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Acute myeloid leukaemia (AML) is a neoplasm of immature myeloid cells characterized by various cytogenetic alterations. The present study showed that in addition to the FLT3-ITD and NPM1 mutation status, telomere length (TL) and telomerase reverse transcriptase (TERT) gene polymorphisms may affect risk and overall survival (OS) in AML. TL was longer in healthy controls than in AML patients and positively correlated with age in the patients, but not in healthy subjects. TL was found to be independently affected by the presence of the FLT3-ITD mutation. As for the TERT gene polymorphism, AML patients with the TERT rs2853669 CC genotype were characterized by significantly shorter OS than patients carrying the T allele. Another observation in our study is the difference in TL and OS in patients belonging to various risk stratification groups related to the FLT3-ITD and NPM1 mutation status. Patients with adverse risk classification (mutation in FLT3-ITD and lack of mutation in NPM1) presented with the shortest telomeres and significantly worse OS. In conclusion, OS of AML patients appears to be affected by TERT gene variability and TL in addition to other well-established factors such as age, WBC count, or FLT3-ITD and NPM1 mutation status.

Renata Sokolik,1,* Milena Iwaszko,2,* Jerzy Świerkot,1 Barbara Wysoczańska,2 Lucyna Korman,1 Piotr Wiland,1 Katarzyna Bogunia-Kubik2 1Department of Rheumatology and Internal Medicine, Wrocław Medical University, Wrocław, Poland; 2Laboratory of Clinical Immunogenetics and Pharmacogenetics, Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wrocław, Poland*These authors contributed equally to this workCorrespondence: Milena IwaszkoHirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Weigla 12, Wrocław 53-114, PolandTel +48 71 3709960Fax +48 71 3371382Email milena.iwaszko@hirszfeld.plIntroduction: The purpose of the study was to investigate whether single-nucleotide polymorphisms (SNPs) IL-6 − 174 G/C and IL-6R Asp358Ala are associated with susceptibility to psoriatic arthritis (PsA) or affect response to treatment with methotrexate (MTX).Patients and Methods: Seventy-four patients diagnosed with PsA and qualified for MTX treatment were enrolled to the study. The control group consisted of 120 healthy individuals. Polymorphisms IL-6 − 174 G/C and IL-6R Asp358Ala were genotyped using a polymerase chain reaction (PCR) amplification employing LightSNiP assays.Results: A significant association between the IL-6 − 174 CC genotype and an improved clinical outcome of MTX therapy was observed. A good response was more frequently observed among PsA patients bearing the IL-6 − 174 CC genotype than patients with the GC or GG genotypes (P = 0.007). On the other hand, patients carrying the IL-6 − 174 GC genotype less frequently responded to MTX treatment as compared to patients with other genotypes (P = 0.006). With respect to the IL-6R Asp358Ala SNP, there were no significant differences in genotype and allelic frequencies in relation to clinical outcome of MTX treatment. No association was found between the IL-6 − 174 G/C or IL-6R Asp358Ala SNPs and PsA susceptibility.Conclusion: Results from this study provide evidence that the IL-6 − 174 G/C polymorphism might influence efficacy of MTX treatment.Keywords: psoriatic arthritis, methotrexate treatment, IL-6 − 174G/C polymorphism, IL-6R Asp358Ala polymorphism

Background: The human telomerase reverse transcriptase (TERT) gene encodes the catalytic subunit of telomerase that is essential for maintenance of telomere length. We aimed to find out whether variability within the TERT gene could be associated with telomere length and development of the disease in non-treated patients with chronic lymphocytic leukemia (CLL). Materials and methods: Telomere length, rs2736100, rs2853690, rs33954691, rs35033501 single-nucleotide polymorphisms, and variable number of tandem repeats (VNTR-MNS16A) were assessed in patients at diagnosis. In addition, blood donors served as controls for the polymorphism studies. Results: The minor rs35033501 A variant was more frequent among CLL patients than in healthy controls (OR=3.488, p=0.039). CLL patients over 60 years of age were characterized with lower disease stage at diagnosis (p=0.001 and p=0.008, for the Rai and Binet criteria, respectively). The MNS16A VNTR-243 short allele was more frequent in patients with a low disease stage (p=0.020 and p=0.028, for the Rai and Binet staging system) and also among older patients having longer telomeres (p=0.046). Patients with Rai 0-I stage were characterized with longer telomeres than those with more advanced disease (p=0.030). This relationship was especially pronounced in patients carrying the rs2736100 C allele, independently of the criteria used, ie, Binet (p=0.048) or Rai (p=0.001). Conclusion: Our results showed that the genetic variation within the TERT gene seems to play a regulatory role in CLL and telomere length.

BACKGROUND NF-κB is an essential player in cancer biology, especially in tumor development, due to its constitutive activation, and because a four-base deletion (ATTG) in the NF-κB1 promoter region at site -94, alters mRNA stability and regulates translation efficiency. This polymorphism is a good candidate risk marker and modulator of clinical course in chronic lymphocytic leukemia (CLL). As the effect of this NF-κB1 gene polymorphism has not been studied in patients with CLL so far, the present study was undertaken to find out whether the NF-κB1 promoter -94 ins/del ATTG polymorphism might be an essential genetic risk factor and/or modulatory disease player associated with CLL. OBJECTIVES The NF-κB1 -94 ins/del ATTG (rs28362491) polymorphism was investigated as a potential CLL susceptibility and progression factor, along with demonstration of potential modulation of the stage of clinical disease based on Rai classification. MATERIAL AND METHODS The associations of NF-κB1 -94 ins/del ATTG polymorphism with CLL and its clinical manifestation in 282 Polish individuals, including 156 CLL patients, were analyzed using polymerase chain reaction (PCR) with primers including a labeled forward primer, followed by capillary electrophoresis. RESULTS A higher occurrence of the del/del homozygosity was observed among patients when compared to controls, resulting in an increase in CLL risk of more than twofold in patients carrying this homozygous genotype (OR = 2.23, p = 0.02, 95% CI = 1.14-4.37). Moreover, the del/del-positive patients more frequently presented the less aggressive disease phenotype (Rai 0), suggesting a low probability of progression to more advanced disease. CONCLUSIONS The NF-κB1 -94 del/del genetic variant, although associated with increased risk of CLL disease, may be associated with maintenance of disease severity in the early, mildest stage. The likelihood of disease progression may increase as the frequency of wild-type (insertion) alleles for this polymorphism increases.

