Class III β-tubulin expression in advanced-stage serous ovarian carcinoma effusions is associated with poor survival and primary chemoresistance

The objective of this study was to analyze the clinical role of nestin, a stem cell marker, and class III β-tubulin in advanced-stage serous ovarian carcinoma. Nestin and class III β-tubulin protein expression were investigated in 217 effusions using immunohistochemistry. Results were analyzed for association with clinicopathologic parameters including chemotherapy response and survival. Class III β-tubulin and nestin were expressed in tumor cells in 98.6% and 95.6% of specimens, respectively. Staining extent was comparable in prechemotherapy and postchemotherapy effusions. No association was found with patient age, histologic grade, International Federation of Gynecology and Obstetrics stage, primary surgery versus secondary debulking, or residual disease volume. High class III β-tubulin expression in prechemotherapy effusions was significantly associated with primary chemoresistance (progression-free survival <6 months; P = .036) and with a trend for less favorable response to first-line treatment (P = .054). In univariate survival analysis, high class III β-tubulin expression in prechemotherapy effusions was significantly associated with poor overall survival (P = .021), with a trend for poor progression-free survival (P = .067). These associations did not have independent prognostic value in Cox multivariate analysis. Nestin expression was unrelated to chemoresistance or survival. Both class III β-tubulin and nestin are frequently expressed in serous ovarian carcinoma cells in effusions. Nestin does not provide predictive or prognostic data in this patient group, whereas class III β-tubulin expression in prechemotherapy effusions is associated with poor chemoresponse and shorter survival, suggesting that it may be a therapeutic target in ovarian cancer.     

Different clinical roles for p21-activated kinase-1 in primary and recurrent ovarian carcinoma

We recently found amplification of chromosome 11q13.5 in high-grade ovarian serous carcinoma. In the present study, we analyzed the protein expression and clinical significance of p21-activated kinase-1, one of the genes amplified at this site. Formalin-fixed paraffin-embedded sections from 186 effusions (152 peritoneal, 34 pleural) were immunostained. p21-Activated kinase-1 expression in tumor cells was analyzed for possible association with clinicopathologic parameters and survival. The association between protein expression of p21-activated kinase-1 and Rsf-1, a chromatin remodeling protein whose gene colocalizes with p21-activated kinase-1, was additionally studied. p21-Activated kinase-1 protein expression was found in carcinoma cells in 158 (85%) of 186 effusions. Of these, 62 (39%) stained weakly and 96 (61%) strongly. p21-Activated kinase-1 was coexpressed with Rsf-1 (P = .006). Specimens from patients diagnosed with International Federation of Gynecology and Obstetrics stage IV disease had higher staining intensity compared with stage III tumors (P = .014). Univariate survival analysis for patients with primary diagnosis prechemotherapy effusions demonstrated a significant association between higher p21-activated kinase-1 staining extent and longer overall survival (P = .024). In addition, higher staining extent (P = .015) and intensity (P = .013) correlated with better progression-free survival. In contrast, higher p21-activated kinase-1 staining extent correlated with poor overall survival in disease recurrence postchemotherapy effusions (P = .044). In Cox analysis, higher p21-activated kinase-1 staining extent independently correlated with longer progression-free survival (P = .016) and shorter overall survival (.049) in primary diagnosis and disease recurrence effusions, respectively. p21-Activated kinase-1 is frequently expressed in ovarian carcinoma cells in effusions and is associated with opposite prognostic role in primary and recurrent disease. This suggests altered cellular function for this kinase along disease progression, possibly chemotherapy mediated.     

