Tiziana Megha

Cellular kinetic and phenotypic heterogeneity in and among Burkitt's and Burkitt-like lymphomas.

This study asks whether the known genotypic heterogeneity within and between endemic or sporadic Burkitts lymphomas (eBLs and sBLs, n=10 each), and Burkitt‐like lymphomas (BLLs, n‐12), is reflected in divergent cytokinetics and related immunophenotypes. There was strong evidence that eBL and BLL grow markedly faster than sBL, as shown by differences in mitotic and apoptotic indices. Furthermore, in BLL, the median percentage of neoplastic cells immunoreactive for the bcl‐2 protein was much higher than that observed in eBL and sBL. The reverse was true for the median fraction of cells containing c‐myc protein. In eBL and sBL, the median fraction of bcl‐6 protein‐positive cells reached values above 50 per cent, while cells of 8/12 BLLs did not contain detectable amounts of this protein. This observation indicates that in this respect, eBL and sBL resemble normal germinal centres of lymphatic tissue much more than do BLL. Evidence for infection of neoplastic cells by the Epstein‐Barr virus (EBV) was observed in 9/10 cases of eBL and in 3/10 of sBL, but not in BLL. EBV‐positive lymphomas were associated with distinctly lower apoptotic indices and smaller median percentages of bcl‐6‐positive cells than EBV‐negative tumours.

Prognostic relevance of proliferative activity evaluated by Mib-1 immunostaining in node negative breast cancer.

AIM The purpose of this prospective observational study was to analyze the role of Mib-1 immunostaining as a proliferation index in breast cancer. Correlations between Mib-1 expression and clinico-pathological characteristics as well as its prognostic value have been studied in a series of 432 node negative breast cancers. METHODS Mib-1 expression was evaluated by immunohistochemistry. Tumor sections from highly cellular invasive areas of cancer were stained by monoclonal antibody Mib-1 (Dako) and cells whose nuclei stained positive were counted in 10 randomly chosen HPFs and expressed as percentages of all epithelial cells. A minimum of 400 cells were counted. Correlation between Mib-1 staining and clinico-pathological factors was investigated by means of univariate and multivariate analyses. The prognostic impact on actuarial disease free (DFS) and overall survival (OS) was evaluated by univariate analysis using the log-rank test and by multivariate analysis using Cox regression model. RESULTS Tumors were considered as positive for Mib-1 expression when more than 15% of cells counted were stained. Mib-1 positivity was found in 190/432 cases and resulted in being significantly related to tumor grade, tumor size and absence of estrogen receptors at multivariate analysis. With a median follow-up of 66 months, Mib-1 positivity resulted in being the only independent predictor of OS (RR 2.92), and an independent predictor of DFS (RR 2.01) together with absence of estrogen receptors (RR 2.15). CONCLUSIONS Mib-1 index of proliferative activity correlates well to other established prognostic factors of breast cancer. Mib-1 index may improve the tailoring of adjuvant therapy in early breast cancer, and our experience adds evidence to its effectiveness as prognostic factor. Efforts to reach uniformity in the methodology and in the scoring system should be done to warrant a standardized procedure and make Mib-1 determination definitively reliable in the current clinical practice.

Prediction of lymph node status by analysis of prognostic factors and possible indications for elective axillary dissection in T1 breast cancers.

