Application of fluorescence in situ hybridization as a diagnostic tool in melanocytic lesions, using paraffin wax-embedded tissues and imprint-cytology specimens.

BACKGROUND: Accurate histopathological diagnosis of certain melanocytic skin lesions as benign or malignant can be notoriously difficult. Recently, four-colour fluorescence in situ hybridization (FISH) has emerged as an important tool for classifying these lesions. AIM: To evaluate the sensitivity and specificity of a melanoma FISH probe kit for accurate diagnosis of melanocytic tumours, and to validate its use with imprint-cytology specimens from the cut surface of tumours. METHODS: In total, 50 melanocytic skin lesions (31 malignant melanomas, 10 benign melanocytic naevi, and 9 histologically challenging benign melanocytic skin lesions) were evaluated. The samples comprise 47 tissue specimens embedded in paraffin wax, and three imprint-cytology specimens from the cut surface of melanomas. FISH was performed using four locus-specific identifier probes [Ras responsive element binding protein (RREB)1, myeloblastosis viral oncogene homologue (MYB), cyclin (CCN)D1 and centromere of chromosome (CEP)6], and results were compared with the clinical long-term follow-up and histopathological diagnosis data. RESULTS: The melanoma FISH probe distinguished between naevi and melanomas with a sensitivity of 100% and a specificity of 94.1%. The most sensitive criterion was a gain in 6p25 (RREB1), seen in 100% of cases, followed by CEP6-related MYB loss (48.1%), CCND1 gain (37%) and MYB gain (22.2%). More than three-quarters (77.8%) of melanomas were positive for two or more criteria. Positive FISH results were also obtained for the imprint-cytology specimens. CONCLUSIONS: FISH is a valuable diagnostic tool for differentiating between benign and malignant melanocytic lesions, providing a high degree of sensitivity and specificity. The probes displayed exceptional discriminative capacity in difficult or ambiguous lesions. To our knowledge, his is the first reported use of imprint-cytology specimens for FISH diagnosis.

The cadherin-catenin complex in laryngeal squamous cell carcinoma.

Abnormal Wnt signaling and impaired cell-cell adhesion due to abnormal E-cadherin and ß-catenin function have been implicated in many cancers, but have not been fully explored in laryngeal squamous cell carcinoma. In this study, ß-catenin cellular location and E-cadherin expression levels were analyzed in 16 laryngeal squamous cell carcinomas (LSCCs) (9 glottic and 7 supraglottic) and 11 samples of non-tumoral inflammatory larynx tissue, using immunohistochemical methods. All non-tumoral tissues showed equally strong membranous expression of ß-catenin, while cytoplasmic expression was found in only 3 of the 11 samples. By contrast, whereas 8/9 glottic LSCCs exhibited only membranous expression of ß-catenin, 6/7 supraglottic LSCCs displayed both membranous and cytoplasmic expression (p = 0.003). Strong E-cadherin staining was observed in 9/11 non-tumoral tissues and 7/9 glottic LSCCs, whereas 4/7 supraglottic LSCCs exhibited weak expression. Reduced membrane expression of E-cadherin and cytoplasmic retention of ß-catenin in supraglottic LSCC seems to be related with more aggressive biological behavior which has been described in clinical studies. Further research is required to clarify the involvement of ß-catenin in the mechanism associated with malignant transformation in laryngeal tissues.

The cadherin-catenin complex in nasopharyngeal carcinoma.

