Minichromosome maintenance proteins 2 and 5 in non-benign epithelial ovarian tumours: relationship with cell cycle regulators and prognostic implications.

Minichromosome maintenance proteins (MCM) have recently emerged as novel proliferation markers with prognostic implications in several tumour types. This is the first study investigating MCM-2 and MCM-5 immunohistochemical expression in a series of ovarian adenocarcinomas and low malignant potential (LMP) tumours aiming to determine possible associations with clinicopathological parameters, the conventional proliferation index Ki-67, cell cycle regulators (p53, p27(Kip1), p21(WAF1) and pRb) and patients' outcome. Immunohistochemistry was applied in a series of 43 cases of ovarian LMP tumours and 85 cases of adenocarcinomas. Survival analysis was restricted to adenocarcinomas. The median MCM-2 and MCM-5 labelling indices (LIs) were significantly higher in adenocarcinomas compared to LMP tumours (P<0.0001 for both associations). In adenocarcinomas, the levels of MCM-2 and MCM-5 increased significantly with advancing tumour stage (P=0.0052 and P=0.0180, respectively), whereas both MCM-2 and MCM-5 increased significantly with increasing tumour grade (P=0.0002 and P=0.0006, respectively) and the presence of bulky residual disease (P<0.0001 in both relationships). A strong positive correlation was established between MCM-2 or MCM-5 expression level and Ki-67 LI (P<0.0001) as well as p53 protein (P=0.0038 and P=0.0500, respectively). Moreover, MCM-2 LI was inversely correlated with p27(Kip-1) LI (P=0.0068). Finally, both MCM-2 and MCM-5 were associated significantly with adverse patients' outcome in both univariate (> or =20 vs >20%, P=0.0011 and > or =25 vs <25%, P=0.0100, respectively) and multivariate (P=0.0001 and 0.0090, respectively) analysis. An adequately powered independent group of 45 patients was used in order to validate our results in univariate survival analysis. In this group, MCM-2 and MCM-5 expression retained their prognostic significance (P<0.0001 in both relationships). In conclusion, MCM-2 and MCM-5 proteins appear to be promising as prognostic markers in patients with ovarian adenocarcinomas.

c-FLIP expression in colorectal carcinomas: association with Fas/FasL expression and prognostic implications.

AIMS: Disruption of apoptotic cell death has been implicated in tumour aggressiveness in colonic carcinogenesis. The Fas-Fas ligand (FasL) system is involved in the execution of apoptosis induced by the immune system. c-FLIP protein constitutes an inhibitor of Fas and other (TRAIL) death receptor-mediated apoptosis. The aim of this study was to investigate the simultaneous expression of Fas, FasL and c-FLIP in relation to standard clinicopathological parameters and patients' outcome in colorectal cancer. METHODS AND RESULTS: Levels of Fas, FasL and c-FLIP protein expression were quantified immunohistochemically in paraffin-embedded tissues from 90 patients. Immunopositivity was detected for Fas, FasL and c-FLIP in 71%, 35.5% and 68.8% of cases, respectively. Concurrent expression of Fas/FasL was seen in 28 samples (31%), of which 24 (85.7%) also displayed c-FLIP positivity (P = 0.04). c-FLIP overexpression (> 10%) tended to prevail marginally in higher stage tumours (P = 0.09). Additionally, FasL and c-FLIP adversely affected survival on both univariate (P = 0.001 and P = 0.0024, respectively) and multivariate analysis [hazard ratio (HR) 3.491, P = 0.005 and HR 2.960, P = 0.036, respectively]. CONCLUSIONS: The frequent expression and coexpression of Fas, FasL and c-FLIP in colorectal carcinoma implicates c-FLIP as an inhibitor of the Fas-FasL-induced death pathway in these tumours. Moreover, c-FLIP conveys independent prognostic information in the presence of classical prognosticators.

Angiogenesis in Hodgkin's lymphoma: a morphometric approach in 286 patients with prognostic implications.