OBJECTIVE To identify those patients with T1 breast cancers with lower risk of nodal metastases who can safely be spared axillary dissection. DESIGN Retrospective study. SETTING University hospital, Italy. SUBJECTS Review of clinical records and histopathological slides of 547 patients with T1 breast cancer, operated on between 1984 and 1997. MAIN OUTCOME MEASURES Incidence of axillary metastases in relation to age, menopausal status, diameter and grade of tumour, vascular invasion, DNA ploidy, S-phase fraction and hormone receptor state, by univariate and multivariate analysis. RESULTS Axillary metastases were present in 159 patients (29%). On univariate analysis, diameter of tumour 10 mm or less (pT1a/pT1b cancers), no vascular invasion, and grade 1 tumour were significantly correlated with a lower risk of nodal metastases, but only vascular invasion (p = 0.0001, odds ratio = 3.1) and diameter of tumour (p = 0.04, odds ratio = 1.6) were independent predictors on multivariate analysis. Among 34 pT1a/pT1b cancers, with low grade of tumour and no vascular invasion, only 2 (6%) had axillary metastases. When only one favourable predictive factor was associated with diameter of tumour of 10 mm or less, the incidence of axillary metastases ranged from 12% for 43 patients with grade 1 cancers to 13% for 76 patients with no vascular invasion. CONCLUSIONS Axillary dissection may be avoided in pT1a and pT1b breast cancers (< or = 10 mm), with low grade of tumour or no vascular invasion. T1 breast cancers 10 mm or less in diameter should be treated by a two-step approach, first wide excision of the tumour and then axillary dissection or not depending on pathological examination of the primary tumour.

Cellular kinetic differences between Hodgkin's and anaplastic large cell lymphomas: relation to the expression of p34cdc2 and cyclin B-1

Our study was designed to compare cellular kinetic parameters of classical Hodgkins disease (HD) with those of anaplastic large cell lymphomas (ALCL‐C, common type; and ALCL‐HL, Hodgkins like), with a particular focus on the G2/M transition. These disorders share some phenotypic properties, e.g., CD30 positivity of putative neoplastic cells. The percentages of cells expressing p34cdc2 (p34) and cyclin B‐1 (cyclin‐B), which form a complex (maturation/mitosis promoting factor, MPF) regulating the G2‐M phases of the cell cycle, were also registered. Highly significant differences between HD and ALCL‐C were recognized: a) in HD, evidence for abortive mitosis (i.e., difficulty to proceed beyond the metaphase stage) and consequent multinucleation and/or deletion of CD30+ cells was prominent, in contrast to ALCL‐C. This was associated with a markedly lower fraction of large atypical cells (LAC) expressing cyclin‐B in the cytoplasm and the nucleus (C + N) in HD than in ALCL‐C; b) the extent of multinucleation of CD30+ cells in HD, but not in ALCL‐C, was correlated with the %p34+ LAC; c) the proportions of LAC expressing p34 and/or cyclin‐B (C) were positively related to the percentages of cyclin‐B (C + N)+ LAC in ALCL‐C but not in HD; d) in HD, in contrast to ALCL‐C, the size of the fraction of cyclin‐B (C + N)+ LAC did not correlate with the ana/telophase indices (ATI, reflecting successful completion of mitosis) and the magnitude of cell loss; e) in ALCL‐C, the percentages of p34+ LAC were positively correlated with ATI or the degree of CD30+ cell deletion, but inversely in HD. With regard to all parameters mentioned above, ALCL‐HL tended to take an intermediate position between HD and ALCL‐C, but sided more with the latter. In conclusion, our present results suggest a derangement of MPF kinetics and functions that is more profound in HD than in ALCL‐C. Int. J. Cancer 77:408–414, 1998.

Expression of p34cdc2 and cyclins A and B compared to other proliferative features of non-Hodgkin's lymphomas: A multivariate cluster analysis

