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Aims Pancreatic cancer (PC) is a heterogeneous disease in terms of histological and molecular subtypes. The aim of this study was to evaluate the prognostic impact of different histological subtypes on cancer-specific survival (CSS) in a large single-centre Middle European cohort. Methods We retrospectively studied the records of 400 consecutive PC patients who were treated from 2004 to 2010 at a single tertiary academic centre. The association of histological subtypes and parameters such as tumour stage, tumour grade, levels of tumour markers carcinoembryonic antigen and CA19-9 at diagnosis, was studied. CSS was calculated using the Kaplan-Meier method, and the influence of each parameter on CSS was assessed with univariate and multivariable Cox proportional models. Results The survival time was significantly shorter in the ductal adenocarcinoma and acinar histological subtypes compared to neuroendocrine differentiation (p<0.001). No survival difference was observed between ductal adenocarcinomas and patients with a histological variant of ductal adenocarcinoma, namely, mucinous non-cystic adenocarcinoma (p=0.7). In multivariable analysis, ductal adenocarcinoma (HR=3.1, CI 1.6 to 6.1, p=0.001) and acinar carcinoma (HR=3.2, CI 1.3 to 8.5, p=0.016) were identified as independent predictors for CSS. Conclusions Our findings suggest that the main histological subtype is an independent predictor of CSS in patients with PC. Thus, our data underline the importance of routine assessment of histological type in PC for individual risk assessment. However, no clinical rationale for the subdivision of ductal adenocarcinoma and mucinous non-cystic adenocarcinoma can be supported by our study. [ABSTRACT FROM AUTHOR]

Study Type - Aetiology (case control) Level of Evidence 2b What's known on the subject? and What does the study add? The multikinase inhibitor sunitinib causes cardiotoxic side effects in a substantial number of treated patients. Previously, data from clinical studies suggested that these side effects are more frequently found in patients with a lower body mass index, but the reasons for this toxic effect remain elusive. In this study we determined, in a human multicellular model, that sunitinib causes an impairment of the contractile function of cardiomyocytes in a dose-dependent manner. Furthermore, we found that sunitinib induces alterations in calcium homeostasis in cardiomyocytes. In addition, an increased production of reactive oxygen species after exposure of cardiomyocytes to this drug was detected. These findings suggest that measurement of plasma concentration might help to control cardiotoxic side effects and optimize a more personalized application of this drug. OBJECTIVES To examine the acute effects of sunitinib on inotropic function, intracellular Ca2+ transients, myofilament Ca2+ sensitivity and generation of reactive oxygen species (ROS) in human multicellular myocardium and isolated mouse cardiomyocytes., To search for microRNAs as suitable biomarkers for indicating toxic cardiac effects., PATIENTS AND METHODS After exposure to sunitinib (0.1-10 µg/mL) developed force, diastolic tension and kinetic variables were assessed in isolated human myocardium., Changes in myocyte sarcomere length, whole-cell calcium transients, myofilament force-Ca2+ relationship, and ROS generation were examined in isolated ventricular mouse cardiomyocytes., Microarray and realtime-PCR were used to screen for differentially expressed microRNAs in cultured cardiomyocytes that were exposed for 24 h to sunitinib., RESULTS We found that higher concentrations of sunitinib (1 and 10 µg/mL) decreased developed force at 30 minutes 76.9 + 2.8 and 54.5 + 6.3%, compared to 96.1 + 2.6% in controls ( P < 0.01)., Sunitinib exposure significantly decreased sarcomere shortening and Ca2+ transients., Myofilament Ca2+ sensitivity was not altered, while ROS levels were significantly increased after exposure to the drug., MicroRNA expression patterns were not altered by sunitinib., CONCLUSIONS Sunitinib elicits a dose-dependent negative inotropic effect in myocardium, accompanied by a decline in intracellular Ca2+ and increased ROS generation., In clinical practice, these cardiotoxic effects should be considered in cases where cardiac concentrations of sunitinib could be increased. [ABSTRACT FROM AUTHOR]

To elucidate the role of predictive factors on individual's drug response, based on genetic variation, we examined the association between eight germline polymorphisms in genes involved in protection against oxidative stress, apoptosis, oncogenic transformation, proliferation, immune response and DNA repair (TP53, NQO1, IL6, TLR4 and XRCC1) and the pathological response to anthracycline-based neoadjuvant chemotherapy in 70 patients with breast cancer. The DNA was genotyped for eight polymorphisms in five genes (TP53, NQO1, IL6, TLR4 and XRCC1) by 5′-exonuclease (TaqMan™) technology. Fisher's exact test was used to evaluate the association between genotype, clinicopathological parameters and pathological response. A good pathological response, defined as a pathological complete response or residual isolated invasive tumor cells, was found significantly more frequently for estrogen (ER) and progesterone receptor (PR) negative breast carcinomas compared to ER and PR positive and ER or PR positive carcinomas, respectively (43.5 vs. 37.5 and 10.3 %, p = 0.006), and was significantly associated with high tumor grade (G3) ( p = 0.002). A non-significant trend towards a good pathological response was shown in patients carrying the Arg/Arg or Arg/Pro TP53 codon 72 gene variant compared to those harboring the Pro/Pro variant (17.6 or 37.9 % vs. 0; p = 0.071). No association was found between NQO1 Pro187Ser, IL6 −174G>C, TLR4 Asp299Gly and Thr399Ile, and XRCC1 Arg194Trp, Arg399Gln and Arg280His and pathological response. The present study shows hormone receptor status and tumor grade as predictors for pathological response to neoadjuvant anthracycline-based chemotherapy. Among various functional germline polymorphisms, a potential predictive value was only found for the TP53 Arg72Pro gene variant. [ABSTRACT FROM AUTHOR]

Purpose: Women with breast cancer that initially involves local lymph nodes have a higher risk for local recurrence or developing metastases. Recent data suggest that germline polymorphism is a significant, previously unrecognized factor in breast cancer progression and metastasis. We assessed the influence of 16 selected common germline polymorphisms in disease-free survival and overall survival among 216 women diagnosed with lymph node-positive breast cancer. Results: The rare allele of FAS 1377G>A was significantly associated with prolonged disease-free survival ( P = 0.012, risk ratio of recurrence (RR) = 0.557, 95% confidence interval (CI) = 0.353-0.878) in univariate analysis. After adjusting for known breast cancer prognostic factors the association remained significant ( P = 0.050, RR = 0.500, CI = 0.309-0.809). In overall survival analysis we found a significant association of the FAS 1377G>A ( P = 0.040, RR = 0.451, CI = 0.496-1.188) and IL10 592C>A polymorphisms ( P = 0.020, RR = 1.707, CI = 1.087-2.680) in the univariate Cox regression. The effect remained statistically significant in the multivariate analysis for the IL10 592C>A polymorphism ( P = 0.013, RR 1.841, CI 1.140-2.973). No association was found for MTHFR 677C>T, VEGF 936C>T, CCND1 870G>A, TGFB1 29T>C, FASLG 844C>T, FAS 670A>G, GPB3 825C>T, ITGA2 807C>T, ITGA2 1648G>A, ITGB3 176T>C, MMP1 -1607 1G/2G, MMP3 5A/6A, PTGS2 8473T>C, IL10 592C>A and SULT1A1 638G>A polymorphisms and disease-free survival or overall survival. Conclusions: Our data suggest that the FAS 1377G>A and IL10 592C>A polymorphisms could modify disease-free and overall survival in women with lymph node-positive breast cancer. [ABSTRACT FROM AUTHOR]

Abstract: Objectives: The aim of the present study was to evaluate the precision and reproducibility of the LINE-1 high-resolution melting (HRM) assay to detect LINE-1 hypomethylation. Design and methods: We first evaluated a methylated DNA dilution matrix and a panel of human cancer cell lines. We then applied this LINE-1 HRM assay to a set of 37 archival prostate cancer tissue samples. Results: Our LINE-1 HRM assay revealed small and reproducible run-to-run and bisulfite-to-bisulfite variations. As expected, we found a large variation in methylation levels between different cancer cell lines. All results were confirmed with MethyLight and pyrosequencing as indicated by the high correlation coefficient. Finally, we successfully applied the LINE-1 HRM assay to archival prostate cancer tissues. Conclusions: The present LINE-1 HRM assay represents a novel, accurate, and cost-effective method to measure global hypomethylation, which makes it suitable for high- and low-throughput laboratories. [Copyright &y& Elsevier]

