António E. Pinto

High Prevalence of RAS Mutations in RET-Negative Sporadic Medullary Thyroid Carcinomas

CONTEXT Sporadic medullary thyroid carcinomas (MTC) frequently harbor mutations in the RET protooncogene. We have earlier reported a series of 51 sporadic MTC with 64.7% of RET-positive and 35.3% of RET-negative cases. OBJECTIVE In the present study, we investigated the possible involvement of RAS and BRAF protooncogenes in the development of sporadic RET-negative MTC. PATIENTS AND DESIGN We performed PCR amplification and sequencing analysis of the three mutational hotspots (codons 12, 13, and 61) of the H-, K-, and N-RAS genes, and of the mutational hotspot (codon 600) and exon 11 of the BRAF gene in 65 sporadic MTC, of which 40 were RET positive and 25 were RET negative. RESULTS Somatic H-RAS and K-RAS mutations were detected in 14 of 25 (56.0%) and three of 25 (12.0%) of RET-negative sporadic MTC, respectively. On the other hand, only one of 40 (2.5%) RET-positive sporadic MTC had a RAS mutation, namely in H-RAS. One of the H-RAS mutations was novel (c.32_37dupCCGGCG). No mutations of N-RAS or BRAF were detected in all assessed tumor samples. CONCLUSIONS Overall, our results showed that RAS mutations were present in 68.0% (17 of 25) of the RET-negative MTC and in only 2.5% of the RET-positive MTC (P < 0.0001), suggesting that activation of the protooncogenes RAS and RET represents alternative genetic events in sporadic MTC tumorigenesis.

Global DNA hypomethylation in breast carcinoma

The global DNA methylation of 136 breast lesions (117 primary invasive carcinomas, 5 benign phyllodes tumors, 11 fibroadenomas, and 3 sclerosing adenosis) and their respective adjacent parenchyma was analyzed using an in vitro enzyme assay.

c-erbB-2 oncoprotein overexpression identifies a subgroup of estrogen receptor positive (ER+) breast cancer patients with poor prognosis

PURPOSE To investigate the predictive value of c-erbB-2 oncoprotein expression as compared with established histopathological and cytometric indicators of disease evolution in breast carcinoma. PATIENTS AND METHODS A short-term retrospective study was conducted on a series of 306 breast cancer patients. Classic prognostic factors included tumour size, nodal involvement, histological grading, and hormone receptor status. Flow cytometric DNA ploidy and S-phase fraction (SPF) were also assessed. A Cox proportional hazards regression model was used for multivariate statistical analysis. RESULTS c-erbB-2 overexpression was present in 43 out of 295 (14.6%) tumours, and showed a statistically significant correlation with high histological grade, DNA aneuploidy, high SPF and lack of estrogen receptors (ER). Univariate analysis revealed its association with worse disease-free survival (DFS) and overall survival (OS). The combined evaluation of c-erbB-2 with ploidy and SPF defines a variable (P + S + c) that showed a significant correlation with disease outcome. By multivariate analysis, only nodal status (P < 0.001) and P + S + c subgrouping (group 2: P = 0.002; group 3: P = 0.001) in relation to DFS, and nodal status (P = 0.001) and DNA ploidy (P = 0.006) in relation to OS, retained independent prognostic significance. Subset analyses showed that cytometric parameters, P + S + c subgrouping and hormone receptors were significantly correlated with disease outcome in node-positive patients, whereas in node-negative subgroup no prognostic indicators were found. c-erbB-2 overexpression exhibited a trend in node-positive breast cancer (DFS: P = 0.068; OS: P = 0.086), and significant correlation with poor clinical evolution in ER positive patients (DFS: P = 0.015; OS: P = 0.004), mostly receiving tamoxifen. CONCLUSIONS c-erbB-2 is an independent prognostic indicator of DFS when evaluated in conjunction with ploidy and SPE. It also seems to predict response to tamoxifen therapy, by identifying a subgroup of ER positive (ER+) breast cancer patients with poor prognosis.

