BACKGROUND:Neoadjuvant chemotherapy (NAC) has been controversial for patients with colorecal cancer liver metastasis (CRLM) with resectable disease. Chemotherapy associated liver injury (CALI) may explain the lack of overall survival (OS) benefit in some studies. It remains unclear why CALI severity varies between patients despite receiving the same duration and type of NAC. Single nucleotide polymorphisms (SNPs) have been associated with liver disease and could account for these interindividual differences. Within this study we used the APRI + ALBI score, a non-invasive liver function marker that increases in response to CALI development during NAC, to assess whether preoperative liver function affects OS in CRLM patients undergoing hepatectomy and explore the impact of SNPs on CALI development. METHODS:551 patients with CRLM undergoing liver surgery after NAC were included. In 149 patients, DNA from histological specimens was genotyped and the presence of SNPs associated with liver disease was assessed. FINDINGS:Patients with APRI + ALBI scores (≥-2.46), associated with the development of CALI, showed decreased OS after hepatectomy (median OS APRI + ALBI < -2.46 = 46.1 months; median OS APRI + ALBI ≥ -2.46 = 34.3, p = 0.027). The mutated rs738409 variant on the patatin-like phospholipase domain-containing protein 3 (PNPLA3) displayed a close association with chemotherapy-associated steatohepatitis (p = 0.007) as well as intrahepatic fat (p = 0.004). Additionally, all PNPLA3 homozygotes shifted into the high-risk APRI + ALBI group during NAC. INTERPRETATION:CALI and its effects on liver function not only impact immediate postoperative outcomes but also significantly affect OS in CRLM patients undergoing liver resection. PNPLA3 polymorphism was associated with CALI development. Considering that PNPLA3 polymorphisms are significantly higher in Asian populations, these results could partly explain the heterogeneity in reported effects of NAC in CRLM patients and might improve patient selection. FUNDING:None of the authors received funding related to the writing of this manuscript.

Thoracic aortic aneurysm (TAA) is an age-related and life-threatening vascular disease. Telomere shortening is a predictor of age-related diseases, and its progression is associated with premature vascular disease. The aim of the present work was to investigate the impacts of chronic hypoxia and telomeric DNA damage on cellular homeostasis and vascular degeneration of TAA. We analyzed healthy and aortic aneurysm specimens (215 samples) for telomere length (TL), chronic DNA damage, and resulting changes in cellular homeostasis, focusing on senescence and apoptosis. Compared with healthy thoracic aorta (HTA), patients with tricuspid aortic valve (TAV) showed telomere shortening with increasing TAA size, in contrast to genetically predisposed bicuspid aortic valve (BAV). In addition, TL was associated with chronic hypoxia and telomeric DNA damage and with the induction of senescence-associated secretory phenotype (SASP). TAA-TAV specimens showed a significant difference in SASP-marker expression of IL-6, NF-κB, mTOR, and cell-cycle regulators (γH2AX, Rb, p53, p21), compared to HTA and TAA-BAV. Furthermore, we observed an increase in CD163+ macrophages and a correlation between hypoxic DNA damage and the number of aortic telocytes. We conclude that chronic hypoxia is associated with telomeric DNA damage and the induction of SASP in a diseased aortic wall, promising a new therapeutic target.

liver metastasis patients, YB-1 protein expression was a negative predictor for overall survival. Oxaliplatin-based chemotherapy induced SG formation in CRC cell-lines and primary tumour tissue culture. Pre-treat-ment with the HDACi MS-275 prevented stress-granule aggre-gation and increased the sensitivity of CRC cell lines to oxaliplatin.

