Background Recurrent genetic lesions provide basis for risk assessment in pediatric acute lymphoblastic leukemia (ALL). However, current prognostic classifiers rely on a limited number of predefined sets of alterations. Methods Disease-relevant copy number aberrations (CNAs) were screened genome-wide in 260 children with B-cell precursor ALL. Results were integrated with cytogenetic data to improve risk assessment. Results CNAs were detected in 93.8% ( n = 244) of the patients. First, cytogenetic profiles were combined with IKZF1 status ( IKZF1 normal , IKZF1 del and IKZF1 plus ) and three prognostic subgroups were distinguished with significantly different 5-year event-free survival (EFS) rates, IKAROS-low ( n = 215): 86.3%, IKAROS-medium ( n = 27): 57.4% and IKAROS-high ( n = 18): 37.5%. Second, contribution of genetic aberrations to the clinical outcome was assessed and an aberration-specific score was assigned to each prognostically relevant alteration. By aggregating the scores of aberrations emerging in individual patients, personalized cumulative values were calculated and used for defining four prognostic subgroups with distinct clinical outcomes. Two favorable subgroups included 60% of patients ( n = 157) with a 5-year EFS of 96.3% (excellent risk, n = 105) and 87.2% (good risk, n = 52), respectively; while 40% of patients ( n = 103) showed high ( n = 74) or ultra-poor ( n = 29) risk profile (5-year EFS: 67.4% and 39.0%, respectively). Conclusions PersonALL, our conceptually novel prognostic classifier considers all combinations of co-segregating genetic alterations, providing a highly personalized patient stratification.

BackgroundThe clinical and genetic heterogeneity of diffuse large B-cell lymphoma (DLBCL) presents distinct challenges in predicting response to therapy and overall prognosis. The main objective of this study was to assess the application of the immunohistochemistry- and interphase fluorescence in situ hybridization (FISH)-based molecular markers in the diagnosis of DLBCL and its prognostic value in patients treated with rituximab-based immunochemotherapy.MethodsThis is a multicenter, retrospective study, which analyzed data from 7 Hungarian hematology centers. Eligible patients were adults, had a histologically confirmed diagnosis of DLBCL, were treated with rituximab-based immunochemotherapy in the first line, and had available clinicopathological data including International Prognostic Index (IPI). On the specimens, immunohistochemistry and FISH methods were performed. Germinal center B-cell like (GCB) and non-GCB subtypes were classified by the Hans algorithm. Outcomes included overall survival (OS), event-free survival (EFS), and EFS at 2 years (EFS24). For survival analysis, we used Kaplan-Meier curves with the log-rank test and multivariate Cox regression.ResultsA total of 247 DLBCL cases were included. Cases were positive for MYC, BCL2, BCL6, and MUM1 expression in 52.1%, 66.2%, 72.6%, and 77.8%, respectively. BCL6 translocation, BCL2 gene copy number (GCN) gain, IGH::MYC translocation, MYC GCN gain, IGH::BCL2 translocation, and BCL6 GCN gain were detected in 21.4%, 14.1%, 7.3%, 1.8%, 7.3%, and 0.9%, respectively. At a median follow-up of 52 months, 140 patients (56.7%) had disease progression or relapse. The Kaplan-Meier estimate for EFS24 was 56.2% (CI: 50.4–62.8%). In univariate analysis, only IPI and BCL6 expression were significant predictors of both OS and EFS, whereas MUM1 predicted EFS only. In multivariate analysis, the IPI score was a significant independent negative, whereas MIB-1 and BCL6 protein expressions were significant independent positive predictors of both OS and EFS.ConclusionIn our study, we found that only IPI, BCL6 protein expression and MIB-1 protein expression are independent predictors of survival outcomes in DLBCL. We did not find any difference in survival by GCB vs. non-GCB subtypes. These findings may improve prognostication in DLBCL and can contribute to designing further research in the area.

