Investigation of pericytes, hypoxia, and vascularity in bladder tumors: association with clinical outcomes

The contribution of endothelial cell growth to angiogenesis has been widely studied; however, the involvement of pericytes is less well documented, especially in human tumors. In this study we aimed to quantify and assess the prognostic significance of pericyte coverage, the extent of hypoxia, and microvessel density (MVD) in normal bladder mucosa and urothelial carcinoma. Antibody to alpha-smooth muscle actin was used to assess the distribution of pericytes (mural/smooth muscle cells) in the microvessels of normal human bladder (n = 4) mucosa and in urothelial carcinoma (n = 47) samples; this was quantitated using microvessel pericyte index (MPI). The MVD was measured using two different methods (n = 47) and hypoxia was assessed using glucose transporter-1 (Glut-1) staining (n = 30). There was a 70% reduction in MPI in urothelial carcinomas compared to normal bladder mucosa (p < 0.0012); MPI did not correlate with tumor stage or grade. Ta and T1 superficial tumors were divided into two groups with a MPI of <15% or >15%. Progression-free survival was significantly shorter for tumors with MPI >15% (p = 0.0036). MVD had no prognostic value using either evaluation method. Glut-1 immunoreactivity was not prognostic in superficial urothelial carcinoma samples. Tumors with a higher MPI showed a greater Glut-1 immunoreactivity (p = 0.0051). Microvessels in urothelial carcinoma have a considerable loss of pericyte coverage compared to normal bladder mucosa. The data from this preliminary study indicate that progression-free survival was shorter in patients whose superficial tumors had higher pericyte coverage of the microvessels. This may be due to increased levels of hypoxia, as demonstrated by a significant increase in Glut-1 staining.     

An immunohistochemical assessment of hypoxia in prostate carcinoma using pimonidazole: implications for radioresistance

PURPOSE: To investigate the presence of hypoxia in human prostate carcinoma by using pimonidazole immunohistochemical labeling in radical prostatectomy specimens. METHODS AND MATERIALS: Forty-three patients (median age, 69 years; range, 49-83 years) with localized prostate adenocarcinoma received 0.5 gm/m2 i.v. pimonidazole 16-24 h before radical prostatectomy. Hypoxia was detected with a monoclonal antibody directed against pimonidazole and scored in formalin-fixed, paraffin-embedded sections. Median and maximal vessel counts were measured with CD34. RESULTS: Thirty-seven patients completed the study. Pimonidazole binding was present in prostate carcinomas in 34 of 37 patients (92%) and in benign prostatic hyperplasia in 35 of 37 patients (95%). A positive correlation of 3+ pimonidazole binding with Gleason score was demonstrated (Spearman's rank, p = 0.044). Vascularity scores did not correlate with hypoxic status or clinical prognostic parameters. CONCLUSION: Prostate carcinoma and benign prostatic hyperplasia have significant areas of hypoxia; greater hypoxia scores are seen with more aggressive prostate cancer. It is postulated that a hypoxic microenvironment within the prostate might be responsible for the promotion of secondary genetic alterations and angiogenic stimulation, leading to malignant progression, a more aggressive cell phenotype, and greater radioresistance. Modification of radiation regimens to specifically target hypoxia might improve local tumor control.     

GLUT1 and CAIX as intrinsic markers of hypoxia in bladder cancer: relationship with vascularity and proliferation as predictors of outcome of ARCON

