Advertisement

Assessing copy number aberrations and copy-neutral loss-of-heterozygosity across the genome as best practice: An evidence-based review from the Cancer Genomics Consortium (CGC) working group for chronic lymphocytic leukemia

      Highlights

      • Review of abnormalities detectable by metaphase chromosome analysis, FISH and microarray.
      • Clinical utility of genomic microarray.
      • Incorporation of array into clinical practice.

      Abstract

      The prognostic role of cytogenetic analysis is well-established in B-cell chronic lymphocytic leukemia (CLL). Approximately 80% of patients have a cytogenetic aberration. Interphase FISH panels have been the gold standard for cytogenetic evaluation, but conventional cytogenetics allows detection of additional abnormalities, including translocations, complex karyotypes and multiple clones. Whole genome copy number assessment, currently performed by chromosomal microarray analysis (CMA), is particularly relevant in CLL for the following reasons: (1) copy number alterations (CNAs) represent key events with biologic and prognostic significance; (2) DNA from fresh samples is generally available; and (3) the tumor burden tends to be relatively high in peripheral blood. CMA also identifies novel copy number variants and copy-neutral loss-of-heterozygosity (CN-LOH), and can refine deletion breakpoints. The Cancer Genomics Consortium (CGC) Working Group for CLL has performed an extensive literature review to describe the evidence-based clinical utility of CMA in CLL. We provide suggestions for the integration of CMA into clinical use and list recurrent copy number alterations, regions of CN-LOH and mutated genes to aid in interpretation.

      Keywords

      Introduction/background

      B-cell chronic lymphocytic leukemia (CLL), a mature B cell neoplasm, is the most common adult leukemia in the Western world. In the United States, CLL represents approximately 40% of adult leukemias, with an annual incidence of 2–6 per 100,000 and median age of diagnosis of 70 years of age. Incidence increases with age; however, 30% of patients are younger than 60 at the time of diagnosis and 15% are younger than 50 [
      • Campo E.
      • et al.
      Chronic lymphocytic leukaemia/small lymphocytic lymphoma.
      • Rai K.R.
      • Jain P.
      Chronic lymphocytic leukemia (CLL)-Then and now.
      . CLL has the highest genetic predisposition of all hematologic neoplasms; approximately 5–10% of cases have a family history of CLL or other non-Hodgkin lymphoma [
      • Goldin L.R.
      • et al.
      Familial risk of lymphoproliferative tumors in families of patients with chronic lymphocytic leukemia: results from the Swedish Family-Cancer Database.
      ].
      CLL exhibits a highly variable clinical course, with life expectancies ranging from a few months to decades. Approximately one-third of patients experience an indolent course with normal survival, one-third experience an initially indolent disease that eventually progresses, and one-third experience an aggressive disease course. Stratifying these patients, particularly in early-stage or asymptomatic disease when most patients are diagnosed, is part of the challenge of this hematologic malignancy. The Rai and Binet clinical staging systems, established in the mid-1970 s, remain useful in defining disease extent and prognosis; however, these systems fall short in distinguishing those who will experience an aggressive clinical course, particularly patients with early stage disease.
      For the past 25 years, the incorporation of genetic markers has become increasingly important in stratifying patients (reviewed in Zenz [
      • Zenz T.
      • et al.
      Importance of genetics in chronic lymphocytic leukemia.
      ]), and cytogenetic analysis is well-established as playing a key role in both diagnosis and prognosis. As many as 80% of patients have a cytogenetic aberration. Since the publication of the Dohner hierarchical classification in 2000 [
      • Dohner H.
      • et al.
      Genomic aberrations and survival in chronic lymphocytic leukemia.
      ], interphase fluorescence in situ hybridization (FISH) with a four- or five-assay panel has been the gold standard for cytogenetic evaluation [
      • Wierda W.G.
      • et al.
      NCCN guidelines insights: chronic lymphocytic leukemia/small lymphocytic leukemia, version 1.2017.
      ]; however, metaphase chromosome analysis, which provides a whole genome assessment, allows detection of abnormalities not in the panel, including translocations, complex karyotypes and multiple clones. Chromosomal microarray analysis (CMA) can interrogate the same aberrations as the FISH panel and, like metaphase chromosome analysis, can provide a whole genome assessment, although it will miss balanced translocations and low level clones. In addition, CMA can detect copy-neutral loss-of-heterozygosity (CN-LOH) and chromothripsis, which the other technologies are not able to do. This document focuses on the clinical utility of CMA in CLL based on a review of peer-reviewed publications.

      Methods

      A systematic literature search was performed for peer-reviewed manuscripts focusing on CNAs and CN-LOH assessment in CLL published between 2000 and 2017. Workgroup members reviewed 72 well-powered studies. The level of evidence for clinical significance of CNAs was assigned as follows: Level 1, present in current WHO classification and/or professional practice guidelines (NCCN); Level 2, recurrent in well-powered studies with suspected clinical significance based upon expert review; and Level 3, recurrent, but uncertain prognostic significance. Single case aberrations were not included. The list of clinically significant and/or recurrent CNAs selected and evaluated based on this process is provided in Table 1.
      Table 1Regions of recurrent copy number change in CLL.
      Chromosome/AbnormalityPrevalenceRelevant genesStrength ofPrognosticStrength of evidenceCommentsReferences
      regiontype(%)evidence forsignificancefor prognosis
      gene(Level
      Level 1: present in WHO classification or professional practice guidelines; Level 2: recurrent in well-powered studies with suspected clinical significance; Level 3: recurrent, but uncertain prognostic significance.
      )
      1pGain?2–5UnknownN/AFavorableSuspected (2)
      • Pfeifer D.
      • et al.
      Genome-wide analysis of DNA copy number changes and LOH in CLL using high-density SNP arrays.
      ,
      • Houldsworth J.
      • et al.
      Genomic imbalance defines three prognostic groups for risk stratification of patients with chronic lymphocytic leukemia.
      ,
      • Chapiro E.
      • et al.
      Gain of the short arm of chromosome 2 (2p) is a frequent recurring chromosome aberration in untreated chronic lymphocytic leukemia (CLL) at advanced stages.
      1q23.2q23.3Loss15UnknownN/AUnknownN/A (3)
      • Edelmann J.
      • et al.
      High-resolution genomic profiling of chronic lymphocytic leukemia reveals new recurrent genomic alterations.
      ,
      • Ma D.
      • et al.
      Array comparative genomic hybridization analysis identifies recurrent gain of chromosome 2p25.3 involving the ACP1 and MYCN genes in chronic lymphocytic leukemia.
      ,
      • Tyybakinoja A.
      • Vilpo J.
      • Knuutila S.
      High-resolution oligonucleotide array-CGH pinpoints genes involved in cryptic losses in chronic lymphocytic leukemia.
      2p12p25.3Gain5–30ACP1, MYCN, ALK, REL, BCL11AMYCN (Established), REL, BCL11A (Candidate)UnfavorableEstablished (if MYCN included) (1)
      • Pfeifer D.
      • et al.
      Genome-wide analysis of DNA copy number changes and LOH in CLL using high-density SNP arrays.
      ,
      • Edelmann J.
      • et al.
      High-resolution genomic profiling of chronic lymphocytic leukemia reveals new recurrent genomic alterations.
      ,
      • Patel A.
      • et al.
      Validation of a targeted DNA microarray for the clinical evaluation of recurrent abnormalities in chronic lymphocytic leukemia.
      ,
      • Stevens-Kroef M.J.
      • et al.
      Identification of prognostic relevant chromosomal abnormalities in chronic lymphocytic leukemia using microarray-based genomic profiling.
      ,
      • Schweighofer C.D.
      • et al.
      Genomic variation by whole-genome SNP mapping arrays predicts time-to-event outcome in patients with chronic lymphocytic leukemia: a comparison of CLL and HapMap genotypes.
      ,
      • Houldsworth J.
      • et al.
      Genomic imbalance defines three prognostic groups for risk stratification of patients with chronic lymphocytic leukemia.
      ,
      • Shao L.
      • et al.
      Array comparative genomic hybridization detects chromosomal abnormalities in hematological cancers that are not detected by conventional cytogenetics.
      ,
      • Chapiro E.
      • et al.
      Gain of the short arm of chromosome 2 (2p) is a frequent recurring chromosome aberration in untreated chronic lymphocytic leukemia (CLL) at advanced stages.
      ,
      • Ma D.
      • et al.
      Array comparative genomic hybridization analysis identifies recurrent gain of chromosome 2p25.3 involving the ACP1 and MYCN genes in chronic lymphocytic leukemia.
      ,
      • Fabris S.
      • et al.
      Chromosome 2p gain in monoclonal B-cell lymphocytosis and in early stage chronic lymphocytic leukemia.
      ,
      • Forconi F.
      • et al.
      Genome-wide DNA analysis identifies recurrent imbalances predicting outcome in chronic lymphocytic leukaemia with 17p deletion.
      ,
      • Jarosova M.
      • et al.
      Gain of chromosome 2p in chronic lymphocytic leukemia: significant heterogeneity and a new recurrent dicentric rearrangement.
      3p21.31Loss1–5ATRIP, CDC25ACandidateUnknownN/A (3)
      • Edelmann J.
      • et al.
      High-resolution genomic profiling of chronic lymphocytic leukemia reveals new recurrent genomic alterations.
      ,
      • Kolquist K.A.
      • et al.
      Evaluation of chronic lymphocytic leukemia by oligonucleotide-based microarray analysis uncovers novel aberrations not detected by FISH or cytogenetic analysis.
      ,
      • Salaverria I.
      • et al.
      Detection of chromothripsis-like patterns with a custom array platform for chronic lymphocytic leukemia.
      3qGain2–19UnknownN/AUnfavorableSuspected (2)Appears to be particularly prevalent in Japanese
      • Houldsworth J.
      • et al.
      Genomic imbalance defines three prognostic groups for risk stratification of patients with chronic lymphocytic leukemia.
      ,
      • Kawamata N.
      • et al.
      Genetic differences between Asian and Caucasian chronic lymphocytic leukemia.
      ,
      • Tsukasaki K.
      • et al.
      Comparative genomic hybridization analysis of Japanese B-cell chronic lymphocytic leukemia: correlation with clinical course.
      4p15.2p16.3Loss14UnknownN/AUnfavorable (occurred with del(11q) or del(17p))Suspected (2)
      • Gunnarsson R.
      • et al.
      Array-based genomic screening at diagnosis and during follow-up in chronic lymphocytic leukemia.
      6p25.3Gain1UnknownN/AUnknownN/A (3)
      • Edelmann J.
      • et al.
      High-resolution genomic profiling of chronic lymphocytic leukemia reveals new recurrent genomic alterations.
      6p22.1Loss1Histone cluster, HFECandidateUnknownN/A (3)
      • Edelmann J.
      • et al.
      High-resolution genomic profiling of chronic lymphocytic leukemia reveals new recurrent genomic alterations.
      6qLoss3–6FOXO3CandidateIntermediateSuspected (2)
      • Cuneo A.
      • et al.
      Chronic lymphocytic leukemia with 6q- shows distinct hematological features and intermediate prognosis.
      ,
      • Wang D.M.
      • et al.
      Intermediate prognosis of 6q deletion in chronic lymphocytic leukemia.
      ,
      • Jarosova M.
      • et al.
      Chromosome 6q deletion correlates with poor prognosis and low relative expression of FOXO3 in chronic lymphocytic leukemia patients.
      ,
      • Nabhan C.
      • Raca G.
      • Wang Y.L.
      Predicting prognosis in chronic lymphocytic leukemia in the contemporary era.
      7pGain5–6UnknownN/AIntermediateSuspected (2)
      • Houldsworth J.
      • et al.
      Genomic imbalance defines three prognostic groups for risk stratification of patients with chronic lymphocytic leukemia.
      7qLoss1–2UnknownN/AUnknownN/A (3)
      • Schweighofer C.D.
      • et al.
      Genomic variation by whole-genome SNP mapping arrays predicts time-to-event outcome in patients with chronic lymphocytic leukemia: a comparison of CLL and HapMap genotypes.
      ,
      • Houldsworth J.
      • et al.
      Genomic imbalance defines three prognostic groups for risk stratification of patients with chronic lymphocytic leukemia.
      8p21Loss2–5TRIM35CandidateUnfavorableSuspected (2)Associated with established unfavorable changes (11q- and 17p-). Not established as an independent prognosticator
      • Houldsworth J.
      • et al.
      Genomic imbalance defines three prognostic groups for risk stratification of patients with chronic lymphocytic leukemia.
      ,
      • Grubor V.
      • et al.
      Novel genomic alterations and clonal evolution in chronic lymphocytic leukemia revealed by representational oligonucleotide microarray analysis (ROMA).
      8q24.1Gain5MYCCandidateUnfavorableSuspected (2)Often associated with 11q and 17p deletion; may not be independent
      • Houldsworth J.
      • et al.
      Genomic imbalance defines three prognostic groups for risk stratification of patients with chronic lymphocytic leukemia.
      • Edelmann J.
      • et al.
      High-resolution genomic profiling of chronic lymphocytic leukemia reveals new recurrent genomic alterations.
      ,
      • Houldsworth J.
      • et al.
      Genomic imbalance defines three prognostic groups for risk stratification of patients with chronic lymphocytic leukemia.
      9q13q21.11Loss1UnknownN/AUnknownN/A (3)
      • Edelmann J.
      • et al.
      High-resolution genomic profiling of chronic lymphocytic leukemia reveals new recurrent genomic alterations.
      10q24Loss2UnknownN/AUnknownN/A (3)Clustered around NFKB2 gene locus
      • Edelmann J.
      • et al.
      High-resolution genomic profiling of chronic lymphocytic leukemia reveals new recurrent genomic alterations.
      ,
      • Stevens-Kroef M.J.
      • et al.
      Identification of prognostic relevant chromosomal abnormalities in chronic lymphocytic leukemia using microarray-based genomic profiling.
      ,
      • Schwaenen C.
      • et al.
      Automated array-based genomic profiling in chronic lymphocytic leukemia: development of a clinical tool and discovery of recurrent genomic alterations.
      11q22.3Loss10–20ATM, BIRC3, MRE11, H2AFXATM established, Others CandidateUnfavorableEstablished (1)
      • Wierda W.G.
      • et al.
      NCCN guidelines insights: chronic lymphocytic leukemia/small lymphocytic leukemia, version 1.2017.
      12Gain10–20UnknownN/AIntermediateEstablished (1)Unfavorable if NOTCH1 mutation is present
      • Wierda W.G.
      • et al.
      NCCN guidelines insights: chronic lymphocytic leukemia/small lymphocytic leukemia, version 1.2017.
      13q14Loss50–60DLEU2, miR-15a/16–1, DLEU1EstablishedFavorableEstablished (1)Co-deletion of RB1 may negatively impact time to treatment
      • Wierda W.G.
      • et al.
      NCCN guidelines insights: chronic lymphocytic leukemia/small lymphocytic leukemia, version 1.2017.
      ,
      • Dal Bo M.
      • et al.
      13q14 deletion size and number of deleted cells both influence prognosis in chronic lymphocytic leukemia.
      ,
      • Malek S.N.
      The biology and clinical significance of acquired genomic copy number aberrations and recurrent gene mutations in chronic lymphocytic leukemia.
      14q24.1q32.3Loss2UnknownN/AUnknownN/A (3)Associated with trisomy 12
      • Edelmann J.
      • et al.
      High-resolution genomic profiling of chronic lymphocytic leukemia reveals new recurrent genomic alterations.
      ,
      • Greipp P.T.
      • et al.
      Patients with chronic lymphocytic leukaemia and clonal deletion of both 17p13.1 and 11q22.3 have a very poor prognosis.
      ,
      • Cosson A.
      • et al.
      14q deletions are associated with trisomy 12, NOTCH1 mutations and unmutated IGHV genes in chronic lymphocytic leukemia and small lymphocytic lymphoma.
      15q15.1Loss4MGACandidateUnknownN/A (3)
      • Edelmann J.
      • et al.
      High-resolution genomic profiling of chronic lymphocytic leukemia reveals new recurrent genomic alterations.
      ,
      • Stevens-Kroef M.J.
      • et al.
      Identification of prognostic relevant chromosomal abnormalities in chronic lymphocytic leukemia using microarray-based genomic profiling.
      17p13.1Loss5–15TP53EstablishedUnfavorableEstablished (1)
      • Wierda W.G.
      • et al.
      NCCN guidelines insights: chronic lymphocytic leukemia/small lymphocytic leukemia, version 1.2017.
      17qGain1UnknownN/AUnfavorableSuspected (2)
      • Houldsworth J.
      • et al.
      Genomic imbalance defines three prognostic groups for risk stratification of patients with chronic lymphocytic leukemia.
      18pLoss3UnknownN/AUnfavorableSuspected (2)
      • Edelmann J.
      • et al.
      High-resolution genomic profiling of chronic lymphocytic leukemia reveals new recurrent genomic alterations.
      ,
      • Houldsworth J.
      • et al.
      Genomic imbalance defines three prognostic groups for risk stratification of patients with chronic lymphocytic leukemia.
      18Gain4UnknownN/AUnfavorableEstablished (1)Associated with trisomy 12
      • Ibbotson R.
      • et al.
      Coexistence of trisomies of chromosomes 12 and 19 in chronic lymphocytic leukemia occurs exclusively in the rare IgG-positive variant.
      19Gain2–5UnknownN/AUnfavorableEstablished (1)Associated with trisomy 12
      • Edelmann J.
      • et al.
      High-resolution genomic profiling of chronic lymphocytic leukemia reveals new recurrent genomic alterations.
      ,
      • Stevens-Kroef M.J.
      • et al.
      Identification of prognostic relevant chromosomal abnormalities in chronic lymphocytic leukemia using microarray-based genomic profiling.
      ,
      • Gunnarsson R.
      • et al.
      Array-based genomic screening at diagnosis and during follow-up in chronic lymphocytic leukemia.
      ,
      • Ibbotson R.
      • et al.
      Coexistence of trisomies of chromosomes 12 and 19 in chronic lymphocytic leukemia occurs exclusively in the rare IgG-positive variant.
      ,
      • Schwaenen C.
      • et al.
      Automated array-based genomic profiling in chronic lymphocytic leukemia: development of a clinical tool and discovery of recurrent genomic alterations.
      Genomic complexity3 or more CNAs10–15N/AUnfavorableEstablished (1)
      • Gunn S.R.
      • et al.
      The HemeScan test for genomic prognostic marker assessment in chronic lymphocytic leukemia.
      ,
      • Kolquist K.A.
      • et al.
      Evaluation of chronic lymphocytic leukemia by oligonucleotide-based microarray analysis uncovers novel aberrations not detected by FISH or cytogenetic analysis.
      ,
      • Stevens-Kroef M.J.
      • et al.
      Identification of prognostic relevant chromosomal abnormalities in chronic lymphocytic leukemia using microarray-based genomic profiling.
      ,
      • Schweighofer C.D.
      • et al.
      Genomic variation by whole-genome SNP mapping arrays predicts time-to-event outcome in patients with chronic lymphocytic leukemia: a comparison of CLL and HapMap genotypes.
      Chromothripsis(> 10 copy number states of 2 and 3)5SETD2, other markers across genome not definedEstablishedUnfavorableEstablished (1)
      • Edelmann J.
      • et al.
      High-resolution genomic profiling of chronic lymphocytic leukemia reveals new recurrent genomic alterations.
      ,
      • Parker H.
      • et al.
      Genomic disruption of the histone methyltransferase SETD2 in chronic lymphocytic leukaemia.
      ,
      • Malek S.N.
      The biology and clinical significance of acquired genomic copy number aberrations and recurrent gene mutations in chronic lymphocytic leukemia.
      low asterisk Level 1: present in WHO classification or professional practice guidelines; Level 2: recurrent in well-powered studies with suspected clinical significance; Level 3: recurrent, but uncertain prognostic significance.