A diffúz nagy B-sejtes limfóma 5 éves általános túlélése a mai kezelések mellett 60–70%, melynek hátterében a betegség komplex heterogenitása állhat. Célkitűzés Magyarországi multicentrikus tanulmányban a betegség fenotipikus, citogenetikai, genomexpressziós profil- és geográfiai heterogenitásának vizsgálata. Módszer A 276 formol-paraffinos betegmintát Hans' algoritmus és dupla protein expresszor státusz alapján klasszifikáltuk. A IGH::MYC , IGH::BCL2 , BCL6 gén átrendeződéseket, valamint a MYC , BCL2 és BCL6 gének nyerését interfázis citogenetikával vizsgáltuk. Az RNA-seq-alapú génexpressziós profilvizsgálat 173 formol-paraffinos mintán volt elvégezhető. Eredmények A Hans' fenotípus alapján 103 (37,3%) germinális centrum B-sejt-szerű és 173 (62,7%) nem germinalis B-sejt-szerű limfómát azonosítottunk, mely besorolás 82,6%-os megegyezést mutatott a genomexpressziós profilstratifikációval. Tripla aberrációt mutató limfóma nem fordult elő. Izolált IGH :: MYC átrendeződés, valamint MYC , BCL2 , BCL6 géntöbblet mindkét Hans' csoportban jelen volt. Az IGH::BCL2 átrendeződés izoláltan vagy kombinációban, szignifikánsan és kizárólag a germinális centrum B-sejt-szerű csoportban volt azonosítható, míg a BCL6 átrendeződés szignifikáns halmozódást mutatott a nem germinális centrum B-sejt-szerű Hans' csoportban. A dupla protein expresszor fenotípus pozitív prediktív értéke mindkét Hans' csoport molekuláris alcsoportjaira alacsony, a 0,04–0,19, illetve a 0,12–0,30, tartományba esett, míg negatív prediktív értéke mindkét főcsoport összes releváns molekuláris alcsoportjában 1,00-nek felelt meg. Következtetések A IGH :: MYC átrendeződés nem Hans' csoport specifikus genotípus. Az IGH::BCL2 átrendeződés germinalis centrum, a BCL6 átrendeződés pedig a nem germinalis centrum B-sejt-szerű limfóma fémjele. A dupla protein expresszor negatív fenotípus mellett IGH::MYC és/vagy IGH::BCL2 átrendeződés nem fordult elő.

Plasma cell disorders are clonal outgrowths of pre-malignant or malignant plasma cells, characterized by extensive chromosomal aberrations. Centrosome abnormalities are a major driver of chromosomal instability in cancer but their origin, incidence, and composition in primary tumor cells is poorly understood. Using cutting-edge, semi-automated high-throughput electron tomography, we characterized at nanoscale 1386 centrioles in CD138pos plasma cells from eight healthy donors and 21 patients with plasma cell disorders, and 722 centrioles from different control populations. In plasma cells from healthy individuals, over-elongated centrioles accumulated with age. In plasma cell disorders, centriole over-elongation was notably frequent in early, pre-malignant disease stages, became less pronounced in overt multiple myeloma, and almost entirely disappeared in aggressive plasma cell leukemia. Centrioles in other types of patient-derived B cell neoplasms showed no over-elongation. In contrast to current belief, centriole length appears to be highly variable in long-lived, healthy plasma cells, and over-elongation and structural aberrations are common in this cell type. Our data suggest that structural centrosome aberrations accumulate with age in healthy CD138pos plasma cells and may thus play an important role in early aneuploidization as an oncogenic driver in plasma cell disorders.

