High CD38 expression in childhood T-cell acute lymphoblastic leukemia is not associated with prognosis

Abstract

BACKGROUND:

Prognostic factors are not well exploited in childhood T-cell acute lymphoblastic leukemia (T-ALL).

OBJECTIVE:

The aim of this study was to analyze the prognostic role of CD38 as well as minimal residual disease (MRD) and other biological factors in T-ALL.

METHODS:

Immunophenotyping of bone marrow (BM) at diagnosis and MRD levels were determined using a standard panel of antibodies by 4-colour flow cytometry. A total of 96 children with T-ALL were enrolled.

RESULTS:

The results showed that 97.9% of T-ALL patients were positive for CD38 with a median level of 85.3%. CD38-high group had a worse early treatment response than the CD38-low group. However, CD38 levels were not associated with prognosis, albeit CD38-high group had a worse 5-year event free survival rate (55.1% vs. 66.6%, $P>$ 0.05) and a higher 5-year cumulative incidence of relapse (35.6% vs. 19.8%, $P>$ 0.05). Very high MRD levels ( $>$ 10%) were related to the worse survival. Neither flow cytometry based minimal residual disease (MRD) levels nor CD38 expression levels showed significant relation to the hazard of relapse ( $P>$ 0.05).

CONCLUSIONS:

We conclude that T-ALL has a high level of CD38 expression which is not associated with prognosis. Very high MRD level ( $>$ 10%) is related to the worse survival, however, FCM based MRD detection does not convey a significant prognostic value.

Keywords

CD38 T-ALL childhood survival relapse

1. Introduction

Outcomes of childhood T-cell acute lymphoblastic leukemia (T-ALL) have been steadily improved with current risk-adapted therapy, as T-ALL patients are normally assigned to the intermediate-risk (IR) or high-risk (HR) arms of contemporary clinical trials. However, relapse is still the most common cause of treatment failure and patients with refractory and relapsed T-ALL have a particularly dismal outcome compared to the B-cell ALL (B-ALL) patients [1, 2, 3, 4]. Few effective therapies exist for refractory and relapsed T-ALL patients yet. So far, the most independent determinant in risk classification and prognostic prediction is minimal residual disease (MRD). Nevertheless, a number of patients with favourable MRD still experience relapse. Therefore, it is necessary to further improve the identification of patients with high risk of relapse.

CD38 is a 45 kDa type II transmembrane protein expressed on thymocytes, activated T cells and terminally differentiated B cells, etc. [5]. Some hematologic malignancies also express different levels of CD38 [6, 7, 8], such as multiple myeloma (MM), chronic lymphocytic leukemia (CLL), non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). In hairy cell leukemia, CD38 is a marker of poor prognosis [9], and CD38 ${}^{+}$ CLL patients also have more aggressive clinical behaviour [10]. CD38 monoclonal antibody (Daratumumab, Isatuximab, etc.) has achieved promising results in patients with MM recently [11, 12, 13, 14], and CD38-specific chimeric antigen receptor T cells (CAR-T) therapy also showed significant anti-tumor effects in a MM xenotransplant model [15]. In the latest report by Bride et al. [16], they tested Daratumumab in T-ALL patients-derived xenografts (PDX) and found 14 of 15 different PDX models responded. The only one non-responder was the one with low to negative CD38 expression. This study suggested that CD38 monoclonal antibody has clinical efficacy in T-ALL.

However, the reports regarding CD38 expression and the association with progression in pediatric ALL are scarce, and even less in T-ALL. In 1993, St. Jude Children’s Research Hospital reviewed 325 ALL patients, found T-cell ALL cases were more likely than B-lineage ALL cases to express CD38 and the expression of CD38 was not related to any clinical or biologic feature [17]. Bras et al. [6] showed CD38 was significantly higher in the pediatric B-ALL than that of AML patient and lower than that of NHL patients. They evaluated 28 pediatric T-ALL patients and indicated that CD38 expression levels were comparable to T-cell lymphoblastic lymphoma. However, there are also some different opinions. Karawajew et al. [18] reported that CD38 was generally under-expressed in childhood ALL.

In this study, the flow cytometric data of 96 cases of childhood T-ALL was analyzed and the prognostic role of CD38 expression was assessed. The results showed that 97.9% of T-ALL patients were positive ( $>$ 20%) for CD38, and CD38 expression in T-ALL patients was significantly higher than in B-ALL and AML patients. Patients with high CD38 expression had a worse early treatment response, including more patients with poor prednisone response and higher level of MRD on day 15 of remission induction. However, CD38 levels were not associated with survival in T-ALL.

