Expression of Interferons Lambda 3 and 4 Induces Identical Response in Human Liver Cell Lines Depending Exclusively on Canonical Signaling.

Lambda interferons mediate antiviral immunity by inducing interferon-stimulated genes (ISGs) in epithelial tissues. A common variant rs368234815TT/∆G creating functional gene from an IFNL4 pseudogene is associated with the expression of major ISGs in the liver but impaired clearance of hepatitis C. To explain this, we compared Halo-tagged and non-tagged IFNL3 and IFNL4 signaling in liver-derived cell lines. Transfection with non-tagged IFNL3, non-tagged IFNL4 and Halo-tagged IFNL4 led to a similar degree of JAK-STAT activation and ISG induction; however, the response to transfection with Halo-tagged IFNL3 was lower and delayed. Transfection with non-tagged IFNL3 or IFNL4 induced no transcriptome change in the cells lacking either IL10R2 or IFNLR1 receptor subunits. Cytosolic overexpression of signal peptide-lacking IFNL3 or IFNL4 in wild type cells did not interfere with JAK-STAT signaling triggered by interferons in the medium. Finally, expression profile changes induced by transfection with non-tagged IFNL3 and IFNL4 were highly similar. These data do not support the hypothesis about IFNL4-specific non-canonical signaling and point out that functional studies conducted with tagged interferons should be interpreted with caution.

Cryptic aberrations may allow more accurate prognostic classification of patients with myelodysplastic syndromes and clonal evolution.

The karyotype of bone-marrow cells at the time of diagnosis is one of the most important prognostic factors in patients with myelodysplastic syndromes (MDS). In some cases, the acquisition of additional genetic aberrations (clonal evolution [CE]) associated with clinical progression may occur during the disease. We analyzed a cohort of 469 MDS patients using a combination of molecular cytogenomic methods to identify cryptic aberrations and to assess their potential role in CE. We confirmed CE in 36 (8%) patients. The analysis of bone-marrow samples with a combination of cytogenomic methods at diagnosis and after CE identified 214 chromosomal aberrations. The early genetic changes in the diagnostic samples were frequently MDS specific (17 MDS-specific/57 early changes). Most progression-related aberrations identified after CE were not MDS specific (131 non-MDS-specific/155 progression-related changes). Copy number neutral loss of heterozygosity (CN-LOH) was detected in 19% of patients. MDS-specific CN-LOH (4q, 17p) was identified in three patients, and probably pathogenic homozygous mutations were found in TET2 (4q24) and TP53 (17p13.1) genes. We observed a statistically significant difference in overall survival (OS) between the groups of patients divided according to their diagnostic cytogenomic findings, with worse OS in the group with complex karyotypes (P = .021). A combination of cytogenomic methods allowed us to detect many cryptic genomic changes and identify genes and genomic regions that may represent therapeutic targets in patients with progressive MDS.

Implication of cytogenetic and molecular cytogenetic analysis in diagnosis of hematological malignancies in the era of the new sequencing techniques.

In patients with hematological malignancies one of the most substantial findings is the karyotype of bone marrow cells at the time of diagnosis. The detection of clonal chromosome aberrations in diagnostic samples not only confirms a neoplastic or premalignant process but also provides important diagnostic and prognostic information essential for precise disease classification and choice of suitable therapy. Karyotype analysis during the disease course also allows monitoring of the treatment success reflected as well in the revised WHO classification where patients are often classified into the different diagnostic subtypes based on the finding of specific chromosome and/or genetic changes. Recently, also increases the number of advanced treatment approaches that directly or indirectly target the genetic aberrations present in tumor cells. Despite the large development of new sequencing technologies in recent years, cytogenetic analysis supplemented by the molecular cytogenetic methods still remains a very important part of diagnostics of hematological malignancies.

Lenalidomide treatment in lower risk myelodysplastic syndromes-The experience of a Czech hematology center. (Positive effect of erythropoietin ± prednisone addition to lenalidomide in refractory or relapsed patients).

