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Clinical utility of chromosomal microarray in establishing clonality and high risk features in patients with Richter transformation

  • Author Footnotes
    1 These authors are co-first authors of this manuscript and contributed equally to this body of work.
    Brian Hess
    Footnotes
    1 These authors are co-first authors of this manuscript and contributed equally to this body of work.
    Affiliations
    Department of Hematology/Oncology, Walton Research Building, Medical University of South Carolina, 39 Sabin Street, Charleston SC, 29425, USA
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  • Author Footnotes
    1 These authors are co-first authors of this manuscript and contributed equally to this body of work.
    James Kalmuk
    Correspondence
    Corresponding author at: Department of Hematology/Oncology, Medical University of South Carolina, Walton Research Building, 39 Sabin St., Charleston, SC 29425, USA
    Footnotes
    1 These authors are co-first authors of this manuscript and contributed equally to this body of work.
    Affiliations
    Department of Hematology/Oncology, Walton Research Building, Medical University of South Carolina, 39 Sabin Street, Charleston SC, 29425, USA
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  • Iya Znoyko
    Affiliations
    Department of Pathology and Laboratory Medicine, Medical University of South Carolina, 171 Ashley Avenue, Charleston SC, 29425, USA
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  • Cynthia A. Schandl
    Affiliations
    Department of Pathology and Laboratory Medicine, Medical University of South Carolina, 171 Ashley Avenue, Charleston SC, 29425, USA
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  • Nina Wagner-Johnston
    Affiliations
    Department of Hematology/Oncology, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, 1650 Orleans Street, Baltimore MD, 21287, USA
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  • Sandra Mazzoni
    Affiliations
    Department of Hematology/Oncology, Cleveland Clinic, 9500 Euclid Avenue, Cleveland OH, 44195, USA
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  • Lindsey Hendrickson
    Affiliations
    Department of Hematology/Oncology, Walton Research Building, Medical University of South Carolina, 39 Sabin Street, Charleston SC, 29425, USA
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  • Zane Chiad
    Affiliations
    Department of Hematology/Oncology, Levine Cancer Institute, Atrium Health, 1021 Morehead Medical Drive, Charlotte NC, 28204, USA
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  • Irl Brian Greenwell
    Affiliations
    Department of Hematology/Oncology, Walton Research Building, Medical University of South Carolina, 39 Sabin Street, Charleston SC, 29425, USA
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  • Daynna J. Wolff
    Affiliations
    Department of Pathology and Laboratory Medicine, Medical University of South Carolina, 171 Ashley Avenue, Charleston SC, 29425, USA
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  • Author Footnotes
    1 These authors are co-first authors of this manuscript and contributed equally to this body of work.
Open AccessPublished:October 28, 2021DOI:https://doi.org/10.1016/j.cancergen.2021.10.003

      Highlights

      • Aggressive lymphoma (DLBCL) arising in CLL/SLL is a Richter transformation.
      • Clonal relationship between the DLBCL and CLL/SLL carries a poor prognosis.
      • CMA can help establish clonality and detect high risk aberrations.
      • Molecular profiling with CMA has potential prognostic and therapeutic implications.

      Abstract

      Richter transformation (RT) refers to the development of an aggressive lymphoma in patients with pre-existing chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL). It carries a poor prognosis secondary to poor response to therapy or rapid disease relapse. Currently there are no randomized trials to guide treatment. Therapeutic decisions are often influenced by the presence or absence of a clonal relationship between the underlying CLL/SLL and the new lymphoma given the poor prognosis of patients with clonally related RT. Chromosomal microarray analysis (CMA) can help to establish clonality while also detecting genomic complexity and clinically relevant genetic variants such as loss of CDKN2A and/or TP53. As a result, CMA has potential prognostic and therapeutic implications. For this study, CMA results from patients with Richter transformation were evaluated in paired CLL/SLL and transformed lymphoma samples. CMA revealed that 86% of patients had common aberrations in the two samples indicating evidence of common clonality. CMA was also useful in detecting aberrations associated with a poor prognosis in 71% of patients with RT. This study highlights the potential clinical utility of CMA to investigate the clonal relationship between CLL/SLL and RT, provide prognostic information, and possibly guide therapeutic decision making for patients with Richter transformation.