2. Methods

2.1 Patients

Between January 2009 and December 2017, 96 evaluable children with newly diagnosed T-ALL treated at our hospital were enrolled. The diagnosis of T-ALL was based on morphology and immunophenotyping. The genetic abnormalities were examined by PCR and the conventional karyotypes were determined by standard methods at diagnosis. Patients who had white blood cell (WBC) count more than 100 $\times$ 10 ${}^{9}$ /L, or had poor response to prednisone or did not reach complete remission (CR) at the end of remission induction therapy were classified as HR group, while the remaining patients were classified to the IR group. In addition, 182 patients with newly diagnosed B-ALL and 90 patients with newly diagnosed AML treated at our hospital during this period were included as control groups. The protocol was approved by the Medical Ethics Committee of the Children’s Hospital of Zhejiang University School of Medicine and the written informed consent was obtained from the parents or guardians.

2.2 Immunophenotyping and minimal residual disease detection

Flow cytometric immunophenotyping of bone marrow (BM) at diagnosis was performed by 4-colour flow cytometry (FACSCalibur with CellQuest software, Becton Dickinson, San Jose, CA, USA) using a standard panel of antibodies. The panel of monoclonal antibodies (BD Biosciences) included the following: CD10, CD19, CD20, cytoplasmic (c) and surface (s) CD22, sIgM for B-lineage, CD1a, CD2, cCD3, sCD3, CD4, CD5, CD7, and CD8 for T-lineage, CD11b, CD13, CD14, CD15, CD33, CD117, and MPO for myeloid lineage, CD34, CD38, CD45, HLA-DR, CD56, and nTdT as non-lineage markers. Isotype antibodies were used as negative controls. CD45 gating strategy was used to identify the blast population. The threshold of $\geqslant$ 10% was used to define positive blasts for cytoplasmic markers while that of $\geqslant$ 20% was defined as positive for surface markers.

Table 1
The characteristics of the T-ALL, B-ALL and AML patients

Characteristics	T-ALL		B-ALL		AML
N	96		182		90
Gender (M/F)	3/1		1.3/1 ${}^{\ast}$		1.1/1 ${}^{\ast}$
Male	72	(75.0%)	102	(56.0%)	46	(51.1%)
Female	24	(25.0%)	80	(44.0%)	43	(47.8%)
Age (years) ${}^{\#}$	9.5	(1.0–16.0)	4.2	(1.1–14.3) ${}^{\ast\ast}$	7.2	(0.4–15.8)
WBC count ( $\times$ 10 ${}^{9}$ /L) ${}^{\#}$	117.8	(0.7–620.9)	7.7	(0.1–546.9) ${}^{\ast\ast}$	12.1	(1.1–235.4) ${}^{\ast\ast}$
Hemoglobin (g/L) ${}^{\#}$	105	(45–151)	78	(31–139) ${}^{\ast\ast}$	77	(34–110) ${}^{\ast\ast}$
Platelet ( $\times$ 10 ${}^{9}$ /L) ${}^{\#}$	69	(4–650)	59	(3–399)	51	(7–219) ${}^{\ast}$

${}^{\#}$ Median (Range). ${}^{\ast}P<$ 0.05, ${}^{\ast\ast}P<$ 0.001, compared to the T-ALL group.

After the bone marrow was labelled with antibodies, a lysing reagent (BD lyse, Becton Dickinson, USA) was added to the bone marrow and then washed twice by PBS, followed by adding 0.5% paraformaldehyde to fix the cells. Then the BM minimal residual disease (MRD) in mononuclear cells was determined by 4-colour flow cytometry [19] at the following time points (TP): TP1, on day 15 of remission induction; TP2, on day 33 at the end of remission induction and before consolidation therapy; TP3, on week 10 before the early intensification; TP4, on month 6 before the first late intensification; TP5, on month 12 before the second late intensification; TP6, on month 24 during the maintenance therapy; TP7, on month 36 of maintenance therapy for those remaining in clinical remission.

