SIRT1 and HSP90α feed-forward circuit safeguards chromosome segregation integrity in diffuse large B cell lymphomas.

Diffuse large B-cell lymphoma (DLBCL) is the most common aggressive non-Hodgkin lymphoma in adults, exhibiting highly heterogenous clinical behavior and complex molecular background. In addition to the genetic complexity, different DLBCL subsets exhibit phenotypic features independent of the genetic background. For example, a subset of DLBCLs is distinguished by increased oxidative phosphorylation and unique transcriptional features, including overexpression of certain mitochondrial genes and a molecular chaperone, heat shock protein HSP90α (termed "OxPhos" DLBCLs). In this study, we identified a feed-forward pathogenetic circuit linking HSP90α and SIRT1 in OxPhos DLBCLs. The expression of the inducible HSP90α isoform remains under SIRT1-mediated regulation. SIRT1 knockdown or chemical inhibition reduced HSP90α expression in a mechanism involving HSF1 transcription factor, whereas HSP90 inhibition reduced SIRT1 protein stability, indicating that HSP90 chaperones SIRT1. SIRT1-HSP90α interaction in DLBCL cells was confirmed by co-immunoprecipitation and proximity ligation assay (PLA). The number of SIRT1-HSP90α complexes in PLA was significantly higher in OxPhos- dependent than -independent cells. Importantly, SIRT1-HSP90α interactions in OxPhos DLBCLs markedly increased in mitosis, suggesting a specific role of the complex during this cell cycle phase. RNAi-mediated and chemical inhibition of SIRT1 and/or HSP90 significantly increased the number of cells with chromosome segregation errors (multipolar spindle formation, anaphase bridges and lagging chromosomes). Finally, chemical SIRT1 inhibitors induced dose-dependent cytotoxicity in OxPhos-dependent DLBCL cell lines and synergized with the HSP90 inhibitor. Taken together, our findings define a new OxPhos-DLBCL-specific pathogenetic loop involving SIRT1 and HSP90α that regulates chromosome dynamics during mitosis and may be exploited therapeutically.

Psychological responses and associated factors during the initial stage of the coronavirus disease (COVID-19) epidemic among the adult population in Poland - a cross-sectional study.

INTRODUCTION: The study aimed to assess the emotional state, the occurrence of symptoms of depression, anxiety, and stress, as well as the quality of life of adults living in Poland during the first weeks of the COVID-19 pandemic. METHOD: The study was conducted on a group of 700 people aged 18 and over living in Poland. An anonymous online questionnaire was used in this cross-sectional study. The psychological impact of COVID-19 was measured using the Revised Event Impact Scale (IES-R) and the Depression, Anxiety, and Stress Scale (DASS - 21). The quality of life was assessed using the WHOQOL-BREF. RESULTS: In Poland, a high average level of post-traumatic stress was found as a result of the COVID-19 pandemic, with at least the minimum level occurring in all surveyed people. There was also a high incidence of depression (48.00%), anxiety (39.29%), and stress (54.86) in the first phase of the pandemic. The average level of quality of life in Poland was the lowest for the physical domain and amounted to 49.56 (SD = 11.71). The standard of living in the psychological domain was 60.26 (SD = 13.14). CONCLUSIONS: The pandemic is having a significant impact on human mental health. The very high average levels of post-traumatic stress, stress, anxiety, and depression as well as low quality of life make it necessary to consider interventions that will favor the use of more adaptive defense mechanisms and build mental resilience during an infectious disease pandemic and its long-term consequences.

Inhibition of PIM Kinases in DLBCL Targets MYC Transcriptional Program and Augments the Efficacy of Anti-CD20 Antibodies.