Glucose transporter-1 protein (GLUT1) and carbonic anhydrase IX (CAIX) are regulated by hypoxia inducible factor-1 (HIF-1) and have been studied as putative intrinsic cellular markers for hypoxia. This study directly compares CAIX and GLUT1 with pimonidazole binding in a prospective series of bladder cancer patients and also studies the prognostic significance of the markers, in combination with vascularity and proliferation, in a retrospective series of bladder cancer patients treated in a phase II trial of radical radiotherapy with carbogen and nicotinamide (ARCON). A total of 21 patients with a diagnosis of transitional cell carcinoma of the bladder received 0.5 g m(-2) pimonidazole. Serial tumour sections were stained for pimonidazole, GLUT1 and CAIX and compared. Tissue sections obtained from a series of 64 patients previously treated for invasive bladder cancer using ARCON were stained for GLUT1 and CAIX together with Ki-67 and CD31/34. There was a good geographical colocalisation of both intrinsic markers with pimonidazole and a highly significant agreement in individual patients; correlation coefficients were 0.82 (P=0.0001) for GLUT1 and 0.74 (P<0.0001) for CAIX. In both series of patients, the intrinsic hypoxia markers were highly correlated with each other and a correlation with proliferation was also evident in the retrospective study. In univariate and multivariate analyses, GLUT1 and CAIX were independent predictors for overall and cause specific survival. The hypoxia markers did not predict for local control or metastases-free survival although higher Ki-67 indices showed a trend towards local failure. The data suggest that both hypoxia modification and accelerated treatment may be valid treatment options in bladder cancer.     

Interrelationship of proliferation and hypoxia in carcinoma of the cervix

PURPOSE: In human cervix cancer treated with radiotherapy, we have previously shown from separate groups of patients that tumor hypoxia and proliferation rate as measured by bromodeoxyuridine (BrdU) labeling index (LI) are important determinants of clinical outcome. We now examine the relationship of these two pre-treatment predictive assays in 43 patients studied prospectively from 1994-98 where both tests were performed for each patient. MATERIAL AND METHODS: Newly diagnosed patients with carcinoma of the cervix were examined under anesthesia for staging purposes. Patients were given BrdU (200 mg) by intravenous route prior to the procedure. Tumor oxygenation was measured with the Eppendorf pO2 histograph. Biopsy of tumor was then performed and the BrdU LI was obtained by flow cytometry. The degree of tumor hypoxia for each tumor was expressed as median pO2 values, and as the percentage of pO2 readings <5 mm Hg (HP5). RESULTS: The median age was 53 years (range 23-79 years). There were 32 squamous, and 11 non-squamous carcinomas. FIGO stages were: IB and IIA, 8; IIB, 17; IIIB, 18; with a median tumor size of 6 cm (range 2-10 cm). The patients received uniform treatment with radical radiation therapy. There were 22 diploid and 21 aneuploid tumors. The median LI, pO2, and HP5 were 8.0%, 5.4 mm Hg, and 46.8%, respectively. Tests for linear associations showed no significant correlation between median pO2 vs. LI (r = 0.078, p = 0.62), and HP5 vs. LI (r = -0.14, p = 0.38). CONCLUSIONS: The clinical outcome in this group of patients is immature, but these results suggest that tumor hypoxia and proliferation measurements are independent and potentially complementary predictive assays in cervix carcinoma. Further investigations are required to examine the distribution of proliferating tumor cells and its relationship with hypoxic tumor cells in tissue sections with the use of immunohistological techniques and image analysis systems.     

Small cell carcinoma of the urinary bladder. A clinicopathologic, morphometric, immunohistochemical, and ultrastructural study of 18 cases

A multiinstitutional review of 3778 patients with a primary malignancy of the urinary bladder revealed 18 cases (0.48%) of small cell carcinoma which were histologically and morphometrically identical to pulmonary small cell undifferentiated carcinoma. Age, sex, and symptoms at first presentation were comparable to that known in transitional cell carcinoma. Sixteen patients (89%) developed metastatic disease, with most frequent involvement of regional lymph nodes, liver, skeleton, and abdominal cavity. The unfavorable clinical outcome was worse as compared with that reported in advanced stage poorly differentiated transitional cell carcinoma, and was similar to the rapidly fatal outcome of pulmonary small cell undifferentiated carcinoma. Fourteen patients (78%) died by tumor at a mean follow-up period of 9.4 months, and only one patient was free of recurrent disease more than 5 years after cystectomy. This apparent aggressive tumor behavior was independent of the presence of neuroendocrine differentiation characteristics at immunohistochemical (13 cases, 72%) or electron microscopic study (eight cases, 44%). The prolonged survival periods (15-38 months) of the five patients who received combination chemotherapy suggested that, just as in small cell lung carcinoma, chemotherapy may be profitable. A unified concept of histogenesis of bladder cancer with a common origin from a multipotent mucosal stem cell is proposed.     

