequently in this subtype (76.5% versus 31.1%, P=0.002 and 82.4% versus 31.1%, P<0.001, respectively). No correlation was found between the aberrant expression of these and clinical behaviour while mucinous adenocarcinomas had a significantly worse prognosis, with shorter disease-free interval and overall survival (P=0.005 and P<0.001, respectively). CONCLUSIONS: Mucinous ITACs appear to follow a distinct molecular pathway(s) from the non-mucinous variants, and pursue an aggressive clinical behaviour.

Tumour suppressor gene TP53 mutations in atypical vascular lesions of breast skin following radiotherapy.

AIMS: Atypical vascular lesions (AVL) occurring at the site of radiotherapy represent an uncommon but well-documented complication in the setting of breast-conserving therapy for breast carcinoma. Although the biological behaviour of AVL has been regarded as benign, it has been suggested that AVL may represent a precursor of angiosarcoma. A better understanding of the biology of AVL is essential in order to assess appropriate patient management. The aim of the present study was to investigate alterations of tumour suppressor gene TP53 in a series of radiation-induced AVL and angiosarcomas (AS). METHODS AND RESULTS: Direct sequencing analysis of the TP53 gene showed the presence of at least one variation in 10 of 12 (83.3%) AVL and in seven of eight (87.5%) AS. The most common alteration in both categories was the P72R polymorphism in exon 4. One angiosarcoma sample carried a pathogenetically relevant disruptive mutation c.592delG, a frameshift deletion in exon 6, causing a premature stop codon. CONCLUSIONS: The presence of TP53 alterations suggests that its mutational inactivation may be implicated in the pathogenesis of radiation-associated vascular proliferations. The common mutational pathway suggested by our data supports the hypothesis that AVL and AS are biologically related entities, most probably representing the extremes of a morphological continuum.

Atypical Spitzoid melanocytic tumors: a morphological, mutational, and FISH analysis.

BACKGROUND: Identification of the clinical behavior of atypical Spitzoid tumors with conflicting histopathologic features remains controversial. OBJECTIVE: We sought to assess whether molecular findings may be helpful in the diagnostic and prognostic assessment of atypical Spitzoid tumors. METHODS: A total of 38 controversial, atypical Spitzoid lesions (≥ 1 mm in thickness) were analyzed for clinicopathological features, chromosomal alterations by fluorescence in situ hybridization (FISH) analysis (RREB1/MYB/CCND1/CEP6), BRAF(V600E) mutation by allele-specific real-time polymerase chain reaction confirmed by sequencing, and H-RAS gene mutation by direct sequencing. RESULTS: Atypical Spitzoid lesions developed in 21 female and 17 male patients (mean age 22 years). Nine patients underwent sentinel lymph node biopsy and a sentinel lymph node micrometastasis was detected in 4 of these 9 cases. Four additional patients, who did not receive a sentinel lymph node biopsy, experienced bulky lymph node metastases and one experienced visceral metastases and death. Lesions from patients with lymph node involvement showed more deep mitoses (P < .01), less inflammation (P = .05), and more plasma cells (P = .04). FISH analysis demonstrated the presence of chromosomal alterations in 6 of 25 cases. Correlation with follow-up data showed that the only case with fatal outcome showed multiple chromosomal alterations by FISH analysis. BRAF(V600E) mutation was detected in 12 of 16 cases (75%) and H-RAS mutation on exon 3 was found in 3 of 11 cases (27%). LIMITATIONS: Our results require validation in a larger series with longer follow-up information. CONCLUSIONS: FISH assay may be of help in the prognostic evaluation of atypical Spitzoid tumors. Diagnostic significance of BRAF(V600E) and H-RAS mutations in this setting remains unclear.

Primary cerebral myxofibrosarcoma: clinical, morphologic, immunohistochemical, molecular, and ultrastructural study of an infrequent tumor in an extraordinary localization.