The family of PIM serine/threonine kinases includes three highly conserved oncogenes, PIM1, PIM2, and PIM3, which regulate multiple prosurvival pathways and cooperate with other oncogenes such as MYC. Recent genomic CRISPR-Cas9 screens further highlighted oncogenic functions of PIMs in diffuse large B-cell lymphoma (DLBCL) cells, justifying the development of small-molecule PIM inhibitors and therapeutic targeting of PIM kinases in lymphomas. However, detailed consequences of PIM inhibition in DLBCL remain undefined. Using chemical and genetic PIM blockade, we comprehensively characterized PIM kinase-associated prosurvival functions in DLBCL and the mechanisms of PIM inhibition-induced toxicity. Treatment of DLBCL cells with SEL24/MEN1703, a pan-PIM inhibitor in clinical development, decreased BAD phosphorylation and cap-dependent protein translation, reduced MCL1 expression, and induced apoptosis. PIM kinases were tightly coexpressed with MYC in diagnostic DLBCL biopsies, and PIM inhibition in cell lines and patient-derived primary lymphoma cells decreased MYC levels as well as expression of multiple MYC-dependent genes, including PLK1. Chemical and genetic PIM inhibition upregulated surface CD20 levels in an MYC-dependent fashion. Consistently, MEN1703 and other clinically available pan-PIM inhibitors synergized with the anti-CD20 monoclonal antibody rituximab in vitro, increasing complement-dependent cytotoxicity and antibody-mediated phagocytosis. Combined treatment with PIM inhibitor and rituximab suppressed tumor growth in lymphoma xenografts more efficiently than either drug alone. Taken together, these results show that targeting PIM in DLBCL exhibits pleiotropic effects that combine direct cytotoxicity with potentiated susceptibility to anti-CD20 antibodies, justifying further clinical development of such combinatorial strategies. SIGNIFICANCE: These findings demonstrate that inhibition of PIM induces DLBCL cell death via MYC-dependent and -independent mechanisms and enhances the therapeutic response to anti-CD20 antibodies by increasing CD20 expression.

IDH2 mutations in patients with normal karyotype AML predict favorable responses to daunorubicin, cytarabine and cladribine regimen.

Mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) genes occur in about 20% patients with acute myeloid leukemia (AML), leading to DNA hypermethylation and epigenetic deregulation. We assessed the prognostic significance of IDH1/2 mutations (IDH1/2+) in 398 AML patients with normal karyotype (NK-AML), treated with daunorubicine + cytarabine (DA), DA + cladribine (DAC), or DA + fludarabine. IDH2 mutation was an independent favorable prognostic factor for 4-year overall survival (OS) in total NK-AML population (p = 0.03, censoring at allotransplant). We next evaluated the effect of addition of cladribine to induction regimen on the patients' outcome according to IDH1/2 mutation status. In DAC group, 4-year OS was increased in IDH2+ patients, compared to IDH-wild type group (54% vs 33%; p = 0.0087, censoring at allotransplant), while no difference was observed for DA-treated subjects. In multivariate analysis, DAC independently improved the survival of IDH2+ patients (HR = 0.6 [0.37-0.93]; p = 0.024; censored at transplant), indicating that this group specifically benefits from cladribine-containing therapy. In AML cells with R140Q or R172K IDH2 mutations, cladribine restrained mutations-related DNA hypermethylation. Altogether, DAC regimen produces better outcomes in IDH2+ NK-AML patients than DA, and this likely results from the hypomethylating activity of cladribine. Our observations warrant further investigations of induction protocols combining cladribine with IDH1/2 inhibitors in IDH2-mutant.

Predictive significance of selected gene mutations in relapsed and refractory chronic lymphocytic leukemia patients treated with ibrutinib.

BACKGROUND: Ibrutinib, an inhibitor of the Bruton's kinase (BTK), is characterized by high efficacy in the therapy of patients with relapsed and refractory chronic lymphocytic leukemia (RR-CLL). AIMS: To analyze the potential significance of the mutational status of selected 30 genes on the disease outcome in 45 patients with RR-CLL using custom-made gene panel and sequencing on Illumina MiSeq FGx platform. RESULTS: The highest rate of mutations was observed in TP53 (n = 18; 40.0%), NOTCH1 (n = 13; 28.8%), SF3B1 (n = 11; 24.4%), ATM (n = 7; 15.6%), MED12 (n = 6, 13.3%), CHD2 (n = 5; 11.1%), XPO1 (n = 5; 11.1%), NFKBIE (n = 5; 11.1%), BIRC3 (n = 4; 8.9%), SPEN (n = 4; 8.9%), POT1 (n = 4; 8.9%), EGR2 (n = 3; 6.7%), and RPS15 (n = 3; 6.7%). With a median observation time of 45.9 months, the median progression-free survival (PFS) and overall survival (OS) were not reached. The 36-month estimated rate of PFS and OS were 64% and 68.2%, respectively. The overall response rate was noted in 23 patients (51.1%), while twenty (44.4%) patients achieved stability. Progression was noted in 2 (4.5%) cases. Analyzed molecular factors had no impact on PFS and OS. CONCLUSION: Despite accumulation of several poor prognostic factors in our real-life cohort of heavily pretreated patients with CLL, ibrutinib treatment showed long-term clinical benefit.