Quantitative assessment of bladder carcinoma by acid labile DNA assay.

BACKGROUND: Effective noninvasive methods for monitoring patients with bladder carcinoma and screening for bladder carcinoma that show better performance than the methods currently in use would be desirable for detecting urothelial carcinoma at an early, easily treatable stage. A rapidly hydrolyzed component of nuclear DNA has been described, the increase of which has been linked to malignancy. Quantitative determination of acid labile DNA has been applied successfully to detect other neoplasms. This study investigates the potential of this method to detect transitional cell carcinomas. METHODS: Touch imprints of transurethral resection material from 62 cases of nonmalignant urothelium (control group including reactive changes) and 94 cases of bladder carcinoma were analyzed. The full Feulgen hydrolysis profiles of the nonmalignant and malignant urothelial cells were compared by measuring the staining density of the nuclei using digital image analysis after various hydrolysis times. Twenty cells were sampled randomly from among the cells measured for calculation of the mean optical density (MOD). The MOD of each time was used to build the hydrolysis profile of a case. RESULTS: A hydrolysis time of 10 minutes was found to be the most discriminative between control and carcinoma cases. Applying a single threshold MOD value of 101 resulted in a test sensitivity of 95.7%, a specificity of 94.4%, a positive predictive value of 98.3%, a negative predictive value of 95.9%, and an area under the receiver operating characteristic curve of 0.97. CONCLUSIONS: The results of this pilot study suggest that measurement of the rapidly hydrolyzed component of DNA present in the nuclei of bladder urothelium offers a highly sensitive and reliable supplement to the qualitative and subjective cytologic procedures currently in use.     

Tumor vascularity in the prognostic assessment of primary cutaneous melanoma.

PURPOSE: The vascular supply of the primary tumor is recognized to play an important role in the progression of a number of solid tumors. However, the role of tumor vascularity in the prognostic assessment of melanoma remains unclear. The purpose of this study was to determine the prognostic impact of patterns of vascularity on the outcome associated with cutaneous melanoma. PATIENTS AND METHODS: Tumor vascularity was documented prospectively using routine histopathologic analysis of 417 primary cutaneous melanomas from the University of California at San Francisco Melanoma Center database. Four patterns of tumor vascularity were recorded: absent, sparse, moderate, and prominent. RESULTS: Increasing tumor vascularity significantly increased the risk of relapse and death associated with melanoma, corresponding to reduced relapse-free and overall survival. By multivariate analysis, tumor vascularity was the most important determinant of overall survival, surpassing tumor thickness. Increasing tumor vascularity was associated with increased incidence of ulceration in the primary tumor. CONCLUSION: Tumor vascularity is an important prognostic factor in melanoma, rivaling tumor thickness. Increasing tumor vascularity is highly correlated with ulceration, possibly helping to explain the biologic basis of this known prognostic factor.     

