https://orcid.org/0000-0003-3904-1101
TO THE EDITOR:
Lenalidomide is an oral thalidomide analog, approved for the treatment of multiple myeloma, 5q– syndrome, mantle cell lymphoma, and follicular lymphoma. In the phase 3 CLLM1 trial, lenalidomide maintenance after first-line chemoimmunotherapy (CIT) was shown to improve progression-free survival in patients with chronic lymphocytic leukemia (CLL) compared with placebo.1
Here, we report clinical and molecular data on 3 patients with B-cell acute lymphoblastic leukemia (B-ALL) observed in 56 patients (5.4%) who had been exposed to lenalidomide within this trial. Upon safety evaluations of those patients, an independent data safety monitoring board and the German CLL Study Group (GCLLSG) decided to terminate lenalidomide treatment early in the remaining CLLM1 study patients.
Overall, the CLLM1 study enrolled 89 patients with CLL who had responded to first-line CIT and had a high risk of early disease progression. Thus, 60 patients were randomly assigned to receive lenalidomide maintenance therapy, 56 of whom were treated with at least 1 dose, and 29 patients were assigned to the placebo arm. After a median observation period of 47.7 months (interquartile range, 39.3-60.1 months) from first-line treatment, diagnosis of B-ALL was confirmed in 3 (5.4%) of the 56 patients exposed to lenalidomide 15, 33, and 48 months after the start of maintenance therapy. No cases of ALL were observed in the control arm.
Cumulative dosing of lenalidomide was similar among the 3 patients who developed B-ALL and the remaining 53 patients treated with lenalidomide (supplemental Table 5, available on the Blood Web site). All 3 patients with B-ALL had a history of high or very high-risk CLL according to assessment using the Chronic Lymphocytic Leukemia International Prognostic Index (CLL-IPI) before first-line CIT (supplemental Table 3). Before lenalidomide maintenance was started, all 3 patients exhibited detectable minimal residual disease (MRD; >10−4 leukemic cells) in peripheral blood; MRD levels decreased during lenalidomide therapy (Figure 1A).
Treatment and disease characteristics. (A) Absolute lymphocyte counts (ALCs) and MRD of the patients during lenalidomide maintenance therapy. Red lines show the time point of ALL diagnosis, and green lines show the threshold for undetectable MRD (10−4 leukemic cells). Patient 1: CLL MRD levels in peripheral blood (PB) decreased steadily from 8.6 × 104 to 1.6 × 104 after 16 months of lenalidomide treatment. BCR-ABL1–positive common B-ALL was diagnosed but no bone marrow (BM) infiltration of CLL cells could be detected morphologically (panel C). Patient 2 received lenalidomide for 15 months before discontinuing because of increased creatinine levels. Undetectable MRD in PB (<10−4) was achieved after 6 months and maintained at month 12 of lenalidomide therapy. When BCR-ABL1–positive common ALL was diagnosed, there were no signs of CLL cells in the PB or BM by cytomorphology or immunophenotyping. Patient 3 received lenalidomide for 45 months before treatment was stopped after a data safety monitoring board recommendation. Although his MRD level in PB had dropped sustainably below 10−4 after receiving lenalidomide for 6 months, a reciprocal increase of residual CLL cells to 8 × 10−3 was observed 2 months after lenalidomide was discontinued. Three months after the end of maintenance treatment, Philadelphia chromosome–negative common ALL was diagnosed after a BM biopsy prompted by persisting pancytopenia. (B) Summary of selected genetic alterations at CLL and ALL time points, including fluorescence in situ hybridization (FISH) analysis for recurrent aberrations in CLL, IGHV status, cancer-related mutations according to next-generation sequencing (NGS), and BCR/ABL status. We performed BCR-ABL1 reverse transcriptase quantitative polymerase chain reaction (PCR) in earlier PB samples from patients 1 and 2. No BCR-ABL1 fusion transcripts were detected in a blood sample 12 months after initiation of maintenance in patient 2 and 11 months after start of lenalidomide treatment in patient 1. (C) Giemsa stains of BM samples from all 3 patients at the time of ALL diagnosis (magnification ×100). BR, bendamustine, rituximab; FCR, fludarabine, cyclophosphamide, rituximab.
