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CCR Translation for This Article from A Comprehensive Analysis of Human Gene Expression Profiles Identifies Stromal Immunoglobulin κ C as a Compatible Prognostic Marker in Human Solid Tumors

Purpose: Although the central role of the immune system for tumor prognosis is generally accepted, a single robust marker is not yet available.Experimental Design: On the basis of receiver operating characteristic analyses, robust markers were identified from a 60-gene B cell–derived metagene and analyzed in gene expression profiles of 1,810 breast cancer; 1,056 non–small cell lung carcinoma (NSCLC); 513 colorectal; and 426 ovarian cancer patients. Protein and RNA levels were examined in paraffin-embedded tissue of 330 breast cancer patients. The cell types were identified with immunohistochemical costaining and confocal fluorescence microscopy.Results: We identified immunoglobulin κ C (IGKC) which as a single marker is similarly predictive and prognostic as the entire B-cell metagene. IGKC was consistently associated with metastasis-free survival across different molecular subtypes in node-negative breast cancer (n = 965) and predicted response to anthracycline-based neoadjuvant chemotherapy (n = 845; P < 0.001). In addition, IGKC gene expression was prognostic in NSCLC and colorectal cancer. No association was observed in ovarian cancer. IGKC protein expression was significantly associated with survival in paraffin-embedded tissues of 330 breast cancer patients. Tumor-infiltrating plasma cells were identified as the source of IGKC expression.Conclusion: Our findings provide IGKC as a novel diagnostic marker for risk stratification in human cancer and support concepts to exploit the humoral immune response for anticancer therapy. It could be validated in several independent cohorts and carried out similarly well in RNA from fresh frozen as well as from paraffin tissue and on protein level by immunostaining. Clin Cancer Res; 18(9); 2695–703. ©2012 AACR.

Purpose: The prognostic and predictive relevance of epidermal growth factor receptor 2 (ERBB2) and topoisomerase II α (TOP2A) have long been a matter of debate. However, the correlation of DNA amplification, RNA levels, and protein expression and their prognostic role and association with anthracycline responses in node-negative breast cancer have not yet been evaluated.Experimental Design: We first analyzed TOP2A and ERBB2 at the levels of gene amplification, and RNA and protein expression, and studied their correlations. Additionally, TOP2A and ERBB2 were analyzed in 782 node-negative breast carcinomas in patients who did not receive systemic therapy and in 80 patients treated with epirubicin and cyclophosphamide (EC) prior to surgery.Results: TOP2A gene amplification did not correlate with protein expression (P = 0.283) and showed an association with gene expression with only borderline significance (P = 0.047). By contrast, TOP2A RNA levels correlated with protein expression (P < 0.001). TOP2A gene expression was significantly associated with the metastasis-free interval (MFI; P < 0.001) and was associated with complete remission in patients treated with EC (P = 0.002). In contrast to TOP2A, ERBB2 gene amplification correlated with RNA level (P < 0.001) and protein expression (P < 0.001). ERBB2 gene expression was associated with the MFI only in estrogen receptor–positive carcinomas, whereas ERBB2 protein expression (P = 0.032) was associated with MFI in the entire cohort.Conclusions: Overall, our study indicates that the TOP2A RNA level is a good prognostic marker and is also associated with a favorable response to anthracyclin-based therapy. By contrast, ESR1 was associated with poorer responses to anthracyclin-based therapy, whereas the association with ERBB2 RNA was not significant. Clin Cancer Res; 16(8); 2391–401. ©2010 AACR.

Supplementary Data from ERBB2 and TOP2A in Breast Cancer: A Comprehensive Analysis of Gene Amplification, RNA Levels, and Protein Expression and Their Influence on Prognosis and Prediction

An amendment to this paper has been published and can be accessed via the original article.

The prognosis of triple-negative breast cancer with metastases after chemotherapy remains dismal. We report the case of a 50-year-old female with first disease recurrence at the axillary lymph node and, later on, bilateral pulmonary metastases with severe shortness of breath. The patient received low-dose immune checkpoint blockade (concurrent nivolumab and ipilimumab) weekly over 3 weeks with regional hyperthermia 3 times a week, followed by systemic fever-range hyperthermia induced by interleukin-2 for 5 days. She went into complete remission of her pulmonary metastases with transient WHO I-II diarrhea and skin rash. The patient remained alive for 27 months after the start of treatment, with recurrence of metastases as a sternal mass, and up to 3 cm pleural metastases. This exceptional response should instigate further research efforts with this protocol, which consists only of approved drugs and treatments.

Background: An earlier published series of neoadjuvant radio-chemotherapy (NRT-CHX) in locally advanced non-inflammatory breast cancer (LABC) has now been updated with a follow up of more than 15 years. Long- term outcome data and predictive factors for pathologic complete response (PCR) were analyzed. Patients and Methods: 315 LABC patients (cT1-cT4/cN0-N1) were treated during 1991–1998 with NRT-CHX. Preoperative radiotherapy (RT) consisted of external beam radiation therapy (EBRT) of 50 Gy (5 × 2 Gy/week) to the breast and the supra-/infraclavicular lymph nodes combined with an electron boost in 214 cases afterwards or – in case of breast conservation – an 10-Gy interstitial boost with 192Ir afterloading before EBRT. Chemotherapy was administered prior to RT in 192 patients, and concomitantly in 113; 10 patients received no chemotherapy. The update of all follow up ended in November 2011. Age, tumor grade, nodal status, hormone receptor status, simultaneous vs. sequential CHX and the time interval between end of RT and surgery were examined in multivariate terms with as endpoint pCR and overall survival. Results: The total PCR rate after neoadjuvant RT-CHX reached 29.2 % with LABC breast conservation becoming possible in 50.8%. In initially node-positive cases (cN+), a complete nodal response (pN0) after NRT-CHX was observed in 56% (89/159). The multivariate analysis revealed that a longer time interval to surgery increased the probability for a pCR (HR 1,17 [95% CI 1,05-1,31], p < 0,01). However, in large tumors (T3-T4) a significantly reduced pCR rate (HR 0.89 [95% CI 0.80 to 0.99], p = 0.03) could be obtained. Importantly, a pCR was the strongest prognostic factor for long term survival (HR 0.28 [95% CI 0.19–0.56], p < 0.001). Conclusions: A PCR identifies patients with a significant better prognosis for long-term survival. However, a long time interval to surgery (> 2 months) increases the probability of a pCR after NRT-CHX. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-14-04.