Inconsistency of the results regarding the genetic variability within genes coding for receptor activator of nuclear factor κB (RANK) and its ligand (RANKL) in rheumatoid arthritis (RA) prompted us to study the RANK and RANKL polymorphisms as potential biomarkers associated with disease predisposition and response to anti-TNF treatment in a group of Polish patients with RA. This study enrolled 318 RA patients and 163 controls. RANK (rs8086340, C > G; rs1805034, C > T) and RANKL (rs7325635, G > A; rs7988338 G > A) alleles were determined by real-time PCR with melting curve analysis and related with clinical parameters. In addition, RANKL serum levels were measured by ELISA. The RANK rs8086340-G allele was overrepresented among patients as compared to controls (OD = 1.777, p = 0.038). C-reactive protein (CRP) levels were significantly (p RANK rs8086340 polymorphism and were higher in the CC-homozygotes at the baseline while lower in the GG-carriers at the 12th week of the treatment. At the latter time point RANKL rs7325635-GG-positive patients also showed significantly lower CRP concentrations. Higher alkaline phosphatase levels before induction of anti-TNF therapy were observed in RANK rs8086340 and RANK rs1805034 CC homozygotes (p = 0.057 and p = 0.035, respectively). The GG homozygosity of both RANKL single nucleotide polymorphisms was significantly associated with the number of swollen joints (rs7988338 and rs7325635, before and at the 12th week of therapy, respectively, p RANK and RANKL genes affect RA susceptibility and anti-TNF treatment outcome.

Promoter region of the telomerase reverse transcriptase gene (TERTp) constitutes a regulatory element capable to affect TERT expression (TE), telomerase activity (TA) and telomere length (TL). TERTp mutation status, TL, TA and TE were assessed in 27 in vitro cultured human cell lines, including 11 solid tumour, 13 haematological and 3 normal cell lines. C228T and C250T TERTp mutations were detected in 5 solid tumour and none of haematological cell lines (p = 0.0100). As compared to other solid tumour cell lines, those with the presence of somatic mutations were characterized by: shorter TL, lower TA and TE. Furthermore, cell lines carrying TERTp mutations showed a linear correlation between TE and TA (R = 0.9708, p = 0.0021). Moreover, haematological cell lines exhibited higher TE compared to solid tumour cell lines (p = 0.0007). TL and TA were correlated in both solid tumour (R = 0.4875, p = 0.0169) and haematological (R = 0.4719, p = 0.0095) cell lines. Our results based on the in vitro model suggest that oncogenic processes may differ between solid tumours and haematological malignancies with regard to their TERT gene regulation mechanisms.

The present study aimed to analyse and compare the distribution of MICA (rs1051792) and NKG2D/KLRK1 (rs1154831, rs1049174, rs2255336) polymorphisms in 61 Greek and 100 Polish patients with rheumatoid arthritis in relation to the presence of the HLA-DRB1 shared epitope and clinical parameters. Genotyping of selected polymorphism was performed using real-time PCR. HLA-DRB1 shared epitope alleles segregated differently in Greek and Polish patients but in both populations were detected in over 60% of cases. The rs1051792-A variant was more common among SE-positive Polish patients (p = 0.003) while the rs1049174-G allele was more frequently observed in Greeks than in Poles (p < 0.001). Moreover, among Greek patients, the rs1051792-GG homozygotes more frequently presented with anti-CCP antibodies and rheumatoid factor (RF), while carriers of the rs1049174-G variant and rs1154831-CC homozygotes were characterized by lower disease activity scores (p < 0.05 in all cases). These results imply that, in addition to the HLA-DRB1 SE alleles, MICA and NKG2D polymorphisms may also play a role in rheumatoid arthritis.

MicroRNA-146a (miR-146a) has been shown to play an important role in the regulation of inflammatory innate immune responses, and found to be differentially expressed in rheumatoid arthritis (RA). Through NF-κB pathway, this molecule is able to stimulate the release of pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-17. It has been also suggested that single-nucleotide polymorphisms (SNPs) in miRNA sequences may alter miRNA expression and that miR-146a rs2910164 SNP may contribute to RA development. These observations prompted us to analyze the potential associations between the miR-146a-3p (rs2910164, G > C) and NFkB1 (rs28362491, ins/del ATTG) polymorphisms and miR-146a-5p expression in patients’ sera in relation to clinical outcome of the treatment as well as predisposition to RA. Genotyping was performed in 111 patients and 130 healthy individuals while 16 controls and 13 RA patients (before and after three months of therapy with TNF-α inhibitors (TNFi)) were studied for the circulating miR-146a-5p serum expression level. Patients carrying the NFkB1 ins/ins genotype were characterized by worse response to TNFi treatment (p = 0.023). In patients, before TNFi therapy, expression levels of miR-146a-5p were less (0.422 ± 0.171) as compared to those detected after three months of treatment (1.809 ± 0.658, p = 0.033) and observed for healthy controls (5.302 ± 2.112, p = 0.048). Moreover, patients with higher circulating miR-146a-5p levels after three months of TNFi administration were more frequently carrying the rs2910164-C allele (p = 0.032). These results support the hypothesis that miR-146a might be involved in pathogenesis of RA and imply that miR-146a-3p polymorphism may be associated with miR-146a-5p levels in serum after anti-TNF-α treatment.

