Cytodiagnosis of endometrial carcinoma and hyperplasia on imprint smears with additional immunocytochemistry using Ki-67 and p53 biomarkers.

OBJECTIVE: It is said to be difficult to interpret the different endometrial lesions by cytomorphology; however, evaluation of the microarchitecture of the cell clumps and application of immunocytochemistry can improve diagnostic accuracy. The aim of the present study was to evaluate cytolomorphological features and correlate them with the histological diagnosis of benign and malignant endometrial lesions, and to investigate certain immunocytochemical biomarkers to achieve a more accurate cytodiagnosis. METHODS: In the present study, we graded the cytomorphology on imprint smears of 35 low-grade endometrial endometrioid carcinomas compared with 23 cases of endometrium ranging from disordered proliferative to benign hyperplastic. Additionally, 10 cases of high-grade endometrial carcinoma and 11 cases of atrophic endometrium were evaluated. Ki-67 and p53 biomarkers were applied to the cytological smears. RESULTS: A total cytological score less than six, resulting from nuclear overlapping, nuclear/cytoplasmic ratio, the presence of a branched pattern, vesicular cytoplasm and loss of cohesiveness, distinguished all the cases of disordered proliferative and benign hyperplastic endometrium from low-grade endometrioid carcinomas of endometrium (P < 0.0001). The application of different cut-off values for Ki-67 and p53 helped differentiate certain endometrial lesions in our study. The integration of the immunocytochemical score of Ki-67 and p53 into the cytological score resulted in a final score that was also diagnostically useful. CONCLUSIONS: The results of the present study demonstrated that evaluation of certain cytological features along with specific immunocytochemical findings could improve the accuracy of endometrial cytodiagnosis but our findings need to be tested in a routine clinical situation, using pre-operative cytological samples, to ascertain whether the diagnostic criteria are reproducible.

c-MYC and h-TERT co-expression in colon adenocarcinoma: a tissue microarray digitized image analysis.

PURPOSE: c-MYC oncogene is frequently deregulated by amplification in colon adenocarcinoma. c-MYC also activates telomerase by inducing expression of its catalytic subunit (h-TERT). Furthermore, telomerase activation plays a crucial role in tumorigenesis by sustaining cellular immortality. Our aim was to evaluate the significance of c-MYC and h-TERT co-expression in colon adenocarcinoma. METHODS: Sixty paraffin embedded primary colon adenocarcinomas were cored at 1.5 mm diameter and transferred to one microarray block. Immunohistochemistry was performed using anti-h-TERT, and c - MYC antibodies. A quantitative digitized macro was performed to evaluate their expression. RESULTS: c-MYC and h-TERT overexpression was observed in 27 (45%) and 28 (46.6%) cases, respectively. Co-over expression of those genes was observed in 17 (28.3%) cases and found to be statistically significant (p=0.001). The results also showed a strong association between c-MYC and grade of differentiation of the examined neoplasms (p=0.0217rpar;. CONCLUSION: Simultaneous c-MYC and h-TERT deregulation is a relatively frequent genetic event in colon adenocarcinoma. Because c-MYC overexpression is correlated with progressive disease - due to colon adenocarcinoma dedifferentiation - inhibition of its activity combined with h-TERT regulated expression is a new target for novel therapeutic regimens.

Estimation of maspin's subcellular localization in invasive ductal breast cancer via light microscopy and computerized image analysis: a comparative study.

PURPOSE: Contradictory results have been reported concerning the role of maspin and its cellular distribution in breast cancer. The purpose of this study was to examine the subcellular localization (nuclear-cytoplasmic) of maspin in breast cancer and to compare the evaluation of maspin immunostaining via light microscopy (LM) to the estimation via computerized image analysis (CIA) system. We also examined correlations between maspin expression and several clinicopathological parameters. METHODS: The sample consisted of 48 primary invasive ductal carcinomas (IDC) of the breast. Maspin immunostaining was quantified and graded via LM by two pathologists, separately in the nuclear and cytoplasmic compartments. Total maspin expression was also estimated via CIA system. Univariate non-parametric statistics and stepwise multivariate ordinal logistic regression were performed. RESULTS: Both maspin components (nuclear and cytoplasmic) were closely associated with each other (p<0.001). Total maspin score was positively and closely associated with nuclear maspin (p<0.001) and cytoplasmic maspin (p<0.001). Total maspin , nuclear maspin and cytoplasmic maspin did not correlate significantly with either age, grade, T, N and M status, stage, micro vessel density (MVD) (CD34), ki-67, p53, estrogen receptor (ER) and HER-2 status, or with any of the 4 groups of the molecular classification. The only factor that showed a borderline inverse correlation with nuclear maspin (p=0.059) was progesterone receptors (PR) positivity. CONCLUSION: The cytoplasmic and nuclear fractions of maspin seem to be closely interwoven. Evidently, both mutually intertwined counterparts were independently reflected upon the total maspin levels measured by CIA. Future studies should ideally encompass all three approaches (nuclear, cytoplasmic, total) adopted herein.