Abnormal Wnt signaling and impaired cell-cell adhesion due to abnormal E-cadherin and ß-catenin function have been implicated in many cancers, but have not been fully explored in nasopharyngeal carcinoma. The aim of this study was to analyze ß-Catenin cellular location and E-cadherin expression levels in nasopharyngeal carcinoma. E-cadherin expression levels were also correlated with clinical data and underlying pathology. ß-Catenin and E-cadherin expression were examined in 18 nasopharyngeal carcinoma and 7 non-tumoral inflammatory pharynx tissues using immunohistochemical methods. Patient clinical data were collected, and histological evaluation was performed by hematoxylin/eosin staining. ß-catenin was detected in membrane and cytoplasm in all cases of nasopharyngeal carcinoma, regardless of histological type; in non-tumoral tissues, however, ß-catenin was observed only in the membrane. As for E-cadherin expression levels, strong staining was observed in most non-tumoral tissues, but staining was only moderate in nasopharyngeal carcinoma tissues. E-cadherin expression was associated with ß-catenin localization, study group, metastatic disease, and patient outcomes. Reduced levels of E-cadherin protein observed in nasopharyngeal carinoma may play an important role in invasion and metastasis. Cytoplasmic ß-catenin in nasopharyngeal carcinoma may impair cell-cell adhesion, promoting invasive behavior and a metastatic tumor phenotype.

Multiple non-metastatic gastrointestinal stromal tumors. Differential features.

INTRODUCTION: gastrointestinal stromal tumors (GISTs) are specific, generally KIT (CD117)-positive, mesenchymal tumors of the digestive tract displaying KIT or PDGFRA gene mutations. Clinically, they tend to present as solitary tumors of the intestinal wall; more rarely, multiple tumors may occur in one or more organs. OBJECTIVE: to review the morphological, immunohistochemical and molecular features of multiple, non-metastatic forms of GIST. SOURCES: review of the literature on Medline, and authors own experience. CONCLUSIONS: multiples GISTs may occur in three different contexts: as spontaneous lesions (in both adults and children); due to familial GIST syndrome (autosomal dominant inheritance); or in association with specific syndromes (e.g. Carney s triad, Carney-Stratakis syndrome, type I neurofibromatosis). Outside these contexts, the existence of multiple GISTs is deemed to be the result of tumor metastasis, and therefore indicative of advanced-stage disease. Clinicians need to be aware of these variants, whose prognosis and treatment differ.

Clinicopathological and immunohistochemical analysis of oral melanocytic nevi and review of the literature.

INTRODUCTION: Oral melanocytic nevi (OMNs) are uncommon benign melanocytic tumors, histologically similar to their cutaneous counterparts. The aim of this study was twofold: to contribute to the epidemiology with a literature review with the first Spanish series of OMNs, and to report on clinicopathological, immunohistochemical and demographic findings. MATERIALS AND METHODS: A retrospective analysis of cases attended over the period 1999-2010 was carried out using data drawn from the pathology unit files at two public hospitals in the Spanish region of Andalusia, serving between them a population of 823.614 inhabitants (11%). RESULTS: Ten cases of OMNs were retrieved, accounting for 0.18% of the total 5499 oral biopsies performed over the period. The female-to-male ratio was 1.5:1; mean patient age was 30. The palate was the most common location (70%). Relative frequencies of histologic types were as follows: subepithelial (40%), common blue (30%), compound (20%) and junctional (10%). Immunohistochemical examination showed strong S-100 protein expression, variable reactivity to HMB-45 and high c-Kit expression by junctional melanocytes. Ki-67 was ≤3 in all cases. CONCLUSIONS: Although this first clinicopathologic analysis of OMNs reported in Spain was based on a small patient series, the results are in line with those reported in larger series and additionally provide new demographic data. Since OMNs and early melanomas are usually detected at routine dental examination, detailed oral exploration should always be performed, and in case of doubt a biopsy should be taken to ensure an accurate diagnosis.

Gastrointestinal stromal tumors (GISTs): SEAP-SEOM consensus on pathologic and molecular diagnosis.