      Evidence review

      Non-Cytogenetic prognostic markers

      Although the diagnosis of CLL in most cases can be made relatively easily by morphology and flow cytometry, other information is critical to determine prognosis for the patient. Prognostic indicators have included mutational status of the variable region of the immunoglobulin heavy chain (IGHV), expression of biomarkers including ZAP-70 and CD38, and cytogenetic aberrations. Molecular analysis of the immunoglobulin genes indicates that 50–60% of cases exhibit somatic hypermutation (mutIGHV, > 2% deviation from germline), while the remainder are unmutated (unmutIGHV, > 98% homology with germline). Patients with mutated IGHV have a better prognosis than those with unmutated IGHV, at least for those with low stage disease [
      • Campo E.
      • et al.
      Chronic lymphocytic leukaemia/small lymphocytic lymphoma.
      ]. Most recently, an international CLL working group has proposed a prognostic index, CLL-IPI, that includes TP53 status, IGHV mutational status, serum B2-microglobulin concentration, clinical stage, and patient age (Hallek, for the International CLL-IPI working group) [
      International, C.L.L.I.P.I.w.g.
      An international prognostic index for patients with chronic lymphocytic leukaemia (CLL-IPI): a meta-analysis of individual patient data.
      ].

      Detection of cytogenetic markers by metaphase chromosome analysis and FISH

      Cytogenetic analysis is a key component in diagnosis, prognosis and determination of optimal treatment strategies in CLL. Metaphase chromosome analysis provides a genome-wide view of abnormalities, but historically this method was hampered by poor growth of B-cells in culture. With the publication of the Dohner hierarchical classification in 2000 [
      • Dohner H.
      • et al.
      Genomic aberrations and survival in chronic lymphocytic leukemia.
      ], interphase FISH became the gold-standard test for cytogenetic evaluation in CLL.
      With the use of four FISH assays [for the detection of trisomy 12, and deletion of 13q14, 11q22 (ATM) and 17p13 (TP53)], FISH has an approximately 80% abnormality detection rate in CLL. Deletion of 13q14 is the most common finding, observed in approximately 50% of cases; trisomy 12, ATM deletion and TP53 deletion are seen in about 20%, 15–20% and 5–10%, respectively, of patients, with the distribution of these abnormalities varying with IGHV mutational status (WHO 2017, Table 13.01) [
      • Campo E.
      • et al.
      Chronic lymphocytic leukaemia/small lymphocytic lymphoma.
      ]. The four-probe assay FISH panel also provides useful prognostic information, with deletion of 13q14 as the sole abnormality conferring a favorable prognosis, while trisomy 12 confers an intermediate prognosis and deletion of either ATM or TP53 confers an unfavorable prognosis. Although more than 15 years have passed since the Dohner publication of 2000, the hierarchy has recently been revisited and reaffirmed [
      • Van Dyke D.L.
      • et al.
      The Dohner fluorescence in situ hybridization prognostic classification of chronic lymphocytic leukaemia (CLL): the CLL Research Consortium experience.
      ].
      Deletion of 6q, seen in approximately 5% of cases, was identified as an unfavorable marker in the original Dohner classification [
      • Dohner H.
      • et al.
      Genomic aberrations and survival in chronic lymphocytic leukemia.
      ]. However, the poor prognostic significance of this aberration has not borne out, and this deletion may be better categorized as an intermediate finding [
      • Cuneo A.
      • et al.
      Chronic lymphocytic leukemia with 6q- shows distinct hematological features and intermediate prognosis.
      ,
      • Dalsass A.
      • et al.
      6q deletion detected by fluorescence in situ hybridization using bacterial artificial chromosome in chronic lymphocytic leukemia.
      ,
      • Setlur S.R.
      • et al.
      Comparison of familial and sporadic chronic lymphocytic leukaemia using high resolution array comparative genomic hybridization.
      ,
      • Wang D.M.
      • et al.
      Intermediate prognosis of 6q deletion in chronic lymphocytic leukemia.
      ]. Two deletion regions, 6q12-q23.3 and 6q25-q27, have been observed, only one of which includes the MYB gene (6q23.3), the typical locus targeted by commercial FISH assays [
      • Dalsass A.
      • et al.
      6q deletion detected by fluorescence in situ hybridization using bacterial artificial chromosome in chronic lymphocytic leukemia.
      ,
      • Stilgenbauer S.
      • et al.
      Incidence and clinical significance of 6q deletions in B cell chronic lymphocytic leukemia.
      ]. A 6q putative tumor suppressor gene has not been identified, although a recent study suggests that FOXO3 may be involved [
      • Jarosova M.
      • et al.
      Chromosome 6q deletion correlates with poor prognosis and low relative expression of FOXO3 in chronic lymphocytic leukemia patients.
      ].
      With the fairly recent identification of more effective mitogens for CLL cells (CpG oligodeoxynucleotides and CD40 ligands), metaphase chromosome analysis has resurged in the past 5–10 years. Chromosome analysis has the advantage over targeted FISH by providing a whole genome analysis. The detection rate of abnormalities that are not targeted by the FISH panel ranges from 25 to 35% [
      • Haferlach C.
      • et al.
      Comprehensive genetic characterization of CLL: a study on 506 cases analysed with chromosome banding analysis, interphase FISH, IgV(H) status and immunophenotyping.
      ,
      • Heerema N.A.
      • et al.
      Stimulation of chronic lymphocytic leukemia cells with CpG oligodeoxynucleotide gives consistent karyotypic results among laboratories: a CLL Research Consortium (CRC) Study.
      ,
      • Rigolin G.M.
      • et al.
      Chromosome aberrations detected by conventional karyotyping using novel mitogens in chronic lymphocytic leukemia with "normal" FISH: correlations with clinicobiologic parameters.
      ,
      • Shi M.
      • et al.
      Improved detection rate of cytogenetic abnormalities in chronic lymphocytic leukemia and other mature B-cell neoplasms with use of CpG-oligonucleotide DSP30 and interleukin 2 stimulation.
      ] and includes the detection of complex karyotypes and multiple clones, both of which are unfavorable prognostic findings that may be missed by FISH alone [
      • Rigolin G.M.
      • et al.
      In CLL, comorbidities and the complex karyotype are associated with an inferior outcome independently of CLL-IPI.
      ].
      The adoption of these new mitogens has also led to the identification of translocations in CLL. Previously not considered to play a significant role in CLL, they are now reported in 30–40% of cases [
      • Baliakas P.
      • et al.
      Chromosomal translocations and karyotype complexity in chronic lymphocytic leukemia: a systematic reappraisal of classic cytogenetic data.
      ,
      • Mayr C.
      • et al.
      Chromosomal translocations are associated with poor prognosis in chronic lymphocytic leukemia.
      • Van Den Neste E.
      • et al.
      Chromosomal translocations independently predict treatment failure, treatment-free survival and overall survival in B-cell chronic lymphocytic leukemia patients treated with cladribine.
      . Less favorable prognosis has been associated with translocations; however, studies are limited by small patient numbers and/or an over-representation of patients with advanced disease [
      • Baliakas P.
      • et al.
      Chromosomal translocations and karyotype complexity in chronic lymphocytic leukemia: a systematic reappraisal of classic cytogenetic data.
      • Heerema N.A.
      • et al.
      Presence of a translocation is associated with short time to treatment from diagnosis in ighv mutated chronic lymphocytic leukemia.
      . Thus, the prognostic value is currently uncertain, and the translocations likely represent a diverse group with different implications.
      Many apparently balanced translocations are actually unbalanced and have CMA-detectable deletions associated with breakpoints in known regions of genomic imbalance, including 13q14 and 17p13 [
      • Higgins R.A.
      • Gunn S.R.
      • Robetorye R.S.
      Clinical application of array-based comparative genomic hybridization for the identification of prognostically important genetic alterations in chronic lymphocytic leukemia.
      ,
      • Put N.
      • Wlodarska I.
      • Vandenberghe P.
      • Michaux L.
      Genetics of Chronic Lymphocytic Leukemia: Practical Aspects and Prognostic Significance.
      ]. Another significant percentage of the apparently balanced translocations identified by metaphase chromosome analysis are those involving immunoglobulin genes; these may confer a poor prognosis [
      • Nowakowski G.S.
      • et al.
      Interphase fluorescence in situ hybridization with an IGH probe is important in the evaluation of patients with a clinical diagnosis of chronic lymphocytic leukaemia.
      ]. The t(14;18)(q32;q21) resulting in IGH/BCL2 recombination and the variants t(18;22)(q21;q11.2) BCL2/IGL and t(2;18)(p12;q21) IGK/BCL2 are observed as secondary changes in CLL patients, often with trisomy 12, but may also be observed in patients with monoclonal lymphocytosis [
      • Reiner S.
      • Aukema S.M.
      Mature B- and T-neoplasms and Hodgkin lymphoma.
      ]. The t(14;19)(q32;q13) IGH/BCL3, which has been identified in a variety of B-cell neoplasms, is a recurrent translocation in CLL often found with trisomy 12 that may identify a subset of CLL patients with distinctive genetic and pathologic features [
      • Huh Y.O.
      • et al.
      Chronic lymphocytic leukemia with t(14;19)(q32;q13) is characterized by atypical morphologic and immunophenotypic features and distinctive genetic features.
      ]. Translocations involving MYC [t(8;14)(q24.1;q32) IGH/MYC as well as the variants t(2;8)(p12;q24.1) IGK/MYC and t(8;22)(q24.1;q11.2) MYC/IGL] are observed infrequently in CLL and when present, are typically acquired during disease progression [
      • Li Y.
      • et al.
      The clinical significance of 8q24/MYC rearrangement in chronic lymphocytic leukemia.
      ]. Note that the t(11;14)(q13;q32) IGH/CCND1 is diagnosed as mantle cell lymphoma according to current diagnostic criteria.

