The prospective prognostic value of the immune checkpoint BTLA expression in adult acute myeloid leukemia patients

One of the crucial functions of the immune system is to prevent tumorigenesis, yet cancer occurs when malignant cells manage to evade immune surveillance via multiple strategies. Accordingly, this study aimed at assessing the potential significance of the novel immune checkpoint B and T lymphocyte attenuator (BTLA) as a prognostic marker in acute myeloid leukemia (AML), in addition to how it relates to response to treatment and patients’ survival. Thus, mRNA expression of BTLA was investigated on peripheral blood in 60 AML patients and 15 healthy controls. BTLA expression was found to be significantly elevated (p = 0.024) in the tested AML cases in comparison with healthy controls. Moreover, BTLA was over-expressed in the CD13, CD33, and HLA-DR positive cases as compared to their negative counterparts (p = 0.003; p < 0.001, and p = 0.001, respectively), and cases showing BTLA over-expression had significantly poorer overall survival times (p = 0.001) as confirmed by Kaplan–Meier survival analysis. These observations suggest that BTLA over-expression may be associated with reduced immunity against tumors and could be recommended as a promising biomarker for unfavorable prognosis in AML.


Background
Progress in understanding the pathophysiology and improving the therapy of acute myeloid leukemia (AML) is now occurring at a rapid pace [1]. AML is well known for including a wide range of gene mutations and chromosomal anomalies [2]. It may respond to high-dose chemotherapy in some patients; however, the mainstream succumb to resistance when eradication does not occur after induction chemotherapy [3]. Successful management of AML needs proper understanding of its pathophysiology at the cellular and molecular level, as well as its cytogenetic markers. It is essential to identify novel biomarkers that may provide better understanding of the molecular basis of AML. This could notably be helpful in diagnosis, prognosis, management and monitoring of patients [4].
Cancer involves the inability of the immune system to eradicate tumor cells. Dysfunctional antitumor T-cell responses actively participate in the development of cancer. Co-stimulatory and co-inhibitory mediators adjust T-cell proliferation, half-life, and cytokine release and enable effective T-cell responses to malignancy, yet controlling autoimmunity [5]. The balance between both these co-stimulatory and co-inhibitory signaling pathways acts as a molecular switch between activation and inhibition [6]. Nevertheless, this balance may be interrupted by continual antigen stimulation, as well as release of mediators, that suppress the immune response in the tumor microenvironment, causing T-cell dysfunction, called "T-cell exhaustion, " leading to its failure of cancer eradication responses [7]. Tumor cells manage diverse approaches to escape the immune response; these include expressing co-inhibitory receptors which inhibits T-cell reactions and cytokine production. Thus, this upregulated expression of co-inhibitory receptors is coupled to T-cell failure in malignancy [8]. The expanding immunotherapeutic approaches markedly offer several treatment options but should be engaged sensibly and cautiously. AML cells develop a variety of mechanisms to evade T-cell-mediated immunity, leading to its progression and relapse. These mechanisms include activation of immune checkpoints pathways that interfere with effective T-cell antitumor immunity [9]. Recent reports demonstrated over-expression of cytotoxic T lymphocyte antigen-4 (CTLA-4) and lymphocyte activation gene-3 (LAG-3) as major immune checkpoints expressed on T-cells [10]. Moreover, it was lately reported that over-expression of programmed cell death protein-1 (PD-1) and its ligands on leukemia cells is associated with more aggressive disease and AML relapse [9].
B and T lymphocyte attenuator (BTLA) or CD272 is a type I transmembrane co-signaling receptor that belongs to the CD28 superfamily and is mainly expressed on immune cells [5]. It is a ligand for tumor necrosis factor receptor superfamily member 14 (TNFRSF14), where their interaction inhibits T-cell immune responses. Besides, BTLA + T-cells have a less-differentiated phenotype, lower cytolytic function, and higher potential to proliferate compared with BTLA − T-cells [11]. Despite the critical role BTLA plays in immune tolerance and immune response, research is needed concerning its expression in AML. Accordingly, this study aimed at assessing the prognostic potential of BTLA in AML patients and how it relates to immune cell populations and response to therapy.