2.3 Treatment

All the patients were treated in our hospital with National Protocol of Childhood Leukemia in China (NPCLC)-ALL2008 protocol [20], which was modified from protocol NPCAC97. After 7 days of prednisone pre-test, all patients received a 4-week remission induction regimen with vincristine, daunorubicin, L-asparaginase and dexamethason (VDLD). Additional doses of L-asparaginase were given to HR patients. A 2-week consolidation therapy consisting of cyclophosphamide, cytarabine, 6-mercaptopurine (6-MP) and a 4-week extramedullary leukemia prophylaxis including 3 courses of high-dose methotrexate (HD-MTX, 5 g/m ${}^{2}$ ) were followed. Then the early intensification with 2-week VDLD followed by one week of 3 successive courses of etoposide and cytarabine (VP16 $+$ AraC) was performed. After that, all patients went into the risk-directed maintenance therapy for 2.5 to 3 years. Two more courses of HD-MTX were given to HR patients. All patients received triple intrathecal therapy with cytarabine, dexamethason and methotrexate. Additional doses were given to HR patients. None of the patients received prophylactic cranial irradiation. The patients who did not achieve CR at the end of remission induction, or relapsed were offered the option of allogeneic hematopoietic stem cell transplantation (HSCT) when they eventually achieved CR. Six patients received HSCT.

2.4 Statistical analysis

The survival rates of event-free survival (EFS) and overall survival (OS) were estimated with Kaplan-Meier analysis and log-rank test. The duration of EFS was defined as the time from the date of diagnosis until the first event (including relapse, development of a second malignancy or death) or until the date of last contact. The duration of EFS of patients who did not reach CR was considered as 0. Cumulative incidence of relapse (CIR) was compared with Gray’s test. Comparison of continuous and categorical variables was performed by Mann-Whitney U test and Chi-square test. Multivariate analysis was conducted by using Cox proportional hazards regression. A $P$ -value $<$ 0.05 (two tailed) was considered to be statistically significant. All calculations were performed with SPSS 19.0 software. The final data updated on December 20, 2018 was used. The median follow-up time was 5.7 years (ranging from 1.0 to 9.7 years).

3. Results

For the cohort of 96 T-ALL patients, 72 male and 24 female, the median age was 9.5 years, range from 1.0 year to 16.0 years. The majority of the characteristics were different compared to the B-ALL patients and AML patients (Table 1). T-ALL had more male patients ( $P<$ 0.05) and higher WBC count at diagnosis ( $P<$ 0.001). Of the 96 patients enrolled, 19 patients were classified to IR group, and the remaining 77 patients were classified to HR group. Translocations were found in 27 patients (28.1%), including SIL-TAL1 (N $=$ 17), MLL-ENL (N $=$ 4), HOX11 rearrangements (N $=$ 2), MLL-AF10 (N $=$ 1), SET/CAN (N $=$ 1), EVI1 (N $=$ 1), and CBF $\beta$ -MYH11 (N $=$ 1).

Figure 1.

The survival of T-ALL patients. A, The 5-year EFS and OS rates of the T-ALL patients; B, The 5-year OS rate of the T-ALL patients compared between CD38-high and CD38-low groups; C, The 5-year EFS rate of the T-ALL patients compared between CD38-high and CD38-low groups; D, The 5-year CIR rate of the T-ALL patients compared between CD38-high and CD38-low groups.

Of the 96 patients, 92 (95.8%) patients achieved CR, of whom, 74 (77.1%) achieved CR on the Day 15 of remission induction and 88 (91.7%) on the Day 33 after remission induction and before consolidation therapy. The 5-year EFS and OS rates were 61.3 $\pm$ 5.8% and 69.8 $\pm$ 5.5%, respectively (Fig. 1A). Relapse occurred in 18 cases, including 13 isolated hematologic, 3 isolated CNS, 1 combined hematologic and CNS, 1 combined hematologic and testicular relapses. The 5-year cumulative incidence of relapse (CIR) rate was 26.8 $\pm$ 5.7%, and the CIR of any CNS relapse was 6.6 $\pm$ 3.3%. Twelve relapses occurred very early which were within 18 months after first diagnosis, while 5 relapses occurred after 18 months of first diagnosis but within 6 months after the end of therapy, and only one relapse occurred late which was more than 6 months after the end of therapy. The 5-year OS rate of patients with any relapses was 26.0 $\pm$ 13.0%, which was extremely worse than those without relapse (81.1 $\pm$ 5.1%, $P<$ 0.001). Twenty-four patients died, 12 of whom died of disease related factors and the other 12 died of treatment related factors such as fatal infections and hemorrhage. Ninety-four (97.9%) patients were positive ( $>$ 20%) for CD38, which was more than CD2, CD5 and CD7 (74.7%, 83.3% and 93.5%, respectively). The median level of CD38 expression was 85.3% (ranging from 4.38% to 98.75%). The median level of CD38 expression on normal T cells was 72.9% (ranging from 61.4% to 81.4%). The percentage of CD38 ${}^{+}$ cells was evaluated for T-ALL, B-ALL and AML patients. CD38 expression in T-ALL patients (median level: 85.3%) was significantly higher than that in B-ALL and AML patients (median level: 71.6% and 75.1%, respectively, $P<$ 0.05, Fig. 2). The percentage of patients with at least 85% of CD38 ${}^{+}$ cells was highest in T-ALL (51.0%), which was lower in B-ALL (26.9%, $P<$ 0.001) and AML (33.3%, $P<$ 0.05). 85% was used as the cut-off value to discriminate CD38-high and CD38-low expression groups.

