Eduard Schulz

Therapy-related myeloid neoplasms: pathobiology and clinical characteristics

Therapy‐related myeloid neoplasms (t‐MNs) are serious long‐term consequences of cytotoxic treatments for an antecedent disorder. t‐MNs are observed after ionizing radiation as well as conventional chemotherapy including alkylating agents, topoisomerase‐II‐inhibitors and antimetabolites. In addition, adjuvant use of recombinant human granulocyte‐colony stimulating factor may also increase the risk of t‐MNs. There is clinical and biological overlap between t‐MNs and high‐risk de novo myelodysplastic syndromes and acute myeloid leukaemia suggesting similar mechanisms of leukaemogenesis. Human studies and animal models point to a prominent role of genetic susceptibilty in the pathogenesis of t‐MNs. Common genetic variants have been identified that modulate t‐MN risk, and t‐MNs have been observed in some cancer predisposition syndromes. In either case, establishing a leukaemic phenotype requires acquisition of somatic mutations – most likely induced by the cytotoxic treatment. Knowledge of the specific nature of the initiating exposure has allowed the identification of crucial pathogenetic mechanisms and for these to be modelled in vitro and in vivo. Prognosis of patients with t‐MNs is dismal and at present, the only curative approach for the majority of these individuals is haematopoietic stem cell transplantation, which is characterized by high transplant‐related mortality rates. Novel transplantation strategies using reduced intensity conditioning regimens as well as novel drugs – demethylating agents and targeted therapies – await clinical testing and may improve outcome. Ultimately, individual assessment of genetic risk factors may translate into tailored therapies and establish a strategy for reducing t‐MN incidences without jeopardizing therapeutic success rates for the primary disorders.

Germline mutations in the DNA damage response genes BRCA1, BRCA2, BARD1 and TP53 in patients with therapy related myeloid neoplasms

Background Therapy related myeloid neoplasms (t-MNs) are complex diseases originating from an interplay between exogenous toxicities and a susceptible organism. It has been hypothesised that in a subset of cases t-MNs develop in the context of hereditary cancer predisposition syndromes. Methods The study systematically evaluated pedigrees of patients with t-MNs for cancer incidences and the possibility of a hereditary cancer predisposition syndrome. In addition, mutational analyses were performed using constitutional DNA from index patients, and deleterious heterozygous germline mutations were assessed for loss of heterozygosity in sorted leukaemic cells by single nucleotide polymorphism array. Results A nuclear pedigree was obtained in 51/53 patients with t-MNs resulting in a total of 828 individuals analysed. With a standardised incidence ratio of 1.03 (95% CI 0.74 to 1.39), the tumour incidence of first- degree relatives was not increased. However, six pedigrees were suggestive for a hereditary breast and ovarian cancer syndrome, three of a Li-Fraumeni like syndrome, and three index patients showed multiple primary neoplasms. Mutational analysis revealed two BRCA1 (c.3112G→T, c.5251C→T), one BRCA2 (c.4027A→G), two BARD1 (C557S) and four TP53 germline mutations (g.18508_18761delinsGCC, c.847C→T, c.845_848dupGGCG, c.1146delA) in nine of 53 (17%) index patients with t-MNs. Loss of heterozygosity in leukaemic cells was demonstrated for the BRCA1c.3112G→T and TP53c.845_848dupGGCG mutations, respectively. Conclusion It is concluded that a proportion of patients with t-MNs carry cancer susceptibility mutations which are likely to contribute to therapy related leukaemogenesis.

Germline variants in the SEMA4A gene predispose to familial colorectal cancer type X

Familial colorectal cancer type X (FCCTX) is characterized by clinical features of hereditary non-polyposis colorectal cancer with a yet undefined genetic background. Here we identify the SEMA4A p.Val78Met germline mutation in an Austrian kindred with FCCTX, using an integrative genomics strategy. Compared with wild-type protein, SEMA4AV78M demonstrates significantly increased MAPK/Erk and PI3K/Akt signalling as well as cell cycle progression of SEMA4A-deficient HCT-116 colorectal cancer cells. In a cohort of 53 patients with FCCTX, we depict two further SEMA4A mutations, p.Gly484Ala and p.Ser326Phe and the single-nucleotide polymorphism (SNP) p.Pro682Ser. This SNP is highly associated with the FCCTX phenotype exhibiting increased risk for colorectal cancer (OR 6.79, 95% CI 2.63 to 17.52). Our study shows previously unidentified germline variants in SEMA4A predisposing to FCCTX, which has implications for surveillance strategies of patients and their families.

