Correlation of RET somatic mutations with clinicopathological features in sporadic medullary thyroid carcinomas.

Screening of REarranged during Transfection (RET) gene mutations has been carried out in different series of sporadic medullary thyroid carcinomas (MTC). RET-positive tumours seem to be associated to a worse clinical outcome. However, the correlation between the type of RET mutation and the patients' clinicopathological data has not been evaluated yet. We analysed RET exons 5, 8, 10-16 in fifty-one sporadic MTC, and found somatic mutations in thirty-three (64.7%) tumours. Among the RET-positive cases, exon 16 was the most frequently affected (60.6%). Two novel somatic mutations (Cys630Gly, c.1881del18) were identified. MTC patients were divided into three groups: group 1, with mutations in RET exons 15 and 16; group 2, with other RET mutations; group 3, having no RET mutations. Group 1 had higher prevalence (P=0.0051) and number of lymph node metastases (P=0.0017), and presented more often multifocal tumours (P=0.037) and persistent disease at last control (P=0.0242) than group 2. Detectable serum calcitonin levels at last screening (P=0.0119) and stage IV disease (P=0.0145) were more frequent in group 1, than in the other groups. Our results suggest that, among the sporadic MTC, cases with RET mutations in exons 15 and 16 are associated with the worst prognosis. Cases with other RET mutations have the most indolent course, and those with no RET mutations have an intermediate risk.

Frequent 7q gains in flow cytometric multiploid/hypertetraploid breast carcinomas: a study of chromosome imbalances by comparative genomic hybridisation.

OBJECTIVE: To investigate underlying genetic events associated with complex DNA ploidy breast carcinomas. METHODS: Screening for chromosome imbalances was carried out using comparative genomic hybridisation (CGH) in 14 frozen samples of tumour from a series of 13 breast cancer patients with multiploid (n = 11) and hypertetraploid (n = 2) tumours. They had previously been analysed by DNA flow cytometry and also assessed immunohistochemically for p53 tissue expression. Ploidy status was determined on frozen samples using the Multicycle software program. RESULTS: The total number of copy gains (n = 242) was significantly greater than the number of copy losses (n = 51). The mean (SD) number of gains per sample was 17.3 (5.7), and of losses, 3.6 (4.2) (p = 0.0001). Gains of chromosomal regions at 1q (14/14; 100%), 7q (12/14; 85.7%), and 3q (11/14; 78.6%), as well as 1p, 2q, 5p, 8q, and 13q (10/14; 71.4%) were the most frequent aberrations in this series. Losses were most commonly found on 17p (5/14; 35.7%). Three patients dying of the disease had tumours with high level amplifications at 1q12-qter, 3q22-q25, and 8q22-q23 regions. Six cases had p53 overexpression, of whom four showed 12q gains and two showed 17p losses. CONCLUSIONS: There is a very high incidence of genetic aberrations, mainly related to chromosomal gains, in this subgroup of aneuploid breast cancer patients, associated with a poor clinical outcome. The 7q locus, not previously reported as showing frequent changes in breast cancer, was found to be a potential site for some candidate oncogenes.

Prognostic biomarkers in renal cell carcinoma: relevance of DNA ploidy in predicting disease-related survival.

OBJECTIVE: To investigate the prognostic value of DNA ploidy, Ki-67 index and p53 expression in relation to disease-related survival in a consecutive series of patients with renal cell carcinoma (RCC). MATERIAL AND METHODS: The study group consisted of 64 RCC patients treated by radical nephrectomy. Histological type, pathological staging and nuclear anaplasia were assessed according to the WHO classification, TNM system and Fuhrman grading criteria, respectively. Ploidy was determined by DNA flow cytometry using two sampling methods (frozen vs paraffin-embedded tissue). Ki-67 and p53 were evaluated by immunohistochemistry techniques using two cutoff points (10% vs mean value) for staining interpretation. Kaplan-Meier and Cox regression analyses were used for prognostic evaluation. RESULTS: Thirty-one tumors (48.4%) showed DNA diploidy and 33 (51.6%) were DNA aneuploid. Concordance between both ploidy measurement methods was found in 85.5% of cases (p=0.0455). The mean values for Ki-67 and p53 immunostaining were 3.65% (0-23.5%) and 5.90% (0-55.9%), respectively. DNA ploidy significantly correlated with staging, tumor size (pT), nuclear grading, and Ki-67 (mean value cutoff). Ki-67 (10% cutoff) correlated with staging and pT, while p53 (mean value cutoff) was associated with Ki-67 (mean value cutoff). There were significant differences between survival curves for pathological stage, pT, nuclear grade, ploidy, Ki-67 (both cutoffs), and p53 (10% cutoff). By univariate regression analysis, stage III and stage IV, pT3, aneuploidy, high Ki-67 (both cutoffs), and p53 overexpression (10% cutoff) showed significant correlations with worse disease-related survival. In addition, DNA aneuploidy significantly correlated with poor prognosis within stages I/II (p=0.0355) and stages III/IV (p=0.0138) of the disease. CONCLUSION: The results indicate that DNA ploidy has relevant prognostic value in RCC, adding useful information to the classic histopathological indicators of clinical outcome.