      CMA in CLL

      CLL is particularly amenable to the detection of CNAs by CMA for the following reasons: (1) genomic gains and losses represent key events with biologic and prognostic significance, with balanced rearrangements being less common and currently of uncertain prognostic value; (2) DNA from fresh samples is generally available, obviating the technical difficulties associated with analysis of DNA from paraffin-embedded tissue; (3) the tumor burden is generally known from flow cytometry studies and can be used to guide the downstream analysis; and (4) the tumor burden tends to be relatively high in peripheral blood. In instances where FISH and CMA data are discrepant, CMA analysis may help to further refine deletion breakpoints and determine the clinical relevance of atypical deletions.
      Clinically validated CMA assays have potential utility for individualized patient risk stratification in CLL. Microarray studies of CLL using bacterial artificial chromosome (BAC), oligonucleotide, targeted oligonucleotide and single nucleotide polymorphism (SNP)-based arrays have been reported. Techniques, technical limitations, clinical applications and challenges have been reviewed by Higgins et al. [
      • Higgins R.A.
      • Gunn S.R.
      • Robetorye R.S.
      Clinical application of array-based comparative genomic hybridization for the identification of prognostically important genetic alterations in chronic lymphocytic leukemia.
      ] and Hagenkord and Chang [
      • Hagenkord J.M.
      • Chang C.C.
      The rewards and challenges of array-based karyotyping for clinical oncology applications.
      ]. Comparison of platform performance in CLL has been reviewed by Gunnarsson et al. [
      • Gunnarsson R.
      • et al.
      Screening for copy-number alterations and loss of heterozygosity in chronic lymphocytic leukemia–a comparative study of four differently designed, high resolution microarray platforms.
      ], recognizing that this is a rapidly changing technology. Overall, copy number alterations have been reported in > 90% of cases when CMA assays have been performed for CLL patients, with the number of copy number alterations per patient generally low (0–2). The lower limit of disease involvement required for detection of clonal aberrations varies by report, but likely is around 10–15% with SNP arrays [
      • Biesecker L.G.
      • Spinner N.B.
      A genomic view of mosaicism and human disease.
      ]. SNP arrays can additionally detect acquired CN-LOH; studies indicate that CN-LOH occurs in regions of the genome with prognostic relevance in CLL [
      • Hagenkord J.M.
      • et al.
      Array-based karyotyping for prognostic assessment in chronic lymphocytic leukemia: performance comparison of Affymetrix 10K2.0, 250 K Nsp, and SNP6.0 arrays.
      ,
      • Pfeifer D.
      • et al.
      Genome-wide analysis of DNA copy number changes and LOH in CLL using high-density SNP arrays.
      ].
      These studies have validated the ability of CMA platforms to detect abnormalities detected by FISH panels as well as to identify novel regions of gain or loss and to identify genomic instability. In general, results of array-based analyses have reported high concordance with FISH results [
      • Edelmann J.
      • et al.
      High-resolution genomic profiling of chronic lymphocytic leukemia reveals new recurrent genomic alterations.
      ,
      • Gunn S.R.
      • et al.
      Whole-genome scanning by array comparative genomic hybridization as a clinical tool for risk assessment in chronic lymphocytic leukemia.
      ,
      • Gunn S.R.
      • et al.
      The HemeScan test for genomic prognostic marker assessment in chronic lymphocytic leukemia.
      ,
      • Kolquist K.A.
      • et al.
      Evaluation of chronic lymphocytic leukemia by oligonucleotide-based microarray analysis uncovers novel aberrations not detected by FISH or cytogenetic analysis.
      ,
      • Patel A.
      • et al.
      Validation of a targeted DNA microarray for the clinical evaluation of recurrent abnormalities in chronic lymphocytic leukemia.
      ,
      • Sargent R.
      • et al.
      Customized oligonucleotide array-based comparative genomic hybridization as a clinical assay for genomic profiling of chronic lymphocytic leukemia.
      ,
      • Schultz R.A.
      • et al.
      Evaluation of chronic lymphocytic leukemia by BAC-based microarray analysis.
      ,
      • Stevens-Kroef M.J.
      • et al.
      Identification of prognostic relevant chromosomal abnormalities in chronic lymphocytic leukemia using microarray-based genomic profiling.
      ]. Instances of non-concordance may be due to array resolution, which allows detection of smaller sized abnormalities that cannot be detected with standard commercial FISH probes. Discordance may also be due to cases with low tumor cell percentage or low level subclones, as FISH analyses may be able to identify smaller populations of abnormal cells than can CMA (the lower limit of sensitivity for FISH is typically 5–7% for detection of deletions, while for CMA it may be closer to 10–15%, although several laboratories report sensitivity down to 5%, depending on the size of the aberration and the platform used [
      • Biesecker L.G.
      • Spinner N.B.
      A genomic view of mosaicism and human disease.
      ] (Wolff and Chun, personal communication). With the high tumor burden and high intratumoral level of abnormalities in most untreated CLL patients, the sensitivity of CMA is adequate to detect prognostically significant abnormalities. Most often, CMA analysis in CLL is used at time of diagnosis. However, it may also be used on post-treatment specimens, particularly when disease transformation is suspected.

      Detection of established prognostic CNVs by CMA

      Del(13q)

      Deletions of 13q14 are the most common genetic change in CLL, usually mono-allelic and occurring more frequently in patients with mutIGHV. The mono-allelic deletion is associated with a good prognosis when present as the sole abnormality [
      • Dohner H.
      • et al.
      Genomic aberrations and survival in chronic lymphocytic leukemia.
      ], while bi-allelic loss in a high proportion of cells is associated with a less favorable prognosis [
      • Garg R.
      • et al.
      The prognostic difference of monoallelic versus biallelic deletion of 13q in chronic lymphocytic leukemia.
      ,
      • Van Dyke D.L.
      • et al.
      A comprehensive evaluation of the prognostic significance of 13q deletions in patients with B-chronic lymphocytic leukaemia.
      ]. Detection of a deletion of 13q, discrimination of mono-allelic and bi-allelic losses, and determination of the extent of the deletion region are readily accomplished by CMA analysis.
      Deletion size is heterogeneous across patients. The minimal deletion region contains the DLEU2 (deleted in lymphocytic leukemia 2) locus, which encodes a long-noncoding RNA (lncRNA), TRIM13, miR-3613, KCNRG, the micro RNA cluster miR-15a/miR-16-1 and the DLEU1 lncRNA gene [
      • Grygalewicz B.
      • et al.
      Monoallelic and biallelic deletions of 13q14 in a group of CLL/SLL patients investigated by CGH Haematological Cancer and SNP array (8x60K).
      ]. In some cases, the deletion includes the DLEU7 gene. DLEU7, which functions as an NF-kB and NFAT inhibitor, resides within a minimal deletion region for miR-15a/16–1 [
      • Palamarchuk A.
      • et al.
      13q14 deletions in CLL involve cooperating tumor suppressors.
      ]. Deletions are seen in two clusters, one of which occurs around DLEU2 and the second of which is distal to GUCY1B2. Several studies have associated larger deletion size with worse prognosis [
      • Gunnarsson R.
      • et al.
      Array-based genomic screening at diagnosis and during follow-up in chronic lymphocytic leukemia.
      ,
      • Parker H.
      • et al.
      13q deletion anatomy and disease progression in patients with chronic lymphocytic leukemia.
      ]. Using the Affymetrix 6.0 array, Mian et al. [
      • Mian M.
      • et al.
      Del(13q14.3) length matters: an integrated analysis of genomic, fluorescence in situ hybridization and clinical data in 169 chronic lymphocytic leukaemia patients with 13q deletion alone or a normal karyotype.
      ] examined a cohort of 169 patients to further refine 13q deletions. By CLL FISH, the patients had been found to be either normal or to have 13q deletion as the sole abnormality. The 13q deletions were sub-classified into three types. Type 1 deletions were the smallest and encompassed DLEU2/miR-15a/16–1. Type 2 deletions were less than 10 Mb in total size and included the Type 1 region as well as RB1. Type 3 deletions were greater than 10 Mb and included the deleted regions in Type 2. Type 1 deletions were more often bi-allelic and showed longer time to first treatment (TTFT), while types 2 and 3 experienced a less favorable clinical course, with larger deletions conferring a worse prognosis. A major advantage of CMA versus FISH is the ability to detect and distinguish between the different deletion types in a single assay, as opposed to using multiple FISH probes. See Fig. 1B for region anatomy.
      Fig. 1.
      Fig. 1Chromosomal locations and genomic coordinates of genes in regions with prognostic significance in CLL.

      Trisomy 12

      Trisomy 12 is considered an intermediate risk prognostic factor, independent of IGHV mutational status. Trisomy 12 cases with concurrent NOTCH1 mutations are associated with a less favorable prognosis. Trisomy 18 and trisomy 19 are not common in CLL, but may be seen together or with trisomy 12 [
      • Ibbotson R.
      • et al.
      Coexistence of trisomies of chromosomes 12 and 19 in chronic lymphocytic leukemia occurs exclusively in the rare IgG-positive variant.
      ,
      • Van Dyke D.L.
      +18 or trisomy 18 in lymphoproliferative disorders.
      ]. Concurrent trisomy 12 and trisomy 19 have been associated with mutIGHV and with the rare IgG-switched variant of CLL.

      Del(11q)

      Deletion of the ATM gene remains the most important marker of poor outcome in patients with del(11q) [
      • Strefford J.C.
      The genomic landscape of chronic lymphocytic leukaemia: biological and clinical implications.
      ]. Alternative targets on 11q include ZW10, PLZF and TSLC1, each of which may be co-deleted with ATM. Whether the poor prognosis in patients with 11q loss reflects loss of multiple genes remains a question. Atypical 11q deletions or concurrent deletion of additional tumor suppressor gene(s) with ATM may contribute to the poor prognosis [
      • Gunn S.R.
      • et al.
      Atypical 11q deletions identified by array CGH may be missed by FISH panels for prognostic markers in chronic lymphocytic leukemia.
      ]. ATM and BIRC3 lesions can be found in the same patient. Some 11q deletions include the BIRC3 gene, but not ATM, supporting BIRC3 as a key player [
      • Fabbri G.
      • Dalla-Favera R.
      The molecular pathogenesis of chronic lymphocytic leukaemia.
      ,
      • Strefford J.C.
      The genomic landscape of chronic lymphocytic leukaemia: biological and clinical implications.
      ]. See Fig. 1A for region anatomy.

      Del(17p)

      The TP53 deletion/mutation on 17p is considered a highly adverse marker, and its prognostic impact has been discussed extensively [
      • Rossi D.
      • et al.
      Clinical impact of small TP53 mutated subclones in chronic lymphocytic leukemia.
      ,
      • Zenz T.
      • et al.
      TP53 mutation and survival in chronic lymphocytic leukemia.
      ]. Patients with 17p loss identified by FISH were examined using an Affymetrix 50 K Xba array, which indicated varied breakpoints. These results suggested the loss of multiple tumor suppressor genes in addition to TP53 and showed that multiple genes may be contributing to the highly adverse prognosis associated with TP53 loss [
      • Fabbri G.
      • Dalla-Favera R.
      The molecular pathogenesis of chronic lymphocytic leukaemia.
      ,
      • Strefford J.C.
      The genomic landscape of chronic lymphocytic leukaemia: biological and clinical implications.
      ,
      • Fabris S.
      • et al.
      Molecular and transcriptional characterization of 17p loss in B-cell chronic lymphocytic leukemia.
      ,
      • Nabhan C.
      • Raca G.
      • Wang Y.L.
      Predicting prognosis in chronic lymphocytic leukemia in the contemporary era.
      ]. Highlighting the importance of identifying these lesions in CLL is the recent FDA premarket approval for the Abbott Molecular/Vysis TP53 FISH probe as a companion diagnostic for Venclexta (venetoclax), a BCL2 inhibitor, to treat TP53 deletion patients who fail previous therapy. See Fig. 1D for region anatomy.
      Note that patients with deletion of both 11q and 17p have an exceptionally poor outcome, significantly worse than either alone [
      • Greipp P.T.
      • et al.
      Patients with chronic lymphocytic leukaemia and clonal deletion of both 17p13.1 and 11q22.3 have a very poor prognosis.
      ].

      Del(14q)

      Deletions of 14q are seen rarely at diagnosis and in ∼5% of patients with established CLL; they have been associated with shortened TTFT [
      • Reindl L.
      • et al.
      Biological and clinical characterization of recurrent 14q deletions in CLL and other mature B-cell neoplasms.
      ]. FISH studies indicated the presence of deletion of 14q in 1.9% of CLL patients studied, with the deletions observed being of variable size but with breakpoints clustered at the centromeric side in 14q24.1 (∼60% of cases) and at the telomeric side within the IGH locus at 14q32.3 (45% of cases). In agreement with these results, using FISH and SNP arrays to study 81 CLL patients with del(14q), Cosson et al. demonstrated that while 14q deletion size varies, 48% of patients had the same 14q24.1q32.33 deletion [
      • Cosson A.
      • et al.
      14q deletions are associated with trisomy 12, NOTCH1 mutations and unmutated IGHV genes in chronic lymphocytic leukemia and small lymphocytic lymphoma.
      ]. Del(14q) is associated with several unfavorable markers, including trisomy 12, NOTCH1 mutations and unmutIGHV. Note that these 14q deletions can involve one-third of chromosome 14 and are much larger than deletions that occur during normal physiological IGH gene rearrangement. See Fig. 1D for region anatomy.