Herein, we describe an intracerebral primary low-grade myxofibrosarcoma occurring in a 9-year-old boy. The lesion measured 7 cm and occupied the left parieto-occipital region. A gross-total removal of the tumor was performed. Nine months later, radiologic follow-up revealed a local recurrence which was again surgically removed. The patient then underwent radiotherapy and chemotherapy. He was well and disease-free at 6 months follow-up. The tumor was composed of spindle, stellated, and multinucleated cells embedded in a myxoid background. Foci of increased cellularity, pleomorphism, and high mitotic rate were present. The tumor borders were sharply demarcated from the non-neoplastic nervous parenchyma. Immunohistochemical staining showed that the neoplastic cells were vimentine and CD34 positive. Fluorescence in-situ hybridization analyses did not show FUS and EWSR1 gene rearrangements. Primary intracranial myxofibrosarcomas are very rare (to the best of our knowledge, less than 10 published cases in the international literature). We believe each new case should be recorded to produce a better clinical, pathologic, molecular, prognostic, and therapeutic characterization of this lesion.

S100A13 is a new angiogenic marker in human melanoma.

Angiogenesis is critical in melanoma progression and metastasis and relies on the synthesis and release of proangiogenic molecules such as vascular endothelial growth factor (VEGF)-A and fibroblast growth factors (FGFs). S100A13 is a small calcium-binding protein that facilitates the release of FGF-1, the prototype of the FGF family. S100A13 is upregulated in astrocytic gliomas, in which it correlates with VEGF-A expression, microvessel density and tumor grading, and promotes a more aggressive, invasive phenotype in lung cancer-derived cell lines. To investigate the involvement of S100A13 in human cutaneous melanoma, we analyzed a series of 87 cutaneous melanocytic lesions: 14 common acquired melanocytic nevi, 14 atypical, so-called 'dysplastic' nevi, 45 melanomas (17 radial growth phase and 28 vertical growth phase) and 14 melanoma metastases. Main clinical and pathological features, including histotype, Breslow thickness, Clark's level and outcome were recorded. Microvessel density was determined with CD105/endoglin staining. Semiquantitative determination of S100A13, FGF-1 and VEGF-A protein expression was obtained by immunostaining. Quantification of S100A13 mRNA was achieved by real-time PCR. We found that S100A13 was expressed in melanocytic lesions; compared with benign nevi, S100A13 protein expression was significantly upregulated in melanomas (P=0.024), in which it correlated positively with the intensity of VEGF-A staining (P=0.041) and microvessel density (P=0.007). The level of expression of S100A13 mRNA also significantly increased with progression of disease, from radial growth phase (0.7+/-0.7) to vertical growth phase (3.6+/-3.1) to metastases (7.0+/-7.0) (P<0.001). Furthermore, S100A13 mRNA correlated positively with VEGF-A (P=0.023), TNM stage (P=0.05), risk of relapse (P=0.014) and status at follow-up (P=0.024). In conclusion, S100A13 is expressed in melanocytic lesions when the angiogenic switch occurs and it may cooperate with VEGF-A in supporting the formation of new blood vessels, favoring the shift from radial to vertical tumor growth. Therefore, S100A13 may represent a new angiogenic and prognostic marker in melanoma.

Preoperative assessment of HER-2/neu status in breast carcinoma: the role of quantitative real-time PCR on core-biopsy specimens.

OBJECTIVES: Knowledge of HER-2/neu status is mandatory to identify breast cancer patients amenable to trastuzumab treatment. We evaluated the diagnostic performance of quantitative real-time polymerase chain reaction (qRT-PCR) in the preoperative determination of HER-2/neu status in breast cancer, using core biopsy material. METHODS: In a prospective series, qRT-PCR was performed on fresh core biopsy specimens taken preoperatively in 87 patients with breast carcinoma. Cases with qRT-PCR ratio > or = 2.0 were considered to have HER-2/neu amplification. The results of RT-PCR analysis were compared with those of the standard immunohistochemistry (IHC) and Fluorescence in situ hybridization (FISH) methods. Cases with IHC 3+ or with IHC 2+ and FISH showing amplification were considered HER-2/neu positive. All other cases were considered HER-2/neu negative. RESULTS: qRT-PCR showed HER-2/neu amplification in 13 cases (14.9%), while the standard IHC-FISH combined approach identified 17 HER-2/neu-positive cases (19.5%). Overall, there was concordance between methods in 83 of 87 patients (95.4%). The Spearman's rho correlation coefficient was 0.851; p<0.001. The diagnostic performance for preoperative diagnosis of HER-2/neu status using RT-PCR on core biopsy specimens as compared to standard approach was as follows: sensitivity 76.5%; specificity 100%; positive predictive value 100%; negative predictive value 94.6%. CONCLUSIONS: Quantitative RT-PCR determination of HER-2/neu status from core biopsy specimens provided results comparable to those given by the standard IHC and FISH methods. The use of qRT-PCR on core biopsy material may represent a very useful and easy tool to enhance early identification of HER-2/neu-positive breast cancer patients who, possibly can benefit from trastuzumab treatment.