      Keywords

      Introduction

      Richter transformation (RT) refers to transformation of chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL) into an aggressive lymphoma, often diffuse large B-cell lymphoma (DLBCL) and rarely Hodgkin or other lymphomas. RT patients have a worse prognosis compared to de novo DLBCL [
      • Rossi D.
      • Spina V.
      • Deambrogi C.
      • Rasi S.
      • Laurenti L.
      • Stamatopoulos K.
      • et al.
      The genetics of Richter syndrome reveals disease heterogeneity and predicts survival after transformation.
      ,
      • Parikh S.A.
      • Rabe K.G.
      • Call T.G.
      • Zent C.S.
      • Habermann T.M.
      • Ding W.
      • et al.
      Diffuse large B-cell lymphoma (Richter syndrome) in patients with chronic lymphocytic leukaemia (CLL): a cohort study of newly diagnosed patients.
      ].
      The described incidence of RT is variable, ranging from 2 to 16% [
      • Parikh S.A.
      • Rabe K.G.
      • Call T.G.
      • Zent C.S.
      • Habermann T.M.
      • Ding W.
      • et al.
      Diffuse large B-cell lymphoma (Richter syndrome) in patients with chronic lymphocytic leukaemia (CLL): a cohort study of newly diagnosed patients.
      ,
      • Tsimberidou A.M.
      • O'Brien S.
      • Khouri I.
      • Giles F.J.
      • Kantarjian H.M.
      • Champlin R.
      • et al.
      Clinical outcomes and prognostic factors in patients with Richter's syndrome treated with chemotherapy or chemoimmunotherapy with or without stem-cell transplantation.
      ,
      • Rossi D.
      • Cerri M.
      • Capello D.
      • Deambrogi C.
      • Rossi F.M.
      • Zucchetto A.
      • et al.
      Biological and clinical risk factors of chronic lymphocytic leukaemia transformation to Richter syndrome.
      ,
      • Thompson P.A.
      • Tam C.S.
      • O'Brien S.M.
      • Wierda W.G.
      • Stingo F.
      • Plunkett W.
      • et al.
      Fludarabine, cyclophosphamide, and rituximab treatment achieves long-term disease-free survival in IGHV-mutated chronic lymphocytic leukemia.
      ]. CLL/SLL is typically characterized by an indolent disease course, and patients can be observed until treatment is indicated based on IWCLL criteria [
      • Hallek M.
      • Cheson B.D.
      • Catovsky D.
      • Caligaris-Cappio F.
      • Dighiero G.
      • Döhner H.
      • et al.
      iwCLL guidelines for diagnosis, indications for treatment, response assessment, and supportive management of CLL.
      ]. Treatment indications include cytopenias, bulky lymphadenopathy, B-symptoms, and significant fatigue. Lately, the treatment landscape for CLL/SLL has rapidly advanced with novel agents targeting the B-cell pathway, including inhibitors of Bruton Tyrosine Kinase (BTK), B-cell Lymphoma 2 (BCL-2), and Phosphoinositide 3-kinase (PI3K) [
      • Ding W.
      Richter transformation in the era of novel agents.
      ,
      • Maddocks K.J.
      • Ruppert A.S.
      • Lozanski G.
      • Heerema N.A.
      • Zhao W.
      • Abruzzo L.
      • et al.
      Etiology of ibrutinib therapy discontinuation and outcomes in patients with chronic lymphocytic leukemia.
      ,
      • Byrd J.C.
      • Furman R.R.
      • Coutre S.E.
      • Burger J.A.
      • Blum K.A.
      • Coleman M.
      • et al.
      Three-year follow-up of treatment-naive and previously treated patients with CLL and SLL receiving single-agent ibrutinib.
      ,
      • Jain P.
      • Thompson P.A.
      • Keating M.
      • Estrov Z.
      • Ferrajoli A.
      • Jain N.
      • et al.
      Long-term outcomes for patients with chronic lymphocytic leukemia who discontinue ibrutinib.
      ,
      • Anderson M.A.
      • Tam C.
      • Lew T.E.
      • Juneja S.
      • Juneja M.
      • Westerman D.
      • et al.
      Clinicopathological features and outcomes of progression of CLL on the BCL2 inhibitor venetoclax. Blood.
      ,
      • Petrackova A.
      • Turcsanyi P.
      • Papajik T.
      • Kriegova E.
      Revisiting Richter transformation in the era of novel CLL agents.
      ]. Unfortunately, the risk of developing RT has not decreased despite significant improvements in outcomes with novel agents [
      • Ding W.
      Richter transformation in the era of novel agents.
      ,
      • Maddocks K.J.
      • Ruppert A.S.
      • Lozanski G.
      • Heerema N.A.
      • Zhao W.
      • Abruzzo L.
      • et al.
      Etiology of ibrutinib therapy discontinuation and outcomes in patients with chronic lymphocytic leukemia.
      ,
      • Byrd J.C.
      • Furman R.R.
      • Coutre S.E.
      • Burger J.A.
      • Blum K.A.
      • Coleman M.
      • et al.
      Three-year follow-up of treatment-naive and previously treated patients with CLL and SLL receiving single-agent ibrutinib.
      ,
      • Jain P.
      • Thompson P.A.
      • Keating M.
      • Estrov Z.
      • Ferrajoli A.
      • Jain N.
      • et al.
      Long-term outcomes for patients with chronic lymphocytic leukemia who discontinue ibrutinib.
      ,
      • Anderson M.A.
      • Tam C.
      • Lew T.E.
      • Juneja S.
      • Juneja M.
      • Westerman D.
      • et al.
      Clinicopathological features and outcomes of progression of CLL on the BCL2 inhibitor venetoclax. Blood.
