Tim Beissbarth

Preoperative Versus Postoperative Chemoradiotherapy for Locally Advanced Rectal Cancer: Results of the German CAO/ARO/AIO-94 Randomized Phase III Trial After a Median Follow-Up of 11 Years

PURPOSE Preoperative chemoradiotherapy (CRT) has been established as standard treatment for locally advanced rectal cancer after first results of the CAO/ARO/AIO-94 [Working Group of Surgical Oncology/Working Group of Radiation Oncology/Working Group of Medical Oncology of the Germany Cancer Society] trial, published in 2004, showed an improved local control rate. However, after a median follow-up of 46 months, no survival benefit could be shown. Here, we report long-term results with a median follow-up of 134 months. PATIENTS AND METHODS A total of 823 patients with stage II to III rectal cancer were randomly assigned to preoperative CRT with fluorouracil (FU), total mesorectal excision surgery, and adjuvant FU chemotherapy, or the same schedule of CRT used postoperatively. The study was designed to have 80% power to detect a difference of 10% in 5-year overall survival as the primary end point. Secondary end points included the cumulative incidence of local and distant relapses and disease-free survival. RESULTS Of 799 eligible patients, 404 were randomly assigned to preoperative and 395 to postoperative CRT. According to intention-to-treat analysis, overall survival at 10 years was 59.6% in the preoperative arm and 59.9% in the postoperative arm (P = .85). The 10-year cumulative incidence of local relapse was 7.1% and 10.1% in the pre- and postoperative arms, respectively (P = .048). No significant differences were detected for 10-year cumulative incidence of distant metastases (29.8% and 29.6%; P = .9) and disease-free survival. CONCLUSION There is a persisting significant improvement of pre- versus postoperative CRT on local control; however, there was no effect on overall survival. Integrating more effective systemic treatment into the multimodal therapy has been adopted in the CAO/ARO/AIO-04 trial to possibly reduce distant metastases and improve survival.3516 Background: CAO/ARO/AIO-94 was published in 2004 with a median follow-up of 46 months (Sauer et al., N Engl J Med 2004). This trial established preoperative CRT as standard treatment for rectal cancer based on an improved local control rate at 5 years, however, no survival benefit could be shown. We here report results with a median follow-up of 134 months. METHODS We randomly assigned 823 patients with stage II or III rectal cancer to preoperative CRT (50.4 Gy) with 5-FU (1 g/msq/days 1-5, 29-33), surgery, and adjuvant 5-FU (500 mg/msq/days 1-5, 4 cycles), or the same schedule applied postoperatively. The study was designed to have 80% power to detect a difference of 10% in the 5-year overall survival as primary endpoint. Secondary endpoints included the cumulative incidence of local and distant relapses and disease-free survival. RESULTS Of 823 patients, 404 and 395 were randomized to preoperative and postoperative CRT, respectively; 24 were ineligible, and 38 requested a change in treatment group. Thus, 406 patients received preoperative CRT, 393 were treated in the postoperative arm. As of 12/2010, updated data for life and tumor status were available for 791 and 783 of 799 eligible patients, respectively. Overall survival at 10 years was 59.9 years (95% CI, 55.0-64.8%) in the preoperative arm, and 59.5% (95% CI, 54.6-64.4%) in the postoperative arm (p=0.86, log-rank test, according to intention to treat). The 10-year cumulative incidence of local relapse after macroscopically complete resection was 5.7% (95% CI, 3.2-8.2%) and 10.4% (95% CI, 7.1-13.4%) in the pre- and postoperative arms, respectively (p=0.009, log-rank test, according to actual treatment). No significant differences were detected for 10-year cumulative incidence of distant metastases (25.5% both, p=0.88) and DFS. CONCLUSIONS There is a persisting significant improvement of pre- vs. postoperative CRT on local control, however, no effect on overall survival. Integrating more effective systemic treatment into the combined modality treatment has been adopted in trial CAO/ARO/AIO-04 to possibly reduce distant metastases and improve survival.

