Search

Purpose:Mantle cell lymphoma (MCL) is an aggressive subtype of B-cell non-Hodgkin lymphomas characterized by (over)expression of BCL2. A BCL2-targeting drug, venetoclax, has promising anticancer activity in MCL. We analyzed molecular mechanisms of venetoclax resistance in MCL cells and tested strategies to overcome it.Experimental Design:We confirmed key roles of proapoptotic proteins BIM and NOXA in mediating venetoclax-induced cell death in MCL. Both BIM and NOXA are, however, differentially expressed in cell lines compared with primary cells. First, NOXA protein is significantly overexpressed in most MCL cell lines. Second, deletions of BIM gene harbored by three commonly used MCL cell lines (JEKO-1, MINO, and Z138) were not found by array comparative genomic hybridization using a validation set of 24 primary MCL samples.Results:We demonstrated that MCL1 and NOXA play important roles in mediating resistance to venetoclax. Consequently, we tested an experimental treatment strategy based on cotargeting BCL2 with venetoclax and MCL1 with a highly specific small-molecule MCL1 inhibitor S63845. The combination of venetoclax and S63845 demonstrated synthetic lethality in vivo on a panel of five patient-derived xenografts established from patients with relapsed MCL with adverse cytogenetics.Conclusions:Our data strongly support investigation of venetoclax in combination with S63845 as an innovative treatment strategy for chemoresistant MCL patients with adverse cytogenetics in the clinical grounds.

Supplemental Figure 1. Next-generation sequencing of PDXs and primary MCL cells Supplemental Figure 2. IHC analysis of primary MCL samples and murine xenografts Supplemental Figure 3. Array comparative genomic hybridization of 24 primary MCL samples Supplemental Figure 4. Sensitivity of HBL2 and MAVER-1 cells resistant to venetoclax to BCL-XL inhibitors WEHI-539 and A1155463 Supplemental Table 1. Complete list of protein coding variants Supplemental Table 2. FISH analyses of the established murine PDXs of MCL Supplemental Table 3. Baseline characteristics of patients Supplemental Table 4. IHC analysis of MCL samples and xenografts Supplemental Table 5. Tumor and spleen weights at the end of in vivo experiments

BACKGROUND Increased expression of fibroblast activation protein (FAP) is characteristic for several cancer types including human glioblastomas (GBM). FAP is expressed on both cancer as well as stromal cells which were demonstrated in extracranial tumors to maintain a microenvironment permissive for tumor growth and thus to contribute to tumor progression. FAP is considered a potential diagnostic and therapeutic target and several approaches for its targeting have been recently developed. In this work, we investigated the role of FAP+ stromal cells in glioblastoma angiogenesis. MATERIAL AND METHODS Expression of FAP and other phenotypic markers was assessed by immunocytochemistry and immunohistochemistry. FAP+ stromal cells and primary microvascular endothelial cells were isolated using magnetic activated cell sorting (MACS). Genetic aberrations were assayed by comparative genomic hybridization/single-nucleotide polymorphism analysis. Angiogenesis was evaluated using a 3D spheroid-based sprouting assay and a chorioallantoic membrane assay. A cytokine array was used to analyze soluble mediators released by FAP+ stromal cells. RESULTS FAP+ stromal cells were predominantly localized around activated CD105+ endothelial cells and their quantity positively correlated with glioblastoma vascularization. FAP+ stromal cells derived from human GBMs had a mesenchymal phenotype, were non-tumorigenic and in most cases lacked cytogenetic aberrations characteristic of GBMs. Conditioned media derived from FAP+ stromal cells induced angiogenic sprouting of both macrovascular HUVEC as well as microvascular primary endothelial cells derived from human GBMs. In a chorioallantoic membrane assay, admixture of FAP+ stromal cells to glioma cells was associated with increased angiogenesis and more frequent occurrence of hemorrhages. Cytokine array revealed a significant disbalance between several proangiogenic and antiangiogenic mediators compared to normal pericytes, and an increased Angiopoietin 2/1 ratio in conditioned media from FAP+ stromal cells. CONCLUSION Our results bring new evidence that GBM associated FAP+ stromal cell promote angiogenesis by changing the balance between proangiogenic and antiangiogenic mediators and thus they may contribute to glioblastoma progression. ACKNOWLEDGEMENT Supported by Progres Q28/1LFUK and grant LM2015064 of the EATRIS-CZ and the Center for Tumor Ecology (CZ.02.1.01/0.0/0.0/16_019/0000785).