In view of recent knowledge on proteins regulating the cell cycle, we re‐evaluated proliferative features of 98 diffusely growing non‐Hodgkins lymphomas. The combined use of 5 proliferation–associated variables (mitotic indices and percentages of Ki‐67+, p34cdc2+, cyclin A+ and cyclin B+ cells) and their entry into a multivariate cluster analysis separated, without overlaps, the entire cohort into 3 groups (clusters) with (1) low, (2) intermediate and (3) high proliferative activity. Conversely, bivariate plots exposed considerable cluster overlaps. Multivariate stepwise discriminant analysis of all cases revealed a decreasing order of discriminant power for % Ki‐67+ cells > % p34cdc2+ cells > mitotic index > % cyclin A+ cells > % cyclin B+ cells. The combined use of 2 variables only, mitotic index and % p34cdc2+ cells, allowed a clear‐cut separation of clusters 2 and 3. In bivariate plots, correlations were best between % Ki‐67+ cells and % cyclin A+ cells and between mitotic indices and % cyclin B+ cells. Except for chronic lymphocytic leukemias, immunocytomas and marginal zone lymphomas (all in cluster 1), individual lymphoma entities were distributed among at least 2 clusters. There was, however, a marked preponderance of mantle cell lymphomas and diffuse follicular center lymphomas in cluster 1 and of diffuse large B‐cell lymphomas and peripheral T‐cell lymphomas in cluster 2. Anaplastic large‐cell lymphomas predominated in cluster 3 and responded best to therapy. Int. J. Cancer 83:203–209, 1999.

Cell kinetics, morphology, and molecular IgVH gene rearrangements in Hodgkin's disease.

The present study dealt with the question of whether any cellular kinetic patterns correlate with clonal rearrangement of the IgVH gene as revealed by polymerase chain reaction on DNA extracted from lymph nodes with classical Hodgkins disease (HD) and/or from single CD30+ cells (Hodgkin [H] and Reed-Sternberg [RS] cells). In 15/4 cases with H-RS cells of B or Null phenotype, signs of such monoclonality could be detected (group I) but not in the others (group II). CD30+/H-RS cells in group I differed slightly but significantly from those in group II in that they a) exhibited a larger fraction of cells attaining the anaphase/telophase stage of mitosis, and b) produced relatively more mononucleated cells (H) at the expense of multinucleated (RS) cells. In addition, reactive lymphoid cell (CD30-) infiltrates were considerably less dense in group I that in group II. These findings suggest that the cytokinesis of H-RS cells in group I was moderately more efficient than in group II. However, signs of monoclonality were not associated with the normalization of the mitotic process, which also proved to be disturbed in group I.

Is tissue inhibitor of metalloproteinase-1 a new prognosticator for breast cancer? An analysis of 266 cases

Overexpression of tissue inhibitor of metalloproteinase-1 at either the messenger RNA or protein level has been related to a poorer prognosis in breast cancer. We investigated the role of tissue inhibitor of metalloproteinase-1 tissue expression, which was evaluated by immunohistochemistry staining of paraffin-embedded samples, as a possible prognostic indicator in breast cancer. The study included 266 patients treated by primary surgery. Tumors were scored tissue inhibitor of metalloproteinase-1 positive when at least 10% of the cells showed moderate or strong staining. Staining was observed in 76 (28.6%) patients; by multivariate analysis, factors independently associated with tissue inhibitor of metalloproteinase-1 positivity included more than 9 metastatic axillary nodes, high Mib-1 expression, and positivity for plasminogen activator inhibitor and CD44. With a median follow-up of 125 months, tissue inhibitor of metalloproteinase-1 expression showed a significant prognostic role in disease-free and overall survival by univariate analysis. Multivariate analysis confirmed an independent negative prognostic impact of tissue inhibitor of metalloproteinase-1 on overall but not disease-free together with high values of Mib-1. The number of involved axillary nodes, and triple negativity were independent predictors of either poorer disease-free or overall survival. In our study, tissue inhibitor of metalloproteinase-1 expression was significantly related to markers of tumor aggressiveness and was a powerful indicator of poorer prognosis, with a difference in 10-year disease-free and overall survival of 14% and 28%, respectively, between tissue inhibitor of metalloproteinase-1-negative and tissue inhibitor of metalloproteinase-1-positive cases. Expression of tissue inhibitor of metalloproteinase-1 also was an independent prognostic factor in node-positive cases, indicating a possible role of tissue inhibitor of metalloproteinase-1 as a marker of reduced chemosensitivity. Thus, tissue inhibitor of metalloproteinase-1 may have a role in clinical practice as a prognostic and predictive factor and a possible target for future therapies.

