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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: 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

An earlier published series of neoadjuvant radiochemotherapy (NRT-CHX) in locally advanced noninflammatory 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.During 1991-1998, 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 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 pCR and overall survival as end point.The total pCR rate after neoadjuvant RT-CHX reached 29.2%, with LABC breast conservation becoming possible in 50.8% of cases. 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 2 months) increases the probability of pCR after NRT-CHX.

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.

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.

About 5%-10% of breast cancers are considered to be hereditary and associated with germline mutations of specific genes. As yet, the most frequently affected genes identified are BRCA1 and BRCA2, but also other genes such as TP53 are supposed to influence the predisposition toward breast cancer. In the present study, we analyzed patients of 19 German families with early onset breast cancer and/or a family history of breast and/or ovarian cancer for the presence of mutations in BRCA1 and TP53. In addition, we screened for germline mutations in the putative tumor suppressor gene TSG101. For this purpose we used direct sequence analysis of the entire coding regions for all three genes and, in the case of BRCA1, single-strand conformation polymorphism analysis and protein transcription-translation assays. We identified eight previously described polymorphisms and several aberrations in BRCA1: 1 unclassified missense mutation, 3 small protein truncating mutations, 1 novel pseudoexon, and 5 splicing variants. No mutation was detected in TP53. Analysis of TSG101 transcripts revealed an aberrant transcript in two breast cancer patients belonging to the same family, suggesting TSG101 as a predisposing gene in hereditary breast cancer.

Background and purpose : The response of squamous cell carcinomas of the head and neck (HNSCC) to radio/chemotherapy is accompanied by complex changes in patterns of gene expression. It is highly probable that a better understanding of molecular and genetic changes can help to optimize the treatment of HNSCC. cDNA arrays provide a powerful tool for high-throughput monitoring of gene expression in small clinical specimens. Materials and methods : We used tumour biopsies from four patients with HNSCC which have been taken prior to and during radio/chemotherapy. The patterns of gene expression obtained from clinical samples were compared with gene expression profiles of two squamous cell carcinoma cell lines (FaDU and UD-7A). Results : The experimental data analysis revealed changes in expression levels of several genes during radio/chemotherapy. Despite treatment, independent samples taken from the same cell line or tumour in situ were more similar to each other than either was to other specimens. The data indicate a high gene heterogeneity of HNSCC that is preserved during treatment. Conclusions : From our preliminary results we conclude that the cDNA array experimental approach can detect differences in gene expression between treated and untreated small tumour biopsies, as well as inter-individual differences in expression profiles between HNSCC tumours. The examination of a greater sample size will be needed to make this preliminary evaluation useful to elucidate the functional significance of individual genes which exhibit altered levels of expression under radiation 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.

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

PURPOSE In 1984, the German Breast Cancer Study Group (GBSG) started a multicenter randomized clinical trial to compare the effectiveness of three versus six cycles of 500 mg/m2 cyclophosphamide, 40 mg/m2 methotrexate, and 600 mg/m2 fluorouracil (CMF) on day 1 and 8 starting perioperatively with or without tamoxifen (TAM) (3 x 10 mg/d for 2 years). The aim of the trial was to compare recurrence-free and overall survival between the different treatment modalities. PATIENTS AND METHODS During 5 years, 41 institutions randomized 473 patients (3 x CMF: 145; 3 x CMF + TAM: 93; 6 x CMF 144; 6 x CMF + TAM: 91). Until March 31, 1992, median follow-up time was 56 months with 197 events for disease-free survival and 116 deaths observed. This provides a power of approximately 80% to detect a potential treatment difference corresponding to a relative risk (RR) of 0.67 for recurrence-free survival. Treatment modalities and various patient characteristics were evaluated by means of a multivariate Cox regression analysis. RESULTS No significant difference in recurrence-free survival was observed with respect to hormonal therapy (RR = 0.75 TAM v no TAM; 95% confidence interval [CI], 0.54 to 1.04; P = .08) as well as duration of chemotherapy (RR = 0.90 of 6 x CMF v 3 x CMF; 95% CI, 0.67 to 1.19; P = .45). Similar results were obtained for overall survival. The multivariate analysis revealed a significant prognostic impact of the number of positive lymph nodes and the progesterone receptor level on recurrence-free survival. Compliance with chemotherapy within the range of 85% to 115% of the target dose was achieved in 94% and 78% of the patients randomized to 3 x CMF and 6 x CMF, respectively. Sufficient compliance with TAM was reported for 141 patients (93%). CONCLUSION At this stage of follow-up, six courses of CMF are not superior to three courses with respect to recurrence-free survival.