Purpose:For the identification of high-risk patients in diffuse large B-cell lymphoma (DLBCL), we investigated the prognostic significance of in vivo radiomics derived from baseline [18F]FDG PET/CT and clinical parameters.Methods:Pre-treatment [18F]FDG PET/CT scans of 85 patients diagnosed with DLBCL were assessed. The scans were carried out in two clinical centers. Two-year event-free survival (EFS) was defined. After delineation of lymphoma lesions, conventional PET parameters and in vivo radiomics were extracted. For 2-year EFS prognosis assessment, the Center 1 dataset was utilized as the training set and underwent automated machine learning analysis. The dataset of Center 2 was utilized as an independent test set to validate the established predictive model built by the dataset of Center 1.Results:The automated machine learning analysis of the Center 1 dataset revealed that the most important features for building 2-year EFS are as follows: max diameter, neighbor gray tone difference matrix (NGTDM) busyness, total lesion glycolysis, total metabolic tumor volume, and NGTDM coarseness. The predictive model built on the Center 1 dataset yielded 79% sensitivity, 83% specificity, 69% positive predictive value, 89% negative predictive value, and 0.85 AUC by evaluating the Center 2 dataset.Conclusion:Based on our dual-center retrospective analysis, predicting 2-year EFS built on imaging features is feasible by utilizing high-performance automated machine learning.

Background: The genomic landscape of pediatric acute lymphoblastic leukemia (ALL) is heterogeneous with distinct copy number aberrations (CNAs) being detectable in vast majority of the patients. A subset of these alterations provides prognostic and/or predictive information; therefore, various CNA-based patient classifiers were introduced in the past. Aims: We applied a next-generation sequencing (NGS) based method to comprehensively screen for recurrent, disease-relevant CNAs in a cohort of Hungarian patients, allowing us to establish novel patient risk stratification approaches. Methods: Diagnostic bone marrow samples from 261 children with B-ALL and 22 matching samples drawn from 21 patients at first or second relapse were investigated by digital multiplex ligation-dependent probe amplification (digitalMLPA) using the ALL-specific D007 probemix. DigitalMLPA libraries were sequenced on an NGS platform. Whole chromosome gains and losses, as well as subchromosomal CNAs were simultaneously profiled. Survival rates were estimated using the Kaplan-Meier method and compared by log-rank tests in R version 4.1.2. Results: In total, 1,400 CNAs including numerical chromosomal aberrations and subchromosomal CNAs were detected in 93.5% of the diagnostic samples. On average, 5.36 CNAs were observed per patient with a mean of 2.45 subchromosomal alterations. Subtype-defining aberrations were identified in 36.0% of the patients with hyperdiploidy and iAMP21 detected in 32.2% and 3.8% of the cases, respectively. Numerous CNAs in disease-relevant genes responsible for cell cycle control, lymphoid development, signaling, or tumor suppression were identified. Considering all affected exons and an upstream region, 10 distinct patterns of IKZF1 deletion were observed. Unbiased co-segregation analysis revealed 15 positive and 3 negative correlations between the various CNAs, e.g. common co-occurrence of iAMP21 and CDKN2A/B deletion, and negative correlation between ETV6 and BTG1 deletions. Comparative analysis of diagnostic and matching relapse samples revealed characteristic temporal changes of copy number profiles with additional aberrations (65%), as well as both emerging and disappearing CNAs (30%) detected at relapse as compared with diagnosis. One patient did not show CNAs in the analyzed samples (5%). Prognostic subgroups determined based on cytogenetic findings were combined (i) with IKZF1del/IKZF1plus status and (ii) with CNA subgroups defined by a comprehensive digitalMLPA profiling. The combined genetic classification methods identified 3 and 4 patient subgroups, respectively, with significantly different 5-year progression-free survival rates. Summary/Conclusion: Comprehensive and highly optimized CNA profiling with digitalMLPA revealed subtype-defining gross-chromosomal changes and additional disease-relevant subchromosomal CNAs in a large cohort of Hungarian patients treated with ALL IC BFM protocols. Comparative scrutiny of matching diagnostic and relapse samples from 21 patients unveiled two different patterns of temporal evolution. Two novel risk stratification approaches have been established by combining cytogenetic data with digitalMLPA-based copy number profiling, with one of those laying more emphasis on CNA data than seen in previously introduced classifications, and the other one combining cytogenetic data with IKAROS status for the first time. DigitalMLPA offers a fast, reliable and standardizable DNA copy number analysis which is easily implementable in the diagnostic workflow of pediatric ALL.