Mathematical modeling for Delta and Omicron variant of SARS-CoV-2 transmission dynamics in Greece.

A compartmental, epidemiological, mathematical model was developed in order to analyze the transmission dynamics of Delta and Omicron variant, of SARS-CoV-2, in Greece. The model was parameterized twice during the 4th and 5th wave of the pandemic. The 4th wave refers to the period during which the Delta variant was dominant (approximately July to December of 2021) and the 5th wave to the period during which the Omicron variant was dominant (approximately January to May of 2022), in accordance with the official data from the National Public Health Organization (NPHO). Fitting methods were applied to evaluate important parameters in connection with the transmission of the variants, as well as the social behavior of population during these periods of interest. Mathematical models revealed higher numbers of contagiousness and cases of asymptomatic disease during the Omicron variant period, but a decreased rate of hospitalization compared to the Delta period. Also, parameters related to the behavior of the population in Greece were also assessed. More specifically, the use of protective masks and the abidance of social distancing measures. Simulations revealed that over 5,000 deaths could have been avoided, if mask usage and social distancing were 20% more efficient, during the short period of the Delta and Omicron outbreak. Furthermore, the spread of the variants was assessed using viral load data. The data were recorded from PCR tests at 417 Army Equity Fund Hospital (NIMTS), in Athens and the Ct values from 746 patients with COVID-19 were processed, to explain transmission phenomena and disease severity in patients. The period when the Delta variant prevailed in the country, the average Ct value was calculated as 25.19 (range: 12.32-39.29), whereas during the period when the Omicron variant prevailed, the average Ct value was calculated as 28 (range: 14.41-39.36). In conclusion, our experimental study showed that the higher viral load, which is related to the Delta variant, may interpret the severity of the disease. However, no correlation was confirmed regarding contagiousness phenomena. The results of the model, Ct analysis and official data from NPHO are consistent.

PTEN expression in non small cell lung carcinoma based on digitized image analysis.

PURPOSE: HER2 depended signalling pathway is dereg-ulated in a subset of non small cell lung carcinoma (NSCLC). The tumor suppressor gene PTEN (10q21) regulates the HER2/PI3K/Akt signalling pathway. Our aim was to evaluate PTEN protein expression in NSCLC based on a quantitative analysis method correlating also the results with clinicopathological parameters. METHODS: Protein expression was determined by immunohistochemistry (IHC) in 61 paraffin-embedded cases of patients with NSCLC. Digital image analysis (staining intensity levels) was performed in the corresponding immunostained slides. RESULTS: Loss of PTEN expression was observed in 24 (39.34%) cases, low expression in 29 (47.54%) and overexpression in 8 (13.12%) cases. Multivariate analysis determined that PTEN overexpression was associated with lower risk to develop metastases (p=0.05). CONCLUSION: PTEN deregulation is a relatively frequent genetic event in NSCLC, associated with progressive metastatic process in those patients. Because of binding to the ErbB2 receptor, trastuzumab stabilizes and activates PTEN gene, and loss of its expression negatively affects the response rates in such patients.

Increased vein wall apoptosis in varicose vein disease is related to venous hypertension.

