Matthias Kolberg

Survival meta-analyses for >1800 malignant peripheral nerve sheath tumor patients with and without neurofibromatosis type 1

There are conflicting reports as to whether malignant peripheral nerve sheath tumor (MPNST) patients with neurofibromatosis type 1 (NF1) have worse prognosis than non-NF1 MPNST patients. Large clinical studies to address this problem are lacking due to the rareness of MPNST. We have performed meta-analyses testing the effect of NF1 status on MPNST survival based on publications from the last 50 years, including only nonoverlapping patients reported from each institution. In addition, we analyzed survival characteristics for 179 MPNST patients from 3 European sarcoma centers. The meta-analyses including data from a total of 48 studies and >1800 patients revealed a significantly higher odds ratio for overall survival (OROS) and disease-specific survival (ORDSS) in the non-NF1 group (OROS = 1.75, 95% confidence interval [CI] = 1.28–2.39, and ORDSS = 1.68, 95% CI = 1.18–2.40). However, in studies published in the last decade, survival in the 2 patient groups has been converging, as especially the NF1 group has shown improved prognosis. For our own MPNST patients, NF1 status had no effect on overall or disease-specific survival. The compiled literature from 1963 to the present indicates a significantly worse outcome of MPNST in patients with NF1 syndrome compared with non-NF1 patients. However, survival for the NF1 patients has improved in the last decade, and the survival difference is diminishing. These observations support the hypothesis that MPNSTs arising in NF1 and non-NF1 patients are not different per se. Consequently, we suggest that the choice of treatment for MPNST should be independent of NF1 status.

Connexin43 acts as a colorectal cancer tumor suppressor and predicts disease outcome

This article is the first to show that loss of connexin43 (Cx43) expression in colorectal tumors is correlated with significantly shorter relapse‐free and overall survival. Cx43 was further found to negatively regulate growth of colon cancer cells, in part by enhancing apoptosis. In addition, Cx43 was found to colocalize with β‐catenin and reduce Wnt signaling. The study represents the first evidence that Cx43 acts as a colorectal cancer tumor suppressor and that loss of Cx43 expression during colorectal cancer development is associated with reduced patient survival. The study has important implications for the assessment of Cx43 as a prognostic marker and target in colorectal cancer prevention and therapy. Gap junctions consist of intercellular channels that permit direct transfer of ions and small molecules between adjacent cells. The gap junction channel protein Cx43 plays important roles in cell growth control and differentiation and is frequently dysregulated in human cancers. However, the functional importance and clinical relevance of Cx43 in cancer development has remained elusive. Here, we show that Cx43 is downregulated or aberrantly localized in colon cancer cell lines and colorectal carcinomas, which is associated with loss of gap junction intercellular communication. The in situ protein expression of Cx43 was analyzed in colorectal tumors in a cohort of 674 patients and related to established clinicopathological variables and survival. A subgroup of the patients had weak or no expression of Cx43 in tumors. Loss of Cx43 expression was significantly correlated with shorter relapse‐free and overall survival. Loss of Cx43 further identified a high‐risk subgroup among stage I and stage II patients with reduced relapse‐free and overall survival. Ectopic expression of Cx43 in the colon cancer cell line HT29 was associated with reduced growth in monolayer and soft agar cultures and in tumor xenografts. Cx43 was found to colocalize with β‐catenin and negatively regulate the Wnt signaling pathway, and expression of Cx43 was associated with increased levels of apoptosis. Altogether, these data indicate that Cx43 is a colorectal cancer tumor suppressor protein that predicts clinical outcome.

Hypermethylated MAL gene - a silent marker of early colon tumorigenesis.

BackgroundTumor-derived aberrantly methylated DNA might serve as diagnostic biomarkers for cancer, but so far, few such markers have been identified. The aim of the present study was to investigate the potential of the MAL (T-cell differentiation protein) gene as an early epigenetic diagnostic marker for colorectal tumors.MethodsUsing methylation-specific polymerase chain reaction (MSP) the promoter methylation status of MAL was analyzed in 218 samples, including normal mucosa (n = 44), colorectal adenomas (n = 63), carcinomas (n = 65), and various cancer cell lines (n = 46). Direct bisulphite sequencing was performed to confirm the MSP results. MAL gene expression was investigated with real time quantitative analyses before and after epigenetic drug treatment. Immunohistochemical analysis of MAL was done using normal colon mucosa samples (n = 5) and a tissue microarray with 292 colorectal tumors.ResultsBisulphite sequencing revealed that the methylation was unequally distributed within the MAL promoter and by MSP analysis a region close to the transcription start point was shown to be hypermethylated in the majority of colorectal carcinomas (49/61, 80%) as well as in adenomas (45/63, 71%). In contrast, only a minority of the normal mucosa samples displayed hypermethylation (1/23, 4%). The hypermethylation of MAL was significantly associated with reduced or lost gene expression in in vitro models. Furthermore, removal of the methylation re-induced gene expression in colon cancer cell lines. Finally, MAL protein was expressed in epithelial cells of normal colon mucosa, but not in the malignant cells of the same type.ConclusionPromoter hypermethylation of MAL was present in the vast majority of benign and malignant colorectal tumors, and only rarely in normal mucosa, which makes it suitable as a diagnostic marker for early colorectal tumorigenesis.