Lenalidomide therapy represents meaningful progress in the treatment of anemic patients with myelodysplastic syndromes with del(5q). We present our initial lenalidomide experience and the positive effect of combining erythropoietin and steroids with lenalidomide in refractory and relapsed patients. We treated by lenalidomide 55 (42 female; 13 male; median age 69) chronically transfused lower risk MDS patients with del(5q) (45) and non-del(5q) (10). Response, meaning transfusion independence (TI) lasting ≥ eight weeks, was achieved in 38 (90%) of analyzed patients with del(5q), of whom three achieved TI only by adding erythropoietin ±â€¯prednisone. Another five patients responded well to this combination when their anemia relapsed later during the treatment. In the non-del(5q) group only one patient with RARS-T reached TI. Cytogenetic response was reached in 64% (32% complete, 32% partial response). The TP53 mutation was detected in 7 (18%) patients; four patients progressed to higher grade MDS or acute myeloid leukemia (AML). All seven RAEB-1 patients cleared bone marrow blasts during lenalidomide treatment and reached complete remission (CR); however, three later progressed to higher grade MDS or AML. Lenalidomide represents effective treatment for del(5q) group and combination with prednisone and erythropoietin may be used for non-responders or therapy failures.

High frequency of dicentric chromosomes detected by multi-centromeric FISH in patients with acute myeloid leukemia and complex karyotype.

Dicentric chromosomes (DCs) are considered markers of cancer in various malignancies. However, they can be overlooked when conventional analysis or multicolor fluorescence in situ hybridization (mFISH) is used to detect complex karyotypes. We analyzed the karyotypes of 114 patients with acute myeloid leukemia (AML) and complex karyotypes and verified the presence of monosomies by FISH using multi-centromeric probes. Monosomy was detected in 63% of patients by G-banding/mFISH and confirmed in 55% of patients by centromeric FISH. FISH analysis indicated a high frequency of DCs that were previously considered monosomies. In some cases, it was apparent that the derivative monocentric chromosome was a primary DC. DCs were formed mostly by chromosomes 17 and 20. In conclusion, chromosome loss and unbalanced translocation suggest the presence of a hidden DC or its previous existence. DCs undergo several stabilizing changes and can induce other chromosomal aberrations and/or the formation of new DCs. This can result in the clonal evolution of abnormal cells, which is considered an independent prognostic marker of an unfavorable disease course and short survival.

Alternating R-CHOP and R-cytarabine is a safe and effective regimen for transplant-ineligible patients with a newly diagnosed mantle cell lymphoma.

Implementation of cytarabine into induction therapy became standard of care for younger patients with mantle cell lymphoma (MCL). On the basis of its beneficial impact, many centers incorporated cytarabine at lower doses also into first-line treatments of elderly patients. We conducted a multicenter observational study that prospectively analyzed safety and efficacy of alternating 3 + 3 cycles of R-CHOP and R-cytarabine for newly diagnosed transplant-ineligible MCL patients. A total of 73 patients were enrolled with median age 70 years. Most patients had intermediate (39.7%) and high-risk (50.7%) disease according to MCL international prognostic index. Rituximab maintenance was initiated in 58 patients. Overall response rate reached 89% by positron emission tomography-computed tomography, including 75.3% complete remissions. Two patients (2.7%) did not complete the induction therapy because of toxicity. Three patients (4.1%) were considered nonresponders, which led to therapy change before completion of induction. Estimated progression-free survival and overall survival were 51.3% and 68.6% at 4 years, respectively. Mantle cell lymphoma international prognostic index, bulky disease (≥ 5 cm), and achievement of positron emission tomography-negativity independently correlated with progression-free survival. Grade 3 to 4 hematologic and nonhematologic toxicity was documented in 48% and 20.5% patients, respectively. Alternation of R-CHOP and R-cytarabine represents feasible and very effective regimen for elderly/comorbid MCL patients. This study was registered at GovTrial (clinicaltrials.gov) NCT03054883.

Rare congenital chromosomal aberration dic(X;Y)(p22.33;p11.32) in a patient with primary myelofibrosis.