OBJECTIVES: The study aimed to evaluate a wide range of apoptotic markers in the vein wall of patients with superficial chronic venous disease (SCVD) compared with normal veins. DESIGN: This was an observational study. METHODS: Vein specimens were obtained from 19 patients suffering from SCVD. From each patient, a specimen of the proximal part of the great saphenous vein (GSV), a specimen of the distal part of the vein and a specimen of a varicose tributary were obtained. Immunohistochemical analysis was used to localise the expression of BAX, p53, Caspase 3, BCL-2, BCL-6, BCL-xs, BCL-xl and Ki-67. Vein specimens from 10 healthy GSVs were used as controls. RESULTS: Saphenous vein specimens from patients with SCVD showed increased BAX, Caspase 3, BCL-xl and BCL-xs (p < 0.01 for all) and Ki-67 (p = 0.02) compared with healthy GSVs. In the venous disease group, GSV specimens from the distal ankle area showed increased BAX (p < 0.01) and BCL-xs (p = 0.031) compared with varicose tributaries specimens, which subsequently showed increased BAX (p = 0.044), Caspase 3 (p = 0.028) and BCL-xs (p = 0.037) compared with specimens from the proximal GSV. In addition, in the venous disease group, specimens from distal GSV showed increased BAX (p < 0.01), Caspase 3 (p = 0.019) and BCL-xs (p = 0.014) compared with the proximal GSV. CONCLUSION: Varicose veins exhibit increased apoptotic activity, by means of increased BAX, Caspase 3, BCL-xl and BCL-xs, compared with normal veins. Patients with varicose vein disease show increased apoptosis in the distal saphenous trunk compared with the proximal saphenous trunk, suggesting an association between chronic venous hypertension and apoptosis.

The significance of DNA image cytometry and Edmondson-Steiner grading on prognosis after curative resection of hepatocellular carcinoma.

PURPOSE: Hepatocellular carcinoma (HCC) is the commonest primary cancer of the liver. Hepatic resection remains the main curative option, although the incidence of disease recurrence in the remaining hepatic parenchyma is high and accounts for the leading cause of death post resection. For this reason, the need to identify prognostic factors which may determine treatment response and survival is of paramount importance. In this study we assessed whether DNA image cytometry and Edmondson-Steiner grading could be used as prognostic factors in a cohort of patients with HCC undergoing radical hepatic resection. METHODS: Forty-four patients with HCC who underwent radical resection were retrospectively analyzed. Histological grading according to Edmondson and Steiner and DNA ploidy using DNA image cytometry, were the two parameters analyzed. Pearson's x(2) or Fisher's exact tests were used to test for any associations between categorical variables. Univariate semi-parametric Cox proportional hazard regression models were used to assess the effect of explanatory variables on death. All reported p values were based on two-sided tests and compared to a significance level of 0.05. RESULTS: In univariate Cox regression analysis, adverse survival outcome was strongly associated with high DNA score and advanced histological grading. Patients with ploidy score >2.2 had 3.95 times higher probability of death, as compared to those with ploidy score ≤ 2.2. Edmondson-Steiner grades III and IV were also associated with 20.49 and 34.47 higher probability of death respectively as compared to grade I. CONCLUSION: Our results validate the prognostic significance of DNA image cytometry and Edmondson-Steiner grading following curative resection of HCC.

Deregulation of EGFR/VEGF/HIF-1a signaling pathway in colon adenocarcinoma based on tissue microarrays analysis.

PURPOSE: Overexpression of epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF) in colon adenocarcinoma (CA) is a frequent event, whereas specific deregulation mechanisms in the corresponding signaling pathways remain under investigation. Our aim was to co-evaluate their expression correlated to the hypoxia inducible factor 1alpha (HIF-1a), which activates the transcription of VEGF gene. METHODS: 60 paraffin-embedded primary CAs were cored at 1.5 mm diameter and transferred to the microarray block. Immunohistochemistry (IHC) was performed using anti-EGFR, -VEGF, and -HIF 1a monoclonal antibodies. Concerning EGFR, quantitative evaluation was based on a semi-automated analysis system. Chromogenic in situ hybridization (CISH) was performed using EGFR gene and chromosome 7 centromeric probes. RESULTS: Protein overexpression was observed in 13/60 (21.6%), 45/60 (75%) and 7/60 (11.6%) cases regarding EGFR, VEGF, and HIF 1a, respectively. CISH analysis detected 4/60 (6.6%) EGFR gene amplified cases, whereas chromosome 7 aneuploidy was identified in 11/60 (18.3%) cases. Significant associations raised correlating stage to chromosome 7 (p=0.024), HIF 1a expression to tumor anatomical location (p=0.019) and also VEGF to HIF 1a expression (p=0.001), whereas EGFR expression was not associated to EGFR gene copies. CONCLUSION: According to our results, chromosome 7 instability is correlated to advanced disease, whereas a significant subset of CAs demonstrates an alternative, non- HIF 1a depended mechanism of VEGF overexpression. Furthermore, EGFR protein overexpression does not predict a specific gene deregulation mechanism.