BackgroundTumor-associated macrophages (TAM) constitute the most abundant immune cells in the tumor stroma initiating pro-inflammatory (M1) or immunosuppressive (M2) responses depending on their polarization status. Advances in tumor immunotherapy call for a detailed understanding of potential immunogenic mechanisms of irradiation routinely applied in rectal cancer patients.MethodsTo test the effects of radiotherapy on TAM, we ex vivo irradiated tissue samples of human rectal cancer and assessed the phenotype by flow cytometry. We furthermore evaluated the distribution of leucocyte subsets in tissue sections of patients after short-course radiotherapy and compared findings to non-pretreated rectal cancer using an immunostaining approach. Organotypic assays (OTA) consisting of macrophages, cancer-associated fibroblast and cancer cell lines were used to dissect the immunological consequences of irradiation in macrophages.ResultsWe demonstrate that short-course neoadjuvant radiotherapy in rectal cancer patients is associated with a shift in the polarization of TAM towards an M1-like pro-inflammatory phenotype. In addition, ex vivo irradiation caused an increase in the phagocytic activity and enhanced expression of markers associated with stimulatory signals necessary for T-cell activation. In OTA we observed that this alteration in macrophage polarization could be mediated by extracellular vesicles (EV) derived from irradiated tumor cells. We identified high mobility group box 1 in EV from irradiated tumor cells as a potential effector signal in that crosstalk.ConclusionsOur findings highlight macrophages as potential effector cells upon irradiation in rectal cancer by diminishing their immunosuppressive phenotype and activate pro-inflammation. Our data indicate that clinically applied short-term radiotherapy for rectal cancer may be exploited to stimulate immunogenic macrophages and suggest to target the polarization status of macrophages to enhance future immunotherapeutic strategies.

Background: Tumor-associated macrophages initiate anti-tumoral (M1) or immunosuppressive (M2) responses depending on their polarization status.

Malignant cells ensure telomere maintenance by the alternative lengthening of telomeres (ALT) in the absence of telomerase activity (TA). The retrotransposons "long interspersed nuclear element-1" (LINE-1, L1) are expressed in malignant cells and are primarily known to contribute to complex karyotypes. Here we demonstrate that LINE-1 ribonucleoprotein particles (L1-RNPs) expression is significantly higher in ALT+- versus in TA+-human glioma. Analyzing a role of L1-RNP in ALT, we show that L1-RNPs bind to telomeric repeat containing RNA (TERRA), which is critical for telomere stabilization and which is overexpressed in ALT+ cells. In turn, L1-RNP knockdown (KD) abrogated the nuclear retention of TERRA, resulted in increased telomeric DNA damage, decreased cell growth and reduced expression of ALT characteristics such as c-circles and PML-bodies. L1-RNP KD also decreased the expression of Shelterin- and the ALT-regulating protein Topoisomerase IIIα (TopoIIIα) indicating a more general role of L1-RNPs in supporting telomeric integrity in ALT. Our findings suggest an impact of L1-RNP on telomere stability in ALT+ dependent tumor cells. As L1-RNPs are rarely expressed in normal adult human tissue those elements might serve as a novel target for tumor ablative therapy.

Long interspersed nuclear elements (LINE-1) are well known as retrotransposons. A number of reports indicate that down-regulation of LINE-1 substantially affects growth of malignant cells and epithelial mesenchymal transition, which is difficult to be explained by its function as retrotransposon. More recent data indicate that LINE-1 is broadly involved in the regulation of telomere maintenance. This explains the essential role of LINE-1 for survival of malignant cells and further supports a global function of active LINE-1 elements in cell proliferation. We further discuss the implications of LINE-1-associated telomere regulation on evolution of telomeric structures, on embryogenesis and on therapy of malignancies.

In operable esophageal cancer patients, neoadjuvant therapy benefits only those who respond to the treatment. The • Pancho trial represents the first prospective randomized trial evaluating the relevance of the mark53 status for predicting the effect of two different neoadjuvant chemotherapies.