Embryonal tumor with abundant neuropil and true rosettes: morphological, immunohistochemical, ultrastructural and molecular study of a case showing features of medulloepithelioma and areas of mesenchymal and epithelial differentiation.

Embryonal tumors are a group of malignant neoplasms that most commonly affect the pediatric population. Embryonal tumor with abundant neuropil and true rosettes is a recently recognized rare tumor. It is composed of neurocytes and undifferentiated neuroepithelial cells arranged in clusters, cords and several types of rosettes in a prominent neuropil-rich background. We describe a new case of this tumor. The patient, a 24-month-old female infant, was referred to the Meyer Children's Hospital with a history of right brachio-crural deficit associated with occasional episodes of headache and vomiting. Computed tomography scan and MRI revealed a large bihemispheric mass. The patient underwent two consecutive surgeries. The resultant surgical resection of the tumor was macroscopically complete. The postoperative period was uneventful. On light microscopy the tumor showed a composite morphology: embryonal tumor with abundant neuropil and true rosettes (specimen from the first surgery); medulloepithelioma with mesenchymal and epithelial areas (specimen from the second surgery). The immunohistochemistry evidenced the heterogeneous (neuronal, mesenchymal and epithelial) immunoprofile of tumoral cells. By real-time polymerase chain reaction (RT-PCR), the PTEN gene expression in the tumor was lower than in the five non-neoplastic brain tissues used as control. Mutation analysis did not show any variation in INI-1 and PTEN sequence while P53 analysis showed the presence of homozygote P72R variation. Fluorescent in situ hybridization analysis showed polysomy of chromosome 2 while amplification of N-MYC was not detected. Owing to the rarity of embryonal tumor with abundant neuropil and true rosettes, each new case should be recorded to produce a better clinical, pathological and molecular characterization of this lesion.

Expression of Notch-1 and alteration of the E-cadherin/beta-catenin cell adhesion complex are observed in primary cutaneous neuroendocrine carcinoma (Merkel cell carcinoma).

Increasing evidence indicates that Notch signaling contributes to physiological processes, including development and differentiation, as well as tumorigenesis, either as a tumor promoter or suppressor, depending on cellular context, expression levels and cross talk with other signaling systems. Recent studies reported absent or minimal Notch-1 expression in neuroendocrine tumors of the lung and gastrointestinal tract, suggesting a tumor-suppressor function of Notch-1. Merkel cell carcinoma is a rare and highly aggressive primary cutaneous neuroendocrine carcinoma. Because no information is available on Notch-1 expression in this tumor, we have investigated a series of 31 Merkel cell carcinoma for Notch-1 immunoreactivity. Immunoreactivities for E-cadherin and beta-catenin were also analyzed. All but 1 Merkel cell carcinoma (30 of 31) retained cytoplasmic and membrane Notch-1 expression in more than 50% of cells. beta-Catenin displayed a prevalent membrane-associated staining in 30 of 31 cases, and 22 cases showed more than 50% of immunoreactive cells whereas nuclear beta-catenin was seen only in 2 of 31 cases. E-cadherin membranous expression was remarkably low, as only 1 of 26 cases was found positive in more than 50% of cells. In contrast with neuroendocrine tumors in other tissues, evident Notch-1 expression was found in Merkel cell carcinoma. This finding does not support a tumor-suppressor function of Notch-1 in Merkel cell carcinoma. Downregulation of E-cadherin and diffuse membranous beta-catenin expression suggest a dysregulation of the E-cadherin/beta-catenin complex in Merkel cell carcinoma. This may contribute to local invasion and distant metastasis.

Androgen receptor regulation of the seladin-1/DHCR24 gene: altered expression in prostate cancer.