SYK inhibition targets acute myeloid leukemia stem cells by blocking their oxidative metabolism.

Spleen tyrosine kinase (SYK) is an important oncogene and signaling mediator activated by cell surface receptors crucial for acute myeloid leukemia (AML) maintenance and progression. Genetic or pharmacologic inhibition of SYK in AML cells leads to increased differentiation, reduced proliferation, and cellular apoptosis. Herein, we addressed the consequences of SYK inhibition to leukemia stem-cell (LSC) function and assessed SYK-associated pathways in AML cell biology. Using gain-of-function MEK kinase mutant and constitutively active STAT5A, we demonstrate that R406, the active metabolite of a small-molecule SYK inhibitor fostamatinib, induces differentiation and blocks clonogenic potential of AML cells through the MEK/ERK1/2 pathway and STAT5A transcription factor, respectively. Pharmacological inhibition of SYK with R406 reduced LSC compartment defined as CD34+CD38-CD123+ and CD34+CD38-CD25+ in vitro, and decreased viability of LSCs identified by a low abundance of reactive oxygen species. Primary leukemic blasts treated ex vivo with R406 exhibited lower engraftment potential when xenotransplanted to immunodeficient NSG/J mice. Mechanistically, these effects are mediated by disturbed mitochondrial biogenesis and suppression of oxidative metabolism (OXPHOS) in LSCs. These mechanisms appear to be partially dependent on inhibition of STAT5 and its target gene MYC, a well-defined inducer of mitochondrial biogenesis. In addition, inhibition of SYK increases the sensitivity of LSCs to cytarabine (AraC), a standard of AML induction therapy. Taken together, our findings indicate that SYK fosters OXPHOS and participates in metabolic reprogramming of AML LSCs in a mechanism that at least partially involves STAT5, and that SYK inhibition targets LSCs in AML. Since active SYK is expressed in a majority of AML patients and confers inferior prognosis, the combination of SYK inhibitors with standard chemotherapeutics such as AraC constitutes a new therapeutic modality that should be evaluated in future clinical trials.

BRAF inhibition curtails IFN-gamma-inducible PD-L1 expression and upregulates the immunoregulatory protein galectin-1 in melanoma cells.

Although melanoma is considered one of the most immunogenic malignancies, spontaneous T-cell responses to melanoma antigens are ineffective due to tumor cell-intrinsic or microenvironment-driven immune evasion mechanisms. For example, oncogenic BRAF V600E mutation in melanoma cells fosters tumor immune escape by modulating cell immunogenicity and microenvironment composition. BRAF inhibition has been shown to increase melanoma cell immunogenicity, but these effects are transient and long-term responses are uncommon. For these reasons, we aimed to further characterize the role of BRAF-V600E mutation in the modulation of PD-L1, a known immunoregulatory molecule, and galectin-1 (Gal-1), a potent immunoregulatory lectin involved in melanoma immune privilege. We report herein that vemurafenib downregulates IFN-γ-induced PD-L1 expression by interfering with STAT1 activity and by decreasing PD-L1 protein translation. Surprisingly, melanoma cells exposed to vemurafenib expressed higher levels of Gal-1. In coculture experiments, A375 melanoma cells pretreated with vemurafenib induced apoptosis of interacting Jurkat T cells, whereas genetic inhibition of Gal-1 in these cells restored the viability of cocultured T lymphocytes, indicating that Gal-1 contributes to tumor immune escape. Importantly, Gal-1 plasma concentration increased in patients progressing on BRAF/MEK inhibitor treatment, but remained stable in responding patients. Taken together, these results suggest a two-faceted nature of BRAF inhibition-associated immunomodulatory effects: an early immunostimulatory activity, mediated at least in part by decreased PD-L1 expression, and a delayed immunosuppressive effect associated with Gal-1 induction. Importantly, our observations suggest that Gal-1 might be utilized as a potential biomarker and a putative therapeutic target in melanoma patients.