OBJECTIVES:Targeted therapies in the management of patients with lung cancer provide significantly better outcome compared to chemotherapy. Detection of the anaplastic lymphoma kinase (ALK) gene rearrangement has great predictive value for treatment with small molecule tyrosine kinase inhibitor (crizotinib and alectinib commonly). Fluorescent in situ hybridisation (FISH) assay is a basic diagnostic test designed for detecting ALK gene rearrangements. Although being considered as gold standard method by IASLC's guideline, it is often regarded as difficult and error prone. Our aim was to examine a unique atypical ALK FISH pattern, revealed during a systematic large-scale monitoring, which carries the great risk of misinterpretation, hence may result in loss of patients eligible for targeted therapy. MATERIALS AND METHODS:Tissue and cytology samples from nearly one thousand patients with advanced stage non-small cell lung cancer (NSCLC, n = 996) were routinely examined by ALK FISH and immunohistochemistry (Ventana ALK-D5F3-CDx assay). Anchored Multiplex PCR based Next Generation Sequencing (AMP-NGS) was used to detect fusion gene transcripts in ambiguous cases. RESULTS:Fifty-nine (5,9%) of the cases were positive with ALK FISH test. Three cases showed atypical pattern with a significantly reduced sized red (3') signal and complete loss of green signals. Digital signal measurement confirmed this finding, showing consistent attenuation of 3' signals throughout the tumours. In all three cases AMP-NGS and ALK IHC verified the presence of a fusion gene and expressed oncoprotein, respectively. CONCLUSION:Approximately 5% of the 59 ALK positive cases exhibited atypical attenuated isolated 3' signal pattern. The immunohistochemistry and AMP-NGS examinations helped to clarify the presence of oncoprotein and the fusion gene, respectively. Our results emphasize the importance of extensive exploration of the genetic background of any unexpected FISH finding to avoid false diagnosis. This enables clinicians to indicate the adequate therapy with higher efficiency for patients suffering from NSCLC.

Introduction: Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy characterized by a heterogeneous genomic landscape. Copy number aberrations (CNA) emerge during the development, progression and treatment resistance of ALL, and can serve as genomic markers for prognostic classification of patients or for scrutinizing clonal evolution associated with relapse. While identification of distinct CNAs with well-characterized prognostic significance has its own value, uncovering the co-segregation of driver aberrations in individual patient samples could allow for a more personalized risk assessment and treatment response prediction. Methods: Disease-relevant CNAs were profiled in children with B- or T-cell precursor ALL using a next-generation sequencing based digital multiplex ligation-dependent probe amplification (digitalMLPA) assay containing 598 probes specific for 54 genes with key relevance in ALL. Besides the diagnostic samples of 91 patients treated according to the BFM protocols, 14 matching samples drawn at the time of first or second relapse were comparatively analyzed. Clonal relationship between B-cell precursor cell populations prevailing at different time points during the disease course was also investigated by screening immunoglobulin heavy-chain gene rearrangements in matching diagnostic and relapse samples using Illumina deep-sequencing with >20,000x coverage. Results: Whole chromosome gains and losses, subchromosomal CNAs as well as alterations conferring intrachromosomal gene fusions were simultaneously identified by digitalMLPA with results available within 36 hours. Aberrations were observed in 96% of diagnostic patient samples and increased numbers of CNAs were detected in individual samples at the time of relapse as compared to diagnosis. DigitalMLPA results were successfully validated by conventional MLPA, FISH and PCR data. Comparative scrutiny of 24 matching diagnostic and relapse samples from 11 patients harboring CNAs revealed three different patterns of clonal relationships with (i) one patient displaying identical CNA profiles at diagnosis and relapse, (ii) six patients showing clonal evolution with all lesions detected at diagnosis being present at relapse and (iii) four patients displaying conserved as well as lost or gained CNAs at the time of relapse, suggestive of the presence of a common ancestral cell compartment giving rise to clinically manifest leukemia at different time points during the disease course. Time between diagnosis and first relapse of T-ALL patients displaying altered CNA profiles suggested a prolonged time requirement of clonal evolution, and of the development of manifest leukemia from an ancestral clone compared to the quick return of an identical clone at the time of relapse. Comparison of the IGH gene rearrangements identified at diagnoses and relapse revealed identical compositions of the most abundant clonotypes in all but one B-ALL patients analyzed; hence, IGH repertoire did not reveal an additional depth of clonal history in our cohort, e.g. by demonstrating the presence of an ancestral clone as the major source of clonal expansion at disease progression in a patient with altered CNA profiles suggesting direct clonal evolution between diagnosis and relapse. Copy number profiles acquired by digitalMLPA were used for determining CNA-based risk groups (Table 1) which were combined with karyotyping and molecular cytogenetic data in order to establish an extended prognostic classifier for patients with B-cell precursor ALL. This novel classifier distinguished four combined genetic risk groups showing significantly different 5-year survival rates (GR: 97%, IR: 84%, IHR: 63% and PR: 13%). Conclusions: DigitalMLPA allows for a rapid, scalable and highly optimized copy number profiling of genomic regions recurrently altered by driver aberrations in pediatric ALL. Based on the comparison of CNA profiles at diagnosis and relapse, clonal evolution and emergence of relapse from an ancestral clone are the predominant driving mechanisms of disease progression. Comprehensive copy number profiling by digitalMLPA identifies distinct prognostic groups for risk assessment in B-cell precursor ALL. Supporting grants : LP95021, K_16 #119950, NVKP_16-1-2016-0004, KH17-126718, BO/00320/18/5, FK_19 #131476, ÚNKP-19-4-SE-77 Disclosures Benard-Slagter: MRC Holland: Employment. de Groot:MRC Holland: Employment. Savola:MRC Holland: Employment.

