Sivaramakrishna Rachakonda

TERT promoter mutations: a novel independent prognostic factor in primary glioblastomas.

BACKGROUND Activating somatic mutations in the promoter region of the telomerase reverse transcriptase gene (TERT) have been detected in several cancers. In this study we investigated the TERT promoter mutations and their impact on patient survival in World Health Organization grade IV glioblastoma multiforme (GBM). METHODS The TERT core promoter region containing the previously described mutations and a common functional polymorphism (rs2853669) was sequenced in tumors and blood samples from 192 GBM patients. O(6)-methylguanine-DNA methyltransferase (MGMT) promoter methylation status was assessed by pyrosequencing in 177 (92.2%) cases. Relevant clinical data were obtained from a prospectively maintained electronic database. RESULTS We detected specific (-124 C>T and -146 C>T) TERT promoter mutations in 143/178 (80.3%) primary GBM and 4/14 (28.6%) secondary GBM (P < .001). The presence of TERT mutations was associated with poor overall survival, and the effect was confined to the patients who did not carry the variant G-allele for the rs2853669 polymorphism. An exploratory analysis suggested that TERT mutations might be prognostic only in patients who had incomplete resections and no temozolomide chemotherapy. CONCLUSIONS In this study, specific TERT promoter mutations were markers of primary GBM and predicted patient survival in conjunction with a common functional polymorphism. The prognostic impact of TERT mutations was absent in patients with complete resections and temozolomide chemotherapy. If confirmed in additional studies, these findings may have clinical implications, that is, TERT mutations appear to characterize tumors that require aggressive treatment.

TERT promoter mutations in melanoma survival.

Despite advances in targeted therapies, the treatment of advanced melanoma remains an exercise in disease management, hence a need for biomarkers for identification of at‐risk primary melanoma patients. In this study, we aimed to assess the prognostic value of TERT promoter mutations in primary melanomas. Tumors from 300 patients with stage I/II melanoma were sequenced for TERT promoter and BRAF/NRAS mutations. Cumulative curves were drawn for patients with and without mutations with progression‐free and melanoma‐specific survival as outcomes. Cox proportional hazard regression models were used to determine the effect of the mutations on survivals. Individually, presence of TERT promoter and BRAF/NRAS mutations associated with poor disease‐free and melanoma‐specific survival with modification of the effect by the rs2853669 polymorphism within the TERT promoter. Hazard ratio (HR) for simultaneous occurrence of TERT promoter and BRAF/NRAS mutations for disease‐free survival was 2.3 (95% CI 1.2–4.4) and for melanoma‐specific survival 5.8 (95% CI 1.9–18.3). The effect of the mutations on melanoma‐specific survival in noncarriers of variant allele of the polymorphism was significant (HR 4.5, 95% CI 1.4–15.2) but could not be calculated for the carriers due to low number of events. The variant allele per se showed association with increased survival (HR 0.3, 95% CI 0.1–0.9). The data in this study provide preliminary evidence that TERT promoter mutations in combination with BRAF/NRAS mutations can be used to identify patients at risk of aggressive disease and the possibility of refinement of the classification with inclusion of the rs2853669 polymorphism within TERT promoter.

Single-Nucleotide Polymorphisms Within the Thrombomodulin Gene (THBD) Predict Mortality in Patients With Graft-Versus-Host Disease

PURPOSE Steroid-refractory graft-versus-host disease (GVHD) is a major and often fatal complication after allogeneic stem-cell transplantation (alloSCT). Although the pathophysiology of steroid refractoriness is not fully understood, evidence is accumulating that endothelial cell stress is involved, and endothelial thrombomodulin (THBD) plays a role in this process. Here we assess whether single-nucleotide polymorphisms (SNPs) within the THBD gene predict outcome after alloSCT. PATIENTS AND METHODS Seven SNPs within the THBD gene were studied (rs1962, rs1042579, rs1042580, rs3176123, rs3176124, rs3176126, and rs3176134) in a training cohort of 306 patients. The relevant genotypes were then validated in an independent cohort (n = 321). RESULTS In the training cohort, an increased risk of nonrelapse mortality (NRM) was associated with three of seven SNPs tested: rs1962, rs1042579 (in linkage disequilibrium with rs3176123), and rs1042580. When patients were divided into risk groups (one v no high-risk SNP), a strong correlation with NRM was observed (hazard ratio [HR], 2.31; 95% CI, 1.36 to 3.95; P = .002). More specifically, NRM was predicted by THBD SNPs in patients who later developed GVHD (HR, 3.03; 95% CI, 1.61 to 5.68; P < .001) but not in patients without GVHD. In contrast, THBD SNPs did not predict incidence of acute GVHD. Multivariable analyses adjusting for clinical variables confirmed the independent effect of THBD SNPs on NRM. All findings could be reproduced in the validation cohort. CONCLUSION THBD SNPs predict mortality of manifest GVHD but not the risk of acquiring GVHD, supporting the hypothesis that endothelial vulnerability contributes to GVHD refractoriness.