Overall survival in advanced breast cancer: relevance of progesterone receptor expression and DNA ploidy in fine-needle aspirates of 392 patients.

Fine-needle aspiration cytology (FNAC) is essential for making a diagnosis in advanced breast cancer. The determination of hormone receptors in the material obtained is useful for predicting patient response to endocrine therapy, but the prognostic value of hormone receptor expression as well as the clinical utility of DNA flow cytometry are controversial. The aim of this prospective study with long-term follow-up (median: 81 months) was to evaluate these biomarkers in relation to overall survival in a series of 392 patients with advanced breast cancer (stage IIB, n=106; IIIA, n=66; IIIB, n=174; and IV, n=46) using FNAC. Estrogen and progesterone receptor expression was found in 65.1% and 46.1% of the tumors, respectively. Hormone receptors were not found to be associated with clinical staging. DNA aneuploidy was present in 70.9% of the cases and the median S-phase fraction (SPF) was 9.4%. There was a significant correlation of aneuploidy and high SPF with lack of hormone receptors. In univariate analysis, advanced disease stage, absence of hormone receptors, DNA aneuploidy and high SPF showed a statistically significant correlation with poor clinical outcome. In multivariate analysis, disease stage, progesterone receptors and DNA ploidy retained independent prognostic significance in relation to overall survival. These data indicate that progesterone receptor expression and DNA ploidy are independent prognostic factors in advanced breast cancer.

Male breast cancer--a reappraisal of clinical and biologic indicators of prognosis.

Between 1970 and 1998, 90 cases of male breast cancer with available pathological material were retrieved. The disease often presented in aged patients (median--66 years) and as advanced stage (stage III/IV-51%). Excluding stage IV disease, the neoplasia were predominantly ductal invasive carcinomas. NOS (not otherwise specified) (92%), grade 1 and grade 2 (94%), positive for estrogen and progesterone receptors (72% and 74%), negative for androgen receptors (100%), p53 negative (95%), c-erbB-2 negative (88%) and DNA aneuploid (73%). Assessment of disease outcome is determined by stage at time of diagnosis, and axillary lymph node status was the only parameter found to have a statistically significant correlation with either disease-free interval or overall survival (p < 0.001) by multivariate analysis. Clinically useful information on the probability of relapse can be added by determining c-erbB-2 (p = 0.02) and progesterone receptors (p = 0.04) in stage III and tumor ploidy (p = 0.04) in pN1 subgroups of patients.

Prognostic comparative study of S-phase fraction and Ki-67 index in breast carcinoma.

AIMS: To investigate the prognostic value of recently proposed flow cytometric S-phase fraction (SPF) variables (average SPF and SPF tertiles) compared with conventional SPF, and to compare the one with the best predictive value with the immunohistochemical Ki-67 index in breast carcinoma. METHODS: A short term follow up study (median, 39.6 months) of a large series of patients (n = 306) was conducted. DNA ploidy was analysed on fresh/frozen tumour samples by flow cytometry, and the SPF was calculated from the DNA histogram using an algorithm. The Ki-67 index was assessed on paraffin wax embedded material by immunohistochemistry (cut off point, 10%). The two methods were compared by means of kappa statistics, and the prognostic significance of both in relation to disease free survival (DFS) and overall survival (OS) was determined. RESULTS: SPF and Ki-67 analysis was performed on 234 (76.5%) and 295 (96.4%) tumours, respectively. The two assessments were simultaneously available in 230 cases. All SPF variables analysed in the whole series significantly correlated with disease evolution, with the conventional median SPF (cut off point, 6.1%) showing the highest predictive value in relation to both DFS (p = 0.0001) and OS (p = 0.0003). SPF tertiles and median SPF evaluated according to DNA ploidy status had no prognostic significance. The Ki-67 index showed a trend in relation to DFS (p = 0.086) that did not reach significance, and no correlation with OS was found (p = 0.264). The comparative analysis of SPF and Ki-67 revealed some agreement between the two methods (agreement, 69.13%; kappa statistic, 0.3844; p < 0.001), especially in the subgroup of diploid tumours. CONCLUSIONS: Flow cytometric SPF is a better prognosticator than the Ki-67 index, but only SPF variables applied in the whole series show potential clinical usefulness.

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.

Objective biologic parameters and their clinical relevance in assessing salivary gland neoplasms.

