From June 1st, 2008, to May 10th, 2024, patients aged 40-74 years who were diagnosed with multiple myeloma were screened. The diagnosis criteria refer to the International Myeloma Working Group Criteria for Diagnosis of Multiple Myeloma (19), and the prognosis stratification refers to mSMART (20). Patients undergoing MST must meet the following criteria: 1. Voluntary participation and signing of informed consent form; 2. Peripheral blood hematopoietic stem cells provided by healthy donors are available; 3. Diagnosed with multiple myeloma and in initial diagnosis or post-induction phase (PR, VGPR, CR or relapse). Exclusion criteria include: 1. Severe organ dysfunction including NYHA grade III-IV heart failure, liver failure, renal failure, etc.; 2. Severe embolism or thrombotic events; 3. Pregnancy and lactation; 4. Severe infectious diseases such as aspergillosis.

Donor selection criteria include: HLA (human leucocyte antigen) typing ≤7/10 or ≤4/6 matched relative or unrelated donors, physical examination meets the conditions for donating hematopoietic stem cells, informed consent obtained and able to tolerate peripheral blood mononuclear cell collection procedure. Donors with HLA typing containing 3 or more homozygous loci are excluded.

This study included a total of 20 patients. Table 1 summarizes the characteristics and risk stratification of the enrolled patients. Of the 20 patients, 9 had high-risk genetic features. 14 were in VGPR or CR status, 2 in PR, and 3 in relapse status before MST. One patient was newly diagnosed who had not received any chemotherapy. 17 patients received GPBMC infusion from related donors, and 3 patients from unrelated donors. The median number of MST cycles received by each patient was 3, with a maximum of 13. The median single cell infusion doses of MNC, CD34, and CD3 were 2.75×10⁸/Kg, 2.03×10⁶/Kg, and 1.02×10⁸/Kg, respectively.

A total of 76 cycles of MST were performed on 20 patients. The hematopoietic recovery and adverse events (AEs) are shown in Table 2 . Grade IV lymphocytopenia was relatively common (59.2%), while Grade III-IV anemia was not pronounced (8%). The overall incidence of Grade IV neutropenia and Grade IV thrombocytopenia was 27.6% and 38.1%, respectively. Six patients did not experience Grade IV neutropenia or thrombocytopenia during treatment of MST. In the remaining 14 patients, the median duration of Grade IV neutropenia and thrombocytopenia was 3.5 days and 9 days, respectively. Total time courses of hematopoietic recovery, including neutrophils, lymphocytes, monocytes, eosinophils, basophils, hemoglobin, and platelet, are presented in Figure 1 . The degree of bone marrow suppression was relatively mild (24). No immature neutrophils or other peripheral blood cells were observed in 76 cycles of MST.

Fever and donor cell infusion-related cytokine release syndrome (CRS) were the most common AEs. Grade I and Grade II fevers were observed in 23 (30.2%) and 8 (10.5%) cases, respectively. All fevers could not be ruled out as being related to CRS. Among them, 20 fever events occurred during the period of Grade IV neutropenia, and 4 of these had definitive positive blood culture results, indicating that fever was also associated with severe infection ( Figure 2A ). Here, two typical cases were shown to distinguish the causes of fever. Patient 1 and Patient 2 both experienced febrile neutropenia three to five days after donor cell infusion, and the increase of C-reactive protein (CRP), lactate dehydrogenase (LDH), alanine aminotransferase (ALT), and cytokines such as interleukin-2r (IL-2r), IL-6, IL-8, were observed ( Figures 2B, C ). However, these indicators could elevate both in CRS and infection. Patient 2 had a positive blood culture result that indicated an infection with Staphylococcus hominis, accompanied by significantly enhanced CRP. Despite the use of carbapenem antibiotics and glucocorticoid, fever was hardly controlled until the neutrophil recovered. Compared to Patient 2, fever in Patient 1 was effectively controlled after the administration of third-generation cephalosporins and glucocorticoid, and there was insufficient evidence of infection based on clinical manifestations, pathogenic microorganism testing, as well as imaging examinations. Therefore, fever of Patient 1 was more likely associated with CRS, while Patient 2’s fever had a close relationship with both CRS and infection.

Despite the occurrence of CRS after donor cell infusion, accompanied by mild clinical manifestations such as skin rash (2.6%), liver function injury (2.6%), and gastrointestinal disorder (3.9%), the absence of a high proportion of donor chimerism after MST suggested that these immune responses induced by donor cell infusion were probably not sufficient to cause GVHD.