Background: Biomarkers predictive of pathological complete response (pCR) to neoadjuvant chemotherapy (NACT) of breast cancer are urgently needed. Methods: Using a training/validation approach for detection of predictive biomarkers in HER2-negative breast cancer, pre-therapeutic core biopsies from four independent cohorts were investigated: Gene array data were analysed in fresh frozen samples of two cohorts (n=86 and n=55). Quantitative reverse transcription polymerase chain reaction (qRT–PCR) was performed in formalin-fixed, paraffin-embedded (FFPE) samples from two neoadjuvant phase III trials (GeparTrio, n=212, and GeparQuattro, n=383). Results: A strong predictive capacity of thymosin beta 15 (TMSB15A) gene expression was evident in both fresh frozen cohorts (P

Purpose: Although the central role of the immune system for tumor prognosis is generally accepted, a single robust marker is not yet available. Experimental Design: On the basis of receiver operating characteristic analyses, robust markers were identified from a 60-gene B cell–derived metagene and analyzed in gene expression profiles of 1,810 breast cancer; 1,056 non–small cell lung carcinoma (NSCLC); 513 colorectal; and 426 ovarian cancer patients. Protein and RNA levels were examined in paraffin-embedded tissue of 330 breast cancer patients. The cell types were identified with immunohistochemical costaining and confocal fluorescence microscopy. Results: We identified immunoglobulin κ C (IGKC) which as a single marker is similarly predictive and prognostic as the entire B-cell metagene. IGKC was consistently associated with metastasis-free survival across different molecular subtypes in node-negative breast cancer (n = 965) and predicted response to anthracycline-based neoadjuvant chemotherapy (n = 845; P < 0.001). In addition, IGKC gene expression was prognostic in NSCLC and colorectal cancer. No association was observed in ovarian cancer. IGKC protein expression was significantly associated with survival in paraffin-embedded tissues of 330 breast cancer patients. Tumor-infiltrating plasma cells were identified as the source of IGKC expression. Conclusion: Our findings provide IGKC as a novel diagnostic marker for risk stratification in human cancer and support concepts to exploit the humoral immune response for anticancer therapy. It could be validated in several independent cohorts and carried out similarly well in RNA from fresh frozen as well as from paraffin tissue and on protein level by immunostaining. Clin Cancer Res; 18(9); 2695–703. ©2012 AACR.

This retrospective study compares patients treated between 1991 and 1998 with neoadjuvant radiotherapy +/- chemotherapy (RCT) or adjuvant RCT for locally advanced noninflammatory breast cancers (LABC) in terms of pathologic complete response (pCR), 10-year relapse-free (RFS), and overall survival (OS).Preoperative RCT in 315 and adjuvant RCT in 329 cases consisted in 50 Gy (5 x 2 Gy/week) to the breast and the supra-/infraclavicular lymph nodes. 101 neoadjuvant patients received - in case of breast conservation - a 10-Gy interstitial boost with (192)Ir afterloading before and 214 neoadjuvant patients a preoperative electron boost after external-beam radiotherapy. In the neoadjuvant RCT group, chemotherapy was applied prior to radiotherapy in 192 patients, and simultaneously in 113; ten had no chemotherapy. In the adjuvant RCT group, chemotherapy was applied to 44 patients before surgery and to 166 after surgery; 119 had no chemotherapy.Breast conservation became possible in 50.8% after neoadjuvant RCT for LABC with a pCR rate at surgery of 29.2%. A complete nodal remission (pN0) after RCT was observed in 56% (89/159) of the cN+ (clinically node-positive) neoadjuvant patients. There were trends in favor of preoperative RCT for RFS and OS (hazard ratio [HR] = 0.85; p = 0.09 for RFS; HR = 0.8130; p = 0.1037 for OS). For patients with cT2 tumors the RFS and OS were statistically significantly better (HR = 0.5090; p = 0.0130 for RFS; HR = 0.4390; p = 0.0026 for OS) after neoadjuvant compared to adjuvant RCT.Neoadjuvant RCT achieved a pCR rate of 29.2% and a statistically significantly better RFS and OS in patients with cT2-category breast cancer.

Purpose: The prognostic and predictive relevance of epidermal growth factor receptor 2 (ERBB2) and topoisomerase II α (TOP2A) have long been a matter of debate. However, the correlation of DNA amplification, RNA levels, and protein expression and their prognostic role and association with anthracycline responses in node-negative breast cancer have not yet been evaluated. Experimental Design: We first analyzed TOP2A and ERBB2 at the levels of gene amplification, and RNA and protein expression, and studied their correlations. Additionally, TOP2A and ERBB2 were analyzed in 782 node-negative breast carcinomas in patients who did not receive systemic therapy and in 80 patients treated with epirubicin and cyclophosphamide (EC) prior to surgery. Results: TOP2A gene amplification did not correlate with protein expression (P = 0.283) and showed an association with gene expression with only borderline significance (P = 0.047). By contrast, TOP2A RNA levels correlated with protein expression (P < 0.001). TOP2A gene expression was significantly associated with the metastasis-free interval (MFI; P < 0.001) and was associated with complete remission in patients treated with EC (P = 0.002). In contrast to TOP2A, ERBB2 gene amplification correlated with RNA level (P < 0.001) and protein expression (P < 0.001). ERBB2 gene expression was associated with the MFI only in estrogen receptor–positive carcinomas, whereas ERBB2 protein expression (P = 0.032) was associated with MFI in the entire cohort. Conclusions: Overall, our study indicates that the TOP2A RNA level is a good prognostic marker and is also associated with a favorable response to anthracyclin-based therapy. By contrast, ESR1 was associated with poorer responses to anthracyclin-based therapy, whereas the association with ERBB2 RNA was not significant. Clin Cancer Res; 16(8); 2391–401. ©2010 AACR.