Natural killer (NK) cell licensing status depends on clonal expression of inhibitory killer cell immunoglobulin-like receptors (iKIR) and short term HLA environment. Licensed NK cells are more efficient in tumor killing than unlicensed NK cells. Cognate KIR-HLA pairs in hematopoietic stem cell transplant (HSCT) donor and recipient are decisive for the possible change in the NK cell licensing status after HSCT. We assessed clinical outcomes in 297 patients with lymphoproliferative or myeloproliferative malignancies, or myelodysplastic syndrome in a model with upward licensing, downward resetting, and unchanged licensing genetics status after T cell replate HSCT from unrelated donors. We found extremely low (0%) relapse/progression incidence (RI), and better (59%) event-free survival (EFS) in recipients with upward licensing status and highly increased RI (37.5%), and reduced EFS (8%) among patients with the downward resetting status of repopulated donor NK cells after HSCT, as compared with unchanged NK cell licensing (RI 23%, EFS 47%). These trends were confirmed in adjusted multivariable models (for RI p = 6.66E-09, OR = 1.47, 95% CI 1.29-1.66 and for EFS p = 3.79E-13, OR = 1.67, 95% CI 1.50-1.84). Differences in the incidence of acute graft versus host disease (GvHD 62, 69, and 47%) and chronic GvHD (24, 44, and 15%, respectively) in three groups were insignificant. It would be rationale the preferential selection of the donors with upward licensing over downward resetting inhibitory KIR:HLA constellation and inclusion of the KIR genotyping in the donor selection algorithm for malignant patients. Further studies using enlarged cohorts of patients with more homogenous diagnosis are essential to reliably verify these preliminary data.

Background Genetic variability in Toll-like receptor 10 (TLR10) may change the balance between pro- and anti-inflammatory responses, and hence modulate the susceptibility to infection and to autoimmune disease including rheumatoid arthritis (RA). Objectives Therefore we aimed to assess the possible associations of the TLR10 genetic variants with RA susceptibility and/or response to treatment Methods TLR10 gene (rs 11096957, N241H, A>C) polymorphism was genotyped by LightSNiP assay in 303 RA patients (237F/66M) and in 140 healthy individuals from Polish population. Results RA patients with the AC genotype showed predisposition to disease development [OR 1.99 (1.32–3.01); p=0.001], while the AA homozygosity seemed to play a protective role [OR 0.63 (0.42–0.95); p=0.034]. Response to treatment with TNF-alpha inhibitors was more effective after 6 months as compared to 3 months (p=0.001), especially in female patients (p=0.05). Women carrying the A allele responded better to treatment after 6 months of anti-TNF treatment as compared to those with the CC genotype (p=0.053). Response to biological treatment was more effective in patients with low stage of disease (p=0,01), with rheumatoid factor (RF) positivity (p=0.01) and with double positivity against cytrulinated (CCP) protein and RF (p=0.003). RF-positive patients (especially women, p=0.001) characterised with a higher degree of the disease as compared to RF-negative cases (p=0.01). Men had a higher activity of the disease before anti-TNF treatment (p=0.05), therefore the remission of the disease was more common in women (p=0.04). Conclusions These results imply that the TLR10 polymorphism has an important role in RA and may potentially influence risk of the disease and effectiveness of biological treatment. Acknowledgements Supported by the National Science Centre grant No. 2016/21/B/NZ5/01901. Disclosure of Interest None declared

Genes involved in regulation of the nuclear factor-κB (NF-κB)—pathway are suggested to play a role in pathogenesis of rheumatoid arthritis (RA). In the present study, genetic polymorphisms of TLR2, TLR4, TLR9 and NF-κB1 genes were investigated to assess their associations with RA susceptibility, progression and response to anti-TNF-α therapy. A group of 110 RA patients and 126 healthy individuals were genotyped for TLR2 (rs111200466), TLR4 (rs4986790, rs4986791), TLR9 (rs5743836, rs187084) and NF-κB1 (rs28362491) alleles. The presence of the TLR9 −1486 T variant (p < 0.0001) and its homozygosity (p < 0.0001) were found to be associated with disease susceptibility. The TLR9 −1237 C allele was associated with predisposition to RA in females only (p = 0.005). Moreover, the TLR4 rs4986791 G (rs4986790 T) alleles were more frequently detected among patients with the stage IV disease (p = 0.045), and were associated with more effective response to anti-TNF-α therapy (p = 0.012). More efficient response to anti-TNF-α treatment was also observed in patients with del within the NF-κB1 gene (p = 0.047), while for the TLR9 −1486 T homozygotes, the treatment was ineffective (p = 0.018). TLR polymorphisms affect disease susceptibility and response to therapy with TNF-α inhibitors in RA patients of Caucasian origin.

Among cancers treated with allogeneic hematopoietic stem-cell transplantation (HSCT), some are sensitive to natural killer (NK) cell reactivity, described as the “missing self” recognition effect. However, this model disregarded the NK cell licensing effect, which highly increases the NK cell reactivity against tumor and is dependent on the coexpression of inhibitory killer cell immunoglobulin-like receptor (iKIR) and its corresponding HLA Class I ligand. We assessed clinical data, HLA and donor iKIR genotyping in 283 patients with myelo- and lymphoproliferative malignancies who underwent HSCT from unrelated donors. We found dramatically reduced overall survival (OS), progression free survival (PFS), and time to progression (TTP) among patients with malignant diseases with the lack of HLA ligand cognate with this iKIR involved in NK cell licensing in corresponding donor (events 83.3% vs. 39.8%, P = 0.0010; 91.6% vs. 47.7%, P = 0.00010; and 30.0% vs. 17.3%, P = 0.013, for OS, PFS, and TTP, respectively). The extremely adverse PFS have withstand the correction when patient group was restricted to HLA mismatched donor-recipient pairs. The incidence of aGvHD was comparable in two groups of patients. In malignant patients after HSCT the missing HLA ligand for iKIR involved in NK cell licensing in corresponding donor (“missing licensing proof”) induced extremely adverse survival of the patients due to the progression of malignancy and not to the aGvHD. Avoiding the selection of HSCT donors with the “missing licensing proof” in the malignant patient is strongly advisable.Am. J. Hematol. 89:E176–E183, 2014. © 2014 Wiley Periodicals, Inc.