      Genomic complexity

      Increased genomic complexity (widespread gains and losses of chromosomal regions in many chromosomes) reflects genomic instability and is a marker independent of ZAP-70, IGHV status and Rai stage for identification of patients with aggressive CLL and a poor outcome [
      • Gunn S.R.
      • et al.
      The HemeScan test for genomic prognostic marker assessment in chronic lymphocytic leukemia.
      ,
      • Braggio E.
      • et al.
      Longitudinal genome-wide analysis of patients with chronic lymphocytic leukemia reveals complex evolution of clonal architecture at disease progression and at the time of relapse.
      ,
      • Knight S.J.
      • et al.
      Quantification of subclonal distributions of recurrent genomic aberrations in paired pre-treatment and relapse samples from patients with B-cell chronic lymphocytic leukemia.
      ,
      • Knight S.J.
      • et al.
      Quantification of subclonal distributions of recurrent genomic aberrations in paired pre-treatment and relapse samples from patients with B-cell chronic lymphocytic leukemia.
      ,
      • Ouillette P.
      • et al.
      Acquired genomic copy number aberrations and survival in chronic lymphocytic leukemia.
      ,
      • Rudenko H.C.
      • et al.
      Characterising the TP53-deleted subgroup of chronic lymphocytic leukemia: an analysis of additional cytogenetic abnormalities detected by interphase fluorescence in situ hybridisation and array-based comparative genomic hybridisation.
      • Schweighofer C.D.
      • et al.
      Genomic variation by whole-genome SNP mapping arrays predicts time-to-event outcome in patients with chronic lymphocytic leukemia: a comparison of CLL and HapMap genotypes.
      . Genomic complexity was observed for patients both with favorable and with adverse FISH markers [
      • Rudenko H.C.
      • et al.
      Characterising the TP53-deleted subgroup of chronic lymphocytic leukemia: an analysis of additional cytogenetic abnormalities detected by interphase fluorescence in situ hybridisation and array-based comparative genomic hybridisation.
      ,
      • Kay N.E.
      • et al.
      Progressive but previously untreated CLL patients with greater array CGH complexity exhibit a less durable response to chemoimmunotherapy.
      ]. Of note, Gunn et al. observed that 21% (37/174) of cases had three or more aberrations not interrogated by the common FISH panel [
      • Gunn S.R.
      • et al.
      The HemeScan test for genomic prognostic marker assessment in chronic lymphocytic leukemia.
      ]. In another study, Kujawski et al. showed that TTFT was 79 months for non-complex cases and 23 months for complex cases, using their own published complexity algorithm [
      • Kujawski L.
      • et al.
      Genomic complexity identifies patients with aggressive chronic lymphocytic leukemia.
      ]. Greater complexity has also been associated with worse progression-free-survival (PFS) and response to therapy, and patients with TP53 deletion have been shown to have a higher frequency of large (>5 Mb) aberrations [
      • Gunnarsson R.
      • et al.
      Large but not small copy-number alterations correlate to high-risk genomic aberrations and survival in chronic lymphocytic leukemia: a high-resolution genomic screening of newly diagnosed patients.
      ,
      • Rudenko H.C.
      • et al.
      Characterising the TP53-deleted subgroup of chronic lymphocytic leukemia: an analysis of additional cytogenetic abnormalities detected by interphase fluorescence in situ hybridisation and array-based comparative genomic hybridisation.
      ,
      • Puiggros A.
      • Blanco G.
      • Espinet B.
      Genetic abnormalities in chronic lymphocytic leukemia: where we are and where we go.
      ]. Increased genomic complexity appears to be an independent marker for identification of patients with aggressive CLL and shorter survival [
      • Braggio E.
      • et al.
      Longitudinal genome-wide analysis of patients with chronic lymphocytic leukemia reveals complex evolution of clonal architecture at disease progression and at the time of relapse.
      ,
      • Ouillette P.
      • et al.
      Acquired genomic copy number aberrations and survival in chronic lymphocytic leukemia.
      ]. It should be noted that the phrase “complex karyotype” awaits a formal definition by NCCN, ISCN or another body [
      • Peterson J.F.
      The complexities of defining a complex karyotype in hematological malignancies: a need for standardization.
      ].

      Clonal diversity

      Clonal diversity, a surrogate marker for clonal evolution, is defined as the presence of two or more clonal populations of cells at different levels of tumor involvement as detected by CMA. For patients with CLL, clonal evolution and an increase in the percentage of cells with CNAs are associated with disease progression [
      • Landau D.A.
      • et al.
      Evolution and impact of subclonal mutations in chronic lymphocytic leukemia.
      ]. Although both metaphase chromosome analysis and CMA can detect clonal diversity, CMA is more sensitive, has higher resolution and can better define percentages of specific clonal abnormalities [
      • Zhang L.
      • et al.
      Clonal diversity analysis using SNP microarray: a new prognostic tool for chronic lymphocytic leukemia.
      ]. CMA-defined clonal diversity has been associated with progressive disease, relapse, need for therapy and an adverse prognosis [
      • Landau D.A.
      • et al.
      Evolution and impact of subclonal mutations in chronic lymphocytic leukemia.
      ,
      • Zhang L.
      • et al.
      Clonal diversity analysis using SNP microarray: a new prognostic tool for chronic lymphocytic leukemia.
      • Del Giudice I.
      • et al.
      Inter- and intra-patient clonal and subclonal heterogeneity of chronic lymphocytic leukaemia: evidences from circulating and lymph nodal compartments.
      .

      Richter transformation

      Richter syndrome (RS), which is associated with poor outcome, is the transformation from CLL to a more aggressive lymphoma, most commonly diffuse large B-cell lymphoma (DLBCL). CMA is emerging as a useful methodology both to identify CLL patients at risk for Richter transformation and to offer prognostic information. The most common changes in RS patients are losses at 17p (TP53), 13q14.3 (DLEU2/miR15a/miR16-1), 9p21 (CDKN2A) and trisomy 12. While deletions at 17p and 13q14.3 as well as trisomy 12 are already present during the CLL phase, 9p21 loss is the most frequent lesion acquired during Richter transformation, mostly occurring concomitantly with TP53 inactivation. TP53 inactivation and/or 9p21 loss appear to be mutually exclusive to trisomy 12, suggesting that RS may develop through two main genetic pathways [
      • Landau D.A.
      • et al.
      Evolution and impact of subclonal mutations in chronic lymphocytic leukemia.
      ,
      • Chigrinova E.
      • et al.
      Two main genetic pathways lead to the transformation of chronic lymphocytic leukemia to Richter syndrome.
      ,
      • Fabbri G.
      • et al.
      Genetic lesions associated with chronic lymphocytic leukemia transformation to Richter syndrome.
      ]. In addition, it has been well established that one of the most important prognostic factors for Richter transformation is the clonal relationship between the CLL and the lymphoma clones [
      • Rossi D.
      • Gaidano G.
      Richter syndrome: pathogenesis and management.
      ]. By assessing the clonal changes by CMA, it is often possible to detect the original CLL clonal changes that reveal a linear progression of the CLL to DLBCL, which has been associated with a less favorable outcome, compared to identification of an apparent new independent clone that would represent non-linear progression and a better overall prognosis [
      • Chigrinova E.
      • et al.
      Two main genetic pathways lead to the transformation of chronic lymphocytic leukemia to Richter syndrome.
      ,
      • Fabbri G.
      • et al.
      Genetic lesions associated with chronic lymphocytic leukemia transformation to Richter syndrome.
      ,
      • Rossi D.
      • Gaidano G.
      Richter syndrome: pathogenesis and management.
      ,
      • Jamroziak K.
      • et al.
      Richter syndrome in chronic lymphocytic leukemia: updates on biology, clinical features and therapy.
      • Mao Z.
      • et al.
      IgVH mutational status and clonality analysis of Richter's transformation: diffuse large B-cell lymphoma and Hodgkin lymphoma in association with B-cell chronic lymphocytic leukemia (B-CLL) represent 2 different pathways of disease evolution.
      .

      Concurrent myelodysplastic syndrome (MDS) related changes

      The concurrent presence or development of myeloid disorders (MDS or AML) is relatively uncommon in CLL [
      • Robertson L.E.
      • et al.
      Therapy-related leukemia and myelodysplastic syndrome in chronic lymphocytic leukemia.
      ]; however, some CLL chemotherapies are known to increase the risk for dysplasia [
      • Skarbnik A.P.
      • Faderl S.
      The role of combined fludarabine, cyclophosphamide and rituximab chemoimmunotherapy in chronic lymphocytic leukemia: current evidence and controversies.
      ]. MDS-associated cytogenetic abnormalities are readily defined by CMA because, as for CLL, the critical cytogenetic aberrations are copy number changes. Metaphase chromosome analysis also readily identifies MDS-associated abnormalities; CMA has the additional advantage of defining percentages of the abnormal clones, while metaphase chromosome analysis can unambiguously ascribe a particular abnormality to a specific clone (e.g. 13q deletion can be seen in both CLL and MDS). Targeted CLL FISH will not identify MDS-associated abnormalities.

      Additional abnormalities identified by CMA analysis

      Gain of 2p

      Various CMA studies in heterogeneous CLL patient populations have indicated gain of 2p as a recurrent aberration; BCL11A, REL and MYCN have been cited as potential targets [
      • Pfeifer D.
      • et al.
      Genome-wide analysis of DNA copy number changes and LOH in CLL using high-density SNP arrays.
      ,
      • Gunnarsson R.
      • et al.
      Array-based genomic screening at diagnosis and during follow-up in chronic lymphocytic leukemia.
      ,
      • Rudenko H.C.
      • et al.
      Characterising the TP53-deleted subgroup of chronic lymphocytic leukemia: an analysis of additional cytogenetic abnormalities detected by interphase fluorescence in situ hybridisation and array-based comparative genomic hybridisation.
      ,
      • Schweighofer C.D.
      • et al.
      Genomic variation by whole-genome SNP mapping arrays predicts time-to-event outcome in patients with chronic lymphocytic leukemia: a comparison of CLL and HapMap genotypes.
      ,
      • Houldsworth J.
      • et al.
      Genomic imbalance defines three prognostic groups for risk stratification of patients with chronic lymphocytic leukemia.
      • Fabris S.
      • et al.
      Chromosome 2p gain in monoclonal B-cell lymphocytosis and in early stage chronic lymphocytic leukemia.
      . In a recently published study, Cosson et al. [
      • Cosson A.
      • et al.
      Gain in the short arm of chromosome 2 (2p+) induces gene overexpression and drug resistance in chronic lymphocytic leukemia: analysis of the central role of XPO1.
      ] identified two minimally gained regions on 2p and implicate XPO1 as a critical player. Their findings also support earlier publications of the association of 2p gain with unfavorable markers, including del(11q), del(17p) and unmutated status of IGHV, and show that 2p gain promotes resistance to several therapeutic drugs. 2p gain also can be present in early stage disease, especially in those patients with other poor prognosis markers.

      Chromothripsis

      The phenomenon of chromothripsis (which can be detected by CMA, but not by FISH or metaphase chromosome analysis) was first identified following a genome-wide screen of 10 CLL patients [
      • Stephens P.J.
      • et al.
      Massive genomic rearrangement acquired in a single catastrophic event during cancer development.
      ]. Since that time, two larger scale studies have detected chromothripsis in 4–5% of CLL patients studied by CMA [
      • Edelmann J.
      • et al.
      High-resolution genomic profiling of chronic lymphocytic leukemia reveals new recurrent genomic alterations.
      ,
      • Salaverria I.
      • et al.
      Detection of chromothripsis-like patterns with a custom array platform for chronic lymphocytic leukemia.
      ]. In both of these large-scale studies, chromothripsis was associated with a poor prognosis. In the Edelman study [
      • Edelmann J.
      • et al.
      High-resolution genomic profiling of chronic lymphocytic leukemia reveals new recurrent genomic alterations.
      ], 74% of patients with chromothripsis had unmutated IGHV status and 79% had high-risk genomic aberrations, including a TP53 mutation in 31%, but univariate analysis still showed patients with chromothripsis to have inferior PFS and OS. Specific chromosomes may be preferentially involved; in three of eight cases in the Salaverria series [
      • Salaverria I.
      • et al.
      Detection of chromothripsis-like patterns with a custom array platform for chronic lymphocytic leukemia.
      ], the chromothripsis involved chromosome 5 and resulted in gain of the TERT locus. A more recent publication on the role of SETD2 showed that deletions of this locus were associated with chromosome 3 chromothripsis, as well as TP53 deletion and genomic complexity [
      • Parker H.
      • et al.
      Genomic disruption of the histone methyltransferase SETD2 in chronic lymphocytic leukaemia.
      ].
      See Table 1 for recurring copy number variations identified by CMA.