Purpose: Mantle cell lymphoma (MCL) is an aggressive subtype of B-cell non-Hodgkin lymphomas characterized by (over)expression of BCL2. A BCL2-targeting drug, venetoclax, has promising anticancer activity in MCL. We analyzed molecular mechanisms of venetoclax resistance in MCL cells and tested strategies to overcome it. Experimental Design: We confirmed key roles of proapoptotic proteins BIM and NOXA in mediating venetoclax-induced cell death in MCL. Both BIM and NOXA are, however, differentially expressed in cell lines compared with primary cells. First, NOXA protein is significantly overexpressed in most MCL cell lines. Second, deletions of BIM gene harbored by three commonly used MCL cell lines (JEKO-1, MINO, and Z138) were not found by array comparative genomic hybridization using a validation set of 24 primary MCL samples. Results: We demonstrated that MCL1 and NOXA play important roles in mediating resistance to venetoclax. Consequently, we tested an experimental treatment strategy based on cotargeting BCL2 with venetoclax and MCL1 with a highly specific small-molecule MCL1 inhibitor S63845. The combination of venetoclax and S63845 demonstrated synthetic lethality in vivo on a panel of five patient-derived xenografts established from patients with relapsed MCL with adverse cytogenetics. Conclusions: Our data strongly support investigation of venetoclax in combination with S63845 as an innovative treatment strategy for chemoresistant MCL patients with adverse cytogenetics in the clinical grounds.

Mantle cell lymphoma (MCL) is a subtype of B-cell lymphoma with a large number of recurrent cytogenetic/molecular aberrations. Approximately 5&ndash, 10% of patients do not respond to frontline immunochemotherapy. Despite many useful prognostic indexes, a reliable marker of chemoresistance is not available. We evaluated the prognostic impact of seven recurrent gene aberrations including tumor suppressor protein P53 (TP53) and cyclin dependent kinase inhibitor 2A (CDKN2A) in the cohort of 126 newly diagnosed consecutive MCL patients with bone marrow involvement &ge, 5% using fluorescent in-situ hybridization (FISH) and next-generation sequencing (NGS). In contrast to TP53, no pathologic mutations of CDKN2A were detected by NGS. CDKN2A deletions were found exclusively in the context of other gene aberrations suggesting it represents a later event (after translocation t(11, 14) and aberrations of TP53, or ataxia telangiectasia mutated (ATM)). Concurrent deletion of CDKN2A and aberration of TP53 (deletion and/or mutation) represented the most significant predictor of short EFS (median 3 months) and OS (median 10 months). Concurrent aberration of TP53 and CDKN2A is a new, simple, and relevant index of chemoresistance in MCL. Patients with concurrent aberration of TP53 and CDKN2A should be offered innovative anti-lymphoma therapy and upfront consolidation with allogeneic stem cell transplantation.

Mantle cell lymphoma (MCL) is a subtype of B-cell lymphoma with a large number of recurrent cytogenetic/molecular aberrations. Approximately 5–10% of patients do not respond to frontline immunochemotherapy. Despite many useful prognostic indexes, a reliable marker of chemoresistance is not available. We evaluated the prognostic impact of seven recurrent gene aberrations including tumor suppressor protein P53 (TP53) and cyclin dependent kinase inhibitor 2A (CDKN2A) in the cohort of 126 newly diagnosed consecutive MCL patients with bone marrow involvement ≥5% using fluorescent in-situ hybridization (FISH) and next-generation sequencing (NGS). In contrast to TP53, no pathologic mutations of CDKN2A were detected by NGS. CDKN2A deletions were found exclusively in the context of other gene aberrations suggesting it represents a later event (after translocation t(11;14) and aberrations of TP53, or ataxia telangiectasia mutated (ATM)). Concurrent deletion of CDKN2A and aberration of TP53 (deletion and/or mutation) represented the most significant predictor of short EFS (median 3 months) and OS (median 10 months). Concurrent aberration of TP53 and CDKN2A is a new, simple, and relevant index of chemoresistance in MCL. Patients with concurrent aberration of TP53 and CDKN2A should be offered innovative anti-lymphoma therapy and upfront consolidation with allogeneic stem cell transplantation.

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.

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.