Prostate cancer (CaP) represents a major leading cause of morbidity and mortality in the Western world. Elevated cholesterol levels, resulting from altered cholesterol metabolism, have been found in CaP cells. Seladin-1 (SELective Alzheimer Disease INdicator-1)/DHCR24 is a recently described gene involved in cholesterol biosynthesis. Here, we demonstrated the androgen regulation of seladin-1/DHCR24 expression, due to the presence of androgen responsive element sequences in its promoter region. In metastatic androgen receptor-negative CaP cells seladin-1/DHCR24 expression and cholesterol amount were reduced compared to androgen receptor-positive cells. In tumor samples from 61 patients who underwent radical prostatectomy the expression of seladin-1/DHCR24 was significantly higher with respect to normal tissues. In addition, in cancer tissues mRNA levels were positively related to T stage. In tumor specimens from 23 patients who received androgen ablation treatment for 3 months before surgery seladin-1/DHCR24 expression was significantly lower with respect to patients treated by surgery only. In conclusion, our study demonstrated for the first time the androgen regulation of the seladin-1/DHCR24 gene and the presence of a higher level of expression in CaP tissues, compared to the normal prostate. These findings, together with the results previously obtained in metastatic disease, suggest an involvement of this gene in CaP.

Arginine metabolism in tumor-associated macrophages in cutaneous malignant melanoma: evidence from human and experimental tumors.

Tumor-associated macrophages (TAMs) may elicit contrasting effects on tumor growth, depending on their biological activities. Macrophages use arginine either to synthesize nitric oxide (NO) through the inducible NO synthase (iNOS) or to produce ornithine through arginase activity. Although the effects of NO are primarily cytotoxic, production of ornithine may promote tumor cell proliferation. Thus, iNOS/arginase balance in TAMs may be crucial in tumor progression. The aim of this study was (a) to explore iNOS and arginase expression in TAMs associated with human melanoma at different stages of tumor progression and (b) to explore whether melanoma cells influence iNOS and/or arginase expression in TAMs under basal condition and in the presence of interferon gamma and/or lipopolysaccharide. Immunohistochemical analyses performed on tissue sections from in situ melanoma, invasive melanoma of different pT categories, and metastatic melanoma revealed that (a) the percentage of iNOS-positive TAMs was significantly higher in in situ and thin melanomas in comparison with more advanced, thicker tumors; (b) the percentage of arginase-positive TAMs did not change among the pT categories analyzed; and (c) the percentage of iNOS-positive TAMs was greater than that of arginase-positive TAMs in peritumoral and intratumoral locations of thin melanomas (pT1). Moreover, by the use of an in vitro experimental protocol represented by B16 murine melanoma cells cocultivated with inflammatory macrophages, we found that melanoma cells stimulate iNOS expression and NO production in macrophages. In conclusion, our in vivo and in vitro results suggest that, mainly in early melanoma lesions, iNOS prevails over arginase in TAMs, a phenomenon possibly stimulated by contact with tumor cells. However, macrophages stimulated by murine melanoma cells secreted a level of NO compatible with an antitumor activity only in the presence of interferon gamma.

Expression of cyclooxygenase-2 in osteosarcoma of bone.

Several studies indicate that cyclooxygenase-2 (COX-2) is overexpressed in human malignancies, where it produces high levels of prostaglandins and contributes to tumor growth. In this study we have analyzed the expression of COX-2 in a series of 48 skeletal osteosarcomas of different subtypes by immunohistochemistry. In addition, we examined the effects of the specific COX-2 inhibitor Celecoxib on the growth of the human osteosarcoma cell line SaOS-2. Immunoreactivity for COX-2 was observed in 39 out of 48 tumors (81.2%), 30 (76.9%) of which showed a moderate or diffuse immunostaining. Considering the group of 42 primary osteosarcomas, COX-2 immunoreactivity was significantly higher in high grade osteosarcomas, where moderate or diffuse expression was detected in 23 out of 32 cases (71.8%), than in low grade osteosarcomas, where moderate or diffuse expression was detected in 2 out of 10 cases (20%) (P = 0.008, Fisher exact test). In addition, low COX-2 expression was always associated with a good response to chemotherapy (5 out of 5 cases), whereas moderate or diffuse COX-2 expression was associated with a good response in 11 out of 20 cases (55%) (P = 0.12, Fisher exact test). In SaOS-2 osteosarcoma cells, which express COX-2, treatment with Celecoxib determined inhibition of cell proliferation and induction of apoptosis. These results indicate that COX-2 is expressed at high levels in high grade osteosarcomas and support the use of COX-2 inhibitors to improve both the tumor response to chemotherapy and the outcome of osteosarcoma patients.

Real-time PCR analysis of RNA extracted from formalin-fixed and paraffin-embeded tissues: effects of the fixation on outcome reliability.