Tumor vascularity: a histological measure of angiogenesis and hypoxia

In this study we sought to clarify the relationship between tumor vascularity, hypoxia, and angiogenesis in human cervix tumors. Two hypotheses were established: first, that measurement of tumor vascularity can provide a histological assessment of both hypoxia and angiogenesis; and second, that expression of angiogenesis-related proteins will provide a surrogate measure of tumor hypoxia. To test the first hypothesis, we studied the prognostic significance of tumor vascularity measured as both intercapillary distance (ICD; thought to reflect tumor oxygenation) and microvessel density (MVD; the hotspot method that provides a histological assessment of tumor angiogenesis). The relationship was also examined of tumor hypoxia, measured using an Eppendorf needle electrode [percentage of values less than 5 mm Hg (HP5)], with ICD and MVD. To test the second hypothesis we examined the relationship between HP5 and the expression of angiogenesis-associated proteins [vascular endothelial growth factor (VEGF) and platelet-derived endothelial cell growth factor (PD-ECGF)]. All of the biological measurements were made on pretreatment tumors. Analysis of data was carried out using log-rank statistics, Cox multivariate analysis, and Spearman's rank correlation. Both ICD and MVD were significant independent prognostic factors for local control. Patients with poorly vascularized tumors (long ICD) had poor local control (P = 0.042). However, patients with poorly vascularized tumors, measured as low MVD, had good local control (P = 0.036). For 107 patients in whom both of the measurements were obtained on the same tumor sections, ICD and MVD provided independent prognostic information in multivariate analysis. There was a significant correlation between tumor hypoxia and ICD (P < 0.005) but not MVD (P = 0.41). There was no relationship between hypoxia and the expression of angiogenic factors (VEGF, PD-ECGF). These analyses show that measurement of tumor vascularity can provide different biological information that is dependent on the method used. It is, therefore, important that studies measuring vascularity should include an appropriate definition. There is no relationship between hypoxia and angiogenesis in advanced carcinoma of the cervix and examining the levels of angiogenic proteins may not have a role in assessing hypoxia in cervix cancer.     

Breast carcinoma in pregnant women: assessment of clinicopathologic and immunohistochemical features

BACKGROUND: Breast carcinoma is one of the most common carcinomas in pregnant women. The incidence of breast carcinoma may increase in the future because of the trend toward delayed childbearing and increased screening. However, very few contemporary studies have attempted to identify the combined histopathologic and immunohistochemical features of breast carcinoma in these patients. METHODS: The authors evaluated 39 patients with breast carcinoma occurring coincident with pregnancy. This was comprised of a critical histologic review and immunohistochemical evaluation to determine the status of prognostic and predictive markers including estrogen receptor (ER), progesterone receptor (PR), HER-2/neu, Ki-67, and p53. RESULTS: The mean age at presentation was 33 years (range, 24-44 years). Densities and/or masses were noted on mammograms in 14 of 16 patients with available radiographic information. The primary tumors were a mean of 4.5 cm in greatest dimension (range, 0.1-13.5 cm). Two of the 39 patients had clinical (American Joint Committee on Cancer) Stage I disease, 19 patients had Stage II disease, 16 had Stage III disease, and 2 patients had Stage IV disease at the time of presentation. Histologically, high-grade invasive ductal carcinomas were found in 32 of 38 patients. The primary tumor was not available for review in one patient. A predominantly solid pattern of growth was observed in nine patients. Lymphovascular invasion was identified in 61% of cases. Ductal carcinoma in situ was identified in 72% of tumors and was high grade in all cases. Of the 25 patients tested, ER positivity was found in 7 patients, PR positivity was found in 6 patients, HER-2/neu positivity was found in 7 patients, and p53 positivity was found in 12 patients. The proliferation rate as shown by Ki-67 staining was high in 60% of the cases. Follow-up information was available for 35 patients and the mean follow-up period was 43 months (range, 2-163 months). Distant metastasis occurred in seven patients. The mean time to disease recurrence was 20.4 months (range, 10-33 months). Of 35 patients, 4 have died, 22 were alive with no evidence of disease, and 9 were alive with disease at the last follow-up. The remaining four patients died of unknown causes. CONCLUSIONS: Pregnant women with breast carcinomas generally present with advanced-stage disease and the tumors have poor histologic and prognostic features. The findings from the follow-up indicated that these tumors do not follow a very aggressive clinical course as was proposed in earlier reports. Breast carcinomas occurring during pregnancy share many histologic and prognostic similarities with breast carcinoma occurring in other young women.     