Chromosome imbalances in thyroid follicular neoplasms: A comparison between follicular adenomas and carcinomas

The underlying genetic events associated with follicular thyroid tumorigenesis are still ill defined. In this study, we performed a screening for chromosome imbalances by comparative genomic hybridization (CGH) in a group of 12 follicular adenomas (FAs) and 20 follicular carcinomas (FTCs) previously characterized by conventional cytogenetics and flow cytometry analysis. In general, a great similarity was observed between the CGH profiles of the FAs and FTCs. In both benign and malignant tumors, a combination of gains affecting 5, 7, 12, 17, 19, and 20 was observed. Chromosome 7 was the most frequently affected chromosome, with three regions of consensus gains: 7p11–12, 7q11.3–q21, and 7q31. Recurrent gains of chromosomes 5 and 12 involved 5p11, 5p15, 5q13–q22, 5q21–q23, 12p11, and 12q11–q12. DNA sequence losses were also observed in both tumor groups. Chromosomal arms deleted in at least five of the neoplasms were (in order of frequency): in adenomas, 15q, 2p/2q, 3q, 6p/6q, 11q, and 22q; and in FTCs, 3p, 2p, 8q, 1p, 2q, 3q, 6q, 8p, 9p, 11q, 13q, 6p, and 18q. The statistical evaluation of the CGH data demonstrated that 15q loss was significantly associated with FA. Two regions of minimal common loss were defined by CGH at 15: 15q11–q21 and 15q26‐qter. The identification of these regions provides a basis for further molecular studies.

Malignant salivary gland neoplasms: a cytogenetic study of 19 cases

A group of 19 malignant salivary gland neoplasms of various histological types (mucoepidermoid carcinoma, acinic cell carcinoma, adenoid cystic carcinoma, epithelial-myoepithelial carcinoma, myoepithelial carcinoma, basal cell adenocarcinoma, carcinoma ex-pleomorphic adenoma, ductal carcinoma, adenocarcinoma not otherwise specified and undifferentiated carcinoma) were cytogenetically investigated. Previous karyotypic information revealed deletion of the long arm of chromosome 6, loss of chromosome Y and the gain of chromosome 8 as the most recurrent deviations found in these neoplasms. Clonal chromosome aberrations were detected in 11 cases of this series. In 7 of them there were only numerical deviations (gain of chromosomes 2, 7, 8, 10 and X and loss of chromosomes 18, 21 and Y) without concomitant structural anomalies. Structural rearrangements such as t(2;7), t(6;16), t(6;9) and t(1;1) translocations were found in two mucoepidermoid carcinomas, one adenoid cystic carcinoma and one ductal carcinoma, respectively. The wide spectrum of changes found in this group of neoplasms may reflect the diversity in their histogenesis and differentiation phenotypes.

Short term significance of DNA ploidy and cell proliferation in breast carcinoma : a multivariate analysis of prognostic markers in a series of 308 patients

AIM: To determine the importance of tumour DNA ploidy and cell proliferation, as measured by the S phase fraction (SPF), in relation to other established clinicopathological indicators of prognosis in breast cancer. METHODS: A prospective study of 308 patients. Tumours were staged following the TNM system criteria and were classified according to the histological type and grade. DNA flow cytometry was performed on fresh/frozen samples stained with propidium iodide. Hormone receptors were analyzed by immunocytochemistry. A Cox proportional hazards regression model was used for statistical evaluation of the prognostic factors. RESULTS: Median follow up time was 39.6 months (range 3 to 84). A DNA diploid pattern was found in 134 tumours (43.5%) and aneuploid in 174 (56.5%). Median SPF value was 6.1% (range 1% to 27.8%). DNA ploidy and SPF were strongly correlated (p < 0.001), and both were related to histological type (p < 0.001), grade of differentiation (p < 0.001), tumour size (p = 0.006 and p = 0.002), and hormone receptor activity (p < 0.001). DNA ploidy was also related to node status (p = 0.022), but SPF was not. In univariate analysis, there were significant correlations between disease-free survival and age, histological grade, tumour size, node status, DNA ploidy, SPF, and hormone receptor activity; age, tumour size, node status, DNA ploidy, and hormone receptors were predictors of overall survival. In multivariate analysis, only node status (p = 0.001) and DNA ploidy (p = 0.006) retained independent prognostic significance in relation with overall survival, while node status (p < 0.001) and SPF (p < 0.001) were predictors of disease-free survival. DNA ploidy and SPF continued to predict disease-free and overall survival in lymph node positive (pN1) patients but not in the lymph node negative (pN0) group. CONCLUSIONS: DNA ploidy and SPF are strongly intercorrelated and have independent prognostic value for predicting the short term clinical outcome of breast carcinoma patients.