BACKGROUND: Constitutional translocations between sex chromosomes are rather rare in humans with breakpoints at Xp11 and Yq11 as the most frequent. Breakpoints on the short arm of the Y chromosome form one subgroup of t(X;Y), giving rise to a derived chromosome with the centromeres of both the X and Y chromosomes, dic(X;Y). Here, we report a rare congenital chromosomal aberration, 46,X,dic(X;Y)(p22.33;p11.32)[20]/45,X[10], in an adult male. CASE PRESENTATION: Primary myelofibrosis, a malignant haematological disease, was diagnosed in a 63-year-old man following liver transplantation after hepatocellular carcinoma. By the analysis of the bone marrow sample, the karyotype 46,X,dic(X;Y)(p22.33;p11.32) was detected in all the mitoses analysed and verified with multicolour fluorescence in situ hybridization (mFISH). A cytogenetic examination of stimulated peripheral blood cells revealed the constitutional karyotype 46,X,dic(X;Y)(p22.33;p11.32)[20]/45,X[10]. The cell line 45,X was confirmed with FISH in 35 % of interphase nuclei. The SRY locus was present on the dicentric chromosome. A CGH/SNP array (Illumina) revealed a gain of 153,7 Mbp of the X chromosome and a 803-kbp microdeletion (including the SHOX gene), which were also confirmed with FISH. SHOX encodes a transcriptional factor that regulates the growth of the long bones. The deletion of the SHOX gene together with the Madelung deformity of the forearm and the short stature of the proband led to a diagnosis of Léri-Weill dyschondrosteosis (LWD). The gain of almost the whole X chromosome (153,7 Mbp) was considered a variant of Klinefelter syndrome (KS). The levels of gonadotropins and testosterone were consistent with gonadal dysfunction. A malformation of the right external ear was detected. CONCLUSIONS: We have reported a structural aberration of the sex chromosomes, dic(X;Y)(p22.33;p11.32). The related genomic imbalance is associated with two known hereditary syndromes, LWD and a KS variant, identified in our proband at an advanced age. Because the breakpoints did not involve cancer genes, we inferred that the two malignancies in the proband were not caused by this abnormality. The possible influence of SHOX haploinsufficiency on the growth regulation of auricular chondrocytes is discussed.

Mantle cell lymphoma-variant Richter syndrome: Detailed molecular-cytogenetic and backtracking analysis reveals slow evolution of a pre-MCL clone in parallel with CLL over several years.

Richter syndrome represents the transformation of the chronic lymphocytic leukemia (CLL) into an aggressive lymphoma, most frequently the diffuse large B-cell lymphoma (DLBCL). In this report we describe a patient with CLL, who developed a clonally-related pleomorphic highly-aggressive mantle cell lymphoma (MCL) after five cycles of a fludarabine-based second-line therapy for the first relapse of CLL. Molecular cytogenetic methods together with whole-exome sequencing revealed numerous gene alterations restricted to the MCL clone (apart from the canonical t(11;14)(q13;q32) translocation) including gain of one copy of ATM gene or emergence of TP53, CREBBP, NUP214, FUBP1 and SF3B1 gene mutations. Similarly, gene expression analysis revealed vast differences between the MCL and CLL transcriptome, including overexpression of cyclin D1, downregulation of cyclins D2 and D3, or downregulation of IL4R in the MCL clone. Backtracking analysis using quantitative PCR specifically detecting an MCL-restricted focal deletion of TP53 revealed that the pre-MCL clone appeared in the bone marrow and peripheral blood of the patient approximately 4 years before the clinical manifestation of MCL. Both molecular cytogenetic and sequencing data support the hypothesis of a slow development of the pre-MCL clone in parallel to CLL over several years, and thereby exclude the possibility that the transformation event occurred at the stage of the CLL relapse clone by mere t(11;14)(q13;q32) acquisition.

TP53 mutation variant allele frequency is a potential predictor for clinical outcome of patients with lower-risk myelodysplastic syndromes.