缺氧调控端粒酶活性及抑制鼻咽癌细胞增殖实验研究

目的探讨缺氧及siRNA沉默缺氧诱导因子-1α(hypoxia inducible factor-1α,HIF-1α)后对鼻咽癌细胞中端粒酶催化亚单位(telomerase catalytic subunit,hTERT)、细胞周期和化疗耐药的影响。方法采用三气培养箱对鼻咽癌细胞5-8F和CNE2进行缺氧处理(1%O2),蛋白质印迹法检测不同乏氧时相(0~72h)HIF-1α和hTERT蛋白的表达。将HIF-1α基因特异性siRNA分别转染鼻咽癌细胞株5-8F和CNE2,筛选出沉默效率最高的siRNA,实验分为未处理组(常氧)、未处理组(缺氧)、Negative-siRNA(缺氧)和HIF-1α-siRNA(缺氧),荧光定量PCR及蛋白质印迹检测瞬时转染后hTERT及HIF-1α的表达。流式细胞术(flow cytometry,FCM)分析缺氧或沉默HIF-1α后对细胞周期的影响。MTT法检测缺氧或沉默HIF-1α后,鼻咽癌细胞对顺铂(DDP)和5-氟尿嘧啶(5-FU)的化疗敏感性。结果缺氧处理0~72h后鼻咽癌5-8F细胞HIF-1α(F=37.147,P<0.001)和hTERT(F=70.069,P<0.001)蛋白的表达上调,差异有统计学意义。HIF-1α-siRNA对5-8F细胞瞬时转染率>98%。HIF-1α-siRNA组hTERT mRNA表达量为0.37±0.05,显著低于未处理组(缺氧)的1.00±0.00和Negative-siRNA(缺氧)的0.95±0.01,F=360.339,P<0.001;hTERT蛋白表达量为(0.27±0.05),显著低于未处理组(缺氧)0.54±0.00和Negative-siRNA组(缺氧)0.53±0.01,F=24.010,P<0.001。未处理组(缺氧)G0/G1期细胞比例明显增加(45.63±2.01)%,显著高于未处理组(常氧)的(26.75±1.28)%,P<0.001。5-8F细胞未处理组(常氧)对5-FU的IC50分别为(17.30±3.31)μg/mL,未处理组(缺氧)为(32.04±12.75)μg/mL,Negative-siRNA组为(33.90±0.87)μg/mL,HIF-1α-siRNA组为(13.72±2.36)μg/mL,F=3.704,P<0.001。5-8F细胞缺氧组对DDP的化疗敏感性也降低,沉默HIF-1α后,5-8F细胞对DDP的化疗敏感性明显提高。除细胞周期外,CNE2与5-8F的结果均一致。结论缺氧促使鼻咽癌细胞发生G1/S阻滞及化疗耐药,可能与上调hTERT表达,进而上调端粒酶活性有关;沉默HIF-1α显著逆转缺氧诱导的肿瘤耐药并下调端粒酶活性。     

An immunohistochemical assessment of cellular proliferation markers in head and neck squamous cell cancers

Prognostic information is essential for the evaluation, judgement and optimal treatment of patients with squamous cell cancers (SCCs) of the upper aerodigestive tract. Using immunohistochemical and flow cytometric techniques, we have studied the significance of cellular expression of the Ki-67 antigen, epidermal growth factor receptor (EGFR), the transferrin receptor (TFR) and DNA ploidy status in a prospective analysis of patients with SCCs of the head and neck region. All 42 fresh tumour samples (five well differentiated; 28 moderately differentiated; nine poorly differentiated) expressed both EGFR and TFR to varying degrees. Receptor expression was most marked on the peripheral invading margin of cancer cell islands although staining was also demonstrated in a random fashion within cellular islands and consistently along the basal cell layer of overlying stratified squamous epithelium. The percentage of cancer cells that reacted with the Ki-67 monoclonal antibody was assessed as low (less than 10%) in 15 samples (35.8%), intermediate (10-30%) in 19 samples (45.2%) and high (greater than 30%) in eight samples (19.0%). Eleven of 15 samples (73%) with a low percentage reactivity were DNA diploid, whereas seven of eight samples (87.5%) with a high percentage reactivity were DNA aneuploid. Poorly differentiated SCCs were significantly more often aneuploid than were either moderately or well differentiated tumours. Our results suggest that EGFR and TFR are widely distributed on SCCs, especially on proliferating cells at the invading tumour margin. In addition, there is a close spatial correlation between cells expressing EGFR, TFR and those expressing the Ki-67 antigen. Tumours in which the staining intensity for both EGFR and TFR was intense invariably expressed the Ki-67 antigen in a high proportion of cells. Further patient follow-up will be important in determining whether intense EGFR and TFR staining, combined with a high percentage reactivity with Ki-67 antibody and DNA aneuploidy, will ultimately define a subset of head and neck cancer patients with a poor clinical outcome.     