      ,
      • Petrackova A.
      • Turcsanyi P.
      • Papajik T.
      • Kriegova E.
      Revisiting Richter transformation in the era of novel CLL agents.
      ].
      RT typically presents with aggressive features, including marked increase in lymphadenopathy, B symptoms, elevated LDH, and/or cytopenias [
      • Robertson L.E.
      • Pugh W.
      • O'Brien S.
      • Kantarjian H.
      • Hirsch-Ginsberg C.
      • Cork A.
      • et al.
      Richter's syndrome: a report on 39 patients.
      ]. Prognostic features include number of lines of therapy prior to transformation, TP53/CDKN2A abnormalities, trisomy 12, genomic complexity, elevated LDH, advanced ECOG scores, and clonal relationship between CLL/SLL and RT [
      • Rossi D.
      • Spina V.
      • Deambrogi C.
      • Rasi S.
      • Laurenti L.
      • Stamatopoulos K.
      • et al.
      The genetics of Richter syndrome reveals disease heterogeneity and predicts survival after transformation.
      ,
      • Tsimberidou A.M.
      • O'Brien S.
      • Khouri I.
      • Giles F.J.
      • Kantarjian H.M.
      • Champlin R.
      • et al.
      Clinical outcomes and prognostic factors in patients with Richter's syndrome treated with chemotherapy or chemoimmunotherapy with or without stem-cell transplantation.
      ,
      • Messina M.
      • Del Giudice I.
      • Khiabanian H.
      • Rossi D.
      • Chiaretti S.
      • Rasi S.
      • et al.
      Genetic lesions associated with chronic lymphocytic leukemia chemo-refractoriness.
      ,
      • Chigrinova E.
      • Rinaldi A.
      • Kwee I.
      • Rossi D.
      • Rancoita P.M.
      • Strefford J.C.
      • et al.
      Two main genetic pathways lead to the transformation of chronic lymphocytic leukemia to Richter syndrome.
      ,
      • Rogers K.A.
      • Huang Y.
      • Ruppert A.S.
      • Salem G.
      • Stephens D.M.
      • Heerema N.A.
      • et al.
      A single-institution retrospective cohort study of first-line R-EPOCH chemoimmunotherapy for Richter syndrome demonstrating complex chronic lymphocytic leukaemia karyotype as an adverse prognostic factor.
      ,
      • Eyre T.A.
      • Clifford R.
      • Bloor A.
      • Boyle L.
      • Roberts C.
      • Cabes M.
      • et al.
      NCRI phase II study of CHOP in combination with ofatumumab in induction and maintenance in newly diagnosed Richter syndrome.
      ,
      • Chun K.
      • Wenger G.D.
      • Chaubey A.
      • Dash D.P.
      • Kanagal-Shamanna R.
      • Kantarci S.
      • et al.
      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.
      ].
      There are no randomized trials to direct RT treatment. Many centers use clonal relationship to direct treatment decisions, including whether patients should receive consolidation with autologous/allogeneic stem cell transplantation [
      • Ding W.
      Richter transformation in the era of novel agents.
      ,
      • Parikh S.A.
      • Kay N.E.
      • Shanafelt T.D.
      How we treat Richter syndrome.
      ]. The majority (∼70–80%) of RT-DLBCL patients are clonally related to the CLL. These patients have a considerably worse prognosis (median survival 6–14 months) as opposed to 5 years if clonally unrelated [
      • Rossi D.
      • Spina V.
      • Deambrogi C.
      • Rasi S.
      • Laurenti L.
      • Stamatopoulos K.
      • et al.
      The genetics of Richter syndrome reveals disease heterogeneity and predicts survival after transformation.
      ,
      • Mao Z.
      • Quintanilla-Martinez L.
      • Raffeld M.
      • Richter M.
      • Krugmann J.
      • Burek C.
      • 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.
      ,
      • Abrisqueta P.
      • Delgado J.
      • Alcoceba M.
      • Oliveira A.C.
      • Loscertales J.
      • Hernández-Rivas J.A.
      • et al.
      Clinical outcome and prognostic factors of patients with Richter syndrome: real-world study of the Spanish Chronic Lymphocytic Leukemia Study Group (GELLC).
      ]. Clonality is typically assessed by sequencing the immunoglobulin heavy chain (IGHV) V-d-J region [
      • Mao Z.
      • Quintanilla-Martinez L.
      • Raffeld M.
      • Richter M.
      • Krugmann J.
      • Burek C.
      • 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.
      ]. Array comparative genomic hybridization has also been reported as a tool to study clonal relationship [
      • Beà S.
      • López-Guillermo A.
      • Ribas M.
      • Puig X.
      • Pinyol M.
      • Carrió A.
      • et al.
      Genetic imbalances in progressed B-cell chronic lymphocytic leukemia and transformed large-cell lymphoma (Richter’s syndrome).
      ]. Unfortunately, clonality testing is not performed in many RT cases because of the need to analyze both the CLL and RT samples and the complicated nature of immunoglobulin gene rearrangement testing (which often necessitates send out of both samples) [
      • Wang Y.
      • Ding W.
      Richter transformation of chronic lymphocytic leukemia in the era of novel agents.
      ]. Given both the clinical significance related to clonality testing as well as the difficulty in performing reliable testing across centers, there is a need for reliable and expedient clonality testing in RT patients [