Genomic Changes in Chromosomes 10, 16, and X in Malignant Peripheral Nerve Sheath Tumors Identify a High-Risk Patient Group

PURPOSE The purpose of this study was to identify genetic aberrations contributing to clinical aggressiveness of malignant peripheral nerve sheath tumors (MPNSTs). PATIENTS AND METHODS Samples from 48 MPNSTs and 10 neurofibromas were collected from 51 patients with (n = 31) or without (n = 20) neurofibromatosis type 1 (NF1). Genome-wide DNA copy number changes were assessed by chromosomal and array-based comparative genomic hybridization (CGH) and examined for prognostic significance. For a subset of 20 samples, RNA microarray data were integrated with the genome data to identify potential target genes. RESULTS Forty-four (92%) MPNSTs displayed DNA copy number changes (median, 18 changes per tumor; range, 2 to 35 changes). Known frequent chromosomal gains at chromosome arms 8q (69%), 17q (67%), and 7p (52%) and losses from 9p (50%), 11q (48%), and 17p (44%) were confirmed. Additionally, gains at 16p or losses from 10q or Xq identified a high-risk group with only 11% 10-year disease-specific survival (P = .00005). Multivariate analyses including NF1 status, tumor location, size, grade, sex, complete remission, and initial metastatic status showed that the genomic high-risk group was the most significant predictor of poor survival. Several genes whose expression was affected by the DNA copy number aberrations were identified. CONCLUSION The presence of specific genetic aberrations was strongly associated with poor survival independent of known clinical risk factors. Conversely, within the total patient cohort with 34% 10-year disease-specific survival, a low-risk group was identified: without changes at chromosomes 10q, 16p, or Xq in their MPNSTs, the patients had 74% 10-year survival.

Identification of p53 as a strong predictor of survival for patients with malignant peripheral nerve sheath tumors

The purpose of this study was to identify new prognostic biomarkers with clinical impact in malignant peripheral nerve sheath tumor (MPNST), a highly aggressive malignancy for which no consensus therapy exists besides surgery. We have used tissue microarrays (TMAs) to assess in situ expression of 14 cell-cycle-regulating proteins in 64 well-characterized MPNST patients: 36 sporadic and 28 with neurofibromatosis type 1 (NF1). We developed a new software application for evaluation and logistics of the TMA images and performed a literature survey of cell cycle proteins in MPNST. For NF1-associated patients, there was a clear association between nuclear expression of p53 and poor survival (p = 0.004). Among the other proteins analyzed, we also found significant associations between survival and clinical variables, but none were as strong as that for p53. For the total series of MPNSTs, p53 was shown to be an independent predictor of survival, and patients without remission, with tumor size larger than 8 cm, and with positive p53 expression had a 60 times greater risk of dying within the first 5 years compared with the remaining patients (p = 0.000002). This is the most comprehensive study of in situ protein expression in MPNST so far, and expressed p53 was found to be a strong surrogate marker for outcome. Patients in complete remission with a primary p53-positive MPNST diagnosis may be considered in a high-risk subgroup and candidates for adjuvant treatment.

Prognostic Significance of β-Catenin, E-Cadherin, and SOX9 in Colorectal Cancer: Results from a Large Population-Representative Series.

Robust biomarkers that can precisely stratify patients according to treatment needs are in great demand. The literature is inconclusive for most reported prognostic markers for colorectal cancer (CRC). Hence, adequately reported studies in large representative series are necessary to determine their clinical potential. We investigated the prognostic value of three Wnt signaling-associated proteins, β-catenin, E-cadherin, and SOX9, in a population-representative single-hospital series of 1290 Norwegian CRC patients by performing immunohistochemical analyses of each marker using the tissue microarray technology. Loss of membranous or cytosolic β-catenin and loss of cytosolic E-cadherin protein expression were significantly associated with reduced 5-year survival in 903 patients who underwent major resection (722 evaluable tissue cores) independently of standard clinicopathological high-risk parameters. Pre-specified subgroup analyses demonstrated particular effect for stage IV patients for β-catenin membrane staining (P = 0.018; formal interaction test P = 0.025). Among those who underwent complete resection (714 patients, 568 evaluable), 5-year time-to-recurrence analyses were performed, and stage II patients with loss of cytosolic E-cadherin were identified as an independent high-risk subgroup (P = 0.020, formal interaction test was not significant). Nuclear β-catenin and SOX9 protein, regardless of intracellular location, were not associated with prognosis. In conclusion, the protein expression level of membranous or cytosolic β-catenin and E-cadherin predicts CRC patient subgroups with inferior prognosis.