Circulating miRNAs are correlated with tumor progression in prostate cancer

Circulating miRNAs have recently been indicated as practicable and promising biomarkers for noninvasive diagnosis in various tumor entities. However, cell‐free miRNAs have not been found to correlate with clinicopathological variables in epithelial carcinomas. To learn more about the potential clinical relevance of circulating miRNAs in prostate cancer, we screened 667 miRNAs in serum samples from patients with metastatic (n = 7) and localized prostate cancer (n = 14). Various miRNAs were highly abundant in the sera of patients with metastatic disease, and five upregulated miRNAs (miRNA‐375, miRNA‐9*, miRNA‐141, miRNA‐200b and miRNA‐516a‐3p) were selected for further validation. In the first validation study (n = 45), selected miRNAs were analyzed in a prospectively collected serum set taken from different prostate cancer risk groups. Most of the selected miRNAs were significantly correlated with adverse risk factors when different clinicopathological variables were analyzed. Circulating miRNA‐375 and miRNA‐141 turned out to be the most pronounced markers for high‐risk tumors. Their levels also correlated with high Gleason score or lymph‐node positive status in a second independent validation study (n = 71). In addition, the expression levels of miRNA‐375 and miRNA‐141 were monitored in 72 prostate tissue samples (36 tumor vs. 36 benign). Both miRNAs were highly expressed in all samples and significantly upregulated in the tumors compared to normal tissues. Overall, our observations suggest that miRNA‐375 and miRNA‐141 expression is enhanced in prostate cancer specimens and their release into the blood is further associated with advanced cancer disease.

Comprehensive, Quantitative Mapping of T Cell Epitopes in Gluten in Celiac Disease

Three highly immunogenic peptides from gluten are primarily responsible for celiac disease, suggesting a rational immunotherapeutic approach that could replace the need for strict, lifelong dietary gluten avoidance. Taming of the Sprue Gluten, a complex protein in wheat, barley, and rye, forms the elastic network responsible for the airy texture of bread. But gluten can also trigger a prevalent inflammatory disorder—celiac disease (sprue)—which afflicts sufferers with problems such as gastrointestinal upset, fatigue, and anemia, and confers increased risks of osteoporosis, autoimmune disease, and cancer. The current therapy consists of strict lifelong avoidance of all foods containing gluten. The development of alternatives has been hampered by the inability to fully characterize the immune response to the toxic peptides within these grains. Several immunotoxic peptides from wheat have been implicated, but it has remained unclear how they contribute to the overall immune response in celiac disease, or whether other potentially toxic peptides from barley and rye exist. Tye-Din and colleagues have now comprehensively assessed the more than 16,000 potentially toxic peptides contained within wheat, barley, and rye, and identified which ones stimulate T cells from celiac disease patients. By feeding doses of wheat, barley, or rye to more than 200 people with celiac disease, the authors were able to examine the induced T cells appearing in the bloodstream several days afterward. These T cells were then tested for recognition of peptides from large libraries encompassing every possible toxic peptide from wheat, barley, and rye. Surprisingly, they found that just three highly active peptides were responsible for most of the immune response seen in patients with celiac disease after eating any of the toxic grains. Although the range of highly stimulatory or dominant peptides was very consistent between individuals, it was dependent on which grain was consumed. A previously described peptide from wheat α-gliadin was dominant only after wheat ingestion; another distinct peptide was dominant after wheat, barley, or rye ingestion. Of most interest was the fact that a combination of these peptides, plus another from barley, could elicit 90% of the response induced by the full complement of wheat, barley, and rye proteins. Because the authors assessed every possible toxic peptide from wheat, as well as barley and rye, they can be confident that their data paint a comprehensive picture of the immune response in celiac disease. This is important because alternative therapies to the complex, costly, and inconvenient gluten-free diet are likely to require a detailed molecular understanding of the peptides driving the immune response in celiac disease. Multiple doses of peptides corresponding to immunodominant T cell epitopes are effective in treating a mouse version of celiac disease, and the discovery that a small number of peptides can elicit the disease in patients suggests that a similar approach may be successful in humans as well. Celiac disease is a genetic condition that results in a debilitating immune reaction in the gut to antigens in grain. The antigenic peptides recognized by the T cells that cause this disease are incompletely defined. Our understanding of the epitopes of pathogenic CD4+ T cells is based primarily on responses shown by intestinal T-cells in vitro to hydrolysates or polypeptides of gluten, the causative antigen. A protease-resistant 33-amino acid peptide from wheat α-gliadin is the immunodominant antigen, but little is known about the spectrum of T cell epitopes in rye and barley or the hierarchy of immunodominance and consistency of recognition of T-cell epitopes in vivo. We induced polyclonal gluten-specific T cells in the peripheral blood of celiac patients by feeding them cereal and performed a comprehensive, unbiased analysis of responses to all celiac toxic prolamins, a class of plant storage protein. The peptides that stimulated T cells were the same among patients who ate the same cereal, but were different after wheat, barley and rye ingestion. Unexpectedly, a sequence from ω-gliadin (wheat) and C-hordein (barley) but not α-gliadin was immunodominant regardless of the grain consumed. Furthermore, T cells specific for just three peptides accounted for the majority of gluten-specific T cells, and their recognition of gluten peptides was highly redundant. Our findings show that pathogenic T cells in celiac disease show limited diversity, and therefore suggest that peptide-based therapeutics for this disease and potentially other strongly HLA-restricted immune diseases should be possible.

