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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.

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.

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.

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.

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

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.

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.

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.

PURPOSE: Treatment of childhood acute lymphoblastic leukemia (ALL) was unified in the year 1986 in the Czech Republic using BFM protocols. PATIENTS AND METHODS: Children were treated in 10 and later on in 8 centers localized at the pediatric departments of the biggest hospitals. More than 1.000 children and adolescents up to the age of 18 years were treated between 1986 and 2002 on three consecutive studies ALL-BFM 83, ALL-BFM 90 and ALL-BFM 95. RESULTS: Event-free survival and overall survival improved gradually from 58/62% on ALL-BFM 83 through 70.5/76.6% on ALL-BFM 90 until 72.1/80.2% on ALL-BFM 95. At the same time the incidence of toxic deaths was decreasing and the success in the achievement of complete remission was increasing. Diagnostics in the Czech Republic improved remarkably in the 1990 with gradual introduction of centralized flow cytometry and molecular genetic analysis in one reference laboratory (CLIP = Childhood Leukemia Investigation Prague), which since then has become an internationally respected research center. CONCLUSIONS: Building up a network of closely collaborating leukemia centers covering the whole country, together with the establishment of reference and research laboratories has paved the way for the implementation of the Czech Pediatric Hematology Working Group (CPH) into the international studies Interfant 99, EsPhALL and for the active role in ALL IC-BFM 2002, the I-BFM-SG international randomized trial for treatment of children and adolescents with non-B ALL starting in 2002.

Posterior polymorphous corneal dystrophy (PPCD) is a rare, bilateral autosomal dominant disorder affecting primarily the corneal endothelium and descemet membrane (DM). The aim of this study was to establish the origin of abnormal endothelium in a patient with PPCD exhibiting cornea graft failure after keratoplasty surgery. A sex-mismatched graft obtained from a patient with PPCD who underwent repeat penetrating keratoplasty and the patient's original cornea were investigated. Combined fluorescent immunohistochemistry for cytokeratin (CK) 19 (a marker of aberrant PPCD endothelium) with fluorescence in situ hybridization (FISH) of the sex chromosomes were used in order to characterize the cells on the posterior graft surface. The pathological endothelium of the failed PPCD cornea revealed strong positivity for CK19 using fluorescent immunohistochemistry. In all the CK19-positive cells, both X and Y chromosomes were simultaneously detected using FISH. The results clearly showed the original cells of the patient (XY), within 3.5 years, almost totally overgrown the posterior corneal surface of the graft (XX). Moreover, an abnormal posterior collagenous layer populated by fibroblast-like cells was observed between DM and the endothelium in the failed graft, but its exact origin could not be established due to the low number of cells. Simultaneous detection of CK19 using fluorescent immunohistochemistry together with the detection of gonosomes using FISH was performed for the first time in the cornea and allowed us to prove that the recurrence of PPCD was caused by pathological abnormal proliferation and migration of recipient cells into donor graft.

Patients with near-tetraploid acute myeloid leukemia (NT-AML) typically have poor survival. We present the case of a 67-year-old Caucasian male with NT-AML M0 who had an unusually long first complete remission of 51 months and an overall survival of 80 months. The only characteristic distinguishing him from other previously described patients with NT-AML was the absence of erythroblastic and/or megakaryocytic dysplasia (EMD) at diagnosis. Molecular-genetic testing for AML fusion transcripts associated with a favorable prognosis (PML/RARα,AML1/ETO, and CBFβ/MYH11) were negative, as were other prognostic markers like MLL-PTD,FLT3-ITD, or mutations of FLT3-D835,NPM1, or CEBPA. Expression studies of ERG,MN1, and EVI1 revealed overexpression of ERG only. The absence of EMD may be a useful prognostic/diagnostic feature of this new rare subtype of NT-AML.