Altered signaling pathways are key regulators of cellular functions in tumor cells. Constitutive activation of signal transducer and activator of transcription (STAT)3 and -5 may be involved in tumor formation and progression. We have investigated the role of STAT5 in cutaneous melanoma metastases using various RNA and protein techniques. In melanoma specimens, Stat5b transcripts were upregulated approximately 3.8-fold. In 13 of 21 (62%) human melanoma metastases, STAT5 was phosphorylated in comparison to normal human melanocytes and benign nevi. The STAT5 target gene Bcl-2 was frequently upregulated. The investigation of the underlying mechanism revealed specific STAT5 activation by recombinant human epidermal growth factor (rEGF). rEGF-induced activation of STAT5 occurred in vitro through the non-receptor tyrosine kinases transforming gene (src) of Rous Sarcoma virus and Janus kinase 1. Inhibition of Stat5b expression by small interfering RNA strongly reduced the expression of Bcl-2 and led to decreased cell viability and increased apoptosis in the melanoma cell lines A375 and BLM. Transfection with dominant-negative Stat5b caused enhanced cell death and G1 arrest in A375 cells. Our study identifies phosphorylated STAT5 in melanoma and shows regulation through rEGF; STAT5 may thus act as a survival factor for growth of human melanoma and may represent a potential target for molecular therapy.

Purpose: Expression profiling by DNA microarray technology permits the identification of genes underlying clinical heterogeneity of bladder cancer and which might contribute to disease progression, thereby improving assessment of treatment and prediction of patient outcome. Experimental Design: Invasive (20) and superficial (22) human bladder tumors from 34 patients with known outcome regarding disease recurrence and progression were analyzed by filter-based cDNA arrays (Atlas Human Cancer 1.2; BD Biosciences Clontech) containing 1185 genes. For 9 genes, array data were confirmed using real-time reverse transcription-PCR. Additionally, Atlas array data were validated using Affymetrix GeneChip oligonucleotide arrays with 22,283 human gene fragments and expressed sequence tags sequences in a subset of three superficial and six invasive bladder tumors. Results: A two-way clustering algorithm using different subsets of gene expression data, including a subset of 41 genes validated by the oligonucleotide array (Affymetrix), classified tumor samples according to clinical outcome as superficial, invasive, or metastasizing. Furthermore, (a) a clonal origin of superficial tumors, (b) highly similar gene expression patterns in different areas of invasive tumors, and (c) an invasive-like pattern was observed in bladder mucosas derived from patients with locally advanced disease. Several gene clusters that characterized invasive or superficial tumors were identified. In superficial bladder tumors, increased mRNA levels of genes encoding transcription factors, molecules involved in protein synthesis and metabolism, and some proteins involved into cell cycle progression and differentiation were observed, whereas transcripts for immune, extracellular matrix, adhesion, peritumoral stroma and muscle tissue components, proliferation, and cell cycle controllers were up-regulated in invasive tumors. Conclusions: Gene expression profiling of human bladder cancers provides insight into the biology of bladder cancer progression and identifies patients with distinct clinical phenotypes.

In this study, we elucidated the potential of recombinant adeno-associated virus type-2 (rAAV-2) vectors for lung cancer gene therapy. Cell lines of the three major histological subtypes of non-small cell lung cancer (NSCLC) were highly susceptible for rAAV-2 showing transduction rates between 63.4 and 98.9%. In contrast, cell lines of small cell carcinomas were resistant to rAAV-2 infection. For restoration of p53 function in p53 deficient NSCLC, a rAAV-2 vector was constructed containing wt p53 cDNA. Following transduction with rAAV-p53, cell growth of all NSCLC cell lines was significantly reduced in a dose-dependent manner between 44 and 71.7% in comparison with rAAV-GFP transduced cells. The reduction of tumor cell growth was associated with increased apoptosis. Adding cisplatin to rAAV-p53-infected cells led to a significant growth inhibition between 81 and 91% indicating a synergistic effect between cisplatin and rAAV-p53. Interestingly, the tumor cells surviving cisplatin and rAAV-p53 treatment were inhibited in their ability to form colonies as reflected by a reduction of colony growth between 57 and 90.4%. In conclusion, rAAV-2 vectors exhibit a strong tropism for NSCLC. Successful inhibition of tumor cell growth following transduction with a rAAV-p53 vector underlines the potential role of rAAV-2 in cancer gene therapy.

Prognostic multigene expression assays have become widely available to provide additional information to standard clinical parameters and to support clinicians in treatment decisions. In this study, we analyzed the impact of variations in tissue handling on the diagnostic EndoPredict test results. EndoPredict is a quantitative reverse transcription PCR assay conducted on RNA from formalin-fixed, paraffin-embedded (FFPE) tissue that predicts the likelihood of distant recurrence in patients with ER-positive/HER2-negative breast cancer. In this study, we performed a total of 138 EndoPredict assays to study the effects of preanalytical variables such as time to fixation, fixation time, tumor cell content, and section storage time on the EndoPredict test results. A time to fixation of up to 12 h and fixation of up to 5 days did not affect the results of the gene expression test. Paired samples of FFPE sections with tumor cell content ranging from 15 to 95 % and tumor-enriched samples showed a correlation coefficient of 0.97. Test results of tissue sections that have been stored for 12 months at +4 or +20 °C showed a correlation of 0.99 when compared to results of nonstored sections. In conclusion, preanalytical tissue handling is not a critical factor for diagnostic gene expression analysis with the EndoPredict assay. The test can therefore be easily integrated into the standard workflow of molecular pathology.

In 1984 the GBSG started a multicenter randomized trial to compare the effectiveness of 6 cycles of cyclophosphamide, methotrexate and fluorouracil (CMF) with or without radiotherapy (RT) as adjuvant treatment in node-positive breast-cancer patients treated by mastectomy. During 5 years, 199 patients were randomized. After a median follow-up of about 9 years, the treatment groups 6 × CMF and 6 × CMF + RT were compared regarding time to recurrence and death. As the first event of failure, we observed locoregional recurrence in 22 patients, distant metastases in 66 patients, a secondary malignancy in 9 patients and death without previous event in 5 patients. For event-free survival (EFS), no significant difference was observed [relative risk (RR) 6 × CMF + RT vs. 6 × CMF 0.82, 95% confidence interval (CI) 0.55–1.21]. Event-specific analysis showed a significant decreased risk after radiotherapy for locoregional recurrence. The risk for distant metastases was estimated as slightly decreased and the risk for secondary malignancy and for death without previous event was estimated as increased in treatment group 6 × CMF + RT in comparison with treatment group 6 × CMF, but these effects were not significant. For overall survival (OS) and breast-cancer-specific survival (BCS), no significant treatment effect could be demonstrated. There is a beneficial effect of radiotherapy on locoregional recurrence. For EFS and BCS, a tendency in favour of radiotherapy is observed, but this is not significant; for OS, no difference can be demonstrated, but the power of the study is too low to detect small treatment effects. Int. J. Cancer 86:408–415, 2000. © 2000 Wiley-Liss, Inc.