TP53 mutations are frequently detected in patients with higher-risk myelodysplastic syndromes (MDS); however, the clinical impact of these mutations on the disease course of patients with lower-risk MDS is unclear. In this study of 154 lower-risk MDS patients, TP53 mutations were identified in 13% of patients, with prevalence in patients with del(5q) (23.6%) compared to non-del(5q) (3.8%). Two-thirds of the mutations were detected at the time of diagnosis, and one-third were detected during the course of the disease. Multivariate analysis demonstrated that a TP53 mutation was the strongest independent prognostic factor for overall survival (OS) (HR: 4.39) and progression-free survival (PFS) (HR: 3.74). Evaluation of OS determined a TP53 variant allele frequency (VAF) threshold of 6% as an optimal cut-off for patient stratification. The median OS was 43.5 months in patients with mutations detected at the time of diagnosis and a mutational burden of > 6% VAF compared to 138 months (HR 12.2; p = 0.003) in patients without mutations; similarly, the median PFS was 20.2 months versus 116.6 months (HR 79.5; p < 0.0001). In contrast, patients with a mutational burden of < 6% VAF were stable for long periods without progression and had no significant impact on PFS or OS. Additionally, we found a high correlation in the mutational data from cells of the peripheral blood and those of the bone marrow, indicating that peripheral blood is a reliable source for mutation monitoring. Our results indicate that the clinical impact of TP53 mutations in lower-risk MDS patients depends on the level of mutational burden.

Copy number neutral loss of heterozygosity at 17p and homozygous mutations of TP53 are associated with complex chromosomal aberrations in patients newly diagnosed with myelodysplastic syndromes.

Complex karyotypes are seen in approximately 20% of patients with myelodysplastic syndromes (MDS) and are associated with a high risk of transformation to acute myeloid leukemia and poor outcomes in patients. Copy number neutral loss of heterozygosity (CN-LOH, i.e., both copies of a chromosomal pair or their parts originate from one parent) might contribute to increased genomic instability in the bone-marrow cells of patients with MDS. The pathological potential of CN-LOH, which arises as a clonal aberration in a proportion of somatic cells, consists of tumor suppressor gene and oncogene homozygous mutations. The aim of our study was to evaluate the frequency of CN-LOH at 17p in bone-marrow cells of newly diagnosed MDS patients with complex chromosomal aberrations and to assess its correlation with mutations in the TP53 gene (17p13.1). CN-LOH was detected in 40 chromosomal regions in 21 (29%) of 72 patients analyzed. The changes in 27 of the 40 regions identified were sporadic. The most common finding was CN-LOH of the short arm of chromosome 17, which was detected in 13 (18%) of 72 patients. A mutational analysis confirmed the homozygous mutation of TP53 in all CN-LOH 17p patients, among which two frameshift mutations are not registered in the International Agency for Research on Cancer TP53 Database. CN-LOH 17p correlated with aggressive disease (median overall survival 4 months) and was strongly associated with a complex karyotype in the cohort studied, which might cause rapid disease progression in high-risk MDS. No other CN-LOH region previously recorded in MDS or AML patients (1p, 4q, 7q, 11q, 13q, 19q, 21q) was detected in our cohort of patients with complex karyotype examined at the diagnosis of MDS. The LOH region appeared to be balanced (i.e., with no DNA copy number change) when examined with conventional and molecular cytogenetic methods. Therefore, a microarray that detects single-nucleotide polymorphisms is an ideal method with which to identify and further characterize CN-LOH. Our data should specify the prognosis and should lead to the identification of potential targets for therapeutic interventions.

Primary and recurrent diffuse astrocytomas: genomic profile comparison reveals acquisition of biologically relevant aberrations.

BACKGROUND: Diffuse astrocytomas are characterized by their highly variable biological behavior. The possibility that tumors develop novel aberrations, with relevant biological properties, is often neglected. In this study, we present two cases of diffuse astrocytoma in which additional cytogenetic and epigenetic markers with potential influence on cell proliferation or differentiation were detected at relapse. FINDINGS: The biopsies taken from the primary and recurrent tumors of two patients were analyzed with molecular methods to detect copy number variations (CNVs), gene mutations and epigenetic changes. Both cases were characterized by the R132H mutation in the isocitrate dehydrogenase 1 (IDH1) gene. Features typical of astrocytomas, such as copy-neutral loss of heterozygosity at 17p and the deletion of the cyclin-dependent kinase inhibitor 2A (CDKN2A) gene, were also detected in both cases. These markers were present in the primary and recurrent lesions. Other aberrations, predominantly deletions or amplifications of chromosomal segments and the hypermethylation of gene promoters, were detected in the recurrent lesions. CONCLUSIONS: The IDH1 mutation was the primary event, as previously reported. According to our observations, the methylation of promoters constituted later events, which may have further disrupted cell proliferation and/or differentiation, together with additional CNVs.

