Cytoplasmic CD79a is a promising biomarker for B lymphoblastic leukemia follow up post CD19 CAR-T therapy

Abstract

Minimal residual disease (MRD) detection is an important prognostic parameter in patients with refractory or relapsed B-cell acute lymphoblastic leukemia (R/R B-ALL). CD79a has been reported to exhibit a high degree of linage-specificity for B-cell differentiation, with a specificity of 88% and a sensitivity of 100%. In this study, we investigated the efficiency and prognostic role of cytoplasmic CD79a (cCD79a) antibody-gated multicolor flow cytometry (MFC) in MRD detection in patients with B-ALL who received CD19-targeted chimeric antigen receptor (CAR) T-cell therapy bridging to allogeneic hematopoietic stem cell transplantation (allo-HSCT). The retrospective analysis was carried on to 59 patients who accepted allo-HSCT after CD19-CAR-T infusion from June 2016 to May 2017. The MFC MRD statuses before and after allo-HSCT were both strongly correlated with the transplantation prognosis, the MFC panel with cCD79a gating can effectively monitor MRD after CD19 CAR T-cell therapy and predict the prognosis after allo-HSCT. Trial registration: ClinicalTrials#: ChiCTR-IIh-16008711.gov: NCT03173417. Registered 30 May 2017 – retrospectively registered, https://www.clinicaltrials.gov/

Keywords:

• Chimeric antigen receptor T cells
• allogeneic hematopoietic stem cell transplantation
• refractory or relapsed B-cell lymphoblastic leukemia
• flow cytometry
• minimal residual disease

Introduction

B-cell acute lymphoblastic leukemia (B-ALL) is the most common hematologic malignancy. The treatment of relapsed/refractory B-cell acute lymphoblastic leukemia (R/R B-ALL) remains a major challenge. Patients with R/R B-ALL often have an unfavorable prognosis [1]. Recently, chimeric antigen receptor (CAR) T cell therapy is a novel method in the immunotherapy of hematological malignancies. CAR T cells can recognize cell-surface tumor antigens, which results in antigen-specific T-cell proliferation and activation, thereby fighting tumors [2]. CD19- and CD22-targeted CAR T-cell therapies have achieved remarkable progress in refractory or relapsed R/R B-ALL [3–7]. Minimal residual disease (MRD) detection by multicolor flow cytometry (MFC) has been recognized as an effective and promising prognostic predictor in acute leukemia. It can provide reference for the formulation of personalized treatment plan [8,9]. However, with the widespread use of CD19 CAR T-cell therapy, CD19 expression on malignant and benign B cells might decrease or be lost in some patients after CD19 CAR T-cell therapy. Thus, the classic B-ALL MRD panel, which uses CD19 to set a rough B-cell gate, might lead to missed diagnosis [6,10]. Therefore, a new MRD panel for monitoring prognosis is necessary.

CD79a is identified as a B cell-specific antigen and CD79a expression starts at the earliest pre-B stage of development and across B cell differentiation to the plasma cell stage [11]. CD79a is a key markers used in routine immunophenotypic analysis. CD79a has been reported to exhibit a high degree of linage-specificity for B-cell differentiation, with a specificity of 88% and a sensitivity of 100% [12]. In classic Hodgkin lymphoma, CD79a-positivity exhibits unique clinicopathological features [13]. It was found that CD79a was related to CNS infiltration and – relapse in B-cell precursor – ALL patients [14]. However, little is known about the role of CD79a in MRD detection in B-ALL.

Here, we investigated the efficiency and prognostic role of cytoplasmic CD79a (cCD79a) antibody-gated MFC in the MRD detection of patients with R/R B-ALL who received CD19 CAR T-cell therapy bridging to allogeneic hematopoietic stem cell transplantation (allo-HSCT). This study would provide a novel method for MRD detection.

Methods

Subjects

For the consistency analysis, we collected 203 bone marrow (BM) specimens from patients with B-ALL who had not received CD19 CAR T-cell therapy, including 103 MRD-negative specimens with a status of complete response (CR) and 100 MRD-positive specimens. We also collected 15 BM specimens from healthy donors for use as normal controls in the specificity analysis of the new panel. To assess the role of the new panel in the prognosis of CD19 CAR T-cell therapy, we collected BM specimens from 59 patients who had received CD19 CAR T-cell therapy bridging to allo-HSCT at Hebei Yanda Ludaopei Hospital, Hebei Province, China, from June 2016 to May 2017. These patients were 33 males and 26 females, median age 10 years old (2–51 years), and median follow-up 46 m (1–55 m). The deadline for the follow-up was February 2021.

