Myeloid leukemia switch as immune escape from CD19 chimeric antigen receptor (CAR) therapy

Gardner et al. recently reported on two cases of mixed lineage leukemia (MLL)⁺ acute myeloid leukemia (AML) occurring after CD19 chimeric antigen receptor (CAR) therapy for MLL⁺ B-acute lymphoblastic leukemia (ALL) (1). This report provides new insights into mechanisms of immune escape from targeted immunotherapy and strategies to prevent their occurrence with adapted immune interventions.

Adoptive cell therapy utilizing genetically engineered T cells expressing second generation CARs specific for CD19 has shown remarkable efficacy against a range of chemo-refractory and relapsed B cell malignancies, most remarkably in ALL (2). A single infusion of CD19 CAR T cells may induce a complete remission in subjects whose leukemia has developed drug resistance and is unlikely to respond to allogeneic stem cell transplantation. This novel cell-based immunotherapy was highlighted, together with checkpoint blockade, as the basis for selecting cancer immunotherapy as the scientific breakthrough of the year in 2013 (3). CARs recognize tumor antigens irrespective of human leukocyte antigen (HLA) and can thus target tumor cells that have down-regulated HLA expression or proteasomal antigen processing, two mechanisms that contribute to tumor escape from T cell receptor (TCR)-mediated immunity (4). The advent of second generation CARs not only enables to retarget T cells but also to augment their functional capabilities (5).

The two cases recently reported by Gardner et al. include a 52-yo woman and an 18-mo girl with an initial diagnosis of MLL-rearranged B-ALL, who presented a lineage switch at relapse after CAR therapy. Several facets of leukemia biology and resistance to CAR therapy need to be taken into account in analyzing these observations.

The MLL gene is frequently involved in chromosomal translocations found in human acute leukemias characterized as AML, ALL or biphenotypic (mixed lineage) leukemia (6-8). MLL rearrangements are found in >70% of infant leukemias, whether the immunophenotype is that of ALL or AML (9). MLL translocations are also found in about 10% of AML in adults, and in therapy-related leukemias that develop in patients previously treated with topoisomerase II inhibitors for other malignancies. More than 50 different translocation fusion partners have been identified. Five of them account for 80% of all MLL-translocation-bearing leukemias: t(Jeny 4;11)(q21;q23) or MLL-AF4, as reported here in the adult subject (case 1); t(Jeny 9;11)(p22;q23) or MLL-AF9; t(Jeny 11;19)(q23;p13.3) or MLL-ENL; t(Jeny 10;11)(p12;q23) or MLL-AF10, as reported here in the infant subject (case 2); and t(Jeny 6;11)(q27;q23) or MLL-AF6 (10,11). Children patients with MLL-rearranged ALL have a particularly poor outcome compared to children with other forms of ALL (12). Adult AML with MLL-AF9 is associated with a more aggressive disease that is more likely to exhibit resistance to chemotherapy (13). Leukemias that bear translocations involving the MLL gene on chromosome 11q21 possess unique clinical and biological characteristics, including the activation of self-renewal properties. MLL rearrangements may direct leukemogenic transformation of committed myeloid progenitors (14), suggesting that myeloid leukemias can originate not only from hematopoietic stem cells (HSCs) but also from committed myeloid progenitors lacking self-renewal capabilities. Some MLL fusion proteins direct a partial stem cell gene expression program in committed progenitors, coinciding with a gain of self-renewal properties. This program has been designated a self renewal-associated signature (15). Given that MLL fusions modulate chromatin structure through histone modification (through the loss of H3K4 methyltransferase activity), it is likely that the process of reactivation of self-renewal can be initiated through modulation of the epigenetic state of a cell.

Given these features of MLL⁺ leukemias, the first question to address is whether the MLL⁺ cases reported by Gardner et al. represent a true lineage switch at relapse or biphenotypic/bilineal leukemias at diagnosis. The authors excluded the second possibility based on the following phenotypic characterization:

• Case #1
  • Diagnosis: MLL⁺CD45⁺CD19⁺CD22⁺CD38⁺HLADR⁺CD15⁺CD33⁺CD13^dimTdT^dimlymphoblasts;
  • Relapse: MLL⁺CD19⁻HLA-DR⁺CD64⁺CD15⁺CD33⁺CD13^dimCD71⁺MPO⁺ monocytic cells.
• Case #2
  • Diagnosis: MLL⁺CD45⁺CD19⁺CD38⁺CD58⁺CD22^dimHLA-DR⁺CD34⁺lymphoblasts;
  • Relapse: MLL⁺CD45⁺CD19⁻CD4⁺CD56⁺CD64⁺CD13⁺CD33⁺CD38⁺HLADR⁺CD34⁺CD71⁺myeloblasts.

