PD1 expression on bone marrow T‐cells in newly diagnosed Egyptian AML patients: Correlation with hematological parameters, aberrant antigens expression, and response to induction therapy

Abstract Background Programed cell death protein 1 (PD‐1) is a key mediator for the development of T cell exhaustion that develops in response to persistent antigen stimulation. Aim In this study, we measured PD1 expression on CD3 positive bone marrow T‐lymphocytes in newly diagnosis AML patients and its relation to clinical/ prognostic outcomes in addition to response to induction therapy (day 28). Methods This study was conducted on 59 newly diagnosed AML patients and 20 healthy controls. Complete blood counts, flow cytometry using acute leukemia panel in addition to PD1 monoclonal antibodies were performed on bone marrow lymphocytes (CD3+), whereas cytogenetic/molecular studies were used to determine risk group. The patients’ remission status following induction therapy was determined. Results PD1 was brightly expressed in 91.5% of the cases than control sample with highly significant difference (P = .001). A cutoff of 3.5 for mean fluorescence intensity was used to divide patients into two groups (higher vs normal PD1 expression). A significant difference between the two groups regarding platelet count and aberrant CD7 expression (P = .007 and .023, respectively) was found. Those normally expressed PD1 respond better to induction therapy. Conclusion PD1 expression on BM T‐cells had a predictive value and providing an immunotherapeutic target for AML.


INTRODUCTION
Several malignant tumors are highly refractory to conventional therapies. The survival of tumors in several cases is assisted by checkpoint immunomodulation to maintain the imbalance between immune surveillance and cancer cell proliferation [1]. That is because tumors can adapt to immune pressure through the loss of antigenicity and immunogenicity as well as through their ability to create an immunosuppressive microenvironment. Therefore, distinct therapeutic strategies, depending on the mechanism of immune evasion by cancer cells, may be required for restoring productive cancer immunosurveillance [2].
Acute myeloid leukemia (AML) is a devastating blood cancer with 5-year survival of only 25%. Targeting inhibitory mechanisms to unleash the patient's own antitumor immune response has achieved major success [3]. The two best known inhibitory immune checkpoints are cytotoxic T-lymphocyte antigen-4 (CTLA-4) and the programmed cell death protein 1 receptor (PD-1) [4]. Blockers of these checkpoints are rapidly becoming a highly promising cancer therapeutic approach that yields remarkable antitumor responses with limited side effects [1].
Physiologically, the PD-1/PD-L1 pathway has emerged as a result of the need to control the degree of inflammation at locations expressing the antigen, in order to secure normal tissue from damage. There is a remarkable expression of the PD-1 protein on the surface of all activated T cells. When a T cell recognizes the antigen expressed by the MHC complex on the target cell, inflammatory cytokines are produced, initiating the inflammatory process. These cytokines result in PD-L1 expression in the tissue, activating the PD-1 protein on T cells leading to immune tolerance, a phenomenon where the immune system loses the control to mount an inflammatory response, even in the presence of actionable antigens [5].
Although this pathway is essential for maintaining peripheral T cell tolerance and is critical for attenuating autoimmunity and maintaining T cell homeostasis, it is also a deterrent to antitumor immunity.  [6,7]. Immune checkpoint inhibition therapy can lead to activation of autoreactive T-cells resulting in unique immune-related adverse events, so combining MBG453; anti-T-cell immunoglobulin domain and mucin domain 3(anti-TIM3) with hypomethylating agents was extremely well tolerated with no high grade immune-related toxicities [8].
In this study, we measured PD-1 expression on CD3 positive bone marrow (BM) T-lymphocytes in newly diagnosis adult Egyptian AML patients and its relation to clinical/ prognostic parameters in addition to response to induction therapy (day 28).

BM sampling at diagnosis
About 4-5 mL BM aspirate was obtained and divided into: • 0.5-1 mL for Leishman smears

FLT3-ITD/NPM1/C-Kit mutational analysis
Genomic DNA was extracted using salting out method. PCR products with altered gel mobility were sequenced. All PCR products were sequenced with Rotor Gene PCR Analyzer (Qiagen, Germany) using the BIG DYE terminator V1.1 cycle sequencing kit.