BACKGROUND: To obtain better insight into the biology of acute myeloid leukemia (AML) in various age groups, this study focused on the genetic changes occurring during a lifetime. METHODS: This study analyzed the relation between age and genetics from birth to 100 years in 5564 patients with de novo AML diagnosed from 1998 to 2012 (1192 patients from nationwide pediatric studies [AML Berlin-Frankfurt-Munster studies 98 and 2004] and 4372 adults registered with the Munich Leukemia Laboratory). RESULTS: The frequencies of cytogenetic subgroups were age-dependent. Favorable subtypes (t(8;21), inv(16)/t(16;16), and t(15;17)) decreased in general from the pediatric age group (2 to 70 years; P

Glioblastomas are deadly neoplasms resistant to current treatment modalities. Fibroblast activation protein (FAP) is a protease which is not expressed in most of the normal adult tissues but is characteristically present in the stroma of extracranial malignancies. FAP is considered a potential therapeutic target and is associated with a worse patient outcome in some cancers. The FAP localization in the glioma microenvironment and its relation to patient survival are unknown. By analyzing 56 gliomas and 15 non-tumorous brain samples, we demonstrate increased FAP expression in a subgroup of high-grade gliomas, in particular on the protein level. FAP expression was most elevated in the mesenchymal subtype of glioblastoma. It was neither associated with glioblastoma patient survival in our patient cohort nor in publicly available datasets. FAP was expressed in both transformed and stromal cells; the latter were frequently localized around dysplastic blood vessels and commonly expressed mesenchymal markers. In a mouse xenotransplantation model, FAP was expressed in glioma cells in a subgroup of tumors that typically did not express the astrocytic marker GFAP. Endogenous FAP was frequently upregulated and part of the FAP+ host cells coexpressed the CXCR4 chemokine receptor. In summary, FAP is expressed by several constituents of the glioblastoma microenvironment, including stromal non-malignant mesenchymal cells recruited to and/or activated in response to glioma growth. The limited expression of FAP in healthy tissues together with its presence in both transformed and stromal cells suggests that FAP may be a candidate target for specific delivery of therapeutic agents in glioblastoma.

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.

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.

Data de publicació electrónica: 29-01-2019 Cytogenomic investigations of haematological neoplasms, including chromosome banding analysis, fluorescence in situ hybridisation (FISH) and microarray analyses have become increasingly important in the clinical management of patients with haematological neoplasms. The widespread implementation of these techniques in genetic diagnostics has highlighted the need for guidance on the essential criteria to follow when providing cytogenomic testing, regardless of choice of methodology. These recommendations provide an updated, practical and easily available document that will assist laboratories in the choice of testing and methodology enabling them to operate within acceptable standards and maintain a quality service.

Deletion 20q is a recurrent abnormality in myeloid malignancies. In our previous study, we identified fusion of the additional sex combs-like 1 (ASXL1) and teashirt zinc finger homeobox 2 genes in a patient with myelodysplastic syndrome. The objective of this study was to determine the frequency of ASXL1 breakpoints in a cohort of 36 patients with deletion 20q as the sole cytogenetic aberration. A combination of molecular cytogenetic methods was used to confirm ASXL1 gene alterations in 19 of the 36 patients, and the determination of ASXL1 gene changes in patients with deletion 20q revealed clinical and prognostic impacts.

Mantle cell lymphoma (MCL) is an aggressive subtype of B-cell non-Hodgkin lymphomas characterized by (over)expression of BCL2. A BCL2-targeting drug, venetoclax, has promising anticancer activity in MCL. We analyzed molecular mechanisms of venetoclax resistance in MCL cells and tested strategies to overcome it.We confirmed key roles of proapoptotic proteins BIM and NOXA in mediating venetoclax-induced cell death in MCL. Both BIM and NOXA are, however, differentially expressed in cell lines compared with primary cells. First, NOXA protein is significantly overexpressed in most MCL cell lines. Second, deletions ofWe demonstrated that MCL1 and NOXA play important roles in mediating resistance to venetoclax. Consequently, we tested an experimental treatment strategy based on cotargeting BCL2 with venetoclax and MCL1 with a highly specific small-molecule MCL1 inhibitor S63845. The combination of venetoclax and S63845 demonstrated synthetic lethalityOur data strongly support investigation of venetoclax in combination with S63845 as an innovative treatment strategy for chemoresistant MCL patients with adverse cytogenetics in the clinical grounds.