Treatment and disease characteristics. (A) Absolute lymphocyte counts (ALCs) and MRD of the patients during lenalidomide maintenance therapy. Red lines show the time point of ALL diagnosis, and green lines show the threshold for undetectable MRD (10−4 leukemic cells). Patient 1: CLL MRD levels in peripheral blood (PB) decreased steadily from 8.6 × 104 to 1.6 × 104 after 16 months of lenalidomide treatment. BCR-ABL1–positive common B-ALL was diagnosed but no bone marrow (BM) infiltration of CLL cells could be detected morphologically (panel C). Patient 2 received lenalidomide for 15 months before discontinuing because of increased creatinine levels. Undetectable MRD in PB (<10−4) was achieved after 6 months and maintained at month 12 of lenalidomide therapy. When BCR-ABL1–positive common ALL was diagnosed, there were no signs of CLL cells in the PB or BM by cytomorphology or immunophenotyping. Patient 3 received lenalidomide for 45 months before treatment was stopped after a data safety monitoring board recommendation. Although his MRD level in PB had dropped sustainably below 10−4 after receiving lenalidomide for 6 months, a reciprocal increase of residual CLL cells to 8 × 10−3 was observed 2 months after lenalidomide was discontinued. Three months after the end of maintenance treatment, Philadelphia chromosome–negative common ALL was diagnosed after a BM biopsy prompted by persisting pancytopenia. (B) Summary of selected genetic alterations at CLL and ALL time points, including fluorescence in situ hybridization (FISH) analysis for recurrent aberrations in CLL, IGHV status, cancer-related mutations according to next-generation sequencing (NGS), and BCR/ABL status. We performed BCR-ABL1 reverse transcriptase quantitative polymerase chain reaction (PCR) in earlier PB samples from patients 1 and 2. No BCR-ABL1 fusion transcripts were detected in a blood sample 12 months after initiation of maintenance in patient 2 and 11 months after start of lenalidomide treatment in patient 1. (C) Giemsa stains of BM samples from all 3 patients at the time of ALL diagnosis (magnification ×100). BR, bendamustine, rituximab; FCR, fludarabine, cyclophosphamide, rituximab.
The predicted incidence rate for B-ALL in patients exposed to lenalidomide (derived from observations made in the CLLM1 study) was 1345.5 cases/100 000 patient-years (95% confidence interval, 277.5-3932.2 cases). We performed a comparative retrospective cohort analysis on the cumulative incidence of B-ALL observed in CLL patients treated with first-line alkylator-containing chemotherapy or CIT without lenalidomide across previously reported trials of the GCLLSG. Overall, 1679 patients fulfilled the criteria for inclusion in this analysis (supplemental Table 1).1-5 A significantly lower incidence rate of 12.6 cases/100 000 patient-years (95% confidence interval, 0.3-70.1 cases) was estimated for this patient population (P < .001) (supplemental Table 6).
To assess the clonal relationship between CLL and ALL, we sequenced disease-specific immunoglobulin and T-cell receptor (TR) gene rearrangements.6 In patients 1 and 3, no mutual immunoglobulin or TR gene rearrangements were identified among the paired CLL and ALL samples (Figure 2A). In patient 2, CLL and ALL samples shared the same dominant IGHV1-18(D)/J6 clonotype (Figure 2B), which strongly suggests a common B-cell origin of both diseases. The detection of an identical clonal cross-lineage TR β (TRB) gene rearrangement in both CLL and ALL further supports this assumption. True cross-lineage TRB rearrangements are frequently found in B-ALL but are rather rare in CLL.7,8 Therefore, we confirmed our findings by using TRB clone-specific droplet digital polymerase chain reaction, which not only proved the high abundance of the clone-harboring rearranged TRB genes but also showed high concordance with levels of the dominant IGHV rearrangement described above, which suggests the concurrent presence of rearranged IGHV and TRB genes in the same B-cell clone.
Assessment of clonal relationship of CLL and B-ALL. (A) NGS-based clonality assessment of immunoglobulin rearrangements in CLL (dark blue) and ALL (yellow) samples from each patient. Samples were analyzed for IGH (IGHV-IGHD-IGHJ and IGHD-IGHJ) and IGK gene rearrangements. Clonal rearrangements with an abundance >5% are shown. Productive rearrangements are depicted in dark blue (CLL) and yellow (ALL). Clonotypes of productive IGH rearrangements are indicated. In patient 2, ALL and CLL share the same productive IGH rearrangement. (B) Patient 2: the nucleotide and amino acid sequence of the CDR3 gene fragment of the abundant clonotype is shown. (C) Patient 2: NGS assessment was performed at the time of CLL and ALL diagnosis by using the QIAGEN myeloid panel, which encompasses 141 cancer-related genes. Alterations in cancer-related genes are shown in blue, and variants of unknown significance are shown in yellow. (D) Patient 2: dynamics of variant allele frequencies of cancer-related genes show persistence of a BIRC3-mutated clone, but the NOTCH1 mutation present in the CLL population is not detected in the ALL sample. IKZF1 and DNMT3A mutations occur in the ALL sample and are not present in CLL.