INTRODUCTION Nrf2 (nuclear factor, erythroid-derived 2-like 2 or NF-E2-related factor 2) is a transcription factor involved in antioxidant response by reducing oxidative stress. Erythropoietin (EPO) was described as an inducer of Nrf2 in the brain. Nrf2 binds to the promoter of gene coding for cereblon (CRBN) and stimulates CRBN expression (Lee et al. Biochem.Biphys Res Commun 2010; 399: 711-715). We showed that the high level of full length CRBN mRNA and CRBN protein is important for the efficacy of lenalidomide (LEN) in lower-risk MDS patients (Jonasova et al. Eur J Haematol 2015; 95: 27-34; Fuchs et al. Leuk Res 2017; 55 S1: S132, abstr. 227). Addition of EPO to LEN restored transfusion independence of MDS patients when their anemia relapsed during the course of LEN treatment (Jonasova et al. Leuk Res 2018; 69: 12-17). LEN inhibits E3 ubiquitin ligase RNF41 (ring finger protein 41), which polyubiquitinates EPO receptor (EPOR) and marks it for degradation in proteasomes. (Basiorka et al. Cancer Res 2016; 76: 3531-3540). AIMS The aim of our study was to evaluate the levels of Nrf2 mRNA and CRBN mRNA in mononuclear cells obtained from peripheral blood of lower-risk MDS patients after addition of EPO to lenalidomide in MDS patients who relapsed during LEN monotherapy. The further goal was to study the effect of LEN, EPO and combination of LEN plus EPO in cell lines (SKM-1 leukemic cell line established from a patient with progression of MDS to myelomonocytic leukemia and in MDS-L cells a human myeloblastic cell line, established as a MDS92 subline from the bone marrow of an MDS patient with del(5q) (Matsuoka et al. Leukemia 2010; 24: 748-755). METHODS Nrf2 mRNA and full-length CRBN mRNA levels were quantified in 9 lower-risk MDS patients with del(5q) and in 2 MDS patients with normal chromosome 5 [nondel(5q)] who were previously resistant to EPO treatment. All patients with median hemoglobin level 80 g/l were transfusion dependent before starting the therapy with LEN (5 or 10 mg/day). Recombinant human EPO (rHuEPO) in dose of 40,000 IU s.c. per week was combined with LEN in these MDS patients who relapsed during LEN monotherapy or were resistant to LEN [one non(del5q) patient]. Four lower-risk MDS patients responsive to EPO after diagnosis were also used for comparison of Nrf2 mRNA and full-length CRBN mRNA levels. Mononuclear cells were isolated by Ficoll-Paque PLUS gradient separation, then washed by phosphate buffered saline and remaining red cells were lysed. Both SKM-1 and MDS-L cells were incubated without or with LEN, EPO and combination of LEN plus EPO for 19, 24 and 43 hours. The end concentration of added Epo was 2U/ml and LEN 10 µM. The levels of Nrf2 mRNA and full length CRBN mRNA were measured using the reverse transcription-quantitative TaqMan PCR assay. RESULTS The levels of Nrf2 mRNA and full-length CRBN mRNA in peripheral blood mononuclear cells correspond during rHuEPO monotherapy of four responsive lower-risk MDS patients. In these cases no increase of Nrf2 mRNA and CRBN mRNA levels was found. Increased Nrf2 mRNA and CRBN mRNA levels in peripheral blood mononuclear cells were found after addition of rHuEPO to lenalidomide in six MDS patients with del(5q) and one non(del5q) MDS patient who relapsed during LEN monotherapy. All these patients responded to combination of rHuEPO and lenalidomide by increased hemoglobin levels. Addition of or rHuEPO to lenalidomide was without effect on Nrf2 mRNA and CRBN mRNA levels in one non(del5q) patient who was resistant to LEN. Experiments with SKM-1 and MDS-L cells showed that rHuEPO alone did not increased Nrf2 mRNA and CRBN mRNA levels. However LEN and in a greater extent the combination of rHuEPO and LEN increased Nrf2 mRNA and CRBN mRNA levels. CONCLUSIONS Our findings indicate that transcription factor Nrf2 is involved in CRBN expression in mononuclear cells obtained from peripheral blood of lower-risk MDS patients and in SKM-1 and MDS-L cells in culture. Expression of both Nrf2 and CRBN is stimulated by lenalidomide and in a greater extent by combination of lenalidomide and rHuEPO. Measurement of CRBN mRNA level as an important factor for prediction of the efficacy of not only lenalidomide monotherapy but also of combination of LEN and rHuEPO. This work was supported by the research project for conceptual development of research organization (00023736; Institute of Hematology and Blood Transfusion, Prague) and Grant Agency of Charles University, project number 924616. 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. These patients have more aggressive type of disease than is expected based on their classification according to the International Prognostic Scoring System. Alongside, recent advances in DNA sequencing have enabled the identification of a large number of mutated genes in more than 80% of MDS patients. We focused on the identification of mutational profile that would be predictive of disease progression in LR-MDS patients. Methods We examined samples from 73 LR-MDS patients by TruSight Myeloid Sequencing Panel containing 54 genes (Illumina) to determinate the mutational profile. In 32 (44%) patients, paired samples processed at the time of diagnosis and during progression were analyzed. The median age of patients was 64.5 years (range, 29-83 years). The median observation period was 33.4 months (range, 4.5-195.9 months) from diagnosis, 36 (49%) patients remained alive including 8 who underwent HSCT. The FASTQ files were analyzed by NextGENe V2.4.2.2 (SoftGenetics). The survival distributions were estimated using the Kaplan-Meier method and prognostic factor analysis was performed with Cox's multivariate regression method. The Mann-Whitney test was used to determine the group independence. Results In total, 70% of patients had a recurrent mutation in at least one gene of the panel (87.5% of patients with progression and 56% without progression). The patients, who progressed, carried on average of 2.4 mutations and unprogressed patients 1.9 mutations. Most of the mutations detected in LR-MDS were in SF3B1 (30.1% of patients) and DNMT3A (24.7% of patients) genes but had no effect on overall survival (OS). Multivariate analysis demonstrated that RUNX1 and TP53 mutations were the strongest independent prognostic factors for OS (HR: 14.9; p=0.0001/HR:6.3; p=0.0007) at the time of diagnosis and also for progression-free survival (PFS) (HR: 3.6; p=0.05/HR:3.3; p=0.05). Moreover, mutations in these genes were detectable in 59.4% of patients with disease progression and only in 4.8% of patients without progression, who however were followed shortly (4.7 months). The median OS was 27.9 and 39.6 months in the patients with mutations of RUNX1 and TP53 genes compared to 84.2 months (p Conclusions The goal of the study was to detect mutations in relevant genes that might predict disease progression up to 6 years from the time of diagnosis in LR-MDS patients and comparison of mutant allele frequencies in selected cases between the time of diagnosis and disease progression. We found that 60% of patients who progressed, carried mutations in RUNX1 and TP53 genes at the time of diagnosis. On the contrary, other mutations detectable at the time of diagnosis had no effect on OS and PFS. The VAF of mutations detected at the diagnosis increased at the time of progression. The NRAS and PTPN11 genes contained most of the new mutations acquired during progression. In conclusion, the results indicate that routine monitoring for mutations in LR-MDS should be performed to refine the risk prediction of disease progression. Supported by AZV grants (16-33485A, 16-31689A, 17-31398A and NV18-03-00227), RVO-VFN64165 and the project for conceptual development of research organization (00023736) from the Ministry of Health of the Czech Republic. Disclosures No relevant conflicts of interest to declare.