Connexins in colorectal cancer pathogenesis.

The connexins constitute a family of integral membrane proteins that form channels between adjacent cells. These channels are assembled in plasma membrane domains known as gap junctions and enable cells to directly exchange ions and small molecules. Intercellular communication via gap junctions plays important roles in regulating cell growth and differentiation and in maintaining tissue homeostasis. This type of cell communication is often impaired during cancer development, and several members of the connexin protein family have been shown to act as tumor suppressors. Emerging evidence suggests that the connexin protein family has important roles in colorectal cancer development. In the normal colonic epithelial tissue, three connexin isoforms, connexin 26 (Cx26), Cx32 and Cx43, have been shown to be expressed at the protein level. Colorectal cancer development is associated with loss of connexin expression or relocalization of connexins from the plasma membrane to intracellular compartments. Downregulation of connexins in colorectal carcinomas at the transcriptional level involves cancer‐specific promoter hypermethylation. Recent studies suggest that Cx43 may constrain growth of colon cancer cells by interfering with the Wnt/β‐catenin pathway. There is also increasing evidence that the connexins may have potential as prognostic markers in colorectal cancer. This review discusses the role of connexins in colorectal cancer pathogenesis, as well as their potential as prognostic markers and targets in the prevention and treatment of the disease.

Peritoneal carcinomatosis of colon cancer origin: Highest incidence in women and in patients with right-sided tumors

: The aim of the study was to evaluate the incidence of peritoneal carcinomatosis (PC) in a prospectively recorded series of colon cancer patients from a defined cohort and to compare clinicopathological characteristics, survival, and TP53 mutation status in primary tumors from patients with and without PC.

Regulator of Chromosome Condensation 2 Identifies High-Risk Patients within Both Major Phenotypes of Colorectal Cancer

Purpose: Colorectal cancer has high incidence and mortality worldwide. Patients with microsatellite instable (MSI) tumors have significantly better prognosis than patients with microsatellite stable (MSS) tumors. Considerable variation in disease outcome remains a challenge within each subgroup, and our purpose was to identify biomarkers that improve prediction of colorectal cancer prognosis. Experimental Design: Mutation analyses of 42 MSI target genes were performed in two independent MSI tumor series (n = 209). Markers that were significantly associated with prognosis in the test series were assessed in the validation series, followed by functional and genetic explorations. The clinical potential was further investigated by immunohistochemistry in a population-based colorectal cancer series (n = 903). Results: We identified the cell-cycle gene regulator of chromosome condensation 2 (RCC2) as a cancer biomarker. We found a mutation in the 5′ UTR region of RCC2 that in univariate and multivariate analyses was significantly associated with improved outcome in the MSI group. This mutation caused reduction of protein expression in dual luciferase gene reporter assays. siRNA knockdown in MSI colon cancer cells (HCT15) caused reduced cell proliferation, cell-cycle arrest, and increased apoptosis. Massive parallel sequencing revealed few RCC2 mutations in MSS tumors. However, weak RCC2 protein expression was significantly associated with poor prognosis, independent of clinical high-risk parameters, and stratifies clinically important patient subgroups with MSS tumors, including elderly patients (>75 years), stage II patients, and those with rectal cancer. Conclusions: Impaired RCC2 affects functional and clinical endpoints of colorectal cancer. High-risk patients with either MSI or MSS tumors can be identified with cost-effective routine RCC2 assays. Clin Cancer Res; 21(16); 3759–70. ©2015 AACR.

The Use of Multi-frequency EPR Techniques to Identify the Radicals Produced in Irradiated β-Blockers

The identification of radicals trapped in irradiated drugs can be very intricate. A multi-frequency electron paramagnetic resonance (EPR) study is proposed to resolve this problem. The Q-band (ca. 34 GHz) comparison with X-band (ca. 9 GHz) did not show significant differences for the four β-blockers studied (atenolol, esmolol, nadolol and propranolol). The use of a higher frequency (285 GHz) was required. It enabled us to determine the g-tensor values of the radicals present in atenolol and esmolol, respectively, g1=2.0086, g2=2.0059 and g3=2.0021 and g1=2.0066, g2=2.0044 and g3=2.0021. The latter was assigned as a phenoxyl radical, which can not be the case for the former. Therefore, radicals produced in esmolol may result from a more complex mechanism than the abstraction followed by the diffusion of an H atom inside the solid. In addition, two molecules as similar as atenolol and esmolol hydrochloride do not contain the same radicals after irradiation. These two conclusions drawn from the EPR results on β-blockers show clearly the importance of continuing the investigations on radiolytic mechanisms in solid-state drugs.