Although specific lineages can be identified for most leukemias, there are instances in which both lymphoid- and myeloid-lineage markers, or T-cell and B-cell markers, coexist (16). The diagnosis of biphenotypic (divergent morphologic and immunophenotypic features uniformly present in one blast population)/bilineal (distinct blast populations in a single patient) leukemia was excluded by the authors according to the WHO classification which describes the mixed phenotype acute leukemia (MPAL) based on the expression of strictly specific T-lymphoid (cytoplasmic CD3) and myeloid (MPO) antigens, the latter shown by either flow cytometry or cytochemistry and/or clear evidence of monocytic differentiation. B-cell lineage assignment in MPAL relies on the strong expression of CD19 together with another B cell-associated marker or, in cases with weak CD19, on the expression of at least three B-lineage markers. The European Group for the Immunological Classification of Leukemia (EGIL) defines MPAL by a scoring system based on the number and specificity degree of lymphoid and myeloid markers expressed by leukemic cells, with point values are greater than 2 for myeloid and 1 for lymphoid lineages. 2: B-lymphoid (CD79a, CD22, cyIgM) T-lymphoid (CD3) myeloid (MPO); 1: B-lymphoid (CD10, CD19), T-lymphoid (CD2, CD5), myeloid (CD13, CD33); 0.5: B-lymphoid (TdT), T-lymphoid (TdT, CD7), myeloid (CD14, CD15, CD11b, CD11c). Therefore, the reported cases seemingly represent a lineage switch at relapse, a phenomenon that is occasionally observed (17). By definition, this occurs when acute leukemias that meet the standard FAB criteria for a lineage (lymphoid or myeloid) at the time of initial diagnosis meet the criteria for the opposite lineage upon relapse. A lineage switch is an uncommon type of mixed leukemia, observed in 6–9% of relapsed acute leukemia (18,19). In ALL, most AML relapses occur during treatment within the first year of diagnosis, as is the case in this report.

The second question is how to explain the lineage switch at relapse. A lineage switch may represent either a relapse of the original clone with heterogeneity at the morphologic level or high plasticity (capacity of changing cell fate without altering genotype), or the emergence of a new leukemic clone (20). The relapsed disease maintained the MLL rearrangement in both cases. Lineage switching has been reported to occur more frequently in children than adults with most cases involving the conversion of ALL to AML (17). Changes in cell potentials can be explained by mechanisms operating at different levels: (I) at the cell-intrinsic level, possibly defined by epigenetic cues (which MLL fusions are capable of mediating); and (II) at the cell/environment interface including therapeutic pressure.

Kawamoto and Katsura have suggested that a myeloid potential is retained in erythroid, T-, and B-cell branches even after these lineages have segregated from each other (21,22). MLL-positive B-ALL show expression profiles consistent with early hematopoietic progenitors, raising the possibility that early bipotential or oligopotential progenitor cells may serve as a substrate for leukemogenic translocations and account for lineage switching events (23). Alternatively, the MLL translocation might induce a stem/progenitor cell phenotype, irrespective of the cell lineage targeted by the translocation, enabling the cellular environment to promote lineage conversions (18). Therapy may also facilitate or induce a subsequent lineage switch. Clonal selection induced by therapy might suppress or eradicate the leukemic clone that is apparent at the time of diagnosis, favoring expansion of a subclone with a different phenotype and/or disrupting the hematopoietic environment to add further selective pressure. In a human-based MLL leukemia mouse model, the microenvironment has been shown to be critical to lineage outcome, with manipulation of the in vivo cytokine milieu influencing the commitment of both lineage-restricted and multipotent leukemia initiating cells (24). The severe cytokine release syndrome (CRS) observed in the reported patients with high levels of IL-6 might have contributed to environmental changes that ultimately supports a lineage switch in a MLL⁺ clone (25). An elegant model of MLL-AF9-induced AML showed the importance of the microenvironment in providing instructive signals for leukemic lineage fates (26). It is possible that the human microenvironment and the fusion protein cooperate in promoting the lineage phenotype arising from a primitive leukemic stem cell.

A lineage switch from CD19⁺ to CD19⁻ may thus account for resistance to CD19 CAR therapy. This mechanism stands in contrast to two other previously described immune escape mechanisms, based on either post-transcriptional editing or mutational loss of CD19 itself.