Repeated fecal microbiota transplantations attenuate diarrhea and lead to sustained changes in the fecal microbiota in acute, refractory gastrointestinal graft-versus-host-disease

Acute graft- versus -host disease (aGvHD) is a serious complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT).[1][1] Although aGvHD of any target organ represents morbidity, lower gastrointestinal (GI) tract involvement is complicated by high mortality.[2][2] Here,

D2HGDH regulates alpha-ketoglutarate levels and dioxygenase function by modulating IDH2

Isocitrate dehydrogenases (IDH) convert isocitrate to alpha-ketoglutarate (α-KG). In cancer, mutant IDH1/2 reduces α-KG to D2-hydroxyglutarate (D2-HG) disrupting α-KG-dependent dioxygenases. However, the physiological relevance of controlling the interconversion of D2-HG into α-KG, mediated by D2-hydroxyglutarate dehydrogenase (D2HGDH), remains obscure. Here we show that wild-type D2HGDH elevates α-KG levels, influencing histone and DNA methylation, and HIF1α hydroxylation. Conversely, the D2HGDH mutants that we find in diffuse large B-cell lymphoma are enzymatically inert. D2-HG is a low-abundance metabolite, but we show that it can meaningfully elevate α-KG levels by positively modulating mitochondrial IDH activity and inducing IDH2 expression. Accordingly, genetic depletion of IDH2 abrogates D2HGDH effects, whereas ectopic IDH2 rescues D2HGDH-deficient cells. Our data link D2HGDH to cancer and describe an additional role for the enzyme: the regulation of IDH2 activity and α-KG-mediated epigenetic remodelling. These data further expose the intricacies of mitochondrial metabolism and inform on the pathogenesis of D2HGDH-deficient diseases.

Identification of a novel variant of epsilon‐gamma‐delta‐beta thalassemia highlights limitations of next generation sequencing

LENKA KUBICZKOVA BESSE, LENKA SEDLARIKOVA, FEDOR KRYUKOV, JANA NEKVINDOVA LENKA RADOVA, MARTINA ALMASI, JANA PELCOVA, JIRI MINARIK, TOMAS PIKA ZUZANA PIKALOVA, VLASTIMIL SCUDLA, MARTA KREJCI, ZDENEK ADAM, LUDEK POUR, ROMAN HAJEK AND SABINA SEVCIKOVA* 1 Babak Myeloma Group, Department of Pathological Physiology, Faculty of Medicine, Masaryk University, Brno, Czech Republic; Department of Clinical Hematology, University Hospital Brno, Brno, Czech Republic; Department of Hematooncology, University Hospital Ostrava and Faculty of Medicine, University of Ostrava, Ostrava, Czech Republic; Institute for Clinical Biochemistry and Diagnostics, University Hospital Hradec Kralove, Czech Republic; Central European Institute of Technology, Masaryk University, Brno, Czech Republic; Department of Internal Medicine Hematooncology, University Hospital Brno, Brno, Czech Republic; Department of Hematooncology, University Hospital Olomouc, Olomouc, Czech Republic Conflict of interest: Nothing to report. Contract grant sponsor: The Ministry of Health (IGA Grants); Contract grant numbers: NT12130, NT13190, NT14575; Contract grant sponsor: The Ministry of Health, Czech Republic Conceptual Development of Research Organization; Contract grant number: FNBr, 65269705 *Correspondence to: Sabina Sevcikova; Babak Myeloma Group, Department of Pathological Physiology, Faculty of Medicine, Masaryk University, Kamenice 5, Brno 625 00, Czech Republic. E-mail: sevcik@med.muni.cz Additional Supporting Information may be found in the online version of this article. Received for publication: 20 November 2014; Accepted: 24 November 2014 Published online: 26 November 2014 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/ajh.23910

The TP53 Pro72Arg SNP in de novo acute myeloid leukaemia – results of two cohort studies involving 215 patients and 3759 controls