      CN-LOH

      Acquired CN-LOH may be due to mitotic recombination or to segmental deletion with replacement of the deleted region by a copy of the remaining allele during development of the neoplasm. When a deleterious mutation precedes such events, CN-LOH can act as a second hit resulting in mutation of both copies of a tumor suppressor gene. For loss-of-function mutations, this is equivalent to bi-allelic deletion of a gene. SNP microarrays allow detection of CN-LOH that remains undetected by karyotyping or FISH.
      CN-LOH in CLL has been reported at variable frequencies in the literature [
      • Pfeifer D.
      • et al.
      Genome-wide analysis of DNA copy number changes and LOH in CLL using high-density SNP arrays.
      ,
      • Stevens-Kroef M.J.
      • et al.
      Identification of prognostic relevant chromosomal abnormalities in chronic lymphocytic leukemia using microarray-based genomic profiling.
      ,
      • Lehmann S.
      • et al.
      Molecular allelokaryotyping of early-stage, untreated chronic lymphocytic leukemia.
      ,
      • Li M.M.
      • et al.
      A multicenter, cross-platform clinical validation study of cancer cytogenomic arrays.
      ,
      • Pei J.
      • et al.
      Copy neutral loss of heterozygosity in 20q in chronic lymphocytic leukemia/small lymphocytic lymphoma.
      ,
      • Saddler C.
      • et al.
      Comprehensive biomarker and genomic analysis identifies p53 status as the major determinant of response to MDM2 inhibitors in chronic lymphocytic leukemia.
      ,
      • Sellmann L.
      • et al.
      Shorter telomeres correlate with an increase in the number of uniparental disomies in patients with chronic lymphocytic leukemia.
      ,
      • Sellmann L.
      • et al.
      Gene dosage effects in chronic lymphocytic leukemia.
      • Xu X.
      • et al.
      The advantage of using SNP array in clinical testing for hematological malignancies–a comparative study of three genetic testing methods.
      ]. In untreated CLL patients, two studies reported a frequency of 6–7% [
      • Edelmann J.
      • et al.
      High-resolution genomic profiling of chronic lymphocytic leukemia reveals new recurrent genomic alterations.
      ,
      • Lehmann S.
      • et al.
      Molecular allelokaryotyping of early-stage, untreated chronic lymphocytic leukemia.
      ], which is lower than other malignancies [
      • Maciejewski J.P.
      • Mufti G.J.
      Whole genome scanning as a cytogenetic tool in hematologic malignancies.
      ]. CN-LOH most frequently affects 13q, 17p and 11q [
      • Edelmann J.
      • et al.
      High-resolution genomic profiling of chronic lymphocytic leukemia reveals new recurrent genomic alterations.
      ,
      • Grygalewicz B.
      • et al.
      Monoallelic and biallelic deletions of 13q14 in a group of CLL/SLL patients investigated by CGH Haematological Cancer and SNP array (8x60K).
      ]. Similar to other hematological malignancies, regions affected by CN-LOH encompass genes involved in disease initiation or progression [
      • Hagenkord J.M.
      • et al.
      Array-based karyotyping for prognostic assessment in chronic lymphocytic leukemia: performance comparison of Affymetrix 10K2.0, 250 K Nsp, and SNP6.0 arrays.
      ], and identification of CN-LOH can help uncover these tumor suppressor genes.
      A significant number of CN-LOH could be present as germline variants. In one study that assessed paired CLL tumor and germline samples, 30 of 39 CN-LOH identified regions were germline [
      • Edelmann J.
      • et al.
      High-resolution genomic profiling of chronic lymphocytic leukemia reveals new recurrent genomic alterations.
      ]. The median sizes of tumor-specific and germline CN-LOH were 48.4 Mb and 12.1 Mb, respectively. In the absence of germline testing, strict criteria should be used for identification of tumor-specific CN-LOH. These criteria may include overlap with known deletion regions and whether the region is telomeric, but a minimum cut-off of 10 Mb seems reasonable in the absence of paired analysis.
      The most studied CN-LOH in CLL is 13q. CLL patients with 13q CN-LOH have a high frequency (85–100%) of bi-allelic deletion within the CN-LOH region [
      • Hagenkord J.M.
      • et al.
      Array-based karyotyping for prognostic assessment in chronic lymphocytic leukemia: performance comparison of Affymetrix 10K2.0, 250 K Nsp, and SNP6.0 arrays.
      ,
      • Edelmann J.
      • et al.
      High-resolution genomic profiling of chronic lymphocytic leukemia reveals new recurrent genomic alterations.
      ,
      • Grygalewicz B.
      • et al.
      Monoallelic and biallelic deletions of 13q14 in a group of CLL/SLL patients investigated by CGH Haematological Cancer and SNP array (8x60K).
      ,
      • Gunnarsson R.
      • et al.
      Array-based genomic screening at diagnosis and during follow-up in chronic lymphocytic leukemia.
      ,
      • Parker H.
      • et al.
      13q deletion anatomy and disease progression in patients with chronic lymphocytic leukemia.
      ,
      • Lehmann S.
      • et al.
      Molecular allelokaryotyping of early-stage, untreated chronic lymphocytic leukemia.
      ,
      • Ouillette P.
      • et al.
      The prognostic significance of various 13q14 deletions in chronic lymphocytic leukemia.
      ]. In one rare case without 13q14 deletion, a homozygous deletion of 1 nucleotide in miR16-1 was reported [
      • Ouillette P.
      • et al.
      The prognostic significance of various 13q14 deletions in chronic lymphocytic leukemia.
      ]. Parker et al. suggested that CN-LOH may result in both bi-allelic deletion of genes as well as homozygosity of a cluster of genes associated with progressive disease [
      • Parker H.
      • et al.
      13q deletion anatomy and disease progression in patients with chronic lymphocytic leukemia.
      ]. Pfiefer et al. have shown that the 13q14 CN-LOH region ends telomeric to the miRNA-15a and 16–1 genes [
      • Pfeifer D.
      • et al.
      Genome-wide analysis of DNA copy number changes and LOH in CLL using high-density SNP arrays.
      ] and some authors have suggested that CN-LOH may lead to dysregulation of miR-15a/16–1 and/or other genes [
      • Gunnarsson R.
      • et al.
      Array-based genomic screening at diagnosis and during follow-up in chronic lymphocytic leukemia.
      ].
      As in other malignancies, CLL patients with CN-LOH of 17p were frequently associated with homozygous mutations in the TP53 gene [
      • Hagenkord J.M.
      • et al.
      Array-based karyotyping for prognostic assessment in chronic lymphocytic leukemia: performance comparison of Affymetrix 10K2.0, 250 K Nsp, and SNP6.0 arrays.
      ,
      • Edelmann J.
      • et al.
      High-resolution genomic profiling of chronic lymphocytic leukemia reveals new recurrent genomic alterations.
      ,
      • Stevens-Kroef M.J.
      • et al.
      Identification of prognostic relevant chromosomal abnormalities in chronic lymphocytic leukemia using microarray-based genomic profiling.
      ,
      • Saddler C.
      • et al.
      Comprehensive biomarker and genomic analysis identifies p53 status as the major determinant of response to MDM2 inhibitors in chronic lymphocytic leukemia.
      ]. Less frequent locations include 11q encompassing the ATM gene [
      • Edelmann J.
      • et al.
      High-resolution genomic profiling of chronic lymphocytic leukemia reveals new recurrent genomic alterations.
      ]. Pei et al. identified three CLL patients with lymphadenopathy with CN-LOH of 20q, although mutations in the most common gene on 20q implicated in hematological malignancies, ASXL1, were absent [
      • Pei J.
      • et al.
      Copy neutral loss of heterozygosity in 20q in chronic lymphocytic leukemia/small lymphocytic lymphoma.
      ].
      See Table 2 for recurrent CN-LOH regions identified by CMA studies.
      Table 2Recurring regions of CN-LOH in CLL.
      CN-LOHCandidate geneAssociationStrength of evidence for prognosis (Level
      Level 1: present in WHO classification or professional practice guidelines; Level 2: recurrent in well-powered studies with suspected clinical significance; Level 3: recurrent, but uncertain prognostic significance
      )
      References
      13qmiR15a/16–1Biallelic deletion of 13qEstablished (1)
      • Hagenkord J.M.
      • et al.
      Array-based karyotyping for prognostic assessment in chronic lymphocytic leukemia: performance comparison of Affymetrix 10K2.0, 250 K Nsp, and SNP6.0 arrays.
      ,
      • Pfeifer D.
      • et al.
      Genome-wide analysis of DNA copy number changes and LOH in CLL using high-density SNP arrays.
      ,
      • Edelmann J.
      • et al.
      High-resolution genomic profiling of chronic lymphocytic leukemia reveals new recurrent genomic alterations.
      ,
      • Grygalewicz B.
      • et al.
      Monoallelic and biallelic deletions of 13q14 in a group of CLL/SLL patients investigated by CGH Haematological Cancer and SNP array (8x60K).
      ,
      • Gunnarsson R.
      • et al.
      Array-based genomic screening at diagnosis and during follow-up in chronic lymphocytic leukemia.
      ,
      • Parker H.
      • et al.
      13q deletion anatomy and disease progression in patients with chronic lymphocytic leukemia.
      ,
      • Lehmann S.
      • et al.
      Molecular allelokaryotyping of early-stage, untreated chronic lymphocytic leukemia.
      ,
      • Ouillette P.
      • et al.
      The prognostic significance of various 13q14 deletions in chronic lymphocytic leukemia.
      17p13TP53Homozygous TP53 mutationsEstablished (1)
      • Hagenkord J.M.
      • et al.
      Array-based karyotyping for prognostic assessment in chronic lymphocytic leukemia: performance comparison of Affymetrix 10K2.0, 250 K Nsp, and SNP6.0 arrays.
      ,
      • Edelmann J.
      • et al.
      High-resolution genomic profiling of chronic lymphocytic leukemia reveals new recurrent genomic alterations.
      ,
      • Stevens-Kroef M.J.
      • et al.
      Identification of prognostic relevant chromosomal abnormalities in chronic lymphocytic leukemia using microarray-based genomic profiling.
      ,
      • Parker H.
      • et al.
      13q deletion anatomy and disease progression in patients with chronic lymphocytic leukemia.
      ,
      • Saddler C.
      • et al.
      Comprehensive biomarker and genomic analysis identifies p53 status as the major determinant of response to MDM2 inhibitors in chronic lymphocytic leukemia.
      11q13-qterIncludes ATMMonoallelic ATM deletionSuspected (2)
      • Edelmann J.
      • et al.
      High-resolution genomic profiling of chronic lymphocytic leukemia reveals new recurrent genomic alterations.
      ,
      • Parker H.
      • et al.
      13q deletion anatomy and disease progression in patients with chronic lymphocytic leukemia.
      20q11UnknownNoneN/A (3)
      • Stevens-Kroef M.J.
      • et al.
      Identification of prognostic relevant chromosomal abnormalities in chronic lymphocytic leukemia using microarray-based genomic profiling.
      ,
      • Pei J.
      • et al.
      Copy neutral loss of heterozygosity in 20q in chronic lymphocytic leukemia/small lymphocytic lymphoma.
      1p36UnknownNoneN/A (3)
      • Edelmann J.
      • et al.
      High-resolution genomic profiling of chronic lymphocytic leukemia reveals new recurrent genomic alterations.
      ,
      • Xu X.
      • et al.
      The advantage of using SNP array in clinical testing for hematological malignancies–a comparative study of three genetic testing methods.
      low asterisk Level 1: present in WHO classification or professional practice guidelines; Level 2: recurrent in well-powered studies with suspected clinical significance; Level 3: recurrent, but uncertain prognostic significance

      Gene mutation analysis by next generation sequencing (NGS)