Preclinical models indicate that DNA damage induces type I interferon (IFN), which is crucial for the induction of an anti-tumor immune response.In human cancers, however, the association between DNA damage and an immunogenic cell death (ICD), including the release and sensing of danger signals, the subsequent ER stress response and a functional IFN system, is less clear.Methods: Neoadjuvant-treated colorectal liver metastases (CLM) patients, undergoing liver resection in with a curative intent, were retrospectively enrolled in this study (n=33).DNA damage (γH2AX), RNA and DNA sensors (RIG-I, DDX41, cGAS, STING), ER stress response (p-PKR, p-eIF2α, CALR), type I and type II IFN-induced proteins (MxA, GBP1), mature dendritic cells (CD208), and cytotoxic and memory T cells (CD3, CD8, CD45RO) were investigated by an immunohistochemistry whole-slide tissue scanning approach and further correlated with recurrence-free survival (RFS), overall survival (OS), radiographic and pathologic therapy response.Results: γH2AX is a negative prognostic marker for RFS (HR 1.32, 95% CI 1.04-1.69,p=0.023) and OS (HR 1.61, 95% CI 1.23-2.11,p<0.001).A model comprising of DDX41, STING and p-PKR predicts radiographic therapy response (AUC=0.785,p=0.002).γH2AX predicts prognosis superior to the prognostic value of CD8.CALR positively correlates with GBP1, CD8 and cGAS.A model consisting of γH2AX, p-eIF2α, DDX41, cGAS, CD208 and CD45RO predicts pathological therapy response (AUC=0.944,p<0.001). Conclusion:In contrast to preclinical models, DNA damage inversely correlated with ICD and its associated T cell infiltrate and potentially serves as a therapeutic target in CLM.

Approximately 30 million people currently suffer from late-onset Alzheimer's disease (LOAD) worldwide. Twin studies demonstrated that 60 to 80% of LOAD is genetically determined, 20% of which remaining unassigned. This case-control study included 118 cognitively healthy controls, 52 patients with mild cognitive impairment (MCI; the pre-stage of LOAD) and 71 LOAD patients. The participants were genotyped for the genetic LOAD marker apolipoprotein E4 (APOE4) and the single-nucleotide polymorphism rs4925 in glutathione S-transferase omega-1 (GSTO1). Additive logistic regression showed a novel, statistically significant association of the major allele GSTO1*C with MCI (OR1.9; p = 0.032). However, identification of significant SNP-disease relations required well-defined study groups. When classifying participants solely by the short Mini Mental State examination (MMSE), the associations of GSTO1*C and the reference marker APOE4 with MCI were cancelled. Moreover, even identifying only the control group by MMSE nullified a statistically significant association (OR1.8; p = 0.045) between GSTO1*C and LOAD. In contrast, these statistical relations were retained when the detailed Consortium to Establish a Registry for Alzheimer's Disease (CERAD-Plus) test battery was used. Hence, besides proposing rs4925 as a genetic marker for cognitive impairment, this work also emphasized the importance of carefully characterized controls in addition to well-diagnosed patients in case-control studies.

We investigated the hypothesis that the varying treatment efficacy of adjuvant 5-fluorouracil (5FU) in stage III colon cancer is linked to the TP53 mutational status.ABCSG-90 was a prospective randomized trial in which effect of adjuvant 5FU was studied in stage III colon cancer patients. Tumor material of 70% of these patients (389/572) was available for analysis of the biomarker TP53 using a TP53-gene-specific Sanger sequencing protocol.Median follow-up was 88 months. TP53 mutation frequency was 33%. A significant interaction between TP53 status, outcomes and nodal category was found (P = 0.0095). In the N1 category, TP53 wildtype patients had significantly better overall survival than TP53 mutated (81.0% vs. 62.0% overall survival at 5 years; HR = 2.131; 95% CI: 1.344-3.378; P = 0.0010). In the N2 category, the TP53 status did not affect survival (P = 0.4992). In TP53 wildtype patients, the prognostic significance of N category was significantly enhanced (P = 0.0002). In TP53 mutated patients, survival curves of N1 and N2 patients overlapped and nodal category was no longer prognostic. The biomarker TP53 independently predicted effect of adjuvant 5FU in N1 colon cancer patients. TP53 was not predictive in N2 patients, in whom 5FU is known to have no effect. (C) 2015 The Authors. Published by Elsevier B.V.

