Efficacy and safety of plasmapheresis without plasma transfusion tandem with chemotherapy to treat multiple myeloma

ABSTRACT Objectives This study evaluates the efficacy and safety of plasmapheresis without plasma transfusion tandem with chemotherapy in treating multiple myeloma (MM). Method This retrospective study involves seventy-two patients, newly diagnosed with multiple myeloma, divided into two groups; one with plasmapheresis without plasma transfusion tandem with the chemotherapy group (Trial group), while the second was chemotherapy group (Control group). The levels of Plasma Globulin, β2-microglobulin, Creatinine, Vascular endothelial growth factor (VEGF), and IL-6 were monitored after plasmapheresis, at initial diagnosis, and after four chemotherapy courses. Overall response rate of groups after four courses of chemotherapy was analyzed, and the adverse events were recorded. Results and Discussion The baseline data showed that sixty-seven percent of patients were at the ISS III stage and showed more severe renal insufficiency in the Trial group. The Plasma Globulin, β2-microglobulin, VEGF and IL-6 levels were significantly different between the two groups during the initial diagnosis. After three times plasmapheresis, Plasma Globulin, β2-microglobulin, VEGF, and IL-6 were significantly reduced in the plasmapheresis group. The Creatinine levels were also lowered, but the differences were not statistically significant. After four courses of chemotherapy, the levels of VEGF and IL-6 in the two groups were significantly reduced than the initial diagnosis; the differences were statistically considerable. No adverse events were found in the trial group as compared to the control group. Conclusion Plasmapheresis reduces Globulin, β2-microglobulin, serum VEGF and IL-6 levels in MM, improves renal functions, and releases some patients from dialysis dependence.


Introduction
Multiple myeloma (MM) [1,2] is the second-largest malignant tumor in the hematological system. Damage of an organ or a tissue due to excessive proliferation of plasma cells coupled with abnormal secretions of monoclonal immunoglobulin (M-Ig) or its para protein fragment (M protein/M spike) is the major cause of its pathogenesis. The majority of MM patients were diagnosed with varying degrees of renal impairment, whereas conditions like hyperviscosity, hyperuricemia, and hypercalcemia tend to aggravate renal injury in some MM patients that were further maintained by hemodialysis. Abnormal components of protein and inflammatory factors can be removed rapidly through plasma exchange that enhances the viscosity of blood and helps reduce organ damage caused by the presence of abnormal protein components and inflammatory factors. Traditional plasmapheresis [3][4][5] requires a large quantity of fresh frozen plasma. The incidence of an anaphylactic reaction that can be significantly enhanced when a large amount of plasma is utilized substantially limits the application of plasmapheresis in multiple myeloma (MM). Therefore, according to the plasma exchange principle, we pursued a clinical study of sequential chemotherapy [6,7] without plasma exchange to treat patients with newly diagnosed multiple myeloma and achieved good results.

Materials and methods
Newly diagnosed multiple myeloma (MM) was analyzed in 72 patients from April 2019 to April 2021 in the Taihe Hospital of Shiyan City, China. Seventeen patients with severe heart, lungs, liver failure and other reasons were excluded from 89 patients suffering from MM. Severe heart condition include ACS, middle or severe valvular disease; CHF, BNP UP 1000 ng/L, EF under 50. Severe lung disease was defined as PO 2 under 60 mmol/L AND/OR PCO 2 , 45 mmol/L. Pulmonary edema, pneumonia and/or fever. Liver failure include patients with Cirrhosis /heavy hepatitis. All patients were informed about procedures regarding disease diagnosis, treatment standards for bone marrow cytology, bone marrow biopsy, bone marrow flow analysis, hematuria immunofixation electrophoresis, necessary imaging (MRI/ CT/ X-ray), electrocardiogram, cardiac color Doppler examination evaluation and consent was obtained. The control group patients have a lower risk factors; the experimental group have higher globular proteins, more severe impairment of renal functions, but after four courses of above treatment, an effective evaluation not inferior to the control group was observed. We want to propose plasmapheresis without plasma can benefit MM patients with higher globular proteins and impaired renal function.
A small number of patients were screened for myeloma fish detection and informed. We selected patients with ISS disease stage, and not R-ISS disease stage, because all patients were not tested for myeloma fish probe. The experimental group contained 40 patients who received sequential chemotherapy without plasma exchange, whereas the control group had 32 patients who received chemotherapy without plasma exchange. The clinical baseline data, biochemical blood indexes, serum interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) levels were noted after three sessions of plasma exchange without plasma and four courses of chemotherapy.