      • Petrackova A.
      • Turcsanyi P.
      • Papajik T.
      • Kriegova E.
      Revisiting Richter transformation in the era of novel CLL agents.
      ].
      Whole genome chromosomal microarray analysis (CMA) provides information on copy number abnormalities (CNAs) and loss of heterozygosity, genomic complexity, and clonal diversity [
      • Chun K.
      • Wenger G.D.
      • Chaubey A.
      • Dash D.P.
      • Kanagal-Shamanna R.
      • Kantarci S.
      • et al.
      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.
      ,
      • Zhang L.
      • Znoyko I.
      • Costa L.J.
      • Conlin L.K.
      • Daber R.D.
      • Self S.E.
      • et al.
      Clonal diversity analysis using SNP microarray: a new prognostic tool for chronic lymphocytic leukemia.
      ]. CMA has shown high concordance with standard CLL FISH panel studies and metaphase chromosome analysis as well as an increased yield of clinically relevant recurrent copy number changes that are either not targeted by the FISH panel or are too small to be detected by routine chromosome studies [
      • Chun K.
      • Wenger G.D.
      • Chaubey A.
      • Dash D.P.
      • Kanagal-Shamanna R.
      • Kantarci S.
      • et al.
      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.
      ,
      • Visentin A.
      • Bonaldi L.
      • Rigolin G.M.
      • Mauro F.R.
      • Martines A.
      • Frezzato F.
      • et al.
      The complex karyotype landscape in chronic lymphocytic leukemia allows to refine the risk of Richter syndrome transformation.
      ]. Presence of >3–5 chromosome abnormalities is known as genomic complexity, which is a biomarker for an adverse prognosis in CLL (particularly if the aberrations are structural) and was recently associated with increased risk of RT [
      • Chun K.
      • Wenger G.D.
      • Chaubey A.
      • Dash D.P.
      • Kanagal-Shamanna R.
      • Kantarci S.
      • et al.
      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.
      ,
      • Visentin A.
      • Bonaldi L.
      • Rigolin G.M.
      • Mauro F.R.
      • Martines A.
      • Frezzato F.
      • et al.
      The complex karyotype landscape in chronic lymphocytic leukemia allows to refine the risk of Richter syndrome transformation.
      ]. In addition, CMA detects loss of both TP53 (which is associated with increased risk for transformation) as well as homozygous loss of CDKN2A/B (also observed to be acquired at transformation) [
      • Rossi D.
      • Spina V.
      • Deambrogi C.
      • Rasi S.
      • Laurenti L.
      • Stamatopoulos K.
      • et al.
      The genetics of Richter syndrome reveals disease heterogeneity and predicts survival after transformation.
      ,
      • Rossi D.
      • Cerri M.
      • Capello D.
      • Deambrogi C.
      • Rossi F.M.
      • Zucchetto A.
      • et al.
      Biological and clinical risk factors of chronic lymphocytic leukaemia transformation to Richter syndrome.
      ,
      • Messina M.
      • Del Giudice I.
      • Khiabanian H.
      • Rossi D.
      • Chiaretti S.
      • Rasi S.
      • et al.
      Genetic lesions associated with chronic lymphocytic leukemia chemo-refractoriness.
      ,
      • Chigrinova E.
      • Rinaldi A.
      • Kwee I.
      • Rossi D.
      • Rancoita P.M.
      • Strefford J.C.
      • et al.
      Two main genetic pathways lead to the transformation of chronic lymphocytic leukemia to Richter syndrome.
      ].
      CMA is routinely performed at our institution to establish chromosomal aberrations at diagnosis for all CLL/SLL patients since it provides a cost-effective method to detect the clinically relevant genetic abnormalities [
      • Chun K.
      • Wenger G.D.
      • Chaubey A.
      • Dash D.P.
      • Kanagal-Shamanna R.
      • Kantarci S.
      • et al.
      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.
      ,
      • Zhang L.
      • Znoyko I.
      • Costa L.J.
      • Conlin L.K.
      • Daber R.D.
      • Self S.E.
      • et al.
      Clonal diversity analysis using SNP microarray: a new prognostic tool for chronic lymphocytic leukemia.
      ,
      • Wierda W.G.
      • Zelenetz A.D.
      • Gordon L.I.
      • Abramson J.S.
      • Advani R.H.
      • Andreadis C.B.
      • et al.
      NCCN guidelines insights: chronic lymphocytic leukemia/small lymphocytic lymphoma, version 1.2017.
      ,
      • Dias L.M.
      • Thodima V.
      • Friedman J.
      • Ma C.
      • Guttapalli A.
      • Mendiratta G.
      • et al.
      Cross-platform assessment of genomic imbalance confirms the clinical relevance of genomic complexity and reveals loci with potential pathogenic roles in diffuse large B-cell lymphoma.
      ]. This provides prognostic information at diagnosis and the ability to track aberrations in the event of progression or transformation of disease. In the event of transformation to DLBCL, CMA results of the aggressive lymphoma can be compared to those of the original CLL/SLL to determine clonal relationship, identify high-risk aberrations, and assess for genomic complexity. Taken together, this data can be utilized to inform prognosis and guide treatment decisions for RT patients.