Serine Biosynthesis Pathway Supports MYC-miR-494-EZH2 Feed-Forward Circuit Necessary to Maintain Metabolic and Epigenetic Reprogramming of Burkitt Lymphoma Cells.

Burkitt lymphoma (BL) is a rapidly growing tumor, characterized by high anabolic requirements. The MYC oncogene plays a central role in the pathogenesis of this malignancy, controlling genes involved in apoptosis, proliferation, and cellular metabolism. Serine biosynthesis pathway (SBP) couples glycolysis to folate and methionine cycles, supporting biosynthesis of certain amino acids, nucleotides, glutathione, and a methyl group donor, S-adenosylmethionine (SAM). We report that BLs overexpress SBP enzymes, phosphoglycerate dehydrogenase (PHGDH) and phosphoserine aminotransferase 1 (PSAT1). Both genes are controlled by the MYC-dependent ATF4 transcription factor. Genetic ablation of PHGDH/PSAT1 or chemical PHGDH inhibition with NCT-503 decreased BL cell lines proliferation and clonogenicity. NCT-503 reduced glutathione level, increased reactive oxygen species abundance, and induced apoptosis. Consistent with the role of SAM as a methyl donor, NCT-503 decreased DNA and histone methylation, and led to the re-expression of ID4, KLF4, CDKN2B and TXNIP tumor suppressors. High H3K27me3 level is known to repress the MYC negative regulator miR-494. NCT-503 decreased H3K27me3 abundance, increased the miR-494 level, and reduced the expression of MYC and MYC-dependent histone methyltransferase, EZH2. Surprisingly, chemical/genetic disruption of SBP did not delay BL and breast cancer xenografts growth, suggesting the existence of mechanisms compensating the PHGDH/PSAT1 absence in vivo.

Overcoming Ibrutinib Resistance in Chronic Lymphocytic Leukemia.

Ibrutinib is the first Bruton's tyrosine kinase (BTK) inhibitor, which showed significant clinical activity in chronic lymphocytic leukemia (CLL) and small lymphocytic lymphoma (SLL) patients regardless of cytogenetic risk factors. Recent results of phase III clinical trials in treatment-naïve CLL patients shift the importance of the agent to frontline therapy. Nevertheless, beside its clinical efficacy, ibrutinib possesses some off-target activity resulting in ibrutinib-characteristic adverse events including bleeding diathesis and arrhythmias. Furthermore, acquired and primary resistance to the drug have been described. As the use of ibrutinib in clinical practice increases, the problem of resistance is becoming apparent, and new methods of overcoming this clinical problem arise. In this review, we summarize the mechanisms of BTK inhibitors' resistance and discuss the post-ibrutinib treatment options.

Surface expression of Cytokine Receptor-Like Factor 2 increases risk of relapse in pediatric acute lymphoblastic leukemia patients harboring IKZF1 deletions.

We prospectively examined whether surface expression of Cytokine Receptor-Like Factor 2 (CRLF2) on leukemic blasts is associated with survival and induction treatment response in pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL) patients. Flow cytometric analysis of bone marrow-derived leukemia cells revealed that 7.51% (29/286) of 386 pediatric BCP-ALL patients were CRLF2-positive (CRLF2pos) at diagnosis. The median minimal residual disease (MRD) was lower in CRLF2pos than CRLF2-negative (CRLF2neg) patients on day 15 (MRD15) after induction therapy [0.01% (0.001-0.42%) vs. 0.45% (0.05-3.50%); p=0.001]. By contrast, the MRD15 was higher in Ikaros family Zinc Finger Protein 1 (IKZF1)-deleted BCP-ALL patients than in BCP-ALL patients without IKZF1 deletions [1.18% (0.06-12.0%) vs 0.33% (0.03-2.6%); p=0.003]. Subgroup analysis showed that MRD15 levels were lower in IKZF1Δ/CRLF2pos patients than in IKZF1Δ/CRLF2neg patients [0.1% (0.02-5.06%) vs. 2.9% (0.25-12%); p=0.005]. Furthermore, MRD15 levels were higher in IKZF1WT/CRLF2neg patients than in IKZF1WT/CRLF2pos patients [0.40% (0.04-2.7%) vs. 0.001% (0.001-0.01%)]. Despite the low MRD15 levels, IKZF1Δ/CRLF2pos patients showed poorer relapse-free survival (RFS) than other patient groups (p=0.003). These findings demonstrate that surface CRLF2 expression is associated with increased risk of relapse in pediatric BCP-ALL patients harboring IKZF1 deletions.