Nuclear fractal dimension as a prognostic factor in oral squamous cell carcinoma.

Strong theoretical reasons exist for using fractal geometry in measurements of natural objects, including most objects studied in pathology. Indeed, fractal dimension provides a more precise and theoretically more appropriate approximation of their structure properties and especially their shape complexity. The aim of our study was to evaluate the nuclear fractal dimension (FD) in tissue specimens from patients with oral cavity carcinomas in order to assess its potential value as prognostic factor. Relationships between FD and other factors including clinicopathologic characteristics were also investigated. Histological sections from 48 oral squamous cell carcinomas as well as from 17 non-malignant mucosa specimens were stained with Hematoxylin-Eosin for pathological examination and with Feulgen for nuclear complexity evaluation. The sections were evaluated by image analysis using fractal analysis software to quantify nuclear FD by the box-counting method. Carcinomas presented higher mean values of FD compared to normal mucosa. Well differentiated neoplasms had lower FD values than poorly differentiated ones. FD was significantly correlated with the nuclear size. Patients with FD lower than the median value of the sample had statistically significant higher survival rates. Within the sample of patients studied, FD was proved to be an independent prognostic factor of survival in oral cancer patients. In addition this study provides evidence that there are several statistically significant correlations between FD and other morphometric characteristics or clinicopathologic factors in oral squamous cell carcinomas.

Microvessel density (MVD) and cyclooxygenase-2 (COX-2)/ beta-catenin interaction are associated with relapse in patients with transitional carcinoma receiving adjuvant chemotherapy with paclitaxel/carboplatin: a hellenic cooperative oncology group (HECOG) study.

BACKGROUND: Cycloxygenase (COX)-2 has been associated with proliferation, apoptosis and angiogenesis in urothelial cancer. The prognostic significance of COX-2 in patients who received adjuvant chemotherapy for urothelial cancer was examined. PATIENTS AND METHODS: Expression of COX-2, p53, ki67, beta-catenin, vascular endothelial growth factor (VEGF) and microvessel density (MVD) were studied retrospectively in 59 patients with urothelial cancer (pT3, pT4, N+) who had undergone surgery. The patients had subsequently received adjuvant chemotherapy. RESULTS: Thirty-eight out of 59 cases (64%) were positive for COX-2. COX-2 was not associated either with progression-free survival (PFS) or overall survival (OS). MVD levels > or =47 were associated with longer median PFS compared with lower levels (not reached vs. 13 months [95% CI: 8-18], p=0.048). The median PFS for patients with beta-catenin nuclear accumulation and COX-2 expression was 6 months (95% CI: 4-7) compared with 19 months (95% CI: 14-23) for neither or only one of these factors (p=0.018). CONCLUSION: MVD may be a useful indicator of relapse in high-risk urothelial cancer treated with adjuvant chemotherapy.

The relationship of endothelin-1 and inducible nitric oxide synthase at several stages of the anastomotic healing process.

AIM: The aim of this study was to investigate the interaction between the endothelin-1 (ET-1) and inducible NO synthase (iNOS) in anastomotic healing. METHODS: The expression of ET-1 and iNOS were investigated by immunohistochemistry in a rat end-to-end arterial anastomotic model. The aorta of 50 male Wistar rats was exposed, then transversely divided and re-anastomosed. The animals were sacrificed immediately after the operation (group A, control group), after 24 h (group B), on 7th postoperative day (group C), on 30th day (group D) and at 6 months (group E). Intima and media thickness and their ratio of the anastomotic segments in each group were calculated from computer digitized images of the individual sections. ET-1 and iNOS expression were measured on a semiquantitative scale ranging from 0 to 3. RESULTS: ET-1 was expressed from endothelial and smooth muscle cells (SMCs), while iNOs was expressed from SMCs and inflammatory cells. An intense expression of ET-1 was demonstrated mainly at 1 week and to a lesser degree at 1 month. Yet, at 6 months this expression was significantly weakened (P<0.001). In contrast, an intense iNOS expression was identified at 24 h, substantially regressing at statistical significant lower levels after 1 week (P<0.001). Bivariate correlation test showed a positive correlation between ET-1 and iNOS expression. CONCLUSION: ET-1 appears to play an important role in intimal thickening during anastomotic healing, especially in the late period of the process. Although there is a positive correlation between ET-1 and iNOS production, the activity of the latter is relatively limited after the first postanastomosis week.