In the long-term follow-up observations, hypogammaglobulinemia had the highest incidence (45%) among the total infection events and immune-related adverse events, followed by lower respiratory infection (35%), intestinal infection (30%), upper respiratory infection (30%), mucosal infection (25%), and shingles (20%) ( Table 3 ).

Among Fourteen patients in VGPR/CR status, twelve maintained VGPR/CR after MST. One of two patients in PR status achieved VGPR/CR after MST. All three patients in PD status achieved PR/VGPR/CR. One previously untreated patient achieved CR after one cycle of MST, resulting in an ORR of 17/20 (85%) for all 20 patients.

The overview of 20 patients including the time of first cycle of MST, efficacy, and survival were shown in Figure 3 . The median OS was 91 months (15-161), with a 1-year and 3-year OS of 100% and 94.1%, respectively ( Table 4 ), and a 6-year OS of 64.7%. Subgroup analysis based on age, DS stage, ISS stage, SMART stage, pre-MST disease status, and number of sequential MST showed that age, ISS stage, and SMART stage had no significant correlation with 6-year OS (p=0.224, 0.745, 0.151) ( Figures 4A, C, D ), while DS stage showed a significant correlation with prognosis (P=0.045) ( Figure 4B ). Patients in VGPR and CR before MST had a better prognosis than those in PR and PD states (p=0.042) ( Figure 4E ). More cycles of MST (n>2) were associated with longer OS compared to two or fewer (n ≤ 2) (P=0.039) ( Figure 4F ).

The median PFS for patients receiving MST was 39 months (2-150), with an overall 6-year PFS of 35.3%. Age, DS staging, ISS staging, SMART staging, and the number of MST cycles were not significantly correlated with PFS (p=0.377, 0.121, 0.372, 0.147, 0.324), whereas disease status before MST was significantly correlated with PFS (p=0.017) ( Figure 5 ).

Among the sixteen IgG myeloma patients, the median PFS was 46 months (2-150) and the median OS was 89 months (15-161). For the two IgD patients, PFS was 20 months and 110 months, and OS was 43 months and 118 months, respectively. One IgA patient in disease relapse status achieved CR with an OS of 161 months after MST. Another IgA patient had a total disease course of 26 months, a PFS of 24 months, and is currently still in CR.

No NRM occurred.

We performed univariate and multivariate regression analyses to identify potential prognostic factors on OS and PFS, including patient age, gender, DS stage, ISS stage, SMART stage, disease status before MST, number of MST cycles, and infused cell dose in MST (MNC, CD34, CD3). Multivariate regression analysis revealed that more MST cycles (≥2) were significantly associated with longer OS (p=0.043) ( Table 5 ).

Among these 20 patients, 3 patients had totally 19 available peripheral blood samples for donor microchimerism tests, and all 3 patients had detectable donor microchimerism (<1.2%) after MST. The chimerism levels fluctuated between 0.012-0.091% on day 1, 0.002-0.735% on day 7, 0.012-0.091% on day 14, 0.02-0.65% at 3 months, and 0.006-1.034% at 6 months, respectively after cell infusion ( Figure 6A ). The disease course of these 3 patients was 21, 26, and 31 months, respectively, and they are currently in sustained remission at last follow-up.

We tested lymphocyte subsets and tumor cell-specific T cell killing function on 5 patients before and after MST. The results showed that the proportions of CD4+ T lymphocytes, total CD3+ T lymphocytes, and cytotoxic CD8+ T cells in peripheral blood after MST were 41.3 ± 10.0%, 77.0 ± 5.8%, and 23.2 ± 2.1%, respectively, which were significantly higher than those before MST of 25.4 ± 7.2%, 66.0 ± 10.9%, and 16.8 ± 6.2% (p=0.005, 0.025, 0.015) ( Figure 6B ). The proportions of NKT and NK cells showed tendency to increase after MST, but with no statistical difference (p=0.993, 0.241). The proportions of γ/δ T and B cells decreased after MST (p=0.012, 0.005). The CD4/CD8 ratio after MST was 1.30 ± 0.54, which was significantly higher than that before MST of 0.78 ± 0.32 (p=0.048) ( Figure 6C ). Th0 cells was 0.19 ± 0.15% after MST, which was higher than that before MST of 0.11 ± 0.04% (p=0.017) ( Figure 6D ).