Molecular cytogenetic analysis of dicentric chromosomes in acute myeloid leukemia.

Dicentric chromosomes (DCs) have been described in many hematological diseases, including acute myeloid leukemia (AML). They are markers of cancer and induce chromosomal instability, leading to the formation of other chromosomal aberrations and the clonal evolution of pathological cells. Our knowledge of the roles and behavior of human DCs is often derived from studies of induced DCs and cell lines. It is difficult to identify all the DCs in the karyotypes of patients because of the limitations of metaphase cytogenetic methods. The aim of this study was to revise the karyotypes of 20 AML patients in whom DCs were found with conventional G-banding or multicolor fluorescence in situ hybridization (mFISH) with (multi)centromeric probes and to characterize the DCs at the molecular cytogenetic level. FISH analyses confirmed 23 of the 29 expected DCs in 18 of 20 patients and identified 13 others that had not been detected cytogenetically. Fourteen DCs were altered by other chromosomal changes. In conclusion, karyotypes with DCs are usually very complex, and we have shown that they often contain more than one DC, which can be missed with conventional or mFISH methods. Our study indicates an association between number of DCs in karyotype and very short survival of patients.

Fanconi anemia with biallelic FANCD1/BRCA2 mutations - Case report of a family with three affected children.

Fanconi anemia, complementation group D1 with bi-allelic FANCD1 (BRCA2) mutations, is a very rare genetic disorder characterized by early onset of childhood malignancies, including acute leukemia, brain cancer and nephroblastoma. Here, we present a case report of a family with 3 affected children in terms of treatment outcome, toxicity and characterization of the malignancies using comprehensive cytogenetic analysis. The first child was diagnosed with T-cell acute lymphoblastic leukemia when he was 11 months old. During chemotherapy, he suffered from repeated pancytopenia, sepsis and severe vincristine polyneuropathy, and 18 months after primary diagnosis, he succumbed to secondary acute monocytic leukemia. The second child was diagnosed with stage 2 triphasic nephroblastoma (Wilms tumor), when he was 3 years and 11 months old. During chemotherapy, he suffered from vincristine polyneuropathy. Currently, he is in complete remission, 29 months following the initial diagnosis. The third child was diagnosed with medulloblastoma with classical histology, when she was 4 years and 5 months old. After the first cycle of chemotherapy, she suffered from prolonged pancytopenia, sepsis and severe skin and mucosal toxicity. Six weeks after primary diagnosis, a first relapse in the posterior fossa was diagnosed, and at 7 and half months after primary diagnosis, a second relapse was diagnosed that led to the patient's death. Our case report underscores tumor heterogeneity, treatment toxicity and poor outcome in Fanconi anemia patients of complementation group D1.

Genetic and epigenetic characterization of low-grade gliomas reveals frequent methylation of the MLH3 gene.

Diffuse astrocytomas and oligodendrogliomas (WHO grade II) are the most common histological subtypes of low-grade gliomas (LGGs). Several molecular and epigenetic markers have been identified that predict tumor progression. Our aim was in detail to investigate the genetic and epigenetic background of LGGs and to identify new markers that might play a role in tumor behavior. Twenty-three patients with oligodendroglioma or oligoastrocytoma (LGO) and 22 patients with diffuse astrocytoma (LGA) were investigated using several molecular-cytogenetic and molecular methods to assess their copy number variations, mutational status and level of promoter methylation. The most frequent findings were a 1p/19q codeletion in 83% of LGO and copy-neutral loss of heterozygosity (CN-LOH) of 17p in 72% of LGA. Somatic mutations in the isocitrate dehydrogenase 1 or 2 (IDH1/IDH2) genes were detected in 96% of LGO and 91% of LGA. The O-6-methylguanine-DNA-methyltransferase (MGMT) promoter was methylated in 83% of LGO and 59% of LGA. MutL homolog 3 (MLH3) promoter methylation was observed in 61% of LGO and 27% of LGA. Methylation of the MGMT promoter, 1p/19q codeletion, mutated IDH1, and CN-LOH of 17p were the most frequent genetic aberrations in LGGs. The findings were more diverse in LGA than in LGO. To the best of our knowledge, this is the first time description of methylation of the MLH3 gene promoter in LGGs. Further studies are required to determine the role of the methylated MLH3 promoter and the other aberrations detected.