Microenvironment-induced PIM kinases promote CXCR4-triggered mTOR pathway required for chronic lymphocytic leukaemia cell migration.

Lymph node microenvironment provides chronic lymphocytic leukaemia (CLL) cells with signals promoting their survival and granting resistance to chemotherapeutics. CLL cells overexpress PIM kinases, which regulate apoptosis, cell cycle and migration. We demonstrate that BCR crosslinking, CD40 stimulation, and coculture with stromal cells increases PIMs expression in CLL cells, indicating microenvironment-dependent PIMs regulation. PIM1 and PIM2 expression at diagnosis was higher in patients with advanced disease (Binet C vs. Binet A/B) and in those, who progressed after first-line treatment. In primary CLL cells, inhibition of PIM kinases with a pan-PIM inhibitor, SEL24-B489, decreased PIM-specific substrate phosphorylation and induced dose-dependent apoptosis in leukaemic, but not in normal B cells. Cytotoxicity of SEL24-B489 was similar in TP53-mutant and TP53 wild-type cells. Finally, inhibition of PIM kinases decreased CXCR4-mediated cell chemotaxis in two related mechanisms-by decreasing CXCR4 phosphorylation and surface expression, and by limiting CXCR4-triggered mTOR pathway activity. Importantly, PIM and mTOR inhibitors similarly impaired migration, indicating that CXCL12-triggered mTOR is required for CLL cell chemotaxis. Given the microenvironment-modulated PIM expression, their pro-survival function and a role of PIMs in CXCR4-induced migration, inhibition of these kinases might override microenvironmental protection and be an attractive therapeutic strategy in this disease.

MiR-17-92 represses PTPROt and PP2A phosphatases and amplifies tonic BCR signaling in DLBCL cells.

B-cell receptor (BCR) signaling plays a pivotal role in the pathogenesis of diffuse large B-cell lymphoma (DLBCL) and targeting the BCR pathway is a highly promising therapeutic strategy in this malignancy. The oncogenic microRNA miR-17-92 modulates multiple cellular processes such as survival, proliferation, apoptosis, angiogenesis, and BCR signaling. In the present study, we identified new targets of miR-17-92, PTPROt (protein phosphatase, receptor type O, truncated) and PP2A (protein phosphatase 2A) phosphatases, which regulate the activity of spleen tyrosine kinase (SYK) and AKT, critical components of BCR signal transduction in DLBCL cells. Introduction of miR-17-92 into DLBCL cells dampened the expression of the PTPROt and PP2A regulatory subunits PPP2R2A (protein phosphatase 2, regulatory subunit B, alpha) and PPP2R5E (protein phosphatase 2, regulatory subunit B, epsilon isoform) and increased the magnitude of SYK and AKT phosphorylation upon BCR ligation. Finally, we found that miR-17-92 expression modulates response to inhibitors of BCR signaling because downregulation of miR-17-92 increased SYK inhibitor-mediated toxicity in DLBCL cells. Our study reveals novel posttranscriptional regulatory pathways that contribute to the deregulation of BCR signaling and modulate SYK inhibitor activity in DLBCL.

MEK Inhibition Sensitizes Precursor B-Cell Acute Lymphoblastic Leukemia (B-ALL) Cells to Dexamethasone through Modulation of mTOR Activity and Stimulation of Autophagy.