HMGA2 protein expression in ovarian serous carcinoma effusions, primary tumors, and solid metastases

The objective of this study was to analyze the expression and clinical role of the high mobility group AT hook (HMGA) protein in advanced-stage serous ovarian carcinoma. HMGA2 protein expression was investigated in 199 effusions and in 50 patient-matched primary tumors and solid metastases using immunohistochemistry. Results were analyzed for association with clinicopathologic parameters, including chemotherapy response, and survival. HMGA2 was expressed in tumor cells in 94.5 %, 96 %, and 90 % of specimens, respectively. There was no difference in HMGA2 expression between patient-matched samples from different anatomic sites (p > 0.3). HMGA2 expression in chemo-na?ve samples was significantly higher in older patients (p = 0.006, p = 0.01, and p = 0.005 for effusions, primary tumors, and solid metastases, respectively). No association was found with residual disease volume. Furthermore, HMGA2 expression was not associated with FIGO stage (p > 0.2), except in chemo-na?ve effusions (n = 106, p = 0.016). There was no difference in HMGA2 expression between chemo-na?ve samples and samples obtained post-chemotherapy in effusions (p = 0.2) or primary tumors (p = 0.1). However, solid metastases obtained after chemotherapy exposure had higher HMGA2 expression compared with chemo-na?ve samples (p = 0.032). HMGA2 expression was unrelated to chemotherapy response or survival. However, it was directly related to protein expression of the previously studied cancer stem cell marker Nestin (p = 0.01) and the gap junction protein claudin-7 (p = 0.02) and inversely related to the mRNA level of the E-cadherin repressor SIP1 (p = 0.02). This study provides evidence that HMGA2 is universally expressed in advanced-stage ovarian serous carcinoma irrespective of anatomic site, suggesting that HMGA2 may have a clinical role as therapeutic target.     

Claudin upregulation in ovarian carcinoma effusions is associated with poor survival

Claudins are tight junction proteins that are highly expressed in ovarian carcinoma (OC). The objective of this study was to analyze the anatomic site-related expression and clinical role of claudins in OC. Effusions (n = 218), corresponding primary tumors (n = 81), and solid metastases (n = 164) (total = 463 tumors) were immunostained for claudin-1, claudin-3, claudin-4, and claudin-7. Results were analyzed for association with anatomic site, clinicopathologic parameters, and survival. All 4 claudins were expressed in >85% of tumors at all anatomic sites. However, staining extent of all except claudin-4 was significantly higher in effusions compared with both primary carcinomas and solid metastases (P < .001). In univariate survival analysis of the entire cohort, higher claudin-3 (P = .038) and claudin-7 (P = .035) expression in effusions correlated with shorter overall survival (OS), with similar results for claudin-7 in analysis of progression-free survival (P = .026). In separate analysis for patients with prechemotherapy effusions, higher claudin-7 expression correlated with shorter OS (P = .045). For patients with postchemotherapy effusions, higher claudin-1 (P = .018) and claudin-3 (P = .009) expression correlated with shorter OS. In multivariate survival analysis of the entire cohort, claudin-7 expression was an independent predictor of poor progression-free survival (P = .017). Claudin-3 independently predicted poor OS for patients with postchemotherapy effusions (P = .012). With the exception of claudin-4, claudins are upregulated in OC effusions compared with solid tumors, in agreement with our previous data for cadherins and integrins in this cancer type, suggesting a prosurvival role for these surface molecules. Claudin-3 and claudin-7 expression in effusions independently predicts poor survival in OC.     

BUB1 mRNA is significantly co-expressed with AURKA and AURKB mRNA in advanced-stage ovarian serous carcinoma