Parenteral Nutrition in Cancer Patients Receiving Chemotherapy: Effects on Toxicity and Nutritional Status

The role of total parenteral nutrition (TPN) in reducing toxicity related to cancer chemotherapy (CT) is presently a controversial issue. To evaluate the effectiveness of TPN in reducing CT-associated toxicity and correcting and preventing CT-related impairments of nutritional status, a prospective crossover controlled study was performed in 43 cancer patients (19 normally nourished and 24 malnourished) randomly divided into two groups (A and B). Group A patients received TPN concomitantly with the first course of chemotherapy, and the second course was administered 21 to 28 days later without TPN support; group B patients were treated in the opposite sequence. The rates of myelotoxicities and gastrointestinal toxicities after CT courses with or without TPN were essentially similar in normally nourished and malnourished patients. No changes in nutritional indexes were detected in normally nourished subjects after each course. Conversely, in undernourished subjects, prealbumin, retinol-binding protein, and nitrogen balance increased in CT+TPN courses (p < .02). In CT-only courses, undernourished subjects showed a decrease in prealbumin and nitrogen balance. Significant changes of nitrogen balance in CT vs CT+TPN courses were detected in malnourished subjects. TPN appears to be unable to reduce CT-associated toxicity. CT administration does not result in any impairment of the nutritional status in normally nourished cancer patients. From our study, it appears that TPN should be limited to severely malnourished neoplastic patients undergoing CT, because of its ability to prevent further impairment of nutritional status and to improve the nitrogen balance and the levels of fast-turnover visceral proteins.

Correlations of cell cycle regulators (p53, p21, pRb and mdm2) and c‐erbB‐2 with biological markers of proliferation and overall survival in breast cancer

Aim: The biological impact of cell cycle regulatory proteins on breast cancer progression is widely recognised, although mostly unclear. The aim of this preliminary study was to investigate the correlations of several cell cycle modulators (p53, p21, pRb, and mdm2) and c‐erbB‐2 expression with cell proliferation markers (S‐phase fraction [SPF] and Ki‐67) and overall survival in breast cancer. Methods: The series comprised 50 women with stage I‐II invasive ductal breast carcinoma (median follow‐up 87 months), who were selected for their tumour proliferative characteristics (15 low, 15 high, and 20 intermediate proliferative tumours). Tumour differentiation was assessed following the Nottingham grading criteria. Cell cycle regulators, oestrogen receptor status, and Ki‐67 index were analysed by immunohistochemistry on paraffin embedded material (cut‐offs 10%). c‐erbB‐2 was evaluated according to a standardised immunohistochemical assay and borderline cases were confirmed by FISH analysis. Ploidy and SPF were determined by DNA flow cytometry on frozen samples. Chi‐square test and Fishers exact test were applied to analyse the statistical significance of data. Results: Positive immunostaining was observed in nine (18%) p53+, 30 (60%) p21+, 13 (26%) pRb+, and one (2%) mdm2+ cases. c‐erbB‐2 expression was considered positive in 11 (22%) cases. In the subset of patients dead of the disease, a high incidence of c‐erbB‐2 over‐expression (7/10, 70%) was verified. In general, no significant correlations among cell cycle regulators or between the latter and histopathological or proliferative characteristics were found. Only the p53−/p21+ phenotype significantly correlated with low SPF (p=0.048), and p21 positivity showed a trend to be associated with low SPF (p=0.083). No statistically significant correlations between cell cycle inhibitors and clinical outcome were found. On the contrary, c‐erbB‐2 over‐expression showed significant correlations with DNA aneuploidy (p<0.001), high SPF (p<0.001), high tumour grading (p=0.008), lack of oestrogen receptors (p=0.036), and poor overall survival (p<0.001). Conclusions: The results seem to indicate the lack of correlations of cell cycle regulatory proteins with cell proliferation markers and overall survival in breast cancer, in contrast to c‐erbB‐2 over‐expression which was found to be associated with increased proliferation rate and worse prognosis.