In many pathologic circumstances, quantitative mRNA expression levels are important for evaluation of possible genome mutations. The development of real-time polymerase chain reaction (RT-PCR) technology has facilitated the realization of nucleic acid quantification. Potentially, quantitative PCR offers a number of advantages over traditional methods because it permits the use of small amounts of genetic material. In the present study, we optimize a RNA purification technique on specimens that are formalin-fixed, paraffin-embedded and we examine prolonged formalin fixation effects on quantitative RT-PCR analysis. We compared RNA levels with 70 colic mucosa samples using the cyclooxygenase 2 gene as marker. The difference in amplification successes between formalin-fixed tissues and formalin-fixed, paraffin-embedded tissues was not statistically significant. Moreover, we compared the expression of formalin-fixed samples with the expression of each fresh tissue. Wilcoxon Mann-Whitney test shows that only the difference in the expression levels of 1- or 3-hour formalin-fixed samples is not statistically significant with respect to other fixation times. We found that the mRNA can be reliably extracted from formalin fixed, paraffin-embedded tissue sections but that prolonged formalin fixation produces different results in quantitative RT-PCR. It can be related to difference in RNA sequences length and the generation of secondary structures that are more susceptible to the prolonged formalin fixation. We suppose that the paraffin do not influence the RNA extraction yield because there are no statistical significant differences between amplification success of formalin-fixed tissues and paraffin-embedded tissues. Therefore, in relative expression quantization, we confirm that it is appropriate to use specimens with same protocols and time for formalin fixation.

Expression of p16 in sinonasal malignant melanoma.

The aim of this study was to investigate the expression of p16 in relation with the histopathologic features and the clinical course in patients with sinonasal melanoma. Thirty-seven sinonasal melanomas were immunostained for p16. Seventeen tumours were investigated for loss of the 9p21 region using interphase fluorescence in situ hybridization (FISH). Twenty-seven melanomas (72.9%) showed loss of p16 expression. All cases with spindle or mixed cytology showed loss of p16, whereas this was present in 50% of epithelioid tumours (p=0.01). Loss of p16 expression was more frequently seen in melanomas with alveolar architecture (87.5%) than in tumours with diffuse architecture (68.9%) (p=0.4). There was no correlation between p16 expression and presence of lymph node or distant metastases (p=0.57 and 0.24, respectively). In addition, p16 status did not influence overall survival (p=0.2). The FISH results were in good agreement with immunohistochemistry: 11 tumours out of 17 showed deletion of the 9p21 region and 10 of these showed loss of protein expression. Loss of p16 expression is a frequent event in sinonasal melanoma and it is mainly related to deletion of 9p21 region. At variance from cutaneous melanoma, loss of p16 is not correlated with the prognosis of patients affected by sinonasal melanoma.

HER-2/neu and bcl-2 in ovarian carcinoma: clinicopathologic, immunohistochemical, and molecular study in patients with shorter and longer survival.

The bcl-2 protein is a membrane protein involved in prolonging cell survival by inhibiting apoptosis. The HER-2 oncogene, which is located on chromosome 17 and encodes for a tyrosine-kinase growth factor receptor, is amplified and HER-2/neu is overexpressed in 25% to 30% of breast carcinomas. The authors analyzed the bcl-2 expression and the bcl-2 gene and HER-2/neu overexpression and amplification in FIGO stage IIIC, serous, G3, ovarian carcinomas obtained from living patients who had no evident disease 5 years after primary treatment compared with ovarian carcinomas obtained from patients, matched for stage, grade of differentiation, and treatment, who had died of progression of disease no later than 2 years after primary treatment. bcl-2 overexpression was statistically correlated with progression of disease during first-line chemotherapy (P=0.021). The HER-2/neu status was found not to correlate with progression of disease during first-line chemotherapy. Both bcl-2 and HER-2/neu expression were not statistically associated with the clinical outcome of ovarian cancer patients. Gene amplification of the HER-2/neu chromosome 17 was found in all the HER-2/neu, 3+ score, positive-staining ovarian carcinomas. None of the analyzed samples revealed a translocation t(14;18)(q32;q21) in the bcl-2 gene. The knowledge of additional prognostic or even predictive factors, such as bcl-2 expression, in patients with advanced ovarian carcinoma before the primary chemotherapeutic treatment may help in the management of patients who require a more tailored treatment. In addition, the gene amplification of the HER-2/neu suggests that HER-2 is a potential target for treatment in ovarian cancer.