A retrospective comparative exploratory study on two Methylentetrahydrofolate Reductase (MTHFR) polymorphisms in esophagogastric cancer: the A1298C MTHFR polymorphism is an independent prognostic factor only in neoadjuvantly treated gastric cancer patients

BackgroundMethylentetrahydrofolate reductase (MTHFR) plays a major role in folate metabolism and consequently could be an important factor for the efficacy of a treatment with 5-fluorouracil. Our aim was to evaluate the prognostic and predictive value of two well characterized constitutional MTHFR gene polymorphisms for primarily resected and neoadjuvantly treated esophagogastric adenocarcinomas.Methods569 patients from two centers were analyzed (gastric cancer: 218, carcinoma of the esophagogastric junction (AEG II, III): 208 and esophagus (AEG I): 143). 369 patients received neoadjuvant chemotherapy followed by surgery, 200 patients were resected without preoperative treatment. The MTHFR C677T and A1298C polymorphisms were determined in DNA from peripheral blood lymphozytes. Associations with prognosis, response and clinicopathological factors were analyzed retrospectively within a prospective database (chi-square, log-rank, cox regression).ResultsOnly the MTHFR A1298C polymorphisms had prognostic relevance in neoadjuvantly treated patients but it was not a predictor for response to neoadjuvant chemotherapy. The AC genotype of the MTHFR A1298C polymorphisms was significantly associated with worse outcome (p = 0.02, HR 1.47 (1.06-2.04). If neoadjuvantly treated patients were analyzed based on their tumor localization, the AC genotype of the MTHFR A1298C polymorphisms was a significant negative prognostic factor in patients with gastric cancer according to UICC 6th edition (gastric cancer including AEG type II, III: HR 2.0, 95% CI 1.3-2.0, p = 0.001) and 7th edition (gastric cancer without AEG II, III: HR 2.8, 95% CI 1.5-5.7, p = 0.003), not for AEG I. For both definitions of gastric cancer the AC genotype was confirmed as an independent negative prognostic factor in cox regression analysis. In primarily resected patients neither the MTHFR A1298C nor the MTHFR C677T polymorphisms had prognostic impact.ConclusionsThe MTHFR A1298C polymorphisms was an independent prognostic factor in patients with neoadjuvantly treated gastric adenocarcinomas (according to both UICC 6th or 7th definitions for gastric cancer) but not in AEG I nor in primarily resected patients, which confirms the impact of this enzyme on chemotherapy associated outcome.

Genetic alterations in seborrheic keratoses

Seborrheic keratoses are common benign epidermal lesions that are associated with increased age and sun-exposure. Those lesions despite harboring multiple somatic alterations in contrast to malignant tumors appear to be genetically stable. In order to investigate and characterize the presence of recurrent mutations, we performed exome sequencing on DNA from one seborrheic keratosis lesion and corresponding blood cells from the same patients with follow up investigation of alterations identified by exome sequencing in 24 additional lesions from as many patients. In addition we investigated alterations in all lesions at specific genes loci that included FGFR3, PIK3CA, HRAS, BRAF, CDKN2A and TERT and DHPH3 promoters. The exome sequencing data indicated three mutations per Mb of the targeted sequence. The mutational pattern depicted typical UV signature with majority of alterations being C>T and CC>TT base changes at dipyrimidinic sites. The FGFR3 mutations were the most frequent, detected in 12 of 25 (48%) lesions, followed by the PIK3CA (32%), TERT promoter (24%) and DPH3 promoter mutations (24%). TERT promoter mutations associated with increased age and were present mainly in the lesions excised from head and neck. Three lesions also carried alterations in CDKN2A. FGFR3, TERT and DPH3 expression did not correlate with mutations in the respective genes and promoters; however, increased FGFR3 transcript levels were associated with increased FOXN1 levels, a suggested positive feedback loop that stalls malignant progression. Thus, in this study we report overall mutation rate through exome sequencing and show the most frequent mutations seborrheic keratosis.