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal neoplasms of the digestive tract, with an incidence of 1.1 cases/100,000 inhabitants/year. A group of experts from the Spanish Society of Pathology and the Spanish Society of Oncology met to discuss a brief update on GISTs and agree on aspects relating to the pathological and molecular diagnosis of these tumors. GISTs are generally solitary, well-circumscribed lesions of variable size (<10 mm-35 cm) that may present with intra- or extra-luminal parietal growth or a mixed-type (hourglass) growth pattern. Histologically, they are unencapsulated neoplasms displaying expansive growth and spindle-shaped (70%), epithelioid (20%), or mixed cellularity (10%). Mitotic activity is generally moderate or low and should be evaluated only in areas with high cellularity or higher mitotic frequency. The great majority of GISTs harbour mutually exclusive activating mutations in genes coding for the type III receptor tyrosine kinases KIT and PDGFRA; less commonly, GISTs have also been reported to display mutations elsewhere, including BRAF and NF1 and SDH-complex genes. The method most widely used to detect KIT and PDGFRA mutations is amplification of the exons involved by polymerase chain reaction followed by direct sequencing (Sanger method) of these amplification products. Molecular analyses should always specify the type of analysis performed, the region or mutations evaluated, and the sensitivity of the detection method employed.

Immunohistochemical study of the neural development transcription factors (TTF1, ASCL1 and BRN2) in neuroendocrine prostate tumours. / Estudio inmunohistoquímico de los factores de trancripción de desarrollo neural (TTF1, ASCL1 y BRN2) en los tumores neuroendocrinos de próstata.

OBJECTIVE: Prostatic small-cell neuroendocrine carcinoma is an uncommon malignancy that constitutes 0.5-1% of all prostate malignancies. The median cancer-specific survival of patients with prostatic small-cell neuroendocrine carcinoma is 19 months, and 60.5% of the patients have metastatic disease. Neural development transcription factors are molecules involved in the organogenesis of the central nervous system and of neuroendocrine precursors of various tissues, including the suprarenal gland, thyroid glands, lungs and prostate. MATERIAL AND METHODS: We present 3 cases of this uncommon condition, applying the new World Health Organisation criteria. We conducted studies through haematoxylin and eosin staining and analysed the expression of the neural development transcription factors achaete-scute homolog like 1, thyroid transcription factor 1 and the class III/IV POU transcription factors, as a new research line in the carcinogenesis of prostatic neuroendocrine tumours. RESULTS: In case 1, there was no TTF1 immunoexpression. Cases 2 and 3 had positive immunostaining for ASCL1, and Case 1 had negative immunostaining. BRN2 immunostaining was negative in case 1 and positive in cases 2 and 3. CONCLUSION: The World Health Organisation does not recognise any molecular or genetic marker with prognostic value. ASCL-1 is related to the NOTCH and WNT signalling pathways. ASCL-1, TTF1 and BRN2 could be used for early diagnosis and as prognostic factors and therapeutic targets.

Ki-67 MIB1 labelling index and the prognosis of primary TaT1 urothelial cell carcinoma of the bladder.

AIMS: To evaluate whether ki-67 labelling index (LI) has independent prognostic value for survival of patients with bladder urothelial tumours graded according to the 2004 World Health Organisation classification. METHODS: Ki-67 LI was evaluated in 164 cases using the grid counting method. Non-invasive (stage Ta) tumours were: papilloma (n = 5), papillary urothelial neoplasia of low malignant potential (PUNLMP; n = 26), and low (LG; n = 34) or high grade (HG; n = 15) papillary urothelial carcinoma. Early invasive (stage T1) tumours were: LG (n = 58) and HG (n = 26) carcinoma. Statistical analysis included Fisher and chi2 tests, and mean comparisons by ANOVA and t test. Univariate and multivariate survival analyses were performed according to the Kaplan-Meier method with log rank test and Cox's proportional hazard method. RESULTS: Mean ki-67 LI increased from papilloma to PUNLMP, LG, and HG in stage Ta (p<0.0001) and from LG to HG in stage T1 (p = 0.013) tumours. High tumour proliferation (>13%) was related to greater tumour size (p = 0.036), recurrence (p = 0.036), progression (p = 0.035), survival (p = 0.054), and high p53 accumulation (p = 0.015). Ki-67 LI and tumour size were independent predictors of disease free survival (DFS), but only ki-67 LI was related to progression free survival (PFS). Cancer specific overall survival (OS) was related to ki-67 LI, tumour size, and p27kip1 downregulation. Ki-67 LI was the main independent predictor of DFS (p = 0.0005), PFS (p = 0.0162), and cancer specific OS (p = 00195). CONCLUSION: Tumour proliferation measured by Ki-67 LI is related to tumour recurrence, stage progression, and is an independent predictor of DFS, PFS, and cancer specific OS in TaT1 bladder urothelial cell carcinoma.