This review summarizes research advances of cytometric, proliferation, cytogenetic, and molecular "objective" measurable parameters, as additional aids to prognostic information of salivary gland tumors provided by classical clinicopathologic indicators. Flow cytometric DNA ploidy and S-phase fraction seem to be of value as predictors of tumor behavior, aneuploidy, and high S-phase identifying an unfavorable clinical evolution of salivary gland neoplasms. Cell proliferation markers assessed by immunohistochemistry (e.g., PCNA, Ki-67) also appear to have predictive significance, but some conflicting results, in part related to technical procedures, limit their routine clinical application. Silver-stained methods (AgNORs) show a scarce value in estimating prognosis of salivary gland malignancies. p53 and c-erbB-2 as well as karyotyping, are of disputable benefit for clinical use, but the biologic information they provide give a better understanding on the molecular mechanisms involved in the development and progression of tumors. Further studies, with large databases, long follow-up information, uniformized histologic classification, and standardized methodologies, are needed to establish how these "objective" parameters would be of truly beneficial for the treatment of patients with salivary gland tumors.

The clinical relevance of ploidy and S-phase fraction determination in salivary gland tumors: a flow cytometric study of 97 cases.

BACKGROUND: The authors studied a series of 97 consecutive cases of salivary gland tumors to investigate the correlation between the biologic parameters DNA ploidy and S-phase fraction (SPF) and the presumptive behavior of the neoplasms, as well as their potential clinical utility. METHODS: Histopathologic classification and grading of the tumors were evaluated according to 1991 World Health Organization criteria. DNA analysis was performed by flow cytometry in fresh material after propidium iodide staining. Clinical data and follow-up information were obtained from the clinical charts. RESULTS: All the 71 benign salivary tumors showed a DNA diploid pattern. Seven carcinomas (7.2%) exhibited DNA aneuploidy. Eleven (42.3%) of 26 malignant tumors were considered low grade carcinomas, all of them being DNA diploid. Of the remaining 15 tumors, classified as high grade carcinomas, 7 showed DNA aneuploidy. SPF values ranged from 0.6% to 27.7%. A statistically significant difference was found between the mean SPF values of benign and malignant tumors, diploid and aneuploid tumors, and low grade and high grade carcinomas. When a cutoff value of 3% was used to discriminate histopathologic subgroups with prognostic impact, a significant difference was found between benign and malignant salivary tumors, high grade and low grade carcinomas, and high grade and benign tumors (P < 0.001). CONCLUSIONS: The data from this study confirm the low incidence of DNA aneuploidy in salivary gland tumors and suggest the potential utility of SPF estimation in evaluating the clinical behavior of these neoplasms.

Global DNA hypomethylation in breast carcinoma: correlation with prognostic factors and tumor progression.

BACKGROUND: 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. METHODS: In the group of patients with breast carcinoma, DNA hypomethylation was correlated with clinical and pathologic parameters known to affect disease prognosis. Histopathologic type, disease stage, and tumor grade were evaluated according to the World Health Organization classification, the TNM system, and the criteria of Elston and Ellis' criteria, respectively. DNA flow cytometry was performed in fresh/frozen samples stained with propidium iodide. Hormone receptor (estrogen and progesterone receptor) status was determined by immunocytochemistry. RESULTS: The comparative study of DNA methylation showed that the DNA of breast carcinomas was statistically significantly less methylated than the DNA of the respective adjacent parenchyma (P=0.0001), the DNA of breast benign lesions (P=0.0002), and the DNA of normal parenchyma (P < 0.0001). A statistically significant correlation was found between the global DNA hypomethylation and the disease stage (P=0.0009), tumor size (P=0.0026), and histologic grade (P=0.0097) of malignant neoplasms. A trend for DNA from breast carcinomas with positive axillary lymph nodes (N1) to be more hypomethylated than those without nodal involvement (NO) (P=0.055) was verified. In contrast, no significant association was found between DNA methylation and histologic type of tumors, hormone receptors, DNA ploidy, and S-phase fraction. CONCLUSIONS: The current shows that DNA hypomethylation is increased in breast carcinomas, playing a potentially important role in tumor development. These findings also suggest that DNA methylation status may be a biologic marker with prognostic significance in this group of neoplasms.

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.

Clear cell meningioma variant and clinical aggressiveness.

The few reports about clear cell meningiomas (CCM) point to an inordinate clinical aggressiveness despite their histological benignity. We studied 5 CCM aiming to assess their clinicopathological, cytometric, and ultrastructural features. Two patients were females and 3 males, with a mean age of 36 years. Two tumors were spinal, one of the cerebral convexity, one of the tentorium-clinoid region, and one of the base of the skull. The first 3 were totally removed and have not recurred for a mean follow-up time of 40 months. The tentorium-clinoid and the skull base tumors had radical subtotal and partial resections, and recurred after 16 and 1.5 months, respectively. All tumors but one, a non-recurrent one, presented no signs of histological anaplasia. The proliferative capacity, as assessed by MIB-1 staining index (SI), of recurrent tumors was slightly higher than that of those tumors that did not recur. All cases showed DNA diploid pattern. Amianthoid-type fibers were disclosed on ultrastructural study. CCM arose in patients younger than those with other variants of meningioma, the spinal canal and the posterior fossa were the common sites. Finally, intracranial tumors were linked to an aggressive behavior.