Subjects
Sixty recently diagnosed AML patients from the Internal medicine Department, Clinical Hematology and Stem Cell Transplantation Unit, [Ain Shams] University Hospitals, Cairo, Egypt were recruited in this study. This AML patients group included 24 males and 36 females with a mean age of 53.4 ± 12.9 years. Morphologic findings from Wright-Giemsa-stained smears of bone marrow aspirates and immunophenotype characterization of leukemic cells were used for diagnosis. According to the FAB classification, this AML group included 8 M0, 4 (M1-M2), 12 M2, 12 M3, 24 M4 patients. Prior to receiving any treatment, peripheral blood (PB) samples were collected in vaccutainer tubes containing Na 2 EDTA (1.5 mg/ml final concentration) for full blood count, immunophenotype characterization and for total RNA extraction. All patients were followed up for 12 months. Overall survival (OS) referred to the time between date of diagnosis and death. Subjects who survived till the end of the 12 months were censored. A healthy control group, consisting of 15 healthy, sex and age matched volunteers, was also involved in the study. This healthy control group included 7 males and 8 females with mean age of 46.9 ± 6.3 years.

Treatment plan
All patients in this study have received standard induction chemotherapy as specified by The National Comprehensive Cancer Network (NCCN) 2019 recommendations for AML (3 + 7 protocol ( anthracycline + cytarabine) for all types of AML except M3 subtype who received PETHEMA protocol) [12]. On day 28, assessments were done for all patients who have survived the induction, according to which patients were classified into responders, who achieved Complete Remission (CR), and nonresponders, who were refractory to chemotherapy. CR was defined as an absolute neutrophilic count > 1000/μl, a platelet count ≥ 100,000/μl, and BM blasts < 5% with no evidence of extramedullary disease.

Immunophenotypic characterization
Flow cytometry technique was used for immunophenotypic characterization using diagnostic kits supplied by Beckman Coulter, Fullerton, CA, USA [13].

Cytogenetic analysis
Fluorescence in situ hybridization (FISH) was applied as illustrated by Pinkel et al. [14] and Anastasi et al. [15] to verify cytogenetic abnormalities. Additionally, as stated by Fischer et al. [16] and Frohling et al. [17], fluorescence microscopy was carried out in order to determine the site of mutation or chromosomal defect.

Gene expression analyses
Total RNA extraction and purification from whole blood: using The QIAamp RNA blood mini kit (Qiagen, Hilden, Germany) according to the protocol specified by the manufacturer.
Synthesis of complementary DNA (cDNA): High capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA) was used, and synthesized cDNA was stored at − 20 °C till use.
Gene expression analyses: using Rotor-Gene Q ® Real-Time PCR cycler (Qiagen, Hilden, Germany) using standard thermal cycling conditions and Taqman assays specific for BTLA (Hs00699198_m1). GAPDH (Hs02786624_g1) was utilized as an endogenous control

CD13, CD33 and HLA-DR expression in AML patients
CD13, CD33 and HLA-DR expression in AML patients are listed in Table 2.

BTLA expression levels and response to chemotherapy in AML patients
To assess the correlation between BTLA expression levels and response to chemotherapy, the patients' responsiveness to induction chemotherapy was studied. By the end of the induction phase, 20% (12 patients) of the study group deceased; this may be attributed to respiratory tract infections, septicemia and/or hemorrhage. All the deceased patients have shown high BTLA expression levels. On the other hand, the survivors (80% of the cohort, 48 patients) were further classified into 36 (75%) responders and 12 (20%) non-responders. BTLA fold expression was found to be significantly up-regulated in non-responsive patients as compared to those responsive to chemotherapy (p = 0.022, Fig. 3).

BTLA expression levels concerning AML patients' survival
Regarding patients' survival, BTLA fold expression was found to be significantly negatively correlated with patients' overall survival (r = − 0.538, p < 0.01, Fig. 4). Additionally, Kaplan-Meier survival analysis revealed that AML patients with high BTLA expression showed significantly worse survival than those with low BTLA expression (p = 0.001, Fig. 5). Finally, multivariate analysis was performed using the Cox Proportional Hazards Model adjusted for age and gender. The test revealed significant relation between decreased survival and high BTLA expression (Cox proportional hazards = 1.282, 95% CI = 1.121-1.465, p < 0.001).