Cytogenetic and molecular genetic data are of paramount importance in the diagnosis, prognosis, and risk stratification of patients with malignant diseases. Sometimes they even directly guide the choice of therapy.1 Disease-specific abnormalities, particularly translocations, can provide essential information to assist the Pathologist and/or Oncologist in assigning a diagnosis. In several diseases, tumour genetics correlate strongly with clinical risk; thus, cytogenetic information may help the Oncologist counsel the patient, choose a specific treatment, and/or modulate treatment intensity. Clinical trials may involve cytogenetic classification of patients to the appropriate treatment regimens. Currently, the provision of specific assays for acquired neoplasiaspecific genomic changes varies among and within countries as a range of laboratories offer diagnostic solid tumour genetics; these may include Cytogenetic, Pathology, Haematology, and Molecular Genetics laboratories. Technical standards and general guidelines for the analysis and the report of results on most solid tumours are lacking. To address these deficits, a tumour best practice meeting with invited tumour experts without conflict of interest was held on 23rd April 2013 in Oxford, United Kingdom. The aim was to produce professional guidelines for tumour genetic laboratories and to incorporate the standards imposed by generic European guidelines,2 regulatory bodies (ISO15189, 2012 Medical laboratories – requirements for quality and competence),3 reporting guidelines,4 ISCN,5 and acquired best practice guidelines, while taking into account the current practice in Europe. The guidelines are aimed principally at giving guidance on the minimum, standard cytogenetic analyses, which are applicable to different types of laboratories operating under different regulatory arrangements and are relevant if more specific recommendations are not available. It was universally acknowledged that information on ancillary techniques in use in most cytogenetic laboratories (eg, RTPCR) or advanced techniques not always extensively performed in all laboratories (eg, next-generation sequencing (NGS)) were considered. The process for developing these evidence-based consensus guidelines included agreement on the need of general uniform rules on solid tumour analysis and reporting, discussion on the architecture of the guidelines, working group formation with different tasks (collection, analysis and comparison of any existing guidelines on this subject, type of tumours to be included according to published data and database consultation, method of analysis to be included, report formulation), circulation of the working group activities, formulation and circulation of the initial recommendations, draft and discussion, final consensus, and approval. It is noted that some elements of the tumour diagnostic service not subject to statute may be varied according to local constraints and agreements. Therefore, these guidelines are minimum requirements and additional professional judgment may be of paramount importance under many circumstances. In addition, as new techniques, particularly NGS, as well as clinical evidence, are becoming available all the time, these should be kept under constant review. Notes: The use of ‘must’ in this document indicates a requirement and the use of ‘should’ indicates a recommendation. Where there appears to be contradiction between available guidelines, the most recently published ones should be taken to apply to all. All diagnostic laboratories should be accredited to national or internationally accepted standards (ISO15189).3,6 Laboratories should participate in an External Quality Assessment Scheme7 in all aspects of their service for which a scheme is available.

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.

Despite attempts to improve the definitions of ambiguous lineage leukemia (ALAL) during the last 2 decades, general therapy recommendations are missing. Herein, we report a large cohort of children with ALAL and propose a treatment strategy. A retrospective multinational study (International Berlin-Frankfurt-Munster Study of Leukemias of Ambiguous Lineage [iBFM-AMBI2012]) of 233 cases of pediatric ALAL patients is presented. Survival statistics were used to compare the prognosis of subsets and types of treatment. Five-year event-free survival (EFS) of patients with acute lymphoblastic leukemia (ALL)-type primary therapy (80% ± 4%) was superior to that of children who received acute myeloid leukemia (AML)-type or combined-type treatment (36% ± 7.2% and 50% ± 12%, respectively). When ALL- or AML-specific gene fusions were excluded, 5-year EFS of CD19+ leukemia was 83% ± 5.3% on ALL-type primary treatment compared with 0% ± 0% and 28% ± 14% on AML-type and combined-type primary treatment, respectively. Superiority of ALL-type treatment was documented in single-population mixed phenotype ALAL (using World Health Organization and/or European Group for Immunophenotyping of Leukemia definitions) and bilineal ALAL. Treatment with ALL-type protocols is recommended for the majority of pediatric patients with ALAL, including cases with CD19+ ALAL. AML-type treatment is preferred in a minority of ALAL cases with CD19- and no other lymphoid features. No overall benefit of transplantation was documented, and it could be introduced in some patients with a poor response to treatment. As no clear indicator was found for a change in treatment type, this is to be considered only in cases with ≥5% blasts after remission induction. The results provide a basis for a prospective trial.

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 ± 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.