Introduction: Chromothripsis is a recently identified genomic instability phenomenon that plays a role in the genesis and progression of cancer. It is a one-step catastrophic genomic event involving multiple chromosomal breakages and random DNA rejoining. This genetic abnormality can affect an entire chromosome, a chromosomal arm, or a single chromosomal region. Chromothripsis is associated with highly complex karyotypes and a very poor prognosis, and has been detected in a wide range of tumor entities, including hematological malignancies. However, this complex genomic abnormality has not been comprehensively studied in patients with myelodysplastic syndromes (MDS). The aim of the study was to assess the incidence, associated genetic features, and clinical significance of chromothripsis in a large homogeneous cohort of patients newly diagnosed with high-risk MDS and complex karyotypes. Methods: A detailed genome-wide analysis of fixed bone-marrow cells from adults with complex karyotypes (≥ 3 aberrations), identified with conventional G-banding at the diagnosis of MDS, was performed. The complex rearrangements were studied with integrative genetic methodologies: fluorescence in situ hybridization (FISH) with Vysis DNA probes (Abbott, Des Plaines, IL), multicolor FISH (mFISH) and/or multicolor banding (mBAND) methods with the 24XCyte and the XCyte color kits (MetaSystems, Altlussheim, Germany), and array-based comparative genomic hybridization with CytoChip Cancer SNP 180K (Illumina, San Diego, CA) or the SurePrint G3 Cancer CGH+SNP 4x180K Microarray (Agilent, Santa Clara, CA). A mutational analysis of the TP53 gene was also performed in selected cases using amplicon-based deep sequencing on a 454 GS Junior System (Roche, Basel, Switzerland) or the TruSight Myeloid Sequencing Panel on MiSeq sequencing instruments (Illumina). Results: In total, 265 patients with complex karyotypes and newly diagnosed high-risk MDS were included (131 females, 134 males; median age, 70 years). The hallmarks of chromothripsis were detected in 67.7% of cases, in both the main clones and one or more subclones. At the cytogenetic level, chromothripsis was apparent as multiple deletions, insertions, ring chromosomes, amplification of individual genes or chromosomal regions, and/or the formation of chaotically reassembled chromosomes. Chromothripsis affected almost all the chromosomes, except the Y chromosome. The most frequently involved were chromosomes 5 (33.3% of events), 7 (28.1% of events), 17 (18.9% of events), 11 and 12 (15.6% of events each). In samples with signs of chromothripsis, the higher frequency of aberrations on chromosome 17p was observed (loss of heterozygosity [LOH], copy-number neutral LOH [cnLOH] and/or homozygous mutation of TP53) (p = 0.05). Patients with chromothripsis had significantly worse overall survival (OS; median, 3 months). We also investigated the effect of chromothripsis on the survival of 183 patients treated with azacytidine. In this cohort, the median OS of chromothripsis-positive patients was 10.1 months (mean, 12.2 months), whereas the median OS of chromothripsis-negative patients was 17.3 months (mean, 31.0 months). Conclusions: Our results demonstrate that chromothripsis is a frequent genomic abnormality in patients with high-risk MDS, which influences the patient's prognosis and disease biology. Chromothripsis was associated with a higher frequency of LOH/cnLOH 17p, rapid disease progression, and short survival. The adverse outcomes may be attributable to the effects on the functions of many important genes. The OS of patients with high-risk MDS with chromotripsis may be slightly improved by azacytidine treatment, but the prognosis of these patients remains very poor. Therefore, a better understanding of the mechanistic basis of chromothripsis is extremely important and could lead to the development of new treatment strategies based on drugs that target the genes present in amplified or deleted regions and/or the DNA damage response pathways. This study was supported by research projects RVO-VFN64165, GACR P302/12/G157, AZV 16-27790A , Progres Q26 and Q28/LF1, GACR 18-01687S, MHCR 00023736 and UNCE/MED/016 . Disclosures No relevant conflicts of interest to declare.

The ETV6/ABL1 (TEL/ABL) fusion gene is a rare aberration in malignant disorders. Only 19 cases of ETV6/ABL1-positive hematological malignancy have been published, diagnosed with chronic myeloid leukemia, other types of chronic myeloproliferative neoplasm, acute myeloid leukemia or acute lymphoblastic leukemia (ALL). This study reports three new cases (aged 8 months, 5 years, and 33 years) of ALL with the ETV6/ABL1 fusion found by screening 392 newly diagnosed ALL patients (335 children and 57 adults). A thorough review of the literature and an analysis of all published data, including the three new cases, suggest poor prognosis of ETV6/ABL1-positive acute leukemias. The course of the disease in the two pediatric patients is characterized by minimal residual disease monitoring, using quantification of both the ETV6/ABL1 transcript and immunoreceptor gene rearrangements. Eosinophilia could not be confirmed as a hallmark of the ETV6/ABL1-positive disease. Studies of neonatal blood spots demonstrated that, in the child diagnosed at five years, the ETV6/ABL1 fusion initiating the ALL originated prenatally.

Objectives: Patients with near-tetraploid (karyotype: 81 - 103 chromosomes) acute lymphoblastic leukemia (NT-ALL) constitute about 1% of childhood ALL and data reported on them are limited and controversial. The aim of the study was to enlarge the knowledge on these rarely occurring ALL. Methods: The members of the European Group for Immunophenotyping of Leukemias (EGIL) searched retrospectively their databases for NT-ALL patients. Results: We collected data of 36 European children from seven European countries with NT-ALL diagnosed since 1992. All patients reached complete remission (CR) after induction chemotherapy. Their blasts were negative for peroxidase and BCR-ABL1. Ten children were diagnosed as T-cell ALL (T-ALL) EGIL categories (T-I n = 2, T-II n = 2, T-III n = 3, T-IV n = 3) and four displayed various structural chromosomal abnormalities. Eight of 10 T-ALL remained in 1st CR; one died in CR from sepsis and one is alive in 2nd CR. Median survival was 88 (7-213) months. B-cell precursor (BCP) ALL was diagnosed in 26 children. Thirteen were positive for ETV6-RUNX1 and are alive in 1st CR for 32-147 months. Ten children were ETV6-RUNX1 negative and remained in 1st CR for 16-163 months. One girl with hypodiploid and NT metaphases and ETV6-RUNX1-negative BCP-ALL and one of two boys with NT-BCP-ALL not examined for ETV6-RUNX1 died of infection after stem cell transplantation in 2nd/3rd CR. Secondary myelodysplastic syndrome developed in two patients with NT-BCP-ALL. Conclusions: Our data demonstrate immunophenotypic, cytogenetic, and molecular heterogeneity of NT-ALL and favorable prognosis of most NT-ALL across different immunophenotypic and/or genetic ALL subtypes.