Figure 2.

Comparison of CD38 expression in the T-ALL, B-ALL and AML patients, ${}^{*}P<$ 0.05.

Figure 3.

The comparison of CD38 expression levels between the relapsed ( $n=$ 18) and the newly diagnosed patients ( $n=$ 96).

CD38 expression was not associated with gender, age, WBC at diagnosis, relapsed or not. In relapsed cases, the CD38 expression levels were comparable to the newly diagnosed patients (Median levels were 82.0% and 89.8%, respectively, $P>$ 0.05, Fig. 3). Comparing the early treatment response between the CD38-high and CD38-low expression groups, a high CD38 expression was associated with more patients experiencing poor prednisone response (49.0% vs. 25.5% in low CD38 expression, $P<$ 0.05) and high level of MRD on TP1 (patients with MRD $\geqslant$ 1.0% were 53.1% in high CD38 expression vs. 31.9% in low CD38 expression, $P<$ 0.05). However, no significant differences were observed in terms of CR rate, relapse rate or mortality between the two groups. The 5-year EFS rate of patients with high CD38 expression was 55.1 $\pm$ 8.7%, which seemed to be worse than those with low CD38 expression (66.6 $\pm$ 7.7%, Fig. 1C). This situation was likely related to a higher CIR in CD38-high group (35.6 $\pm$ 9.3% and 19.8 $\pm$ 6.9%, respectively, Fig. 1D). However, the differences did not reach the statistical significance ( $P>$ 0.05). The 5-year OS rates of patients with high CD38 expression and low CD38 expression were comparable (70.3 $\pm$ 7.4% and 71.0 $\pm$ 7.6%, respectively, Fig. 1B). Overall, the CD38 levels were not associated with the survival in T-ALL.

There was also no statistically significant difference in the expression of the markers CD1a, CD2, CD3, CD4, CD5, CD7, CD8, CD13, CD33, CD34, CD56 and CD117 with regard to inferior survival ( $P>$ 0.05). The analysis of other antigen expressions on the T-ALL demonstrated no significant preferential expression of CD38 with CD34, CD2 or CD33 (Table 2). Only CD1a negative patients had high levels of CD38 expression ( $P<$ 0.05).

Table 2

CD38 expression on T-ALL detected by flow cytometry

Immunophenotype	CD38-high	CD38-low	$P$ value
	N (%)	N (%)
CD34
Pos	18 (36.7)	14 (29.8)	0.521
Neg	31 (63.3)	33 (70.2)
CD2
Pos	32 (69.6)	39 (78.0)	0.363
Neg	14 (30.4)	11 (22.0)
CD33
Pos	4 (8.2)	7 (14.9)	0.351
Neg	45 (91.8)	40 (85.1)
CD1a
Pos	19 (26.8)	15 (60.0)	0.004
Neg	52 (73.2)	10 (40.0)

The cut-off point for CD38 expression as high or low was 85%.

Figure 4.

The association analysis of high MRD levels and the survival. A, The 5-year EFS compared between the patients with MRD $>$ 10% and MRD $\leqslant$ 10% on TP1 (on day 15 of remission induction); B, The 5-year OS compared between the patients with MRD $>$ 10% and MRD $\leqslant$ 10% on TP1; C, The 5-year EFS compared between the patients with MRD $>$ 10% and MRD $\leqslant$ 10% on TP2 (on day 33 after completion of remission induction and before consolidation therapy); D, The 5-year OS compared between the patients with MRD $>$ 10% and MRD $\leqslant$ 10% on TP2.