Diabetes may increase risk for oral cancer through the insulin receptor substrate-1 and focal adhesion kinase pathway.

In light of recent epidemiological studies that associate diabetes mellitus with increased risk for oral cancer, we investigated in diabetic (type I) and normal rats with induced oral squamous cell carcinoma whether the molecular basis for that putative association involves insulin receptor substrate-1 (IRS-1) and focal adhesion kinase (FAK). Fourteen diabetic and 12 normal rats developed cancer after 4-nitroquinoline-N-oxide treatment, while six diabetic and six normal animals were used as controls. Oral sections were studied using monoclonal antibodies against IRS-1 and FAK proteins. Expression of IRS-1 was significantly higher in diabetic than normal rats, but it decreased in diabetic animals with tumor, especially in more advanced stages. FAK expression was significantly higher in rats with cancer in comparison to the ones without it, regardless the diabetes status. These data suggest that the IRS-1/FAK pathway is altered by diabetes resulting in reduced cell adhesion and possibly increasing risk for oral cancer.

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.

Effect of green tea on angiogenesis and severity of atherosclerosis in cholesterol-fed rabbit.

BACKGROUND AND AIMS: Since the development of the atherosclerotic plaque requires the growth of new microvessels in the plaque itself (vasa vasorum), we postulated that green tea may exert an anti-atherogenic effect. METHODS AND RESULTS: Thirteen male New Zealand white rabbits were studied for 17 weeks. All rabbits were fed an hypecholesterolemic diet. After 2 weeks of adaptation rabbits were randomly assigned into two groups. Animals in Group A were fed the hypercholesterolemic diet and received plain tap water ad libitum. Animals in Group B were fed with the same diet and furthermore received 2.5% (g/g) green tea for 17 weeks. CONCLUSION: According to our results the atherosclerotic lesions were more severe in Group B than in Group A specimens. Also, the number of VEGF positively stained foam cells and smooth muscle cells of Group B were significantly greater than in Group A. About 30% less plaque was found in Group A than in the control group (Group B). So, our study showed that the consumption of green tea leads to a reduction of atherosclerosis as well as a significant decrease of VEGF expression in the atherosclerotic plaque of rabbit aorta. The hypothesis that probably green tea may produce its anti-atherogenetic effect through an anti-angiogenetic mechanism needs more investigation.

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.

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.

Breast cancer following curative chemotherapy for non-Hodgkin's lymphoma and the effect of drug resistance proteins to the final outcome. A retrospective study.

PURPOSE: To investigate the overall survival (OS) of patients developing breast cancer (BC) after curative chemotherapy for non-Hodgkin's lymphoma (NHL) and to evaluate the possible effect on the patients' outcome of the expression of drug resistance-related proteins (P-glycoprotein-Pgp, multidrug resistance-associated protein-MRP, and multidrug resistance-related vault lung resistance protein-LRP) in BC issue. STUDY GROUP: 25 female patients (median age 60 years, range 37-70) who developed BC after chemotherapy for high/intermediate grade B-cell NHL, treated with CHOP and achieving complete remission (CR). This group was further subdivided in subgroups A and B, according to the time interval between NHL and BC development ( 24 months, respectively). A matched-pair group of de novo BC patients formed the control group. BC tissue was immuno-histochemically stained for Pgp, MRP and LRP. RESULTS: The median interval between NHL diagnosis and BC development was 26 months (range 9-49). In both groups 14 patients had tumor grade II; 16 were negative for steroid receptors; 17 overexpressed c-erbB-2; 14 were stage IIIA/B, and 11 stage IV. CMF or CNF (mitoxantrone instead of doxorubicin) were given for BC. Early progression was noticed in all study group patients for which second-line chemotherapy was instituted. There was a better response for stage IV patients in the control versus the study group (p=0.07). More prolonged OS was demonstrate for patients with stage III in the control group (median 51 months) and in subgroup B (median 47 months) than in subgroup A (median 16 months; p=0.00012), as well as for patients with advanced disease (p=0.0045). Development of BC < 24 months after NHL resulted in reduced OS (p=0.017). No difference was noticed in the expression of drug resistance proteins between the study and control group or between subgroups A and B. CONCLUSION: BC developing shortly after a CR to NHL is an aggressive disease variant with minimal potential for response to conventional chemotherapy. Analysis of Pgp, MRP and LRP failed to demonstrate significant difference between the study and control group, although indications exist that drug resistance mechanisms might be part of the aggressive disease phenotype, contributing to the poor outcome.