Abstract: To address the question whether the higher onset of apoptosis of circulating NK cell subsets might be activation induced in cancer patients, surface expression of NKG2D and serum (s) levels of MHC class I chain-related (MIC) proteins in relation to apoptosis marker and CD95 expression on NK cells were evaluated. Patients showed a significantly higher onset of spontaneous apoptosis of CD56dim NK cells. No difference in the CD95 expression could be detected between patients and normal controls (NCs). Patients’ CD56bright NK cells demonstrated a higher expression of NKG2D compared to CD56dim NK cells. The sMICB levels showed a higher level in patients versus NCs. No correlation between sMIC protein levels with both NKG2D expression and onset of spontaneous apoptosis of NK cell subsets was found. Our data suggest that the higher onset of apoptosis of circulating NK cell subsets of patients is not triggered by activation-induced cell death. [Copyright &y& Elsevier]

Abstract: Background: Death receptor 4 (DR4) and death receptor 5 (DR5) are tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) receptors that activate apoptosis via the extrinsic apoptosis pathway. DcR1 and DcR2 are decoy receptors for TRAIL that act antagonistically. Intracellular trafficking of TRAIL receptors has been described, but the role of the subcellular localization of TRAIL receptors in non-small cell lung cancer (NSCLC) progression is unclear. Methods: Expression and intracellular localization of pro-apoptotic and decoy TRAIL receptors were analyzed by immunohistochemistry in 50 samples of advanced or recurrent NSCLC. Using confocal microscopy, localization of TRAIL receptors was studied in NSCLC cell lines. Results: Cytoplasmic staining for all four TRAIL receptors was observed in the majority of samples. Nuclear staining was infrequent in the case of DR4 (12%) and DcR2 (8%), while DR5 and DcR1 were localized in the nucleus in 27% and 60% of samples. When overall survival was analyzed, cytoplasmic staining for DR5 in tumor cells (P =0.025) and nuclear staining for DR5 in tumor cells (P =0.007) were significant prognostic factors in univariate, as well as in multivariate analysis including clinicopathologic factors (P =0.026 and 0.021, respectively). In A549, NCI-H358, and A427 NSCLC cells all four TRAIL receptors were found to be mainly located perinuclearly, but also in the nucleus. Conclusion: The study for the first time shows that TRAIL receptors are found in different intracellular compartments including the nucleus in NSCLC cells and that both nuclear and cytoplasmic DR5 might predict improved survival in patients with advanced NSCLC. [Copyright &y& Elsevier]

Background: Ovarian suppression plus tamoxifen is a standard adjuvant treatment in premenopausal women with endocrine-responsive breast cancer. Aromatase inhibitors are superior to tamoxifen in postmenopausal patients, and preclinical data suggest that zoledronic acid has antitumor properties. Methods: We examined the effect of adding zoledronic acid to a combination of either goserelin and tamoxifen or goserelin and anastrozole in premenopausal women with endocrine-responsive early breast cancer. We randomly assigned 1803 patients to receive goserelin (3.6 mg given subcutaneously every 28 days) plus tamoxifen (20 mg per day given orally) or anastrozole (1 mg per day given orally) with or without zoledronic acid (4 mg given intravenously every 6 months) for 3 years. The primary end point was disease-free survival; recurrence-free survival and overall survival were secondary end points. Results: After a median follow-up of 47.8 months, 137 events had occurred, with disease-free survival rates of 92.8% in the tamoxifen group, 92.0% in the anastrozole group, 90.8% in the group that received endocrine therapy alone, and 94.0% in the group that received endocrine therapy with zoledronic acid. There was no significant difference in disease-free survival between the anastrozole and tamoxifen groups (hazard ratio for disease progression in the anastrozole group, 1.10; 95% confidence interval [CI], 0.78 to 1.53; P=0.59). The addition of zoledronic acid to endocrine therapy, as compared with endocrine therapy without zoledronic acid, resulted in an absolute reduction of 3.2 percentage points and a relative reduction of 36% in the risk of disease progression (hazard ratio, 0.64; 95% CI, 0.46 to 0.91; P=0.01); the addition of zoledronic acid did not significantly reduce the risk of death (hazard ratio, 0.60; 95% CI, 0.32 to 1.11; P=0.11). Adverse events were consistent with known drug-safety profiles. Conclusions: The addition of zoledronic acid to adjuvant endocrine therapy improves disease-free survival in premenopausal patients with estrogen-responsive early breast cancer. (ClinicalTrials.gov number, NCT00295646.) N Engl J Med 2009;360:679-91. [ABSTRACT FROM AUTHOR]

Integrins such as α2β1, αIIbβ3, and αvβ3 have been suggested as key players for cancer development and progression. Several polymorphisms affecting these molecules, two in integrin α2 (ITGA2 807C>T and 1648G>A) and one in β3 (ITGB3 176T>C), influence their levels, structure, and possibly their function. To analyze the role of ITGA2 and ITGB3 polymorphisms for colorectal cancer risk and clinical presentation, we performed a case–control study. Four hundred thirty-three colorectal cancer patients and 433 healthy sex- and age-matched control subjects were investigated. ITGA2 and ITGB3 polymorphisms were determined by 5′-nuclease assays. The ITGA2 807C>T polymorphism was associated with reduced colorectal cancer risk. In a codominant model, the odds ratio for each additional 807-T allele for colorectal cancer was 0.77 (95% confidence interval 0.64–0.94; p = 0.011). The ITGA2 1648G> and the ITGB3 176T>C polymorphism were not associated with colorectal cancer. None of the three polymorphisms investigated was associated with tumor size, histological grade, presence of primary lymph node metastases, tumor stage, or age at diagnosis. We conclude that the ITGA2 807C>T polymorphism may be associated with reduced colorectal cancer risk. [ABSTRACT FROM AUTHOR]

Summary: Background: The Austrian Breast and Colorectal Cancer Study Group trial-12 (ABCSG-12) bone substudy assesses zoledronic acid for preventing bone loss associated with adjuvant endocrine therapy and reports on long-term findings of bone-mineral density (BMD) during 3 years of treatment and 2 years after completing adjuvant treatment with or without zoledronic acid. The aim of this substudy is to gain insight into bone health in this setting. Methods: ABCSG-12 is a randomised, open-label, phase III, 4-arm trial comparing tamoxifen (20 mg/day orally) and goserelin (3·6 mg subcutaneously every 28 days) versus anastrozole (1 mg/day orally) and goserelin (3·6 mg subcutaneously every 28 days), both with or without zoledronic acid (4 mg intravenously every 6 months) for 3 years in premenopausal women with endocrine-responsive breast cancer. This prospective bone subprotocol measured BMD at 0, 6, 12, 36, and 60 months. The primary endpoint of the bone substudy (secondary endpoint in the main trial) was change in BMD at 12 months, assessed by dual-energy X-ray absorptiometry in assessable patients. Analyses were intention to treat. Statistical significance was assessed by t tests. The ABCSG-12 trial is registered on the ClinicalTrials.gov website, number NCT00295646. Findings: 404 patients were prospectively included in the bone substudy and randomly assigned to endocrine therapy alone (goserelin and anastrozole or goserelin and tamoxifen; n=199) or endocrine therapy concurrent with zoledronic acid (goserelin, anastrozole, and zoledronic acid or goserelin, tamoxifen, and zoledronic acid; n=205). After 3 years of treatment, endocrine therapy alone caused significant loss of BMD at the lumbar spine (−11·3%, mean difference −0·119 g/cm2 [95% CI −0·146 to −0·091], p<0·0001) and trochanter (−7·3%, mean difference −0·053 g/cm2 [−0·076 to −0·030], p<0·0001). In patients who did not receive zoledronic acid, anastrozole caused greater BMD loss than tamoxifen at 36 months at the lumbar spine (−13·6%, mean difference −0·141 g/cm2 [−0·179 to −0·102] vs −9·0%, mean difference −0·095 g/cm2 [−0·134 to −0·057], p<0·0001 for both). 2 years after the completion of treatment (median follow-up 60 months [range 15·5–96·6]), patients not receiving zoledronic acid still had decreased BMD at both sites compared with baseline (lumbar spine −6·3%, mean difference −0·067 g/cm2 [−0·106 to −0·027], p=0·001; trochanter −4·1%, mean difference −0·03 g/cm2 [−0·062 to 0·001], p=0·058). Patients who received zoledronic acid had stable BMD at 36 months (lumbar spine +0·4%, mean difference 0·004 g/cm2 [−0·024 to 0·032]; trochanter +0·8%, mean difference 0·006 g/cm2 [−0·018 to 0·028]) and increased BMD at 60 months at both sites (lumbar spine +4·0%, mean difference 0·039 g/cm2 [0·005–0·075], p=0·02; trochanter +3·9%, mean difference 0·028 g/cm2 [0·003–0·058], p=0·07) compared with baseline. Interpretation: Goserelin plus tamoxifen or anastrozole for 3 years without concomitant zoledronic acid caused significant bone loss. Although there was partial recovery 2 years after completing treatment, patients receiving endocrine therapy alone did not recover their baseline BMD levels. Concomitant zoledronic acid prevented bone loss during therapy and improved BMD at 5 years. Funding: AstraZeneca (London, UK) and Novartis (Basel, Switzerland). [Copyright &y& Elsevier]