Assessment of clonal relationship of CLL and B-ALL. (A) NGS-based clonality assessment of immunoglobulin rearrangements in CLL (dark blue) and ALL (yellow) samples from each patient. Samples were analyzed for IGH (IGHV-IGHD-IGHJ and IGHD-IGHJ) and IGK gene rearrangements. Clonal rearrangements with an abundance >5% are shown. Productive rearrangements are depicted in dark blue (CLL) and yellow (ALL). Clonotypes of productive IGH rearrangements are indicated. In patient 2, ALL and CLL share the same productive IGH rearrangement. (B) Patient 2: the nucleotide and amino acid sequence of the CDR3 gene fragment of the abundant clonotype is shown. (C) Patient 2: NGS assessment was performed at the time of CLL and ALL diagnosis by using the QIAGEN myeloid panel, which encompasses 141 cancer-related genes. Alterations in cancer-related genes are shown in blue, and variants of unknown significance are shown in yellow. (D) Patient 2: dynamics of variant allele frequencies of cancer-related genes show persistence of a BIRC3-mutated clone, but the NOTCH1 mutation present in the CLL population is not detected in the ALL sample. IKZF1 and DNMT3A mutations occur in the ALL sample and are not present in CLL.
To further assess clonal relationship in patient 2, we applied next-generation sequencing for selected somatic mutations in 141 cancer-related genes. In the CLL sample taken before the start of lenalidomide therapy, a dominant clone harboring a NOTCH1 (p.P2514fs*) frameshift deletion could be detected as well as a subclone carrying a BIRC3 (p.L421fs*) frameshift deletion (Figure 2C). The NOTCH1 mutation could not be found in the ALL sample, but CLL and ALL shared the presence of an identical subclonal BIRC3 mutation.
In the ALL sample, additional mutations in DNMT3A (p.V716D) and IKZF1 (p.G89fs*) were detected as well as several variants of unknown significance and recurrent ALL-associated alterations such as dup(21q22) or monosomy 7 (Figure 1B). Taken together, the mutational profiles of both samples support a common clonal ancestry of CLL and ALL with persistence of a BIRC3-altered subclone and treatment-related suppression of the NOTCH1-mutated clone (Figure 2D). BCR-ABL1(e1-a2) transcripts were detected at ALL diagnosis with a BCR-ABL1:ABL ratio of 0.1%, and BCR-ABL1 fluorescence in situ hybridization analysis was negative, suggesting a subclonal event. If we consider the presence of initiating (but not necessarily CLL-specific) mutations in hematopoietic progenitor cells in CLL patients,9 the transformation toward B-ALL may have occurred from a BIRC3-altered precursor rather than from mature CLL cells.
Increasing knowledge about the mode of action of lenalidomide supports hypotheses that the drug may facilitate clonal evolution toward acute leukemia. In vitro data have shown that lenalidomide induces proteasomal degradation of the transcription factors Ikaros and Aiolos.10 Genetic deletions or mutations in IKZF1, which encodes the Ikaros protein, are frequent driver lesions in ALL and promote leukemogenesis and treatment resistance through various biological pathways.11,12 It is known that lenalidomide can suppress Ikaros protein levels in human CD34+ cells almost equivalently to a genetic loss of IKZF1.13 The drug also protects self-renewal activity, which results in an expansion of the CD34+ progenitor cell pool, while it has anti-proliferative effects on malignant mature B cells.13,14 It has been hypothesized that the lenalidomide-associated increase in cycling CD34+ cells and a deregulated metabolic state resulting from the loss of Ikaros may predispose patients to acquire DNA damage including leukemogenic events, particularly under therapeutic selection pressure.15,16
Interestingly, we found an emerging IKZF1 frameshift deletion when ALL was diagnosed in patient 2, which may be related to a possible selection benefit for this subclone under lenalidomide maintenance. In patient 2, dup(21q22) and associated RUNX1 amplification might have also constituted a selection advantage through RUNX1-mediated inhibition of IKZF1 degradation and consequent desensitization toward lenalidomide, as recently described.17
All patients in the CLLM1 trial were treated with alkylators that have been attributed to increase the risk for second primary malignancies (SPMs) in patients with myeloma.18 Male sex and age have also been identified as independent risk factors for SPMs in CLL.19 Furthermore, more profound remissions induced by the use of anti-CD20 antibody combinations have been associated with accelerated clonal evolution in CLL.20 Of note, the 3 patients reported herein belonged to a subset minority that achieved undetectable or at least strongly decreasing MRD levels during lenalidomide treatment, suggesting an association between depth of response and occurrence of B-ALL.
In the CLLM1 study, all patients were preselected for an unfavorable disease course after CIT first-line therapy. As CLL patients have been shown to carry initiating genetic lesions in hematopoietic progenitor cells,9 preexisting as well as acquired genomic instability induced or selected by CIT might have contributed to genetic hits in lymphatic precursor cells, which finally grew out as acute leukemia during or after lenalidomide maintenance therapy.
In previous reports, ALL has generally been described as an unrelated second malignancy in patients with CLL and only recently as a clearly clonally related transformation.21-24 In this analysis, we report the first clonally related transformation of CLL into a BCR-ABL–positive B-ALL.