Plasma exchange without plasma transfusion
Plasma exchange was performed using a Fresenius Com. Tec ® blood cell separator (Germany) where 20% human serum albumin (100 mL), blood preservation solution ACD (Anticoagulant Citrate Dextrose, 500 mL), hydroxyethyl starch (500 mL), Ringer's solution (500 mL) and normal saline as exchange fluid (instead of traditional plasma exchange that uses fresh frozen plasma). Plasma exchange volume was 30-40 mL/kg, and each exchange interval was around 24-48 h. Three consecutive plasmaphereses without plasma were performed. Blood pressure, hematocrit, plasma albumin and physical status were evaluated before plasma exchange. Patients with severe anemia were infused with red blood cells to improve anemia. Coagulation function, hemoglobin, plasma albumin, globulin and vital signs (blood pressure, oxygen saturation, respiratory rate, and pulse) were monitored after plasma exchange. Fibrinogen (FIB) was supplemented when FIB was lower than 1.0 g/L. The patients with hypotension and dizziness were supplemented with crystals and colloids (albumin or fresh frozen plasma).

Laboratory test indicators
Initial diagnosis of enrolled patients included baseline assessment including liver and kidney function tests, i.e. lactate dehydrogenase (LDH), beta-2-microglobulin (β-2-µG), albumin, erythrocyte sedimentation rate (ESR), NT-proBNP, blood coagulation factors, and infectious diseases markers (HIV, HBV, and HCV). Organ function assessment was completed for patients with complications. After three times of plasmapheresis without plasma and four courses of chemotherapy, all patients were treated with plasma globulin, albumin, and albumin. ELISA detected the serum levels of IL-6 and VEGF.

Chemotherapy
Fifty-three patients were treated with VTD (Velcade, Thalidomide, Dexamethasone), 10 patients were treated with BCD (Bortezomib, Cyclophosphamide, Dexamethasone), seven patients were treated with VRD (Bortezomib, Lenalidomide, Dexamethasone), two patients were treated with traditional VAD (Vinblastine, Adriamycin, Dexamethasone). MM effective evaluation often made every two courses, so median time to first response after treated two courses was 6 weeks, a detailed evaluation was made at four courses or at 12 weeks' time.

Statistical methods
U-test was used to compare the baseline assessment data of two groups. The sample mean values measured the levels of inflammatory factors and biochemical blood indexes of two groups, and a t-test was performed. The total effective rate was evaluated by the chi-square test. The incidence of adverse events was analyzed by nonparametric rank test and SPSS ver. 16.0.

Results
There was no significant difference of age and gender between the experimental and control group in baseline clinical characteristics, but there was a substantial disparity in elevated baseline globulin, renal function damage, and the number of patients with elevated LDH at initial diagnosis. In the ISS stage III patients of the experimental group, the proportion of complications was higher.
The levels of inflammatory factors, globulin, microglobulin and serum creatinine in the experimental group were significantly higher than the control group ( Table 1). The difference was statistically significant, suggesting that the baseline data of the experimental group was more severe than that of the control group.
Compared with the initial diagnosis, the levels of inflammatory factors, globulin, microglobulin, and serum creatinine in the experimental group after three sessions of plasma exchange and four courses of treatment levels were found to be decreased substantially. The differences were statistically prominent ( Table 2), suggesting that plasma exchange and chemotherapy are the effective measures for the treatment of multiple myeloma.
The levels of inflammatory factors, globulin, microglobulin and serum creatinine in the control group after four courses of chemotherapy were significantly lower than those at the initial diagnosis, and the difference was statistically significant (Table 3). After four courses of chemotherapy, the levels of inflammatory factors, globulin, microglobulin and serum creatinine in the two groups decreased more significantly in the experimental group, but there was no significant difference between the two groups (Table 4).
After four courses of chemotherapy, the curative effect of the two groups was evaluated. The total effective rate and response rate (ORR) for both groups were calculated. The differences between the two groups were found to be considerable (Table 5). During the first hospitalization, the cases of adverse events during plasma exchange and chemotherapy without plasma were observed and analyzed by a nonparametric rank test (P > 0.05) ( Table 6). We have mentioned side effects with lower and then 3 grade ratios in Table 7. We have studied Peripheral edema, while gastrointestinal reactions including nausea stomach discomfort. We observed the patients, for allergy to human serum albumin and blood preservation solution ACD, when treated with plasmapheresis. Coagulation abnormalities and cirrate reaction were the highly occurred allergic reactions, where citrate reactions were found only in experimental group. Side effects of ≤ 2 grade were 32% in experimental while 16% in control group, while ≥3 grade side effects were not observed for both groups.