      Gene mutations in CLL have been studied by next generation sequencing to determine drivers of tumorigenesis and progression and have been extensively reviewed elsewhere [
      • Fabbri G.
      • Dalla-Favera R.
      The molecular pathogenesis of chronic lymphocytic leukaemia.
      ,
      • Strefford J.C.
      The genomic landscape of chronic lymphocytic leukaemia: biological and clinical implications.
      ,
      • Nabhan C.
      • Raca G.
      • Wang Y.L.
      Predicting prognosis in chronic lymphocytic leukemia in the contemporary era.
      ,
      • Alsolami R.
      • Knight S.J.
      • Schuh A.
      Clinical application of targeted and genome-wide technologies: can we predict treatment responses in chronic lymphocytic leukemia.
      ,
      • Landau D.A.
      • et al.
      Mutations driving CLL and their evolution in progression and relapse.
      ]. Briefly, the most commonly mutated genes are TP53, ATM, NOTCH1, SF3B1 and MYD88. DDX3X, chromatin regulators (CHD2, HIST1C1), B-cell transcription factors (EGR2, IKZF3), RNA export factors (XPO1, RANBP2), ribosomal proteins (RPS15), telomere-associated proteins (POT1) and signal transducers (RAS, MAP2K1, MAP2K3) have also been implicated [
      • Fabbri G.
      • Dalla-Favera R.
      The molecular pathogenesis of chronic lymphocytic leukaemia.
      ]. NOTCH1 mutations are found in approximately 10% of CLL patients at diagnosis, primarily those with unmutIGHV [
      • Fabbri G.
      • Dalla-Favera R.
      The molecular pathogenesis of chronic lymphocytic leukaemia.
      ]. NOTCH1 and SF3B1 mutations appear to be mutually exclusive and are each associated with adverse prognosis [
      • Nabhan C.
      • Raca G.
      • Wang Y.L.
      Predicting prognosis in chronic lymphocytic leukemia in the contemporary era.
      ,
      • Alsolami R.
      • Knight S.J.
      • Schuh A.
      Clinical application of targeted and genome-wide technologies: can we predict treatment responses in chronic lymphocytic leukemia.
      ]. The frequency of BIRC3 mutations in CLL ranges between 0.4% and 14% [
      • Baliakas P.
      • et al.
      Recurrent mutations refine prognosis in chronic lymphocytic leukemia.
      ,
      • Brown J.R.
      • et al.
      Extended follow-up and impact of high-risk prognostic factors from the phase 3 RESONATE study in patients with previously treated CLL/SLL.
      ,
      • Chiaretti S.
      • et al.
      NOTCH1, SF3B1, BIRC3 and TP53 mutations in patients with chronic lymphocytic leukemia undergoing first-line treatment: correlation with biological parameters and response to treatment.
      ,
      • Cortese D.
      • et al.
      On the way towards a 'CLL prognostic index': focus on TP53, BIRC3, SF3B1, NOTCH1 and MYD88 in a population-based cohort.
      ,
      • Nadeu F.
      • et al.
      Clinical impact of clonal and subclonal TP53, SF3B1, BIRC3, NOTCH1, and ATM mutations in chronic lymphocytic leukemia.
      • Xia Y.
      • et al.
      Frequencies of SF3B1, NOTCH1, MYD88, BIRC3 and IGHV mutations and TP53 disruptions in Chinese with chronic lymphocytic leukemia: disparities with Europeans.
      . The prognostic significance of BIRC3 mutations is unclear at this time. In one study, BIRC3 lesions (both mutations and deletions) were associated with CLL refractory to fludarabine [
      • Rossi D.
      • et al.
      Disruption of BIRC3 associates with fludarabine chemorefractoriness in TP53 wild-type chronic lymphocytic leukemia.
      ]. In total, over 70% of patients with treatment- (fludarabine) resistant CLL have one or more mutations in the TP53, NOTCH1, SF3B1 and BIRC3 genes, confirming their importance in treatment-resistant CLL pathogenesis [
      • Puiggros A.
      • Blanco G.
      • Espinet B.
      Genetic abnormalities in chronic lymphocytic leukemia: where we are and where we go.
      ].
      By integrating cytogenetic and mutational data, it has been shown that TP53 and/or BIRC3 abnormalities are associated with a high risk, NOTCH1 and/or SF3B1 mutations and/or del(11q) are associated with an intermediate risk, trisomy 12 or normal karyotype is associated with a low risk, and del(13q) is associated with a very low risk [
      • Fabbri G.
      • Dalla-Favera R.
      The molecular pathogenesis of chronic lymphocytic leukaemia.
      ,
      • Strefford J.C.
      The genomic landscape of chronic lymphocytic leukaemia: biological and clinical implications.
      ,
      • Guieze R.
      • Wu C.J.
      Genomic and epigenomic heterogeneity in chronic lymphocytic leukemia.
      ,
      • Ojha J.
      • et al.
      Deep sequencing identifies genetic heterogeneity and recurrent convergent evolution in chronic lymphocytic leukemia.
      ]. Importantly, approximately 20% of patients who would be assigned to low risk categories based solely on FISH prognostic markers would be reclassified to higher risk categories due to the presence of NOTCH1, TP53 or SF3B1 mutations and BIRC3 disruption [
      • Puiggros A.
      • Blanco G.
      • Espinet B.
      Genetic abnormalities in chronic lymphocytic leukemia: where we are and where we go.
      ].
      See Table 3 for gene mutations in CLL.
      Table 3Recurrent mutated genes in CLL.
      GeneLocusFunctionMutation typePrevalence (%)PrognosticStrength ofCommentsReferences
      significanceevidence
      (Level
      Level 1: present in WHO classification or professional practice guidelines; Level 2: recurrent in well-powered studies with suspected clinical significance; Level 3: recurrent, but uncertain prognostic significance
      )
      ATM11q22.3DNA repair and cell-cycle controlMissense, nonsense, indel10–14UnfavorableEstablished (1)Associated with unmutIGHV and 11q-; Candidate driver gene
      • Wierda W.G.
      • et al.
      NCCN guidelines insights: chronic lymphocytic leukemia/small lymphocytic leukemia, version 1.2017.
      ,
      • Campregher P.V.
      • Hamerschlak N.
      Novel prognostic gene mutations identified in chronic lymphocytic leukemia and their impact on clinical practice.
      BIRC311q22.2Apoptosis inhibitorFrameshift, nonsense, whole gene deletion1–10 (higher in previously treated patients)UnfavorableEstablished (1)In ∼25% of fludarabine-refractory CLL; Candidate driver gene
      • Strefford J.C.
      The genomic landscape of chronic lymphocytic leukaemia: biological and clinical implications.
      ,
      • Alsolami R.
      • Knight S.J.
      • Schuh A.
      Clinical application of targeted and genome-wide technologies: can we predict treatment responses in chronic lymphocytic leukemia.
      ,
      • Baliakas P.
      • et al.
      Recurrent mutations refine prognosis in chronic lymphocytic leukemia.
      ,
      • Rossi D.
      • et al.
      Integrated mutational and cytogenetic analysis identifies new prognostic subgroups in chronic lymphocytic leukemia.
      CHD215q26.1Chromatin remodelerMissense, truncation5–10UnknownN/A (3)
      • Strefford J.C.
      The genomic landscape of chronic lymphocytic leukaemia: biological and clinical implications.
      ,
      • Rodriguez D.
      • et al.
      Mutations in CHD2 cause defective association with active chromatin in chronic lymphocytic leukemia.
      FBXW74q31.3Ubiquitin ligase subunit/targets include NOTCH1Missense4UnknownN/A (3)Exclusive to NOTCH1 mutation patients; Negatively regulates NOTCH1
      • Wang L.
      • et al.
      SF3B1 and other novel cancer genes in chronic lymphocytic leukemia.
      MYD883p22.2Inflammatory pathway signal transducerMissense2–10Favorable/ No effectSuspected (2)Candidate driver gene
      • Baliakas P.
      • et al.
      Recurrent mutations refine prognosis in chronic lymphocytic leukemia.
      ,
      • Rossi D.
      • et al.
      Integrated mutational and cytogenetic analysis identifies new prognostic subgroups in chronic lymphocytic leukemia.
      ,
      • Filip A.A.
      New boys in town: prognostic role of SF3B1, NOTCH1 and other cryptic alterations in chronic lymphocytic leukemia and how it works.
      NOTCH19q34.3Intercellular signalingMissense, nonsense, insertion, duplication, frameshift4–10 (diagnosis) 12–30 (progression)UnfavorableEstablished (1)Associated with + 12; Candidate driver gene
      • Nabhan C.
      • Raca G.
      • Wang Y.L.
      Predicting prognosis in chronic lymphocytic leukemia in the contemporary era.
      ,
      • Campregher P.V.
      • Hamerschlak N.
      Novel prognostic gene mutations identified in chronic lymphocytic leukemia and their impact on clinical practice.
      ,
      • Rossi D.
      • et al.
      Integrated mutational and cytogenetic analysis identifies new prognostic subgroups in chronic lymphocytic leukemia.
      ,
      • Balatti V.
      • et al.
      NOTCH1 mutations in CLL associated with trisomy 12.
      ,
      • Del Giudice I.
      • et al.
      NOTCH1 mutations in +12 chronic lymphocytic leukemia (CLL) confer an unfavorable prognosis, induce a distinctive transcriptional profiling and refine the intermediate prognosis of +12 CLL.
      POT17q31.33Telomere protector/ stabilizer; component of telomerase RNP complexMissense, frameshift, splicing5–10UnfavorableSuspected (2)Associated with familial CLL
      • Herling C.D.
      • et al.
      Complex karyotypes and KRAS and POT1 mutations impact outcome in CLL after chlorambucil-based chemotherapy or chemoimmunotherapy.
      ,
      • Ramsay A.J.
      • et al.
      POT1 mutations cause telomere dysfunction in chronic lymphocytic leukemia.
      ,
      • Speedy H.E.
      • et al.
      Germline mutations in shelterin complex genes are associated with familial chronic lymphocytic leukemia.
      SF3B12q33.1Spliceosome componentMissense10 −18UnfavorableEstablished (1)Enriched in patients with del(11q) and unmutIGHV; Candidate driver gene for disease progression
      • Baliakas P.
      • et al.
      Recurrent mutations refine prognosis in chronic lymphocytic leukemia.
      ,
      • Rossi D.
      • et al.
      Integrated mutational and cytogenetic analysis identifies new prognostic subgroups in chronic lymphocytic leukemia.
      ,
      • Mitsui T.
      • et al.
      SF3B1 and IGHV gene mutation status predict poor prognosis in Japanese CLL patients.
      ,
      • Quesada V.
      • et al.
      Exome sequencing identifies recurrent mutations of the splicing factor SF3B1 gene in chronic lymphocytic leukemia.
      ,
      • Wan Y.
      • Wu C.J.
      SF3B1 mutations in chronic lymphocytic leukemia.
      TP5317p13.1DNA repair and cell-cycle controlMissense5–10 (higher with progressive disease)UnfavorableEstablished (1)
      • Wierda W.G.
      • et al.
      NCCN guidelines insights: chronic lymphocytic leukemia/small lymphocytic leukemia, version 1.2017.
      ,
      • Strefford J.C.
      The genomic landscape of chronic lymphocytic leukaemia: biological and clinical implications.
      ,
      • Rossi D.
      • et al.
      Clinical impact of small TP53 mutated subclones in chronic lymphocytic leukemia.
      ,
      • Zenz T.
      • et al.
      TP53 mutation and survival in chronic lymphocytic leukemia.
      ,
      • Stilgenbauer S.
      • et al.
      Gene mutations and treatment outcome in chronic lymphocytic leukemia: results from the CLL8 trial.
      XPO12p15Exports proteins/RNA fragments from nucleus into cytoplasmMissense5–7.5Unfavorable/ high risk of progressionSuspected (2)Associated with unmutIGHV
      • Cosson A.
      • et al.
      Gain in the short arm of chromosome 2 (2p+) induces gene overexpression and drug resistance in chronic lymphocytic leukemia: analysis of the central role of XPO1.
      ,
      • Filip A.A.
      New boys in town: prognostic role of SF3B1, NOTCH1 and other cryptic alterations in chronic lymphocytic leukemia and how it works.
      ,
      • Jain N.
      • O'Brien S.
      Initial treatment of CLL: integrating biology and functional status.
      low asterisk Level 1: present in WHO classification or professional practice guidelines; Level 2: recurrent in well-powered studies with suspected clinical significance; Level 3: recurrent, but uncertain prognostic significance

      Technical considerations

      For general considerations on the use of CMA in cancer, refer to the American College of Medical Genetics and Genomics guidelines [
      • Cooley L.D.
      • et al.
      American College of Medical Genetics and Genomics technical standards and guidelines: microarray analysis for chromosome abnormalities in neoplastic disorders.
      ]. Additional considerations specific to CLL are discussed below.

      CMA design

      Laboratories performing CMA analysis should choose a platform with adequate probe coverage for the detection of copy number aberrations associated with CLL [
      • Cooley L.D.
      • et al.
      American College of Medical Genetics and Genomics technical standards and guidelines: microarray analysis for chromosome abnormalities in neoplastic disorders.
      ]. If a targeted platform design is preferred, it should minimally include enough probe coverage to detect deletions at 11q22.3 (ATM), 13q14.2q14.3 (RB1, DLEU2 and DLEU1), and 17p13.1 (TP53), as well as trisomy 12, as these regions correspond to the four prognostic FISH markers, currently the gold standard in CLL analysis. Beyond this, however, a genome-wide platform with SNPs should be designed to detect other copy number changes as well as regions of CN-LOH, which are becoming increasingly important in cancer [
      • Pfeifer D.
      • et al.
      Genome-wide analysis of DNA copy number changes and LOH in CLL using high-density SNP arrays.
      ,
      • Pei J.
      • et al.
      Copy neutral loss of heterozygosity in 20q in chronic lymphocytic leukemia/small lymphocytic lymphoma.
      ,
      • Xu X.
      • et al.
      The advantage of using SNP array in clinical testing for hematological malignancies–a comparative study of three genetic testing methods.
      ]. Commercially available platforms such as Affymetrix, Illumina and Agilent have been validated and are commonly used for clinical testing. Tables 13 list recurrent copy number abnormalities, regions of CN-LOH and gene mutations identified in CLL.

      FISH analysis

      In certain cases, concurrent FISH analysis for the IGH/CCND1 rearrangement should be considered to rule out mantle cell lymphoma. This test would also detect an IGH rearrangement with a gene other than CCND1. An IGH break-apart probe can also be used to detect all IGH rearrangements, including fusions with BCL2 and BCL3.

      Sample type

      A peripheral blood specimen is sufficient for CLL CMA analysis at diagnosis; bone marrow specimens can also be used. DNA can be extracted directly from the specimen, cultured cells or fixed cell pellets. Direct specimen may be preferred over the cultured cells to provide true clonality levels and avoid potential culture bias of a clone; however, for specimens with limited disease content, culture with oligonucleotide mitogen or B-cell enrichment may allow for increased sensitivity. Whichever method is chosen should be made clear to the ordering providers. Additionally, it is important to validate each specimen type on the chosen CMA platform.

      Analysis

      Size cut-off and backbone threshold parameters should be determined for genome-wide platforms. A .bed file containing important cancer genes can also be used to highlight copy number changes in these regions. The laboratory should establish methods for the detection of clinically relevant CNVs that fall below the established cut-offs.
      Thresholds to identify clinically important regions of homozygosity consistent with CN-LOH should be established. As mentioned above, however, a minimum cut-off of 10 Mb seems reasonable in the absence of paired CLL tumor and germline sample analysis.

      Reporting considerations

      CMA nomenclature

      Current International System for Human Cytogenomic Nomenclature (ISCN) [
      International Standing Committee on Human Cytogenomic Nomenclature
      ISCN: an international system for human cytogenomic nomenclature (2016).
      ] should be used to describe relevant abnormalities in the patient. This is critical for understanding exactly what the abnormality is, not only for the testing laboratory, but for other laboratories who may receive a copy of the original report.

      Interpretation

      Since ISCN nomenclature is specialized, it is important to describe relevant changes in lay terms in the report. Clinically important genes within the CNVs should be provided. It is also critical to provide prognostic or clinically relevant information on the observed changes to guide the clinician in the management of their patient. References, whenever possible, would be helpful as well.
      If a suspected clinically significant constitutional finding is observed, additional studies may be recommended in the report.
      Reporting and interpretation should conform to guidelines provided by the College of American Pathologists (CAP) and the American College of Medical Genetics and Genomics (ACMG) [
      • Cooley L.D.
      • et al.
      American College of Medical Genetics and Genomics technical standards and guidelines: microarray analysis for chromosome abnormalities in neoplastic disorders.
      ].

      Integration of CMA analysis into clinical use

      Per Clinical Laboratory Improvement Amendments of 1988 (CLIA 88) regulations, laboratories using CMA for clinical testing must validate the procedure for the intended use, define specimen acceptability, define cut-off values for determining abnormalities, report results in a meaningful manner including use of ISCN nomenclature, participate in proficiency testing and monitor quality measures. Like any laboratory-developed test, the clinical approach for implementation of CMA for CLL is laboratory-dependent but should include establishing the limit of detection, reproducibility, sensitivity and specificity. While each laboratory will need to establish the appropriate approach, one group has proposed integration of CMA for CLL in clinical practice including the use of CMA as a first-line test in patients with > 30% tumor cells as determined by flow cytometry and use of FISH in those with < 30% tumor cells, to assess risk as normal, low or high [
      • Higgins R.A.
      • Gunn S.R.
      • Robetorye R.S.
      Clinical application of array-based comparative genomic hybridization for the identification of prognostically important genetic alterations in chronic lymphocytic leukemia.
      ], and for each method to reflex to the other if no abnormalities are found [
      • Gunn S.R.
      • et al.
      Whole-genome scanning by array comparative genomic hybridization as a clinical tool for risk assessment in chronic lymphocytic leukemia.
      ]. Using a strategy in which CMA was performed as a first-line test for patients with > 30% tumor cells (as determined by flow cytometry), 89% of cases had abnormalities detected by CMA and were completed without further testing; cases with negative results on CMA were reflexed to FISH. With the combination of CMA and FISH analyses, 96% of CLL cases had clinically significant genomic imbalances [
      • Gunn S.R.
      • et al.
      Whole-genome scanning by array comparative genomic hybridization as a clinical tool for risk assessment in chronic lymphocytic leukemia.
      ] and CMA analysis was able to simultaneously reveal prognostic marker status and the level of genomic complexity in > 85% of cases [
      • Gunn S.R.
      • et al.
      The HemeScan test for genomic prognostic marker assessment in chronic lymphocytic leukemia.
      ,
      • Braggio E.
      • Fonseca R.
      • Kay N.E.
      CGH protocols: chronic lymphocytic leukemia.
      ,
      • Gunn S.R.
      The vanguard has arrived in the clinical laboratory: array-based karyotyping for prognostic markers in chronic lymphocytic leukemia.
      ]. Typically, disease burden is high enough at time of diagnosis that the percentage of tumor cells is not an issue. If there is a suspicion of mantle cell lymphoma, FISH for IGH/CCND1 should be considered.

