T-cell acute lymphoblastic leukemia (T-ALL) is a genetically heterogeneous aggressive hematological malignancy of precursor T-cells that accounts for 10%–15% of pediatric and 20%–25% of adult patients with ALL.1 2 Despite advancements in therapeutic strategies in ALL, the clinical outcome of T-ALL remains inferior to B-cell lymphoblastic leukemia (B-ALL).2–4 Unlike the refractory/relapsed B-ALL, it is challenging to treat the patients with refractory/relapsed T-ALL, especially after allogeneic hematopoietic stem cell transplant.5–7 Further, the early thymic precursor subtype of T-ALL (ETPALL), which accounts for 10%–15% of T-ALL, has been shown to have a poorer clinical outcome in adult patients.2 4 6 8 9 Unfortunately, since the approval of nelarabine, no new agent has been approved for T-ALL and efforts are undergoing for evaluating the newer agents.6

An exciting recent achievement of modern cancer therapy is the targeted immunotherapy which has broadened the spectrum of therapeutic options for various malignancies, and monoclonal antibody (Mab) therapy is one of them. Mab specifically binds to a target molecule (epitope) expressed on the surface of cancer cells. In the last few years, daratumumab, a Mab against CD38, has been shown to be the most successful agent for the treatment of multiple myeloma (MM, a plasma cell malignancy with high levels of surface CD38 expression) and has gained rapid approval for the same.10–13 It is also being considered as an exciting option for other hematological neoplasms expressing CD38.14 CD38, a transmembrane glycoprotein and an ectoenzyme, is expressed in a variety of hematopoietic cells like plasma cells, myeloid/B-cells progenitor cells, monocytes, germinal center lymphocytes, natural killer (NK) cells, dendritic cells, and so on.15 It is variably expressed in many hematological neoplasms such as B-ALL, acute myeloid leukemia, Burkitt lymphoma, chronic lymphocytic leukemia, MM, and so on,14 15 which makes anti-CD38 Mab therapy as a potential therapeutic option in these malignancies. In early 2018, our bone marrow (BM) transplant unit has demonstrated the potential use of daratumumab in a patient with T-ALL with post-transplant refractory disease who remains disease-free for the last two and a half years.7 More recent preclinical studies of anti-CD38 therapy in T-ALL have also demonstrated promising results making it a potential therapeutic agent for future clinical trials.16–18 Hence, anti-CD38 Mab therapy is being considered as an exciting potential therapeutic option for high-risk T-ALL.

The mechanism of action of anti-CD38 Mab-like daratumumab involves Fc-dependent immunological reactions like complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity, antibody-dependent cellular phagocytosis, and so on.13 These mechanisms are also dependent on the levels of target antigen on tumor cells.19–22 In 2016, Nijhof et al have shown that the low levels of CD38 expression can affect the antitumor effect of daratumumab therapy in multiple myeloma.23 Interestingly, it has been also demonstrated that CD38 expression can be upregulated using drugs like all-trans retinoic acid and Panobinostat which can improve the antitumor efficacy of anti-CD38 Mab therapy.24 25 Therefore, the data on expression levels of CD38 in a tumor of interest is a pre-requisite for considering CD38 targeted therapy. Data on the expression level of CD38 in leukemic blasts of T-ALL are scarce and limited to the recently published small series of (21 and 8) patients.14 17 Moreover, the data on CD38 expression levels in leukemic blasts from the refractory T-ALL lack entirely. In view of the recent focus on the potential role of anti-CD38 Mab therapy in T-ALL, we have performed an in-depth study on the CD38 expression in leukemic blasts at diagnosis, in measurable residual disease (MRD), refractory disease, and relapsed disease in a large cohort of patients with T-ALL .

We studied CD38 expression levels in childhood as well as adolescent and adult patients with T-ALL treated at Tata Memorial Centre, India, between October 2017 and December 2019. The study was approved by the Hospital Ethics Committee. The diagnosis of T-ALL was established based on the morphology, cytochemistry (myeloperoxidase) and flow cytometric immunophenotyping (online supplementary table S1) in accordance to WHO 2016 guidelines.26 Patients were classified into two groups based on the immunophenotype at diagnosis that is, ETPALL27 and non-ETPALL. Pediatric patients were treated with MCP841 protocol28 29 and adolescent/adult patients were treated with BFM90 protocol.30 31 Treatment response was monitored at the end of induction (EOI) and subsequent time points for complete remission on BM aspirate morphological examination and MRD assessment.

The study included 347 samples (188 diagnostic, 124 MRD and 35 relapse samples; refer to figure 1) from 196 patients (85 children with age <15 years; and 111 adolescents and adults with age ≥15 years) with an overall median age of 15 years (range, 1 to 53 years). Out of 196, 188 patients were newly diagnosed and 8 patients had samples only at relapse. Characteristics of the newly diagnosed patients (n=188) with T-ALL are described in table 1. Patients were immunophenotypically classified as ETPALL (54/188, 28.7%), and non-ETPALL (134/188, 71.3%). Of 188 new patients with T-ALL, EOI-MRD assessment was available in 152, EOC-MRD was available in 96 and subsequent follow-up (SFU)-MRD was available in 14 patients. EOI-MRD was measurable (≥0.01%) in 90 (59.2%) samples including 24/152 (15.8%) patients who were not in morphological remission (blasts>5%) and were categorized as ‘refractory’ disease. EOC-MRD assessment was available mostly in patients with EOI-MRD positive status (96/152) and was detectable (≥0.01%) in 30/96 (31.25%) samples. Of 14 SFU-MRD samples, MRD was detectable in 4/14 (28.57%) samples. Thus, we included 124 MRD samples including 24 with refractory disease. We also included 35 samples with medullary relapse (27, in 188 new T-ALL and 8, only relapses).