Discussion
Multiple Myeloma (MM) is a kind of plasma cell tumor mainly caused by abnormal clonal plasma cell proliferation, secretion of a monoclonal immunoglobulin or its fragment (M protein), and inflammatory factors leading to organ or tissue damage. Its clinical manifestations include renal insufficiency, bone destruction, anemia, hyper-viscosity syndrome, multiple organ amyloidosis, and other life-threatening complications. Most common injuries include acute and chronic kidney damage. Research shows that kidney injury in Newly Diagnosed Multiple Myeloma (NDMM) patients [8,9] is mainly related to the direct deposition of abnormal M protein and injury mediated by an indirect immune mechanism. Plasma exchange can rapidly reduce abnormal proteins and inflammatory factors in a patient's plasma. After more than three times of plasma exchange, patients' level of free light chain can be reduced by more than 90%.
The level of IL-6 in plasma of MM patients was significantly higher than that of healthy people, which was positively correlated with the stage of disease [10,11]. Plasma IL-6 in MM patients can promote the proliferation of myeloma cells, activate osteoclasts, and promote the occurrence of bone destruction. Plasma VEGF can lead to tumor tissue vascular  proliferation and promote tumor cell proliferation. In MM patients, the plasma VEGF level will increase, which is related to the prognosis of the disease. Plasma exchange is mainly used to separate patients' plasma and blood cells through a blood cell separator and discard the pathogenic factors in plasma such as autoantibodies, immune complexes, poisons, etc. To eliminate or reduce the pathogenic factors in patients, the blood cells and the same amount of fresh frozen plasma and a certain proportion of exchange fluid are infused back to patients. In the treatment of plasma cell diseases, plasma exchange can remove abnormal M protein, inflammatory factors, and other pathogenic factors from patients' plasma and achieve an excellent therapeutic effect [10,11]. Traditional plasmapheresis requires a large amount of fresh plasma as the exchange fluid. However, as a blood product for rescue, the limited resources greatly limit the application of plasmapheresis in MM. Korttila [12] used albumin, low molecular dextran, hydroxyethyl starch, normal saline, and other colloids as exchange fluid to replace fresh frozen plasma for plasma exchange, which achieved good results and good safety in the treatment of hyperviscosity syndrome. Marder [13] used plasma exchange without plasma to treat severe ITP, and the technology development was not limited by plasma, which saved plasma resources and achieved an excellent therapeutic effect. However, the application of plasma exchange without plasma in Multiple Myeloma has not been reported.
Our study shows sequential chemotherapy of plasma exchange without plasma is effective, safe, and feasible in the treatment of MM. By analyzing clinical characteristics, in the experimental group patients with later stages, globulin levels increased significantly, whereas more than 90% of patients had apparent renal dysfunction. Through the plasma free plasma exchange sequential chemotherapy treatment, the total response rate of patients reached 75%. In comparison, the control group's 63% response suggests that plasma exchange treatment without plasma may improve the efficacy of high-risk MM patients. By comparing the levels of plasma globulin, microglobulin, VEGF, and IL-6 levels in the experimental group before and after the exchange, it is found that the plasma exchange without plasma can significantly reduce the levels of globulin, microglobulin, VEGF, and IL-6 β Microglobulin, VEGF and IL-6 in plasma were measured. The two groups of patients through chemotherapy can significantly reduce the level of globulin, serum albumin. The microglobulin levels, VEGF, and IL-6 in the experimental group were significantly lower than those in the control group. The results show that sequential chemotherapy without plasma exchange can substantially benefit patients with multiple high-risk myeloma, especially in patients with renal damage. The mechanism is related to the fact that plasma exchange without plasma transfusion can rapidly reduce M protein and inflammatory factors, VEGF, and IL-6 in plasma. It can reduce the Table 4. Comparison of inflammatory factors and biochemical indexes in the control group before and after treatment. Groups IL-6 (pg/mL) VEGF (pg/mL) GLO (g/L) β2MG (mg/L) Scr (mmol/L)  Table 5. Comparison of levels of inflammatory factors and biochemical indexes between the two groups after four courses of chemotherapy.