      Summary

      CLL represents a model hematologic neoplasm for integration of CMA analysis into clinical testing for the following reasons: genetic lesions with known clinical relevance are primarily gains and losses rather than balanced translocations and inversions; DNA from fresh samples is readily available; tumor burden tends to be relatively high in the peripheral blood and can be assessed by flow cytometry.
      Based on the evidence identified through review of the literature, CMA analysis may be sufficient to replace the standard CLL FISH panel and metaphase chromosome analysis in a clinical diagnostic setting (see Table 4 for Comparison of Cytogenetic Technologies). CMA readily detects the gold standard FISH panel abnormalities as well as the genomic imbalances identified by metaphase chromosome analysis. Additionally, CMA has identified the presence of 10–15 second tier abnormalities, present in 1–5% of CLL patients, that are not targeted by FISH panels and may be too small to be seen by metaphase chromosome analysis [
      • Ouillette P.
      • Malek S.
      Acquired genomic copy number aberrations in CLL.
      ]. CMA is more powerful than either FISH or metaphase chromosome analysis at specifically defining regions of imbalance. It will detect deletions that may be missed by FISH panels, elucidate abnormalities that cannot be characterized by analysis of banded metaphase chromosomes and identify potential imbalances in translocations that appear balanced by metaphase chromosome analysis.
      Table 4Comparison of cytogenetic methods for detecting genetic changes in CLL.
      MethodStrengthsWeaknesses
      Metaphase chromosome analysis• Whole genome scan.• Requires cultured cells.
      • Detect balanced rearrangements.• Requires B-cell mitogen (CpG) for increased sensitivity.
      • Detect clonal evolution.• Analysis is slow – one case at a time.
      • Discover novel abnormalities.• Resolution limit ∼ 10 Mb.
      • Exact rearrangements may not be evident by G-banding alone.
      • Cannot detect regions of homozygosity.
      FISH• Sensitivity – detect low level clones.• Only detect abnormalities where FISH probes bind.
      • Batch cases.• Multiple FISH probes required to look at different abnormalities.
      • Does not require cultured cells.• Clonal evolution may not be apparent.
      • Can perform on archival specimens (e.g. FFPE).• Cannot detect regions of homozygosity or genomic instability.
      • Detect specific balanced translocations.• Cannot detect genomic complexity.
      CMA• Whole genome scan.• Cannot detect balanced rearrangements.
      • Batch cases.• Multiple clones not evident.
      • Does not require cultured cells.• Clonal evolution may not be apparent.
      • Can perform on archival specimens (e.g. FFPE).• Less sensitive than FISH.
      • Resolution of 50 kb or less depending on platform.• May require B-cell enrichment if tumor burden is low.
      • Discover novel imbalances in the genome (e.g. genes involved).
      • Detect regions of homozygosity.
      • Detect chromothripsis/genomic instability.
      Both metaphase chromosome and CMA analyses can identify genomic complexity, an independent marker for identification of patients with aggressive CLL. However, chromothripsis, another marker of aggressive disease, is detectable only by CMA. Similarly, CMA is the only one of the three technologies that can detect CN-LOH.
      Limitations of CMA analysis mainly are decreased performance at low levels of tumor involvement and the inability to detect balanced chromosome rearrangements. Both limitations can be readily overcome in CLL. Flow cytometry information is typically available for CLL patients, so the presence of low level disease should be known to the laboratory and CMA analysis should be deferred. Specific translocations of known clinical significance often involve the IGH locus. As such, addition of an IGH break-apart probe or an IGH/CCND1 probe set can be considered to complement CMA analysis in CLL.
      Currently, CMA is evolving for clinical application in CLL. Many laboratories are establishing its effectiveness as a stand-alone method that is likely more efficient and cost-effective than the combination of metaphase chromosome analysis and FISH. At this point in time, many clinical trials require FISH for eligibility and those patients with positive CMA results must have redundant FISH analysis performed to be eligible. Widespread acceptance of CMA technology could eliminate the cost of extra testing. Lastly, CMA technology provides the opportunity for discovery of clinically significant genomic alterations that have not been previously identified by other methodologies.