The objective of this study was to investigate the expression and clinical role of the spindle checkpoint kinase budding uninhibited by benzimidazole 1 (Bub1) in primary and metastatic advanced-stage ovarian serous carcinoma. BUB1 mRNA expression was analyzed in 178 tumors (88 effusions, 38 primary carcinomas, and 52 solid metastases) from 144 patients with advanced-stage disease using quantitative real-time polymerase chain reaction (PCR). Bub1 protein expression by Western blotting was studied in 63 carcinomas (30 effusions and 33 solid lesions). BUB1 mRNA expression at different anatomic sites was studied for association with clinicopathologic parameters, including chemotherapy resistance and survival. BUB1 mRNA was universally expressed in serous carcinomas, irrespective of anatomic site. BUB1 mRNA levels were uniformly low in six ovarian surface epithelium specimens analyzed for comparative purposes. Bub1 protein was expressed in 22/30 effusions and 28/33 solid lesions. BUB1 mRNA expression was significantly higher in chemo-naïve primary carcinomas and solid metastases compared to specimens obtained following neoadjuvant chemotherapy (p?<?0.001) and was unrelated to chemotherapy exposure in effusions nor to chemoresponse or survival at any anatomic site. BUB1 mRNA levels in both effusions and solid lesions were strongly related to the mRNA levels of AURKA and AURKB previously studied in this cohort (p?<?0.001 for both). Bub1 is widely expressed in primary and metastatic OC, suggesting a biological role in this cancer. BUB1 mRNA levels are lower following chemotherapy exposure in solid lesions, though its presence is unrelated to clinical behavior including response to chemotherapy and survival. BUB1 is co-expressed with AURKA and AURKB suggesting biological relationship between these spindle cell components.     

Heat shock protein 90 is a putative therapeutic target in patients with recurrent advanced-stage ovarian carcinoma with serous effusions

Heat shock protein 90 (HSP90) has anti-apoptotic properties exerted through its cytoprotective function of chaperone activity and increased expression in response to stress. The present study analyzed the clinical role of HSP90 in effusions from patients with advanced-stage ovarian carcinoma. HSP90 protein expression was investigated in 265 effusions using immunohistochemistry. Results were analyzed for association with clinicopathologic parameters, including chemotherapy response and survival. The correlation between HSP90 and a panel of previously-studied antiapoptotic proteins was additionally investigated. HSP90 was expressed in the cytoplasm and nucleus of tumor cells in 97% and 18% of specimens, respectively. Nuclear HSP90 expression was significantly higher in post-chemotherapy compared to pre-chemotherapy effusions (P = .005), significantly related to previous treatment with both platinol (P = .024) and paclitaxel (P = .007). Cytoplasmic HSP90 expression was significantly higher in effusions from patients with complete compared to incomplete/no response after second-line chemotherapy (P = .016). No association was found between HSP90 expression and other clinicopathologic parameters or survival. Cytoplasmic HSP90 expression was significantly associated with that of Bcl-2 in pre-chemotherapy effusions (P = .04), and marginally associated with cytoplasmic Survivin expression in post-chemotherapy effusions (P = .05). HSP90 is upregulated along tumor progression from primary diagnosis to recurrent effusion. HSP90 does not provide prognostic data in patients with advanced ovarian carcinoma effusions. However, HSP90 may be of predictive value as to who will benefit from treatment with HSP90 inhibitors to potentiate the effectiveness of platinol and paclitaxel in patients with recurrent advanced ovarian carcinoma effusions. We propose HSP90 as a potential therapeutic target in this patient group.     

软组织平滑肌肉瘤的流式细胞术研究

目的　研究软组织平滑肌肉瘤 (LMS)中DNA倍体与S期分数 (SPF)及二者与临床病理的联系。方法　应用流式细胞术 (FCM )检测 33例LMS的DNA指数及SPF。结果　LMS中异倍体率为 39 4 % ,平均SPF值为 12 2 36。低分化LMS组的异倍体率 (70 0 % )高于高分化LMS组 (2 6 1% ) (P <0 0 5 ) ;异倍体LMS组的SPF均值 (15 5 92 )高于二倍体组 (10 0 5 5 ) (P<0 0 5 ) ;瘤体最大径 >6cm组的SPF均值 (13 6 36 )明显高于 <6cm组 (9 4 36 ) (P <0 0 5 )。结论　LMS的SPF与DNA倍体和体积均呈现出相关关系。LMS的DNA倍体与肿瘤的分化程度具有相关性     

Expression and clinical role of the bric-a-brac tramtrack broad complex/poxvirus and zinc protein NAC-1 in ovarian carcinoma effusions