Jacoby et al. reported that murine E2a:PBX1-driven pre-B ALL CD19⁺ cells treated with CD19 CAR T cells may undergo early and late relapse following CD19 CAR therapy. Early post CAR relapses retained a pre-B phenotype with isolated loss of CD19 extracellular expression by flow cytometry, loss in CD19 exon1 and 2 mRNA but intact mRNA for all other CD19 exons. In contrast, late relapses demonstrated complete loss of CD19 protein and mRNA expression with concomitant loss of the major B cell transcription factors PAX5 and EBF1 suggesting loss of the B-cell developmental program. Late post CD19 CAR relapses typically gained myeloid, stem cell or T cell phenotypic markers, consistent with a lineage switch, which was confirmed by RNA-seq of multiple late relapse samples. The authors demonstrated intermediate phenotype of post-CD19 CAR relapse in vivo with co-expression of both myeloid (Gr1, CD11b) and B cell markers (B220, CD22) on the same cells, suggesting a differentiation rather than a selection process. Leukemic relapse with CD19 expression loss that retain a B-cell program rapidly regain CD19 upon in vivo passage in the absence of CD19 CAR pressure. However, relapses due to lineage reprogramming retained a stable myeloid phenotype upon serial passage without regain of CD19 or other B cell markers (27).

Another example of CD19 post-transcriptional editing involves the clustering of nonsense and missense mutations in exon2 of CD19. Frameshift mutations clustered in the non-constitutive exon2 eliminate full-length CD19, but allow expression of the isoform that is mostly cytosolic (hidden from T cells) and its membrane fraction does not trigger killing by CART-19. However this isoform rescues defects in cell proliferation and pre-BCR signaling associated with CD19 loss (28). A case report of lineage switch from CD19⁺ALL to CD19⁻AML was also described following CD19-targeted therapy with blinatumomab (29).

In general, acquired resistance of tumor cells to immunotherapy may proceed as a Darwinian selection for genetic or epigenetic heritable traits that pre-exist in the tumor mass before a therapeutic intervention. This outcome is likely the consequence of genomic and epigenetic instability of transformed cells (MLL translocation for instance) as well as some therapeutic interventions that affect mutational load or tumor microenvironmental conditions. In the case of immunotherapy, tumor cells may also alter their gene expression in response to interactions with immune cells, such as PDL1 up-regulation in response of IFN-gamma, which may alter the risk of selecting for resistant clones.

The multiplicity of mechanisms accounting for antigen escape calls for adapted modifications to CAR therapy. The use of CD19 CARs targeting CD19 essential exons has been previously suggested (30), but this would only address a subset of CD19 losses. A more promising approach entails the targeting of a second, independently expressed antigen. Thus, most CD19-negative relapses appear to retain expression of CD22, for which CARs have now been developed (31). For the cases reviewed here, this approach would require the identification of suitable AML targets. Four AML CAR targets have been reported to date. Lewis (Le)-Y, a difucosylated carbohydrate antigen, was targeted in a phase I study in four patients with relapsed AML. Infusion of a second generation CD28-based CAR resulted in stable/transient remission of three patients, who ultimately progressed despite T cell persistence (32). CD123 is the high-affinity interleukin-3 receptor α-chain. A partial remission was induced in a patient with FLT3-ITD⁺ AML treated with a third generation CD123-CD28/CD137/CD27/CD3z CAR (33). Preclinical studies have however revealed significant myeloablation attributed to CD123 expression in early hematopoietic stem/progenitor cells (34). CD33 is a myeloid-specific sialic acid-binding receptor, that has been targeted by gentuzumab ozogamicin (35), which has shown survival benefit (36,37). Preclinical activity of CD33 CAR⁺ CIK cells resulted in slowing disease progression (38) and CD33 CAR⁺ T showed significant effector functions in vitro and in vivo with reduction of myeloid progenitors in mice (39). One AML patient treated with CD33 CAR T cells at the Chinese PLA General Hospital, showed transient efficacy and mild fluctuations in bilirubin (40). Folate receptor-β is another myeloid-lineage antigen, that has also been proposed as a target in AML (41,42). None of these CARs has yet been thoroughly evaluated in clinical trials, and all pose varying concerns owing to their expression in normal tissues. The quest for suitable AML targets is still awaiting its champions.

Acknowledgments

Funding: None.

Footnote

Provenance and Peer Review: This article was commissioned and reviewed by the Section Editor Xia Fang (Department of hematology, Shanghai Tongji Hospital, Tongji University School of Medicine, Shanghai, China).