This study was approved by the Ethics Committee of Hebei Yanda Ludaopei Hospital.

Reagents and panel

The classical panel of our lab was two-tube seven-color panel (Supplementary Table 1). They were CD38 FITC/CD10 PE/CD34PerCP Cy5.5/CD19 PE CY7/CD13 + CD33 APC/CD20 APC CY7/CD45 V500 in the first tube, and TdT FITC/CD81 PE/CD34 PerCP Cy5.5/CD10 PE CY7/cCD79a APC/CD45 V500 in the second tube. In the new panel, CD13 + CD33 APC was substituted by cCD79a in the first tube and the second tube was the same as that of the classical panel. TdT FITC was purchased from Beckman Coulter Inc. (Brea, CA), cCD79a APC and other antibodies were all purchased from Becton Dickinson Inc. (Franklin Lakes, NJ). Lysing solution and Fix & Perm were purchased from Becton Dickinson Inc. (Franklin Lakes, NJ). Panels contain the classical panel (CD19 gating) and the new panel (cCD79a gating). They were used to detect the recovery of CD19 and verify each other.

Specimen handling

The specimens were processed using a standard lyse/wash surface or surface/intracellular staining procedure with FACS lysing solution (BD Biosciences, San Jose, CA) and a FACS Fix Perm kit (BD Biosciences, San Jose, CA), according to the manufacturer’s protocol. All MRD specimens were analyzed using a 3 laser/8 color FACSCanto II cytometer (BD Biosciences, San Jose, CA). One million events were acquired for each tube, and data were analyzed using FACSDiva 8.0.2 software (BD Biosciences, San Jose, CA). The instrument set-up and compensation matrix were established using CS&T beads (BD Biosciences, San Jose, CA), as per the manufacturer’s recommendations.

MFC analysis

All data are shown as dot plots. Adherent cells were depleted by gating P1, which was set using forward-scatter (FSc)-area (A) and FSc-height. The live-cell gate (P2) was set according to the FSc and side scatter (SSc) in P1. Lymphocytes, monocytes, maturing granulocytes, and nucleated red blood cells were gated according to the SSc/CD45 dot plots in the P2 gate. Rough B-cell gates were set by SSc-A/cCD79a and SSc-A/CD19. If the sample was MRD-positive, sequential gates were used to analyze the cells in rough B-cell gates with dot plots of SSc/CD45, CD20/CD34, CD34/CD10, CD38/CD10, CD19/cCD79a, CD20/CD10, CD34/CD38, CD20/CD38, CD34/terminal deoxynucleotidyl transferase (TdT), CD10/CD81, CD34/CD81, and CD19/CD45. The CD19/cCD79a dot plot was substituted with CD34/CD13 + CD33 in the classic panel.

Definitions

CR was defined in agreement with the National Comprehensive Cancer Network (NCCN) guidelines, version 1.2016. MRD was defined as the ratio of leukemia cells in BM evaluated by flow cytometry. MRD-positive was defined as positive results for all MRD tests. MRD-negative CR was defined as morphologic remission and negative results for all MRD tests, including flow cytometry. OS events were defined as from the first CAR-T cell infusion to death. DFS was defined as the survival period with continuous CR. Relapse was defined as the reemergence of blasts in the BM after CR.

Statistical analysis

Statistical analysis was performed using SPSS version 25.0 (SPSS Inc., Chicago, IL). Fisher’s exact test was used to compare rates. The effects of MRD status after CD19 CAR T-cell therapy before and after allo-HSCT on the prognosis and the relapse time after allo-HSCT were analyzed using a homogeneity test. Overall survival (OS) was analyzed using the Kaplan–Meier method. p<.05 was considered statistically significant.