Evaluation of cell proliferation in breast carcinoma. Comparison of Ki-67 immunohistochemical study, DNA flow cytometric analysis, and mitotic count

Growth kinetics of 102 breast carcinomas were studied by immunohistochemical analysis with the monoclonal antibody Ki-67, which reacts with a nuclear antigen in proliferating cells. The results were correlated with ploidy and S-phase fraction (SPF) analyzed by DNA flow cytometric study and with mitotic count analyzed by light microscopic study. The proportion of Ki-67-positive cells (Ki-67 score) in breast carcinomas varied from 0.6% to 80% (median, 6.3%). Ki-67 scores were significantly higher in the DNA aneuploid than in the DNA diploid tumors. Ki-67 scores correlated significantly with tumor SPF in DNA aneuploid tumors. In DNA diploid tumors SPF showed no correlation with Ki-67 score. High Ki-67 scores were associated with high mitotic counts (P less than 0.0001) and histologic grade (P less than 0.0001). Nuclear pleomorphism, tubule formation, or lymph node status were not correlated with Ki-67 score. In conclusion, Ki-67 immunostaining correlates with other measures of cell proliferation (SPF, mitotic count) supporting its clinical significance.     

Anaplastic thyroid carcinoma. A clinical, histologic, and immunohistochemical study

Twenty-six cases of anaplastic thyroid tumor were investigated and reclassified using immunoperoxidase techniques. Sections of the neoplasms were stained immunohistologically for the following thyroid associated antigens: (1) thyroglobulin, which shows a positive reaction with follicular cells of the thyroid; (2) calcitonin, which is positive in medullary carcinoma of the thyroid; and (3) leucocyte common antigen (LC), which identifies lymphomata and Factor VIII-related antigen for hemangioendothelioma. Using these methods, five cases were reclassified. Three cases were identified as lymphomata, one case was reclassified as medullary carcinoma of the thyroid, and one case was identified as hemangioendothelioma. Eleven cases were confirmed to be anaplastic carcinoma of the thyroid and ten cases were negative for all the antigens tested. There was a significant difference in the survival of the groups of patients mentioned above. Prognostic data support the suggestion that immunohistochemical methods should be used for the precise classification of anaplastic carcinoma. In this way, tumors such as malignant lymphoma and medullary carcinoma, which resemble anaplastic carcinoma histologically but have a better prognosis, can be identified. This is important for planning surgical procedures and choosing chemotherapy and/or radiotherapy.     

缺氧诱导对人结肠癌SW480细胞增殖和缺氧诱导因子-1α、血红素氧合酶-1基因表达的影响

目的构建缺氧诱导模型和观察缺氧对人结肠癌SW480细胞系生长和缺氧诱导因子-1α(hypoxiainduciblefactor-1alpha,HIF-1α),血红素氧合酶-1(hemeoxygenase-1,HO-1)表达的影响。方法利用氯化钴(cobaltchloride,CoCl2)构建缺氧诱导模型,以MTT试验观察不同程度缺氧条件对SW480细胞生长曲线的影响;利用RT-PCR方法测定氯化钴缺氧诱导与SW480细胞HIF-1α和HO-1基因mRNA表达的时效和量效关系。结果适度缺氧(CoCl2浓度<200μmol/L)可刺激肿瘤细胞增殖,过度缺氧(CoCl2浓度>250μmol/L)则抑制其生长;HIF-1α、HO-1基因mRNA表达与氯化钴缺氧呈明显的量效关系和时效关系;HIF-1α与HO-1基因mRNA的表达在缺氧诱导量效关系(相关系数r=0.786,P<0.05)和时效关系(相关系数r=0.863,P<0.05)中均存在显著的正相关性。结论缺氧可以刺激SW480细胞增殖;HIF-1α和HO-1基因的表达与肿瘤缺氧程度密切相关;HO-1的适量表达对缺氧细胞具有细胞保护作用,其过度表达可能导致细胞损伤和凋亡。     