Occasional reports have suggested an unfavorable effect of high estrogen receptor (ER) concentrations in primary breast cancer. In a population-based study we identified a subgroup explicitly exhibiting this seemingly paradoxical effect. ER concentrations were prospectively measured in a single laboratory by multipoint DCC assay. The relative risk of death in relation to the concentration of the interval-scaled variables ER and PgR was continually estimated by serial Cox regression analyses. Thus we circumvented loss of information due to primary categorization and avoided assumptions about relations between factor and risk. Based on 2,735 consecutively accrued primary breast cancer cases (median follow-up 56 months) we identified node-negative patients up to 60 years of age as the relevant subpopulation. High (>/=300 fmol/mg protein) ER concentrations exhibited an even more unfavorable impact (p < 0.03) on overall survival than ER concentrations of less than 10 fmol/mg protein. The well-known association of age and ER concentration was definitely excluded as an underlying biological cause for the increased risk. Differences in the distribution of other prognostic factors (HER-2/neu, Ki-67, DNA ploidy) were also excluded. As we observed a preponderance of pT2 tumors in the high ER group, we repeated the analysis, selectively focusing on pT2 tumors in the relevant subgroup, but the effect remained unchanged. In contrast, node-positive patients adjusted for age significantly (p = 0.02) profited from high ER concentrations as compared to the ER-negative group. As the phenomenon did not occur in node-positive patients, receptor defects in the high-ER group seem unlikely. To the contrary, we suspect that ER overexpressing cells are hypersensitive even to low levels of estrogens. Once they have sneaked past local barriers prior to primary surgery, they may cause early death in the absence of appropriate adjuvant endocrine therapy.

Prognostic models that predict the clinical course of a breast cancer patient are important in oncology. We propose an approach to constructing such models based on fractional polynomials in which useful transformations of the continuous factors are determined. The idea may be applied with all types of regression model, including Cox regression, the method of choice for survival-time data. We analyse a prospective study of node-positive breast cancer. Seven standard prognostic factors – age, menopausal status, tumour size, tumour grade, number of positive lymph nodes, progesterone and oestrogen receptor concentrations – were investigated in 686 patients, of whom 299 had an event for recurrence-free survival and 171 died. We determine a final model with transformations of prognostic factors and compare it with the more traditional approaches using categorized variables or assuming a straight line relationship. We conclude that analysis using fractional polynomials can extract important prognostic information which the traditional approaches may miss. © 1999 Cancer Research Campaign

Breast cancer can exist for long periods as non-invasive or invasive but non-metastatic disease. Early detection of breast cancer, for example by mammography screening, significantly improves a woman ’ s chances of survival. If breast cancer is diagnosed and treated while it is confined to the breast, the cure rate can approach 100%. However, mammography screening is far from perfect in terms of sensitivity and specificity of cancer detection. There is a significant clinical need for accurate and highly sensitive tests for early detection of breast cancer. Target applications are screening of high-risk groups, particularly younger women (under the age of 50) from high-incidence families, and breast cancer patients to detect the primary metastasis and migratory route, to direct treatment and the early detection of recurrence when screening breast cancer patients.

Objective Insulin-like growth factor (IGF)-1, -2 and Insulin like growth factor binding proteins (IGFBP) are involved in the proliferation and differentiation of cells. It has never been evaluated, if the IGF-system can serve as a tumor marker in neoplasms. Methods In our prospective study 163 patients with colorectal cancer (22), prostate cancer (21), head and neck tumors (17), lymphomas (20), lung cancer (34) and other entities (49) were analysed for their IGF and IGFBP serum levels at the beginning and the end of radiotherapy and compared to 13 healthy people. Subgroups of patients with local tumor disease versus metastatic disease, primary and recurrent therapy and curative versus palliative therapy were compared. Results The serum levels of IGF-2 were significantly elevated in patients with prostate and colorectal cancer. However, sensitivity and specificity were only 70%. IGFBP-2 serum levels were elevated in patients with head and neck tumors. Again sensitivity and specificity were only 73%. A difference between local disease and metastatic disease could not be found. A difference between IGF serum levels before and after radiotherapy could not be detected. Conclusion The IGF-system cannot serve as a new tumor marker. The detected differences are very small, sensitivity and specificity are too low. IGF measurement is not useful for the evaluation of the success of radiotherapy in malignancies.

Background Tumor-related methylated DNA and circulating tumor cells (CTC) in the peripheral blood might be of prognostic importance in breast cancer. Thus, the aim of our study was to examine free methylated DNA and CTC in the blood from breast cancer patients and to correlate it with clinicopathological features known to influence prognosis. Materials and methods We prospectively obtained serum samples from 85 patients with breast cancer and 22 healthy volunteers. Sera were analysed by methylation specific PCR (MethyLight PCR) for five genes: adenomatous polyposis coli (APC), ras association domain family protein 1A (RASSF1A), estrogen receptor 1 (ESR1), CDKN2A (p16) and glutathione s-transferase pi 1 (GSTP1). Beta actin (ACTB) served as control. In parallel matched peripheral blood of 63 patients was used to assay for circulating tumor cells in the peripheral blood by a modified immunomagnetic AdnaTest BreastCancerSelect with PCR detection for EPCAM, MUC1, MGB1 and SPDEF. Results We found a hypermethylation in the APC gene in 29% (25/85), in RASSF1A in 26% (22/85), in GSTP1 in 18% (14/76) and in ESR1 in 38% (32/85) of all breast cancer patients. No hypermethylation of CDKN2A was found (0/25). Blood samples of patients were defined CTC positive by detecting the EPCAM 13% (8/63), MUC1 16% (10/63), MGB 9% (5/55), SPDEF 12% (7/58) and in 27% detecting one or more genes (15/55). A significant difference was seen in methylated APC DNA between cancer patients and healthy volunteers. Moreover, methylated APC, RASSF1 and CTC were significantly different in metastatic versus non-metastatic disease. In addition, the presence of methylated APC, RASSF1A and CTC correlated significantly with AJCC-staging (p = 0.001, p = 0.031 and 0.002, respectively). High incidences of methylations were found for the genes RASSF1 and ESR1 in healthy individuals (both 23% 5/22). Methylated GSTP1 was predominantly found in the serum of patients with large primaries (p = 0.023) and was highly significantly correlated with positive Her2/neu status (p = 0.003). Elevated serum CA15.3 was strongly correlated with methylated APC and CTC detection (both p = 0.000). Methylated ESR1 failed to exhibit significant correlations with any of the above mentioned parameters. The presence of CTC in peripheral blood was significantly associated with methylated APC (p = 0.012) and methylated GSTP1 (p = 0.001). Conclusion The detection of methylated APC and GSTP1 DNA in serum correlated with the presence of CTC in the blood of breast cancer patients. Both methylated DNA and CTC correlated with a more aggressive tumor biology and advanced disease.