TERT promoter mutations

BACKGROUND Activating somatic mutations in the promoter region of the telomerase reverse transcriptase gene (TERT) have been detected in several cancers. In this study we investigated the TERT promoter mutations and their impact on patient survival in World Health Organization grade IV glioblastoma multiforme (GBM). METHODS The TERT core promoter region containing the previously described mutations and a common functional polymorphism (rs2853669) was sequenced in tumors and blood samples from 192 GBM patients. O(6)-methylguanine-DNA methyltransferase (MGMT) promoter methylation status was assessed by pyrosequencing in 177 (92.2%) cases. Relevant clinical data were obtained from a prospectively maintained electronic database. RESULTS We detected specific (-124 C>T and -146 C>T) TERT promoter mutations in 143/178 (80.3%) primary GBM and 4/14 (28.6%) secondary GBM (P < .001). The presence of TERT mutations was associated with poor overall survival, and the effect was confined to the patients who did not carry the variant G-allele for the rs2853669 polymorphism. An exploratory analysis suggested that TERT mutations might be prognostic only in patients who had incomplete resections and no temozolomide chemotherapy. CONCLUSIONS In this study, specific TERT promoter mutations were markers of primary GBM and predicted patient survival in conjunction with a common functional polymorphism. The prognostic impact of TERT mutations was absent in patients with complete resections and temozolomide chemotherapy. If confirmed in additional studies, these findings may have clinical implications, that is, TERT mutations appear to characterize tumors that require aggressive treatment.

Bleomycin-induced chromosomal damage and shortening of telomeres in peripheral blood lymphocytes of incident cancer patients.

Disruption of genomic integrity due to deficient DNA repair capacity and telomere shortening constitute hallmarks of malignant diseases. Incomplete or deficient repair of DNA double‐strand breaks (DSB) is manifested by chromosomal aberrations and their frequency reflects inter‐individual differences of response to exposure to mutagenic compounds. In this study, we investigated chromosomal integrity in peripheral blood lymphocytes (PBL) from newly diagnosed cancer patients, including 47 breast (BC) and 44 colorectal cancer (CRC) patients and 90 matched healthy controls. Mutagen sensitivity was evaluated by measuring chromatid breaks (CTAs) induced by bleomycin and supplemented by the chemiluminescent measurement of γ‐H2AX phosphorylation in 19 cancer patients (11 BC, 8 CRC). Relative telomere length (RTL) was determined in 22 BC, 32 CRC, and 64 controls. We observed statistically significant increased level of CTAs (P = .03) and increased percentage of aberrant cells (ACs) with CTAs (P = .05) in CRC patients compared with controls after bleomycin treatment. No differences were observed between BC cases and corresponding controls. CRC and BC patients with shorter RTL (below median) exhibited significantly higher amount of ACs (P = .02), CTAs (P = .02), and cells with high frequency of CTAs (≥12 CTAs/PBL; P = .03) after bleomycin treatment. No such associations were observed in healthy controls. γ‐H2AX phosphorylation after bleomycin treatment in PBL did not differ between CRC and BC patients. Our results suggest that altered DSB repair measured by sensitivity towards mutagen in PBL occurs particularly in CRC carcinogenesis. Irrespective of cancer type, telomere shortening may be associated with a decreased capacity to repair DSB.

Genome-wide association study identifies susceptibility loci for B-cell childhood acute lymphoblastic leukemia.