We further analysed the relationship between the very high MRD levels ( $>$ 10%) and the survival (Fig. 4). The 5-year OS rates of patients with MRD $>$ 10% on TP1 was 57.3 $\pm$ 15.2%, which was much worse than those with MRD $\leqslant$ 10% (71.9 $\pm$ 5.9%, $P<$ 0.05, Fig. 4B). However, there was no significant difference between the 5-year EFS rates (57.3 $\pm$ 15.2% vs. 62.1 $\pm$ 6.2%, $P>$ 0.05, Fig. 4A). Patients with very high MRD $>$ 10% on TP2 had significantly worse 5-year EFS and OS than those with MRD $\leqslant$ 10% ( $P<$ 0.05, Fig. 4C and D).

Multivariate cox regression analysis was performed for the risk of relapse in T-ALL patients. However, none of the clinical and biological features including gender, age, WBC at diagnosis, risk group, response to prednisone, MRD levels, or immunophenotype, including CD38 expression level showed significant relation to the hazard of relapse ( $P>$ 0.05).

4. Discussion

Patients with T-ALL especially refractory and relapsed T-ALL have particularly dismal outcomes compared to the B-ALL. Considering the high incidence of relapse and poor survival after relapse, it is important to identify patients early who are at risk of relapse. MRD monitoring has been proven valuable to predict the prognosis in ALL by many studies, especially in B-ALL [21, 22, 23]. The importance of MRD analyses is exemplified by the AIEOP-BFM-ALL 2000 study that showed that MRD $\geqslant$ 10 ${}^{-3}$ at day 79 constituted the most powerful predictor for relapse of T-ALL [24]. Furthermore, when the MRD combined with oncogenetic mutation profiles, it can improve the discrimination of MRD-defined risk categories [25]. These studies monitored the MRD levels by the method of PCR. Our study showed that very high MRD levels ( $>$ 10%) on TP1 and TP2 were related to the worse survival. However, our study also showed flow cytometry based MRD levels had no significant relation to the hazard of relapse in T-ALL. This result is consistent with the COG report which found that the end of induction minimal residual disease alone was not a useful determinant for risk stratified therapy in pediatric T-ALL [26]. This may be due to the fact that the flow cytometry based MRD is less sensitive than the PCR based MRD. So we would suggest it necessary to monitor the MRD in T-ALL patients through PCR method to define the risk stratification strategies in our future study.

Recently, CD38 targeting monoclonal antibodies, such as Daratumumab and Isatuximab, have been successfully applied in the therapy of multiple myeloma patients. CD38 has a rather unique pattern of expression in lymphocytes. Naïve lymphocytes and peripheral B and T lymphocytes have weak to negative expression of CD38 while this molecule is strongly expressed on lymphocyte precursors and activated lymphocytes. The high level expression of CD38 on T-ALL cell surface makes the molecule an attractive target for antibody-based treatment strategy in childhood T-ALL.

Our study have showed that 97.9% of the T-ALL patients are positive ( $>$ 20%) for CD38, with a median level of 85.3%, which is significantly higher than those in B-ALL and AML patients (71.6% and 75.1%, respectively, $P<$ 0.05). T-ALL patients present a significantly older age, higher WBC count and male predominance compared to the B-ALL and AML patients. However, CD38 expression is not associated with gender, age or WBC at diagnosis. More importantly, CR, EFS or CIR are not different significantly between CD38-high and CD38-low patients. Only the early treatment responses such as prednisone response and the level of MRD on TP1 are different between CD38-high and CD38-low patients. A high CD38 expression is associated with more patients with poor prednisone response and high level of MRD on TP1.

In conclusion, CD38 levels are not associated with the survival in pediatric T-ALL and the high expression of CD38 on this malignancy indicates that CD38 might be a novel molecular target for the antibody-based treatment of this disease.

Footnotes

Acknowledgments

This study was supported in part by grants from National Natural Science Foundation of China (Nos. 81770202 and 81470304), the Pediatric Leukemia Diagnostic and Therapeutic Technology Research Center of Zhejiang Province, the Doctoral Program of Higher Education (No: 20130101120057), and the General Program of Health department of Zhejiang Province (2014KYB145). We would also like to thank Mr. Hong-Qiang Shen, Mrs. Bai-Qin Qian and Dr. Si-Si Li for their excellent technical support on flow cytometry.

Supplementary data

The supplementary files are available to download from http://dx.doi.org/10.3233/CBM-190946.

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