Conflicts of Interest: Both authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/tcr.2016.08.15). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

References

1. Gardner R, Wu D, Cherian S, et al. Acquisition of a CD19-negative myeloid phenotype allows immune escape of MLL-rearranged B-ALL from CD19 CAR-T-cell therapy. Blood 2016;127:2406-10. [Crossref] [PubMed]
2. Sadelain M. CAR therapy: the CD19 paradigm. J Clin Invest 2015;125:3392-400. [Crossref] [PubMed]
3. Couzin-Frankel J. Breakthrough of the year 2013. Cancer immunotherapy. Science 2013;342:1432-3. [Crossref] [PubMed]
4. Zhou G, Levitsky H. Towards curative cancer immunotherapy: overcoming posttherapy tumor escape. Clin Dev Immunol 2012;2012:124187.
5. van der Stegen SJ, Hamieh M, Sadelain M. The pharmacology of second-generation chimeric antigen receptors. Nat Rev Drug Discov 2015;14:499-509. [Crossref] [PubMed]
6. Krivtsov AV, Armstrong SA. MLL translocations, histone modifications and leukaemia stem-cell development. Nat Rev Cancer 2007;7:823-33. [Crossref] [PubMed]
7. Dimartino JF, Cleary ML. Mll rearrangements in haematological malignancies: lessons from clinical and biological studies. Br J Haematol 1999;106:614-26. [Crossref] [PubMed]
8. De Braekeleer M, Morel F, Le Bris MJ, et al. The MLL gene and translocations involving chromosomal band 11q23 in acute leukemia. Anticancer Res 2005;25:1931-44. [PubMed]
9. Biondi A, Cimino G, Pieters R, et al. Biological and therapeutic aspects of infant leukemia. Blood 2000;96:24-33. [PubMed]
10. Huret JL, Dessen P, Bernheim A. An atlas of chromosomes in hematological malignancies. Example: 11q23 and MLL partners. Leukemia 2001;15:987-9. [Crossref] [PubMed]
11. Meyer C, Schneider B, Jakob S, et al. The MLL recombinome of acute leukemias. Leukemia 2006;20:777-84. [Crossref] [PubMed]
12. Chen CS, Sorensen PH, Domer PH, et al. Molecular rearrangements on chromosome 11q23 predominate in infant acute lymphoblastic leukemia and are associated with specific biologic variables and poor outcome. Blood 1993;81:2386-93. [PubMed]
13. Schoch C, Schnittger S, Klaus M, et al. AML with 11q23/MLL abnormalities as defined by the WHO classification: incidence, partner chromosomes, FAB subtype, age distribution, and prognostic impact in an unselected series of 1897 cytogenetically analyzed AML cases. Blood 2003;102:2395-402. [Crossref] [PubMed]
14. Cozzio A, Passegué E, Ayton PM, et al. Similar MLL-associated leukemias arising from self-renewing stem cells and short-lived myeloid progenitors. Genes Dev 2003;17:3029-35. [Crossref] [PubMed]
15. Krivtsov AV, Twomey D, Feng Z, et al. Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9. Nature 2006;442:818-22. [Crossref] [PubMed]
16. Bennett JM, Catovsky D, Daniel MT, et al. Proposed revised criteria for the classification of acute myeloid leukemia. A report of the French-American-British Cooperative Group. Ann Intern Med 1985;103:620-5. [Crossref] [PubMed]
17. Dorantes-Acosta E, Pelayo R. Lineage switching in acute leukemias: a consequence of stem cell plasticity? Bone Marrow Res 2012;2012:406796.
18. Stass S, Mirro J, Melvin S, et al. Lineage switch in acute leukemia. Blood 1984;64:701-6. [PubMed]
19. Imataki O, Ohnishi H, Yamaoka G, et al. Lineage switch from precursor B cell acute lymphoblastic leukemia to acute monocytic leukemia at relapse. Int J Clin Oncol 2010;15:112-5. [Crossref] [PubMed]
20. Park M, Koh KN, Kim BE, et al. Lineage switch at relapse of childhood acute leukemia: a report of four cases. J Korean Med Sci 2011;26:829-31. [Crossref] [PubMed]
21. Kawamoto H, Katsura Y. A new paradigm for hematopoietic cell lineages: revision of the classical concept of the myeloid-lymphoid dichotomy. Trends Immunol 2009;30:193-200. [Crossref] [PubMed]
22. Bell JJ, Bhandoola A. The earliest thymic progenitors for T cells possess myeloid lineage potential. Nature 2008;452:764-7. [Crossref] [PubMed]