e14060Background: Diffuse gliomas represent heterogeneous group of tumors with highly variable biological behavior. Despite of radical surgical resection and intensive therapy, recurrent lesions wi...

e19025Background: The interstitial deletion of the long arm of chromosome 5—del(5q)—is the most common cytogenetic abnormality in MDS occurring either as a sole aberration or as a part of complex k...

The prognostic significance of 1q21 gain, del(13)(q14), del(17)(p13), t(4;14)(p16.3;q32), and t(11;14)(q13;q32) detected by interphase fluorescein in situ hybridization (FISH) was studied in a cohort of 91 patients with newly diagnosed multiple myeloma (MM). 1q21 gain was detected in 37 of 91 patients (40.7%). In comparison with patients lacking 1q21 gain, patients with 1q21 gain had significantly shorter progression-free survival (PFS) (14.9 versus 27.4 months; P = .044) and worse 4-year overall survival (OS) (40.1% versus 76.2% of patients; P =

Background Mixed phenotype acute leukemia (MPAL) represents a diagnostic and therapeutic dilemma. The European Group for the Immunological Classification of Leukemias (EGIL) scoring system unambiguously defines MPAL expressing aberrant lineage markers. Discussions surrounding it have focused on scoring details, and information is limited regarding its biological, clinical and prognostic significance. The recent World Health Organization classification is simpler and could replace the EGIL scoring system after transformation into unambiguous guidelines. Design and Methods Simple immunophenotypic criteria were used to classify all cases of childhood acute leukemia in order to provide therapy directed against acute lymphoblastic leukemia or acute myeloid leukemia. Prognosis, genotype and immunoglobulin/T-cell receptor gene rearrangement status were analyzed. Results The incidences of MPAL were 28/582 and 4/107 for children treated with acute lymphoblastic leukemia and acute myeloid leukemia regimens, respectively. In immunophenotypic principal component analysis, MPAL treated as T-cell acute lymphoblastic leukemia clustered between cases of non-mixed T-cell acute lymphoblastic leukemia and acute myeloid leukemia, while other MPAL cases were included in the respective non-mixed B-cell progenitor acute lymphoblastic leukemia or acute myeloid leukemia clusters. Analogously, immunoglobulin/T-cell receptor gene rearrangements followed the expected pattern in patients treated as having acute myeloid leukemia (non-rearranged, 4/4) or as having B-cell progenitor acute lymphoblastic leukemia (rearranged, 20/20), but were missing in 3/5 analyzed cases of MPAL treated as having T-cell acute lymphobastic leukemia. In patients who received acute lymphoblastic leukemia treatment, the 5-year event-free survival of the MPAL cases was worse than that of the non-mixed cases (53±10% and 76±2% at 5 years, respectively, P =0.0075), with a more pronounced difference among B lineage cases. The small numbers of MPAL cases treated as T-cell acute lymphoblastic leukemia or as acute myeloid leukemia hampered separate statistics. We compared prognosis of all subsets with the prognosis of previously published cohorts. Conclusions Simple immunophenotypic criteria are useful for therapy decisions in MPAL. In B lineage leukemia, MPAL confers poorer prognosis. However, our data do not justify a preferential use of current acute myeloid leukemia-based therapy in MPAL.

Translocations involving chromosome 11q23 frequently occur in pediatric acute myeloid leukemia (AML) and are associated with poor prognosis. In most cases, the MLL gene is involved, and more than 50 translocation partners have been described. Clinical outcome data of the 11q23-rearranged subgroups are scarce because most 11q23 series are too small for meaningful analysis of subgroups, although some studies suggest that patients with t(9;11)(p22;q23) have a more favorable prognosis. We retrospectively collected outcome data of 756 children with 11q23- or MLL-rearranged AML from 11 collaborative groups to identify differences in outcome based on translocation partners. All karyotypes were centrally reviewed before assigning patients to subgroups. The event-free survival of 11q23/MLL-rearranged pediatric AML at 5 years from diagnosis was 44% (± 5%), with large differences across subgroups (11% ± 5% to 92% ± 5%). Multivariate analysis identified the following subgroups as independent prognostic predictors: t(1;11)(q21;q23) (hazard ratio [HR] = 0.1, P = .004); t(6;11)(q27;q23) (HR = 2.2, P < .001); t(10;11)(p12;q23) (HR = 1.5, P = .005); and t(10;11)(p11.2;q23) (HR = 2.5, P = .005). We could not confirm the favorable prognosis of the t(9;11)(p22;q23) subgroup. We identified large differences in outcome within 11q23/MLL-rearranged pediatric AML and novel subgroups based on translocation partners that independently predict clinical outcome. Screening for these translocation partners is needed for accurate treatment stratification at diagnosis.