Background/purpose: In this study we assessed the applicability of image analysis and a machine learning algorithm on diagnostic discrimination of benign and malignant melanocytic skin tumours in in vivo confocal laser-scanning microscopy (CLSM). Methods: A total of 857 CLSM tumour images including 408 benign nevi and 449 melanoma images was evaluated. Image analysis was based on features of the wavelet transform. For classification purposes we used a classification tree software (CART). Moreover, automated image analysis results were compared with the prediction success of an independent human observer. Results: CART analysis of the whole set of CLSM tumour images correctly classified 97.55% and 96.32% of melanoma and nevi images. In contrast, sensitivity and specificity of 85.52% and 80.15% could be reached by the human observer. When the image set was randomly divided into a learning (67% of the images) and a test set (33% of the images), overall 97.31% and 81.03% of the tumour images in the learning and test set could be classified correctly by the CART procedure. Conclusion: Provided automated decisions can be used as a second opinion. This can be valuable in assisting diagnostic decisions in this new and exciting imaging technique. [ABSTRACT FROM AUTHOR]

BACKGROUND Recently, a wide range of new noninvasive therapies has been introduced for the treatment of actinic keratoses. As these treatment options do not provide tissue for histopathologic examination, in vivo confocal laser scanning microscopy may become an important method for obtaining a reliable diagnosis. OBJECTIVE The objective was to validate the diagnostic confocal examination of actinic keratoses. METHODS Thirty actinic keratoses and skin fields from the contralateral sides of the patients were consecutively sampled and examined using a confocal microscope. Stored images were rated by four independent observers. RESULTS Distinct diagnostic morphologic features could be visualized. Overall, sensitivity of 93.34% and specificity of 88.34% could be achieved by two clinical dermatooncologists (positive predictive value 88.94%, negative predictive value 93.15%). Assessment of distinct confocal microscopy features showed a moderate interobserver correlation (κ= 0.4–0.6 in five of seven criteria). Classification and regression tree analysis yielded a one-step algorithm based on only one criterion (irregular keratinocyte cell borders), facilitating a correct classification in 86.67% of actinic keratoses and 85% of normal skin. LIMITATIONS Hyperkeratotic actinic keratoses were excluded from the study set. CONCLUSIONS This study provides a set of morphologic confocal microscopy criteria showing promise as a noninvasive monitoring tool in the treatment of actinic keratoses. [ABSTRACT FROM AUTHOR]

Tumor growth requires the formation of new blood vessels, a phenomenon known as angiogenesis. The most important regulator of angiogenesis is vascular endothelial growth factor (VEGF). Several common polymorphisms in the VEGF-gene have been associated with different VEGF expression, production and plasma levels according to allele status, and influence the risk of developing different types of cancer. Therefore, these variants might be risk factors for colorectal cancer (CRC). In the present case–control study, VEGF genotypes of the +936 C > T, −2578 C > A and −634 G > C polymorphisms were determined in 427 patients with histologically verified CRC and 427 age and sex-matched healthy control subjects. Genotypes were analyzed by a fluorogenic exonuclease assay (TaqMan™). P-value for age at diagnosis was analyzed by student’s t test, P-values for tumor characteristics were determined by Pearson’s Chi-square test. Threshold for significance was P < 0.05. At the time of diagnoses, patients were between 29 and 83 years of age, with a mean age of 61 ± 10.9 years. VEGF −2578 C > A and VEGF −634 G > C genotype frequencies were similar among patients and controls. Carriers of the 936T-allele were found slightly more frequent among controls (27.2%) than among patients (22.5%), but this difference did not reach statistical significance ( P = 0.07). Furthermore, no correlation was found between all these variants and tumor characteristics like size, histological grading, positive regional lymph node metastases or tumor stage. We conclude that the investigated polymorphisms are not associated with individual susceptibility to colorectal cancer. [ABSTRACT FROM AUTHOR]

Background Clinical trial data have shown that among breast cancer patients who were disease free after 5 years of adjuvant treatment with tamoxifen, further extended treatment with the nonsteroidal aromatase inhibitor letrozole reduces breast cancer recurrence. We examined the efficacy and tolerability of extended adjuvant therapy with another aromatase inhibitor, anastrozole, for 3 years among women who had completed 5 years of adjuvant therapy. Methods Austrian Breast and Colorectal Cancer Study Group (ABCSG) Trial 6a is an extension of ABCSG Trial 6, in which hormone receptor-positive postmenopausal patients received 5 years of adjuvant tamoxifen, with or without the aromatase inhibitor aminoglutethimide, for the first 2 years of therapy. For ABCSG Trial 6a, patients who were disease free at the end of Trial 6 were randomly assigned to receive either 3 years of anastrozole or no further treatment. Efficacy data were analyzed with the use of a Cox proportional hazards regression model with two-sided P values and Kaplan-Meier curves, and tolerability data were estimated using logistic regression analysis with odds ratios and 95% confidence intervals (CIs). Results ABCSG Trial 6a included 856 patients. At a median follow-up of 62.3 months, women who received anastrozole (n = 387) had a statistically significantly reduced risk of recurrence (locoregional recurrence, contralateral breast cancer, or distant metastasis) compared with women who received no further treatment (n = 469; hazard ratio = 0.62; 95% CI = 0.40 to 0.96, P = .031). Anastrozole was well tolerated, and no unexpected adverse events were reported. Conclusions These data confirm the benefit of extending adjuvant tamoxifen therapy beyond 5 years with anastrozole compared with no further treatment. Further research is required to define the optimum length of extended adjuvant therapy and to investigate the possibility of tailoring this period to suit different disease types. [ABSTRACT FROM AUTHOR]

Aim: To evaluate feasibility and safety of neoadjuvant chemotherapy with capecitabine, oxaliplatin and bevacizumab followed by concomitant standard chemoradiation and surgical resection in patients with high-risk locally advanced rectal cancer., Patients and Methods: Magnetic resonance imaging (MRI)-defined high-risk cT3/4 rectal cancer patients were treated with 3 cycles of neoadjuvant chemotherapy with capecitabine (1,000 mg/m 2 twice daily days 1-14, 22-35, 43-56), oxaliplatin (130 mg/sqm on days 1, 22, 43) and bevacizumab (7.5 mg/kg on days 1, 22, 43) followed by capecitabine (825 mg/m 2 twice daily on radiotherapy days week 1-4) concomitantly with radiotherapy (1.8 Gy daily up to 45 Gy in 5 weeks) and surgical resection by total mesorectal excision. Feasibility, safety, response rate and postoperative morbidity were evaluated., Results: Twenty-five patients were recruited. Median age was 62 years (range=24-78 years) and all patients had Eastern Cooperation Oncology Group (ECOG) performance status 0. From all patients, 79.2% finished neoadjuvant chemotherapy. Twenty patients underwent surgery. Pathologic complete remission rate, R0 resection and T-downstaging were achieved in 25%, 95% and 54.2% of the "intention to treat" (ITT) patients. The most common grade 3 adverse events (AEs) during neoadjuvant chemotherapy were diarrhea (16.6%) and mucositis (12.5%). In one patient, a grade 4 acute renal failure occurred (4.2%). During chemoradiation, skin reactions (5.3%) were the most common grade 3 AEs. Two major perioperative complications required re-intervention., Conclusion: Neoadjuvant chemotherapy with bevacizumab, capecitabine and oxaliplatin followed by concomitant standard chemoradiation is feasible in patients with high-risk locally advanced rectal cancer (LARC) and resulted in complete pathologic remission (pCR) rate of 25% and neoadjuvant chemotherapy completion rate of 80%., (Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Expression of miR-96-5p is frequently altered in various types of cancer and the KRAS oncogene has been identified as one of its potential targets. However, the biological role of miR-96-5p expression in colorectal cancer (CRC) and its ability to predict the clinical course of patients have not been investigated yet. In this study, we explored miR-96-5p expression in 80 CRC patients and evaluated the impact on clinical outcome by Kaplan-Meier curves and multivariate Cox proportional models. In vitro miR-96-5p inhibition and overexpression were performed in CRC cells and the effects on cellular growth, anchorage-independent growth, apoptosis, and epithelial-mesenchymal transition (EMT)-related gene expression were explored. Low miR-96-5p expression levels in tumor tissue were associated with distant metastasis (P = 0.025) and multivariate Cox regression analysis identified low levels of miR-96-5p as an independent prognostic factor with respect to cancer-specific survival (hazard ratio = 1.78, 95%CI = 1.03-3.03, P < 0.038). In vitro overexpression of miR-96-5p led to a reduced cellular growth rate (P < 0.05), reduced colonies in soft agar (P < 0.05), corroborated by a decreased cyclin D1 and increased p27-CDKN1A expression (P < 0.05). Forced expression of miR-96-5p in CRC cells entailed no effects on apoptosis or EMT-related genes but decreased the expression levels of the KRAS oncogene (P < 0.05). Despite regulating KRAS expression, there was no significant association in miR-96-5p expression levels and response rates to EGFR-targeting agents. In conclusion, our data suggest that miR-96-5p influences cellular growth of CRC cells and low expression of miR-96-5p seems to be associated with poor clinical outcome in CRC patients., (© 2014 Wiley Periodicals, Inc.)

