CAR T-cell Therapy Meets Clonal Hematopoiesis

Clonal hematopoiesis (CH) is used as a general term and refers to any outgrowth of a single mutated stem cell in the hematopoietic system. These mutations can occur in genes associated with hematologic malignancies, whereas full diagnostic criteria for a malignancy is lacking in these individuals. This state is called “clonal hematopoiesis of indeterminate potential” (CHIP). Some common somatic mutations in CHIP-associated genes include tet methylcytosine dioxygenase 2 (TET2), DNA (cytosine-5)-methyltransferase 3A (DNMT3A), tumor protein P53 (TP53), and ASXL1, which are more frequently seen in older people and in cancer patients who underwent chemotherapy or radiotherapy. This might—but does not necessarily have to—lead to hematologic malignancies in due course. In addition, many reports conclude that CHIP may have effects on nonneoplastic processes such as inflammation and cardiovascular diseases, as well as on therapies including adoptive cellular immunotherapy (1). For instance, patients with hematologic malignancies who received allogeneic hematopoietic stem cell transplantation (ASCT) from CHIP carriers showed lower relapse rates, whereas rates for graft-versus-host disease (GvHD) were higher (2). Thus, one could also suggest a potential association of CHIP with other cellular immunotherapies.

To date, T cells genetically engineered to express CD19 or BCMA-specific chimeric antigen receptors (CAR) have been approved for leukemia, lymphoma, and multiple myeloma (3). We expect advances in cell biology and synthetic biology will further enhance engineered T-cell functionality, and this approach will likely be used for an increasingly broader scope of hematologic malignancies (4). Because CAR T-cell patients are usually heavily pretreated, the incidence of somatic mutations in CHIP-associated genes is much higher than in the general population (1). For example, two recent studies have found that the incidence of preexisting CHIP in adult patients enrolled on CAR T trials was 34% (5) and 48% (6), whereas the incidence is 5% to 10% in a similarly aged healthy population (7). The frequent coexistence of CHIP and CAR T-cell therapy makes it important to understand the implications of CHIP in cellular therapy. Fortunately, three recent studies have investigated the impact of preexisting CHIP on the safety and efficacy of CAR T-cell therapy (5, 6, 8).

Saini and colleagues report in the current issue of Blood Cancer Discovery on the possible association of CH and CAR T-cell treatment-related cytokine release syndrome (CRS) as well as immune effector-cell-associated neurotoxicity syndrome (ICANS) (8). A total of 114 large B-cell lymphoma patients treated with approved autologous CD19-specific CAR T-cell products following lymphodepleting chemotherapeutic regimen were analyzed. Somatic mutations were detected in pretreatment peripheral blood samples of 42 patients (36.8% of the patient population). The median age of the study population was 63 years, with a slightly higher median age of CAR T cell–treated patients with CHIP when compared with patients where CHIP could not be detected (64 and 62 years, respectively). Although no difference in the incidence of all grades of CRS or ICANS between the two patient populations (CH vs. no CH) was seen, the rate of grade ≥3 ICANS was significantly higher in patients with CH. Also, more total cases of grade ≥3 CRS were seen in patients with CH while statistical significance was not achieved. Higher toxicities in patients with CH were primarily associated with somatic mutations in the genes DNMT3A and TET2 (8). No differences in CAR T-cell response rates or overall survival were observed between cohorts (8).

To date, two additional observational studies have been published analyzing the possible association of CHIP status and CAR T-cell therapy outcome and related toxicities (5, 6). Miller and colleagues reported on 154 CAR T cell–treated non-Hodgkin lymphoma (NHL) and multiple myeloma patients and found that somatic mutations in CHIP-associated genes were detected in 48% of the study population [mainly protein phosphatase 1D (PPM1D), DNMT3A, TP53, and TET2; ref. 6]. The presence of CHIP was associated with increased rates of CRS severity, but also with a higher rate of complete responses. The former, however, was only seen in patients younger than 60 years. No differences between cohorts were reported regarding overall survival following CAR T-cell treatment at all ages (6). In another study with a smaller cohort of 32 B-cell non-Hodgkin lymphoma patients (B-NHL), Teipel and colleagues found that 34% of the study population had mutations in CHIP-associated genes, mainly in DNMT3A and TP53 (5); however, no significant differences were observed in terms of the occurrence and severity of CRS or ICANS, and therapy outcome and overall survival were also not different. The median age of the study populations was 63 years (6) and 62 years (5). In both reported studies, older age was associated with higher rates of somatic mutations in CHIP-associated genes.

All three previously cited observational studies were concordant in that they failed to find an association of CHIP status and patients’ overall survival following CAR T-cell therapy (5, 6, 8). This is in accordance with a retrospective multicenter analysis that suggests a potential adverse impact of matched sibling donor CHIP on relapse incidence and chronic GvHD following ASCT, whereas no impact on overall survival was observed (2). A subsequent study of patients with acute myelogenous leukemia/myelodysplastic syndrome undergoing donor ASCT also found higher rates of acute GvHD but no difference in relapse or progression-free survival when donors with CH were used (9).

These observational studies were published within the last year and provide new insights into the toxicity and efficacy of CAR T-cell therapies for blood cancer. More studies will most likely follow soon. However, the results of the three studies have some discrepancies, likely due to differences in the underlying patient characteristics, the treatment with various CAR T-cell products that have distinct signaling domains, and underpowering in the smaller studies, highlighting the complexity of this topic. General conclusions should therefore be drawn with caution, and further clinical trials with larger cohorts and mechanistic studies will be needed to draw more accurate conclusions.