      Appendix. Supplementary materials

      References

        • Campo E.
        • et al.
        Chronic lymphocytic leukaemia/small lymphocytic lymphoma.
        in: Swerdlow S.H. WHO classification of tumours of haematoppoietic and lymphoid tissues. International Agency for Research on Cancer (IARC), Lyon, France2017: 216-221
        • Fabbri G.
        • Dalla-Favera R.
        The molecular pathogenesis of chronic lymphocytic leukaemia.
        Nat Rev Cancer. 2016; 16: 145-162
        • Rai K.R.
        • Jain P.
        Chronic lymphocytic leukemia (CLL)-Then and now.
        Am J Hematol. 2016; 91: 330-340
        • Goldin L.R.
        • et al.
        Familial risk of lymphoproliferative tumors in families of patients with chronic lymphocytic leukemia: results from the Swedish Family-Cancer Database.
        Blood. 2004; 104: 1850-1854
        • Zenz T.
        • et al.
        Importance of genetics in chronic lymphocytic leukemia.
        Blood Rev. 2011; 25: 131-137
        • Dohner H.
        • et al.
        Genomic aberrations and survival in chronic lymphocytic leukemia.
        N Engl J Med. 2000; 343: 1910-1916
        • Wierda W.G.
        • et al.
        NCCN guidelines insights: chronic lymphocytic leukemia/small lymphocytic leukemia, version 1.2017.
        J Natl Compr Canc Netw. 2017; 15: 293-311
        • International, C.L.L.I.P.I.w.g.
        An international prognostic index for patients with chronic lymphocytic leukaemia (CLL-IPI): a meta-analysis of individual patient data.
        Lancet Oncol. 2016; 17: 779-790
        • Van Dyke D.L.
        • et al.
        The Dohner fluorescence in situ hybridization prognostic classification of chronic lymphocytic leukaemia (CLL): the CLL Research Consortium experience.
        Br J Haematol. 2016; 173: 105-113
        • Cuneo A.
        • et al.
        Chronic lymphocytic leukemia with 6q- shows distinct hematological features and intermediate prognosis.
        Leukemia. 2004; 18: 476-483
        • Dalsass A.
        • et al.
        6q deletion detected by fluorescence in situ hybridization using bacterial artificial chromosome in chronic lymphocytic leukemia.
        Eur J Haematol. 2013; 91: 10-19
        • Setlur S.R.
        • et al.
        Comparison of familial and sporadic chronic lymphocytic leukaemia using high resolution array comparative genomic hybridization.
        Br J Haematol. 2010; 151: 336-345
        • Wang D.M.
        • et al.
        Intermediate prognosis of 6q deletion in chronic lymphocytic leukemia.
        Leuk Lymphoma. 2011; 52: 230-237
        • Stilgenbauer S.
        • et al.
        Incidence and clinical significance of 6q deletions in B cell chronic lymphocytic leukemia.
        Leukemia. 1999; 13: 1331-1334
        • Jarosova M.
        • et al.
        Chromosome 6q deletion correlates with poor prognosis and low relative expression of FOXO3 in chronic lymphocytic leukemia patients.
        Am J Hematol. 2017;
        • Haferlach C.
        • et al.
        Comprehensive genetic characterization of CLL: a study on 506 cases analysed with chromosome banding analysis, interphase FISH, IgV(H) status and immunophenotyping.
        Leukemia. 2007; 21: 2442-2451
        • Heerema N.A.
        • et al.
        Stimulation of chronic lymphocytic leukemia cells with CpG oligodeoxynucleotide gives consistent karyotypic results among laboratories: a CLL Research Consortium (CRC) Study.
        Cancer Genet Cytogenet. 2010; 203: 134-140
        • Rigolin G.M.
        • et al.
        Chromosome aberrations detected by conventional karyotyping using novel mitogens in chronic lymphocytic leukemia with "normal" FISH: correlations with clinicobiologic parameters.
        Blood. 2012; 119: 2310-2313
        • Shi M.
        • et al.
        Improved detection rate of cytogenetic abnormalities in chronic lymphocytic leukemia and other mature B-cell neoplasms with use of CpG-oligonucleotide DSP30 and interleukin 2 stimulation.
        Am J Clin Pathol. 2013; 139: 662-669
        • Rigolin G.M.
        • et al.
        In CLL, comorbidities and the complex karyotype are associated with an inferior outcome independently of CLL-IPI.
        Blood. 2017; 129: 3495-3498
        • Baliakas P.
        • et al.
        Chromosomal translocations and karyotype complexity in chronic lymphocytic leukemia: a systematic reappraisal of classic cytogenetic data.
        Am J Hematol. 2014; 89: 249-255
        • Mayr C.
        • et al.
        Chromosomal translocations are associated with poor prognosis in chronic lymphocytic leukemia.
        Blood. 2006; 107: 742-751
        • Van Den Neste E.
        • et al.
        Chromosomal translocations independently predict treatment failure, treatment-free survival and overall survival in B-cell chronic lymphocytic leukemia patients treated with cladribine.
        Leukemia. 2007; 21: 1715-1722
        • Heerema N.A.
        • et al.
        Presence of a translocation is associated with short time to treatment from diagnosis in ighv mutated chronic lymphocytic leukemia.
        Blood. 2016; 128: 4372
        • Higgins R.A.
        • Gunn S.R.
        • Robetorye R.S.
        Clinical application of array-based comparative genomic hybridization for the identification of prognostically important genetic alterations in chronic lymphocytic leukemia.
        Mol Diagn Ther. 2008; 12: 271-280
        • Put N.
        • Wlodarska I.
        • Vandenberghe P.
        • Michaux L.
        Genetics of Chronic Lymphocytic Leukemia: Practical Aspects and Prognostic Significance.
        Chronic Lymphocytic Leukemia. 2012; (Available from)
        • Nowakowski G.S.
        • et al.
        Interphase fluorescence in situ hybridization with an IGH probe is important in the evaluation of patients with a clinical diagnosis of chronic lymphocytic leukaemia.
        Br J Haematol. 2005; 130: 36-42
        • Reiner S.
        • Aukema S.M.
        Mature B- and T-neoplasms and Hodgkin lymphoma.
        in: Heim S. Mitelman F. Cancer cytogenetics, chromosomal and molecular genetic aberrations of tumor cells. Wiley-Blackwell, West Sussex, UK2015 (Editors)
        • Huh Y.O.
        • et al.
        Chronic lymphocytic leukemia with t(14;19)(q32;q13) is characterized by atypical morphologic and immunophenotypic features and distinctive genetic features.
        Am J Clin Pathol. 2011; 135: 686-696
        • Li Y.
        • et al.
        The clinical significance of 8q24/MYC rearrangement in chronic lymphocytic leukemia.
        Mod Pathol. 2016; 29: 444-451
        • Hagenkord J.M.
        • Chang C.C.
        The rewards and challenges of array-based karyotyping for clinical oncology applications.
        Leukemia. 2009; 23: 829-833
        • Gunnarsson R.
        • et al.
        Screening for copy-number alterations and loss of heterozygosity in chronic lymphocytic leukemia–a comparative study of four differently designed, high resolution microarray platforms.
        Genes Chromosomes Cancer. 2008; 47: 697-711
        • Biesecker L.G.
        • Spinner N.B.
        A genomic view of mosaicism and human disease.
        Nat Rev Genet. 2013; 14: 307-320
        • Hagenkord J.M.
        • et al.
        Array-based karyotyping for prognostic assessment in chronic lymphocytic leukemia: performance comparison of Affymetrix 10K2.0, 250 K Nsp, and SNP6.0 arrays.
        J Mol Diagn. 2010; 12: 184-196
        • Pfeifer D.
        • et al.
        Genome-wide analysis of DNA copy number changes and LOH in CLL using high-density SNP arrays.
        Blood. 2007; 109: 1202-1210
        • Edelmann J.
        • et al.
        High-resolution genomic profiling of chronic lymphocytic leukemia reveals new recurrent genomic alterations.
        Blood. 2012; 120: 4783-4794
        • Gunn S.R.
        • et al.
        Whole-genome scanning by array comparative genomic hybridization as a clinical tool for risk assessment in chronic lymphocytic leukemia.
        J Mol Diagn. 2008; 10: 442-451
        • Gunn S.R.
        • et al.
        The HemeScan test for genomic prognostic marker assessment in chronic lymphocytic leukemia.
        Expert Opin Med Diagn. 2008; 2: 731-740
        • Kolquist K.A.
        • et al.
        Evaluation of chronic lymphocytic leukemia by oligonucleotide-based microarray analysis uncovers novel aberrations not detected by FISH or cytogenetic analysis.
        Mol Cytogenet. 2011; 4: 25
        • Patel A.
        • et al.
        Validation of a targeted DNA microarray for the clinical evaluation of recurrent abnormalities in chronic lymphocytic leukemia.
        Am J Hematol. 2008; 83: 540-546
        • Sargent R.
        • et al.
        Customized oligonucleotide array-based comparative genomic hybridization as a clinical assay for genomic profiling of chronic lymphocytic leukemia.
        J Mol Diagn. 2009; 11: 25-34
        • Schultz R.A.
        • et al.
        Evaluation of chronic lymphocytic leukemia by BAC-based microarray analysis.
        Mol Cytogenet. 2011; 4: 4
        • Stevens-Kroef M.J.
        • et al.
        Identification of prognostic relevant chromosomal abnormalities in chronic lymphocytic leukemia using microarray-based genomic profiling.
        Mol Cytogenet. 2014; 7: 3
        • Garg R.
        • et al.
        The prognostic difference of monoallelic versus biallelic deletion of 13q in chronic lymphocytic leukemia.
        Cancer. 2012; 118: 3531-3537
        • Van Dyke D.L.
        • et al.
        A comprehensive evaluation of the prognostic significance of 13q deletions in patients with B-chronic lymphocytic leukaemia.
        Br J Haematol. 2010; 148: 544-550
        • Grygalewicz B.
        • et al.
        Monoallelic and biallelic deletions of 13q14 in a group of CLL/SLL patients investigated by CGH Haematological Cancer and SNP array (8x60K).
        Mol Cytogenet. 2016; 9: 1
        • Palamarchuk A.
        • et al.
        13q14 deletions in CLL involve cooperating tumor suppressors.
        Blood. 2010; 115: 3916-3922
        • Gunnarsson R.
        • et al.
        Array-based genomic screening at diagnosis and during follow-up in chronic lymphocytic leukemia.
        Haematologica. 2011; 96: 1161-1169
        • Parker H.
        • et al.
        13q deletion anatomy and disease progression in patients with chronic lymphocytic leukemia.
        Leukemia. 2011; 25: 489-497
        • Mian M.
        • et al.
        Del(13q14.3) length matters: an integrated analysis of genomic, fluorescence in situ hybridization and clinical data in 169 chronic lymphocytic leukaemia patients with 13q deletion alone or a normal karyotype.
        Hematol Oncol. 2012; 30: 46-49
        • Ibbotson R.
        • et al.
        Coexistence of trisomies of chromosomes 12 and 19 in chronic lymphocytic leukemia occurs exclusively in the rare IgG-positive variant.
        Leukemia. 2012; 26: 170-172
        • Van Dyke D.L.
        +18 or trisomy 18 in lymphoproliferative disorders.
        Atlas Genet Cytogenet Oncol Haematol. 2003; (Available from)
        • Strefford J.C.
        The genomic landscape of chronic lymphocytic leukaemia: biological and clinical implications.
        Br J Haematol. 2015; 169: 14-31
        • Gunn S.R.
        • et al.
        Atypical 11q deletions identified by array CGH may be missed by FISH panels for prognostic markers in chronic lymphocytic leukemia.
        Leukemia. 2009; 23: 1011-1017
        • Rossi D.
        • et al.
        Clinical impact of small TP53 mutated subclones in chronic lymphocytic leukemia.
        Blood. 2014; 123: 2139-2147
        • Zenz T.
        • et al.
        TP53 mutation and survival in chronic lymphocytic leukemia.
        J Clin Oncol. 2010; 28: 4473-4479
        • Fabris S.
        • et al.
        Molecular and transcriptional characterization of 17p loss in B-cell chronic lymphocytic leukemia.
        Genes Chromosomes Cancer. 2008; 47: 781-793
        • Nabhan C.
        • Raca G.
        • Wang Y.L.
        Predicting prognosis in chronic lymphocytic leukemia in the contemporary era.
        JAMA Oncol. 2015; 1: 965-974
        • Greipp P.T.
        • et al.
        Patients with chronic lymphocytic leukaemia and clonal deletion of both 17p13.1 and 11q22.3 have a very poor prognosis.
        Br J Haematol. 2013; 163: 326-333
        • Reindl L.
        • et al.
        Biological and clinical characterization of recurrent 14q deletions in CLL and other mature B-cell neoplasms.
        Br J Haematol. 2010; 151: 25-36
        • Cosson A.
        • et al.
        14q deletions are associated with trisomy 12, NOTCH1 mutations and unmutated IGHV genes in chronic lymphocytic leukemia and small lymphocytic lymphoma.
        Genes Chromosomes Cancer. 2014; 53: 657-666
        • Braggio E.
        • et al.
        Longitudinal genome-wide analysis of patients with chronic lymphocytic leukemia reveals complex evolution of clonal architecture at disease progression and at the time of relapse.
        Leukemia. 2012; 26: 1698-1701
        • Gunnarsson R.
        • et al.
        Large but not small copy-number alterations correlate to high-risk genomic aberrations and survival in chronic lymphocytic leukemia: a high-resolution genomic screening of newly diagnosed patients.
        Leukemia. 2010; 24: 211-215
        • Knight S.J.
        • et al.
        Quantification of subclonal distributions of recurrent genomic aberrations in paired pre-treatment and relapse samples from patients with B-cell chronic lymphocytic leukemia.
        Leukemia. 2012; 26: 1564-1575
        • Ouillette P.
        • et al.
        Acquired genomic copy number aberrations and survival in chronic lymphocytic leukemia.
        Blood. 2011; 118: 3051-3061
        • Rudenko H.C.
        • et al.
        Characterising the TP53-deleted subgroup of chronic lymphocytic leukemia: an analysis of additional cytogenetic abnormalities detected by interphase fluorescence in situ hybridisation and array-based comparative genomic hybridisation.
        Leuk Lymphoma. 2008; 49: 1879-1886
        • Schweighofer C.D.
        • et al.
        Genomic variation by whole-genome SNP mapping arrays predicts time-to-event outcome in patients with chronic lymphocytic leukemia: a comparison of CLL and HapMap genotypes.
        J Mol Diagn. 2013; 15: 196-209
        • Kay N.E.
        • et al.
        Progressive but previously untreated CLL patients with greater array CGH complexity exhibit a less durable response to chemoimmunotherapy.
        Cancer Genet Cytogenet. 2010; 203: 161-168
        • Kujawski L.
        • et al.
        Genomic complexity identifies patients with aggressive chronic lymphocytic leukemia.
        Blood. 2008; 112: 1993-2003
        • Puiggros A.
        • Blanco G.
        • Espinet B.
        Genetic abnormalities in chronic lymphocytic leukemia: where we are and where we go.
        Biomed Res Int. 2014; 2014435983
        • Peterson J.F.
        The complexities of defining a complex karyotype in hematological malignancies: a need for standardization.
        Acta Haematol. 2017; 138: 65-66
        • Landau D.A.
        • et al.
        Evolution and impact of subclonal mutations in chronic lymphocytic leukemia.
        Cell. 2013; 152: 714-726
        • Zhang L.
        • et al.
        Clonal diversity analysis using SNP microarray: a new prognostic tool for chronic lymphocytic leukemia.
        Cancer Genet. 2011; 204: 654-665
        • Del Giudice I.
        • et al.
        Inter- and intra-patient clonal and subclonal heterogeneity of chronic lymphocytic leukaemia: evidences from circulating and lymph nodal compartments.
        Br J Haematol. 2016; 172: 371-383
        • Chigrinova E.
        • et al.
        Two main genetic pathways lead to the transformation of chronic lymphocytic leukemia to Richter syndrome.
        Blood. 2013; 122: 2673-2682
        • Fabbri G.
        • et al.
        Genetic lesions associated with chronic lymphocytic leukemia transformation to Richter syndrome.
        J Exp Med. 2013; 210: 2273-2288
        • Rossi D.
        • Gaidano G.
        Richter syndrome: pathogenesis and management.
        Semin Oncol. 2016; 43: 311-319
        • Jamroziak K.
        • et al.
        Richter syndrome in chronic lymphocytic leukemia: updates on biology, clinical features and therapy.
        Leuk Lymphoma. 2015; 56: 1949-1958
        • Mao Z.
        • et al.
        IgVH mutational status and clonality analysis of Richter's transformation: diffuse large B-cell lymphoma and Hodgkin lymphoma in association with B-cell chronic lymphocytic leukemia (B-CLL) represent 2 different pathways of disease evolution.
        Am J Surg Pathol. 2007; 31: 1605-1614
        • Robertson L.E.
        • et al.
        Therapy-related leukemia and myelodysplastic syndrome in chronic lymphocytic leukemia.
        Leukemia. 1994; 8: 2047-2051
        • Skarbnik A.P.
        • Faderl S.
        The role of combined fludarabine, cyclophosphamide and rituximab chemoimmunotherapy in chronic lymphocytic leukemia: current evidence and controversies.
        Ther Adv Hematol. 2017; 8: 99-105
        • Houldsworth J.
        • et al.
        Genomic imbalance defines three prognostic groups for risk stratification of patients with chronic lymphocytic leukemia.
        Leuk Lymphoma. 2014; 55: 920-928
        • Shao L.
        • et al.
        Array comparative genomic hybridization detects chromosomal abnormalities in hematological cancers that are not detected by conventional cytogenetics.
        J Mol Diagn. 2010; 12: 670-679
        • Cosson A.
        • et al.
        Gain in the short arm of chromosome 2 (2p+) induces gene overexpression and drug resistance in chronic lymphocytic leukemia: analysis of the central role of XPO1.
        Leukemia. 2017; 31: 1625-1629
        • Chapiro E.
        • et al.
        Gain of the short arm of chromosome 2 (2p) is a frequent recurring chromosome aberration in untreated chronic lymphocytic leukemia (CLL) at advanced stages.
        Leuk Res. 2010; 34: 63-68
        • Ma D.
        • et al.
        Array comparative genomic hybridization analysis identifies recurrent gain of chromosome 2p25.3 involving the ACP1 and MYCN genes in chronic lymphocytic leukemia.
        Clin Lymphoma Myeloma Leuk. 2011; 11: S17-S24
        • Fabris S.
        • et al.
        Chromosome 2p gain in monoclonal B-cell lymphocytosis and in early stage chronic lymphocytic leukemia.
        Am J Hematol. 2013; 88: 24-31
        • Stephens P.J.
        • et al.
        Massive genomic rearrangement acquired in a single catastrophic event during cancer development.
        Cell. 2011; 144: 27-40
        • Salaverria I.
        • et al.
        Detection of chromothripsis-like patterns with a custom array platform for chronic lymphocytic leukemia.
        Genes Chromosomes Cancer. 2015; 54: 668-680
        • Parker H.
        • et al.
        Genomic disruption of the histone methyltransferase SETD2 in chronic lymphocytic leukaemia.
        Leukemia. 2016; 30: 2179-2186
        • Lehmann S.
        • et al.
        Molecular allelokaryotyping of early-stage, untreated chronic lymphocytic leukemia.
        Cancer. 2008; 112: 1296-1305
        • Li M.M.
        • et al.
        A multicenter, cross-platform clinical validation study of cancer cytogenomic arrays.
        Cancer Genet. 2015; 208: 525-536
        • Pei J.
        • et al.
        Copy neutral loss of heterozygosity in 20q in chronic lymphocytic leukemia/small lymphocytic lymphoma.
        Cancer Genet. 2014; 207: 98-102
        • Saddler C.
        • et al.
        Comprehensive biomarker and genomic analysis identifies p53 status as the major determinant of response to MDM2 inhibitors in chronic lymphocytic leukemia.
        Blood. 2008; 111: 1584-1593
        • Sellmann L.
        • et al.
        Shorter telomeres correlate with an increase in the number of uniparental disomies in patients with chronic lymphocytic leukemia.
        Leuk Lymphoma. 2016; 57: 590-595
        • Sellmann L.
        • et al.
        Gene dosage effects in chronic lymphocytic leukemia.
        Cancer Genet Cytogenet. 2010; 203: 149-160
        • Xu X.
        • et al.
        The advantage of using SNP array in clinical testing for hematological malignancies–a comparative study of three genetic testing methods.
        Cancer Genet. 2013; 206: 317-326
        • Maciejewski J.P.
        • Mufti G.J.
        Whole genome scanning as a cytogenetic tool in hematologic malignancies.
        Blood. 2008; 112: 965-974
        • Ouillette P.
        • et al.
        The prognostic significance of various 13q14 deletions in chronic lymphocytic leukemia.
        Clin Cancer Res. 2011; 17: 6778-6790
        • Alsolami R.
        • Knight S.J.
        • Schuh A.
        Clinical application of targeted and genome-wide technologies: can we predict treatment responses in chronic lymphocytic leukemia.
        Per Med. 2013; 10: 361-376
        • Landau D.A.
        • et al.
        Mutations driving CLL and their evolution in progression and relapse.
        Nature. 2015; 526: 525-530
        • Baliakas P.
        • et al.
        Recurrent mutations refine prognosis in chronic lymphocytic leukemia.
        Leukemia. 2015; 29: 329-336
        • Brown J.R.
        • et al.
        Extended follow-up and impact of high-risk prognostic factors from the phase 3 RESONATE study in patients with previously treated CLL/SLL.
        Leukemia. 2018; 32: 83-91
        • Chiaretti S.
        • et al.
        NOTCH1, SF3B1, BIRC3 and TP53 mutations in patients with chronic lymphocytic leukemia undergoing first-line treatment: correlation with biological parameters and response to treatment.
        Leuk Lymphoma. 2014; 55: 2785-2792
        • Cortese D.
        • et al.
        On the way towards a 'CLL prognostic index': focus on TP53, BIRC3, SF3B1, NOTCH1 and MYD88 in a population-based cohort.
        Leukemia. 2014; 28: 710-713
        • Nadeu F.
        • et al.
        Clinical impact of clonal and subclonal TP53, SF3B1, BIRC3, NOTCH1, and ATM mutations in chronic lymphocytic leukemia.
        Blood. 2016; 127: 2122-2130
        • Xia Y.
        • et al.
        Frequencies of SF3B1, NOTCH1, MYD88, BIRC3 and IGHV mutations and TP53 disruptions in Chinese with chronic lymphocytic leukemia: disparities with Europeans.
        Oncotarget. 2015; 6: 5426-5434
        • Rossi D.
        • et al.
        Disruption of BIRC3 associates with fludarabine chemorefractoriness in TP53 wild-type chronic lymphocytic leukemia.
        Blood. 2012; 119: 2854-2862
        • Guieze R.
        • Wu C.J.
        Genomic and epigenomic heterogeneity in chronic lymphocytic leukemia.
        Blood. 2015; 126: 445-453
        • Ojha J.
        • et al.
        Deep sequencing identifies genetic heterogeneity and recurrent convergent evolution in chronic lymphocytic leukemia.
        Blood. 2015; 125: 492-498
        • Cooley L.D.
        • et al.
        American College of Medical Genetics and Genomics technical standards and guidelines: microarray analysis for chromosome abnormalities in neoplastic disorders.
        Genet Med. 2013; 15: 484-494
        • International Standing Committee on Human Cytogenomic Nomenclature
        ISCN: an international system for human cytogenomic nomenclature (2016).
        Basel. vi. Karger, New York2016 (139 pages)
        • Braggio E.
        • Fonseca R.
        • Kay N.E.
        CGH protocols: chronic lymphocytic leukemia.
        Methods Mol Biol. 2013; 973: 87-98
        • Gunn S.R.
        The vanguard has arrived in the clinical laboratory: array-based karyotyping for prognostic markers in chronic lymphocytic leukemia.
        J Mol Diagn. 2010; 12: 144-146