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.

Liposomal vectors based on cationic lipids have been proven to be an attractive alternative to viral vectors in gene therapy protocols with regard to safety and manufacturing concerns. In order to improve the transfection efficiency we have synthesized two novel carboxycholesteryl-modified chloroquine analogues. Due to their potential endosomal buffering capacity these compounds enable the efficient transfection of various gynecological tumors and therefore are promising reagents in gene therapy applications.

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.

The available clinical prognostic tools show an obvious limitation in predicting the outcome of breast cancer patients, and pathological features cannot classify tumours accurately. Microarray-based molecular classification of breast tumours or selection of gene expression panels to improve risk prediction or treatment outcomes are thought to be theoretically superior to established clinical and pathological criteria, based on guidelines such as the St Gallen and National Institute of Health consensus, or which use specific prognostic tools, such as the Nottingham Prognostic Index or Adjuvant-Online algorithm. Although two diagnostic tests based on gene expression profiling of breast cancer are commercially available, a new molecular classification and molecular forecasting of breast cancer based on expression profiling cannot outperform the standard tumour diagnostic at present. This review focuses on some important problems in the practical application of molecular profiling of breast cancer for clinical purposes.

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.

Cytokines play important roles in the expression of adhesion molecules and the function of anti-tumor effector cells in the immune system. In this study, the influence of interleukin-12 (IL-12) and IL-18 on the expression of ICAM-1 and natural killer (NK)-cell mediated lysis in a human osteosarcoma cell line (HOS) was evaluated. ICAM-I expression of HOS cells were analyzed by flow cytometry following treatment with IL-12, IL-18 or both, and in co-cultures with peripheral lymphocytes. NK-cell activation in response to IL-12 and IL-18 was investigated by selective flow cytometry using propidium iodide. ICAM-1 expression on HOS cells was significantly enhanced by IL-12, but only when co-cultured in cell-to-cell contact with peripheral lymphocytes. Antibodies to interferon-gamma abrogated this effect. If HOS cells and peripheral lymphocytes were separated in co-cultures, IL-18 could substitute for cell-to-cell contact, facilitating IL-12-mediated enhancement of ICAM-1. Addition of IL-18 also enhanced NK-mediated cytolysis of HOS cells. These findings demonstrate that IL-12 can enhance the expression of ICAM-1 in the presence of IFN-gamma and, with IL-18, enhances NK anti-tumor activity. Immunomodulation via cytokine therapy may lead to improved eradication of chemotherapy-resistant osteosarcomas.

Tamoxifen (TAM) is a well-tolerated compound in the treatment of breast cancer and is primarily considered to act by competition with estrogen receptors (ER). Here we investigated the in vitro efficacy and potentially underlying mechanisms of TAM in established cell lines of squamous cell carcinomas of the head and neck (SCCHN). Using proliferation and apoptosis assays the antitumor activity of TAM in five SCCHN and the breast carcinoma line MCF-7 (positive control) was determined. MCF-7 was more sensitive to low-dose TAM (below 1 microM), whereas SCCHN showed significant growth inhibition at higher TAM concentrations (5-10 microM). Growth curve analysis and apoptosis assays were indicative for a cytostatic effect of low-dose TAM and high-dose TAM led to cell loss by apoptosis in sensitive SCCHN. In order to further characterize the observed antitumor effects we determined the amount of steroid hormone receptors with the dextran-coated charcoal method and immunocytochemistry. In addition, production of transforming growth factor (TGF-)-alpha, -beta1 and -beta2 was measured by ELISA, and protein kinase C (PKC) activity was assessed with a radioligand assay. Except MCF-7, none of the SCCHN lines was positive for ER. TAM caused decreased TGF-alpha and increased TGF-beta levels in MCF-7, but not in SCCHN supernatants. Furthermore, the antiestrogen reduced PKC activity in MCF-7, but not in SCCHN. In the present in vitro system, the observed antitumor activity of high-dose TAM in SCCHN cannot be explained by estrogen antagonism, alterations of TGF-alpha/beta levels or decreased PKC activity.