      Materials/Methods

      We performed a retrospective analysis of all patients at our institution with confirmed CLL/SLL between 01/01/2010 and 02/14/2019. Patients with CLL/SLL were then evaluated for RT by reviewing clinical documentation and pathologic data in the electronic medical record. We collected baseline demographic, clinical, laboratory, pathology, and outcomes data on patients with CLL/SLL and subsequent DLBCL and who had CMA performed on both the CLL/SLL and DLBCL samples. One patient did exhibit transformation to Hodgkin Lymphoma; however, CMA data was not available on both samples thus was excluded.
      CMA was performed using the Infinium HD Human Omni1 BeadChip or the CytoSNP-850 K v1.1 BeadChip Array (Illumina, Inc., San Diego, CA) on genomic DNA extracted from peripheral blood/marrow and fresh or formalin fixed paraffin-embedded (FFPE) lymph node samples. Copy number and genotype data were analyzed using NxClinical software (BioDiscovery, El Segundo, CA). Aberrations that were in 100% of cells and were present in public databases of germline variants were considered constitutional and not included in analysis; whereas, aberrations found in less than 100% of cells were considered clonal changes. Genome build hg19 (February 2009) was used for probe locations and data interpretation.

      Results

      We identified 670 patients with CLL, of which 24 (3.5%) developed RT (consistent with reported incidence rates of RT) [
      • Parikh S.A.
      • Rabe K.G.
      • Call T.G.
      • Zent C.S.
      • Habermann T.M.
      • Ding W.
      • et al.
      Diffuse large B-cell lymphoma (Richter syndrome) in patients with chronic lymphocytic leukaemia (CLL): a cohort study of newly diagnosed patients.
      ,
      • Tsimberidou A.M.
      • O'Brien S.
      • Khouri I.
      • Giles F.J.
      • Kantarjian H.M.
      • Champlin R.
      • et al.
      Clinical outcomes and prognostic factors in patients with Richter's syndrome treated with chemotherapy or chemoimmunotherapy with or without stem-cell transplantation.
      ,
      • Rossi D.
      • Cerri M.
      • Capello D.
      • Deambrogi C.
      • Rossi F.M.
      • Zucchetto A.
      • et al.
      Biological and clinical risk factors of chronic lymphocytic leukaemia transformation to Richter syndrome.
      ,
      • Thompson P.A.
      • Tam C.S.
      • O'Brien S.M.
      • Wierda W.G.
      • Stingo F.
      • Plunkett W.
      • et al.
      Fludarabine, cyclophosphamide, and rituximab treatment achieves long-term disease-free survival in IGHV-mutated chronic lymphocytic leukemia.
      ]. Of these patients, seven had CMA performed on both the CLL/SLL and RT samples and were used for analysis. Patient age at the time of RT ranged 51–75 while median time from CLL/SLL diagnosis to RT was 52 months (range 22–192 months).
      The most common symptoms prior to diagnosis were progressive and/or palpable lymphadenopathy (5/7 patients; 71%). Median labs at time of RT included WBC 11.47 (range 2.57–103.18 K/cumm; normal 4.8–10.8 K/cumm), platelets 153 (range 75–305 K/cumm; normal 140–440 K/cumm), and LDH 301 (range 209–607 U/L; normal 100–240 U/L).
      Histology following transformation was consistent with DLBCL in all patients. Although there is no standard regimen, the most common therapies received for CLL/SLL were chemoimmunotherapy such as bendamustine/rituximab and ibrutinib, while the most common therapy for DLBCL was R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone) (Table 1).
      Table 1Clinical characteristics of study patients including age, gender, timing of RT, lactate dehydrogenase values at time of RT diagnosis, sample source, therapy received, and overall survival. Also included is chromosomal microarray data including status of TP53, CDKN2A, and whether chromosomal microarray demonstrated evidence of common clonality.
      PatientGender/Age at RT DxTime Lapse CLL -> RTLDH at RT Dx(ULN 240 U/L)Microarray Sample SourceTherapyTP53 loss or LOHCLL/RTHomozygous CDKN2A loss CLL/RTCMA proven clonal relationshipOS survival from RT diagnosis
      01M/5552 months337CLL/SLL: BM; RT: LNCLL/SLL: Ibrutinib, Venetoclax; RT: R-CHOP + Ibrutinib, Ibrutinib + Venetoclax+/+-/-NoDeceased; 1 month
      02M/55156 months209CLL/SLL: PB; RT: LNCLL/SLL: BR; RT: R-CHOP + Lenalidomide, Allo-STC+/+-/-YesAlive as of last follow-up at 3 months