Currently, most CAR T-cell patients are treated with autologous T cells, although there is an increasing use of CAR in other cell types (NK cells and macrophages), as well as allogeneic cells derived from a variety of sources such as cord and peripheral blood, and induced pluripotent stem cells. When CAR T cells are manufactured, the patient's own T cells are ex vivo transduced with a tumor antigen–specific CAR, expanded, and finally reinfused into patients. CHIP could, therefore, affect therapy response and overall survival through modifications in CHIP-harboring engineered immune cells itself, as well as through an interplay with the host immune system and tumor microenvironment. Additionally, the size of the CHIP clone may matter with respect to its influence on outcomes, so treating CHIP as a unified entity may be misleading given heterogeneity in both specific alleles and clonal architecture, and the size of the somatic mutant cell population.

Cell-intrinsic enhancement of CAR T-cell expansion following disruption of the CHIP-associated genes TET2 and CBL has been reported by us (10) and others (11). In a patient with refractory chronic lymphocytic leukemia (CLL) that we treated with a CD19 CAR T-cell product, we discovered a profound delayed clonal expansion of a single CAR-transduced T cell with biallelic TET2 dysfunction that resulted in complete remission in an initially nonresponding patient; the TET2 deficiency was due to compound heterozygosity with one somatic allele having loss of function and the other allele-induced loss of function during CAR T manufacturing and insertional mutagenesis by the lentiviral vector. The reexpansion of CAR T cells was accompanied by high-grade CRS in this patient. These TET2-deficient CAR T-cell clones displayed a central memory phenotype, resulting in higher T-cell proliferation, long-term persistence, increased functionality, and sustained antitumor response (10). A subsequent analysis of a larger patient cohort at our center showed evidence that lentiviral insertion mutagenesis data from preinfusion CAR T-cell products may be used to forecast response in CLL (12). Similarly, exceptional reexpansion of a CD22-specific CAR T-cell clone was reported in another patient with acute lymphocytic leukemia (ALL) (11). In this patient, insertion of the CAR vector in an allele of the CBL gene was detected, leading to a rapid clonal expansion with subsequent complete remission. Unlike in our patient, CRS was not observed (11). In a recently published preclinical study, deletion of DNMT3A in CAR T cells could prevent CAR T-cell exhaustion and enhance antitumor activity in animal models (13). Although these are only a few reports, the disruption of certain CHIP-associated genes in the process of ex vivo CAR T-cell generation could represent a promising strategy for the regeneration of exhausted or dysfunctional T cells and to shape immune responses toward tumor entities where CAR T cells have shown limited durable success, e.g., in patients with CLL or solid tumors. Preclinical as well as clinical studies of these and other CHIP-associated gene disruptions in CAR T cells will most likely follow.

An established finding is that patients with CH and CHIP have increased clinical and laboratory findings of inflammation. The presence of CHIP with somatic mutations in certain genes has been associated with increased levels of IL1 and IL6, cytokines that also play a crucial role in the development of CAR T cell therapy-associated systemic inflammatory syndromes CRS and ICANS (14). Thus, it seems in theory likely that the presence of CHIP with certain somatic mutations in CAR T cell–treated patients may result in an increased risk of toxicity development as well as in more severe toxicities. In the previously cited article by Saini and colleagues, a trend toward elevated baseline IL6 levels prior to CAR T-cell treatment in the CH cohort was observed. IL1 cytokine levels were not analyzed (8). However, it is perhaps noteworthy that Saini and colleagues found an increased frequency of ICANS but not CRS in patients with CH (8). In a recent study of children with pre–B-cell ALL treated with CD19-targeted CAR T cells, we used serum proteomic profiling to identify biomarkers of CRS and ICANS (15). Interestingly, we found that IL18 was associated with the development of ICANS, whereas IL1 and IL6 elevations in the serum were universally observed in the serum of patients with severe CRS (15). Thus, CAR T cells or myeloid cells with genes commonly mutated in CHIP may preferentially alter pathways leading to IL18 elevation and ICANS. This hypothesis needs to be tested in further studies, ideally by comparing young versus adult patients where somatic mutations in CHIP-associated genes are more frequently seen. In addition, further research is needed to understand the implications of CHIP on preexisting and subsequently inflammatory pathways in general.

The recent exciting studies of CAR T-cell therapy in patients with CH and CHIP are providing important insights into the mechanisms of efficacy and toxicity after cellular therapies. Further studies are required to confirm the initial observations in larger cohorts with single-cell analysis of various immune cells including the CAR T cells. This will give mechanistic insights to understand not only the role of CHIP-harboring transfused engineered cells itself but also the potential interplay with the patient's immunity harboring CHIP, e.g., in the peripheral lymph node or solid tumor microenvironment. The possibility to increase CAR T-cell efficacy through cellular engineering of CHIP-associated genes may represent a promising approach and could augment responses in difficult-to-treat CLL or solid tumor patients. Manipulation of genes involved in CH may also have the potential to alter the development and severity of treatment-related inflammatory side effects, e.g., CRS and ICANS.

C.H. June reports other support from Tmunity Therapeutics and Capstan Therapeutics outside the submitted work; in addition, C.H. June has a patent for Novartis issued, licensed, and with royalties paid and a patent for Tmunity issued and licensed. No disclosures were reported by the other authors.

C.H. June is supported by NIH grants (P01CA214278 and U54CA244711) and the Parker Institute for Cancer Immunotherapy. U. Uslu is supported by a Mildred Scheel Postdoctoral Fellowship of the German Cancer Aid. We thank R. Young, J. Tchou, J. Scholler, and members of the laboratory for helpful suggestions.