Mitotic activity of the endocrine cells in rat thyroid glands during postnatal life.

This paper presents the results of investigations into the mitotic rates of thyroid endocrine cells in normal postnatal rats, aged 1-120 days. Our study revealed considerable age-dependent shifts in the mean mitotic activity of follicular cells and C-cells. The maximum indices of endocrine cell renewal were reached during the first 10 days of life, decreasing gradually and significantly until 25 days. At 1 month, there was a significant recuperation of the division rate for both cell types, which declined in the adult rat. These results mean that the proliferation of C-cells and follicular cells is inversely proportional to age. The preferential zone of localization of mitoses of both cell types is the central region of the thyroid lobe. The present paper provides new evidence for the postnatal origin of C-cells and follicular cells from the preexisting endocrine cells.

Blue nevus: classical types and new related entities. A differential diagnostic review.

Blue nevus is an uncommon pigmented lesion of dermal melanocytes. By convention, two well defined histologic variants, designated as "common" and "cellular", have been recognised. In the last few years, these lesions have attracted much attention due to the recognition of news entities and to its confusion with malignant melanoma. In the present review, we point out the more striking features of new related entities (combined nevus, deep penetrating nevus, compound blue nevus) and establish the differential diagnosis with conflictive lesions such as atypical blue nevus, locally aggressive blue nevus, congenital giant melanocytic nevus with nodular growth and melanocytic dermal tumor of unpredictable outcome. We also review the concept of malignant blue nevus and the significance of lymph node metastases. The blue nevus is an uncommon pigmented lesion consisting of dermal melanocytes that can appear in diverse forms: dendritic, spindle-shaped, oval-shaped, or polyhedral. Although it usually occurs in skin, it has been reported in other locations, such as oral mucosa, sclera, uterine cervix, vagina, prostate, spermatic cord, pulmonary hilus, orbit, conjunctiva, maxillary sinus, breast, and lymph nodes 3,8,42,49. Generally, it occurs in adults as a single, acquired, intensely pigmented lesion, although familial and multiple nevi have been reported 7,39. By convention, there are two well-defined histologic variants, designated as "common" and "cellular", but lesions often manifest intermediate features. In the last few years, blue nevus has attracted much attention due to the recognition of new (clinical and histologic) entities and to its confusion with malignant melanoma.(ABSTRACT TRUNCATED AT 250 WORDS)

Apoptosis in breast carcinoma.

Apoptosis may play a major role in determining tumor growth and aggressiveness. The aim of this study was to examine the relationship between apoptosis, expression of bcl-2 and p53 proteins, proliferation index, and other clinicopathological features of breast carcinoma. Sixty-five formalin-fixed paraffin-embedded tissue sections from invasive ductal breast carcinomas were studied for the presence of apoptosis by the terminaldeoxynucleotidyl-transferase-mediated dUTP-FITC nick end-labeling (TUNEL) method. Immunohistochemical methods were also used to determine the expression of estrogen receptor, Ki67, bcl-2 and p53 proteins. The number of apoptotic cells ranged from 2.0 to 236.0/10HPF (mean 36.26, median 28.0). The observation of 30 apoptotic cells/10HPF was more common in tumors > 3 cm, of histological grade III, with a high mitotic index, Ki67 index > or = 300, and p53 positivity; however, statistical significance was found only for the histological grade. Grade I and III tumors displayed an inverse association between the apoptotic index and bcl-2 and p53 protein expressions; grade I tumors frequently expressed bcl-2 (19/28), lacked p53 (20/28), and presented a low number of apoptotic cells (18/28), whereas grade III tumors tended to express p53 (12/17), lacked bcl-2 (13/17), and displayed a high number of apoptotic cells/10HPF (12/17). Multivariate analysis for survival revealed that estrogen receptors and apoptosis were independent variables. These data suggest that apoptosis, rather than proliferation index or expression of bcl-2 or p53 proteins, is an independent factor for the prognosis of survival.