Resistance to glucocorticosteroids (GCs) is a major adverse prognostic factor in B-ALL, but the molecular mechanisms leading to GC resistance are not completely understood. Herein, we sought to elucidate the molecular background of GC resistance in B-ALL and characterize the therapeutic potential of targeted intervention in these mechanisms. Using exploratory bioinformatic approaches, we found that resistant cells exhibited significantly higher expression of MEK/ERK (MAPK) pathway components. We found that GC-resistant ALL cell lines had markedly higher baseline activity of MEK and small-molecule MEK1/2 inhibitor selumetinib increased GCs-induced cell death. MEK inhibitor similarly increased in vitro dexamethasone activity in primary ALL blasts from 19 of 22 tested patients. To further confirm these observations, we overexpressed a constitutively active MEK mutant in GC-sensitive cells and found that forced MEK activity induced resistance to dexamethasone. Since recent studies highlight the role GC-induced autophagy upstream of apoptotic cell death, we assessed LC3 processing, MDC staining and GFP-LC3 relocalization in cells incubated with either DEX, SEL or combination of drugs. Unlike either drug alone, only their combination markedly increased these markers of autophagy. These changes were associated with decreased mTOR activity and blocked 4E-BP1 phosphorylation. In cells with silenced beclin-1 (BCN1), required for autophagosome formation, the synergy of DEX and SEL was markedly reduced. Taken together, we show that MEK inhibitor selumetinib enhances dexamethasone toxicity in GC-resistant B-ALL cells. The underlying mechanism of this interaction involves inhibition of mTOR signaling pathway and modulation of autophagy markers, likely reflecting induction of this process and required for cell death. Thus, our data demonstrate that modulation of MEK/ERK pathway is an attractive therapeutic strategy overcoming GC resistance in B-ALL patients.

FOXO1 activation is an effector of SYK and AKT inhibition in tonic BCR signal-dependent diffuse large B-cell lymphomas.

Inhibition of spleen tyrosine kinase (SYK) in tonic B-cell receptor (BCR) signal-dependent diffuse large B-cell lymphomas (DLBCLs) inhibits cellular proliferation, decreases cholesterol biosynthesis, and triggers apoptosis, at least in part via a mechanism involving decreased activity of phosphatidylinositol 3-kinase/AKT axis. Because forkhead box O1 (FOXO1) is a major effector of this pathway, we investigated the role of FOXO1 in toxicity of BCR pathway inhibition. Inhibition of SYK in DLBCL cells with tonic BCR signaling decreased phospho-AKT and phospho-FOXO1 levels and triggered FOXO1-driven gene expression. Introduction of constitutively active FOXO1 mutant triggered cell cycle arrest and apoptosis, indicating that increased FOXO1 activity is toxic to these DLBCL cells. Depletion of FOXO1 with short hairpin RNA led to almost complete resistance to chemical SYK inhibitor R406, demonstrating that FOXO1 is also required for R406-induced cell death. FOXO1 in these cells is also involved in regulation of expression of the critical master regulator of cholesterol biosynthesis, SREBP1. Because HRK is the key effector of SYK inhibition, we characterized a mechanism linking FOXO1 activation and HRK induction that involves caspase-dependent cleavage of HRK's transcriptional repressor DREAM. Because AKT in lymphoma cells can be regulated by other signals than BCR, we assessed the combined effects of the AKT inhibitor MK-2206 with R406 and found markedly synergistic FOXO1-dependent toxicity. In primary DLBCLs, FOXO1 expression was present in 80% of tumors, correlated with SYK activity, and was associated with longer overall survival. These results demonstrate that FOXO1 is required for SYK and AKT inhibitor-induced toxicity.

Comparison of high-resolution melting analysis with direct sequencing for the detection of recurrent mutations in DNA methyltransferase 3A and isocitrate dehydrogenase 1 and 2 genes in acute myeloid leukemia patients.

Acute myeloid leukemia (AML) cells harbor frequent mutations in genes responsible for epigenetic modifications. Increasing evidence of clinical role of DNMT3A and IDH1/2 mutations highlights the need for a robust and inexpensive test to identify these mutations in routine diagnostic work-up. Herein, we compared routinely used direct sequencing method with high-resolution melting (HRM) assay for screening DNMT3A and IDH1/2 mutations in patients with AML. We show very high concordance between HRM and Sanger sequencing (100% samples for IDH2-R140 and DNMT3-R882 mutations, 99% samples for IDH1-R132 and IDH2-R172 mutations). HRM method reported no false-negative results, suggesting that it can be used for mutations screening. Moreover, HRM displayed much higher sensitivity in comparison with DNA sequencing in all assessed loci. With Sanger sequencing, robust calls were observed when the sample contained 50% of mutant DNA in the background of wild-type DNA. In marked contrast, the detection limit of HRM improved down to 10% of mutated DNA. Given the ubiquitous presence of wild-type DNA background in bone marrow aspirates and clonal variations regarding mutant allele burden, these results favor HRM as a sensitive, specific, labor-, and cost-effective tool for screening and detection of mutations in IDH1/2 and DNMT3A genes in patients with AML.