We recently identified NAC-1, member of the bric-a-brac tramtrack broad complex/poxvirus and zinc domain family, as an overexpressed gene in ovarian serous carcinoma and found more frequent NAC-1 protein expression in recurrent compared to primary tumors. In the present study, we assessed the clinical significance of NAC-1 expression in ovarian carcinoma effusions. Formalin-fixed, paraffin-embedded sections from 176 effusions (137 peritoneal, 39 pleural) and 197 corresponding solid tumors (69 primary tumors, 128 solid metastases) were analyzed for NAC-1 expression using immunohistochemistry. Staining intensity and extent results were analyzed for possible association with clinicopathologic parameters and survival. Nuclear NAC-1 immunoreactivity was found in carcinoma cells in 98% of (173/176) effusions, 94% (65/69) of primary tumors, and 95% (121/128) of metastases. Staining intensity and extent were significantly higher in effusions compared with matched solid tumors (P = .002 for intensity, P = .003 for extent compared with primary tumors; P < .001 for both intensity and extent compared with metastases). Furthermore, NAC-1 expression intensity was significantly higher in specimens obtained after the administration of chemotherapy (P = .002) and correlated with shorter progression-free survival (PFS) in analysis of 62 patients with post-chemotherapy effusions (P = .039). International Federation of Gynecology and Obstetrics stage (IV versus III) was the only clinical parameter associated with PFS in this group (P = .004). In Cox analysis, only the International Federation of Gynecology and Obstetrics stage was an independent predictor of shorter PFS (P = .009). In conclusion, NAC-1 expression is higher in ovarian carcinoma cells in effusions compared with their solid tumor counterparts. NAC-1 is up-regulated in tumor cells after chemotherapy, suggesting a role for this protein in tumor progression and in the development of chemotherapy resistance in ovarian cancer.     

MUC4 is upregulated in ovarian carcinoma effusions and differentiates carcinoma cells from mesothelial cells

Using gene expression arrays, we recently showed that MUC4 expression is significantly higher in ovarian/primary peritoneal serous carcinoma (OC/PPC) compared to diffuse peritoneal malignant mesothelioma (DMPM). In the present study, we analyzed the anatomic site-related expression of MUC4 in OC/PPC and studied its prognostic role. We additionally studied the ability of MUC4 to differentiate between OC/PPC and reactive mesothelial cells (RMC). OC/PPC effusions (n = 142) and benign reactive effusions (n = 10) were immunostained for MUC4 expression. Immunoreactivity was scored in carcinoma cells and RMC and was compared with tumor cell expression in 60 previously studied primary carcinomas and solid metastases and analyzed for association with clinicopathologic parameters, including survival. MUC4 was detected in carcinoma cells in 141/142 (99%) effusions, with comparable expression in peritoneal and pleural effusions. RMC were present in 72 malignant effusions and were MUC4-negative in all specimens, as well as in the 10 reactive effusions. MUC4 expression in carcinoma cells in effusions was significantly higher than in primary carcinomas and solid metastases (P < 0.001). Higher MUC4 expression was seen in tumors from older (>60 year) patients (P = 0.049). No association was found between MUC4 expression and other clinicopathologic parameters, including survival. MUC4 is universally expressed in OC/PPC effusions and is upregulated at this anatomic site compared to primary carcinomas and solid metastases. The data in the present study, together with our earlier report, show that MUC4 is an excellent marker for differentiating OC/PPC from both benign and malignant mesothelial cells.     

Survivin, a member of the inhibitors of apoptosis family, is down-regulated in breast carcinoma effusions

We analyzed the expression and prognostic role of inhibitors of apoptosis in breast carcinoma effusions. We used immunoblotting to analyze 22 effusions for XIAP, survivin, and livin expression. Based on immunoblotting results, 49 effusions and 46 corresponding solid tumors were immunostained for XIAP and survivin. Results were analyzed for association with anatomic site, clinicopathologic parameters, and survival. Immunoblotting showed frequent expression of XIAP and survivin and no expression of livin. Carcinoma cells in effusions showed lower survivin immunostaining compared with lymph node metastases (P = .008) and primary carcinomas (P = .041). Higher cytoplasmic survivin expression correlated with poor disease-free survival for patients with postchemotherapy effusions (P = .035). XIAP and survivin, but not livin, are frequently expressed in advanced breast carcinoma. Survivin is down-regulated in effusions compared with solid tumors, possibly in relation to the different cellular economy at this anatomic site. Survivin expression may predict disease-free survival for patients with postchemotherapy effusions.     