The success story of daratumumab in multiple myeloma (MM) has attracted major interest in its potential utility for the treatment of other hematological neoplasms expressing CD38 antigen on tumor cells including T-ALL.7 15 35–38 Several studies have shown that the levels of target antigen can affect the antitumor efficacy of Mab therapy including daratumumab therapy.19–21 23 Since daratumumab is being considered as a potential therapeutic option for T-ALL, the data on the levels and patterns of CD38 expression in T-ALL leukemic blasts and its subtypes like ETPALL are urgently required. However, studies reporting the expression levels of CD38 in T-ALL at diagnosis and kinetics of its expression during the course of chemotherapy and at relapse are sparse and limited to a very small number of patients.8 17 18 Moreover, there is no report highlighting CD38 expression pattern in adult patients. We studied the expression of CD38 in a large cohort of T-ALL that included pediatric (85 children<15 years of age) as well as adult (111 adolescents and adults) patients. The study was performed in T-ALL leukemic samples at diagnosis, refractory disease, MRD assessment, and relapse. CD38 expression was evaluated for the percentages of leukemic cells positive for CD38, levels of CD38 expression (antigen density) with MFI and pattern of its expression (homogenous or heterogeneous) with CVIF.

The level of CD38 expression (MFI) on T-ALL blasts was not as strong as plasma cells but was of intermediate-level, that is closer to that of monocytes and NK cells (figure 2A). The CD38 expression in T-ALL was found predominantly between dim to intermediate levels. No difference was noted between the levels of CD38 expression between pediatric and adult patients as well as ETPALL and non-ETPALL. These findings are closely comparable to the recently published findings by Bride et al in a series of 21 pediatric patients.17 Using the conventional cut-off of 20% positive expression, CD38 was positive in almost all (>97%) of patients with T-ALL with median CD38-PBP of 85.9%. There was no significant difference between CD38-PBP in samples from pediatric versus adult, male versus female and patients with ETPALL versus non-ETPALL. These findings were similar to those reported by Bras et al and Naik et al in the recently published series of 8 and 12 cases of T-lymphoblastic lymphoma, respectively.14 16 Thus, we confirmed that CD38 was positive in the majority of leukemic blasts from newly diagnosed patients with T-ALL in a large cohort. Furthermore, our data demonstrated that the expression pattern of CD38 was prominently homogeneous with CVIF ranging between 61.8 and 66.4.

Anti-CD38 Mab therapy has been shown effective in combination with chemotherapy in newly diagnosed as well as in refractory or relapsed MM.10–12 39 Hence, we also evaluated the effect of cytotoxic chemotherapy (MCP841 in children and BFM90 in adults) on CD38 expression in residual tumor cells in refractory and MRD samples. The levels of CD38 expression (CD38-MFI), as well as the pattern of CD38 (CVIF), did not show any significant shift between paired diagnostic and EOI samples. These findings are in line with recently published data by Bride et al and Naik et al.16 17 Naik et al also demonstrated similar findings in acute myeloid leukemia (AML) samples.16 These findings highlighted the post cytotoxic chemotherapy stability in the expression levels of CD38 in the leukemic blasts of T-ALL. However, in our data, we also noted a mild reduction in the CD38-PBP in MRD samples (p=0.016) indicating induction therapy might have some effect on CD38-PBP, and a potential mechanism of daratumumab resistance in these patients. Nevertheless, the CD38-PBP was still sufficiently high (median 74%) in most of the MRD samples (>20% of blasts in 88.7% of MRD samples). To the best of our knowledge, this finding has not been reported in any of the earlier published studies. Although the percentage of CD38 positive blasts demonstrated a mild drop, we did not observe significant downregulation in the CD38 expression levels (CD38-MFI) in leukemic blasts from diagnostic to postinduction MRD samples. Similar findings were noted in refractory samples too. These findings highlighted the stability in the expression of CD38 in leukemic blasts even after the initiation of chemotherapy. Our data also included 35 samples from patients with relapsed T-ALL. Out of 35 patients, diagnostic and relapsed paired samples were available in 29 patients. We did not find any significant difference in the CD38-PBP, MFI and CVIF levels between the diagnostic and relapsed paired samples indicating consistent expression of CD38 in T-ALL blasts.

To summarize, we have demonstrated the details of CD38 positive percentages, levels, and patterns in leukemic blasts from a large cohort of patients with T-ALL. To the best of our knowledge, this is the first report on CD38 expression in adult patients. Our data showed that CD38 is uniformly expressed in the leukemic blasts of T-ALL across all groups of samples including ETPALL. We have also shown that cytotoxic chemotherapy has little effect on the expression of CD38 as demonstrated at various time points during the course of therapy. Thus, we conclude that the CD38 expression in leukemic blasts of T-ALL is robust and stable, making it a potentially effective target for anti-CD38-Mab therapy in patients with high-risk T-ALL.