Results

Good consistency between CD19 and cCD79a gating panel

Similar MRD results were observed between classical and cCD79a gating panels in 103 samples with MRD-negative CR R/R B-ALL and 100 samples with MRD-positive R/R B-ALL. The percentage of B cells in the nucleated cells detected by the classical gating panel correlated well with that detected by the cCD79a gating panel (Figure 1). Genetic testing results of B-ALL patients are shown in Supplementary Table 2. For MRD-negative CR R/R B-ALL patients, BCR-ABL1 positive expression accounted for the highest proportion, followed by EP300-ZNF384 and EBF1-PDGFRB. For MRD-positive R/R B-ALL patients, BCR-ABL1 positive expression accounted for the highest proportion, followed by MEF2D-BCL9 and EP300-ZNF384.

Figure 1. Linearity between the classical and cCD79a gating panels for detecting minimal residual disease (MRD). (A) A good linear correlation was observed between the two panels for MRD negative CR B-ALL patients, with R²=0.9990 and y= 0.9957x + 0.0448. (B) A good linear correlation was observed between the two panels for MRD positive B-ALL patients, with R²=0.9904 and y= 1.0137x + 0.1423.

Excellent specificity of the new panel

When the rough B-cell gates were set with cCD79a in the new panel, all BM specimens from healthy donors were MRD negative. We discovered that the percentage of cCD79a-positive B cells in nucleated cells were 9.86%. A small number of CD19-negative cCD79a-positive B cells were observed in the nucleated cells of the normal control specimens (median, 0.03%; range, 0.0–0.18%, Figure 2). The percentages of CD19-negative subsets in B cells were not associated with the percentages of B cells in the nucleated cells (p=.152). The immunophenotyping of CD19-negative B cells showed mainly CD34-positive early hematogones, but they differed from their CD19-positive counterparts, showing dimmer CD10 and CD38, brighter TdT and CD34, and larger FSc and SSc (Figure 3). In addition, MRD positive case after CD19-CAR-T was also detected by the new panel. The malignant cells were demonstrated as red events gated by cCD79a, which were cCD79a, CD38 and dim CD45 positive, CD19, CD10,CD34, and CD20 negative (Figure 4). Moreover, the results of patients who relapsed after CD19-CART infusion are shown in Supplementary Fig. S1. All cCD79a positive B cells were found in P3, including red malignant naive B cells, which accounted for 1.29% of the nuclear cells, expressed cCD79a, lost CD19, strongly expressed CD10 and CD34, weakly expressed CD38 and CD81, and unexpressed CD20. Blue cells are normal differentiated B-line cells, including B-progenitor cells, naive B-cells and mature B-cells, which all expressed CD19 and cCD79a. Furthermore, BM samples of patients after CD19 CAR T-cell therapy were detected using the new panel at different time. The results are shown in Supplementary Fig. S2. Before the patient was infused with CD19-CAR-T, cCD79a positive cell mass accounted for 0.62% of nuclear cells, expressing CD19, CD79a, CD10bri, but not expressing CD20 and CD38, indicating malignant tumor cells. When the patient was on day 30 after infusion, cCD79a positive B cells accounted for 0.06% of the nuclear cells and were normal B cells, among which 65.45% of the B cells had no recovery of CD19 expression. When the patient was on day 120 after the infusion, cCD79a positive B cells accounted for 0.74% of the nuclear cells and were normal B cells. CD19 was recovered in almost all B cells.

Figure 2. The percentages of cCD79a-positive B cells and cCD79a-positive CD19-negative B cells in nucleated cells.

Figure 3. Immunophenotyping of CD19-negative B cells (dark blue events) in normal specimens. Lymphocytes, monocytes, maturing granulocytes, and nucleated red blood cells were gated according to the SSC-A/CD45 dot plots in the P2 gate. Rough B-cell gates were set by SSc-A/cCD79a and SSc-A/CD19. B cells circled with a CD19 gate (rose red) accounted for 0.17% of the nuclear cells. Lymphocytes (green) accounted for 4.75% of the nuclear cells. P4 gates were B cells circled with cCD79a, showing CD19-negative cCD79a positive B cells in blue and CD19-positive cCD79a positive B cells in red after CAR T-cell therapy.