We investigated the hypothesis that the varying treatment efficacy of adjuvant 5-fluorouracil (5FU) in stage III colon cancer is linked to the TP53 mutational status. ABCSG-90 was a prospective randomized trial in which effect of adjuvant 5FU was studied in stage III colon cancer patients. Tumor material of 70% of these patients (389/572) was available for analysis of the biomarker TP53 using a TP53-gene-specific Sanger sequencing protocol. Median follow-up was 88 months. TP53 mutation frequency was 33%. A significant interaction between TP53 status, outcomes and nodal category was found (P = 0.0095). In the N1 category, TP53 wildtype patients had significantly better overall survival than TP53 mutated (81.0% vs. 62.0% overall survival at 5 years; HR = 2.131; 95% CI: 1.344-3.378; P = 0.0010). In the N2 category, the TP53 status did not affect survival (P = 0.4992). In TP53 wildtype patients, the prognostic significance of N category was significantly enhanced (P = 0.0002). In TP53 mutated patients, survival curves of N1 and N2 patients overlapped and nodal category was no longer prognostic. The biomarker TP53 independently predicted effect of adjuvant 5FU in N1 colon cancer patients. TP53 was not predictive in N2 patients, in whom 5FU is known to have no effect.

Surgical excision of colorectal cancer at early clinical stages is highly effective, but 20-30% of patients relapse. Therefore, it is of clinical relevance to identify patients at high risk for recurrence, who would benefit from adjuvant chemotherapy. The objective of this study was to identify prognostic and/or predictive methylation markers in stage II colorectal cancer patients. Therefore, we selected six gene promoters (FZD9, PCDH10 (protocadherin 10), SFRP2, SPARC (secreted protein acidic and rich in cysteine), UCHL1 (ubiquitin carboxyl-terminal hydrolase 1), and WIF1) for methylation analysis in formalin-fixed, paraffin-embedded primary tumor samples of colorectal cancer patients (n=143) who were enrolled in a prospective randomized phase III trial of the Austrian Breast and Colorectal cancer Study Group. Patients were randomized to adjuvant chemotherapy with 5-fluorouracil and leucovorin or surveillance only. Survival analyses revealed that combined evaluation of three promoters (PCDH10, SPARC, and UCHL1) showed differential effects with regard to disease-free survival and overall survival in the two treatment groups (significance level 0.007). In the chemotherapy arm, a statistically insignificant trend for patients without methylation toward longer survival was observed (P=0.069 for disease-free survival and P=0.139 for overall survival). Contrary, patients in the surveillance arm without methylation in their gene promoters had shorter disease-free survival and overall survival (P=0.031 for disease-free survival and P=0.003 for overall survival), indicating a prognostic effect of methylation in this group (test for interaction, P=0.006 for disease-free survival and P=0.018 for overall survival). These results indicate that promoter methylation status of PCDH10, SPARC, and UCHL1 may be used both as prognostic and predictive molecular marker for colorectal cancer patients and, therefore, may facilitate treatment decisions for stage II colorectal cancer.

Background and Objectives: Accumulating evidence indicates an important pathophysiological role of fibrinogen on tumor cell progression and metastases in different types of cancer. The aim of the present study was to evaluate the prognostic relevance of pre-operative fibrinogen levels on clinical outcome in a large cohort of STS patients., Methods: Two hundred ninety-four consecutive STS patients were retrospectively evaluated. Cancer-specific survival (CSS), disease-free survival (DFS), and overall survival (OS) were assessed using the Kaplan-Meier curves and Cox regression models. Finally, we supplemented the well-established Kattan nomogram by the fibrinogen level and evaluated the gain of predictive accuracy of this novel nomogram by Harrell's concordance index (c-index)., Results: An elevated plasma fibrinogen level was significantly associated with established prognostic factors, including age, tumor grade, size, and depth (P<0.05). Furthermore, in multivariate analysis, increased fibrinogen levels were significantly associated with a poor outcome for CSS (HR=2.48; 95% CI=1.28-4.78; P=0.007), DFS (HR=2.00; 95% CI=1.11-3.60; P=0.021), and OS (HR=2.20; 95% CI=1.39-3.47; P<0.001). The estimated c-index was 0.747 using the original Kattan nomogram and 0.779 when the fibrinogen levels was added., Conclusion: The pre-operative plasma fibrinogen level may represent a strong and independent unfavorable prognostic factor for CSS, DFS and OS in STS patients., (© 2013 Wiley Periodicals, Inc.)

Angiogenesis and cell cycle control play critical roles in breast cancer susceptibility and clinical outcome and are mainly controlled by vascular endothelial growth factor (VEGF) and cyclin-dependent kinases, respectively. Functional germline polymorphisms in these genes alter the function, thereby causing inter-individual differences in breast cancer risk and clinical outcome. In this study, we investigated the influence of the functional polymorphisms VEGF-A rs3025039 C > T and CCND1 rs9344 G > A on risk and clinical outcome in early-stage breast cancer. DNA of 539 female patients with histologically confirmed early-stage breast cancer and 804 control subjects was genotyped for these polymorphisms. Genotypes were tested for associations with breast cancer risk and clinical outcome. There was no significant association between the polymorphisms and breast cancer risk. However, the minor allele of VEGF-A rs3025039 C > T was significantly associated with decreased recurrence-free survival (HR 1.845; 95% confidence interval [CI] 1.035-3.290; P = 0.038) and remained significant in multivariate analysis (HR 1.880; 95% CI 1.020-3.465; P = 0.043). Patients carrying at least one A-allele in CCND1 rs9344 G > A showed a trend towards decreased recurrence-free survival in univariate analysis (HR 2.379; 95% CI 0.841-6.728; P = 0.068). This study provides evidence that the functional VEGF-A rs3025039 C > T polymorphism influences recurrence-free survival in early-stage breast cancer., (© 2013 Wiley Periodicals, Inc.)

Unlabelled: AIM/ BACKGROUND: The neutrophil-to-lymphocyte ratio (NLR), a combined indicator of inflammation and immunology, is as yet unidentified regarding the clinical outcome of stage II and III colon cancer patients. We evaluated the effect of NLR on time-to-recurrence (TTR) and overall survival (OS) in selected patients., Patients and Methods: A total of 504 patients with stage II and III colon cancer were included in this retrospective study. Preoperative NLR with a cut-off level of 4 was associated with TTR and OS., Results: In univariate analysis, elevated NLR was significantly associated with decreased TTR (p=0.001) and remained significant in multivariate analysis (p=0.006). Patients with NLR >4 showed a median TTR of 62.2 months. In contrast, patients with NLR ≤ 4 had a median TTR of 92.6 months., Conclusion: This study suggests that preoperative NLR may be an independent prognostic marker for TTR in stage II and III colon cancer patients.