We conducted a cytogenetic analysis of 642 children with de novo acute myeloid leukemia (AML) treated on the AML-Berlin-Frankfurt-Münster (BFM) 04 protocol to determine the prognostic value of specific chromosomal aberrations including monosomal (MK+), complex (CK+) and hypodiploid (HK+) karyotypes, individually and in combination. Multivariate regression analysis identified in particular MK+ (n=22) as a new independent risk factor for poor event-free survival (EFS 23±9% vs 53±2% for all other patients, P=0.0003), even after exclusion of four patients with monosomy 7 (EFS 28±11%, P=0.0081). CK+ patients without MK had a better prognosis (n=47, EFS 47±8%, P=0.46) than those with MK+ (n=12, EFS 25±13%, P=0.024). HK+ (n=37, EFS 44±8% for total cohort, P=0.3) influenced outcome only when t(8;21) patients were excluded (remaining n=16, EFS 9±8%, P

Analyses at nucleotide resolution reveal unexpected complexity of seemingly simple and balanced chromosomal rearrangements. Chromothripsis is a rare complex aberration involving local shattering of one or more chromosomes and reassembly of the resulting DNA segments. This can influence gene expression and cause abnormal phenotypes. We studied the structure and mechanism of a seemingly balanced de novo complex rearrangement of four chromosomes in a boy with developmental and growth delay. Microarray analysis revealed two paternal de novo deletions of 0.7 and 2.5 Mb at two of the breakpoints in 1q24.3 and 6q24.1-q24.2, respectively, which could explain most symptoms of the patient. Subsequent whole-genome mate-pair sequencing confirmed the chromothriptic nature of the rearrangement. The four participating chromosomes were broken into 29 segments longer than 1 kb. Sanger sequencing of all breakpoint junctions revealed additional complexity compatible with the involvement of different repair pathways. We observed translocation of a 33 bp long DNA fragment, which may have implications for the definition of the lower size limit of structural variants. Our observations and literature review indicate that even very small fragments from shattered chromosomes can be detected and handled by the repair machinery during germline chromothriptic chromosome reassembly.

BACKGROUND The high incidence of mutations and cytogenetic abnormalities in patients with myelodysplastic syndrome (MDS) suggests that defects in DNA repair mechanisms. We monitored DNA repair pathways in MDS and their alterations during disease progression. METHODS Expression profiling of DNA repair genes was performed on CD34+ cells, and paired samples were used for monitoring of RAD51 and XRCC2 gene expression during disease progression. Immunohistochemical staining for RAD51 was done on histology samples. RESULTS RAD51 and XRCC2 showed differential expression between low-risk and high-risk MDS (P

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.

Chromosomal translocations are acquired genetic rearrangements in human cancers. Jumping translocations are rare nonreciprocal rearrangements involving the same donor chromosome segment translocated to two or more recipient chromosomes. In this report, we describe a patient with Burkitt lymphoma/leukemia (BL) and a complex karyotype including a t(2;8)(p12;q24), copy-neutral loss of heterozygosity at 17p13.1-p13.3 and 19q13.1-q13.2, trisomy 20, and two uncommon chromosomal aberrations. The first uncommon aberration was a complex rearrangement of chromosome 15 (probably the consequence of chromothripsis) masked by an apparently balanced reciprocal translocation, t(11;15)(p11.2;q21). The second one was a special type of unbalanced "vice versa" jumping translocation, which involved the same acceptor chromosome arm (13q) and various donor chromosome segments. It is unclear whether both atypical rearrangements are the consequence of the TP53 alteration or whether assumed chromothripsis influenced the development of the jumping-like translocation. However, the presence of the t(11;15)(p11.2;q21) in all pathological cells suggests that it occurred in the early stage of the disease, whereas the jumping-like translocation, as an additional change, subsequently accelerated the progression of the disease.

MDS with complex chromosomal aberrations (CCA) are characterized by short survival and a high rate of transformation to AML. A comprehensive genome-wide analysis of bone-marrow cells of 157 adults with newly diagnosed MDS and CCA revealed a large spectrum of nonrandom genomic changes related to the advanced stages of MDS. Chromosome shattering, probably resulting from chromothripsis, was found in 47% of patients. Deleted chromosome 5 was unstable and often involved in different types of cryptic unbalanced rearrangements. No true monosomy 5 was observed. Patients with CCA involving deleted chromosome 5 had an extremely poor prognosis (median overall survival, 2 months).