Background Breast cancer (BC) represents one of the leading causes of cancer related deaths worldwide. New tools for diagnostic staging and therapeutic monitoring are needed to improve individualized therapies and improve clinical outcome. The analyses of circulating tumour cells may provide important prognostic information in the clinical setting. Materials and methods Circulating tumour cells (CTC) of 63 BC patients were isolated from peripheral blood (PB) through immunomagnetic separation. Subsequently, RT-PCR or mPCR for the genes ga733.2, muc-1, c-erbB2, mgb-1, spdef and c-erbB2 were performed. Subsequently, expression data were correlated with the tumour stages. Fourteen healthy individuals served as controls. Results Significant correlations with tumour stages were found in single gene analyses of ga733.2, muc-1 and in multi-gene analyses of ga733.2/muc-1/mgb1/spdef. Furthermore, a significant correlation of Ca 15-3 and all studied genes was also observed. Conclusion Herein, we demonstrated a positive correlation of a gene signature consisting of ga733.2, muc-1, mgb1 and spdef and advanced stages of BC. Moreover, all studied genes and gene patterns revealed a significant correlation with Ca 15-3 positive cases.

BACKGROUND. Cleavage of membrane-anchored heparin-binding epidermal growth factor-like growth factor (proHB-EGF) yields a soluble HB-EGF isoform (sHB-EGF), which is an activating epidermal growth factor receptor (EGFR) ligand and a C-terminal fragment HB-EGF-C acting directly in the nucleus. In bladder cancer, overexpression of both HB-EGF and EGFR have been observed, but to the authors' knowledge the prognostic significance of different modes of HB-EGF signaling have remained unclear. METHODS. Expression and intracellular localization of HB-EGF and EGFR were examined by immunohistochemistry in paraffin-embedded specimens from 121 patients who underwent cystectomy for bladder cancer. Tumor stage was pTis/pT1 in 7 patients, pT2 in 41 patients, pT3 in 55 patients, and pT4 in 18 patients. Lymph node metastases were present in 32 patients. RESULTS. Using an antibody directed against the C-terminal domain, HB-EGF expression was detected in the cytoplasm or in the nucleus of tumor cells. EGFR staining was uniform at the plasma membrane. The actuarial 5-year cancer-specific survival of patients with tumors with predominant nuclear HB-EGF staining was 28% compared with 57% if HB-EGF staining was predominantly cytoplasmic (P = .027). Disease outcome of patients with a ‘mixed’ HB-EGF staining pattern was found to be between that of the 2 former groups. In agreement with previous studies, strong EGFR expression was associated with poor prognosis. Despite strong EGFR expression, predominant cytoplasmic HB-EGF staining was associated with a more favorable outcome, whereas a predominant nuclear pattern defined a subgroup with extremely poor prognosis (5-year tumor-specific survival of 55% vs 13%, respectively; P = .026). CONCLUSIONS. The current study results confirm that EGFR expression is significantly correlated with disease-specific mortality but that the outcome is also influenced by the mode of HB-EGF signaling. Additional nuclear HB-EGF signaling, indicative of increased cleavage of proHB-EGF, appears to enhance the adverse activities. Cytoplasmic HB-EGF staining likely reflects proHB-EGF, which may also exert antiproliferative effects. Cancer 2007. © 2007 American Cancer Society.

Despite tremendous effort and progress in the diagnostics of pancreatic cancer with respect to imaging techniques and molecular genetics, only very few patients can be cured by surgery leading to a 5-year survival rate of only 3%. Especially the lack of chemotherapeutical options in this entity requires a better understanding of the molecular mechanisms leading to pancreatic carcinoma growth and progression in order to develop novel treatment regimens. To identify signaling pathways that are critical for this tumor entity, we compared six well-established pancreatic cancer cell lines (Capan-1, Capan-2, HUP-T3, HUP-T4, KCL-MOH, PaTu-8903) with colon cancer cell lines and tumor cell lines of non-epithelial origin by expression profiling. For this purpose we employed Human Genome Focus Arrays representing about 8500 well annotated human genes. We identified 353 genes with significantly high expression in the group of pancreatic carcinomas. Based on Gene Ontology annotations these genes are especially involved in Rho protein signal transduction, proteasome activator activity, cell motility, apoptotic program, and cell-cell adhesion processes indicating these pathways to be interesting candidates for the design of targeted therapies. Most pancreatic carcinomas are characterized by mutations in the TP53 and the KRAS genes and the absence of microsatellite instability, which could also be confirmed for our panel of pancreatic carcinoma cell lines. Looking for individual differences within this group that may be responsible for more or less aggressive behavior, we identified genomic amplifications at the 8q22.1 and the 8q24.22 loci to be associated with enhanced gene transcription. Because we have previously shown that gains of genomic material from the long arm of chromosome 8 have an adverse effect on the outcome of pancreatic carcinoma patients, we conclude that functional analysis of amplified genes at 8q22 and/or 8q24 may lead to an improved understanding of pancreatic carcinoma progression.