Increased expression of angiogenin in hepatocellular carcinoma in correlation with tumor vascularity.

PURPOSE: Neovascularization is known to be one of the major characteristics of human hepatocellular carcinoma (HCC). Angiogenin (ANG), originally discovered in a human colon cancer cell line, is a liver-derived polypeptide that shows strong angiogenic activity in vivo. However, the role of ANG on the development of HCC remains unknown. The present study was designed to examine the implication of ANG in the neovascularization of human HCC. EXPERIMENTAL DESIGN: Forty-one HCC patients who had undergone conventional celiac angiography were used in this study. ANG protein expression and microvessel density (MVD) in HCC specimens obtained by liver biopsy or surgical resection were examined by immunohistochemistry, and the levels were quantified by the KS-400 image analyzing system. ANG mRNA expression in liver tissues was evaluated by in situ hybridization. Serum ANG concentrations were measured by an ELISA. Survival rates were calculated using the Kaplan-Meier method. RESULTS: Immunohistochemistry and in situ hybridization showed greater increments of ANG protein expression and mRNA expression, respectively, in HCC tissues than in the surrounding nontumorous tissues. MVD within tumorous tissues increased according to dedifferentiation of the histological grade of HCC, showing a significant correlation (r = 0.877, P = 0.0009) with ANG expression levels. Mean +/- SD serum ANG levels of healthy subjects and chronic hepatitis (CH) patients were 362.3 +/- 84.1 ng/ml and 331.9 +/- 133.8 ng/ml, respectively, with no significant difference. Serum ANG levels of liver cirrhosis patients (242.4 +/- 126.9 ng/ml) were lower than those of healthy subjects or CH patients and decreased as the fibrosis grade advanced. In HCC patients, despite the cirrhotic background, serum ANG levels increased as the tumor vascularity increased (197.8 +/- 64.9 ng/ml for hypovascular, 326.7 +/- 148.6 ng/ml for hypervascular, and 405.0 +/- 121.3 ng/ml for very hypervascular), in good accordance with histological grading, and significantly decreased (P = 0.015) after successful treatment with transcatheter arterial embolization or percutaneous ethanol injection. HCC patients were conventionally divided into two groups according to the serum level of ANG, those with values higher than the mean level (332.9 +/- 143.8 ng/ml) and those with values lower than the mean,; the 5-year survival rate of the latter group was determined to be significantly higher than that in the former group. CONCLUSIONS: These results suggest that ANG is one of the neovascularization defining factors of HCC. Thus, measuring serum ANG may assist in monitoring the disease, and targeting ANG may provide a new strategy for treating advanced HCC.     

The sensitivity of bladder wash flow cytometry, bladder wash cytology, and voided cytology in the detection of bladder carcinoma