Modeling ERBB receptor-regulated G1/S transition to find novel targets for de novo trastuzumab resistance

BackgroundIn breast cancer, overexpression of the transmembrane tyrosine kinase ERBB2 is an adverse prognostic marker, and occurs in almost 30% of the patients. For therapeutic intervention, ERBB2 is targeted by monoclonal antibody trastuzumab in adjuvant settings; however, de novo resistance to this antibody is still a serious issue, requiring the identification of additional targets to overcome resistance. In this study, we have combined computational simulations, experimental testing of simulation results, and finally reverse engineering of a protein interaction network to define potential therapeutic strategies for de novo trastuzumab resistant breast cancer.ResultsFirst, we employed Boolean logic to model regulatory interactions and simulated single and multiple protein loss-of-functions. Then, our simulation results were tested experimentally by producing single and double knockdowns of the network components and measuring their effects on G1/S transition during cell cycle progression. Combinatorial targeting of ERBB2 and EGFR did not affect the response to trastuzumab in de novo resistant cells, which might be due to decoupling of receptor activation and cell cycle progression. Furthermore, examination of c-MYC in resistant as well as in sensitive cell lines, using a specific chemical inhibitor of c-MYC (alone or in combination with trastuzumab), demonstrated that both trastuzumab sensitive and resistant cells responded to c-MYC perturbation.ConclusionIn this study, we connected ERBB signaling with G1/S transition of the cell cycle via two major cell signaling pathways and two key transcription factors, to model an interaction network that allows for the identification of novel targets in the treatment of trastuzumab resistant breast cancer. Applying this new strategy, we found that, in contrast to trastuzumab sensitive breast cancer cells, combinatorial targeting of ERBB receptors or of key signaling intermediates does not have potential for treatment of de novo trastuzumab resistant cells. Instead, c-MYC was identified as a novel potential target protein in breast cancer cells.

Monitoring the Switch from Housekeeping to Pathogen Defense Metabolism in Arabidopsis thaliana Using cDNA Arrays

Plants respond to pathogen attack by deploying several defense reactions. Some rely on the activation of preformed components, whereas others depend on changes in transcriptional activity. Using cDNA arrays comprising 13,000 unique expressed sequence tags, changes in the transcriptome ofArabidopsis thaliana were monitored after attempted infection with the bacterial plant pathogen Pseudomonas syringae pv. tomato carrying the avirulence geneavrRpt2. Sampling at four time points during the first 24 h after infiltration revealed significant changes in the steady state transcript levels of ∼650 genes within 10 min and a massive shift in gene expression patterns by 7 h involving ∼2,000 genes representing many cellular processes. This shift from housekeeping to defense metabolism results from changes in regulatory and signaling circuits and from an increased demand for energy and biosynthetic capacity in plants fighting off a pathogenic attack. Concentrating our detailed analysis on the genes encoding enzymes in glycolysis, the Krebs cycle, the pentose phosphate pathway, the biosynthesis of aromatic amino acids, phenylpropanoids, and ethylene, we observed interesting differential regulation patterns. Furthermore, our data showed potentially important changes in areas of metabolism, such as the glyoxylate metabolism, hitherto not suspected to be components of plant defense.