The results of repeated interphase fluorescence in-situ hybridization (I-FISH, FISH) examination of 97 CLL patients and correlation of these findings with IgVH hypermutation status, ZAP-70 and CD38 expression are presented. The appearance of new, FISH-detectable, genomic aberrations during disease course, described as clonal evolution (CE), was observed in 26% of patients. The most frequent newly acquired cytogenetic abnormality was 13q deletion in 64% (16/25). In contrast to earlier studies, there was no correlation found between CE and either one of single negative prognostic factors (unmutated IgVH; CD38 positivity; ZAP-70 positivity). However, the combination of all three negative factors correlated with CE highly significantly (p=0.005) and moreover, also with a shift from lower to higher FISH risk category (p=0.010). As the prognostic data were known in all patients, this study represents the complete insight on the association of CE and other risk parameters in CLL.

// Monika Belickova 1 , Jitka Vesela 1 , Anna Jonasova 2 , Barbora Pejsova 1 , Hana Votavova 1 , Michaela Dostalova Merkerova 1 , Zuzana Zemanova 3 , Jana Brezinova 1 , Dana Mikulenkova 1 , Marie Lauermannova 1 , Jan Valka 1 , Kyra Michalova 1, 3 , Radana Neuwirtova 2 , Jaroslav Cermak 1 1 Institute of Hematology and Blood Transfusion, Prague, Czech Republic 2 First Department of Medicine, General University Hospital and First Faculty of Medicine, Charles University, Prague, Czech Republic 3 Center of Oncocytogenetics, General University Hospital and First Faculty of Medicine, Charles University, Prague, Czech Republic Correspondence to: Monika Belickova, email: monika.belickova@uhkt.cz Keywords: myelodysplastic syndrome, mutational status, TP53, prognosis, variant allele frequency Received: January 03, 2016 Accepted: April 16, 2016 Published: May 06, 2016 ABSTRACT 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.

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.

B-chronic lymphocytic leukemia (B-CLL) is the most common adult leukemia. Molecular genetic characterization of B-CLL has made significant progress and typical chromosomal anomalies have been assessed. The most frequent chromosomal abnormalities are deletions at 13q14, 17p13, and 11q22 approximately q23 and trisomy 12. The aim of this study was to establish incidence of chromosomal changes in bone marrow or peripheral blood cells (or both) of B-CLL patients using a molecular cytogenetic method, interphase fluorescence in situ hybridization (I-FISH), and to evaluate the prognostic implications. We performed I-FISH on bone marrow and blood smears from 217 B-CLL patients (124 male, 93 female). Trisomy 12 was found in 35 of the 217 (16%); deletion 13q14 was analyzed in 207 patients and found in 112 (54%). Deletion 17p13 was found in 34 (16%) out of 206 examined. Deletion of 11q23 was analyzed in 56 patients and was present in 7 (12%). Statistical analyses were performed to correlate the molecular-cytogenetic findings with disease status (stable versus progressive), Rai stage, CD38/CD19 antigen coexpression, immunoglobulin variable heavy chain (IgV(H)) mutational pattern, and other clinical and laboratory parameters. No apparent differences in distribution were noted for anomalies +12, del(13)(q14), or del(17)(p13) among patients with stable and progressive disease, and no consistent pattern in the distribution of type of genomic changes were found among various Rai stages and in CD38/CD19-positive or -negative patients. Patients without IgV(H) mutation had a worse prognosis; however, distribution of chromosomal abnormalities identified with FISH was the same for patients with and without IgV(H) mutations.

Telomere length was evaluated by terminal repeat fragment method (TRF) in 50 patients with myelodysplastic syndromes (MDS) and acute myelogenous leukemia (AML) arising from MDS and in 21 patients with untreated primary AML to ascertain, whether telomere erosion was associated with progression of MDS towards overt leukemia. Heterogeneity of TRF among MDS FAB subgroups ( P =0.004) originated from its shortening in increased number of patients during progression of the disease. Chromosomal aberrations were present in 32% MDS patients with more eroded telomeres ( P =0.022), nevertheless a difference between mean TRF in the subgroups with normal and abnormal karyotype diminished during progression of MDS. A negative correlation between individual TRF and IPSS value ( P =0.039) showed that telomere dynamics might serve as a useful prognostic factor for assessment of an individual MDS patient’s risk and for decision of an optimal treatment strategy.

Prenatal acquisition of leukaemia-associated gene rearrangements is a well-established phenomenon. This is the first report of a complex cytogenetic clone, in association with an ETV6/AML1 fusion, developing in utero. Identical twin girls, aged 4 years, developed ETV6/AML1-positive acute lymphoblastic leukaemia (ALL) within 3 months of one another. Both demonstrated an identical four way, variant t(12;21). There was gain of an AML1 signal in twin 1 and loss of an ETV6 one in twin 2 at interphase. This unique case study demonstrates that ETV6/AML1 fusion and the associated complex chromosomal rearrangements occurred in utero. Clonal expansion of the abnormal cell in one twin was followed by metastasis to the other. There was a prolonged preleukaemic phase, which lasted well into childhood. The short time between the two diagnoses of ALL suggests a common precipitating event. The significance of the different secondary markers remains unclear.

The phenotype of Gorlin-Goltz syndrome or basal cell nevus syndrome (BCNS, #109400, OMIM), a Mendelian trait due to PTCH mutations has been reported in a few cases of interstitial deletion of chromosome 9q. We present an 11-year-old girl with clinical features consistent with BCNS including bridging of sella turcica, biparietal bossing, downward slanting palpebral fissures, mandible prognathism, pectus excavatum, thumb abnormalities, occult spina bifida at L5-S4, numerous basal cell nevi, and single basal cell carcinoma. Cytogenetic analysis using high-resolution banding techniques and fluorescence in situ hybridization (FISH) revealed interstitial chromosome deletion 9q22.32-q33.2 involving the PTCH gene as a secondary breakage event to a chromosome translocation t(9;17)(q34.1;p11.2)mat. Further FISH studies showed the translocation breakpoint on 9q34.11 maps proximal to ABL, between the BAC clone RP11-88G17 and the LMX1B gene. The latter gene encodes a transcription factor, in which loss of function mutations are responsible for the nail-patella syndrome (NPS, #161200 OMIM). Interestingly, some features of our proband (e.g., bilateral patellar dysplasia and abnormal clavicular shape), as well as her healthy sister who carries the same translocation, are also found in patients with NPS. The chromosome 17p11.2 breakpoint maps in the Smith-Magenis syndrome common deletion region, within two overlapping BAC clones, CTD-2354J3 and RP11-311F12.