Recent evidence suggests that PLS3 (T-Plastin), an important member of the actin filamentous network, significantly influences cell invasion and metastasis. Germline polymorphisms within the PLS3 gene may impact the gene's function, resulting in inter-individual differences in tumor recurrence capacity. In the present study, we investigated the association of germline polymorphisms in PLS3 to predict time to recurrence (TTR) in patients with stage II and III colon cancer. A total of 264 patients with histologically confirmed colon cancer were included in this retrospective study. Germline DNA was genotyped for rs871773 C>T, rs757124 C>G, rs1557770 G>T, rs6643869 G>A, and rs2522188 C>T in the PLS3 gene by 5'-exonuclease (TaqMan™) technology. As the PLS3 gene is located on the X chromosome, a gender-specific statistical analysis was performed. In univariate analysis, the minor allele of PLS3 rs871773 C>T was significantly associated with decreased TTR in women (hazard ratio (HR) = 5.02; 95 % confidence interval (CI) = 1.251-20.114; p = 0.023) and remained significantly associated in multivariate analysis (HR = 6.165; 95 % CI = 1.538-24.716; p = 0.010). Female patients carrying the C/T genotype in PLS3 rs871773 showed a median TTR of 69 months. In contrast, female patients with homozygous C/C had a median TTR of 112 months. There were no significant associations between PLS3 rs871773 C>T and TTR in male and between the other polymorphisms and TTR in male or female colon cancer patients. In conclusion, we identified a common gene variant in PLS3 as an independent prognostic marker in female patients with stage II and III colon cancer. Larger prospective trials are warranted to confirm these findings.

With the increasing number of available predictive biomarkers, clinical management of cancer is becoming increasingly reliant on the accurate serial monitoring of tumor genotypes. We tested whether tumor-specific copy number changes can be inferred from the peripheral blood of patients with cancer. To this end, we determined the plasma DNA size distribution and the fraction of mutated plasma DNA fragments with deep sequencing and an ultrasensitive mutation-detection method, i.e., the Beads, Emulsion, Amplification, and Magnetics (BEAMing) assay. When analyzing the plasma DNA of 32 patients with Stage IV colorectal carcinoma, we found that a subset of the patients (34.4%) had a biphasic size distribution of plasma DNA fragments that was associated with increased circulating tumor cell numbers and elevated concentration of mutated plasma DNA fragments. In these cases, we were able to establish genome-wide tumor-specific copy number alterations directly from plasma DNA. Thus, we could analyze the current copy number status of the tumor genome, which was in some cases many years after diagnosis of the primary tumor. An unexpected finding was that not all patients with progressive metastatic disease appear to release tumor DNA into the circulation in measurable quantities. When we analyzed plasma DNA from 35 patients with metastatic breast cancer, we made similar observations suggesting that our approach may be applicable to a variety of tumor entities. This is the first description of such a biphasic distribution in a surprisingly high proportion of cancer patients which may have important implications for tumor diagnosis and monitoring., (Copyright © 2013 UICC.)

Recent data suggest that erythropoietin (EPO) plays a substantial role in cancer development and clinical outcome by stimulating cell proliferation, invasion and angiogenesis. A functional polymorphism (rs1617640 G>T) in the promoter region of the EPO gene increases EPO protein expression. In the present study, we investigated the association of EPO rs1617640 G>T with susceptibility and clinical outcome of early-stage breast cancer. Genomic DNA of 539 female patients with histologically confirmed early-stage breast cancer and 804 controls was genotyped for EPO rs1617640 G>T. No association was found between EPO rs1617640 G>T and early-stage breast cancer susceptibility and clinical outcome (hazard ratio=1.24, 95% confidence interval=1.82-1.90, p=0.31). In conclusion, our findings suggest a lack of influence of EPO rs1617640 G>T on early-stage breast carcinogenesis and clinical outcome.

The nuclear factor κ-light-chain enhancer of activated B-cells (NF-κB) signaling pathway is regarded as an important factor in inflammation and carcinogenesis. Recently, a role in hepatocarcinogenesis has been attributed to the NF-κB regulatory subunit IKKγ (NEMO) using knockout mice. However, a detailed investigation of NEMO expression in human hepatocellular carcinomas (HCCs) has not yet been reported. We selected 85 HCC patients who had undergone curative liver resection and analyzed NEMO expression of the respective tumors by immunohistochemistry, Western blotting, and real-time PCR. NEMO expression was correlated with clinicopathological parameters, and the impact on 5-year disease-free survival and 5-year overall survival was calculated using multivariate Cox proportional models. In our study, complete loss of NEMO immunoreactivity was found in 34 (40%) of 85 HCCs compared with their adjacent nonneoplastic tissue (P < .05). NEMO messenger RNA (mRNA) expression was detected in all HCC cases; however, no correlation between NEMO immunoreactivity and mRNA level was found. Five-year overall survival rates for patients with low and high NEMO expression were 22% and 50%, respectively (P = .049). However, high tumor stage, but not level of NEMO expression, was confirmed as an independent poor prognostic factor for 5-year disease-free survival (hazards ratio [HR] = 2.1, 95% confidence interval [CI] = 1.3-3.6, P = .009) and 5-year overall survival (HR = 2.5, CI = 1.4-4.4, P = .002). In conclusion, a loss of NEMO immunoreactivity occurs in a substantial proportion of human HCCs. Although low NEMO expression is correlated with a poor 5-year overall survival in patients with HCC, NEMO cannot be regarded as an independent prognostic marker for predicting the clinical outcome of patients suffering from HCC., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: Integrins influence tumourigenesis, tumor progression and development of metastases. The impact of polymorphisms in integrin genes on relapse-free survival (RFS) and overall survival (OS) for 433 Caucasian patients with colorectal cancer was analysed in this retrospective study., Patients and Methods: A Cox regression model including integrin genotype, age, grading, tumour size, number of lymph nodes examined, number of metastatic lymph nodes, stage and application of fluorouracil-based adjuvant chemotherapy was used to estimate their effect., Results: After a median follow-up of 41 months for RFS and 55 months for OS, no significant correlation between the ITGA2 1648A allele (RFS p=0.618, OS p=0.604), the ITGA2 807T allele (RFS p=0.603, OS p=0.807) and the ITGB3 176C allele (RFS p=0.719, OS p=0.261) and survival was detectable., Conclusion: The investigated integrin polymorphisms are not associated with RFS or OS in colorectal cancer patients.

With an incidence of about 300 000 new cases colorectal cancer (CRC) is the second leading cause of cancer-related death in Europe and the United States. Environmental and genetic factors influence CRC risk. Hypoxia-inducible factor-1 (HIF-1), a heterodimeric protein composed of two subunits, HIF-1 alpha and HIF-1 beta, plays a critical role in oxygen homeostasis and is involved in angiogenesis and cell proliferation. The gene for the HIF-1 alpha subunit (HIF1A) carries two common missense mutations-P582S (rs11549465) and A588T (rs11549467)-which both have been related to increased trans-activation capacity of HIF1A. In our case-control study we investigated the association between these polymorphisms and CRC risk. We investigated 381 patients with histologically confirmed CRC and 2156 control subjects. HIF1A genotypes were determined by exonuclease (TaqMan) assays. For determination of microvessel density (MVD) tumor sections were stained using a mouse monoclonal antibody recognizing the pan-endothelial marker CD31. In a multivariate logistic regression analysis including age and sex neither the HIF1A 582S allele (Odds ratio: 1.204; 95% confidence interval 0.911-1.592; P = 0.193) nor the 588T allele was significantly associated with CRC (Odds ratio: 0.851; 95% confidence interval 0.444-1.631; P = 0.626). However, in an exploratory analysis, the HIF1A 588T allele was associated with tumor localization (P = 0.016) and tumor size (P = 0.003). MVD was similar in tumors of patients carrying HIF1A 588T allele and patients without this rare allele. We conclude that functional polymorphisms in the HIF1A gene do not modify CRC risk but maybe associated with clinic-pathological features of the disease., (2010 Wiley-Liss, Inc.)

Background: Hypoxia inducible factor-1 (HIF-1) is the key regulator of cellular responses to hypoxia and plays a central role in tumour growth. Recently, two single nucleotide polymorphisms (SNPs) in the HIF-1 alpha gene, C1772T and G1790A, were shown to cause significantly higher transcriptional activity than did the wild-type. This study aimed to investigate the effect of these SNPs on the prognosis of colorectal cancer (CRC)., Patients and Methods: DNA from 336 CRC patients was genotyped. Genotypes of each polymorphism were tested for association with disease-free survival (DFS) using univariate and multivariate Cox-regression analysis., Results: Genotype frequencies were: CC 75.6%, CT 18.8% and TT 1.8% for HIF1A1 C1772T and GG 93.2%, GA 2.7% and AA 0% for G1790A. A statistically significant association between DFS and clinicopathological features was observed. However, no association was found between HIF1A1 C1772T (p=0.44; risk ratio of recurrence, RR=1.19, 95% confidence interval, CI=0.77 to 1.83) and G1790A (p=0.89; RR=0.92, 95% CI=0.29 to 2.90) polymorphisms and DFS in univariate and multivariate Cox-regression analysis., Conclusion: These results suggest that HIF1A1 C1772T and G1790A polymorphisms are not involved in the progression or metastasis of CRC.