The type of a biomarker whether it is prognostic or predictive is frequently not known, although such information is crucial for assessing the clinical value of a marker.In order to evaluate the type of marker TP53 is, we identified a cohort of 76 patients with colorectal liver metastases (CLM), homogeneously staged as resectable, who had been treated either with or without fluorouracil-based neoadjuvant chemotherapy. The TP53 genotype was assessed retrospectively from paraffin-embedded, diagnostic tumour biopsies using a standardised, p53 gene-specific sequencing protocol (mark53 (R) kit).The overall median survival was 44.2 months, and the overall TP53 mutation frequency was 55%. A significant interaction was observed between chemotherapy and TP53 status (P = 0.045). To illustrate this effect, the 51 patients with and the 25 patients without neoadjuvant chemotherapy were described separately. In patients with neoadjuvant chemotherapy, mutated TP53 was significantly associated with poor survival (P = 0.0025), resulting in five-year survival rates of 22%, compared to 60% in patients with normal TP53. The hazard ratio was 3.12 (95% confidence intervals (CI): 1.46-6.95) to the disadvantage of TP53-mutated patients and 5.49 (P = 0.0001; 95% CI: 2.28-13.24) after adjustment for known prognostic factors. hi patients treated with surgery alone, a mutated TP53 did not have a negative effect on survival (P = 0.54).A mutated TP53 status independently predicted survival disadvantage in CLM patients in the presence, but not in the absence, of neoadjuvant chemotherapy. Our data suggest that TP53 might be a pure predictive marker. (C) 2015 Elsevier Ltd. All rights reserved.

BACKGROUND:Aberrant DNA methylation is more prominent in proximal compared with distal colorectal cancers. Although a number of methylation markers were identified for colon cancer, yet few are available for rectal cancer. METHODS:DNA methylation differences were assessed by a targeted DNA microarray for 360 marker candidates between 22 fresh frozen rectal tumour samples and 8 controls and validated by microfluidic high-throughput and methylation-sensitive qPCR in fresh frozen and formalin-fixed paraffin-embedded (FFPE) samples, respectively. The CpG island methylator phenotype (CIMP) was assessed by MethyLight in FFPE material from 78 patients with pT2 and pT3 rectal adenocarcinoma. RESULTS:We identified and confirmed two novel three-gene signatures in fresh frozen samples that can distinguish tumours from adjacent tissue as well as from blood with a high sensitivity and specificity of up to 1 and an AUC of 1. In addition, methylation of individual CIMP markers was associated with specific clinical parameters such as tumour stage, therapy or patients' age. Methylation of CDKN2A was a negative prognostic factor for overall survival of patients. CONCLUSIONS:The newly defined methylation markers will be suitable for early disease detection and monitoring of rectal cancer.

LINE-1 elements (L1s, long interspersed nuclear elements-1) are abundant retrotransposons in the eukaryotic genome that are primarily known for facilitating aberrant recombination. Here we show the involvement of L1 in telomere maintenance in cells using the less characterized alternative lengthening of telomeres (ALT) mechanism. Interestingly, ALT cells show significantly higher expression levels of L1 when compared to TA cells. Knock-down (KD) of L1 in ALT cell lines was associated with reduced length of telomeres, an increase in telomere dysfunction foci, a reduced expression of ALT characteristics such as c-circles and ALT associated PML bodies, and a decreased growth. On the other hand, overexpression of L1 in ALT cell lines lead to a higher rate of c-circles and increased length of telomeres. LINE-1 not only bound to the C-strand of telomeric DNA but also to telomere-repeat-containing RNA (TERRA), a multifunctional component of human telomeres that is highly expressed in ALT cells and has been described to be involved in telomere stabilization. Whereas L1-KD decreased overall TERRA, L1 overexpression increased this RNA. Moreover, L1 KD abrogated the nuclear retention of TERRA. Moreover, the L1-ribonucleoprotein can use the polyadenylated form of TERRA as a template to generate telomere-specific DNA. Thus, L1 appears to contribute to telomere maintenance by a mechanism involving TERRA, either by ensuring its nuclear localization or by using this RNA as a template for the generation of telomere-specific DNA. Our findings now render L1 proteins a promising target in cancer therapy by interfering with telomere lengthening in ALT cells. Citation Format: Thomas Aschacher, Brigitte Wolf, Philip Kienzl, Florian Enzmann, Barbara Messner, Klaus Holzmann, Michael M. Bergmann. A role of long interspersed nuclear element-1 (LINE-1) for telomere maintenance in cells with alternative lengthening of telomeres. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-084. doi:10.1158/1538-7445.AM2015-LB-084