Osteopontin expression in ovarian carcinoma effusions is related to improved clinical outcome

Osteopontin, a soluble protein present in all body fluids, is involved in signaling pathways related to adhesion and extracellular matrix interactions, affecting multiple cellular functions, including inflammation, angiogenesis, and tumor metastasis. We studied osteopontin expression by immunohistochemistry and its clinical relevance in 170 effusions (140 peritoneal, 30 pleural) from women with advanced-stage ovarian carcinoma. Carcinoma cells expressed osteopontin in 126 (74%) of 170 effusions. Osteopontin expression was more frequent in effusions from patients with high-grade tumors (P = .036) but was significantly associated with better debulking at primary surgery (P = .019) and complete response to chemotherapy at diagnosis (P = .021). Osteopontin expression was positively associated to that of the previously studied nuclear factor κB inhibitor IκB (P = .019) and negatively related to expression of the inhibitor of apoptosis family member XIAP (P = .008) and the angiogenic marker endoglin (CD105; P = .018). In univariate survival analysis, the presence of osteopontin in carcinoma cells in primary diagnosis prechemotherapy effusions was associated with longer progression-free survival (P = .037), a finding that did not retain its significance in multivariate Cox analysis. This study demonstrates that osteopontin is frequently expressed in ovarian carcinoma effusions. However, its presence in tumor cells at this anatomical site is unexpectedly associated with less aggressive clinical course, suggesting different and yet undefined biological roles for this protein in serous effusions.     

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.     

Two-color, cytokeratin-labeled dna flow cytometric analysis of 332 breast cancers: lack of prognostic value with 12-year follow-up

CONTEXT: DNA flow cytometry of breast cancer is a proposed tumor marker of prognostic significance that is of controversial clinical utility because of lack of standardization and confirmatory studies. OBJECTIVE: To evaluate the prognostic significance of the more informative technique of multiparametric 2-color DNA flow cytometry as recommended by the 1992 DNA Cytometry Consensus Conference. DESIGN: Three hundred thirty-two breast carcinomas with 7 to 12 years of follow-up were prospectively analyzed as fresh tumors that were mechanically dissociated into whole cell suspensions. These suspensions were dual fluorescence-labeled with propidium iodide (DNA) and antibodies to cytokeratin (epithelium) and leukocyte common antigen (internal leukocyte control) for gated analysis of subpopulations. Multicycle software with histogram-dependent algorithms employing background, aggregate, and debris correction were used in DNA and cell-cycle quantitation. Data were analyzed according to the DNA Flow Cytometry Consensus Conference recommendations. RESULTS: DNA ploidy and proliferation stratified into 3 categories were not predictive of overall or disease-free survival. Sixty-five percent of tumors were nondiploid, and 35.4% were diploid. Two hundred six tumors were able to be evaluated for synthesis-phase fraction (SPF) analysis, with 74 of 206 cases in the low range (<13.4%), 36.4% in the intermediate range (>13.5 to <25.4%), and 27.6% in the high SPF (>25.5%) category. Aneuploid tumors tended to have a higher SPF. Univariate survival analysis showed prognostic significance of the following: tumor size, stage, TNM components, vascular invasion, nuclear grade, and histologic grade. Only T classification, presence of positive axillary lymph nodes, and distant metastases were significant independent predictors of survival in multivariate Cox regression models. Age and hormone receptor status showed no prognostic significance. Synthesis-phase fraction was significantly correlated with tumor size, stage, T classification, nuclear and histologic grade, presence of estrogen or progesterone receptors, and axillary lymph node status. None of the histologic parameters showed any significant association with DNA aneuploidy, except for high nuclear and histologic grade and the absence of estrogen receptors. CONCLUSIONS: Despite the use of state-of-the-art processing and flow cytometry analytic techniques, DNA ploidy and proliferation measurements were not predictive of survival in any stage of breast cancer. However, select histopathologic parameters and TNM stage were significant predictors of survival in univariate and multivariate analyses. We conclude that DNA ploidy and proliferation measurements do not provide significant prognostic information for clinicians to integrate into therapeutic decision making for patients with breast cancer.     