Statistical analysis
In addition to descriptive analysis, data were analyzed using SPSS

Laboratory and clinical data of the studied patients
The patients were classified according to the French-American-British

PD1 expression in relation to different clinical/Laboratory parameters and response to induction therapy
Based on the PD-1 expression (MFI) on BM CD3+ T lymphocytes (Table 3)

DISCUSSION
Novel immunotherapeutic strategies are rapidly evolving for treatment of the devastating acute myeloid leukemia. T-cell dysfunction has been noticed in various hematological neoplasm and has been put into the context of T-cell exhaustion as a result of increase expression of several inhibitory receptors in combination with defect effector function and finally apoptosis [10]. The PD-1 is a key mediator for such T-cell exhaustion that develops in response to persistent antigen stimulation, including cancer [3]. In this study, we measured PD-1 expression on CD3 positive BM T-lymphocytes in newly diagnosed adult Egyptian AML patients and its relation to clinical/prognostic parameters in addition to response to induction therapy (day 28).
Limited studies were carried on the immune response in BM of AML patients; although in a majority of patients, AML is derived from myeloid hematopoietic progenitors and rapidly grows in BM before mobilizing to peripheral blood; therefore understanding the anti-leukemia immune response within the BM of AML patients is likely to be a key to develop immune therapeutics for leukemia, and therefore in this study, we investigated the inhibitory checkpoint (PD-1) T cell within BM of AML patients [11].
In our study, examination of CD3 positive T-cells was done without differentiation in reference to the study done by Lion et al [12] who found that T cells of newly diagnosed AML patients were not significantly different from those of age-matched healthy controls, suggesting that increased T-cell differentiation could be induced by a long standing contact with AML cells (i.e, chronic stimulation). Inhibitory molecules (e.g, PD1) on T-cells have not been studied as broadly as on CD8 T-cells; however, the same molecules were also seen to play a role in CD4 T-cells as to CD8 cells. Persistent antigen exposure can induce a dysfunction state in CD4 T-cells that correlate with PD1 expression; it was found that Treg were unlikely to account for the observed increasein PD1 expression on CD4 T-cells, as isolated Treg from healthy controls has been reported to express PD1 intracellular [13]. The increase in inhibitory PD1 molecules was a surrogate for a shift toward different effector T-cells instead of a significant T-cell exhaustion; therefore such exhausted T-cells might be as a result of chronic activation [14].
In this study, on evaluating PD-1 expression on CD3+ BM Patients with normal PD-1 expression responded better to induction therapy (ie, complete remission) than patients with high PD-1 expression with a significant difference. This could be explained by a study done by Tan et al [15] who found a decreased tendency of exhausted T cells (PD1+CD244+, PD1+CD57+) in AML-complete remission group and found a particular influence on CD8+ exhausted T-cells, suggesting a poor anti-leukemia immune response in these patients; however, in our study we did not measure expression of PD1 or exhaustion markers on BM lymphocytes at day 28 for further evaluations.
Regarding platelet count that was significantly lower in those with higher PD-1 expression, a study done by Elif et al [16] who measured serum PD-1 and PD-L1 in patients with idiopathic thrombocytopenic purpura (ITP) revealed that there was a positive correlation between serum PD-1 levels and platelet count, which needs further studies to correlate serum PD1 and platelet count in AML patients. In this study, aberrant CD7 expression on myeloblasts was found in those with higher PD-1 expression on their T-cells; an early study described an increase of some activation markers (HLA-DR, CD69, CD71, and CD57) on T-cells at diagnosis [17]. This was in line with data from gene expression profiling of T-cells providing some hints at aberrant T-cell markers in AML patients [18]. Whether aberrant T-cell markers on myeloblasts of AML patients are associated along with PD L1 and PD1 on T-cells is a question for further larger cohort studies.

CONCLUSION
This study determined a cutoff value of MFI 3.5 to differentiate PD-1 expression level on CD3+ BM T-lymphocytes of newly diagnosed adult Egyptian AML patients. Higher PD-1 expression was associated with lower platelet count, aberrant CD7 expression on their myeloblasts, and poor response to induction therapy.