Genome-wide association studies (GWAS) have advanced our understanding of susceptibility to B-cell precursor acute lymphoblastic leukemia (BCP-ALL); however, much of the heritable risk remains unidentified. Here, we perform a GWAS and conduct a meta-analysis with two existing GWAS, totaling 2442 cases and 14,609 controls. We identify risk loci for BCP-ALL at 8q24.21 (rs28665337, P = 3.86 × 10−9, odds ratio (OR) = 1.34) and for ETV6-RUNX1 fusion-positive BCP-ALL at 2q22.3 (rs17481869, P = 3.20 × 10−8, OR = 2.14). Our findings provide further insights into genetic susceptibility to ALL and its biology.While GWAS have uncovered susceptibility loci for B-cell precursor acute lymphoblastic leukemia (BCP-ALL), much of the heritable risk remains undiscovered. Here, the authors perform a meta-analysis of two existing BCP-ALL GWAS together with an unpublished GWAS to identify risk loci at 8q24.21 and 2q22.3.

Single Nucleotide Polymorphisms in CD40L Predict Endothelial Complications and Mortality After Allogeneic Stem-Cell Transplantation

Purpose Endothelial vulnerability is a potential risk factor for complications after allogeneic stem-cell transplantation (alloSCT). The CD40/CD40 ligand (CD40L) axis contributes to inflammatory diseases and is upregulated in endothelial cells upon activation, suggesting a role in alloSCT biology. Here, we studied single nucleotide polymorphisms (SNPs) in the CD40L gene in recipients of alloSCT. Patients and Methods Three CD40L SNPs (rs3092920, rs3092952, rs3092936) were analyzed for association with transplant-associated thrombotic microangiopathy, overall nonrelapse mortality (NRM), and NRM after acute graft-versus-host disease in 294 recipients of alloSCT without statin-based endothelial prophylaxis (SEP). The significant genotype was then put into perspective with established thrombomodulin ( THBD) gene polymorphisms. Findings were validated in an independent cohort without SEP and in an additional 344 patients who received SEP. Results The rs3092936 CC/CT genotype was associated with an increased risk of transplant-associated thrombotic microangiopathy ( P = .001), overall NRM ( P = .03), and NRM after acute graft-versus-host disease ( P = .01). The rs3092936 CC/CT genotype was largely mutually exclusive of high-risk THBD SNPs. Both CD40L and THBD SNPs predicted adverse overall survival (OS) and overall NRM to a similar extent in training cohort (OS, P = .04; NRM, P < .001) and validation cohort (OS, P = .01; NRM, P = .001) without SEP. In contrast, SEP completely abolished the influence of the high-risk CD40L and THBD SNPs ( P = .40). Conclusion An increased risk of endothelial complications can be predicted before alloSCT by genetic markers in the recipients genome. The normalization of mortality risks in patients treated with SEP suggests a way of overcoming the negative effect of high-risk genotypes and warrants further clinical validation.

Leukocyte telomere length throughout the continuum of colorectal carcinogenesis

Considering the high prevalence of colorectal cancer (CRC) and relatively high mortality there is strong interest in identification of clinically relevant biomarkers. Telomere shortening is supposed to contribute to genomic instability and crucially involved in process of carcinogenesis. Peripheral blood leukocyte (PBL) telomere length was previously investigated in several studies as potential biomarker for CRC but with controversial results. This prompted us to investigate relative PBL telomere length in association with different histological findings throughout the continuum of colorectal carcinogenesis in order to reflect the whole spectrum of putative CRC development in a large study involving 2011 individuals. The study based on the Colorectal Cancer Study of Austria (CORSA), including 384 CRC cases as well as age- and gender-matched 544 high-risk adenomas, 537 low-risk adenoma patients and 546 colonoscopy-negative controls. Relative expression of telomeric repeats and the single copy reference gene, albumin (T/S ratio) was determined using monochrome multiplex quantitative PCR (MMQPCR). Telomeres were found to be significantly longer in CRC patients compared to control subjects (P = 3.61x10-6). Yet, no significant differences in telomere length could be detected for high-risk (P = 0.05956) and low-risk colorectal adenoma patients (P = 0.05224). In addition, results presented in this manuscript highlight the impact of various epidemiological factors on PBL telomere length and its involvement in CRC. However, further large studies also including colorectal adenomas are necessary to confirm these results.