Could Microparticles and PD1 Interplay Add More Effects on Treatment Outcomes of Multiple Myeloma? A Prospective Controlled Study

Background: Although multiple myeloma (MM) is still considered as an incurable disease by current standards, the development of several combination therapies, and immunotherapy approaches has raised the hope towards transforming MM into an indolent, chronic disease, and possibly achieving a cure. Objectives: We tried to shed light on the expression of PD1 and different Microparticles (MPs) in MM and their interplay as a mechanism of resistance to standardized treatments, in addition, nd their associations with prognostic factors of MM. Methods: thirty patients with newly diagnosed and chemotherapy naïve active MM, along with 20 healthy participants of comparable age and sex were recruited, after diagnosis of MM; blood samples were collected from both patients and controls for ow cytometric detection of CD4+, CD8+, CD4+PD1+, and CD8+PD1+ T cells, total MPs, CD138+ MPs, and platelet MPs. Results: MM patients had statistically signicant higher levels of TMPs, CD138+MPs compared to their controls, while PMPs exhibited no signicant difference between both groups. Statistically signicant higher percentages of CD8+, PD1CD8+, PD1CD4+T cells were detected in patients compared to controls, while the latter group had a signicantly higher percentage of CD4+T cells than MM patients, patients who didn't achieve complete response, had signicantly higher percentages of PMPs, CD138+MPs, PD1+CD8+, PD1+CD4+, and CD8+T cells (cutoff values= 61, 10.6, 13.5, 11.3 & 20.1 respectively), (p-values=0.002, 0.003, 0.017, 0.001 & 0.008 respectively). Conclusion: Microparticles and PD1 expressions were associated with proliferative potential and resistance to Bortezomib-based treatments, our results suggested that they played a crucial role in myeloma progression.


Introduction
Multiple myeloma (MM), a type of malignancy arises from plasma cells in bone marrow, represents about 10% of all hematologic malignancies, standard approach of treatment consists of induction therapy followed by high dose chemotherapy and autologous stem cell transplantation (ASCT) in candidate patients, for non-candidate patients, standard doublet, triplet, or quadruplet, agent-containing induction treatment are applied until progression. Although MM is still considered as an incurable disease by current standards, the development of several combination therapies, and immunotherapy approaches has increased the hope towards transforming MM into an indolent, chronic disease, and possibly achieving a cure in some patients (1) with a median survival for all patients of about 3-4 years (2).
In addition to the International and Durie-Salmon staging systems, biological markers, including cytogenetic abnormalities such as presence of translocations including t(4; 14), and t(14;16), hypodiploidy, del(17p), and del (13), serum β2-microglobulin levels greater than 2.5 mg/L, an elevated plasma cell labeling index, and detection of circulating plasma cells, are predictors of poor prognosis in newly diagnosed MM patients (3,4).
Microparticles (MP) are membrane vesicles or extracellular vesicles shed systemically into the circulation, ranging in size from 0.1-1 µm in diameter (5,6). MPs rather, develop as a result of blebbing of plasma membrane (7), previous studies have reported that MPs provide a "non-genetic" basis for the acquirement, progression and supremacy of detrimental tumor behaviors, including enhanced metastatic potentiality of malignant cells and multidrug resistance (MDR) (8,9). CD138, a transmembrane heparin sulfate proteoglycan, is expressed on the surface of mature plasma cells to act as a classical co-receptor for growth factors, angiogenic factors, and chemokines; it is the gold standard marker for detecting MM cells using immunohistochemistry and multiparametric ow cytometry analysis of bone marrow biopsies (10).
Multiple myeloma is a tumor of elderly with longtime antigen exposure, and subsequent over-expression of PD 1 to indicate myeloma antigen-exposed T cells, viral antigen-speci c memory T cells, or immune senescent cells (11), up-regulation of PD1 on CD8+, CD4 + T cells, and NK cells is observed in MM (12), blocking of PD1 will restore T and NK cell functions. PD-L1, a major ligand of PD1, is upregulated on myeloma cells to become more proliferative, resistant to cell-mediated killing and traditional myeloma drugs (13). Engagement of PD-1 by its ligands, PD-L1 or PD-L2, results in the activation of phosphatases that deactivate signals emanating from the Tcell receptor (14), up regulates the expression of basic leucine ATF-like transcription factor (BATF), which in turn impairs T-cell proliferation and cytokine secretion (15).
Preclinical data have con rmed the important role of the PD-1 pathway in immune evasion by MM cells (16), anti-PD1 and anti-PD-L1 monoclonal antibodies exhibited objective responses and antitumor activity in relapsed and refractory MM in phase I studies (17,18).
We tried to shed light on the expression of PD1 and CD138 + microparticles in MM and their possible interplay as a mechanism of resistance to standardized treatments by proteasome inhibitors and immunomodulatory drugs, in addition, nd their associations with other prognostic factors of MM.