Figure 4. MRD-positive patients after CD19 CAR T-cell therapy detected using the new panel. Lymphocytes (green), monocytes (dark orange), maturing granulocytes (blue), and nucleated red blood cells were gated according to the SSC-A/CD45 dot plots in the P2 gate. Rough B-cell gates were set by SSc-A/cCD79a and SSc-A/CD19. The malignant cells were detected as red events gated by cCD79a, which were cCD79a-, CD38-, and dim CD45-positive and CD19-, CD10-, CD34-, and CD20-negative.

Effectiveness of the new panel in predicting prognosis after CD19 CAR T-cell therapy bridging to allo-HSCT

In total, 51 patients we collected achieved MFC MRD-negative CR (Supplementary Table S3). These patients received allo-HSCT at a median of 64 (range, 44–122) days after CD19 CAR T-cell therapy. Of them, 76.47% (39/51) patients had disease-free survival (DFS) and they had a status of MFC MRD-negative CR on the last examination before the end of the follow-up period. Simultaneously, 3.92% (2/51) patients relapsed at a median of 11.5 (range, 5–18) months after allo-HSCT and achieved a second CR and the OS median time was 46 (range, 44–48) months. Additionally, 19.61% (10/51) patients died after allo-HSCT, of whom 5.88% (3/51) died from relapse at a median of 4 (range, 3–15) months after allo-HSCT. Moreover, 13.73% (7/51) MRD-negative patients in CR died from allo-HSCT-related complications.

Eight patients we collected remained MFC MRD-positive after CD19 CAR T-cell therapy. They received allo-HSCT at a median of 61 (range, 51–211) days after CD19 CAR T-cell therapy. Interestingly, 37.5% (3/8) patients were alive with MRD-negative CR, and the OS median time was 46 (range, 46–48) months. One patient with MRD-negative CR and severe graft-versus-host-disease was died in 5 months after allo-HSCT. Additionally, 50% (4/8) patients did not achieve CR after allo-HSCT and died from relapse, the median OS time was 61.5 (range, 40–84) days.

Our data showed a significant difference in OS between the MRD-positive and MRD-negative patients after CD19 CAR T-cell therapy (p=.0049; Figure 5).

Figure 5. The overall survival (OS) curves of MRD-positive and MRD-negative patients after CD19 CAR T-cell therapy. The OS analysis was performed using the Kaplan–Meier method. p<.05 was considered statistically significant.

MRD status as an independent prognostic factor

The new panel was used to evaluate the MRD status after CD19 CAR T-cell therapy. The effects of sex, age, the time between CD19 CAR T-cell therapy and allo-HSCT, and MFC MRD status before and after allo-HSCT on prognosis were evaluated using univariate and Chi-square analyses. The MRD statuses before and after allo-HSCT were strongly correlated with prognosis after CD19 CAR T-cell therapy (Table 1). However, the correlation analysis indicated that prognosis was not associated with sex, age, or the time interval from CD19 CAR T-cell therapy to allo-HSCT.

Table 1. Analysis of prognostic factors.

N: 59	Died of GVHD	Died of relapse	Survival	Value	p	Statistic method
Gender				2.115	.381	Pearson’s test
Male	3	3	27
Female	5	4	17
MFC MRD status between CAR-T and allo-HSCT				9.667	.004	Fisher’s exact test
Negative	7	3	41
Positive	1	4	3
MFC MRD status post allo-HSCT				29.488	.000	Fisher’s exact test
Negative	8	0	42
Positive	0	7	2
Age				0.278	1.0	Fisher’s exact test
Adult	2	2	11
≤1u years	6	5	33
Time from CD19-CAR-T to allo-HSCT				4.326	.310	Fisher’s exact test
<60 d	3	5	13
61–90 d	5	2	27
>90d	0	0	4

MFC: multicolor flow cytometry; MRD: minimal residual disease; CAR-T: chimeric antigen receptor T-cells; allo-HSCT: allogeneic hematopoietic stem cell transplantation; GVHD: graft-versus-host-disease.

After CD19 CAR T-cell therapy, the MFC MRD results before and after allo-HSCT were the only factors that were found to affect prognosis.