The sensitivity of voided urinary cytology (VUC), bladder wash cytology (BWC), and bladder wash flow cytometry (BWFCM) in detecting cancer was studied in 70 patients with biopsy-proven bladder tumors. There were 11 Grade I papillomas, 14 Grade II TA, 18 Grade II-III TIS, 19 Grade II-III T1, and eight Grade II-III T2 carcinomas. One to five VUCs per patient (mean, 2.63) were obtained within the 24 hours preceding biopsy. At endoscopy a bladder wash specimen was obtained and divided for cytologic and flow cytometric examinations. For all tumor categories combined, the sensitivity for one, two, and three voided cytology examinations per patient was 41%, 41%, and 60%, respectively. The sensitivity of a single BWC was 61%, of a single BWFCM, 83%. Thus, one BWFCM is more sensitive than three VUC (binomial test; P = 0.006); one BWC is more sensitive than two VUC (P = 0.01); and one BWFCM is more sensitive than one BWC (P = 0.003). These findings remain significant when papillomas are excluded from the analysis (P less than or equal to 0.03) and when papillomas and T2 tumors are jointly excluded (P less than or equal to 0.02). Only four of 70 patients (6%) had their cancers detected by VUC and/or BWC rather than BWFCM. In summary, irrigation cytology specimens are more sensitive than voided urinary cytology, and bladder wash flow cytometry is more sensitive than either in diagnosing bladder cancer. Flow cytometry is more sensitive because of the better sampling of bladder irrigation compared with voided urine and because of the measurement technique itself.     

The role of Ki67 proliferation assessment in predicting local control in bladder cancer patients treated by radical radiation therapy.

PURPOSE: To assess whether tumour proliferation as measured by Ki67 immunostaining has any predictive value for local control in bladder cancer patients treated by radiotherapy. PATIENTS AND METHODS: Fifty-five patients suffering from infiltrating bladder carcinoma recommended for radical radiotherapy (66 Gy/6-7 weeks) were included in this study. Paraffin-embedded pre-treatment tumour sections were stained with the Ki67 antibody. The percentage of Ki67-positive nuclei was correlated with established prognostic factors, local control and survival. RESULTS: The Ki67 index was not related to local control in our patients when the median was selected as the cut-off value. Patients with tumours with a very low (<27%) Ki67 index had better local control at 5 years (69%) than patients with tumours with greater (>27%) Ki67 expression indices (31.5%) (P<0.05; log-rank test). CONCLUSIONS: Ki67 immunostaining was a feasible method to estimate tumour proliferation. Patients with very low proliferating tumours seemed to achieve better local control after fractionated radiotherapy compared to other patients. Further studies are needed with a greater number of patients to accurately define the role of Ki67 expression in predicting tumour repopulation during fractionated radiotherapy.     

Noninvasive assessment of tumor vascularity by contrast-enhanced ultrasonography and the prognosis of patients with nonresectable pancreatic carcinoma

BACKGROUND: Studies have shown that angiogenesis is one of the factors that influences the prognosis of patients with solid tumors, including pancreatic carcinomas. However, none have assessed noninvasively the relation between angiogenesis and prognosis in patients with pancreatic carcinoma. Contrast-enhanced ultrasonography (US) not only is a convenient, harmless, and noninvasive imaging modality, but it also provides detailed information on tumor vascularity. The objectives of this study were to assess the vascularity of pancreatic carcinoma noninvasively by contrast-enhanced US and to clarify the prognostic value of tumor vascularity in patients with nonresectable pancreatic carcinoma. METHODS: Thirty-five consecutive patients with pathologically confirmed, nonresectable pancreatic carcinoma were examined with contrast-enhanced US before systemic chemotherapy. The correlations among tumor vascularity, clinicopathologic factors, and clinical outcomes then were analyzed statistically to investigate prognostic indicators. RESULTS: The median time to progression (TTP) was longer in patients who had avascular tumors compared with patients who had vascular tumors (110 days vs. 28 days, respectively; P=0.0072; log-rank test). The median survival also was longer in patients who had avascular tumors (267 days vs. 115 days, respectively; P=0.0034; log-rank test). A multivariate analysis using a Cox proportional hazards model revealed that tumor vascularity was a significant, independent factor that influenced TTP (P <0.001) and survival (P=0.022) along with primary tumor size and serum lactate dehydrogenase (LDH) level, which are well known as prognostic factors in patients with pancreatic carcinoma. CONCLUSIONS: The current results indicated that contrast-enhanced US may be useful in assessing the prognosis of patients with nonresectable pancreatic carcinoma who receive systemic chemotherapy.