Acute lymphoblastic leukemia is the most common pediatric cancer characterized by a heterogeneous genomic landscape with copy number aberrations occurring at various stages of pathogenesis, disease progression, and treatment resistance. In this study, disease-relevant copy number aberrations were profiled in bone marrow samples of 91 children with B- or T-cell precursor acute lymphoblastic leukemia using digital multiplex ligation-dependent probe amplification (digitalMLPATM). Whole chromosome gains and losses, subchromosomal copy number aberrations, as well as unbalanced alterations conferring intrachromosomal gene fusions were simultaneously identified with results available within 36 hours. Aberrations were observed in 96% of diagnostic patient samples, and increased numbers of copy number aberrations were detected at the time of relapse as compared with diagnosis. Comparative scrutiny of 24 matching diagnostic and relapse samples from 11 patients revealed three different patterns of clonal relationships with (i) one patient displaying identical copy number aberration profiles at diagnosis and relapse, (ii) six patients showing clonal evolution with all lesions detected at diagnosis being present at relapse, and (iii) four patients displaying conserved as well as lost or gained copy number aberrations at the time of relapse, suggestive of the presence of a common ancestral cell compartment giving rise to clinically manifest leukemia at different time points during the disease course. A newly introduced risk classifier combining cytogenetic data with digitalMLPATM-based copy number aberration profiles allowed for the determination of four genetic subgroups of B-cell precursor acute lymphoblastic leukemia with distinct event-free survival rates. DigitalMLPATM provides fast, robust, and highly optimized copy number aberration profiling for the genomic characterization of acute lymphoblastic leukemia samples, facilitates the decipherment of the clonal origin of relapse and provides highly relevant information for clinical prognosis assessment.

EML4‐ALK gene fusion (inv2(p21p23)) of non‐small cell lung cancer (NSCLC) predisposes to tyrosine kinase inhibitor treatment. One of the gold standard diagnostics is the dual color (DC) break‐apart (BA) FISH technique, however, the unusual closeness of the involved genes has been suggested to raise likelihood of random co‐localization (RCL) of signals. Although this is suspected to decrease sensitivity (often to as low as 40–70%), the exact level and effect of RCL has not been revealed thus far. Signal distances were analyzed to the 0.1 µm precision in more than 25,000 nuclei, via automated high content‐image cytometry. Negative and positive controls were created using conventional DC BA‐, and inv2(p21p23) mimicking probe‐sets, respectively. Average distance between red and green signals was 9.72 pixels (px) (±5.14px) and 3.28px (±2.44px), in positives and negatives, respectively; overlap in distribution being 41%. Specificity and sensitivity of correctly determining ALK status was 97% and 29%, respectively. When investigating inv2(p21p23) with DC BA FISH, specificity is high, but seven out of ten aberrant nuclei are inevitably falsely classified as negative, due to the extreme level of RCL. Together with genetic heterogeneity and dilution effect of non‐tumor cells in NSCLC, this immense analytical false negativity is the primary cause behind the often described low diagnostic sensitivity. These results convincingly suggest that if FISH is to remain a gold standard for detecting the therapy relevant inv(2), either a modified evaluation protocol, or a more reliable probe‐design should be considered than the current DC BA one. © 2018 International Society for Advancement of Cytometry