The significance of angiogenesis in Hodgkin's lymphoma (HL) is not well defined. The aim of this study was to evaluate various morphometric characteristics of microvessels in lymph node sections of 286 patients with HL at diagnosis and investigate their relationship with clinicopathologic parameters and prognosis. Microvessel density (MVD), total vascular area (TVA) and several size- and shape-related microvascular parameters were quantitated--after anti-CD34 immunohistochemical staining--in the region of most intense vascularization, using image analysis. An increase in microvessel caliber parameters (area, perimeter, major and minor axis length) and a decrease in MVD were noted with increasing stage. An inverse relationship was recorded between MVD and the number of involved sites (NIS) and LDH. In univariate analysis, overall disease-specific survival was adversely affected by MVD and TVA, whereas inferior failure-free survival (FFS) was associated with the presence of more flattened vessel sections. Multivariate analysis disclosed that the extent of angiogenesis (MVD/TVA), age and the NIS independently affected overall survival. Accordingly, FFS was independently linked to the shape of microvessels and albumin levels or the NIS. In conclusion, our data support the view that angiogenesis in HL provides independent prognostic information, requiring the concomitant evaluation of quantitative and qualitative aspects of microvascular network.

Hypoxia-inducible factor 1alpha/vascular endothelial growth factor axis in astrocytomas. Associations with microvessel morphometry, proliferation and prognosis.

Hypoxia-inducible factor (HIF)-1alpha is a transcription factor that promotes ischaemia-driven angiogenesis. The aim of this study was to determine the relation of HIF-1alpha to vascular endothelial growth factor (VEGF; an important angiogenic molecule in brain tumours), p53 expression, angiogenesis, proliferative potential and clinical outcome in a large series of diffuse astrocytomas. Expression of HIF-1alpha, VEGF, Ki-67 (a proliferation-associated marker) and p53 was determined immunohistochemically in 83 adult patients with supratentorial diffuse astrocytomas. Microvessels, highlighted by means of anti-CD34 immunohistochemistry, were enumerated with computer-assisted image analysis. Although HIF-1alpha and VEGF were expressed in the majority of cases, their levels increased significantly with increasing grade and proliferative potential. HIF-1alpha positively correlated with microvessel counts and VEGF with total vascular area and the presence of rounder vessel sections. There was a positive correlation of VEGF with p53 expression in astrocytomas and anaplastic astrocytomas. In univariate analysis, both VEGF and HIF-1alpha were associated with shortened survival in the entire cohort, but lost significance when grades II/III and grade IV were analysed separately. Multivariate analysis revealed that the combination of HIF-1alpha with grade was a significant prognostic indicator. HIF-1alpha expression may be used to refine the prognostic information provided by grade in patients with diffuse astrocytomas. Its adverse prognostic effect is most likely mediated by hypoxia, the driving force for HIF-1alpha accumulation.

Evaluation of the cyclin-dependent kinase inhibitor p21Cip1 in epithelial ovarian tumors of low malignant potential and adenocarcinomas.

OBJECTIVE: In view of the controversial information on the significance of the cyclin-dependent kinase inhibitor p21Cip1 in ovarian cancer, we conducted a retrospective investigation to clarify the relationships of this protein to proliferation rate, clinicopathological variables and prognosis of epithelial ovarian tumors. METHODS: Paraffin-embedded tissue from 43 ovarian tumors of low malignant potential (LMP) and 82 primary ovarian adenocarcinomas were stained immunohistochemically for p21Cip1, p53 protein and Ki-67 antigen (a marker of cell proliferation). RESULTS: p21Cip1 levels were significantly higher in LMP tumors (p<0.001) as well as in early stage adenocarcinomas (p=0.021) and those associated with minimal residual disease (p=0.008). However, no relationship existed between p21Cip1 expression and the proliferation rate of adenocarcinomas or LMP tumors. In the vast majority of LMP tumors p21Cip1 expression was not accompanied by p53 accumulation. This p21Cip1-positive/p53-negative phenotype prevailed in the early stage (p=0.026), lower grade (p=0.018) adenocarcinomas as well as in those left with minimal residual disease (p=0.059). In patients with lower grade adenocarcinomas, decreased p21Cip1 expression was adversely related to poor overall survival on its own (p=0.0500) and when combined with p53 protein overexpression (p=0.0323). In multivariate analysis, only the stage remained as the independent predictor of survival. CONCLUSIONS: Decreased p21Cip1 expression is related to several indicators of aggressiveness in ovarian adenocarcinomas and seems to be differentially regulated in LMP tumors and adenocarcinomas. On the contrary, deregulation of p21Cip1 expression does not seem to participate in the pathogenesis of LMP tumors. Furthermore, although p21Cip1 alone or combined with p53 is of prognostic significance in lower grade adenocarcinomas, it does not appear to add to the information gained from traditional prognosticators.

Mitosin, a novel marker of cell proliferation and early recurrence in intracranial meningiomas.