Expression of the Ets transcription factor EHF in serous ovarian carcinoma effusions is a marker of poor survival

The EHF (Ets homologous factor) gene was previously shown to be overexpressed in ovarian/primary peritoneal serous carcinoma compared to malignant mesothelioma using gene expression arrays. The objective of this study was to validate this finding at the mRNA level in a larger series. We analyzed the diagnostic role of EHF in 98 ovarian serous carcinoma effusions, 23 malignant mesothelioma specimens (20 effusions, 3 surgical specimens), and 28 primary ovarian serous carcinomas using quantitative real-time polymerase chain reaction. Expression levels of EHF in ovarian carcinoma were additionally investigated for association with clinicopathologic parameters and survival. Quantitative real-time polymerase chain reaction analysis showed significantly higher expression of EHF mRNA in ovarian carcinoma effusions and in primary ovarian carcinoma compared to malignant mesothelioma effusions (P < .001 for both). EHF mRNA expression was additionally higher in primary ovarian carcinomas compared to effusions of this cancer (P < .001). In univariate analysis for all patients with effusions, higher EHF mRNA levels were associated with a trend for shorter progression-free survival (P = .066), which became significant in analysis of 45 patients with primary diagnosis pre-chemotherapy effusions (P = .01). In Cox multivariate analysis, EHF mRNA expression was an independent predictor of poor progression-free survival for all patients and patients with primary diagnosis pre-chemotherapy effusions (P = .033 and P = .009, respectively). EHF mRNA levels differentiate ovarian carcinoma from malignant mesothelioma and may thus be of diagnostic value in this setting. EHF may be a novel prognostic marker in ovarian carcinoma.     

Expression of the chromatin remodeling factor Rsf-1 is down-regulated in breast carcinoma effusions

We recently identified Rsf-1, a chromatin-remodeling gene, as a potential oncogene that is frequently amplified and overexpressed in ovarian serous carcinoma, and demonstrated that its expression in carcinoma cells in effusions is associated with poor prognosis. In the present study, we assessed the clinical significance of Rsf-1 overexpression in breast carcinoma effusions. Formalin-fixed paraffin-embedded sections from 47 effusions were analyzed for Rsf-1 expression by immunohistochemistry. Matched primary tumors (n = 30) and solid metastases (n = 26) from 30 patients were additionally studied. Rsf-1 expression in tumor cells in effusions was analyzed for association with clinicopathologic parameters and survival. Rsf-1 protein expression was found in carcinoma cells in 34 (72%) of 47 effusions, 24 (80%) of 30 primary carcinomas, and 24 (92%) of 26 metastases. Rsf-1 immunoreactivity in effusions showed no association with HER-2 or hormone receptor status. Rsf-1 expression level was significantly lower in effusions compared with primary tumors (P = .026 and P = .011 for extent and intensity, respectively) and lymph node metastases (P = .023 and P = .013 for extent and intensity, respectively). Staining extent and intensity were both significantly lower in breast compared with ovarian carcinoma effusions (P = .001 for extent, P < .001 for intensity). Rsf-1 expression showed no association with survival. In conclusion, in contrast to ovarian carcinoma, Rsf-1 expression is down-regulated in breast carcinoma cells in effusions compared with the solid counterparts and has no prognostic role at this anatomic site.     