      03M/5122 months301CLL/SLL: BM; RT: LNCLL/SLL: None; RT: R-CHOP, R-ICE, Ibrutinib, Allo-SCT-/--/-YesAlive, 37 months
      04M/6754 months607CLL/SLL: BM; RT: LNCLL/SLL: BR, Ibrutinib, Allo-SCT, DLI; RT: R-CHOP, BR+ARA-C-/+-/-YesDeceased: 5 months
      05M/6484 months239CLL/SLL: LN; RT: LNCLL/SLL: FCR, BR, PCR, Ibrutinib; RT: R-EPOCH+/+-/+YesDeceased: 3 months
      06F/7542 months275CLL/SLL: PB; RT: LNCLL/SLL: None; RT: R-CHOP-/--/-YesAlive, 24 months
      07F/67192 months433CLL/SLL: PB; RT: LNCLL/SLL: FND, Rituximab, BR, Ibrutinib, Venetoclax; RT: R-CHOP + Venetoclax+/+-/+YesAlive >12 months, transitioned to Hospice
      Abbreviations: Dx: Diagnosis; M: Male; F: Female; CLL: Chronic Lymphocytic Leukemia; RT: Richter Transformation; ULN: Upper Limit Normal; Allo-SCT: Allogeneic stem cell transplant; ARA-C: cytarabine; BR: bendamustine, rituximab; FCR: fludarabine, cyclophosphamide, rituximab; R-CHOP: rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone; BM: bone marrow; PB: peripheral blood; LN: lymph node; DLI: donor lymphocyte infusion; R-EPOCH: rituximab, prednisone, etoposide, cyclophosphamide, doxorubicin; FND: fludarabine, mitoxantrone, dexamethasone; PCR: pentostatin, cyclophosphamide, rituximab; OS: overall survival; LOH: Loss of Heterozygosity; CMA: Chromosomal Microarray;.
      CMA established a clonal relationship between the CLL and RT for most samples (Fig. 1). Six of seven patients (86%) at RT had fully or partially retained chromosomal aberrations with shared breakpoints detected in corresponding pre-transformed samples (Fig. 1A; Supplementary Table) providing evidence of a common clonality. Conversely, for patient 1 the clonal relationship of the paired samples could not be unequivocally established as no breakpoints were shared, although some aberrations were similar (Fig. 1B). For this patient, CMA at time of CLL diagnosis showed complex abnormal results with an estimated six abnormal clonal populations, including loss of CDKN2A and TP53. FFPE lymph node biopsy demonstrated two abnormal clones, and although the RT sample also had loss of CDKN2A and TP53 no aberration had identical breakpoints to those identified in the CLL sample (Fig. 1B; Supplementary Table). This patient died within one month of RT diagnosis.
      Fig. 1
      Fig. 1A. Chromosomal microarray analysis of samples for representative cases 2, 3, 5 and 7 revealed clonal relationship between CLL and RT. Blue, red and orange bars represent gains, losses and loss of heterozygosity (LOH), correspondingly. Arrows mark abnormalities with identical breakpoints. B. Comparative analysis of samples for case 1 did not reveal any aberrations with identical breakpoints; thus, clonal relationship could not be established. Aberrations for representative chromosomes 8, 9 and 17 are enlarged in insets.
      CMA was useful in establishing other prognostic factors for transformation. Four patients (1, 2, 4, 7) had TP53 loss in both CLL and RT samples while one patient (5) acquired a TP53 deletion at transformation. CDKN2A loss was noted at transformation in patients 2, 5 and 7 but was not detected in the corresponding CLL samples. Patients 3, 6 and 7 had trisomy 12 in the CLL samples which has been linked to an increased risk for RT typically when paired with NOTCH1 mutation [
      • Ding W.
      Richter transformation in the era of novel agents.
      ]. Genomic complexity was noted in all RT samples except patient 6 (Supplementary Table). Patient 5 only exhibited deletion of 13q and 11q at the CLL stage while at RT exhibited a complex pattern of aberrations including 10q interrupting the ERG2 gene, which has also been associated with transformation to RT [
      • Allan J.N.
      • Furman R.R.
      Current trends in the management of Richter’s syndrome.
      ,
      • Young E.
      • Noerenberg D.
      • Mansouri L.
      • Ljungström V.
      • Frick M.
      • Sutton L.A.
      • et al.
      EGR2 mutations define a new clinically aggressive subgroup of chronic lymphocytic leukemia.
      ,
      • Märklin M.
      • Heitmann J.S.
      • Fuchs A.R.
      • Truckenmüller F.M.
      • Gutknecht M.
      Bugl Set al. NFAT2 is a critical regulator of the anergic phenotype in chronic lymphocytic leukaemia.
      ].