Correlation between relapse time after allo-HSCT and MRD status before allo-HSCT

The homogeneity test was used to analyze differences in the relapse time among patients who received allo-HSCT in MRD-positive status and died from relapse (MRD PD; n = 4), patients who received allo-HSCT in MRD-negative status and died from relapse (MRD ND; n = 3), and patients who received allo-HSCT in MRD-negative status, but achieved a second CR after relapse (MRD NC, n = 2). The median relapse time was 61.5 (range, 40–84) days in the MRD PD group (Supplementary Fig. S3) and 4 (range, 3–15) months in the MRD ND group. The two patients in the MRD NC group relapsed at 11.5 (range, 5–18) months after allo-HSCT and achieved a second CR. However, there was no significant difference in the interval between allo-HSCT and relapse among the three groups.

Discussion

In recent years, a large number of studies have shown that CD19 CAR T-cell therapy effectively prolongs the survival time and improves the survival rate of patients with R/R B-ALL, making it a promising treatment [10,15–17]. Although MFC is effective in detecting MRD after leukemia treatment [18,19], other studies and our unpublished data have found that CD19 expression on normal or malignant B cells can decrease or become lost after CD19 CAR T-cell therapy. Thus, the classic panel, which sets the rough B-cell gate by CD19-positive cells, was not applicable in monitoring MRD in these patients. Therefore, there is an urgent need to find new and effective methods for monitoring MRD. A study at Washington University that used CD22 combined with a CD24-positive/CD66C-negative method to set a rough B-cell gate to detect MRD after CAR T-cell infusion suggested that new effective gating methods were necessary for ongoing CD19 and CD22 dual-target or CD22 CAR T-cell therapy [20]. Although c antibody staining can be more complicated than surface antibody staining, our laboratory found that cCD79a gating was the most promising method after comparing the sensitivity and specificity of various antibodies. CD24 was not an ideal rough B-cell marker because the coverage rate was only 80.6% in B-ALL [20], with an exceptionally high negative rate in B-ALL with MLL translocation [21]. CD22 has poor specificity and could be expressed in basophils, plasmacytoid dendritic cells, and mast cells [22]. Furthermore, CD22 expression is reduced in KMT2A-rearranged B-ALL, which might affect the sensitivity of CD22 gating [23]. Studies have reported that CD19 and CD22-CAR T-cell sequential therapy [24,25], CD19/CD22 bi-target [26,27], CAR T-cell therapy, and CD19 and CD22 CAR T-cell combination therapy can possibly lead to normal and neoplastic cells losing CD19 and CD22 antigens simultaneously, making cCD79a gating a more feasible option.

To the best of our knowledge, this is the first time that we focused on cCD79a antibody-gated MFC in MRD detection. We discovered that MFC MRD detection using cCD79a gate can effectively monitor MRD after CD19-CAR T treatment and predict the prognosis after allo-HSCT. These results suggest that the novel panel can be a potentially effective strategy for MRD detection and optimization of CAR T therapy, thereby improving therapeutic effect and prognosis.

In the present study, we achieved good specificity for MRD detection using the cCD79a panel in 15 BM specimens from healthy donors, and no malignant cells were detected. However, a small number of CD19-negative cCD79a-positive B cells were found in the normal control samples, with a median percentage of 0.03% (range, 0.00–0.18%) nucleated cells. A similar result was reported by Washington University [20], where CD19-negative B lymphoblasts were detected in BM specimens from patients of B-ALL who had not received CD19 CAR T-cell therapy, and these B lymphoblasts were classified into three subsets according to the antigen expression. The earliest hematogones were CD34-positive, dim CD22-positive, and dim CD38-positive B cells that were negative for CD19, CD10, CD20, and CD24. The second group of hematogones was CD34-, CD22-, CD38-, and cCD79a-positive cells, heterogeneously expressing CD10 and CD20 as well as CD24 and CD19. The third group was CD22-positive/CD24-negative or CD22-negative/CD24-positive, with CD19-, CD10-, and CD34-negative cells. In our study, bivariate correlation analysis revealed that there was no correlation between the proportion of CD19-negative B cells in the nucleated cells and the proportion of B cells in the nucleated cells. Additionally, CD19-negative, cCD79a-positive B cells differed from CD19-positive B cells, which mainly expressed TdT and CD34. Therefore, attention should be paid to avoid misdiagnosing as MRD-positive when applying the new panel.