P-glycoprotein, metallothionein and NM23 protein expressions in breast carcinoma.

Cellular drug resistance and increased metastatic potential are the major obstacles in the successful treatment of cancer with chemotherapy. The aim of this study was to investigate whether the immunohistochemical expression of two proteins implicated in drug resistance (P-glycoprotein and metallothionein) and the product of the suppressor gene nm23 could be related to prognosis in breast cancer. Seventy-two patients with palpable or occult breast carcinoma, not treated with chemotherapy or endocrine therapy, were examined. Immunohistochemical methods were used to determine the expression of P-glycoprotein (PG), metallothionein (MT), nm23, as well as the estrogen receptor (ER), the p53 status, and the Ki67 index. The results were correlated with clinical and morphological features. Cytoplasmic and membrane-specific immunostainings of PG were seen exclusively in tumor cells and identified in 14 of 72 cases (19.4%). Only a statistically significant association with metastases, (p = 0.06) and recurrences (p = 0.1) was observed. MT-positive reaction was identified in the cytoplasm of the tumor cells in 47 (65.3%) cases. Statistical significance was associated with metastases (p = 0.07), but not with death or recurrences. Specific immunostaining of nm23 protein was seen only in the cytoplasm of tumor cells. A positive reaction was observed in 55 of 72 (89.3%) cases. Although a significant association between nm23 protein expression and other morphologic and immunohistochemical variables did not exist, we observed a higher morbidity in patients with the MT-positive/nm23-negative tumor phenotype. Univariate analysis for survival selected the following variables: histologic grade (p = 0.001), ER (p = 0.002), mitotic index (p = 0.005), Ki 67 index (p = 0.068), MT (p = 0.046) and PG (p = 0.085). The Cox model provided the following independent variables: histologic grade (p = 0.021) and metallothionein (p = 0.03). These data confirm the prognosis observed in patients with PG or metallothionein expression as well as the independence of these two variables. It also suggests that nm23 is not necessarily involved in the development of an invasive phenotype.

Pulmonary vein myxoid leiomyosarcoma.

A case of myxoid leiomyosarcoma located in the right pulmonary veins is presented. The patient complained of progressive dyspnea, orthopnea, sputum cruentum and right chest pain. Angiography revealed an obliteration of right pulmonary veins by a tumor mass that expanded into the left atrium. Histologically, the lesion contained densely packed fusiform cell areas that alternated with other much less cellular and richer in interstitial myxoid matrix. The tumor cells showed specific immunoreactivity to desmin antibodies and contained abundant thin filaments with focal densities and micropinocytic vesicles.

Prognostic factors in follicular carcinoma of the thyroid.

Although there are numerous publications on the nomenclature, morphological diagnostic criteria, and prognostic factors of follicular carcinoma of the thyroid, these issues remain controversial. We present the findings of a retrospective and comparative study of 82 patients who underwent thyroid surgery for thyroid neoplasms. Of these patients, 58 had follicular carcinoma, 12 had atypical adenoma, and the remaining 12 patients had adenoma with partial capsular invasion in the surgical specimen. The goal of the study was to determine diagnostic criteria and prognostic factors in follicular carcinoma. Our results showed that encapsulated thyroid follicular neoplasms should be considered malignant when they demonstrate total capsular invasion and/or vascular invasion. Statistical analysis of the cases of follicular carcinoma showed that higher morbidity rates were seen in the patients with metastasis at the time of diagnosis, with extensively invasive tumors, with age older than 35 years, and with tumors with simultaneous vascular and capsular invasion. The accumulated survival rate over 5 years based on mortality was 91%, and based on morbidity it was 71% (84% for patients with encapsulated tumors and 56% for patients with extensively invasive tumors). In the univariate survival study based on morbidity, we found a relationship with the following parameters: metastasis at the time of diagnosis, grade of local invasion, presence of capsular and vascular invasion, cellular atypia, histologic pattern (insular vs others), presence of thyroid nodule, and age of the patient at the time of diagnosis. In the multivariate survival study based on morbidity, the factors that showed prognostic value were metastasis at the time of diagnosis, grade of local invasion, and age of the patient at the time of diagnosis.