Traditional and new prognosticators in breast cancer: Nottingham index, Mib-1 and estrogen receptor signaling remain the best predictors of relapse and survival in a series of 289 cases.

Histopathological and immunohistochemical findings on tissue microarrays, overall survival (OS), disease-free survival (DFS) and incidence of relapses (R) were recorded and statistically analyzed in 289 breast cancers. A higher R and a shorter DFS were significantly related to larger tumors, lymph node invasion, higher tumor grade, absence of estrogen receptors (ER), triple negative tumors, and presence of lymphovascular invasion (LVI). Longer OS was observed to be significantly associated with smaller tumor size (T), lymph node negativity, lower tumor grade, absence of LVI, lower Mib-1 expression and with the presence of ER. At multivariate analysis, only T for DFS and lymph node status and triple negativity either for DFS or OS had independent prognostic value. In the 194 lymph node-negative women DFS and OS were inversely related to tumor grade, absence of ER, Mib-1 expression in more than 15% of neoplastic cells and, only for DFS, presence of LVI. In the 95 lymph node-positive the number of involved nodes was the most discriminating parameter either for DFS or OS; T, Her-2 status and presence of LVI were significantly related to DFS. ER negativity was related to higher grade, progesterone receptors (PR) negativity, Her-2 negativity, hence to triple negativity, to basal-like type, Mib-1expression over 15% of neoplastic cells. Her-2 positivity was related to higher grade, ER positivity and PR positivity. Basal-like type was not an independent prognosticator, while triple negative type has a significant relation to shorter OS. The Nottingham prognostic index accurately identifies prognostic groupings and Mib-1 expression and ER signaling are the key biological predictors even in single cases.

Mitotic Activity and Nuclear DNA Damage of Large Cells in Hodgkin's Disease: Comparison with the Expression of p53 and bcl-2 Proteins and the Presence of Epstein-Barr Virus

The roles of the bcl-2 and p53 proteins in Hodgkins disease (HD) are poorly understood. We therefore compared their detected presence in Hodgkin and Reed-Sternberg/large atypical (H-RS/LA) cells immunohistochemically with the percentages of these cells double-labeled for CD30 and DNA strand breaks (DNA fragmentation index, DFI); mitotic indices (MI); and the EBV infection status. We found a highly significant inverse correlation between the fractions per case of H-RS/LA cells expressing bcl-2/p53 proteins and the DFI of CD30+ elements. No marked effect of these two oncoproteins on MI was noticed, although these parameters and DFI of CD30+ cells were linearly related. EBV infection of H-RS/LA cells exerted only a limited effect on the parameters tested. The results of this study suggest that overexpressed bcl-2 and, to some extent, p53 proteins in H-RS/LA cells of HD primarily counteract deletion of these cells.

Localization of laminin chains in the human retina: possible implications for congenital muscular dystrophy associated with α2-chain of laminin deficiency

One recently described form of congenital muscular dystrophy (CMD) is associated with deficiency of the alpha 2-chain of laminin, an extracellular matrix protein that is specifically located in the basement membrane of placental villi, Schwann cells and skeletal muscle in healthy humans. This laminin is also normally present in the skin, kidney and basement membrane of blood vessels of the CNS, though it is absent from the blood vessel walls in other tissues. In this immunohistochemical study, we have explored the presence of the alpha 1, alpha 2, beta 1 and gamma 1 chains of laminin in the normal human retina, which are all localized in the basement membrane of blood vessels. This study adds to the growing evidence that the alpha 2-chain of laminin is selectively expressed in certain tissues, and suggests that CMD associated with a lack of this protein may be a multisystem disorder, with possible direct involvement of the visual system.