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.

Ploidy is an important prognostic factor in the risk stratification of multiple myeloma (MM) patients. Patients with MM can be divided into two groups according to the modal number of chromosomes: nonhyperdiploid (NH-MM) and hyperdiploid (H-MM), which has a more favorable outcome. The two ploidy groups represent two different oncogenetic pathways determined at the premalignant stage. The ploidy subtype also persists during the course of the disease, even during progression after the therapy, with only very rare cases of ploidy conversion. The clinical significance of ploidy conversion and its relation to drug resistance have been previously discussed. Here, we describe a female MM patient with a rare change in her ploidy status from H-MM to NH-MM, detected by cytogenetic and molecular cytogenetic examinations of consecutive bone marrow aspirates. We hypothesize that ploidy conversion (from H-MM to NH-MM) is associated with disease progression and acquired resistance to bortezomib/lenalidomide therapy.

We used comparative genomic hybridization (CGH) and conventional cytogenetics (CC) to define chromosomal changes and to evaluate the usefulness of CGH in 65 patients having childhood acute lymphoblastic leukemia (ALL). Subsequently, fluorescence in situ hybridization (FISH) was used to evaluate the CGH and cytogenetic results. Comparative genomic hybridization revealed DNA copy number changes in 49 (75%) patients (including 7 patients with unsuccessful cytogenetics and 2 patients with normal karyotype). A total of 85 losses and 195 gains were detected. The most commonly gained chromosomes were 21 (35%), X (31%), 18 (27%), 10 (26%), 6 (25%), 17 (25%), 4 (23%), and 14 (22%). Losses were most frequently observed on chromosomes 9p (18%) and 12p (11%). Other losses were detected on chromosomes 13q (9%), 6q (9%), 7p (8%), and chromosome X (6%). Conventional cytogenetics revealed chromosomal changes in 53 (82%) patients. The employment of CGH and FISH together with CC analysis revealed chromosomal changes in 62 (95%) of the childhood ALL patients investigated. The CGH completed CC results in 36 patients; in 9 patients, the changes escaped detection without using CGH. The results of our study were compared to 6 other CGH studies previously reported. Our observations underline the benefits of supplementing routine cytogenetic investigation in childhood ALL by FISH and CGH, because small unbalanced changes may escape detection when conventional cytogenetics is the only diagnostic method used.

Fluorescence in situ hybridization (FISH) using specific probes for the 5q31-32 region and a whole chromosomal painting (WCP) probe for chromosome 5 were used to corroborate the results of classical cytogenetic examinations performed on G-banded chromosomes of 77 patients with hematological malignancies. Using classical cytogenetic methods, we suspected the presence of clones with a deletion 5q in 63 patients, and complex rearrangements with involvement of chromosome 5 in 14 other cases. Fluorescence in situ hybridization proved the occurrence of deletion 5q31 in 23 patients and ascertained translocations of part of the long arms of deleted chromosome 5 with missing region 5q31 in 12 patients. In 2 cases, the 5q31 region was translocated to other chromosomes as a part of complex rearrangements. The combination of classical cytogenetics and FISH with specific probes for the 5q31 band yielded cytogenetic results in 35 cases. Routine FISH detection of deleted regions was possible by commercially available cosmid probes for the 5q31 chromosomal band. The interpretation of small deletions and frequent involvement of the deleted chromosomes 5 in complex translocations were ascertained by WCP probes.

We report three cases of acute myeloid leukemia (AML) with a near-tetraploid karyotype in most metaphases while lacking chromosomal abnormalities typical for AML. All patients, 63, 72 and 81 years old, were female. In two cases, AML was diagnosed 5–7 months after a cytopenic period while the third patient had a secondary AML after therapy for a pleural tumor. Leukemic blasts were classified as AML M0, AML M1 and AML without further specification. Two patients died on the 18th and 52nd day after the start of cytotoxic chemotherapy, the third patient refused chemotherapy and died 22 days after the diagnosis. The three patients may represent a distinct AML category with the following features: (1) the near-tetraploid karyotype in most bone marrow metaphases examined at diagnosis of AML; (2) the presence of very large myeloid blasts in the bone marrow and dysplastic changes in erythroid and/or megakaryocytic lineages pointing to the origin of AML in pluripotent myeloid progenitor cells; (3) the expression of the CD34 antigen; (4) the low growth of granulocyte-macrophage colony forming cells in culture; and (5) the presence of a preleukemic phase, a higher age and a poor prognosis.

240 patients with MDS studied cytogenetically at diagnosis between 1981 and 1990 were followed until death or until April 1992 to evaluate the prognostic significance of FAB classification, age and karyotype. 61 patients (25.4%) subsequently transformed into AML and 176 (73.3%) died during the follow-up period. Patients with blastic MDS types had a shorter survival and a higher probability of leukemic transformation. The younger age increased the probability of leukemic transformation, but was associated with a longer survival. The absence of analyzable mitoses was associated with a shorter survival. The complex chromosomal abnormalities at the initial evaluation identified a subgroup of patients with a high risk of a short survival and/or subsequent leukemia transformation. In refractory anemia the presence of complex chromosomal abnormalities was linked with a relative risk of 3.58 of leukemic transformation and shorter survival as compared with other cytogenetically defined groups.