Aberrant expression of the microRNA cluster in 14q32 is associated with del(5q) myelodysplastic syndrome and lenalidomide treatment.

Lenalidomide is a novel thalidomide analogue with immunomodulatory and antiangiogenic effects that has been successfully used for the treatment of low and intermediate-1 risk myelodysplastic syndromes (MDSs) with a del(5q) aberration. Because information about the influence of lenalidomide on the microRNA (miRNA) transcriptome is limited, we performed miRNA expression profiling of bone marrow CD34+ cells obtained from MDS patients with the del(5q) abnormality who had been subjected to lenalidomide treatment. To define differences in miRNA expression, we performed paired data analysis to compare the miRNA profiles of patients before and during lenalidomide treatment and those of healthy donors. The analysis showed that miRNAs clustering to the 14q32 region had a higher expression level in patient samples before treatment than in the healthy control samples, and this elevated expression was diminished following lenalidomide administration. Because some of the 14q32 miRNAs play important roles in hematopoiesis, stem cell differentiation, and apoptosis induction, the expression of this cluster may be associated with the pathophysiology of the disease.

Characterization of a new human plasma cell leukemia cell line UHKT-944.

OBJECTIVE: A new interleukin-6 (IL-6)-dependent plasma cell leukemia cell line UHKT-944 was established from bone marrow cells derived from a 55-yr-old man with plasma cell leukemia. RESULTS: The cell line possesses phenotypic characteristics of plasma cells including the production of a monoclonal immunoglobulin IgA1-kappa. VH3-9 region of IgVH genes was rearranged and somatically hypermutated. The UHKT-944 cells were found to be negative for most of tested B-cell, T-cell, and myeloid markers. According to cytogenetic analysis, the cells were classified as near tetraploid with several numerical and structural abnormalities including the t(14;20) involving IgH locus. CONCLUSION: The established permanent plasma cell leukemia cell line is a suitable model for the study of cellular and molecular mechanisms of pathogenesis of this rare malignant disease.

High level of full-length cereblon mRNA in lower risk myelodysplastic syndrome with isolated 5q deletion is implicated in the efficacy of lenalidomide.

Downregulation of cereblon (CRBN) gene expression is associated with resistance to the immunomodulatory drug lenalidomide and poor survival outcomes in multiple myeloma (MM) patients. However, the importance of CRBN gene expression in patients with myelodysplastic syndrome (MDS) and its impact on lenalidomide therapy are not clear. In this study, we evaluate cereblon expression in mononuclear cells isolated from bone marrow [23 lower risk MDS patients with isolated 5q deletion (5q-), 37 lower risk MDS patients with chromosome 5 without the deletion of long arms (non-5q-), and 24 healthy controls] and from peripheral blood (38 patients with 5q-, 52 non-5q- patients and 25 healthy controls) to gain insight into, firstly, the role of cereblon in lower risk MDS patients with or without 5q deletion and, secondly, into the mechanisms of lenalidomide action. Patients with 5q- lower risk MDS have the highest levels of CRBN mRNA in comparison with both lower risk MDS without the deletion of long arms of chromosome 5 and healthy controls. CRBN gene expression was measured using the quantitative TaqMan real-time PCR. High levels of CRBN mRNA were detected in all lenalidomide responders during the course of therapy. A significant decrease of the CRBN mRNA level during lenalidomide treatment is associated with loss of response to treatment and disease progression. These results suggest that, similar to the treatment of MM, high levels of full-length CRBN mRNA in lower risk 5q- patients are necessary for the efficacy of lenalidomide.

Genome-wide miRNA profiling in myelodysplastic syndrome with del(5q) treated with lenalidomide.