Polymorphism in IKZF1 gene affects age at onset of childhood acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer, characterized by a peak of incidence between 2 and 5 years. Since recently conducted genome-wide association (GWA) studies revealed that the common low-penetrance susceptibility allele at 7p12.2 (IKZF1 gene) confers an increased risk of pediatric ALL, we investigated whether the risk allele at rs4132601 also coexists with well-established prognostic factors, among 508 Polish pediatric patients with newly diagnosed ALL. Additionally, to verify whether the risk allele is favored by somatic tumor evolution, we examined the incidence of IKZF1 deletions in leukemic clones derived from 153 previously genotyped cases of pediatric ALL. Results of the analysis provide statistically significant support for an association between the rs4132601 polymorphic site and age at diagnosis of childhood ALL (p = 0.04). No association between allele variant and occurrence of IKZF1 deletions was found. These data provide further evidence of a biological role of gene variants in the development of ALL.

Asparagine synthetase (ASNS) gene polymorphism is associated with the outcome of childhood acute lymphoblastic leukemia by affecting early response to treatment.

The polymorphism of 14-bp tandem repeat sequence located in the ASNS gene probably acts as a transcriptional enhancer element and leads to higher expression of the gene in carriers of more than 2 repeats (>R2). We searched for an association with disease outcome in 264 children with ALL. A multivariate proportional hazard regression model adjusted for age at diagnosis (HR (95%CI)=1.05 (1.04-1.09)) and high-risk group (HR(95%CI)=3.47 (1.74-6.88)) revealed that R3 carriers with a poor response at day 15 had an increased risk of events, HR (95%CI)=2.72 (1.06-6.96). These results suggest a conditional interaction between the ASNS polymorphism and an early response to chemotherapy among pediatric patients with ALL.

Influence of genomic variation in FTO at 16q12.2, MC4R at 18q22 and NRXN3 at 14q31 genes on breast cancer risk.

Breast cancer is a major cause of cancer-related deaths in women. It is known that obesity is one of the risk factors of breast cancer. The subject of our interest was genes: FTO, MC4R and NRXN3-associated with obesity. In this study we have analyzed frequencies of genomic variants in FTO, MC4R and NRXN3 in the group of 134 breast cancer patients. We genotyped two polymorphic sites located in FTO gene (rs993909 and rs9930506), one polymorphic site of MC4R gene (rs17782313) and one polymorphic site of NRXN3 gene (rs10146997). Our hypothesis was that above mentioned SNPs could participate in carcinogenesis. Our research has showed that only rs10146997 was significantly (P = 0.0445) associated with higher risk of breast cancer development (OR = 0.66 (95% CI 0.44-0.99)). Moreover, G allele carriers in rs10146997 of the NRXN3 gene were the youngest patients at onset of breast cancer. On the basis of our research we suggest that further functional may elucidate the role of genomic variation in breast cancer development.

Role of 657del5 NBN mutation and 7p12.2 (IKZF1), 9p21 (CDKN2A), 10q21.2 (ARID5B) and 14q11.2 (CEBPE) variation and risk of childhood ALL in the Polish population.

Recent studies have shown that SNPs mapping to 7p12.2 (IKZF1), 9p21 (CDKN2A), 10q21.2 (ARID5B), and 14q11.2 (CEBPE) and carrier status for recessively inherited Nijmegen Breakage syndrome (NBS) influence childhood acute lymphoblastic leukemia (ALL) risk. To examine these relationship, we analysed 398 ALL cases and 731 controls from Poland. Statistically significant association between genotype at 7p12.2 (IKZF1), 10q21.2 (ARID5B) and the NBS associated locus, 8q21.3 (NBN) and ALL risk was found; odds ratios (ORs), 1.34 (P=0.002), 1.33 (P=0.003), and 1325.21 (P=0.0028), respectively. These data provide further insights into the biological basis of ALL highlighting the existence of both common and rare disease susceptibility variants.