Genetic polymorphisms are responsible for inter-individual variation and diversity and have been recently considered as the main genetic elements involved in the development and progression of cancer. We examined associations between common germline genetic variants in 7 genes involved in folate metabolism, cell proliferation and apoptosis, prostaglandin synthesis, detoxification of compounds and inflammation, and disease-free survival among women diagnosed with invasive breast cancer. DNA from up to 432 women was genotyped for 8 polymorphisms. The genotypes of each polymorphism were tested for association with disease-free survival using univariate and multivariate Cox regression analysis. The model was adjusted for known breast cancer prognostic factors. The rare allele of the IL-10 592C>A polymorphism was significantly associated with reduced disease-free survival (P = 0.018, risk ratio of recurrence (RR) = 1.45, 95% confidence interval (CI) = 1.06-1.98), which was not attenuated after adjusting for age at diagnosis, tumor size, lymph node status, clinical stage, histological grade, estrogen receptor status, progesterone receptor status, and treatment modalities (P = 0.019, RR = 1.48, 95% CI = 1.066-2.044). No association was found between MTHFR 677C>T, TGFB1 29T>C, FASLG 844C>T, FAS 1377G>A, FAS 670A>G, PTGS2 8473T>C and SULT1A1 638G>A polymorphisms and disease-free survival. Our data suggest that the rare allele of IL-10 592C>A may be a potential prognostic marker in breast cancer for disease-free survival.

The involvement of the immune system for the course of breast cancer, as evidenced by varying degrees of lymphocyte infiltration (LI) into the tumor is still poorly understood. The aim of this study was to evaluate the prognostic value of LI in breast cancer samples using microarray-based screening for LI-associated genes. Starting from the observation that most published ER gene signatures are heavily influenced by the LI effect, we developed and applied a novel approach to dissect molecular signatures. Further, a meta-analysis encompassing 1,044 hybridizations showed that LI alone is not sufficient to highlight breast cancer patients with different prognosis. However, for ER positive patients, high LI was associated with shorter survival times, whereas for ER negative patients, high LI is significantly associated with longer survival. Annotation of LI, in addition to ER status, is important for breast cancer patient prognosis and may have implications for the future treatment of breast cancer.

Women undergoing treatment for breast cancer often experience a marked decrease in bone mineral density. This decrease is observed with chemotherapy as well as endocrine therapy and is more pronounced and rapid than normal postmenopausal bone loss. Pharmacologic intervention is, therefore, necessary in many cases to preserve bone health and prevent fractures. Many small studies have demonstrated that cancer therapy-induced bone loss (CTIBL) is effectively prevented by bone-targeted therapies, such as bisphosphonates and other inhibitors of bone resorption. Recently, several trials have confirmed the efficacy of bisphosphonates in the prevention of CTIBL in both premenopausal and postmenopausal women with early-stage breast cancer. In addition, concomitant treatment with zoledronic acid 4 mg every 6 months and standard adjuvant endocrine therapy has been reported to significantly improve disease-free survival and decrease disease recurrence in bone as well as other sites compared with standard therapy alone. Zoledronic acid treatment has also decreased residual tumor volume in the neoadjuvant setting. Furthermore, long-term follow-up of a single study in patients with bone marrow micrometastases from breast cancer revealed overall survival benefits for patients receiving clodronate 1600 mg/day compared with placebo; however, combined results from several trials of clodronate are inconclusive. Overall, a large body of evidence is accumulating to support the potential adjuvant benefits of bisphosphonates in the treatment of early-stage breast cancer. Results from ongoing studies are expected to further elucidate the benefits of bisphosphonates in maintaining bone health and improving clinical outcomes in patients with breast cancer.

BRAF V600E is the predominantly occurring mutation of the cytoplasmic kinase BRAF, and, in colorectal cancer, its determination provides a diagnostic exclusion criterion for hereditary nonpolyposis colorectal cancer. The aim of our study was to develop a sensitive BRAF V600E high resolution melting (HRM) assay. We first established and optimized the BRAF HRM assay using a cell line dilution model, enabling us to detect 1% mutant DNA in a background of wild-type DNA. In a comparison, DNA sequencing and real-time allele-specific PCR in the cell line dilution model HRM assay proved to be more sensitive than DNA sequencing and denaturing high performance liquid chromatography, retaining the same sensitivity as real-time allele-specific PCR. In a learning set of 13 patients with known BRAF V600 status, the mutation was detected with high concordance by all four methods. Finally, we validated the HRM assay on 60 formalin-fixed, paraffin-embedded colorectal cancer samples. Although all mutated samples were correctly identified by HRM, the detection limit of the HRM assay decreased when using low-quality DNA derived from formalin-fixed, paraffin-embedded samples. In conclusion, HRM analysis is a powerful diagnostic tool for detection of BRAF V600E mutation with a high sensitivity and high-throughput capability. Despite the expected decrease in sensitivity, HRM can reliably be applied in archival formalin-fixed, paraffin-embedded samples tissues.

High-resolution melting (HRM) analysis is a novel tool for analysis of promoter methylation. The aim of the present study was to establish and validate HRM analysis for detection of promoter methylation on archival formalin-fixed paraffin-embedded tissues from colorectal cancer patients. We first evaluated HRM assays for O(6)-methylguanine-DNA methyltransferase (MGMT) and adenomatous polyposis coli (APC) promoter methylation on a methylated DNA dilution matrix and DNA extracted from eight fresh or formalin-fixed paraffin-embedded human cancer cell lines. Then we used these assays for the analysis of MGMT and APC promoter methylation in a subset of archival formalin-fixed paraffin-embedded colorectal tumor specimens. All samples with promoter methylation of MGMT or APC and randomly selected samples without promoter methylation were analyzed twice. All results generated by HRM were validated with MGMT and APC MethyLight assays. APC and MGMT promoter methylation data were consistent and reproducible throughout the dilutions and all three replicates in the methylated DNA dilution matrix and between two experiments in clinical samples. There was high concordance between HRM and MethyLight results. HRM for APC promoter methylation revealed consistent results between fresh and formalin-fixed paraffin-embedded human cancer cell line DNA. The methylation status in archival tumor specimens from patients with colorectal cancer can therefore be determined with high quality by HRM. The ability to analyze archival tissues greatly facilitates further research and its clinical implementation.

Purpose: To evaluate the impact that pre- and postoperatively administered chemotherapy with cyclophosphamide, methotrexate and fluorouracil (CMF) and postoperative chemotherapy vs. postoperative chemotherapy alone have on long-term prognosis., Patients and Methods: The ABCSG conducted a nationwide randomized phase III trial in high-risk endocrine non-responsive breast cancer patients comparing pre- and postoperative chemotherapy containing CMF as preoperative treatment vs. postoperative chemotherapy alone between 1991 and 1999. From 1996 the ABCSG-07 protocol was amended to also allow randomization of high-risk endocrine-responsive patients. Of 423 eligible patients with high-risk primary breast cancer, 203 patients were randomly assigned to preoperatively receive three cycles of CMF (cyclophosphamide, methotrexate, fluorouracil; 600/40/600 mg/m(2)) intravenously on day 1 and 8, while 195 patients received postoperative chemotherapy alone. In both groups, three cycles of CMF were given initially, and another three cycles of CMF were administered in node-negative patients, whereas node-positive patients received three cycles of EC (epirubicin, cyclophosphamide; 70/600 mg/m(2))., Results: Overall response rate to preoperative chemotherapy with three cycles of CMF was 56.2%; complete pathological response was achieved in 12 patients (5.9%). Recurrence-free survival was significantly better in patients receiving chemotherapy postoperatively (HR 0.7, 0.515-0.955; P = 0.024). No survival difference was observed between the two therapy groups (HR 0.800, 0.563-1.136; P = 0.213)., Discussion: Preoperative chemotherapy with CMF has to be considered as insufficient in high-risk breast cancer patients. Delayed surgery and anthracycline-based chemotherapy result in shorter recurrence-free survival but not overall survival.