In this study we evaluated the ability of inducing activation of the enzyme indoleamine 2,3-dioxygenase (IDO) for the development of a new adjuvant therapy of osteosarcomas. The pathway that has described in the literature states that IDO activity is elevated by IFN-gamma. This mechanism is important because the increased IDO activation induces an intracellular degradation of the essential amino acid tryptophan and thereby activates apoptosis in human osteosarcoma cells.Four different well-established human osteosarcoma cell lines (MNNG/HOS, KHOS-240, HOS and MG-63) were investigated in vitro. Several cytokines were tested for their ability to induce IDO activity. However special emphasis was placed to evaluate the synergistic effects of Interleukin-12 (IL-12) in combination with Interleukin-18 (IL-18). In the first series of experiments IDO induction was investigated by direct application of the cytokines IFN-gamma, TNF-alpha, IL-12 and IL-18 in different concentrations. Secondly, the increase of IDO expression from osteosarcoma cell lines was analysed in the presence of activated lymphocytes with or without cytokine application.Our results demonstrated that the combined application of IL-12 and IL-18 enhanced IDO activity in the human osteosarcoma cell lines HOS and MG-63, in the presence of activated lymphocytes. In the absence of activated lymphocytes, no significant enhancement could be detected. In all our experiments the increase in IDO expression was only partly inhibited by blocking INF-gamma.The presented study demonstrates that IL-12 and IL-18, or even more a combined application of both cytokines, induce IDO expression besides the known pathway via IFN-gamma. These mechanisms have been shown herein for the first time in human osteosacoma cell lines. Since IDO expression could still be shown after complete blocking of IFN-gamma, we conclude that at least a second pathway is responsible for inducing IDO activity. This is in contrast to the present knowledge about IDO activation.

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

The purpose of this investigation was to study the long-term prognosis of breast cancer patients with 10 or more positive lymph nodes after conventional chemotherapy treatment with cyclophosphamide, methotrexate and 5-fluorouracil (CMF). Between 1984 and 1989, 1048 node-positive patients were treated with CMF in two separate trials conducted by the German Breast Cancer Study Group (GBSG). Subgroups either received radiotherapy or tamoxifen in addition. In this study, long-term prognosis in the subgroup of 141 patients with 10 or more positive lymph nodes was investigated. Univariate and multivariate Cox models were used to evaluate the effect of prognostic factors on event-free survival (EFS) and overall survival (OS). Both univariate and multivariate analyses revealed the progesterone receptor (PR) status as the dominating prognostic factor for both EFS and OS, resulting in a strongly increased risk of more than 2-fold for receptor-negative patients. A large number of positive lymph nodes also affected the prognosis for EFS. In univariate analysis, the degree of lymph node involvement (i.e. percentage of positive nodes out of all examined nodes), oestrogen status (ER) status, and tumour grade also showed significant effects. To conclude, the prognosis in the subgroup of patients with 10 or more positive lymph nodes is heterogeneous. Some surprisingly high survival rates have been observed in case series of breast cancer patients treated with high-dose chemotherapy which may be explained by patient selection. From the usual factors investigated in this study, the PR status showed the strongest effect, and, at least this factor should be taken into account in the design and analysis of trials for breast cancer patients with a poor prognosis.

The aim of this study was to evaluate the clinical significance of a dual-parameter immunoflow cytometry (DPI-FCM) assay in the detection of tumor cells in barbotage specimens of bladder cancer patients.DPI-FCM is an automated method, based on the utilization of two monoclonal antibodies (mAbs) either used for a preselection of urothelial cells (mAb Due AUT 2) or the analysis of the expression of a differentiation antigen within the preselected urothelial cells (mAb Due ABC 3). The ratio of ABC 3-positive urothelial cells was used to discriminate between the normal and malignant state of the urothelium. At the time of examination 40 patients had endoscopically overt bladder tumors. Another 30 patients without endoscopically visible tumors were examined before routine rebiopsy. Thirty barbotage specimens from patients with diseases not related to bladder cancer were examined as controls.Overall, the sensitivity of DPI-FCM in patients with endoscopically overt and invisible residual tumors was 95 and 83%, respectively, regardless of concomitant urinary tract infection. The sensitivity of DPI-FCM for both patient groups was 86, 95 and 94% for tumor grades 1, 2 and 3, respectively. The specificity of the method in 30 patients with no history of bladder cancer was 93%.DPI-FCM appears to be a highly reliable method of recognizing tumor cells in bladder barbotage specimens even in patients with concomitant urinary tract infection. The procedure may be of value in monitoring bladder cancer patients for tumor recurrence.