Multiple myeloma (MM) is a genetically heterogeneous disease with a diverse clinical outcome. Copy number alterations (CNAs), including whole chromosome and subchromosomal gains and losses, are common contributors of the pathogenesis and have demonstrated prognostic impact in MM. We tested the performance of digital multiplex ligation-dependent probe amplification (digitalMLPA), a novel technique combining MLPA and next-generation sequencing, to detect disease-related CNAs. Copy number status at 371 genomic loci was simultaneously analyzed in 56 diagnostic bone marrow samples, which were also examined by conventional MLPA and interphase fluorescence in situ hybridization (iFISH). On average, digitalMLPA identified 4.4 subchromosomal CNAs per patient. The increased number of probes compared with conventional MLPA allowed a detailed mapping of CNAs, especially on chromosome 1, where 24 different patterns were observed in 38 patients harboring loss(1p) and/or gain(1q). iFISH, MLPA, and digitalMLPA results at loci investigated by multiple methods showed a congruency of 95%. Besides precise characterization of hyperdiploid karyotypes not efficiently achievable by iFISH or MLPA, digitalMLPA unraveled 156 CNAs not detected by the other two methods in 45 patients (80%). In addition, we provide proof of principle that digitalMLPA can detect known point mutations, in this case the BRAFV600E. Our study demonstrates the robustness of digitalMLPA to profile CNAs and to screen point mutations in MM, which could efficiently be used in myeloma diagnostics.

Acute lymphoblastic leukemia (ALL) is the most frequent malignancy in children. In Hungary 60-70 new cases are diagnosed annually. The survival rate is 85-90% in developed countries with current treatment protocols. The most common genetic category of childhood ALL is the high hyperdiploid subtype (HHD) with chromosome numbers of 51 to 67. It accounts for approximately 25% of all cases. The prognosis is very good, though relapse occurs in ~15% of cases and there are data on the heterogeneity of this subgroup as well. In this paper we give an overview of the cytogenetic, clinical, epidemiological and prognostic features of this subgroup. We also demonstrate our interphase fluorescent in situ hybridization (iFISH) analysis performed retrospectively on 168 untreated bone marrow samples of precursor B pediatric ALL patients to reveal the numerical aberrations of chromosomes 4, 6, 10, 14, 17, 18, 21 and X, which are most frequently affected by gain in HHD ALL. Data from 48 high hyperdiploid patients indicated that high modal number (>55 chromosomes) and specific chromosomal gains (+4, +4/+6, +4/+17, +4/+18) exhibited significance in terms of beneficial overall survival.

Background: High hyperdiploid (HeH) pre-B pediatric acute lymphoblastic leukemia (B-pALL) is known to be heterogeneous by prognosis, but the stratification principals according to conventional cytogenetic analysis (CCA) are equivocal.Procedure: Untreated bone marrow samples of 214 B-pALL patients were previously classified according to the modal numbers (iMN8) based on the gains of the chromosomes 4, 6, 10, 14, 17, 18, 21, and X as revealed by consecutive and correlated 2x4 color interphase fluorescence in situ hybridization, and at least five years of follow up data were analyzed.Results: Data from 48 of the 53 HeH (iMN(8)>50) B-pALL patients indicated that among the age, gender, WBC, and iMN8 parameters, only the last was significantly associated with overall survival (pOS), which allowed the cases to be classified as iMN(8) 51-54 (75%) and iMN(8) >= 55 (95%). Among the specific chromosomal gains of +4, +4/+6, +4/+17 and +4/+18, the first exhibited the most significance in terms of beneficial outcomes. The better prognostic group according to the iMN(8) was associated with a significantly reduced complexity of the subclonal landscape. However, iMN(8) did not prove to be an independent variable but was instead overridden by isolated trisomy of chromosome 4.Conclusions: These data indicate that the better outcomes in the HeH B-pALL group arose from the gain of a specific chromosome that always ranks at the same position in the sequential acquisition of the affected chromosomes. (C) 2016 Elsevier Ltd. All rights reserved.