The expression of mitosin, a novel proliferation-associated molecule was evaluated immunohistochemically in a consecutive series of 47 patients with primary intracranial benign and atypical meningiomas. Mitosin expression was correlated with proliferation markers Ki-67 (MIB-1), proliferating cell nuclear antigen (PCNA), topoisomerase IIalpha (TopoIIalpha) and mitotic index, as well as with standard clinicopathological parameters and patient outcome. Seven tumors recurred (14.8%) following gross total resection, within a follow-up period ranging from 21 to 108 months (median 60 months). The higher proliferation indices were obtained with mitosin and PCNA and the lower ones with TopoIIalpha. Mitosin labeling index (LI) ranged from 0.1 to 57% (median 3%), with a significant overlapping of values between grades. A significant positive correlation was shown between mitosin LI on the one hand and Ki-67 LI (p < 0.001), or the mitotic index (p = 0.027) on the other. The incidence of recurrence was higher in cases with a mitosin LI higher than 3% (p = 0.048). Univariate analysis disclosed mitosin LI (p = 0.033) along with the mitotic index (p = 0.024) and tumor size (p = 0.028) as significant predictors of shortened recurrence-free survival. In multivariate analysis, the labeling indices of mitosin (p = 0.035) and Ki-67 (p = 0.032), along with tumor size, were shown to provide independent prognostic information, beyond that obtained by standard clinical and pathological parameters. However, as indicated by factor analysis, the prognostic information yielded by mitosin was superior to that provided by the remaining proliferation markers (p = 0.041). We conclude that mitosin immunohistochemical expression, although failing to discriminate between benign and atypical meningiomas, may be of use as a novel cell proliferation marker and as a predictor of tumor recurrence.

Clinicopathologic correlations of bone marrow angiogenesis in chronic myeloid leukemia: a morphometric study.

Various morphometric characteristics of microvessels, highlighted by means of anti-CD34 immunohistochemical staining, were evaluated in the bone marrow of 52 patients with chronic myeloid leukemia (CML) in chronic phase, in relation to several clinicopathologic parameters. Twenty control bone marrows and 15 cases of CML in blastic phase were also studied. Microvessel density (MVD), total vascular area (TVA) and several size- and shape-related parameters were quantitated in the region of most intense vascularization using image analysis. Overall, the group of chronic phase CML had higher MVD and size-related parameters and more branching microvessels than controls. Blastic phase was characterized by increased numbers of microvessels with a rounder shape and smaller caliber than chronic phase. A positive correlation emerged between marrow fibrosis and MVD as well as between white blood cell counts and rounder vessel sections. No relationship existed between microvascular parameters and Hasford or Sokal prognostic scores. In univariate analysis, overall and progression-free survival were adversely affected by MVD, size-related parameters, increased platelet count, age and spleen size. Multivariate analysis indicated that microvessel area was related to progression-free survival, whereas both MVD and area were significant prognosticators of overall survival, even when Hasford or Sokal scores are introduced into the model. Our data suggest that changes in angiogenic parameters may participate in the conversion of normal marrow to CML and ultimately to blastic transformation. More importantly, MVD and microvessel caliber are significant predictors of patient survival and progression.

Prognostic evaluation of the microvascular network in myelodysplastic syndromes.

Considering the recently stated suggestion of neovascularization being implicated in myelodysplastic syndromes (MDS) pathogenesis, we evaluated multiple morphometric microvascular characteristics in MDS, in relation to clinicopathologic factors and prognosis. Trephines from 50 newly diagnosed MDS patients were immunostained for factor VIII and compared to those from 20 controls, 10 chronic myelomonocytic leukemia (CMML) and 12 acute myeloid leukemia (AML) patients. Quantitation of microvessel density (MVD), area, total vascular area (TVA), major and minor axis length, perimeter, compactness, shape factor, Feret diameter, and the number of branching vessels was performed by image analysis. Overall, the MDS group had significantly higher MVD, TVA, minor axis and shape factor values and significantly lower compactness than the control group. AML was characterized by increased vascularity compared to MDS and CMML, as well as by the presence of flattened microvessels (lower values of shape factor). Hypercellular MDS showed higher MVD. RA/RARS displayed larger caliber vessels than RAEB, which explains the favorable prognostic effect of increased size-related parameters on progression and/or survival. Moreover, decreased compactness and MVD were independent predictors of longer progression-free survival. It is concluded that angiogenesis is involved in the conversion of normal marrow to MDS and ultimately to AML and that disease progression within MDS is accompanied by qualitative alterations of the microvascular network. Furthermore, size-related parameters affect survival, while shape-related parameters and MVD are more influential with regard to progression-free survival.