Thirty-one patients (19 males and 12 females; mean age 23.9 years, range 4–41 years) were treated with bone marrow transplantation (BMT) after intensive chemoradiotherapy. Their diagnoses were as follows: chronic myeloid leukemia (CML) in 13, acute myeloid leukemia (AML) in seven, acute lymphocytic leukemia (ALL) in six, myelodysplastic syndrome (MDS) in two, aplastic anemia (AA) in two, and Fanconi anemia (FA) in one. Allogeneic BMT was performed in 28 cases (17 donors were of like sex, 11 were of unlike sex), one patient received syngenic transplant, and one received transplant of cells obtained from an unrelated donor through a computerized international registry in London. Autologous BMT was performed in three patients. BM cells were analyzed cytogenetically at diagnosis, before and serially after BMT (three to nine times). Follow-up ranged from 2 to 55.5 months. Cytogenetic examination was a very useful method for monitoring posttransplantation course in patients with CML or in those who received BM cells of unlike sex. Results of concomitant cytogenetic examinations are reported in detail.

Detection of Flt3-internal tandem duplication (ITD) in acute myelogenous leukemia (AML) blasts at the time of diagnosis is associated with an increased risk of leukemia relapse after chemotherapy. In the present report, we describe two patients with Flt3-abnormalities that were only detectable at the time of relapse. One of the patients had an ITD at the time of diagnosis (duplicated region 41-133 numbered from the start of exon 14 plus CGG insertion), but at the time of relapse a new abnormality (duplicated region 70-230 plus insertion GGGGAGT) was detected as the only Flt3-abnormality. The second patient showed no Flt3-ITD at the time of diagnosis, but ITD was detected at the time of relapse (duplicated region 12-96, no insertion). Our observations of independent Flt3-ITD and the detection of new Flt3-abnormalities at the time of relapse suggest that (i) Flt3-gene abnormalities may be even more important in the pathogenesis of chemotherapy-resistant AML than suggested from previous studies of newly diagnosed AML, and (ii) monitoring of minimal residual disease by using patient-specific real-time polymerase chain reaction assays for Flt3-abnormalities may not be reliable.

The prognostic impact of chromosomal abnormalities was evaluated by fluorescence in situ hybridization with cytoplasmic immunoglobulin light chain staining (cIg-FISH) and by classical metaphase cytogenetics in a cohort of 207 patients with newly diagnosed multiple myeloma who were treated with high-dose therapy followed by autologous stem cell transplantation in the CMG2002 clinical trial. The incidence of chromosomal abnormalities detected by FISH was as follows: 52.7% for del(13)(q14), 6.5% for del(17)(p13), 18.6% for t(11;14)(q13;q32), 22.8% for t(4;14)(p16;q32) and 45.7% for gain(1)(q21). Metaphase cytogenetic analysis revealed a complex karyotype in 19.1% and hyperdiploidy in 21.7% of patients. The overall response rate was not influenced by the presence of any studied chromosomal abnormality. Patients with a complex karyotype, those with translocation t(4;14) and those with gain of the 1q21 locus had a shorter time to progression (TTP) and overall survival (OS). Other genomic changes such as translocation t(11;14) and del(13q) had less impact on TTP and OS. In multivariate analysis, complex karyotype, translocation t(4;14) and β(2)-microglobulin level > 2.5 mg/L were independent prognostic factors associated with poor overall survival. Their unfavorable prognostic impact was even more pronounced if they were present in combination. Patients with t(4;14) present together with a complex karyotype had the worst prognosis, with a median OS of only 13.2 months, whereas patients with a normal karyotype or karyotype with ≤ 2 chromosomal changes had the best outcome, with 3-year OS of 85.9%. In conclusion, complex karyotype, gain of 1q21 region and translocation t(4;14) are major prognostic factors associated with reduced survival of patients with newly diagnosed multiple myeloma treated with autologous stem cell transplantation.

Purpose To establish the origin of cells causing the recurrence of posterior polymorphous corneal dystrophy (PPCD). Methods A corneal explant obtained from a male patient suffering from PPCD, who had undergone repeat penetrating keratoplasty (female donor), was examined. Cytokeratin (CK) 19 was detected using indirect immunofluorescence to confirm the presence of abnormal epithelial-like cells on the endothelial layer. The origin of these CK19-positive aberrant cells was then determined using fluorescence in situ hybridization of the X and Y chromosomes. Results Two out of 559 cells evaluated (0.4%) were XX positive and CK19-negative throughout the pathological explant. 99.6% of the endothelial cells were positive for CK19 and exhibited XY staining. These results indicate that the abnormal endothelial cells, which overgrew the posterior surface of the explant within 3.5 years, originated from the patient (XY) but not from the donor tissue (XX). Extreme aggressivity of the cells could be clinically correlated with the finding of retrocorneal membrane on the iris, anterior synechiae, corectopia and secondary glaucoma. Conclusion PPCD recurrence is caused by the abnormal proliferation and migration of the patient’s aberrant endothelium, which remained localized at the periphery of the original cornea, into the donor graft. The presence of retrocorneal membrane prior to the surgery should be considered as an indication of potential aggressive behavior of these cells and it should be taken into consideration prior to the indication of corneal grafting.