Telomeres protect the ends of chromosomes maintaining genome stability. The activity of telomerase enzyme, or alternatively the process of recombination, regulates the length of telomeres. In the absence of these mechanisms, excessive shortening of telomeres reach its critical level. Excessively shortened telomeres do not protect chromosome ends, the cell division cycle is stopped while the inactivity of replication process generates cellular senescence and cell death. On the other hand, critically shortened telomeres may promote chromosomal instability. These changes can lead to the development of carcinogenesis. In this process enzymatic activity of telomerase is reactivated. To maintain the protection of the chromosome ends, telomeres bind the stabilizing protein complex (shelterin). The presence of these protective proteins prevents undesirable DNA damage and initiates the repair system pathways. Molecular technologies enable the evaluation of telomere lengths, the analysis of telomerase expression and activity, and detection of mutations, polymorphic and epigenetic changes in telomere--shelterin--telomerase complex related genes. The purpose of research is to describe new mechanisms that affect the biology of telomere lengths, and to determine the impact on bone marrow failures, development of haematological malignancies, neurodegenerative diseases and others disorders associated with chromosomal instability. The model of modern therapies based on telomere biology explains the significance of the maintenance of telomere lengths in the process of cellular senescence and carcinogenesis.

Background Stem cell transplantation (SCT) is a curative treatment for children with primary immunodeficiencies. Methods The present retrospective analysis describes the long-term outcomes at a median follow-up of 9 years of 29 patients with immunodeficiency after SCT; 5 sibling and 24 alternative donor transplantations. T-cell engraftment emphazed on thymic dependent signal-joint T-cell receptor excision circles (sjTREC) generation and donor chimerism in relation to killer cell immunoglobulin-like receptor genes and their ligands. Results All children except two were reconstituted successfully from grafted material, including 9 and 18 cases of mixed chimerism (MC) and complete chimerism (CC), respectively. Univariate analyses showed that the number of activating KIR genes or HLA-C1/C2 ligand mismatches (P = .048) and possibly transplantation from an alternative donor (P = .054) facilitated CC development. Multivariate analysis showed that the presence of donor KIR haplotype B or incompatibility within C1/C2 ligands (relative risk, 6.1; 95% confidence interval, 1.08–34.69; P = .025) were significantly associated with the development of CC. Conclusions These results suggested that the donor-activating KIR gene repertoire affected successful engraftment.

Introduction Allotransplantation in chronic myeloid leukemia (CML) patients offers long-lasting remissions, which largely depend on immunologic surveillance of alloreactivity. Alloreactivity in CML patients has a durable potential. However a large proportion of relapsing patients, who have to undergo donor lymphocyte treatment is still abundant. Methods We studied a group of 31 CML patients post allogeneic transplantation for their level of T-cell receptor excision circles (TREC) and proportion of naive and memory/effector T cells in the peripheral blood (PB). TREC numbers were determined by quantitative PCR (qPCR) and T-cell subsets CD4+CD27+CD45RO−, CD4+CD27−CD24RO+, CD4+CCR7+, and CD4+CCR7− by flow cytometry. Patients were analyzed for posttransplant chimerism, type of bcr-abl transcripts, and number of TREC in association with the presence of chronic graft-versus-host disease (cGVHD) and relapse. CML patients with TREC+ in PB had a higher proportion of CD4+CD27+CD45RO− cells (3.54 vs 2.45%; P = .105) and CD4+CCR7+ cells (4.85 vs 2.67%; P = .007), and a lower proportion of CD4+CD27−CD45RO+ cells (5.55 vs 9.09%; P = .037). The incidence of cGvHD was reduced among TREC+ CML patients (3/14 vs 11/17; P = .006). Results The 5 out of 31 CML patients who relapsed were characterized by the presence of b2a2, b3/a2 or both type of transcripts, a lack of TREC in the blood, and a lower proportion of naive and effector/memory T cells. No association was observed between any of HLA specificities, type of bcr-abl transcripts and incidence of relapse. Conclusion The presence of TREC is affected by chronic GvHD; TREC negativity may constitute a risk of mixed chimerism and relapse.

Genes involved in regulation of the nuclear factor - kappa B (NF-κB) pathway are suggested to play a role in the pathogenesis of acute myeloid leukaemia (AML). The present study aimed to assess the association between the NF-κB1, TRAF3 and TLRs genes single nucleotide polymorphisms (SNPs) and disease susceptibility as well as progression in patients with AML. For this purpose 62 patients and 126 healthy individuals were genotyped for NF-κB1 (rs28362491), TRAF3 (rs11160707; rs12147254), TLR2 (rs201786064), TLR4 (rs4986790; rs4986791) and TLR9 (rs5743836; rs187084) alleles. Three SNPs were found to be associated with the risk for the AML development. The TRAF3 (rs12147254) AA homozygosity (RR = 2.770, P = 0.0392), TLR9 (rs5743836) C wild-type allele (RR = 2.542, P = 0.0096) as well as TLR9 (rs187084) T allele (RR = 13.396, P < 0.0001) and its homozygosity (RR = 11.805, P < 0.0001) were more frequent among patients with AML than healthy individuals. The associations of the rs187084 SNP were significant for both sexes. Moreover, patients who relapsed were more frequently characterized with the presence of the rs187084 TLR9 TT genotype (P = 0.045) or the rs12147254 TRAF3 A variant (P = 0.066). In conclusion, polymorphisms within the TLR9 and TRAF3 genes are associated with predisposition to AML and may affect the progression of the disease in the Polish population.