Patients And Methods
The study was a prospective non randomized controlled one carried out in Assiut university hospital and south Egypt cancer Institute of Assiut University.

-Ethical statement
All methods were carried out in accordance with declarations of Helsinki, the study was approved by ethical committee of faculty of medicine, Assiut university (IRB no: 17300484). Written informed consents were obtained from all participants after explaining our objectives and study procedures.

-Inclusion and exclusion
A total of 30 patients with newly diagnosed chemotherapy naïve active multiple myeloma were recruited over a period of one year and followed up over a period of 6 months to determine their response to treatments, bisphosphonates and palliative local radiotherapy were allowed, patients with previous history of chemotherapy, targeting therapy, or hormonal therapy for any other malignancies were excluded, also patients with excruciated infections, and non-candidate patients for myeloma therapy were excluded. In addition, 19 healthy participants of comparable age and sex were recruited.

-Methodology
After diagnosis of multiple myeloma by bone marrow aspirate ± biopsy for evaluation of plasma cell in ltration, immunophenotyping, ow cytometry, protein electrophoresis, immuno xation electrophoresis, serum free light chain measurements (done in limited number of cases), blood samples were collected from both patients and controls for ow cytometric detection of CD4+T cells, CD8+T cells, CD4+PD1+ T cells, CD8+PD1+ T cells, total Microparticles (TMP), CD138+ MPs, and platelet MPs (PMP).
Patients were evaluated for different prognostic factors and hematologic parameters including, CBC, β2 microglobulin, LDH, serum calcium, and blood chemistries (CRAB signs).
Evaluation of bone lysis was done based on whole body magnetic resonance imaging; any focal lesion to be considered of value must be ≥5mm in size.
Patients with performance status ≤2 received Bortezomib with dexamethasone or Bortezomib, lenalidomide, and dexamethasone regimens; monthly zoledronic acid was given ±palliative local radiotherapy to alleviate pain.
Other supportive measures including recombinant erythropoietin and darbepoitin alfa were used to treat myeloma-associated anemia to maintain Hb around 12g/dL and below 14 g/dL to avoid thromboembolic complications after measuring their microparticles level, also granulocyte colony-stimulating factor (G-CSF) was used to treat chemotherapy-induced neutropenia.
Patients at high risk of thrombosis or had elevated levels of MPs received high dose of dexamethasone, also patients under lenalidomide treatment received aspirin to avoid thromboembolic complications.
Complete response (CR) was de ned as negative immuno xation on the serum and urine, with disappearance of any soft tissue plasmacytomas and ≤5% plasma cells in BM, deviation from this de nition of CR was considered non CR.