Introduction: Myelodysplastic syndrome (MDS) is characterized by differentiation blockade, cytopenias with commontransfusion dependency and immune defects. Upon progression the myeloblasts accumulate and the patients become vulnerable to severe infection complications. Based on the Prague Charles University General Hospital registry (N=164, median age 73), the AZA therapy in higher-risk MDS patients results in median OS 13.8 Mo with ORR 48.5%. We also noted from our retrospective data that AZA-treated patients with higher G-CSF consumption had significantly reduced occurrence of Grade 4 neutropenias and longer OS (19 vs 16 Mo, p value 0.039). Rationale: To improve poor clinical outcomes we initiated a randomized open labeled academic trial that compares standard AZA therapy (A) with novel AZA-based therapy combination involving use of G-CSF added prior AZA (GA). Both AZA and also decitabine were preclinically shown to induce myeloid differentiation upon G-CSF preincubation. G-CSF binds its receptor in granulocytic precursors and neutrophils to stimulate their survival, proliferation, and differentiation via myeloid master regulator transcription factor and leukemia-suppressor PU.1. We also have previously shown that AZA increases PU.1. expression. Study design & Methods: GA/MDS-2013 (EudraCT No 2013-001639-38). Expected for enrollment are 134 patients, currently enrolled 53 patients (GA arm N=29, A arm N=24) with median age 74 years, M:F ratio 32:21 (GA 16:16, A 13:8),median IPSS-R 6, median follow up 11.2 Mo, median cycles of therapy 6. Diagnosis included:MDS (EB1, EB 2) with IPSS int-2/high (75%), MDS/AML Results: The presented data include 2.5 years since the beginning of the trial. Median survival for GA arm was 11 vs 6 Mo in the A arm. ORR (CR, CRm, PR, HI) was 56% in GA arm vs 33% in the A arm. Transformation to AML for both arms was comparable. The stratified longitudinal Cox proportional hazards model containing time-varying covariates together with the ordinal multilevel logistic mixed model were utilized. From this joint fitted model, a negative coefficient for the G-AZA treatment (significant p-value 0.0442) can be noticed in the case of the Cox Proportional Hazard part of the model. This means that G-AZA treatment improves patient survival. The estimated odds for the GA arm that responded to the therapy with remission rather than progression is 12.4x higher than for the A arm, controlling for the remaining patients' characteristics (p-value 0.0016).Both the GA and A arms are comparably tolerated. Data on serious infections and neutropenia gr4 were not yet available. The levels of G-CSF in sera prior the study in both arms (GA vs A) were comparable. Flow cytometry revealed G-CSF mediated upregulation of FCgRI (CD64) in the GA but not in the A arm. Multivariate analysis indicates the following: mutated genes: DNMT3A (p-value 0.0157), EZH2 (p-value 0.0091), TP53 (borderline p-value 0.0510), & CSF3R (p-value 0.0057) shorten the overall survival. The significant negative effects on response was noted for mutated EZH2 (p-value 0.0208) and CSF3R (p-value 0.0424) genes. Conclusions: The current results supported by different methods and statistics indicates a beneficial effect of G-CSF pre-treatment to standard AZA therapy in higher risk MDS patients. G-CSF pre-treatment to AZA increases OS and ORR. In addition, we identified biomarkers that are negatively associated with patient survival and response including EZH2, DNMT3A, TP53, & CSF3R. Grant Support: Ministry of Health, #16-27790A. Institutional resources: Progres Q49 & Q26, UNCE/MED/016, LQ1604, SVV 260374/2017, RVO-64165. Disclosures No relevant conflicts of interest to declare.

Background and Aims A part of lower-risk myelodysplastic (LR-MDS) patients progress to higher-risk MDS or acute myeloid leukemia (AML). The progression may be predicted in some of these patients by determining mutations associated with myeloid malignancies at the time of diagnosis. We focused to find out dysregulated pathways caused by a well-defined mutation associated with disease progression. Methods We examined 158 samples from LR-MDS patients at diagnosis using TruSight Myeloid Sequencing Panel containing 54 genes (Illumina) to identify the mutational profile. We applied RNA-seq (NEB; Illumina) on bone marrow CD34+ cells of 5 LR-MDS patients with RUNX1 mutation and 6 LR-MDS patients without mutations. We performed differential gene expression analysis, gene set enrichment analysis (GSEA), Reactome biological pathway and Gene Ontology (GO) annotations. Results Forty out of 158 patients (25%) progressed during the follow-up period (median: 45.1 months). Mutation in RUNX1 gene was detected in 10 patients. Using RNA-seq, significantly dysregulated expression was observed in 986 genes (FDR2). Patients with mutation in RUNX1 gene had significant down regulation of expression genes involved in cellular senescence induced by DNA Damage/Telomere Stress (FDR=1.4e-11) and Oxidative Stress (FDR=3.6e-9), HDACs deacetylate histones (FDR =1.1e-13) and Senescence Associated Secretory Phenotype (FDR=1.4e-11) according to Reactome pathway database. There were 21 genes involved in the regulation of these pathways, of which 10 were part of all described pathways and belonged to the Nucleosome Assembly according to GO biological process with 33 down regulated genes (FDR=6.3e-28). According to GSEA, GO chromatin silencing gene set (Figure 1) was down regulated in the patients with RUNX1 mutation (p2); however, they did not form a specific pathway. The up-regulated genes were classified as so-called leukemia-associated antigens (LAAs) such as PRAME (adjusted p=6.9e-09; logFC=6.8), WT1AS (adjusted p=4.4e-07; logFC=5.7), BAALC (adjusted p=3.5e-06; logFC=2.1), WT1 (adjusted p=6.8e-07; logFC=3.2), FLT3 (adjusted p=0.004; logFC=1.3) and LEF1 (adjusted p=5.3e-08; logFC=-4.2). Conclusions To study the transcriptome of the hematopoietic stem cells of LR-MDS patients, we used gene expression profiling and identified biological processes in relation to mutations in RUNX1 gene that were detected in patients with disease progression. According to our results, we suppose that cells of LR-MDS patients with no mutations are maintained in senescent state or apoptosis in opposite to RUNX1 mutated cells. Finding out of dysregulated senescence was reinforced by other dysregulated pathways such as HDACs deacetylate histones, chromatin structural changes and senescence-associated secretory phenotype. About cellular senescence is known that may act as a strong tumor suppression mechanism that restrains proliferation of cells at risk for malignant transformation. We suppose that in precancerous cells of LR-MDS patients apoptosis or cellular senescence limits the replicative capacity of cells, thus preventing the proliferation. The disturbed state of cellular senescence in RUNX1 gene mutated hematopoietic stem cell can escape proliferation control and then can contribute to the disease progression in these patients. Supported by grants NV18-03-00227 and 00023736 from the Ministry of Health of the Czech Republic. Disclosures No relevant conflicts of interest to declare.