Papillary cystic neoplasm of the pancreas with liver metastasis coexisting with thyroid papillary carcinoma.

BACKGROUND: Papillary cystic neoplasm of the pancreas is an uncommon neoplasm that usually appears to be benign or to have a very low potential for metastasis. We report a case of a patient with papillary and cystic neoplasm of the pancreas, which metastasized to the liver and concomitantly presented a stage I thyroid papillary carcinoma. CASE DESCRIPTION: The patient was a 38-year-old woman with a 12-year history of abdominal pain. She was first admitted to the hospital 3 years after symptoms began, and an exploratory laparotomy revealed a pancreatic cystic lesion that was diagnosed as a pancreatic pseudocyst, which drained by cystojejunosotomy. Nine years after onset, a stage I thyroid papillary carcinoma was excised. One-and-a-half years later, the patient was readmitted to the hospital for evaluation of a palpable abdominal mass. Abdominal computerized axial tomography disclosed a large pancreatic tumor and two nodular lesions of the liver. Papillary cystic neoplasm of the pancreas was diagnosed by cytologic, histologic, and ultrastructural studies of samples obtained directly from the pancreatic mass using fine-needle aspiration biopsy. The patient underwent palliative chemotherapy with mitomycin C, 4-epiadriamycin, and 5-fluorouracil. One year after this treatment began, a computerized axial tomography scan showed persistence of the pancreatic mass, with more prominent cystic spaces, increase in size and in number of the lesions in the liver, slight increase in the lesion of the right ovary, and a new lesion that also had solid and cystic areas in the spleen. CONCLUSION: The pathologic and clinical features of patients with papillary cystic neoplasm of the pancreas, based on tumoral extension, reveal localized tumors (88.2%), tumors with local infiltration or recurrence (6.2%), and tumors with metastasis at a distance (5.6%). Although strong similarities exist among the three groups, there are certain differences in age of presentation, previous clinical diagnosis of pseudocyst, and tumor location.

Blood group antigens in differentiated thyroid neoplasms.

Alteration of cell-surface blood group antigens during malignant transformation is a well-known phenomenon that has not yet been sufficiently investigated in thyroid gland neoplasms. We evaluated 50 normal thyroid glands and 141 differentiated thyroid neoplasms (29 follicular adenomas, 30 follicular carcinomas, 56 papillary carcinomas, and 26 medullary carcinomas) both by the immunoperoxidase technique, using monoclonal antibodies against blood group antigens (A, B, H, Le(a), Le(b), Le(x), and Le(y)) and precursor substances (T, Tn, and sTn), and by affinity to the lectin from Arachis hypogea, to determine the usefulness of these antigens as tumor markers and prognostic factors. Neoplastic tissues showed immunostaining with concordant and nonconcordant expression of ABH antigens. There were statistically significant differences between normal and neoplastic tissues but not among the different neoplasms. Statistically significant differences in Lewis antigen expression were noted between normal and neoplastic tissues and between benign and malignant tumors. Tn and sTn antigen expression showed statistically significant differences between normal and neoplastic tissues. In conclusion, blood group antigens are tumor markers that are expressed more frequently in malignant than in benign neoplasms. The presence of metastases was correlated with enhanced peanut lectin receptors and a loss of A or B antigens.

Sarcomatoid carcinoma of the skin: report of a case.

We report the histological, immunohistochemical and ultrastructural findings of an exophytic cutaneous tumor composed of a mixture of typical basal cell carcinoma (BCC) and malignant fibrous histiocytoma. Nine previously reported carcinosarcomas of the skin are reviewed. We prefer the term "sarcomatoid carcinoma" for this rare neoplasm. Only the BCC showed a positive immunoreaction to cytokeratin; the sarcomatous component was negative, but it did express vimentin, and, focally, smooth-muscle-specific actin and KP1 (CD68). Both components showed p53 immunostaining.