Distinct transcriptional MYCN/c-MYC activities are associated with spontaneous regression or malignant progression in neuroblastomas

BackgroundAmplified MYCN oncogene resulting in deregulated MYCN transcriptional activity is observed in 20% of neuroblastomas and identifies a highly aggressive subtype. In MYCN single-copy neuroblastomas, elevated MYCN mRNA and protein levels are paradoxically associated with a more favorable clinical phenotype, including disseminated tumors that subsequently regress spontaneously (stage 4s-non-amplified). In this study, we asked whether distinct transcriptional MYCN or c-MYC activities are associated with specific neuroblastoma phenotypes.ResultsWe defined a core set of direct MYCN/c-MYC target genes by applying gene expression profiling and chromatin immunoprecipitation (ChIP, ChIP-chip) in neuroblastoma cells that allow conditional regulation of MYCN and c-MYC. Their transcript levels were analyzed in 251 primary neuroblastomas. Compared to localized-non-amplified neuroblastomas, MYCN/c-MYC target gene expression gradually increases from stage 4s-non-amplified through stage 4-non-amplified to MYCN amplified tumors. This was associated with MYCN activation in stage 4s-non-amplified and predominantly c-MYC activation in stage 4-non-amplified tumors. A defined set of MYCN/c-MYC target genes was induced in stage 4-non-amplified but not in stage 4s-non-amplified neuroblastomas. In line with this, high expression of a subset of MYCN/c-MYC target genes identifies a patient subtype with poor overall survival independent of the established risk markers amplified MYCN, disease stage, and age at diagnosis.ConclusionsHigh MYCN/c-MYC target gene expression is a hallmark of malignant neuroblastoma progression, which is predominantly driven by c-MYC in stage 4-non-amplified tumors. In contrast, moderate MYCN function gain in stage 4s-non-amplified tumors induces only a restricted set of target genes that is still compatible with spontaneous regression.

Tumor Regression Grading After Preoperative Chemoradiotherapy for Locally Advanced Rectal Carcinoma Revisited: Updated Results of the CAO/ARO/AIO-94 Trial

PURPOSE We previously described the prognostic impact of tumor regression grading (TRG) on the outcome of patients with rectal carcinoma treated with preoperative chemoradiotherapy (CRT) in the CAO/ARO/AIO-94 trial. Here we report long-term results after a median follow-up of 132 months. PATIENTS AND METHODS TRG after preoperative CRT was determined in 386 surgical specimens by the amount of viable tumor cells versus fibrosis, ranging from TRG 4 (no viable tumor cells) to TRG 0 (no signs of regression). Clinicopathologic parameters and TRG were correlated to the cumulative incidence of local recurrence, distant metastasis, and disease-free survival (DFS). RESULTS Ten-year cumulative incidence of distant metastasis and DFS were 10.5% and 89.5% for patients with TRG 4 (complete regression), 29.3% and 73.6% for TRG 2 and 3 (intermediate regression), and 39.6% and 63% for TRG 0 and 1 (poor regression), respectively (P = .005 and P = .008, respectively). On multivariable analysis, residual lymph node metastasis (ypN+) and TRG were the only independent prognostic factors for cumulative incidence of distant metastasis (P < .001 and P = .035, respectively) and DFS (P < .001 and P = .039, respectively), whereas local recurrence was significantly affected by ypN status (P < .001) and lymphatic invasion (P = .026). CONCLUSION Complete and intermediate tumor regressions were associated with improved long-term outcome in patients with rectal carcinoma after preoperative CRT independent of clinicopathologic parameters. This classification system needs to be prospectively tested in multiple data sets to validate its reproducibility in a wider setting.