Introduction: Mantle cell lymphoma (MCL) is rare B-cell lymphoma subtype with usually aggressive behavior. Mutations of TP53 gene and complex karyotype (CK) were described to be poor prognostic factors (Obr A et al, 2018), but their impact on primary resistance to induction has not been investigated. Methods: We analyzed 115 MCL patients (pts) treated in two Czech university centers from 4/2006 to 10/2016. Both classical cytogenetics and next generation sequencing (NGS, MiSeq, Illumina) for TP53 mutation detection were performed on tumoral tissue (peripheral blood, bone marrow) in all patients. Cut-off for TP53 mutation was 1% variant allele frequency (VAF). CK was defined as 3 and more cytogenetic aberrations in one cell population. Pts with stable or progressive disease (SD/PD) during induction or with relapse within 6 months after induction completion were considered as primary refractory (PrR). Variables were compared by chi-squared test. T-test was used to compare the difference between means of TP53 mutation load in PrR+ and PrR- subgroup. Cut-off for TP53 mutation load (27%) was established as median VAF. Overall and progression free survival (OS, PFS) were calculated from the date of diagnosis. Results: The median age at diagnosis was 66 (40-87) years. Ninety-six percent of pts had advanced disease (III and IV). MIPI score was low, intermediate and high in 19.1%, 27.8% and 53.0% pts, respectively. Induction regimens used were as follows: R-CHOP/R-CHOP-like in 47.8%, intensive R-HDAC-containing in 38.3% and non-anthracycline regimen in 9.6% pts. Complete and partial response was achieved in 53.0% and 27.0% pts, resp. SD/PD was observed in 11.3% pts. Overall, 27 (23.5%) pts were considered as primary refractory. TP53 mutation and CK were present in 37 (32.3%) and 15 (13.0%) pts, respectively. Age, disease stage, ECOG score, MIPI, HDAC therapy and CK did not correlate with PrR status. Significantly higher TP53 mutation load was observed in the PrR+ (42%), compared to PrR- subgroup (25%, p=0.03). After median follow-up of 4.6 years, 3-year overall survival (3-y OS) and 3-year progression free survival (3-y PFS) in all pts was 65.8% and 50.9%, resp. Median OS in PrR+ and PrR- pts was 9.2 months, and 4.5 years, resp. Survival analysis stratified according to the TP53 mutation status/mutation load showed 3-y OS and 3-y PFS for wild type (TP53-WT), low mutation load (TP53-low load), high mutation load (TP53-high load) 74.2%, 47.6%, 33.3% and 56.0%, 37.0%, 27.8%, respectively (p=0.001 both). Conclusions: High TP53 mutation load (>27% VAF), but not CK, has significantly correlated with refractory disease in MCL pts. Pts with high mutation load of TP53 gene should be considered to an innovative treatment. Acknowledgement: Supported by IGA_LF_2019_001, MH CZ - DRO (FNOl, 00098892), AZV16-32339A and AZV16-31092A grants Figure Disclosures Trneny: Morphosys: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Incyte: Consultancy, Honoraria; Gilead sciences: Consultancy, Honoraria; Takeda: Consultancy, Honoraria.