Purpose: Vascular endothelial growth factor (VEGF) is a key regulator of tumor-induced angiogenesis and is required for growth of tumors. We tested the hypothesis that VEGF gene polymorphisms may be associated with breast cancer., Experimental Design: We performed a case-control study including 804 female incident breast cancer patients and 804 female age-matched healthy control subjects. We selected seven VEGF candidate polymorphisms and determined genotypes by 5'-nuclease (TaqMan) assays. Furthermore, VEGF plasma levels and genotypes were analyzed in a group of 81 healthy volunteers (64 men and 17 women)., Results: Haplotype analysis showed two separate blocks of high-linkage disequilibrium, formed by five polymorphisms upstream of the coding sequence (promoter and 5' untranslated region) and two polymorphisms downstream of the coding sequence. None of the single polymorphisms or haplotypes was significantly associated with the presence of breast cancer. After Bonferroni correction for multiple testing, only one statistical signifcant association between VEGF genotypes and haplotypes and tumor characteristics was observed (-634C allele and small tumor size; p < 0.001). In a multivariate regression analysis including sex, age, VEGF genotypes, and haplotypes as covariates and VEGF plasma level as dependent variable, none of the VEGF polymorphism or haplotypes was a significant predictor of VEGF plasma levels., Conclusions: Our findings do not support the hypothesis that VEGF polymorphisms are associated with breast cancer risk. The association of the VEGF -634C allele with small tumor size is in clear contrast to a previous publication and should be interpreted with caution until replicated by additional studies.

Purpose: To determine the effects of anemia on local relapse-free, relapse-free, and overall survival (LRFS, RFS, and OS, respectively) in premenopausal, primary breast cancer patients receiving adjuvant polychemotherapy, and to determine which conventional prognostic factors affected these outcomes., Experimental Design: Four hundred twenty-four premenopausal patients with early-stage primary breast cancer and hormone receptor-expressing tumors were treated with i.v. cyclophosphamide/methotrexate/5-fluorouracil (CMF) polychemotherapy as part of an adjuvant phase III trial (Austrian Breast and Colorectal Cancer Study Group Trial 5). The influence of anemia (hemoglobin <12 g/dL) on LRFS, RFS, and OS was evaluated in a retrospective analysis., Results: Of 424 patients, 77 (18.2%) developed anemia on CMF chemotherapy. After a median follow-up time of 5 years, 8.9% of nonanemic patients had local relapse compared with 19.6% of anemic patients (P=0.0006). Although mastectomy was associated with anemia (26% versus 13.7% in breast conserving surgery; P=0.002), multivariate analysis did not show mastectomy per se to be a significant risk factor for LRFS. Age, lymph node status, and hemoglobin had an independent significant influence on LRFS (P<0.005). Anemic patients had a relative risk of 2.96 (95% confidence interval, 1.41-6.23) for developing local relapse in comparison with nonanemic patients., Conclusion: Premenopausal breast cancer patients who developed anemia during the CMF regimen had significantly worse LRFS. In Austrian Breast and Colorectal Cancer Study Group Trial 5, anemia may have contributed to an almost doubled incidence of local recurrence in the chemotherapy arm. Molecular targets associated with tumor hypoxia and distinct from erythropoiesis should receive further attention in experimental and clinical settings.

Regulatory T cells may be crucial in the development of T cell tolerance to malignancies and contribute to immune dysfunctions. We investigated the percentage, activity, and onset of apoptosis of T cell subpopulations by multicolor flow cytometry in metastatic epithelial cancer patients compared to normal controls. Furthermore, a possible relationship between the presence of circulating tumor cells detected by immunocytochemistry and immune cell abnormalities was evaluated. Our study demonstrated a significantly elevated proportion of regulatory T cells in cancer patients (p < 0.001). In contrast to all other T cell subpopulations, regulatory T cells showed comparable Annexin V-binding characteristics in patients and normal controls. No relationship between the detection of circulating tumor cells and immune dysfunction was observed. These results indicate that cancer patients have a higher number of regulatory T cells with resistance to apoptotic stimuli partly responsible for immune dysfunctions as often observed in cancer patients.

In the biology of complex disorders, such as breast cancer, interactions among genetic factors may play an important role and theoretical considerations suggest that gene-gene interactions are quite common in such diseases. In this case-control study with 500 breast cancer patients and 500 population-based healthy sex- and age-matched control subjects, we applied a multigenic approach to examine the associations with breast cancer risk of a comprehensive panel of 16 selected polymorphisms in a variety of pathways using classification tree analysis (CART). Overall, 79.6% of all breast cancer patients and 80.6% of all control subjects were correctly classified on the basis of their individual genetic profile by the classification procedure. CART analysis of the data identified the heterozygous vascular endothelial growth factor (VEGF) and matrix metalloproteinase 3 (MMP3) genotype and homozygous cyclooxygenase-2 (PTGS2) mutant as the initial splits, indicating that these genotypes exert the greatest impact on the classification process. Breast cancer patients were primarily indicated by 30 distinct genetic profiles. The odds ratio of these genetic risk profiles for breast cancer was 16.12 (95% confidence interval 11.09-23.49). Five genetic profiles formed homogenous breast cancer subgroups and represented highest risk genetic profiles. This is the first comprehensive study to use a multigenic analysis for breast cancer and the data suggest that individuals with distinct genetic profiles are at an increased risk for breast cancer, confirming the importance of taking a multigenic approach for risk assessment.

Background: Primary malignant melanoma of the esophagus is an exceedingly rare disease. This tumor is typically aggressive and disseminates early via the lymphatics and the bloodstream with a mean survival time between 10 and 15 months after radical surgical resection. The role of chemotherapy and immunotherapy is unclear. No treatment plan for the disease has yet been established., Case Report: A 78-year-old man came for a checkup with a medical history of reflux esophagitis and chronic gastritis. Esophagogastroscopy showed a bluishgray tumor of the esophagus, and histology revealed features consistent with malignant melanoma. The patient underwent total transhiatal esophagectomy with curative intention, and esophagogastric anastomosis was performed. Immunohistochemistry revealed tumor cells strongly positive for the melanoma-specific antigen HMB45 and protein S-100, and negative for cytokeratin. A proposed postoperative chemotherapy was declined by the patient. Nine months after surgery, the patient's condition deteriorated, and a mediastinal lymph node conglomerate was found. Two months later, he died of bleeding into the cervical soft tissue., Conclusion: Up to date, radical surgical resection is the main treatment. Very little is known about the benefits of chemotherapy and immunotherapy. However, these therapeutic modalities may play an important role in the future.

Goals of Work: This study reflects variables being depicted as predictors of global quality of life in current research. The evaluation was conducted at the Division of Clinical Oncology at the Medical University in Graz, Austria., Patients and Methods: A sample of 210 breast cancer patients between ages 30 and 80 years was assessed 1-5 years after initial diagnosis in a tumor-free stage. Besides the socio-demographic and medical variables, the Brief Symptom Inventory (BSI), Impact of Event Scale (IES), Mental Adjustment to Cancer (MAC), and the Perceived Family Support (PFS) were used. To identify variables related to quality of life, stepwise multiple regression analyses were calculated., Main Results: In a regression analysis, the general severity index (BSI) was identified as the most important and helplessness/hopelessness (MAC) as the second important variable related to QoL. Including the BSI-subscales as predictors, the depression-subscale (BSI) explained 25% of the variance; in addition, somatization (BSI), helplessness/hopelessness (MAC), and having financial problems (semi-structured interview) were significantly related to global quality of life, but the medical variables showed no associations to the measured quality of life. Depression itself is associated with negative the impact of cancer, the number of stressful life events, being uncomfortable with the body, having financial problems and anxious preoccupation (MAC)., Conclusions: The awareness of the role of multi-factorial and associated variables could provide patients, family, and medical staff with appropriate and adequate tools to treat specific symptoms.

Implementation of hospital-based palliative care team is a new subject in German-speaking areas, especially in university hospitals. Our Section of Palliative Care was subsequently built up to a 12-bed palliative care unit, a home-based, and a hospital-based palliative care team. Analysis of the implementation strategies, development of the working profile, and documentation of the hospital-based palliative care team were done. During the first 2 years, quality and number of inquiries for palliative care increased significantly. In our opinion, a high degree of expertise, involvement of the nursing staff, and personal contact play a key role for a successful implementation of a palliative care team in a large university hospital.