OBJECTIVES: Lenalidomide is a potent drug with pleiotropic effects in patients with myelodysplastic syndrome (MDS) with deletion of the long arm of chromosome 5 [del(5q)]. We investigated its effect on regulation of microRNA (miRNA) expression profiles in del(5q) patients with MDS in vivo. METHODS: We used miRNA expression microarrays to study changes in miRNA levels in peripheral blood CD14+ monocytes collected from patients before and during lenalidomide treatment and compared them with those from healthy donors. RESULTS: Before treatment, we observed strong upregulation of pro-apoptotic miR-34a and miR-34a* that diminished during lenalidomide exposure. Upregulation of HOX-related miR-196b and erythroid-specific miR-451 seen in untreated patients remained unchanged after the treatment. At the time of hematologic response, expression of several miRNAs clustering to the 14q32 locus was reduced. Additionally, we focused more deeply on miRNAs from the 5q commonly deleted region and found that levels of miR-378 and miR-378* followed haploinsufficiency trend. CONCLUSIONS: This report describes changes in miRNA expression in del(5q) patients with MDS treated with lenalidomide, likely arising from deregulation of pathways implicated in lenalidomide action.

An unusual case of high hyperdiploid childhood ALL with cryptic BCR/ABL1 rearrangement.

BACKGROUND: Both high hyperdiploidy (HeH) and the translocation t(9;22)(q34;q11) are recurrent abnormalities in childhood B-cell acute lymphoblastic leukemia (ALL) and both are used in current classification to define different genetic and prognostic subtypes of the disease. The coexistence of these two primary genetic aberrations within the same clone is very rare in children with ALL. Here we report a new case of a 17-year-old girl with newly diagnosed ALL and uncommon cytogenetic and clinical finding combining high hyperdiploidy and a cryptic BCR/ABL1 fusion and an inherited Charcot-Marie-Tooth neuropathy detected during the induction treatment. RESULTS: High hyperdiploid karyotype 51,XX,+X,+4,+14,+17,+21 without apparent structural aberrations was detected by conventional cytogenetic analysis and multicolor FISH. A cryptic BCR/ABL1 fusion, which was caused by the insertion of part of the ABL1 gene into the 22q11 region, was proved in HeH clone by FISH, RT-PCR and CGH-SNP array. In addition, an abnormal FISH pattern previously described as the deletion of the 3'BCR region in some BCR/ABL1 positive cases was not proved in our patient. CONCLUSION: A novel case of extremely rare childhood ALL, characterized by HeH and a cryptic BCR/ABL1 fusion, is presented and to the best of our knowledge described for the first time. The insertion of ABL1 into the BCR region in malignant cells is supposed. Clearly, further studies are needed to determine the genetic consequences and prognostic implications of these unusual cases.

CBFB-MYH11 hypomethylation signature and PBX3 differential methylation revealed by targeted bisulfite sequencing in patients with acute myeloid leukemia.

BACKGROUND: Studying DNA methylation changes in the context of structural rearrangements and point mutations as well as gene expression changes enables the identification of genes that are important for disease onset and progression in different subtypes of acute myeloid leukemia (AML) patients. The aim of this study was to identify differentially methylated genes with potential impact on AML pathogenesis based on the correlation of methylation and expression data. METHODS: The primary method of studying DNA methylation changes was targeted bisulfite sequencing capturing approximately 84 megabases (Mb) of the genome in 14 diagnostic AML patients and a healthy donors' CD34+ pool. Subsequently, selected DNA methylation changes were confirmed by 454 bisulfite pyrosequencing in a larger cohort of samples. Furthermore, we addressed gene expression by microarray profiling and correlated methylation of regions adjacent to transcription start sites with expression of corresponding genes. RESULTS: Here, we report a novel hypomethylation pattern, specific to CBFB-MYH11 fusion resulting from inv(16) rearrangement that is associated with genes previously described as upregulated in inv(16) AML. We assume that this hypomethylation and corresponding overexpresion occurs in the genes whose function is important in inv(16) leukemogenesis. Further, by comparing all targeted methylation and microarray expression data, PBX3 differential methylation was found to correlate with its gene expression. PBX3 has been recently shown to be a key interaction partner of HOX genes during leukemogenesis and we revealed higher incidence of relapses in PBX3-overexpressing patients. CONCLUSIONS: We discovered new genomic regions with aberrant DNA methylation that are associated with expression of genes involved in leukemogenesis. Our results demonstrate the potential of the targeted approach for DNA methylation studies to reveal new regulatory regions.