Haematopoietic stem cell transplantation (HSCT) is a curative treatment of many hematological disorders. However, although significant advances have been made in donor-recipient matching or conditioning regimens, HSCT is associated with a risk of post transplant complications. Those include generation of toxic lesions, graft-versus-host-disease (GvHD) and viral reactivations. Recent studies have shown the association between polymorphic features of non-HLA encoding genes and the incidence and severity of post-transplant complications in the recipients of allogeneic HSCT, implying that the donor-recipient genotyping, extended for cytokine loci, may be of prognostic value for the transplantation outcome. Thus, the pre-transplant analysis of the patients' genetic predisposition may be considered as important factor allowing the classification of the transplant recipients as less or more susceptible for developing toxic lesions, severe and/or fatal acute GvHD or viral reactivation. This review focuses on the relationship between the polymorphic patterns of tumor necrosis factor (TNF)- alpha and TNF-beta, IFN-gamma, interleukin (IL)-6, IL-10 and heat shock protein (HSP)70-hom encoding genes and the manifestation of post-transplant complications, acute and chronic GvHD, generation of toxic lesions, viral reactivations and mortality.

A total of 110 patients (71 adults and 39 children) who received allogeneic haematopoietic stem cell transplantation from HLA-matched sibling donors were studied for the incidence of acute graft-versus-host disease (aGvHD) in relation to IFN-gamma gene microsatellite polymorphism. A strong tendency was observed towards the lower incidence of grades II-IV aGvHD in patients having an IFN-gamma 2/2 genotype as compared to the recipients with other IFN-gamma genotypes (0.12 vs 0.33, P=0.06). This relationship was independent of the intensity of conditioning regimen and diagnosis. IFN-gamma polymorphic features, together with other clinical and biological factors (patient's age, donor-recipient gender, diagnosis, conditioning regimen, transplant material and GvHD prophylaxis), were subjected to multivariate analysis for aGvHD manifestation in order to exclude indirect association of the IFN-gamma 2/2 genotype. In multivariate analysis, myeloablative therapy (OR=11.462, P=0.013), recipient age (OR=4.896, P=0.009) and lack of IFN-gamma 2/2 genotype (OR=4.311, P=0.048) were found to significantly contribute to the development of grade II-IV aGvHD, while type of GvHD prophylaxis showed less-strong influence (OR=2.963, P=0.066). Thus, it appeared that the IFN-gamma 2/2 genotype constituted an independent and protective factor associated with a decreased risk of grade II-IV aGvHD. However, this genotype was not found to be associated with the risk of cGvHD or survival.

The present study aimed to assess the impact of the CXCL12 gene polymorphism (rs1801157) on clinical outcome of hematopoietic stem cell transplantation from unrelated donors. Toxic complications were less frequent among patients transplanted from donors carrying the CXCL12-3'-A allele (42/79 vs. 105/151, p=0.014 and 24/79 vs. 73/151, p=0.009, for grade II-IV and III-IV, respectively). Logistic regression analyses confirmed a role of donor A allele (OR=0.509, p=0.022 and OR=0.473, p=0.013 for grade II-IV and III-IV toxicity). In addition, age of recipients (OR=0.980, p=0.036 and OR=0.981, p=0.040, respectively) was independently protective while female to male transplantation and HLA compatibility were not significant. The incidence of aGvHD (grades I-IV) was lower in patients having A allele (52/119 vs. 113/204, p=0.043) and AA homozygous genotype (6/25 vs. 159/298, p=0.005). Independent associations of both genetic markers with a decreased risk of aGvHD were also seen in multivariate analyses (A allele: OR=0.591, p=0.030; AA homozygosity: OR=0.257, p=0.006) in which HLA compatibility seemed to play less protective role (p

Objectives : The aim of this study was to assess the suitability of a technique based on counter-flow centrifugal elutriation (CCE), which should allow one to enrich chronic myeloid leukemia (CML) patients' unstimulated native leukapheresis product (nLP) in CD34 + HLADR – cells and BCR-ABL negative cells. Methods : Six newly diagnosed CML patients were subjected to leukapheresis, and the products were subfractionated with the use of CCE. nLP and all fractions were studied for the presence of CD34 + cells and a proportion of BCR-ABL fluorescence in situ hybridization (FISH) + cells. Results : CCE fractions with a high flow rate contained the highest proportion of CD34 + cells [mean (SEM) 6.89% (3.88)]. However, CD34 + cells present in low-rate CCE fractions showed a higher proportion of HLADR – [49.6% (13.5 in 70 mL min −1 ) and 21.5% (11.6 in 110 mL min −1 )] than those in 170 mL min −1 [3.2% (2.5)] and ‘rotor off’[3.4% (1.9)]. This was associated with lower proportions of BCR-ABL FISH + [8.1% (4.8) and 1.9 (1.7)] and smaller BCR-ABL to ABL transcript ratios [0.58 ( 17 ) and 0.26 (0.08) in 70 and 110 mL min −1 ] fractions as compared to 140 and 170 mL min −1 fractions [21.6% (5.2) and 31.6% (15.3) for BCR-ABL FISH + cells and 0.75 (0.16) and 0.90 (0.24) for BCR - ABL / ABL ]. Fractions with the lowest proportions of BCR - ABL -positive cells and the lowest BCR - ABL / ABL transcript ratios (110 mL min −1 ) contained from 1.3 × 10 6 to 82.7 × 10 6 (median: 3.97 × 10 6 ) CD34 + cells. Conclusions : In the present study we have shown that CCE may be used effectively to obtain nLP fractions enriched in normal hematopoietic progenitors.