BackgroundOur goal was to identify gene signatures predictive of response to preoperative systemic chemotherapy (PST) with epirubicin/cyclophosphamide (EC) in patients with primary breast cancer.MethodsNeedle biopsies were obtained pre-treatment from 83 patients with breast cancer and mRNA was profiled on Affymetrix HG-U133A arrays. Response ranged from pathologically confirmed complete remission (pCR), to partial remission (PR), to stable or progressive disease, "NoChange" (NC). A primary analysis was performed in breast tissue samples from 56 patients and 5 normal healthy individuals as a training cohort for predictive marker identification. Gene signatures identifying individuals most likely to respond completely to PST-EC were extracted by combining several statistical methods and filtering criteria. In order to optimize prediction of non responding tumors Student'st-test and Wilcoxon test were also applied. An independent cohort of 27 patients was used to challenge the predictive signatures. Ak-Nearest neighbor algorithm as well as two independent linear partial least squares determinant analysis (PLS-DA) models based on the training cohort were selected for classification of the test samples. The average specificity of these predictions was greater than 74% for pCR, 100% for PR and greater than 62% for NC. All three classification models could identify all pCR cases.ResultsThe differential expression of 59 genes in the training and the test cohort demonstrated capability to predict response to PST-EC treatment. Based on the training cohort a classifier was constructed following a decision tree.First, a transcriptional profile capable to distinguish cancerous from normal tissue was identified. Then, a "favorable outcome signature" (31 genes) and a "poor outcome signature" (26 genes) were extracted from the cancer specific signatures. This stepwise implementation could predict pCR and distinguish between NC and PR in a subsequent set of patients. Both PLS-DA models were implemented to discriminate all three response classes in one step.ConclusionIn this study signatures were identified capable to predict clinical outcome in an independent set of primary breast cancer patients undergoing PST-EC.

Osteosarcomas are primary malignant tumors of bone or soft parts arising from bone-forming mesenchymal cells. Despite dramatic therapeutic advances, namely neo-adjuvant and adjuvant chemotherapy, progress is at a plateau. Cytokine-mediated gene therapy might represent a further advance in the therapy of the osteosarcoma.We transfected UMR 108 osteosarcoma cells with different plasmids encoding IL-12, IL-23, proIL-18 and ICE (Interleukin-converting enzyme). IFN-gamma induction, which is known to induce antitumor effects mediated by the immune system, and cytotoxic effects of various cytokine combination were investigated.Our results show that local secretion of IL-12 by UMR 108 cells led to an induction of cytotoxic effects mediated by mononuclear cells, which were enhanced by additional administration of recombinant IL-18. In contrast to IL-18, IL-23 showed a moderate increase of IFN-gamma induction when transfected alone and could only slightly increase the IFN-gamma induction mediated by IL-12. IL-18 enhanced IFN-gamma induction when applied alone and was able to increase the IFN-gamma production that was induced by IL-12.IL-23 seems to be a less effective immuno-therapeutic for adjuvant treatment of osteosarcomas than IL-12 and IL-18, when taking only IFN-gamma induction into consideration.

Purpose: Our goal was to identify genes undergoing expressional changes shortly after the beginning of neoadjuvant chemotherapy for primary breast cancer. Experimental Design: The biopsies were taken from patients with primary breast cancer prior to any treatment and 24 hours after the beginning of the neoadjuvant chemotherapy. Expression analyses from matched pair samples representing 25 patients were carried out with Clontech filter arrays. A subcohort of those 25 paired samples were additionally analyzed with the Affymetrix GeneChip platform. All of the transcripts from both platforms were queried for expressional changes. Results: Performing hierarchical cluster analysis, we clustered pre- and posttreatment samples from individual patients more closely to each other than the samples taken from different patients. This reflects the rather low number of transcripts responding directly to the drugs used. Although transcriptional drug response occurring during therapy differed between individual patients, two genes (p21WAF1/CIP1 and MIC-1) were up-regulated in posttreatment samples. This could be validated by semiquantitative and real-time reverse transcription-PCR. Partial least- discriminant analysis based on approximately 25 genes independently identified by either Clontech or Affymetrix platforms could clearly discriminate pre- and posttreatment samples. However, correlation of certain gene expression levels as well as of differential patterns and clusters as determined by a different platform was not always satisfying. Conclusions: This study has demonstrated the potential of monitoring posttreatment changes in gene expression as a measure of the pharmacodynamics of drugs. As a clinical laboratory model, it can be useful to identify patients with sensitive and reactive tumors and to help for optimized choice for sequential therapy and obviously improve relapse- free and overall survival.

Expression profiling by DNA microarray technology permits the identification of genes underlying clinical heterogeneity of bladder cancer and which might contribute to disease progression, thereby improving assessment of treatment and prediction of patient outcome.Invasive (20) and superficial (22) human bladder tumors from 34 patients with known outcome regarding disease recurrence and progression were analyzed by filter-based cDNA arrays (Atlas Human Cancer 1.2; BD Biosciences Clontech) containing 1185 genes. For 9 genes, array data were confirmed using real-time reverse transcription-PCR. Additionally, Atlas array data were validated using Affymetrix GeneChip oligonucleotide arrays with 22,283 human gene fragments and expressed sequence tags sequences in a subset of three superficial and six invasive bladder tumors.A two-way clustering algorithm using different subsets of gene expression data, including a subset of 41 genes validated by the oligonucleotide array (Affymetrix), classified tumor samples according to clinical outcome as superficial, invasive, or metastasizing. Furthermore, (a) a clonal origin of superficial tumors, (b) highly similar gene expression patterns in different areas of invasive tumors, and (c) an invasive-like pattern was observed in bladder mucosas derived from patients with locally advanced disease. Several gene clusters that characterized invasive or superficial tumors were identified. In superficial bladder tumors, increased mRNA levels of genes encoding transcription factors, molecules involved in protein synthesis and metabolism, and some proteins involved into cell cycle progression and differentiation were observed, whereas transcripts for immune, extracellular matrix, adhesion, peritumoral stroma and muscle tissue components, proliferation, and cell cycle controllers were up-regulated in invasive tumors.Gene expression profiling of human bladder cancers provides insight into the biology of bladder cancer progression and identifies patients with distinct clinical phenotypes.