The Rectal Cancer microRNAome – microRNA Expression in Rectal Cancer and Matched Normal Mucosa

Purpose: miRNAs play a prominent role in a variety of physiologic and pathologic biologic processes, including cancer. For rectal cancers, only limited data are available on miRNA expression profiles, whereas the underlying genomic and transcriptomic aberrations have been firmly established. We therefore, aimed to comprehensively map the miRNA expression patterns of this disease. Experimental Design: Tumor biopsies and corresponding matched mucosa samples were prospectively collected from 57 patients with locally advanced rectal cancers. Total RNA was extracted, and tumor and mucosa miRNA expression profiles were subsequently established for all patients. The expression of selected miRNAs was validated using semi-quantitative real-time PCR. Results: Forty-nine miRNAs were significantly differentially expressed (log2-fold difference >0.5 and P < 0.001) between rectal cancer and normal rectal mucosa. The predicted targets for these miRNAs were enriched for the following pathways: Wnt, TGF-beta, mTOR, insulin, mitogen-activated protein kinase, and ErbB signaling. Thirteen of these 49 miRNAs seem to be rectal cancer-specific, and have not been previously reported for colon cancers: miR-492, miR-542-5p, miR-584, miR-483-5p, miR-144, miR-2110, miR-652, miR-375, miR-147b, miR-148a, miR-190, miR-26a/b, and miR-338-3p. Of clinical impact, miR-135b expression correlated significantly with disease-free and cancer-specific survival in an independent multicenter cohort of 116 patients. Conclusion: This comprehensive analysis of the rectal cancer miRNAome uncovered novel miRNAs and pathways associated with rectal cancer. This information contributes to a detailed view of this disease. Moreover, the identification and validation of miR-135b may help to identify novel molecular targets and pathways for therapeutic exploitation. Clin Cancer Res; 18(18); 4919–30. ©2012 AACR.

Integrative analysis of RUNX1 downstream pathways and target genes

BackgroundThe RUNX1 transcription factor gene is frequently mutated in sporadic myeloid and lymphoid leukemia through translocation, point mutation or amplification. It is also responsible for a familial platelet disorder with predisposition to acute myeloid leukemia (FPD-AML). The disruption of the largely unknown biological pathways controlled by RUNX1 is likely to be responsible for the development of leukemia. We have used multiple microarray platforms and bioinformatic techniques to help identify these biological pathways to aid in the understanding of why RUNX1 mutations lead to leukemia.ResultsHere we report genes regulated either directly or indirectly by RUNX1 based on the study of gene expression profiles generated from 3 different human and mouse platforms. The platforms used were global gene expression profiling of: 1) cell lines with RUNX1 mutations from FPD-AML patients, 2) over-expression of RUNX1 and CBFβ, and 3) Runx1 knockout mouse embryos using either cDNA or Affymetrix microarrays. We observe that our datasets (lists of differentially expressed genes) significantly correlate with published microarray data from sporadic AML patients with mutations in either RUNX1 or its cofactor, CBFβ. A number of biological processes were identified among the differentially expressed genes and functional assays suggest that heterozygous RUNX1 point mutations in patients with FPD-AML impair cell proliferation, microtubule dynamics and possibly genetic stability. In addition, analysis of the regulatory regions of the differentially expressed genes has for the first time systematically identified numerous potential novel RUNX1 target genes.ConclusionThis work is the first large-scale study attempting to identify the genetic networks regulated by RUNX1, a master regulator in the development of the hematopoietic system and leukemia. The biological pathways and target genes controlled by RUNX1 will have considerable importance in disease progression in both familial and sporadic leukemia as well as therapeutic implications.

The Histone H2B Monoubiquitination Regulatory Pathway Is Required for Differentiation of Multipotent Stem Cells

Extensive changes in posttranslational histone modifications accompany the rewiring of the transcriptional program during stem cell differentiation. However, the mechanisms controlling the changes in specific chromatin modifications and their function during differentiation remain only poorly understood. We show that histone H2B monoubiquitination (H2Bub1) significantly increases during differentiation of human mesenchymal stem cells (hMSCs) and various lineage-committed precursor cells and in diverse organisms. Furthermore, the H2B ubiquitin ligase RNF40 is required for the induction of differentiation markers and transcriptional reprogramming of hMSCs. This function is dependent upon CDK9 and the WAC adaptor protein, which are required for H2B monoubiquitination. Finally, we show that RNF40 is required for the resolution of the H3K4me3/H3K27me3 bivalent poised state on lineage-specific genes during the transition from an inactive to an active chromatin conformation. Thus, these data indicate that H2Bub1 is required for maintaining multipotency of hMSCs and plays a central role in controlling stem cell differentiation.