Some cancers treated with allogeneic hematopoietic stem cell transplantation (HSCT) are sensitive to natural killer cell (NK) reactivity. NK function depends on activating and inhibitory receptors and is modified by NK education/licensing effect and mediated by coexpression of inhibitory killer-cell immunoglobulin-like receptor (KIR) and its corresponding HLA I ligand. We assessed activating KIR (aKIR)-based HLA I–dependent education capacity in donor NKs in 285 patients with hematological malignancies after HSCT from unrelated donors. We found significantly adverse progression-free survival (PFS) and time to progression (TTP) in patients who received transplant from donors with NKs educated by C1:KIR2DS2/3, C2:KIR2DS1, or Bw4:KIR3DS1 pairs (for PFS: hazard ratio [HR], 1.70; P = .0020, Pcorr = .0039; HR, 1.54; P = .020, Pcorr = .039; HR, 1.51; P = .020, Pcorr = .040; and for TTP: HR, 1.82; P = .049, Pcorr = .096; HR, 1.72; P = .096, Pcorr = .18; and HR, 1.65; P = .11, Pcorr = .20, respectively). Reduced PFS and TTP were significantly dependent on the number of aKIR-based education systems in donors (HR, 1.36; P = .00031, Pcorr = .00062; and HR, 1.43; P = .019, Pcorr = .038). Furthermore, the PFS and TTP were strongly adverse in patients with missing HLA ligand cognate with educating aKIR-HLA pair in donor (HR, 3.25; P = .00022, Pcorr = .00045; and HR, 3.82; P = .027, Pcorr = .054). Together, these data suggest important qualitative and quantitative role of donor NK education via aKIR-cognate HLA ligand pairs in the outcome of HSCT. Avoiding the selection of transplant donors with high numbers of aKIR-HLA-based education systems, especially for recipients with missing cognate ligand, is advisable.

Summary MNS16A is a functional polymorphic tandem repeat within the human telomerase reverse transcriptase (hTERT) gene. To investigate whether any of the MNS16A repeats represents a genetic risk factor for NHL susceptibility, progression of or response to therapy in 75 patients with non-Hodgkin's lymphomas (NHLs) and 126 healthy individuals were genotyped using the PCR-VNTR technique. A slightly higher frequency of the MNS16A VNTR-243 variant was detected among patients who did not respond to treatment (NR) as compared to patients with complete or partial remission (0.83 vs. 0.51, P = 0.055). NR patients more frequently developed aggressive than indolent type of the disease (0.92 vs. 0.41, P = 0.001). The VNTR-243 allele was more frequently detected among patients with an intermediate–high/high International Prognostic Index (IPI 3–4) score (P = 0.063), especially in patients with advanced age and IPI 3–4 (P = 0.040). In multivariate analysis, higher IPI 3–4 score (OR = 11.364, P = 0.051) and aggressive type of the disease (OR = 18.182, P = 0.012) were found to be independent genetic markers associated with nonresponse to treatment. Presence of the MNS16A VNTR-243 variant also strongly tended to affect the risk of a less favourable response to therapy and was more frequently present among nonresponders (OR = 5.848, P = 0.059). Genetic variation within the hTERT gene may affect the progression and treatment of lymphoproliferative disorders.

Among the complex network of inflammatory cells involved in the pathogenesis of rheumatoid arthritis (RA), Th17 cells have recently been identified as key cells in the promotion of autoimmune processes, and joint destruction. The IL-23/Th17 signalling pathway, consisting of IL-23/IL-23R, IL-17A and IL-17F encoding genes, represents a candidate way for RA development with possible involvement in disease susceptibility and effect on disease progression. The present study aimed to determine the association between the polymorphic variants of the IL-17A (rs2275913), IL-17F (rs763780) and IL-23R (rs11209026) genes and RA susceptibility, progression and response to therapy with TNF-α inhibitors. Eighty-nine patients and 125 healthy individuals were investigated. The IL-17A polymorphism was found to affect RA progression and response to anti-TNF treatment. Female patients carrying the IL-17A wild-type genotype more frequently presented with stage 4 (8/24 vs. 6/47; p = 0.058) and were characterized by more active disease (the highest DAS28 score >5.1) after 3 months of therapy with the TNF inhibitors (12/23 vs. 15/45; p = 0.040). The IL-17F polymorphism appeared to be associated with susceptibility to the disease. The presence of the IL-17F minor variant (OR 3.97; p

Among cancers treated with allogeneic hematopoietic stem-cell transplantation (HSCT), some are sensitive to natural killer (NK) cell reactivity, described as the "missing self" recognition effect. However, this model disregarded the NK cell licensing effect, which highly increases the NK cell reactivity against tumor and is dependent on the coexpression of inhibitory killer cell immunoglobulin-like receptor (iKIR) and its corresponding HLA Class I ligand. We assessed clinical data, HLA and donor iKIR genotyping in 283 patients with myelo- and lymphoproliferative malignancies who underwent HSCT from unrelated donors. We found dramatically reduced overall survival (OS), progression free survival (PFS), and time to progression (TTP) among patients with malignant diseases with the lack of HLA ligand cognate with this iKIR involved in NK cell licensing in corresponding donor (events 83.3% vs. 39.8%, P = 0.0010; 91.6% vs. 47.7%, P = 0.00010; and 30.0% vs. 17.3%, P = 0.013, for OS, PFS, and TTP, respectively). The extremely adverse PFS have withstand the correction when patient group was restricted to HLA mismatched donor-recipient pairs. The incidence of aGvHD was comparable in two groups of patients. In malignant patients after HSCT the missing HLA ligand for iKIR involved in NK cell licensing in corresponding donor ("missing licensing proof") induced extremely adverse survival of the patients due to the progression of malignancy and not to the aGvHD. Avoiding the selection of HSCT donors with the "missing licensing proof" in the malignant patient is strongly advisable.