Immunohistochemical detection of cell cycle regulators, Fhit protein and apoptotic cells in parathyroid lesions.

OBJECTIVE: The pathological distinction between parathyroid neoplasms and hyperplasias remains difficult. Changes in cell cycle control may lead to clonal proliferation and precede tumorigenesis. The parathyroid adenoma 1 oncogene, subsequently identified as the gene encoding cyclin D1, has been shown to be important to parathyroid tumour development. In addition to cell proliferation, the mechanisms of parathyroid cell turnover include apoptosis. The tumour-suppressor activity of the fragile histidine triad gene (FHIT) is linked to its proapoptotic function and cell cycle control. We attempted to evaluate the cellular proliferative kinetics and apoptotic function of the parathyroid glands in patients with non-familial hyperparathyroidism (HPT). DESIGN: TIssue specimens were taken from 40 patients with primary HPT (17 adenomas, two carcinomas and 21 primary hyperplasias) and from 30 patients with secondary HPT. Normal glands served as controls. METHODS: In a standard immunohistochemical procedure, monoclonal antibodies to Ki-67 antigen and single-stranded DNA were applied to detect cycling and apoptotic cells respectively; polyclonal antibodies to cyclin D1 and Fhit protein were used. Immunostaining was estimated by image analysis and statistical analysis was subsequently performed. RESULTS: Significantly higher proliferative and apoptotic indexes were detected in the diseased glands in comparison with normal controls. In neoplastic and secondarily hyperplastic glands, apoptotic indexes were higher than in primarily hyperplastic glands; the difference between neoplastic and primarily hyperplastic glands was statistically significant (P=0.034). Cyclin D1 was overexpressed in a considerable proportion of tumours (68.4%). A reduction of Fhit protein immunoreactivity was selectively noticed in carcinomas. CONCLUSIONS: In primary hyperplasia, the remarkable proliferation of parathyroid glands may be due to the reduction of the apoptotic process. FHIT gene abnormalities are worthy of investigation in parathyroid carcinogenesis.

Markers of drug resistance in relapsing colon cancer.

PURPOSE: 5-Fluorouracil failure and drug resistance, which often occurs during chemotherapy, is still a great obstacle to the success of human colon cancer treatment. Thus, the comparative study of markers of drug resistance in cancer cells before and after chemotherapy may be extremely helpful in the selection of the appropriate chemotherapeutic drug in colon cancer patients who fail adjuvant treatment with 5-fluorouracil. In the present study we examined the differential expression of three multidrug resistance-related proteins (i.e., P-glycoprotein, MRP, and LRP) and of topoisomerase IIalpha in a series of 20 primary colon carcinomas and their recurrences. METHODS: All markers were determined at tissue level by three-step immunohistochemistry using appropriate monoclonal antibodies, and the markers' immunopositivity was quantified by image analysis. In addition, Feulgen stain was used for the assessment of nuclear DNA content of malignant cells at their primary location. RESULTS: Some degree of aneuploidy was detected in all primary carcinomas. The immunoexpression of the three multidrug resistance-related proteins did not change significantly, either qualitatively (positivity vs negativity) or quantitatively, after chemotherapy. On the contrary, the percentages of topoisomerase IIalpha-positive malignant cells were significantly increased in the tumour recurrences by comparison to their primary locations ( P=0.011). CONCLUSIONS: According to our results, increased topoisomerase IIalpha immunohistochemical expression appears to be part of the malignant cells' phenotype in recurrent colon cancers. Therapeutic options after failure of 5-fluorouracil-based treatment could therefore include appropriate topoisomerase IIalpha-targeted drugs.

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.