Follicular thyroid carcinoma: Chromosome analysis of 19 cases

Short‐term cultures of 19 follicular thyroid carcinomas were examined cytogenetically. Clonal chromosomal changes were detected in 12 tumors. Two follicular carcinomas had only numerical alterations: one with a hyperdiploid karyotype with trisomies/polysomies of chromosomes 7 and 12, similar to the karyotypes previously identified in a sub‐group of benign thyroid lesions, and the other with monosomy 20. In the remaining ten cases several structural chromosome anomalies were found. Loss of the short arm of chromosome 3 was observed in one tumor. In two widely invasive and metastasizing follicular carcinomas there was a t(7;8)(p15;q24) as the sole abnormality in one case and a der(8)t(7;8)(p15;q24) together with other cytogenetic alterations in the other case. This finding suggests that t(7;8)(p15;q24) may be related to an aggressive behavior of follicular thyroid carcinomas. Genes Chromosomes Cancer 21:250–255, 1998.

B-zone small lymphocytic lymphoma: a morphologic, immunophenotypic, and clinical study with comparison to "well-differentiated" lymphocytic disorders.

The pathologic, immunologic, and clinical features of five cases of B-zone small lymphocytic lymphoma (BZSLL), characterized by a nondestructive growth pattern with a selective and complete replacement of the B-zone areas of lymph nodes, were examined. These findings were compared with those of 13 cases of intermediate differentiated lymphoma/mantle zone lymphoma (ILL/MZL) and 20 cases of typical small lymphocytic lymphoma/chronic lymphocytic leukemia (SLL/CLL). B-zone SLL was characterized histologically by a deceptively benign pattern at a low magnification, the lymph node architecture being substantially preserved, in contrast to the ILL/MZL and SLL/CLL cases, in which complete effacement of the normal architecture usually could be observed. Moreover, in BZSLL the cellular population was rather uniform and lacked either a prolymphocytic component or the small-cleaved lymphoid cells often seen in SLL/CLL and ILL/MZL cases, respectively. The phenotypic profile of the BZSLL clonal cell population studied by the immunoperoxidase method and by single- and double-labeling flow cytometric analyses (SIg+, CD19+, CD20+, CD21+, CD22+, CD24+, CD35+, CD37+, CD74+, CD45+, CD45R+, MB2+, HLA-DR+, Leu-8+, CD9+/-, CDw75+/-, CD5-/+, CD23-/+, CD10-, FMC7-, PCA-1-, CD25-, CD38-, CD43-, CD3-) appeared to be fairly homogeneous and sufficiently distinct from that of ILL/MZL, based on the absence of FMC7 and CD38 molecules, and from that of SLL/CLL due to significantly stronger expression of SIgs (P less than .05), the higher reactivity with anti-CD9 and -CD22 antibodies (P less than .05), the lower reactivity with anti-CD5 and -CD23 antibodies (P less than .05), and the absence of CD25 determinants. Several clinical features of patients with BZSLL, including age group, advanced stage disease, and high frequency of bone marrow and peripheral blood involvement, were similar to those found in the other patients with ILL/MZL and SLL/CLL, but none of the BZSLL patients had an absolute lymphocyte count higher than 15.0 x 10(9)/L at presentation. Based on the architectural pattern, cytologic features, immunophenotypes, and hematologic findings, we conclude that BZSLL is an unusual variant of SLL that is primary in the lymph nodes and should be distinguished from ILL/MZL and CLL.