The significant cumulative incidence of B-ALL in CLL patients exposed to lenalidomide in the CLLM1 study, when compared across GCLLSG first-line trials, points to a potential relationship between lenalidomide and the risk of developing B-ALL. As hematologic SPMs in CLL have been increasing over past decades (according to public registry data25 ), meticulous reporting of such events and identification of independent risk factors in meta-analyses are desirable.
Presented in part at the annual meeting of the American Society of Clinical Oncology, Chicago, IL, 4 June 2018, the 23rd annual meeting of the European Hematology Association, Stockholm, Sweden, 16 June 2018, the annual meeting of the German, Austrian and Swiss Societies for Hematology and Medical Oncology, Vienna, Austria, 30 September 2018, and the 18th meeting of the International Workshop on Chronic Lymphocytic Leukemia, Edinburgh, Scotland, 22 September 2019.
For original data, please contact the corresponding author Moritz Fürstenau by e-mail at moritz.fuerstenau@uk-koeln.de.
The online version of this article contains a data supplement.
Acknowledgments
The authors thank all patients and physicians for their participation in the study. They also thank Aline Zey, Anne Westermann, and Miriam Schüler-Aparicio who served as project managers; Florian Drey, Annette Beer, and Jan-Erik Mittler who served as data managers; and Sabine Frohs, Tanja Annolleck, and Berit Falkowski who served as safety managers in the CLLM1 study. They thank Heinz Diem, Manuela Fernández Guijarro, and Candida Vitale who provided pictures of bone marrow smears and Nima Abedpour for his help in interpreting the genomic data.
This work was supported by funding from Celgene for the CLLM1 study (NCT01556776), which was an investigator-initiated trial sponsored by the German CLL Study Group. Experimental work was supported in part by a grant from the Deutsche Forschungsgemeinschaft (CRU-286) (C.D.H. and M.H.).
Authorship
Contribution: M.F., A.M.F., J.W., S.R., M.H., B.E., and C.D.H. designed the study; R.E., J.d.l.S., M. Crespo, M. Coscia, S.B., S.S., E.T., M.H., and B.E. enrolled patients and contributed data; A.S., J.W., C.V., M.R., G.W., and M.B. contributed and interpreted data; A.S., J.W., G.W., M.B., and C.D.H. performed and analyzed experiments; M.F. and S.R. designed and performed statistical analyses; K.F., M.H., B.E., M.B., and C.D.H. supervised the project; M.F., A.M.F., B.E., M.B., and C.D.H. drafted the first version of the manuscript; and all authors revised and approved the final version of the manuscript.
Conflict-of-interest disclosure: A.M.F. received grants from Celgene during the conduct of the study and personal fees (advisory board) from Janssen outside the submitted work. S.B. received a research grant for minimal residual disease and T-cell and NK cell analyses from Celgene during the conduct of the study, grants and personal fees from Janssen, AbbVie, and Roche, grants from Genentech, and personal fees from Becton Dickinson and Novartis, all outside the submitted work. M.R. received personal fees from Hoffman-La Roche (advisory board) and nonfinancial support from Celgene, both outside the submitted work. S.S. received grants, personal fees, and nonfinancial support from AbbVie, AstraZeneca, Celgene, Gilead, GlaxoSmithKline, Hoffmann-La Roche, Janssen, Novartis, Pharmacyclics, and Sunesis and personal fees and nonfinancial support from Verastem during the conduct of the study and outside the submitted work. E.T. received grants and personal fees (advisory board, speakers bureau) from Roche and AbbVie and personal fees and nonfinancial support (advisory board, speakers bureau, travel support) from Janssen. K.F. received travel grants from Roche and honoraria from Roche and AbbVie outside the submitted work. M.H. received grants, nonfinancial support, and personal fees from Roche, Gilead, Mundipharma, Janssen, Celgene, Pharmacyclics, and AbbVie outside the submitted work. B.E. received grants and personal fees from Janssen-Cilag, Roche, AbbVie, and Gilead, personal fees from Novartis, Celgene, ArQule, AstraZeneca, and Oxford Biomedica (UK), and grants from BeiGene, all outside the submitted work. M.B. received personal fees from Incyte (advisory board) and Roche Pharma AG, financial support for reference diagnostics from Affimed and Regeneron, grants and personal fees from Amgen (advisory board, speakers bureau, travel support), and personal fees from Janssen (speakers bureau), all outside the submitted work. C.D.H. received research funding from Roche and congress support from Gilead outside the submitted work. The remaining authors declare no competing financial interests.
Correspondence: Moritz Fürstenau, German CLL Study Group, Gleueler Straße 176-178, 50935 Köln, Germany; e-mail: moritz.fuerstenau@uk-koeln.de.