BackgroundPathogenesis of the non‐random accumulation of extra chromosomes in the low and high hyperdiploid (HeL, HeH) pre‐B pediatric acute lymphoblastic leukemia (B‐pALL) is largely unknown, and has been clarified with respect only to tetrasomic chromosomes. We analyzed the hierarchy of changes in chromosome number and chromosomal instability, as well as clonal heterogeneity and evolution, in the untreated bone marrow cell samples from 214 B‐pALL patients.ProcedureApplying relocation, 2 × 4 color interphase fluorescence in situ hybridization was used to detect copy number alterations (CNAs) of the most commonly involved chromosomes, 4, 6, 10, 14, 17, 18, 21, and X. This approach allowed us to acquire a dataset correlated for all eight parameters.ResultsBased on chromosome number, an average of 6.9 and 10.2, whereas according to unique constellation 15.3 and 26.7 subclones could be identified in the HeL and HeH subgroups, respectively. Cluster analysis revealed the order of CNAs to chromosomes was highly conserved, and network analysis indicated changes in chromosome number were sequential for 80–90% of all numerical aberrations. Significant chromosome instability was revealed in both subgroups of leukemia.ConclusionsData generated using this new approach indicate that chromosomal instability, which causes heterogeneity in the subclonal landscape, and the sequential changes to chromosome numbers, are both determining factors in the pathomechanism of the hyperdiploid B‐pALL. These new observations could prompt research into the mitotic machinery of leukemic cells to identify new therapeutic targets for treating this disease. Pediatr Blood Cancer 2014;61:2208–2214. © 2014 Wiley Periodicals, Inc.

Multiple myeloma (MM) is a genetically heterogeneous disease with diverse clinical outcomes. Interphase fluorescence in situ hybridization (i‐FISH) is the most commonly used approach to detect recurrent cytogenetic abnormalities in this malignancy. We aimed to assess the performance of multiplex ligation‐dependent probe amplification (MLPA) to reveal copy number abnormalities (CNAs) in MM. Diagnostic bone marrow samples from 81 patients were analyzed using 42 MLPA probes for the following regions: 1p32‐31, 1p21, 1q21.3, 1q23.3, 5q31.3, 12p13.31, 13q14, 16q12, 16q23, and 17p13. All samples were also screened by i‐FISH for the presence of hyperdiploidy, deletion/monosomy of chromosome 13, deletion of TP53, disruption of the immunoglobulin heavy‐chain gene, t(4;14), t(11;14), t(14;16), t(8;14), gain of 5q and abnormalities of chromosome 1. A total of 245 alterations were detected in 79 cases (98%). Investigating the same aberrations, the two methods showed a congruency of higher than 90%. A low proportion of cells with the relevant abnormality, focal CNAs and unmatched probes were responsible for the discrepancies. MLPA revealed 95 CNAs not detected by i‐FISH providing additional information in 53 cases (65%). Scrutiny of CNAs on chromosome 1, using more than 20 probes, revealed significant heterogeneity in size and location, and variable intra‐chromosomal and intra‐clonal rates of loss or gain. Our results suggest that MLPA is a reliable high‐throughput technique to detect CNAs in MM. Since balanced aberrations are key to prognostic classification of this disease, MLPA and i‐FISH should be applied as complementary techniques in diagnostic pathology. © 2013 Wiley Periodicals, Inc.