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The TP53 Pro72Arg SNP in de novo acute myeloid leukemia

In a recent issue of Haematologica, Lucena-Araujo and co-workers report that the germline variation rs1042522 in TP53, which encodes either proline (72Pro) or arginine (72Arg), influences the risk of de novo acute myeloid leukemia (AML) development and overall survival (OS). This stems from a case-control study of patients and healthy volunteers from Brazil. In comparing the frequency of the rs1042522 genotype in 198 AML cases and 224 ageand sex-matched controls with no history of hematological disease, the homozygous 72Pro status was reported to be associated with 2.06 increased AML risk. Genotyping was performed by the polymerase chain reaction restriction fragment length polymorphism (RFLP) method. Peripheral blood was used as DNA source material in healthy volunteers, but the source was not specified for the patients. Importantly, no deviation from Hardy-Weinberg equilibrium (HWE) was detected in patients or controls groups. Univariate survival analysis (n= 119, 63%), after exclusion of patients who did not receive conventional chemotherapy, revealed a 41% decreased risk of death from AML (hazard ratio [HR] 0.59, 95% CI 0.37 to 0.95) for patients with homozygous 72Pro resulting in a significantly higher calculated 5-year OS rate (42%) compared to the other genotypes (12%; P=0.031). This survival benefit was not apparent in multivariate analysis where also established risk factors like age and cytogenetic risk groups failed to show significant associations. The median follow up for the entire cohort was 135 days. Following an analysis of the TP53 Pro72Arg SNP in patients with therapy-related AML, we have very recently performed a case-control study of 215 de novo AML patients and 3759 controls from Austria and Germany. Constitutional DNA from buccal swabs or saliva was used for genotyping of rs1042522 by the TaqMan SNP Genotyping Assay whereby more than 10% of each patient cohort were also genotyped by direct sequencing. No deviation from HWE was detected in any group, and median follow up for all patients was 427 days for overall survival (OS), and 344 days for relapse free survival (RFS). We did not find any evidence to support rs1042522 as a risk factor of de novo AML development and survival; the latter could be assessed in 186 patients who were treated by standard induction and consolidation therapy, including allogeneic stem cell transplantation, according to European LeukemiaNet (ELN) risk groups. Known risk factors such as age and ELN risk groups, but not rs1042522 status, were found to influence survival in univariate and multivariable analyses comparable to previous published studies. How can these obviously conflicting results be explained? There are major differences between both studies which we think must be addressed. Most importantly, the difference with respect to the risk of AML is due to the genetic background of the study populations, which is mixed in the Brazilian study according to Bezerra et al., whereas almost only Caucasians participated in our study. Consequently, genotype frequencies are highly significantly different between both cohorts (Table 1). It is possible that other, yet unknown genetic and environmental modifiers linked to certain population backgrounds, might further modulate the risk of AML caused by rs1042522. Evidence for such a population-specific risk can be found in a recent meta-analysis of 32 case-control studies involving 8,586 cases and 10,275 controls, which revealed an increased risk of colorectal cancer for Asian individuals with homozygous TP53 72Pro, but not for the overall population. Other issues that should be considered in the survival analyses are patient sample size and follow up. As described above, a substantial percentage of patients were excluded from survival analysis in the study of Bezerra et al., which increases the chance of sample bias and type I error, and median follow up was shorter than a year masking late events. Indeed, although the difference in OS between homozygous 72Pro and the other genotypes was modestly significant, other known strong risk factors such as age and cytogenetics were not significantly associated with OS. From a technical point of view, one must note that RFLP was used for genotyping the Brazilian cohorts but no other methods, such as direct sequencing, were applied for validation of the results. Furthermore, DNA source material was not specified for the patient population. Although the authors have stated that chromosome 17p abnormalities were not observed upon karyotyping in their AML cohort, submicroscopic alterations at 17p, the locus of TP53, may, nevertheless, have skewed their results if diagnostic material were used for genotyping. In conclusion, the comparison of these two analyses on the role of the TP53 Pro72Arg SNP with respect to AML risk and survival following intensive treatments again reveal the ongoing challenges of genetic association studies. Importantly, consideration of the genetic background of populations analyzed remains a major issue with respect to interpretation and potential application of their results.