      Discussion

      Despite increasingly effective and well tolerated therapies for CLL, RT prevalence has not decreased [
      • Petrackova A.
      • Turcsanyi P.
      • Papajik T.
      • Kriegova E.
      Revisiting Richter transformation in the era of novel CLL agents.
      ]. RT is a multistep process driven by clonal evolution, and it is unknown if newer therapies may add selective pressure on existing CLL clones over time [
      • Wierda W.G.
      • Zelenetz A.D.
      • Gordon L.I.
      • Abramson J.S.
      • Advani R.H.
      • Andreadis C.B.
      • et al.
      NCCN guidelines insights: chronic lymphocytic leukemia/small lymphocytic lymphoma, version 1.2017.
      ]. The prevalence of RT is likely to increase as patients live longer and experience increased selective pressure. DLBCL clonally related to the CLL/SLL clone has a poor prognosis and presents an enormous clinical challenge [
      • Rossi D.
      • Spina V.
      • Deambrogi C.
      • Rasi S.
      • Laurenti L.
      • Stamatopoulos K.
      • et al.
      The genetics of Richter syndrome reveals disease heterogeneity and predicts survival after transformation.
      ,
      • Abrisqueta P.
      • Delgado J.
      • Alcoceba M.
      • Oliveira A.C.
      • Loscertales J.
      • Hernández-Rivas J.A.
      • et al.
      Clinical outcome and prognostic factors of patients with Richter syndrome: real-world study of the Spanish Chronic Lymphocytic Leukemia Study Group (GELLC).
      ]. An expeditious and reliable way to establish clonality for this patient population is imperative.
      In the current study, CMA was able to provide strong evidence of common clonality in six of seven patients. Our data revealed that typically the transformation process is associated with an increasing number of genetic abnormalities. However, the majority of cases in our cohort retained aberrations from the original CLL clone that could be easily traced to the transformed clone. This suggests clonal evolution as a prevalent mechanism in RT, confirming previous findings [
      • Allan J.N.
      • Furman R.R.
      Current trends in the management of Richter’s syndrome.
      ].
      RT is reportedly enriched in patients harboring high risk molecular characteristics [
      • Rossi D.
      • Spina V.
      • Deambrogi C.
      • Rasi S.
      • Laurenti L.
      • Stamatopoulos K.
      • et al.
      The genetics of Richter syndrome reveals disease heterogeneity and predicts survival after transformation.
      ]. In five of seven patients, CMA identified poor prognostic markers in RT including loss of TP53/CDKN2A1–2. Five of seven (71%) patients exhibited TP53 loss in the RT clone; four acquired at CLL stage and one identified during transformation. TP53 is an established poor prognostic factor in CLL typically observed in a minority of cases. However, retrospective studies of CLL and RT revealed that incidence of TP53 inactivation via loss or mutation is significantly enriched in pre-transformed CLL cases compared to non-transforming CLL cases (up to 8 fold) [
      • Chigrinova E.
      • Rinaldi A.
      • Kwee I.
      • Rossi D.
      • Rancoita P.M.
      • Strefford J.C.
      • et al.
      Two main genetic pathways lead to the transformation of chronic lymphocytic leukemia to Richter syndrome.
      ]. Inactivating TP53 aberrations may be acquired during transformation, with an overall rate of 50% of RT samples having TP53 aberrations [
      • Chigrinova E.
      • Rinaldi A.
      • Kwee I.
      • Rossi D.
      • Rancoita P.M.
      • Strefford J.C.
      • et al.
      Two main genetic pathways lead to the transformation of chronic lymphocytic leukemia to Richter syndrome.
      ]. Loss of TP53 function serves as an independent predictor of overall survival after transformation [
      • Rossi D.
      • Cerri M.
      • Capello D.
      • Deambrogi C.
      • Rossi F.M.
      • Zucchetto A.
      • et al.
      Biological and clinical risk factors of chronic lymphocytic leukaemia transformation to Richter syndrome.
      ,
      • Chigrinova E.
      • Rinaldi A.
      • Kwee I.
      • Rossi D.
      • Rancoita P.M.
      • Strefford J.C.
      • et al.
      Two main genetic pathways lead to the transformation of chronic lymphocytic leukemia to Richter syndrome.
      ]. Loss of TP53 in CLL is associated with refractoriness to chemotherapy, which may contribute to selective pressure on CLL clones further driving clonal evolution towards RT [
      • Rossi D.
      • Spina V.
      • Deambrogi C.
      • Rasi S.
      • Laurenti L.
      • Stamatopoulos K.
      • et al.
      The genetics of Richter syndrome reveals disease heterogeneity and predicts survival after transformation.
      ,
      • Rossi D.
      • Cerri M.
      • Capello D.
      • Deambrogi C.
      • Rossi F.M.
      • Zucchetto A.
      • et al.
      Biological and clinical risk factors of chronic lymphocytic leukaemia transformation to Richter syndrome.