23. Pui CH, Raimondi SC, Behm FG, et al. Shifts in blast cell phenotype and karyotype at relapse of childhood lymphoblastic leukemia. Blood 1986;68:1306-10. [PubMed]
24. Wei J, Wunderlich M, Fox C, et al. Microenvironment determines lineage fate in a human model of MLL-AF9 leukemia. Cancer Cell 2008;13:483-95. [Crossref] [PubMed]
25. Cohen A, Petsche D, Grunberger T, et al. Interleukin 6 induces myeloid differentiation of a human biphenotypic leukemic cell line. Leuk Res 1992;16:751-60. [Crossref] [PubMed]
26. Flynn CM, Kaufman DS. Donor cell leukemia: insight into cancer stem cells and the stem cell niche. Blood 2007;109:2688-92. [PubMed]
27. Jacoby E, Nguyen SM, Fountaine TJ, et al. CD19 CAR immune pressure induces B-precursor acute lymphoblastic leukaemia lineage switch exposing inherent leukaemic plasticity. Nat Commun 2016;7:12320. [Crossref] [PubMed]
28. Sotillo E, Barrett DM, Black KL, et al. Convergence of Acquired Mutations and Alternative Splicing of CD19 Enables Resistance to CART-19 Immunotherapy. Cancer Discov 2015;5:1282-95. [Crossref] [PubMed]
29. Rayes A, McMasters RL, O'Brien MM. Lineage Switch in MLL-Rearranged Infant Leukemia Following CD19-Directed Therapy. Pediatr Blood Cancer 2016;63:1113-5. [Crossref] [PubMed]
30. Mittal D, Gubin MM, Schreiber RD, et al. New insights into cancer immunoediting and its three component phases--elimination, equilibrium and escape. Curr Opin Immunol 2014;27:16-25. [Crossref] [PubMed]
31. Haso W, Lee DW, Shah NN, et al. Anti-CD22-chimeric antigen receptors targeting B-cell precursor acute lymphoblastic leukemia. Blood 2013;121:1165-74. [Crossref] [PubMed]
32. Ritchie DS, Neeson PJ, Khot A, et al. Persistence and efficacy of second generation CAR T cell against the LeY antigen in acute myeloid leukemia. Mol Ther 2013;21:2122-9. [Crossref] [PubMed]
33. Luo Y, Chang LJ, Hu Y, et al. First-in-Man CD123-Specific Chimeric Antigen Receptor-Modified T Cells for the Treatment of Refractory Acute Myeloid Leukemia. Blood 2015;126:3778.
34. Gill S, Tasian SK, Ruella M, et al. Preclinical targeting of human acute myeloid leukemia and myeloablation using chimeric antigen receptor-modified T cells. Blood 2014;123:2343-54. [Crossref] [PubMed]
35. U.S. Food and Drug Administration. Mylotarg (gemtuzumab ozogamicin): Market Withdrawal. 2010.Available online: http://www.fda.gov/Safety/MedWatch/SafetyInformation/SafetyAlertsforHumanMedicalProducts/ucm216458.htm
36. Ravandi F, Estey EH, Appelbaum FR, et al. Gemtuzumab ozogamicin: time to resurrect? J Clin Oncol 2012;30:3921-3. [Crossref] [PubMed]
37. Hills RK, Castaigne S, Appelbaum FR, et al. Addition of gemtuzumab ozogamicin to induction chemotherapy in adult patients with acute myeloid leukaemia: a meta-analysis of individual patient data from randomised controlled trials. Lancet Oncol 2014;15:986-96. [Crossref] [PubMed]
38. Pizzitola I, Anjos-Afonso F, Rouault-Pierre K, et al. Chimeric antigen receptors against CD33/CD123 antigens efficiently target primary acute myeloid leukemia cells in vivo. Leukemia 2014;28:1596-605. [Crossref] [PubMed]
39. Kenderian SS, Ruella M, Shestova O, et al. CD33-specific chimeric antigen receptor T cells exhibit potent preclinical activity against human acute myeloid leukemia. Leukemia 2015;29:1637-47. [Crossref] [PubMed]
40. Wang QS, Wang Y, Lv HY, et al. Treatment of CD33-directed chimeric antigen receptor-modified T cells in one patient with relapsed and refractory acute myeloid leukemia. Mol Ther 2015;23:184-91. [PubMed]
41. Lynn RC, Poussin M, Kalota A, et al. Targeting of folate receptor β on acute myeloid leukemia blasts with chimeric antigen receptor-expressing T cells. Blood 2015;125:3466-76. [Crossref] [PubMed]
42. Lynn RC, Feng Y, Schutsky K, et al. High-affinity FRβ-specific CAR T cells eradicate AML and normal myeloid lineage without HSC toxicity. Leukemia 2016;30:1355-64. [Crossref] [PubMed]