Purpose Because cytogenetic data are essential for risk stratification of childhood acute myeloid leukemia (AML), the impact of chromosomal aberrations is crucial. Patients and Methods Data of a large group of patients younger than 18 years treated according to study AML–Berlin-Frankfurt-Münster (BFM) 98 (n = 454), including their cytogenetics, were analyzed. Results The favorable outcome in the subgroups of patients with t(8;21), inv(16), and t(15;17), with an overall survival of 91% (SE, 4%), 92% (SE, 6%), and 87% (SE, 5%), respectively, was confirmed. Within this group, the 5-year probability of event-free survival (pEFS) of all 17 children with t(8;21) and additional aberrations apart from del(9q) or −X/−Y was 100%. As expected, the cytogenetic finding of a complex karyotype (n = 35; pEFS, 33%; SE, 8%) or a monosomy 7 (n = 12; pEFS, 17%; SE, 11%) was associated with a poor outcome. Compared with remaining patients with cytogenetic data (pEFS, 48%; SE, 2%), prognosis in patients with an MLL rearrangement (n = 91) was inferior (pEFS, 34%; SE, 5%; P = .0005). Particularly, children with t(9;11) and additional aberrations (n = 13; pEFS, 31%; SE, 14%) and MLL rearrangements other than t(9;11) and t(11;19) (n = 41; pEFS, 24%; SE, 7%) had an unfavorable outcome. Nine patients with aberrations in 12p showed an adverse prognosis (pEFS, 11%; SE, 10%). The outcome of patients with aberrations of chromosome 5 (n = 13) was better than expected (pEFS, 50%; SE, 13%). Conclusion Because the prognostic value of rare recurrent chromosomal aberrations still has to be elucidated, these data will contribute to future risk stratification for the treatment of pediatric AML.

UNLABELLED Telomere length was evaluated by terminal repeat fragment method in 66 previously untreated patients with B-chronic lymphocytic leukemia (B-CLL) to ascertain whether telomere shortening was associated with genomic aberrations, immunoglobulin variable heavy chain (IgVH) mutational status, CD38 and ZAP-70 expression, and telomerase activity. Chromosomal aberrations were present in peripheral blood cells of 73% patients (48/66), no difference in telomere length between patients with good and intermediate prognosis according to cytogenetics was found. Association between telomere length and IgVH mutational status, ZAP-70 and CD38 expression was proved as significantly shorter telomeres in patients with unmutated IgVH status (p=0.01) and ZAP-70 positivity (p=0.01) and CD38 positivity (p=0.05) were detected. Telomerase activity was positive in 11 patients out of 21 examined, correlation between telomere length and telomerase activity was found (p=0.05). Telomere length and telomerase activity in combination with other prognostic parameters complete the risk profile of B-CLL patients and might serve for an easy decision on optimal treatment strategy. KEYWORDS B-chronic lymphocytic leukemia, telomere length, telomerase activity, chromosomal aberrations, prognosis.

Abstract 762 Pediatric AML is a heterogeneous disease. We showed that differential outcomes of 11q23/MLL-rearranged AML depend on the fusion partner involved in the translocation in a large international retrospective study from 11 collaborative groups comprising 756 children (Balgobind et al, Blood 2009). In the current analysis we focused on the clinical characteristics and prognostic impact of additional cytogenetic aberrations (ACA). All patients with complete karyotypes (n=733) were centrally reviewed and classified for type and number of aberrations. Cases with 2 or more aberrations (including 11q23/MLL-rearrangement), were included in the ACA group (n=344/733, 47%). Numerical and structural ACA were divided into gains and losses of full or partial chromosomes, and balanced translocations were defined by the breakpoints. A complex karyotype was defined as 3 or more aberrations. ACA occurring in at least 10 patients were considered for statistical analysis, including differences in presenting characteristics (chi-square or Fisher's exact tests) and survival estimates (Kaplan-Meier method and Cox proportional hazards model for Event free survival (EFS), 5 year estimates and 95% confidence intervals (CI) are given). Multivariate analysis included the fusion partners with independent prognostic significance, as identified by Balgobind et al. Patients with ACA were found to be older (median age, 3.0 y vs 1.8 y, p=.001) and had a higher frequency of FAB M7 phenotype (4.5 % vs 1.1 %, p=.007) than those without ACA. For the complete cohort (n=733) estimates of EFS and overall survival (OS) were .44 and .56, and cumulative incidence of relapse (CIR) was .35 (standard errors .03). Patients with ACA showed significantly poorer clinical outcome than cases without ACA (EFS .38 vs .48, p=.002; OS .47 vs .62, p Disclosures: Smith: Pfizer, Inc:.

Near-tetraploid acute myeloid leukemias (NT-AML) constitute an unusual presentation defined by blasts containing 80–104 chromosomes without aberrant near-diploid metaphases. Only about 60 NT-AML cases have been described so far in the literature.1 Reported cases where detailed information was available can be classified as M2 AML with duplication of t(8;21)(q22;q22) (N=13),1, 2 acute promyelocytic leukemias (M3 AML) with duplication of t(15;17)(q22;q21) (N=4)1, 3 – both categories reported mostly from Asian patients – or acute erythroleukemias M6 AML (N=7)1 and M0-M5 AML putatively originating from pluripotent myeloid progenitors characterized by erythroblastic and/or megakaryocytic (EM) dysplasia (N=17).4 Near-tetraploid blast cells are characterized by their large size on bone marrow smears,2, 3, 4 but individual chromosomal abnormalities are often difficult to analyze because of their 'fuzzy appearance'.1, 4

We identified cytogenetically unrelated clones in the bone marrow of 12 of 240 patients with myelodysplastic syndrome (MDS) and in 3 of 232 patients with acute myeloid leukemia (AML). In addition, unrelated single-cell abnormalities were found in six MDS and two AML patients. The most commonly encountered abnormalities present in the unrelated clones in patients with refractory anemia (RA) were del(5q), +8, and -7. In blastic types of MDS and AML trisomy 8 was found in two of eight patients while in the remaining cases the chromosome abnormalities were diverse and nonspecific. The presence of the chromosomally unrelated clones, together with recent data on the early appearance of monoclonality provided by molecular biology studies, support the interpretation that aberrations such as +8 and del(5q) are actually secondary abnormalities that develop during tumor progression in a cell with a primary submicroscopic genomic rearrangement.