Groups IL-6 (pg/mL) VEGF (pg/mL) GLO (g/L) β2MG (mg/L) Scr (µmol/L)  deposition of M protein in the kidney and immune injury. By clearing inflammatory factors, it may inhibit the clonal proliferation and abnormal secretion of myeloma cells.
Traditional plasma exchange has many adverse reactions in rescuing critically ill patients, limiting the wide application of plasma exchange. Kaszuba-Zwoińska [14] showed that the incidence and intensity of the anaphylactic reaction of allogeneic plasma would enlarge with the increase of the number and amount of plasma received and even lead to severe anaphylactic shock. In our study, for 40 patients with MM, 120 plasma exchange treatments were given without plasma transfusion and no allergic reactions. The reason is that crystal and albumin were used as the exchange fluid in plasmapheresis without plasma, which can save plasma resources and significantly reduce allergic reactions. As the patients with establish and diagnosed MM evaluated for heart and lung and liver functions, we started implementation of plasmapheresis, each exchange interval was around 24-48 h. After finishing three times plasmapheresis, the patients were started chemotherapy. Median time to start plasmapheresis from diagnosis need s2.5 ± 1.3 days. Median time to start chemotherapy need 5.6 ± 2.7 days.
The adverse reactions observed during plasma exchange without plasma include hypoproteinemia and hypoproteinemia. Lemaire [15] reported that the incidence of adverse reactions in 260 cases of plasma exchange in ICU was 26.9%, in which low hematocrit related hypotension and allergic reactions associated with the type of exchange fluid, coagulation abnormality associated with the choice of replacement fluid, and decreased fibrinogen levels were more common adverse effects. In our study, adverse events were observed in the experimental group during the first hospitalization, but these effects were not significantly higher than those in the control group (sodium citrate reaction, mild oedema, and hypofibrinogenemia). Furthermore, there was no significant increase in the cardiovascular system, digestive system, coagulation function, nosocomial infection, and thrombotic events compared with the control group showing the process safe and reliable. Sequential chemotherapy without plasma exchange is found effective, safe, and feasible in the early treatment of multiple myeloma. Clearing the abnormal M protein and inflammatory factors in patients may rapidly reduce blood viscosity and blood uric acid and play a protective role in the kidney, heart, and other target organs. In addition, clearing the inflammatory factors IL-6 and VEGF may inhibit the proliferation of myeloma cells and promote the prognosis of the disease. With the innovation of new drugs and treatment methods, improving quality of life, and disease-free survival of multiple myeloma, all of these factors have put forward higher requirements for maintaining heart, kidney, and other vital organs. Due to the small sample size and short observation time, the PFS and OS of the two groups are still in continuous follow-up. A study with a large cohort size is planned to continue evaluating the long-term efficacy of this treatment. Autologous stem cell transplantation (ASCT) is suggested to the patients with very good partial response (VGPR), while this study involved Newly Diagnosed Multiple Myeloma (NDMM). If these patients achieve VGPR, then ASCT can be suggested.

Conclusion
Plasmapheresis without plasma transfusion tandem with chemotherapy can rapidly reduce the Globulin, β2microglobulin, serum VEGF and IL-6 levels in the multiple myeloma, improve renal functions, release some patients from dialysis dependence, and possibly enhance the total response rate of high-risk MM therapy. We can conclude that plasmapheresis without plasma is safe and effective when we select to treat new diagnosis multiple myeloma patients who has high globular proteins and high inflammatory factors.