Li–Fraumeni syndrome (LFS) is a rare autosomal dominant inherited disorder associated with the occurrence of a wide spectrum of early-onset malignancies, the most prevalent being breast cancer and sarcoma. The presence of TP53 germline mutations in the majority of LFS patients suggests a genetic basis for the cancer predisposition. No special recommendations for the treatment of LFS patients have been made to date, except that of minimizing radiation. We hypothesized that TP53 germline mutations may be associated not only with cancer predisposition, but also with lack of response to chemo- and radiotherapy. Here, we present an Austrian LFS family whose members were intensively treated with chemo- and radiotherapy due to cancers that occurred at a predominantly young age, including eight breast cancers in six patients. Material from seven family members was screened for p53 mutation by Sanger sequencing and immunohistochemistry. A rare missense mutation in the tetramerization domain of exon 10 of the TP53 gene was found to segregate with malignant disease in this family. Lack of response to various chemotherapies and radiotherapy could be ascertained by histopathology of surgical specimens after neoadjuvant treatment, by cancer relapse occurring while receiving adjuvant systemic treatment and by the occurrence of second primaries in areas of adjuvant radiation. Our observations suggest that current standards of cancer treatment may not be valid for patients with LFS. In patients with TP53 germline mutation, cytotoxic treatment may bear not only the risk of tumor induction but also the risk of treatment failure.

A hallmark of cancer cells is an activated telomere maintenance mechanism, which allows prolonged survival of the malignant cells. In more than 80% of tumours, telomeres are elongated by the enzyme telomerase, which adds de novo telomere repeats to the ends of chromosomes. Cancer cells are also characterized by expression of active LINE-1 elements (L1s, long interspersed nuclear elements-1). L1 elements are abundant retrotransposons in the eukaryotic genome that are primarily known for facilitating aberrant recombination. Using L1-knockdown (KD), we show for the first time that L1 is critical for telomere maintenance in telomerase-positive tumour cells. The reduced length of telomeres in the L1-KD-treated cells correlated with an increased rate of telomere dysfunction foci, a reduced expression of shelterin proteins and an increased rate of anaphase bridges. The decreased telomere length was associated with a decreased telomerase activity and decreased telomerase mRNA level; the latter was increased upon L1 overexpression. L1-KD also led to a decrease in mRNA and protein expression of cMyc and KLF-4, two main transcription factors of telomerase and altered mRNA levels of other stem-cell-associated proteins such as CD44 and hMyb, as well as a corresponding reduced growth of spheroids. The KD of KLF-4 or cMyc decreased the level of L1-ORF1 mRNA, suggesting a specific reciprocal regulation with L1. Thus, our findings contribute to the understanding of L1 as a pathogenicity factor in cancer cells. As L1 is only expressed in pathophysiological conditions, L1 now appears to be target in the rational treatment of telomerase-positive cancer.