Flow cytometric DNA hypertetraploidy is associated with unfavourable prognostic features in breast cancer.

AIM: Breast tumours with a DNA content higher than 4N (hypertetraploidy) are not well characterised. The aim of this study was to evaluate the clinical and biological characteristics of 51 hypertetraploid breast carcinomas selected from a series of 860 consecutive cases analysed by flow cytometry. METHODS: The clinicopathological characteristics of the hypertetraploid group were compared with those of a control group of 138 non-hypertetraploid breast carcinomas. Breast tumours from patients submitted to surgery as primary therapeutic approach (15 hypertetraploid and the 138 non-hypertetraploid) were TNM staged and classified according to the histological type and grade. The remaining 36 patients had advanced neoplastic disease at presentation and were classified by cytological criteria only. DNA flow cytometric analysis was performed on fresh-frozen samples stained with propidium iodide. Hormone receptors were analysed by immunocytochemistry. RESULTS: The incidence of hypertetraploid breast tumours was 5.9% (51 of 860). All the patients were women and the mean age at diagnosis was 65 years. There was a family history of breast cancer in 21.6% of cases. In the group of operated patients, 33.3% had pT3 tumours and 53.3% had axillary lymph node metastases. All but one tumour were invasive ductal carcinomas; the remaining was an invasive papillary carcinoma. Ten (66.7%) tumours were classified as poorly differentiated carcinomas. Oestrogen and progesterone receptors were negative in 33 (64.7%) and 38 (74.5%) tumours, respectively. At last follow up, 35 (72.9%) patients were alive, while 13 (27.1%) died of disease within three years of diagnosis. Statistical comparison of the clinicopathological features of hypertetraploid v non-hypertetraploid breast carcinomas yielded a significant difference in tumour size (p < 0.001), histological grade (p < 0.001), hormone receptor status (p < 0.001), and overall survival (p < 0.001) between the two groups. CONCLUSION: Flow cytometric DNA hypertetraploidy is related to clinicopathological features of breast cancer usually associated with unfavourable prognosis.

Flow cytometric DNA ploidy and S-phase fraction correlate with histopathologic indicators of tumor behavior in colorectal carcinoma.

BACKGROUND: The clinical behavior of colorectal carcinoma is highly variable without reliable predictive biomarkers. Previous reports have shown that flow cytometric DNA analysis may provide valuable prognostic information in these tumors. PURPOSE AND METHODS: This study evaluates the DNA ploidy and the S-phase fraction (SPF) on frozen samples obtained from 61 patients with colorectal carcinoma by using flow cytometry, and it correlates the data with histopathologic features known to affect disease prognosis. Tumors were classified using the World Health Organization's histologic criteria and were staged according the American Joint Committee on Cancer's classification system. Grade of the neoplasm, vascular invasion, and perineural tumor spread were evaluated in every case. RESULTS: Fifty-nine percent of tumors were aneuploid and showed statistically significant higher S-phase values than diploid tumors (22.5 vs. 11.2 percent; P < 0.00001). Mean SPF of the whole series was 17.9 (range, 4.2-44.2) percent. A statistically significant association was found between SPF values and histologic grade (P < 0.0016), nodal status (P < 0.0007), distant metastasis (P < 0.0001), tumor stage (P < 0.0001), venous invasion (P < 0.0002), and lymphatic permeation (P < 0.01) but not with perineural growth and infiltration of the neoplasm through the bowel wall (T). DNA ploidy correlated positively with tumor stage (P < 0.03), and the association between aneuploidy and advanced stages of the disease was statistically significant. CONCLUSIONS: These findings showed that flow cytometric DNA ploidy and SPF, evaluated in fresh samples, are potentially useful parameters to estimate colorectal carcinoma biopathology. Aneuploidy and high replicative neoplastic activity correlated with histopathologic features that are commonly associated with the prognosis of colorectal carcinoma, being SPF-related to disease dissemination and, therefore, an indicator of clinical relevance.

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.

Homicidal hanging.

We report a case of homicide by hanging of a 29-year-old woman that was committed by her husband, who nearly escaped from justice. We offer some reflections on the rarity of this method of mechanical asphyxia in cases of homicide, and on the difficulties in the diagnosis of the medico-legal etiology of these situations. We also offer a short review of similar cases in the medico-legal literature.