      ,
      • Messina M.
      • Del Giudice I.
      • Khiabanian H.
      • Rossi D.
      • Chiaretti S.
      • Rasi S.
      • et al.
      Genetic lesions associated with chronic lymphocytic leukemia chemo-refractoriness.
      ].
      Two of seven (28%) RT cases in the current study demonstrated focal homozygous loss of CDKN2A not detected in the corresponding CLL samples. This is in agreement with previously published analyses of CLL and RT in which disruption of CDKN2A/B cell cycle regulator genes were not detected in CLL, but were detected in 20–30% of RT cases [
      • Rossi D.
      • Cerri M.
      • Capello D.
      • Deambrogi C.
      • Rossi F.M.
      • Zucchetto A.
      • et al.
      Biological and clinical risk factors of chronic lymphocytic leukaemia transformation to Richter syndrome.
      ,
      • Chigrinova E.
      • Rinaldi A.
      • Kwee I.
      • Rossi D.
      • Rancoita P.M.
      • Strefford J.C.
      • et al.
      Two main genetic pathways lead to the transformation of chronic lymphocytic leukemia to Richter syndrome.
      ,
      • Fabbri G.
      • Khiabanian H.
      • Holmes A.B.
      • Wang J.
      • Messina M.
      • Mullighan C.G.
      • et al.
      Genetic lesions associated with chronic lymphocytic leukemia transformation to Richter syndrome.
      ].
      CMA demonstrated genomic complexity in four CLL and six RT samples, consistent with clonal evolution driving disease progression in the majority of RT cases. The two RT cases with the least complexity (cases 3 and 6) both had trisomy 12. CLL with trisomy 12 demonstrates atypical morphological/immunophenotypic features and increased risk for RT [
      • Matutes E.
      • Oscier D.
      • Garcia-Marco J.
      • Ellis J.
      • Copplestone A.
      • Gillingham R.
      • et al.
      Trisomy 12 defines a group of CLL with atypical morphology: correlation between cytogenetic, clinical and laboratory features in 544 patients.
      ,
      • Strati P.
      • Abruzzo L.V.
      • Wierda W.G.
      • O'Brien S.
      • Ferrajoli A.
      • Keating M.J.
      Second cancers and Richter transformation are the leading causes of death in patients with trisomy 12 chronic lymphocytic leukemia.
      ]. Our data suggest trisomy 12 CLL likely acquires additional aberrations in genes that drive RT (such as NOTCH1) and does not always show increased chromosomal instability/genomic complexity [
      • Abruzzo L.V.
      • Herling C.D.
      • Calin G.A.
      • Oakes C.
      • Barron L.L.
      • Banks H.E.
      • et al.
      Trisomy 12 chronic lymphocytic leukemia expresses a unique set of activated and targetable pathways.
      ]. Of note, clonal diversity in CLL samples with an estimated 7, 3 and 5 clonal populations (cases 1, 2 and 4 respectively) reduced to 1- 2 clonal populations in the RT samples. This suggests selection for aggressive and often highly complex clones upon transformation.
      Methods for testing aberrations in CLL/RT are often predicated on cost-effectiveness and availability. For our patients, use of CMA to assess the whole genome at high resolution (particularly to detect biomarkers that increase the risk for RT or that are present at time of transformation) and establish a clonal relationship provides a cost-effective strategy. Obtaining comparable results using other methods would require multiple samples to undergo a FISH panel, chromosome analysis, and send-out clonality studies using sequencing as opposed to the single-test strategy with CMA. We did not obtain additional studies such as heavy chain sequencing, thus we do not have data to enable direct comparison of CMA to IGH sequencing for clonality assessment. However, CMA detection of clonal changes with identical breakpoints in paired CLL and RT samples supports the utility of microarray in clonality assessment (in addition to detection of high-risk genetic aberrations). While CMA cannot detect low level clonal populations of cells (<10%) or detect truly balanced translocations, the benefits of a single test approach offset such limits and provides comprehensive results at reasonable cost.
      In conclusion, CMA provides important prognostic information in RT which could ultimately influence clinical decision making, such as recommendation for stem cell transplantation consolidation. In the future, CMA may successfully categorize risk for RT by identifying “high risk” features prior to transformation (TP53/CDKN2A, etc.). Risk stratification by CMA could potentially justify closer observation, provoke more frequent imaging assessments, and possibly trigger pre-emptive therapy. Thus, CMA has the capability to provide information of prognostic and therapeutic significance with a single-test that may obviate the need for send-out testing of multiple samples.

      Appendix. Supplementary materials

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