Switches from the lymphoid to myeloid lineage during B-cell precursor acute lymphoblastic leukemia (BCP-ALL) treatment are considered rare and thus far have been detected in MLL-rearranged leukemia. Here, we describe a novel BCP-ALL subset, switching BCP-ALL or swALL, which demonstrated monocytosis early during treatment. Despite their monocytic phenotype, 'monocytoids' share immunoreceptor gene rearrangements with leukemic B lymphoblasts. All swALLs demonstrated BCP-ALL with CD2 positivity and no MLL alterations, and the proportion of swALLs cases among BCP-ALLs was unexpectedly high (4%). The upregulation of CEBPα and demethylation of the CEBPA gene were significant in blasts at diagnosis, prior to the time when most of the switching occurs. Intermediate stages between CD14(neg)CD19(pos)CD34(pos) B lymphoblasts and CD14(pos)CD19(neg)CD34(neg) 'monocytoids' were detected, and changes in the expression of PAX5, PU1, M-CSFR, GM-CSFR and other genes accompanied the switch. Alterations in the Ikaros and ERG genes were more frequent in swALL patients; however, both were altered in only a minority of swALLs. Moreover, switching could be recapitulated in vitro and in mouse xenografts. Although children with swALL respond slowly to initial therapy, risk-based ALL therapy appears the treatment of choice for swALL. SwALL shows that transdifferentiating into monocytic lineage is specifically associated with CEBPα changes and CD2 expression.

Chromosome 11 abnormalities are found in many hematological malignancies. In acute myeloid leukemia (AML), a proto-oncogene MLL (11q23.3) is frequently altered. However, rearrangements involving other regions of chromosome 11 have been reported. Therefore, we have characterized the chromosome 11 breakpoints and common deleted and amplified areas in the bone marrow or peripheral blood cells of newly diagnosed patients with AML. Using molecular-cytogenetic methods (multicolor fluorescence in situ hybridization (mFISH), multicolor banding (mBAND), microarrays, and FISH with bacterial artificial chromosome (BAC) probes, chromosome 11 abnormalities were delineated in 54 out of 300 (18%) newly diagnosed AML patients. At least 36 different chromosome 11 breakpoints were identified; two were recurrent (11p15.4 in the NUP98 gene and 11q23.3 in the MLL gene), and three were possibly nonrandom: 11p13 (ch11:29.31-31.80 Mb), 11p12 (ch11:36.75-37.49 Mb) and 11q13.2 (68.31-68.52 Mb). One new MLL gene rearrangement is also described. No commonly deleted region of chromosome 11 was identified. However, some regions were affected more often: 11pter-11p15.5 (n = 4; ch11:0-3.52 Mb), 11p14.1-11p13 (n = 4; ch11:28.00-31.00 Mb) and 11p13 (n = 4; ch11:31.00-31.50 Mb). One commonly duplicated (3 copies) region was identified in chromosomal band 11q23.3-11q24 (n = 9; ch11:118.35-125.00 Mb). In all eight cases of 11q amplification (>3 copies), only the 5' part of the MLL gene was affected. This study highlights several chromosome 11 loci that might be important for the leukemogeneic process in AML.

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.

Myelodysplastic syndrome (MDS), a clonal disorder originating from hematopoietic stem cell, is characterized by a progressive character often leading to transformation to acute myeloid leukemia. We used single nucleotide polymorphism arrays (SNP-A) to identify previously cryptic chromosomal abnormalities such as copy number alterations and uniparental disomies (UPD) in cytogenetically normal MDS. In the aberrant regions, we attempted to localize candidate genes with potential relevance to the disease. Using SNP-A, we analyzed peripheral blood granulocytes from 37 MDS patients. The analysis identified 13 cryptic chromosomal defects in 10 patients (27%). Four UPD (affecting chromosomes 3q, 7q, 17q, and 20p), 5 deletions and 4 duplications were detected. Gene expression data measured on CD34+ cells were available for 4 patients with and 6 patients without SNP-A lesions. We performed an integrative analysis of genotyping and gene expression microarrays and found several genes with an altered expression located in the aberrant regions. The expression microarrays suggested BMP2 and TRIB3 located in 20p UPD as potential candidate genes contributing to MDS. We showed that the genome-wide integrative approach is beneficial to the comprehension of molecular backgrounds of diseases with incompletely understood etiopathology. © 2012 Wiley Periodicals, Inc.