CD34+ hematopoietic stem cells are used clinically to support cytotoxic therapy, and recent studies raised hope that they could even serve as a cellular source for nonhematopoietic tissue engineering. Here, we examined in 18 volunteers the gene expressions of 1185 genes in highly enriched bone marrow CD34+(BM-CD34+) or granulocyte–colony-stimulating factor–mobilized peripheral blood CD34+(PB-CD34+) cells by means of cDNA array technology to identify molecular causes underlying the functional differences between circulating and sedentary hematopoietic stem and progenitor cells. In total, 65 genes were significantly differentially expressed. Greater cell cycle and DNA synthesis activity of BM-CD34+ than PB-CD34+ cells were reflected by the 2- to 5-fold higher expression of 9 genes involved in cell cycle progression, 11 genes regulating DNA synthesis, and cell cycle–initiating transcription factor E2F-1. Conversely, 9 other transcription factors, including the differentiation blocking GATA2 and N-myc, were expressed 2 to 3 times higher in PB-CD34+ cells than in BM-CD34+cells. Expression of 5 apoptosis driving genes was also 2 to 3 times greater in PB-CD34+ cells, reflecting a higher apoptotic activity. In summary, our study provides a gene expression profile of primary human CD34+ hematopoietic cells of the blood and marrow. Our data molecularly confirm and explain the finding that CD34+ cells residing in the bone marrow cycle more rapidly, whereas circulating CD34+ cells consist of a higher number of quiescent stem and progenitor cells. Moreover, our data provide novel molecular insight into stem cell physiology.

Background New prognostic markers may be of value in determining survival and informing decisions of adjuvant treatment in the heterogeneous group of soft tissue sarcomas known as malignant fibrous sarcomas (MFS). Increased CD44 expression has been associated with a better outcome in cancers such as bladder tumors and could potentially relate to cell-cell interaction as a marker for potential invasion/metastasis. The aim of this pilot study was to determine if there is a correlation between the expression rate of CD44 in adult patients with MFS and clinical outcomes. Methods The clinical outcome of 34 adult MFS patients (19 males and 15 females, average age 62 years, median 63 years, range: 38–88 years) who underwent surgical treatment were evaluated. Twenty-five of these patients had additional adjuvant radiotherapy. Extracted RNA from sarcoma tissues was used to measure the transcripts of CD44s (standard form) and isoform expression. The pooled data for each variant of CD44 was divided in half at the median expression value into two equally sized groups (low and high). Survival modeling and multivariate analysis were used with these two groups to determine if there were differences in survival times and whether this was independent of known factors such as tumor stage/grade, patient age and resection margin status. Results High CD44s and low of CD44v6 expression significantly correlated with an improved outcome (P P 30% difference in survival between low/high expressions at 5 years. These finding were independent of the other measured MFS survival predictors, though the group was homogenous. Conclusions High CD44s and low CD44v6 expression may be an independent predictor of improved survival in MFS patients in this pilot data. This is contrary to other MFS data, which did not account for the CD44 isoforms but is confirmed by data from other cancer types. Further investigation is needed to confirm CD44 isoform expression data as a relevant survival biomarker and whether it could be used to inform clinical decisions such as adjuvant therapy.

Somatic gene therapy with the herpes simplex virus type I thymidine kinase gene/ganciclovir (HSV-Tk/GCV) system and murine retroviral vector producer cells (VPCs) was introduced as a new adjuvant treatment modality to treat tumor bulk and to prevent tumor recurrence in patients harboring malignant glioma. The single-center experience after treatment of 27 patients undergoing tumor resection followed by intracerebral VPC injection for HSV-Tk suicide gene therapy will be presented focused on findings of systematic and close MRI follow-up and a few histological specimens. The data indicate that hemorrhagic necrosis due to endothelial cell transfection mediated vessel necrosis and that local inflammatory immune response occurs frequently after gene therapy. These phenomena seem to be specific because none of the patients of a control group showed any similar features. The prognosis (time to progression, survival) of the patients with “bystander effects” after gene therapy was better, but compared to those patients without bystander effects, they were also privileged by a favorable constellation of prognostic factors. Therefore, the appearance of these neuroradiologic features cannot serve as an indicator for treatment effectiveness and outcome. Cancer Gene Therapy (2001) 8, 843–851

Background: Histopathological grading is an important part for the characterization of breast cancer with regard to prognosis and treatment options. 30-60% of breast cancer patients have a grade 2 (G2) malignancy resulting in an intermediate prognosis and the challenge of finding individualized adequate treatment options. Moreover, the lack of objectivity of the grading system is criticized. With the genetic analysis of breast cancer, the deciphering of its genes and gene expression profiling using microarray technology is a new way of classifying breast cancer Patients, Materials, and Methods. Tumor tissue from 229 breast cancer patients collected from January 2009 - June 2010 was examined. A histological grading of the tumors was determined and gene expression profiling was performed by microarray technology (Agilent Technologies). Gene expression data were reduced and a novel molecular grade index (MGI-9) was defined as a G1 versus G3 classifier by SAM (Significance Analysis of Microarray) and PAM (Prediction Analysis of Microarray). The validity of the newly defined MGI-9 as a prognostic factor was tested by applying it to three published data sets with external follow-up information for a total of 760 patients Results: The significance analysis of gene expression profiles of 229 breast cancer patients using SAM showed more than 11,000 significantly differentially expressed genes according to the grading classes G1-2-3. The number of differentially expressed genes was reduced to 228 genes by SAM analysis using only the G1 vs. G3 cases (63 patients). PAM analysis resulted in a more reduced classifier of 44 molecular probes and we could show that a subset of 9 genes gave similar classification results and could reliably used in FFPE Material. The new MGI-9 index was tested in thee external data sets (GSE1456, GSE2034 and GSE3494) and could subdivide the histological G2 group in two molecular subgroups G2A and G2B with significant impact on survival in all test scenarios. Conclusion: The results show that MGI 9-gene molecular grade index is more informative than the conventional histopathological grading system. In order to validate MGI-9 a prospective, randomized study with long-term follow-up for local recurrence rate and distant metastases is urgently needed. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-05-02.