Tumour proliferation, angiogenesis, and ploidy status in human colon cancer

AIMS: Tumour angiogenesis is essential for carcinogenesis and facilitates the process of tumour development and metastasis. Vascular endothelial growth factor (VEGF) is a well characterised angiogenetic factor and is known to play a crucial role in new vessel development. To gain further insight into the effects of microvessel density and VEGF expression in colon cancer, their relation with tumour proliferation, ploidy status, and p53 expression was investigated in colon cancer. METHODS: Tissue samples of 50 archived colon cancers were analysed by immunohistochemistry for VEGF, p53, and the endothelial cell marker, von Willebrand factor (VWF), using specific antibodies. The same samples were re-cut for flow cytometric studies to obtain S phase fraction (SPF) and ploidy status. RESULTS: A positive significant correlation was found between SPF and angiogenesis. The median microvessel count in high SPF tumours was significantly higher than in low SPF ones. No association was found between VEGF expression and SPF. A positive correlation was found between ploidy status and p53 expression and microvessel count. Furthermore, a positive correlation was established between DNA ploidy, VEGF expression, and microvessel count. CONCLUSION: This study provides evidence that in colon cancer, tumour growth may be stimulated by vascular supply, and the lack of a correlation between tumour cell proliferation and VEGF expression indicates that these two parameters may be regulated by separate mechanisms. Furthermore, the positive correlation between microvessel density, VEGF expression, and ploidy status provides more evidence that genetic alterations are involved in tumour angiogenesis.     

DNA flow cytometry but not telomerase activity as predictor of disease-free survival in pT1-2/N0/G2 breast cancer.

OBJECTIVE: In the subgroup of patients with node-negative (N0) moderately differentiated (G2) breast cancer, the clinical decision of giving adjuvant therapy is critical. The aim of this study was to investigate the prognostic value of biomarkers (DNA flow cytometry and telomerase activity in correlation with routinely used estrogen receptors (ER) and HER oncoprotein) in pT1-2/N0/G2 breast cancer, for improving therapeutic management. METHODS: The series involved 135 patients with pT1-2/N0/G2 breast cancer and median follow-up of 58.5 months. DNA ploidy and S-phase fraction (SPF) (10%) were assessed on frozen samples. Telomerase activity, ER and c-erbB-2 expression were analyzed by standardized immunohistochemistry techniques. A Cox regression analysis was performed for prognostic evaluation. RESULTS: Aneuploidy significantly correlated with high SPF and lack of ER, while high SPF showed significant correlations with high telomerase activity, c-erbB-2 overexpression and absence of ER. Kaplan-Meier curves showed significant differences for ploidy and SPF in relation with disease-free survival (DFS) and overall survival (OS), and a statistical trend for ER. By Cox regression analysis, DNA aneuploidy (RR = 16.7; p = 0.007) and high SPF (RR = 23.1; p = 0.004) revealed significant correlations with worse DFS. Among patients with diploid (n = 76) and low/intermediate SPF (n = 85) tumors, only one had recurrence of the disease. No association between telomerase activity and clinical outcome was observed. CONCLUSION: In pT1-2/N0/G2 breast cancer patients, DNA ploidy and SPF are relevant prognostic biomarkers that should be considered as additional tools in the therapeutic planning.     

The flow-cytometric analysis of DNA content and S-phase fraction (SPF) of human breast cancer

The DNA ploidy of breast cancer tissue from paraffin blocks was measured by flow cytometry in 122 patients. In this material there was a difference in lymph node involvement and in the presence of distant metastases between diploid and aneuploid tumors. Diploid tumors were smaller than aneuploid tumors. Aneuploid tumors were more common in postmenopausal than in premenopausal women. Near tetraploid tumors were found in older patients rather than tumors with other ploidy patterns. The ploidy pattern was not associated with survival during the mean follow-up of 4.1 years. We specially studied the S-phase fraction (SPF) which was distinctly higher in aneuploid tumors than in diploid tumors. Also near tetraploid, hypertetraploid and multiploid tumors showed higher SPF than diploid tumors. The median of SPF in our material was 8.5%. Positive axillary lymph nodes were found in 32% of the patients who had tumors with an SPF below the median and in 46% of those with tumors above the median (SPF greater than 8.5%). The difference, however, was not statistically significant. In our material the SPF of the tumor did not show a significant association with survival. However, longer follow up time is needed for firm conclusions on the predictive value of cell DNA on survival.