The WAF1/p21 gene product is an inhibitor of cyclin-dependent kinases which can be induced by the tumor suppressor p53 and mediate some of its effects, or function in p53-independent pathways of cell cycle regulation. Although a potential tumor suppressor gene, WAF1/p21 is expressed in bladder cancer. To elucidate the function of p21 in tumor cells we have investigated in urothelial carcinoma cell lines: i) WAF1/p21 mRNA and protein expression, ii) the biological effects of p21 overexpression or down-regulation and (iii) whether p21 can be induced by p53. WAF1/p21 mRNA levels examined in four cell lines were comparable to bladder mucosa. One cell line, HT1376, failed to express p21 protein due to a frame shift mutation. Overexpression of WAF1/p21 cDNA inhibited clone formation in three cell lines, whereas transfection with antisense WAF1 increased clone sizes and numbers. WAF1 sense clones showed diminished cell proliferation compared to the parental cell line. Apoptosis- induced wild-type p53 was not inhibited by overexpression of antisense WAF1/p21. In a cell clone derived from line VMCub1 by stable transfection with wild-type p53 under the control of a metallothionein promotor, p21 was induced along with p53 upon activation of the promoter with zinc chloride. This induction was accompanied by a decrease in cell proliferation but by little apoptosis. These data suggest that p21 inhibits proliferation in a p53-dependent or independent manner but does not mediate p53-induced apoptosis in urothelial carcinoma cells.

Purpose : To test the feasibility of recombinant human granulocyte colony stimulating factor application during large-field radiotherapy. Methods and Materials : Fifteen patients with clinically and histologically proven malignancy who received large-field radiotherapy entered this study. Administration of recombinant granulocyte colony stimulating factor (G-CSF) at a dose of 300 μg subcutaneously was started on Friday and was continued on Saturday and Sunday after the first radiotherapy treatment, which began on the Monday before. In this way four courses of G-CSF were applied every Friday, Saturday, and Sunday during the radiotherapy period. Absolute neutrophil cell (ANC) and blood counts were monitored twice a week and compared to a second group of 15 patients who received large-field radiotherapy without G-CSF. Before and at the end of every cycle of G-CSF, ANC, blood counts, and biochemistry were measured. We compared the myelotoxicity of the patients treated with G-CSF with 15 patients without G-CSF treated at the same period with large-field radiotherapy, in match pair technique. Results : G-CSF increased the ANC throughout the period of irradiation, and the treatment time needed for completing radiotherapy was shorter in the group who received G-CSF. Fourteen of 15 patients who received G-CSF treatment completed large-field radiotherapy without pause. Only 1 of 15 patients not receiving G-CSF was able to receive radiation treatment on schedule. Patients receiving G-CSF completed treatment with the mantle-field technique in 24 days and those with the abdominal bath technique in 26.5 days. Conversely, patients treated without G-CSF completed treatment with the mantle-field technique in 30.5 days and those with the abdominal bath technique in 36 days. The most frequent side effect was musculoskeletal pain. Conclusion : The prophylactic application of G-CSF during large-field radiotherapy before the onset of neutropenia was feasible in this schedule. Whether or not this shortening of treatment duration will translate into an improvement in efficacy is not clear.

The introduction of monoclonal antibodies into the diagnosis of transitional cell carcinoma (TCC) has been a further step toward the use of biological parameters to support conventional cytological diagnosis of this tumor entity. Several investigators have demonstrated the high sensitivity and good specificity of immunocytology. Nevertheless this technique is hampered by the inconvenient and time-consuming microscopic analysis. The purpose of this study was to combine the advantages of immunocytology with the capabilities of an automated flow-cytometric system. Since all monoclonal antibodies (mAbs) currently used for immunocytology cross-react with granulocytes a preselection of urothelial cells becomes necessary for immuno-flow cytometry (immuno-FCM). Based on earlier analyses mAb Due AUT 2, reactive against urothelium and not against granulocytes, was chosen to preselect for urothelial cells. mAb Due ABC 3 was used to discriminate between normal and malignant urothelial cells. Initial experiments including 10 barbotage specimens from patients with histologically proven TCC and concomitant urinary tract infection yielded a high sensitivity (90%) of immuno-FCM. In 10 control patients with malignancies other than bladder TCC and benign diseases ABC 3 expression was not increased. Flow-cytometric examination of irrigation specimens from 10 patients with a history of bladder cancer but without cystoscopic and cytologic evidence of tumor recurrence showed abnormal results in 6 patients. Rebiopsy and/or cystectomy confirmed tumor recurrence in 5 of these patients and in 1 patient with negative immuno-FCM. Ongoing prospective trials will further define the clinical impact of this approach in the diagnosis and follow-up of patients with bladder cancer.

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.