Background: Melanoma and nonmelanoma skin cancer are the most frequent malignant tumors by far among whites. Currently, early diagnosis is the most efficient method for preventing a fatal outcome. In vivo confocal laser-scanning microscopy (CLSM) is a recently developed potential diagnostic tool., Methods: One hundred seventeen melanocytic skin lesions and 45 nonmelanocytic skin lesions (90 benign nevi, 27 malignant melanomas, 15 basal cell carcinomas, and 30 seborrheic keratoses) were sampled consecutively and were examined using proprietary CLSM equipment. Stored images were rated by 4 independent observers., Results: Differentiation between melanoma and all other lesions based solely on CLSM examination was achieved with a positive predictive value of 94.22%. Malignant lesions (melanoma and basal cell carcinoma) as a group were diagnosed with a positive predictive value of 96.34%. Assessment of distinct CLSM features showed a strong interobserver correlation (kappa >0.80 for 11 of 13 criteria). Classification and regression tree analysis yielded a 3-step algorithm based on only 3 criteria, facilitating a correct classification in 96.30% of melanomas, 98.89% of benign nevi, and 100% of basal cell carcinomas and seborrheic keratoses., Conclusions: In vivo CLSM examination appeared to be a promising method for the noninvasive assessment of melanoma and nonmelanoma skin tumors., (Copyright 2006 American Cancer Society.)

The purpose of this study was to investigate the influence of androgen receptor (AR) expression in tumour tissue on survival of patients with metastatic breast cancer. Tumour specimens from 232 patients with metastatic breast cancer were examined for presence of AR by immunohistochemistry. According to the extent of immunostaining, AR expression was classified as score 0, 1+, 2+, or 3+. AR positivity was observed in 164 (70.7%) tumours. The median survival after disease recurrence (SAR) of patients with AR-expressing tumours was significantly longer compared to that of patients with AR-negative tumours (21.89 months, 95% CI 17.23-26.55 vs 11.99 months, 95% CI 9.36-14.62; log-rank test 0.0282). In addition, patients with AR score 3+ had a significantly longer disease-free survival (DFS) compared to patients with AR score 0, 1+, and 2+, (24.67 months, 95% CI 13.72-35.62 vs 16.36 months, 95% CI 13.18-19.54, log-rank test 0.0043). Multivariate Cox analyses showed no statistically significant influence of AR expression on DFS or SAR. In conclusion, SAR is significantly longer in patients with AR-expressing breast carcinoma. However, AR expression is not an independent prognostic factor for SAR in metastatic breast cancer.

Integrins are cell surface receptors, which mediate cell-to-cell and cell-to-extracellular matrix adhesion. Some of them, e.g. alpha(V)beta(3), alpha(IIb)beta(3) and alpha(2)beta(1), have been suggested as key players for cancer development and tumor metastasis. Two polymorphisms in the gene for the alpha(2) component, ITGA2 807C>T and 1648G>A, have been associated with the cell-surface density of integrin alpha(2)beta(1). The 176T>C polymorphism in the ITGB3 gene, encoding the beta(3) subunit of integrins alpha(IIb)beta(3) and alpha(V)beta(3), modifies a variety of traits of beta(3) expressing cells. To analyze the role of ITGA2 and ITGB3 polymorphisms for breast cancer risk and prognosis, we performed a case-control study including 500 female breast cancer patients and 500 healthy female age-matched control subjects. All study participants were of Caucasian origin (Austria, Middle-Europe). The ITGA2 1648&#95;AA genotype was significantly associated with breast cancer (odds ratio 3.12; 95% confidence interval 1.11-8.77). Carriers of the most common ITGA2 haplotype (807C&#95;1648G, 'wildtype') were at decreased risk for breast cancer (odds ratio 0.72; 95% confidence interval 0.53-0.98). A histological grade of 3 or 4 was found more often in ITGA2 807TT subjects (p=0.039 compared to CC+CT genotypes) and carriers of an ITGA2 1648A allele (p=0.017 compared to GG genotype). Carriers of the ITGA2 807C&#95;1648G haplotype were less likely to have a histological grade 3 or 4 compared to non-carriers (p=0.003). The ITGB3 176T>C polymorphisms was not associated with breast cancer susceptibility. In a Cox-regression analysis, carriers of the homozygous ITGB3 176-CC genotype had a higher risk for metastasis (relative risk 2.3; 95% CI 1.3-4.2; p=0.005). We conclude that functional polymorphisms in integrin genes ITGA2 and ITGB3 influence the development and progression of breast cancer, respectively. The precise mechanism remains to be determined, but likely involves dysregulated signaling pathways.

We evaluated the usefulness of cytokeratin 20 (CK20) mRNA expression in the quantitative detection of circulating tumor cells in the blood of patients with colorectal cancer (CRC). Blood samples from healthy volunteers (HVs; n = 37), patients with localized (n = 42) and metastatic colorectal cancer (n = 40), and patients with chronic inflammatory bowel disease (CID; n = 15) were examined. After immunomagnetic enrichment using microbeads against human epithelial antigen, total RNA was extracted, reverse transcribed, and analyzed by real-time reverse transcriptase-polymerase chain reaction using the LightCycler instrument. CK20 expression in peripheral blood was found in 46 of 82 (56%) patients with CRC, 8 of 37 (22%) HVs, and 9 of 15 (60%) patients with CID. Levels of CK20 mRNA were significantly higher in blood samples from CRC patients (median 681) than in blood samples from HVs (median 0) (P = 0.001), whereas no difference could be detected between patients with CRC and CID. Although the present technique could not distinguish CRC from CID, the method warrants further efforts to improve sample preparation and tumor cell enrichment, which may render real-time CK20 reverse transcriptase-polymerase chain reaction a feasible technique in identifying circulating tumor cells in peripheral blood of cancer patients.

The aim of this study was to define the frequency and clinical relevance of cytokeratin positive metastatic tumor cells in the peripheral circulation of patients with stage IV breast cancer. Peripheral blood was collected from 32 consecutive patients with metastatic breast cancer and 23 healthy donors. Tumor cells were enriched using positive selection with anti-HEA125-microbeads and cytospins were prepared of the positive selection eluate. Slides were incubated with a Fab2 fragment of the pancytokeratin antibody A45-B/B3 conjugated with alkaline phosphatase (AKP) and a CAM5.2-AKP monoclonal antibody and developed with an alkaline phosphatase anti-alkaline phosphate reaction (APAAP). All samples were evaluated using light microscopy and an automated image analysis system. In 8/32 (25%) patients cytokeratin positive (CK+) cells could be detected after anti-HEA125 enrichment in the peripheral blood whereas in none out of 23 healthy donors. One to 1000 (median 5) positive cells per patient sample were observed and cluster of tumor cells in one patient. Automated image analysis was as powerful in detecting micrometastases as conventional light microscopy. All patients with CK+ cells in the peripheral circulation (8/8, 100%) showed progressive disease at the time-point of blood draw whilst only 9/24 (37.5%) showed disease progression without detection of positive cells. The median overall survival of CK+ patients was 4+/-2 months compared to 13+/-7 months of CK- patients (p<0.001). CK+ cells are detectable in the peripheral circulation of 25% of patients with metastatic breast cancer after positive selection with anti-HEA125. Detection of tumor cells in the peripheral circulation might be correlated with progression of disease and shorter overall survival.

Purpose: Hypoxia-inducible factor (HIF)-1alpha is a transcription factor that supports the adaptation of human cancer cells to hypoxia and is involved in various pathways supporting tumor growth and progression. The aim of this study was to determine the prognostic influence of HIF-1alpha expression in patients with advanced-stage breast cancer, evident by positive lymph nodes., Experimental Design: Expression of HIF-1alpha was determined immunohistochemically in 206 patients with lymph node-positive breast cancer. Furthermore, the interrelationship of HIF-1alpha with p53 and HER-2 protein expression, estrogen receptor density, and survival was analyzed. Colocalization of p53 and HIF-1alpha proteins was analyzed by confocal laser scanning microscopy., Results: Strong nuclear expression of HIF-1alpha by invasive cancer cells was found in 48 patients (23.3%), moderate expression was found in 74 patients (35.9%), and weak expression was found in 35 patients (17%); no expression was observed in 49 patients (23.8%). HIF-1alpha protein overexpression was associated with significantly shorter overall and disease-free survival time (P = 0.003 and P = 0.001, respectively; Cox regression analysis). No correlation of HIF-1alpha and HER-2 expression or estrogen receptor density was observed., Conclusions: This study shows that HIF-1alpha is an independent prognostic factor for an unfavorable prognosis in patients with lymph node-positive breast cancer. Our results indicate that patients with advanced-stage breast cancers might profit from future therapies targeting HIF-1alpha.