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PALINE-1 elements (L1s, long interspersed nuclear elements-1) are abundant retrotransposons in the eukaryotic genome that are primarily known for facilitating aberrant recombination. Here we show the involvement of L1 in telomere maintenance in cells using the less characterized alternative lengthening of telomeres (ALT) mechanism. Interestingly, ALT cells show significantly higher expression levels of L1 when compared to TA cells. Knock-down (KD) of L1 in ALT cell lines was associated with reduced length of telomeres, an increase in telomere dysfunction foci, a reduced expression of ALT characteristics such as c-circles and ALT associated PML bodies, and a decreased growth. On the other hand, overexpression of L1 in ALT cell lines lead to a higher rate of c-circles and increased length of telomeres. LINE-1 not only bound to the C-strand of telomeric DNA but also to telomere-repeat-containing RNA (TERRA), a multifunctional component of human telomeres that is highly expressed in ALT cells and has been described to be involved in telomere stabilization. Whereas L1-KD decreased overall TERRA, L1 overexpression increased this RNA. Moreover, L1 KD abrogated the nuclear retention of TERRA. Moreover, the L1-ribonucleoprotein can use the polyadenylated form of TERRA as a template to generate telomere-specific DNA. Thus, L1 appears to contribute to telomere maintenance by a mechanism involving TERRA, either by ensuring its nuclear localization or by using this RNA as a template for the generation of telomere-specific DNA. Our findings now render L1 proteins a promising target in cancer therapy by interfering with telomere lengthening in ALT cells.Citation Format: Thomas Aschacher, Brigitte Wolf, Philip Kienzl, Florian Enzmann, Barbara Messner, Klaus Holzmann, Michael M. Bergmann. A role of long interspersed nuclear element-1 (LINE-1) for telomere maintenance in cells with alternative lengthening of telomeres. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-084. doi:10.1158/1538-7445.AM2015-LB-084

BACKGROUND:Fluorouracil and cisplatin have been used most frequently as neoadjuvant therapy for esophageal cancer. Both drugs are believed to act via a p53-dependent apoptosis pathway. The TP53 gene is frequently mutated in esophageal cancer.OBJECTIVE:To test the value of TP53 as a biomarker prognosing outcome in patients with neoadjuvantly treated esophageal cancer.PATIENTS AND METHODS:The investigation included 36 patients with primary operable esophageal cancer who were treated neoadjuvantly with cisplatin and fluorouracil. The TP53 genotype was assessed from paraffin-embedded diagnostic tumor biopsies using a standardized gene-specific TP53 sequencing protocol (mark53 kit; mark53 Ltd, Vienna, Austria).RESULTS:Mutations in the TP53 gene were present in 50% of tumors. Two-year overall survival rates were 55.6% in patients with a normal TP53 marker status, compared with 16.7% in those with a mutant TP53 gene. In patients with normal TP53, neoadjuvant treatment resulted in significant advantages in terms of tumor-associated survival (P=.0049) and overall survival (P=.0304) compared with those with mutant TP53. The median tumor-associated survival was 34.2 months for patients with normal TP53, compared with 8.9 months for those with mutant TP53. The latter had a 3-fold higher risk of dying (hazard ratio, 3.01; 95% confidence interval, 1.359-6.86).CONCLUSIONS:The biomarker TP53 divides esophageal cancer patients into 2 categories with markedly different outcomes: patients with a normal TP53 marker status may experience notable benefits from neoadjuvant chemotherapy with cisplatin/fluorouracil, whereas those with a mutant TP53 marker status appear to be at risk for lack of response.

Background: The presented study aims to determine whether the biomarker TP53 is prognostic or predictive.

The following, from the 12th OESO World Conference: Cancers of the Esophagus, includes commentaries on the animal reflux-inflammation models for Barrett's esophagus and esophageal adenocarcinoma; genomic/epigenomic analyses; eflornithine-based combinations; the molecular derangements that promote neoplastic transformation; the role of COX-2 inhibitors, proton pump inhibitors, and phase II trials in Barrett's adenocarcinoma; statins in chemoprevention and treatment of esophageal cancer; and biomarkers as potential targets in Barrett's adenocarcinoma.