Flow cytometric detection of Microparticles
Blood samples were collected into a 5 mL tubes containing 3.2% citrate for MPs isolation

Microparticles Isolation and Characterization
The MPs were isolated within 15 min. after collection. Cells were removed by centrifugation for 20 min at 1550 ×g at 20°C. Then 250 µL of plasma were centrifuged for 30 min at 18,800 ×g at 20°C. After centrifugation, the supernatant was removed and the pellet was resuspended in phosphate-buffered saline (PBS) and centrifuged for 30 min at 18,800 ×g at 20°C. The supernatant was removed again and MPs pellet was suspended in PBS. Total MPs were identi ed on the basis of their forward scatter compared with that of calibrate reference beads of 1.0 µm to calibrate the size range of microparticles (Latex beads, amine-modi ed polystyrene, uorescent red aqueous suspension, 1.0 μm mean particle size, Sigma-Aldrich ChemieGmbh Munich, Germany) and positivity for annexin V. Total MPs were reported as a percentage of the total events. MPs subpopulations were identi ed by their ability to bind cell-speci c monoclonal antibodies. Platelet MPs was detected as CD41+ MPs. Myeloma MPs were CD138+MPs. The percentage platelet and myeloma MPs were expressed as percentage of total MPs ( gure 1).
Flow cytometric detection of T lymphocyte subsets in peripheral blood.

Statistics
It was expected that the percentage of MM in Egypt in 2020 according to Amal et al study (19) was 0.45% to total number of cancers, so the sample size was calculated based on the equation= , where p=0.0045, q=0.995, absolute error e=0.03, and z for 99 % con dence interval was.2.56, so the calculated sample size was 32 patients, and we recruited 30 patients for our study.
Data were analyzed using IBM SPSS version 26, descriptive statistics in the form of mean, median, standard error, standard deviation, and percentages, Shapiro-Wilk test and Q-Q plots were applied to detect normality of variables, and Cook's distance test was plotted against serial number of cases to determine the in uential outliers as shown in gure (3), cases number 2, 19, & 26 could affect our results and may be considered in uential outliers and to con rm this effect, standardized DFFIT was applied and plotted against serial cases showing all cases were not deviated away from±3, so these values were not most probably in uential.
Independent sample t-test and Mann Whitney U test were applied for the associations between continuous and two-groups categorical variables, while one way Manova test was applied for detecting differences of multiple continuous variables on dependent categorical variable (ISS stage) after applying homogeneity test to insure equality of variances (Levene's test), and equality of covariances (Box's test), in addition post hoc analysis was applied using Tukey test for pairwise comparison, multicollinearity diagnostics was done with VIF <4, but the condition index >10 for all immune cells and Microparticles due to the presence of autocorrelations between these variables, so to determine which one of these variables greatly affect the variation of response Logistic regression with forward LR method was applied changing ISS stage into dummy variables. Roc curve was also applied for different variables to nd their cutoff points of achieving non CR, all data were considered signi cant at p-value<5%.