We analyzed 59 BM specimens using MFC with the new panel, which uses cCD79a antibody to set the rough B-cell gate, to verify the efficiency of the new panel in monitoring and predicting OS in patients with R/R B-ALL who received CD19 CAR T-cell therapy bridging to allo-HSCT. The MFC MRD detection results were consistent with those of the status evaluation in clinical patients. The correlations between prognosis and sex, age, allo-HSCT time, and MRD status after CD19 CAR T-cell therapy before and after allo-HSCT were analyzed. A positive MFC MRD status after CD19 CAR T-cell therapy was a strong adverse prognostic marker both before and after allo-HSCT. Furthermore, most patients with R/R B-ALL could achieve MFC MRD-negative CR after CD19 CAR T-cell therapy, and 80.39% (41/51) patients survived for a median of 47 (range, 42–55) months. The OS of R/R B-ALL was greatly improved in MRD-negative R/R-B ALL patients, whereas MRD-positive patients only achieved 37.5% (3/8) OS. This suggested that after CD19 CAR T-cell therapy, allo-HSCT performed on patients with R/R B-ALL and an MFC MRD-negative status enhances the survival rates. Most importantly, it was demonstrated that the new panel could effectively monitor the disease process after CD19 CAR T-cell therapy and predict the survival rate after allo-HSCT.

During the regular patient checkups after allo-HSCT, nine patients relapsed after a median of 90 (range, 40–480) days. The relapse time of the MRD PD group was earlier than that of the MRD ND group, and the MRD NC group showed the longest time to relapse, but these differences were not statistically significant. This may be because of the small number of patients in our study. Therefore, a larger sample is necessary for further research from a long-term perspective. At the same time, two patients in the MRD NC group achieved a second CR, suggesting that an increase in the frequency of MRD detection helps to achieve the aim of early detection, early treatment, and, subsequently, better results. For most hospitals, the regular checkpoints are 1, 2, 3, 6, 9, 12, 18, and 24 months after allo-HSCT.

During follow-up, two patients achieved a second CR. One patient relapsed 5 months after allo-HSCT, and the percentage of malignant cells was 9.48%, with CD19-negative cells. After CD22 CAR T-cell therapy and chemotherapy, MFC MRD was negative by the end of the follow-up period (48 months later). The other patient relapsed 18 months after allo-HSCT; the tumor load was 0.23%, with CD19-positive cells. After CD19 CAR T-cell therapy and chemotherapy, the MFC MRD was negative for 44 months. These results indicated that different CAR T-cell therapies could be chosen according to CD19-positive or -negative relapse, and MFC MRD provided good guidance for a second CAR T-cell therapy.

In conclusion, the new MFC panel using cCD79a gating is a reliable technique to assess prognosis and monitor relapse in patients with R/R B-ALL after CD19 CAR T-cell therapy bridging to allo-HSCT. The MRD status after CD19 CAR T-cell therapy is an exclusive prognostic marker before and after allo-HSCT. The increase in MRD detection frequencies and monitoring time might be helpful in uncovering more valuable treatment methods for R/R B-ALL.

Authors contributions

Conceptualization and funding acquisition: HW. Data curation: MC. Formal analysis: MF, AW, XZ, and JY. Investigation: MC, MF, AW, XZ, JY, YZ, XW, JZ, MG, and PL. Methodology: MC and MF. Writing-original draft: MC, MF, AW, XZ, JY, YZ, XW, JZ, MG, PL, and HW. Writing-review and editing: MC. All authors read and approved the final manuscript.

Hui Wang, corresponding author, designed the research and revise paper. Man Chen, the first author, statistical analysis, analyzed data and wrote the paper. Minjing Fu, co-first authors, gather clinical data, analyzed data and revise paper. Aixian Wang, Xueying Wu, Junyi Zhen, Meiwei Gong, Qing Du, test samples and report MRD results. Xian Zhang, Guanlan Yue, Wei Zhao, Yanli Zhao, and Peihua Lu, gather clinical data and clinical treatment.

Acknowledgements

Ethics approval: All procedures were in accordance with the ethical standards of the institutional research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Informed consent: Written informed consent was obtained from the patient for publication of this case.

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Disclosure statement

The authors declared that they have no conflict of interest.