BACKGROUND: Neoadjuvant radiochemotherapy (NRT-CHX) in locally advanced noninflammatory breast cancer (LABC) is an innovative method in treating patients. Proliferation markers make up the majority of genes included in RNA-based prognostic gene signatures applied for breast cancer patients. In this subgroup analysis long-term clinical outcome data and predictive factors were analyzed. PATIENTS AND METHODS: During 1991-1998, a total of 315 LABC patients (cT1-cT4/cN0-N1) were treated with NRT-CHX. Preoperative radiotherapy (RT) consisted of external beam radiation therapy (EBRT) of 50 Gy (5 × 2 Gy/week) to the breast and the supra-/infraclavicular lymph nodes combined with an electron boost in 214 cases afterwards or -in case of breast conservation- a 10-Gy interstitial boost with (192)Ir after loading before EBRT. Chemotherapy was administered prior to RT in 192 patients, and concomitantly in 113; 10 patients received no chemotherapy. The impact of age, tumor grade, nodal status, hormone and growth factor receptor status (ER, PR, EGFR), p53, ki-67, HER2/neu, and bcl-2 on pathological complete response pCR and disease-free survival were examined in uni- and multivariate terms. RESULTS: Hormone receptor status, proliferative activity, bcl-2, EGFR-status and clinical tumor size had a significant impact on predicting neoadjuvant therapy success. Age, cN, grading, p53, and HER2/neu status failed to reach a significant correlation to complete remission. All examined immunohistochemical factors with the exception of EGFR, and all clinical factors displayed an univariately significant impact on DFS (disease free survival). Particularly, while HER-2/neu had no predictive value for pCR it displayed the highest impact on DFS after complete response (n = 92), even in a multivariate setting with clinical tumor size and nodal status. Complementary, p53 was the most superior immunhistochemical factor for prognosis after neoadjuvant incomplete remission (n = 223). CONCLUSION: Her2/neu is a predictive marker for overall survival independent from the pCR. It has no predictive value for the pCR. P53 is a prognostic marker for patients with incomplete remission. Prospective studies are needed to evaluate their use for decisions to further individualize adjuvant treatment after neoadjuvant radiochemotherapy. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-05-03.

Background We compared gene expression profiles in acutely dissected aorta with those in normal control aorta. Materials and methods Ascending aorta specimen from patients with an acute Stanford A-dissection were taken during surgery and compared with those from normal ascending aorta from multiorgan donors using the BD Atlas™ Human1.2 Array I, BD Atlas™ Human Cardiovascular Array and the Affymetrix HG-U133A GeneChip ®. For analysis only genes with strong signals of more than 70 percent of the mean signal of all spots on the array were accepted as being expressed. Quantitative real-time polymerase chain reaction (RT-PCR) was used to confirm regulation of expression of a subset of 24 genes known to be involved in aortic structure and function. Results According to our definition expression profiling of aorta tissue specimens revealed an expression of 19.1% to 23.5% of the genes listed on the arrays. Of those 15.7% to 28.9% were differently expressed in dissected and control aorta specimens. Several genes that encode for extracellular matrix components such as collagen IV α2 and -α5, collagen VI α3, collagen XIV α1, collagen XVIII α1 and elastin were down-regulated in aortic dissection, whereas levels of matrix metalloproteinases-11, -14 and -19 were increased. Some genes coding for cell to cell adhesion, cell to matrix signaling (e.g., polycystin1 and -2), cytoskeleton, as well as several myofibrillar genes (e.g., α-actinin, tropomyosin, gelsolin) were found to be down-regulated. Not surprisingly, some genes associated with chronic inflammation such as interleukin -2, -6 and -8, were up-regulated in dissection. Conclusion Our results demonstrate the complexity of the dissecting process on a molecular level. Genes coding for the integrity and strength of the aortic wall were down-regulated whereas components of inflammatory response were up-regulated. Altered patterns of gene expression indicate a pre-existing structural failure, which is probably a consequence of insufficient remodeling of the aortic wall resulting in further aortic dissection.

In situ monitoring of early effects of epirubicin-based neoadjuvant chemotherapy in breast cancer by cDNA array technology

The effects of insulin ( 10 −10 −10 −8 mol/l ) on lateral diffusion of three fluorescent lipid probes, 1- acyl -2-(N-4- nitrobenzo -2- oxa -1,3- diazole)aminocaproyl phosphatidylcholine (NBD-PC), 5-(N- hexadecanoyl)aminofluorescein (F-C16), 5-(N- dodecanoyl)aminofluorescein (F-C12), and of fluorescein isothiocyanatelabeled proteins in the plasma membrane of intact rat hepatocytes were studied by the technique of fluorescence recovery after photobleaching. The absolute lateral diffusion coefficients of the lipid analogues NBD-PC, F-C16 and F-C12 at 21°C were 2.5 · 10−9 cm2/s, 5.4 · 10−9 cm2/s and 19 · 10−9 cm2/s, respectively. The diffusion coefficient mean of proteins labeled with fluorescein isothiocyanate was 6.4 · 10−10 cm2/s. Insulin at 10−9 and 10−8 mol/l reduced the lateral diffusion coefficient for F-C12- and F-C16-labeled cells by 20% and for NBD-PC-labeled cells by 30% ( P ). The insulin effect was specific as tested by cell incubation with proinsulin and desoctapeptide insulin (10−8 mol/l) and was detectable after 7 min of insulin preincubation. In contrast to lateral diffusion of lipid probes, lateral mobility of unselected membrane proteins was not altered by insulin. The observed modulation of lipid dynamics in the plasma membrane of intact hepatocytes, by which a variety of membrane functions can be influenced, may be an important step in the mechanism of insulin action.

Objectives: heritable connective tissue abnormalities and arterial hypertension may predispose to aortic dissection. This study evaluates gene expression profiles in the acutely dissected human aorta. Design, Materials and Methods: Atlas™ Human Broad Arrays I, II, and III (Clontech) were used to compare gene expression in acutely dissected (6 patients) and normal ascending aortas (6 multiorgan donors). The tissues were also compared macroscopically. Results: of 3537 genes analysed, 1250 (35%) were expressed in aortic tissue. For statistical analysis we focused on 627 genes, which had an intensity>0.95 of the mean patients or controls. Dissected and adjacent macroscopically intact aorta displayed similar gene expression patterns. On the contrary, 66 genes were expressed significantly different in dissected aorta, compared with undiseased control aorta of multiorgan donors. Genes, predominantly upregulated in dissection, are involved in inflammation, in extracellular matrix proteolysis, in proliferation, translation and transcription. Predominantly downregulated genes code for extracellular matrix proteins, adhesion proteins and cytoskeleton proteins. Conclusion: our results demonstrate for the first time the complexity of the dissecting process on a molecular level. The ultimate dissection seems to be the dramatic endpoint of a long-lasting process of degradation and insufficient remodelling of the aortic wall. Altered patterns of gene expression suggest a pre-existing structural failure of the aortic wall, resulting in dissection.