Results
Thirty patients with MM and 19 healthy controls were enrolled in this study, with signi cant accumulations of TMPs (p<0.0001), and CD138+MPs (p<0.0001) in patients compared to controls, while no signi cant difference in PMPs (p=0.07) among both groups, table (1).   international staging system, CR; complete response. Table 4 described different correlations between MPs, immune cells, and prognostic factors, with positive correlations were reported between PMP and PD1+CD4+T cells, CD138+MPs and CD8+T cells, PD1+CD8+T cells and PD1+CD4+T cells, PD1+CD8+T cells were positively correlated with LDH, M-protein, bone marrow plasma percentage, and β2 microglobulin, and β2 microglobulin was also correlated with PD1+CD4+T cells.    furthermore other variables were removed from the model because of autocorrelation and no added value in the prediction rate of model.  (20). Among all cancers, it represents about 1%, in spite, a minority of them achieved sustained complete response for a prolonged period or the so-called operational cure (21), even if they achieve this cure they will continue on suppressive therapy to ght against the risk of relapse with no clear plateau in overall survival (22).
Our results elucidated signi cant higher levels of TMP, CD138+MP, T cell subsets, PD1 expressed on T cells in MM patients compared with controls, also there were signi cant positive correlations between PMP and PD1+CD4+T cells, CD138+MP and CD8, and PD1+CD8+T cells and all of the followings LDH, M protein, BM plasma level, and β2 microglobulin, furthermore, there were signi cantly elevated levels of PMP, CD138MP, PD1, CD8+T cells in patients who didn't achieve CR with speci c cutoff values as mentioned in table 6.
Immune checkpoint blockade is proposed to be effective in many cancers where immune deregulation due to increased expression of negative co-stimulation of cells in tumor microenvironment plays important role in tumor progression and resistance to treatment, multiple myeloma is an example of these cancers with progressive immune dysregulation characterized by loss of myeloma reactive T-cell population, decreased antigen presenting and effector cell functions, and BM microenvironment that promotes immune escape (23,24 Physiologically, MPs take part in cell signaling and swapping of proteins and nucleic acids between cells, also are involved in the intercellular crosstalk, elevated levels of MPs are detected in many pathological conditions including in ammation, vascular diseases, diabetes, and cancers where they act as a surrogate marker for disease activity especially in poorly accessible tissues (27)(28)(29)(30), it is well documented that immunomodulatory drugs play important role in treatment of MM to be involved in many treatment regimens, however the risk of thromboembolism increased with their use to focus on the clinical signi cance of MP especially PMP (29).
Studies reported that TMP, PMP, and CD138+MP were elevated in patients with MM compared to healthy controls with signi cant prognostic potential of CD138+MPs in predicting the response to treatment and risk of relapse (31), our results agreed with the previous study.
Our results showed no signi cant differences in the total MP among response groups to demonstrate that they may predict the disease state of MM patients compared to healthy controls rather than response state.
Resistance to chemotherapy and immunotherapy is characteristic of MM (32); furthermore myeloma and plasma cells remain restricted to bone marrow secreting abnormal immunoglobulins till advanced stage, therefore continued monitoring of response based on BM examination, protein electrophoresis, free light chain assay immuno xation, and whole body MRI is time consuming and limited especially in non-secretory MM, hence the need for a marker of high sensitivity and speci city for detecting early relapse is important. For that, MPs emerged as a surrogate marker for response in many cancers including MM (33,34), our results failed to consider MPs as a marker for disease progression because of absence of signi cant differences of MPs across different ISS stages, in spite this study adds to this body of research and provides support for the use of MPs as a novel prognostic for response assessment in MM.
Crosstalk between tumor cells and immune cells in tumor microenvironment is critical for tumor progression; in addition, tumor cells are capable to hijack immune cells especially innate immunity, subsequently MPs released from tumor cells, not only contain messenger molecules, enzymes, RNAs, and even DNA, but also are capable of transferring these bioactive molecules from one cell to another, subsequently act as a vector for transferring messages between tumor cells and possibly immune cells to acquire aggressive phenotype (35,36). This crosstalk rather appeared in the current study where signi cant positive correlations between TMP and CD8+T cells, PMP and PD1+CD4+T cells, and CD138+MP and CD8+T cells while negative correlation between TMP and CD8+T cells.
To our knowledge, this study was the rst one to address the relations between Microparticles and immune cells and the added impact of these relations in resistance to treatment and disease progression, further studies with thorough methodology and adequate sample size may augment these relations.

Conclusion
We have demonstrated elevated levels of MPs and immune cells in MM patients compared to healthy controls, also the levels of these cells and particles were lower in patients achieved CR compared to those without CR, Microparticles and PD1 expressions were associated with proliferative potential and resistance to Bortezomibbased treatments, our results suggested that they played a crucial role in myeloma progression, in addition this study provides support for the potential prognostic value of MPs and the possible interplay between MPs and PD1 in multiple myeloma.

Declarations
Con ict of interest: the authors declared that they had no con ict of interest Data availability statement: all data generated or analyzed during this study are included in this submitted article.    signi cant difference between response groups of MM patients regarding CD138MP, PD1+CD8+T, and CD8+T cells