Background Interleukin (IL) - 23 is one of the of cytokines involved in systemic inflammation. Interaction between this cytokine and its receptor (IL-23R) that plays an important role in pathogenesis of psoriatic arthritis (PsA). Objectives The present study aimed to assess the associations between polymorphisms within gene coding IL-23R, IL-23 serum levels and disease activity in patients with PsA. Methods Fifty-two PsA patients (diagnosed by the criteria recommended by CASPAR group) were genotyped for the IL-23R (rs11209026 and rs7530511) polymorphisms. The nuclear factor kappa (NF-kB1 (rs28362491; ins/del)) polymorphism (associated with the promoter activity of this gene and cytokine gene expressions, including IL-23) was also analyzed in PsA patients group. IL-23 serum levels were assessed by ELISA in patients with PsA, and for comparison, 10 healthy individuals. These laboratory data were further related with clinical characteristics of the patients. Disease Activity Score was measured (swollen and tender joints, ESR, CRP) in addition to BASDAI, BASFI, VAS, and PASI scores. Results Significantly (p Conclusions Patients with PsA characterize with higher serum levels of IL-23 than healthy individuals. IL-23 concentrations in serum of PsA patients are associated with the polymorphism (rs7530511) of IL-23 receptor encoding gene. Disclosure of Interest None declared

The thymus is crucial in the reconstitution of the T-cell compartment following lymphodepletion and also in establishing a normal, diverse T-cell receptor (TCR) repertoire after immune response to antigens. TCRalphabeta diversity is generated through rearrangements of the TCR alpha and TCR beta chain genes. The TCR delta chain locus lies within the TCR alpha chain locus and its excision forms the first step in TCR alpha chain gene rearrangement. The intervening excised DNA is circularized by the formation of a "signal joint" forming a DNA episome, termed a signal-joint T-cell receptor excision circle (sjTREC). Approximately 70% of T cells emerging from the thymus contain one or two sjTRECs--depending on whether one or both TCRalpha loci genes are rearranged. As these long-lived naïve T cells mature and proliferate, their sjTRECs are stable and do not divide. The thymus contributes naïve T (CD45RA+) cells with TRECs to the peripheral immune system, but memory T cells (CD45RO+) contain few if any detectable TRECs. Quantification of thymic function is clinically relevant in settings with immunodeficiencies, after HSCT, during HIV therapy as well as in autoimmune patients. In humans there is no specific phenotypic marker for recent thymic emigrants, therefore the use of real-time quantitative PCR methods for absolute TREC quantification provides a good tool for estimating recent thymic function in different clinical situations.

The present study aimed to determine existing associations between single nucleotide polymorphisms within the promoters of interleukin (IL)-6 (-174 G/C) and IL-10 (-1082 G/A, -819 C/T, -592 C/A) genes and the outcome of allogeneic sibling hematopoietic stem cell transplantation. Ninety-three recipients and 74 donors were typed for IL-6 and IL-10 alleles by polymerase chain reaction-sequence specific primer. Then, IL-6 activity in patient serum and the concentration of C-reactive protein were analyzed at various times after transplantation in relation to transplant complications and IL-6 genotype. IL-6 activity in serum was significantly higher in patients who died as a result of toxic complications and after the 6 weeks after transplantation in patients with severe acute graft-versus-host disease (aGVHD). Recipient IL-6 G genotype was associated with increased IL-6 activity and C-reactive protein production. In univariate analyses, recipient IL-6 G and donor IL-6 GG associated or tended to associate with increased risk for aGVHD. In contrast, recipient IL-10 GCC/GCC and donor IL-10 ACC decreased the risk of aGVHD. IL-6 and IL-10 polymorphic features, together with other factors known to affect the risk of aGVHD, were also subjected to multivariate analyses. These analyses confirmed the independent contribution of recipient IL-10 GCC/GCC (odds ratio = 0.085, p = 0.046) and donor IL-6 GG (odds ratio = 3.934, p = 0.034) genotypes to the risk of aGVHD.

In the present study, short tandem repeats (STR) polymorphism within the first intron of IFN-gamma gene in connection with HLA-DRB1*03 specificities was analysed in 43 sarcoidosis patients, 14 of which presented with Lofgren’s syndrome (LS). Four out of 5 known IFN-gamma alleles with 12–15 CA repeats, respectively were detected in sarcoidosis patients. IFN-gamma 3,3 homozygosity was found to constitute a risk factor associating with LS manifestation (0.50 versus 0.19, OD=4.18, P =0.014). As expected, DRB1*03 prevailed in LS cases (9/14 versus 7/29, P =0.014 LS versus non-LS cases and 9/14 versus 35/162, OD=6.20, P =0.001, LS patients versus controls). However, we also showed the presence of a combined association between DRB1*03 and IFN-gamma 3,3 in sarcoidosis ( P =0.017) and LS patients ( P =0.001).

Background The pro-inflammatory cytokines IL-17A and IL-17F, primarily produced by Th17 cells, has been shown to be involved in all stages of the autoimmune and inflammatory diseases [1]. Th17 cells are thought to play an important role in rheumatoid (RA) and psoriatic arthritis (PsA) [2]. Objectives The present study aimed to determine the association between their polymorphic variants with possible involvement in disease susceptibility and effect on disease progression in patients with PsA and RA susceptibility, progression and response to therapy with TNF-alpha inhibitors. Methods Eighty-nine RA and 54 PsA patients and 126 healthy individuals were investigated and genotyped for the IL-17A (rs2275913; G-197A), IL-17F (rs763780; A7488G; His161Arg) and transcription nuclear factor NFkB-1 (rs28362491; -94 ins/del ATTG) alleles. Results Female RA patients carrying the IL-17A wild type genotype more frequently presented with radiographic changes grade 4 (Steinbrocker) (8/24 vs. 6/47, p=0.058) and were characterized by more active disease after 3 months of therapy with the TNF inhibitors (12/23 vs. 15/45, p=0.040). The presence of the IL-17F minor ( G ) variant (OR=3.97, p The NFkB1 del genotype was more frequently observed in patients with PsA onset over 40 year of age (16/20 vs. 8/17, p=0.047). PsA patients lacking this NFkB1 del/del genotype ( ins positive cases) more frequently presented with severe disease (PASI >10, BSA >10, more than five swollen and tender joints) and subjected to anti-TNF-alpha therapy (20/21 vs. 12/18, p=0.035). Conclusions These results suggest that the IL-17A and IL-17F polymoprhisms play a significant role in RA while NFkB-1 variants seem to affect PsA. References Miossec P, Kolls JK. Targeting IL-17 and TH 17 cells in chronic inflammation. Nat Rev Drug Discov 2012; 11:763:776 Shen H., Goodal JC, Hill Ga ston JS. Frequency and phenotype of peripheral blood Th17 cells in ankylosing spodylitis and rheumatoid arthritis. Arthritis Rheum 2009; 60:1647-1656. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.5856