Discussion
Cancer evolution is currently documented as an outcome of developing crosstalk between different tumor cells and the surrounding stroma. While normally stroma is not permissive for cancer development, malignant cells have the ability to transform it. This may include their capability of changing the proportions of effector to regulatory T-cells, as well as altering co-stimulatory and co-inhibitory molecule expression, resulting in immune suppression, tumor evolution and immune evasion [18]. Understanding this immune environment in AML and manipulating coherent approaches to target its immune biology are an area of deep constant research in AML [19]. BTLA has been considered a novel co-inhibitory receptor, similar in structure and function to CTLA-4 and PD-1, and present on most lymphocytes [18]. The present study demonstrated the significant up-regulation of BTLA expression in AML patients versus the control group. Previous data reported AML blasts to be involved in immune suppression and in creating suppressive microenvironments [3]. Accordingly, immune response is inhibited; leading to immune escape by cancer cells and AML progression. Moreover, BTLA is linked with T-cell differentiation; previous studies concerning dysfunctional T-cells in cancers illustrated that exhausted T-cells demonstrate up-regulated expression in inhibitory receptors (IRs) and lost ability to eradicate tumor cells. However, the crucial procedure of T-cell exhaustion in cancer is still unclear [7].
Lately, the up-regulation of BTLA expression has been linked to tumor progression, with the worst prognosis reported for human melanoma [20], chronic and small lymphocytic leukemias [21], colorectal cancer patients [22], as well as gastric adenocarcinoma [23]. Those results come in accordance with the findings of this study, as the up-regulated BTLA expression was correlated with a decrease in the overall survival of AML patients, suggesting its potential use as a prognostic marker. Moreover, the significant up-regulation of BTLA expression in chemotherapy non-responder patients in our study as compared to responders may indicate that high BTLA expression may be related to resistance to chemotherapy. Similarly, other immune checkpoints have also demonstrated prognostic significance in AML patients such as T-cell immunoglobulin and mucin domain 3 (TIM-3) [24]. Additionally, the study of Chen et al. demonstrated that increased immune checkpoints co-expression of PD-1/CTLA-4, PD-L2/CTLA-4 or PD-1/LAG-3 correlated with poor OS in AML patients [25]. Interestingly, BTLA expression level showed a significant increase in CD13 + in comparison with CD13 − patients and in CD33 + as compared to CD33 − patients. CD13 expression is generally regarded as a bad prognostic sign, when it is not detected better outcomes are expected [26], while CD33 is more expressed on AML blasts as compared to healthy donor myeloid progenitors, which makes it an attractive nominee for targeted AML therapy [27]. Furthermore, BTLA expression in HLA-DR + patients was significantly more than its expression in HLA-DR − patients, indicating low chances of complete remission [26]. Accordingly, BTLA can be possibly suggested as an indicator of prognosis and survival in AML patients.
The fact that allogeneic hematopoietic cell transplantation is effective in AML treatment suggests that AML may be immune-responsive and indicates that novel immune-therapies such as immune checkpoint inhibitors might be able to provide a significant disease control [20]. The purpose of AML immunotherapy is enhancing the immune cells' capacity to eliminate leukemic cells. However, chemotherapy provokes several alterations within immune effector cells and might hinder T-cells functionality, thus the optimal timing of immunotherapy with chemotherapy should be taken into consideration [3].
The study of Chen et al. evaluated the prospective of BTLA as targets for ovarian cancer therapy preclinically. Obvious BTLA expression was prognostic for poor survival. Moreover, inhibition of BTLA combined with chemotherapy was found to encourage immune activation and produce influential antitumor effects in an animal model. Therefore, the combination of chemotherapy and anti-BTLA Ab might show clinical potential [28], but still future studies regarding BTLA targetting as a novel therapeutic strategy in AML are necessary. Several monoclonal antibodies targeting the CTLA-4, PD-1 and PD-L1 immune checkpoints on T-cells are now approved for clinical use in several solid tumors and hematological malignancies [29]. Some different immunotherapies are now under evaluation in AML such as PD1 inhibitors nivolumab and pembrolizumab. Early promising signals demonstrated in AML suggest a prospective future role for these targeted checkpoint therapies [19]. Moreover, dual blockade treatments were proposed to have a superior effect [30]. Nevertheless, resistance to treatment necessitates the development of unorthodox approaches. Finally, the up-regulated BTLA expression demonstrated in the present study and its negative impact on prognosis and survival may be a basis for designing future immune-therapies. The use of multiple combinations targeting immune checkpoints may provide superiority in cancer therapy. Accordingly, it is proposed that our findings concerning the prognostic value of BTLA expression in AML would be valuable in the upcoming studies to develop this novel approach.

Conclusions
As a final conclusion, these results demonstrate that BTLA gene expression may be considered as a promising significant determinant of AML patients' prognosis and survival. Additional studies are necessary for evaluating the significance of BTLA inhibitors in designing future immunotherapies.