The thymus is the main organ of T-cell maturation in humans. However, it regresses at puberty, whereas T-cell rearrangement continues even in the elderly,1 and there is a hypothesis that the function of the thymus may partially be replaced by other tissues including the intestinal epithelium. Indeed, intraepithelial lymphocytes located in the small intestinal mucosa were found to express recombination activating genes RAG 1, RAG 2 and pre-T -chain, which are all required for T-cell receptor (TCR) rearrangement.2, 3, 4 Although these findings strongly suggest the presence of extrathymic T-cell maturation (ETCM), no direct evidence of this phenomenon has been provided so far.

Four CML patients were investigated for post transplant chimerism for total marrow (BM) cells and Mesenchymal Stem Cell (MSC) populations purified and propagated from BM cells. Patients were conditioned with BuFluATG and after allogeneic PBPCT were disease free with proven hematological, cytogenetical and genetical remission. For the study 15–30 ml fresh BM aspirates were phenotypically characterized for the presence of cell populations which may contain MSC. We found (median values): 11.7% CD45-CD34− 1.7% CD45-CD34-CD105+, 0.13% CD45-CD34-CD90+ and 0.03% CD45-CD34-CD73+ cells. These BM populations contained 0.75% CD34+ cells. Genetical work showed that BM cells were BCR-ABL negative and their STR informative allele patterns were consistent with those of donors. BM cells were processed as follows: incubation with Glycophorin A, CD3, CD14, CD19, CD66b, CD38 antibody cocktail which by cross-linking unwanted cells with red blood cells led to rosette formation (RosetteSep MSC Enrichment Cocktail, StemCell Technology), unrosetted cells (MSC enriched) were recovered from the interface after buoyant gradient centrifugation and contained (median values): 68.7% CD45-CD34-, 21.6% CD45-CD34-CD105+, 1.1% CD45-CD34-CD90+ and 0.4% CD45-CD34-CD73+. MSC enriched BM populations were cultured in Medium for Human MSC with Stimulatory Supplements (StemCell Technology). After 10–14 days CFU-F colonies were scored (median value was 77 CFU-F/106 cells) and cells were further cultured until >90% confluence of fibroblast-like cells were reached (usually 3 to 4 weeks after culture initiation). The cells were detached with 0.05% trypsin-EDTA and studied for STR allele patterns, the presence of BCR-ABL transcripts (at that time cells showed STR alleles of the recipient pattern for the first time - mixed chimerism) and the bulk of cells were further passaged. Usually after 3–4 passages (within 7–8 weeks) when fibroblast-like stromal cell populations reached the level of 3x106 cells, cultures were terminated and the cells were studied. These cells were in (median values) 27.0% CD45-CD34-, 23.8% CD45-CD34-CD105+, 26.5% CD45-CD34-CD90+ and 24.3% CD45-CD34-CD73+. The cells had a fibroblast-like morphology but only 26% had phenotype features of MSC on average. Therefore, the population consisted of MSC and more differentiated cells originated from CFU-F (MSC). RNA and DNA were isolated from the cells propagated for 7–8 weeks from the MSC enriched BM populations were BCR-ABL negative. However, their STR informative allele patterns were consistent with those of the recipients in variance to primary BM cell populations which was in all 4 cases of donor origin. Conclusions: with the use of the RosetteSep MSC enrichment purification system BM cells can be enriched in CFU-F which paralleled with an increase in the proportion of CD45-CD34-, CD45-CD34-CD90+, CD45-CD34-CD73+ and CD45-CD34-CD105+ cells, CFU-F BM enriched populations can be cultured with Medium for Human MSC with Stimulatory Supplements for successful propagation of fibroblast-like cells with kinetics documenting ex potential growth after 38 days (median) of culture, Cells originated from CFU-F were in contrast to the BM hematopoietic compartment of the recipients origin and were also BCR-ABL negative, MSC were not replaced by allogeneic PBPC-graft derived MSC.

Purpose Recent studies have suggested that Th17 cells may play a role in the pathogenesis of acute myeloid leukemia (AML). This subset of CD4+ cells is characterized by interleukin (IL)-17A and IL-17F production, which share strong homology, and surface expression of the IL-23 receptor (IL-23R). The present study aimed to determine the association between the polymorphic features located within the IL-17A, IL-17F and IL-23R genes and disease susceptibility, progression and response to therapy. In addition, the relationship between the polymorphic variants and the plasma IL-17 levels in patients was analyzed. Methods For this purpose, 187 individuals of Polish origin including 62 AML patients and 125 healthy controls were typed for IL-17A (rs2275913; G-197A), IL-17F (rs763780; A7488G; His161Arg) and IL-23R (rs11209026, G1142A; Arg381Gln) alleles. Results The rs763780 IL-17F polymorphism appeared to be associated with susceptibility to the disease. The presence of the minor (G) variant (RR = 4.76, p