Long-term follow-up results of anti-BCMA CAR-T cell therapy combined with autologous hematopoietic stem cell transplantation in relapsed/refractory multiple myeloma with extramedullary disease

Long-term follow-up results of anti-BCMA CAR-T cell therapy combined with autologous hematopoietic stem cell transplantation in relapsed/refractory multiple myeloma with extramedullary disease | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Long-term follow-up results of anti-BCMA CAR-T cell therapy combined with autologous hematopoietic stem cell transplantation in relapsed/refractory multiple myeloma with extramedullary disease Xin Li, Can Liu, Siyan Niu, Ru Li, Shuquan Gao, Rui Cui, Jia Wang, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8582254/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Background Relapsed/refractory multiple myeloma (R/R MM) with extramedullary disease (EMD) carries a poor prognosis. Responses to current therapies, including autologous stem cell transplantation (auto-HSCT) and chimeric antigen receptor T-cell (CAR-T) therapy, remains unsatisfactory, or with frequent early progression despite initial response. Methods Eighteen patients with R/R MM with EMD were enrolled in clinical trials evaluating anti-BCMA CAR-T therapy. Of these, eight patients were treated with ASCT in combination (T-C group), while the remaining ten underwent CAR-T therapy alone (C group). We systematically compared clinical responses, CAR-T cell expansion kinetics, T-cell subset profiles, serum interleukin-6 (IL-6) levels, treatment-related toxicities, and long-term outcomes between the two cohorts. Results In the T-C group, all 8 patients achieved an overall response (ORR) based on combined hematologic and imaging assessments of EMD. In contrast, among the 10 patients in the C group, 8 met hematologic criteria for ORR, but only 6 demonstrated radiographic response in EMD lesions. Progression-free survival (PFS) and overall survival (OS) were markedly improved in the T-C group. This cohort also exhibited higher peak levels of both CAR-T cells and interleukin-6 (IL-6). On day 28 post-infusion, the proportion of CD3⁺CD4⁺ T cells were significantly greater in the T-C group. While cytokine release syndrome (CRS) tended to be more severe in this group, the incidence and severity of immune effector cell-associated neurotoxicity syndrome (ICANS) were comparable between groups. Hematologic recovery was delayed in the T-C group, and three of eight patients developed poor graft function. Conclusion When followed up for more than 3 years, the combination of anti-BCMA CAR-T cell therapy and auto-HSCT was associated with improved PFS and OS in R/R MM patient with EMD. Moreover, CD4⁺ T cell dynamics might be associated with the durable clinical response observed in the T-C group. Although the T-C group experienced higher-grade CRS and more prolonged hematologic toxicity, no treatment-related deaths due to treatment-related toxicities were observed. (Trial registration: ChiCTR2000033925 ) Relapsed Refractory Multiple myeloma Extramedullary disease Anti-BCMA CAR-T Autologous hematopoietic stem cell transplantation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Multiple myeloma (MM), a malignant neoplasm of plasma cells, accounts for approximately 10% of all hematologic malignancies and has an annual incidence of 6.6 cases per 100,000 individuals [ 1 ]. In recent years, the introduction of novel therapeutic agents, including proteasome inhibitors (PIs), immunomodulatory drugs (IMiDs), monoclonal antibodies, autologous stem cell transplantation (auto-HSCT), and chimeric antigen receptor T-cell (CAR-T) therapy, has substantially improved survival outcomes in patients with multiple myeloma (MM) [ 2 – 4 ]. Despite these advances, multiple myeloma is still considered incurable, with recurrent relapses representing the major therapeutic hurdle [ 5 ]. Extramedullary disease (EMD) is a frequent complication in relapsed/refractory (R/R) MM and is consistently associated with inferior survival outcomes [ 6 ]. At initial diagnosis, EMD is present in approximately 2% to 5% of MM patients, whereas its prevalence rises to 20% to 40% in the R/R MM patients [ 7 – 9 ]. MM patients with EMD have a significantly poorer prognosis compared to those without EMD, with shorter median progression-free survival (PFS) and overall survival (OS) [ 10 , 11 ]. Especially for EMD not related to bones, the prognosis is even worse [ 12 ]. MM patients with EMD still have a poor response to conventional therapies, including PIs, chemotherapy and ASCT, resulting in poor PFS and OS [ 13 ]. In CAR-T cell therapy for R/R MM, EMD is also one of the adverse factors affecting the prognosis of this therapy, especially for MM patients with non-bone-related extramedullary diseases, the prognosis is the worst [ 14 – 16 ]. Effective strategies to improve prognosis and prolong survival in MM patients with EMD remain an unmet clinical need. To address this challenge, we developed a combined therapeutic approach integrating anti-BCMA CAR-T cell therapy with auto-HSCT. To date, long-term outcomes of combining CAR-T therapy with ASCT in MM patients with EMD remain unreported. Our study represents the first clinical observation with three-year follow-up, offering insights into the sustained efficacy and safety of this strategy.Preliminary results indicate that this strategy enhances both PFS and OS in patients with MM with EMD. Patients and Methods Patients enrolled in the study Between June 2020 and December 2022, 18 R/R MM patients with at least one site of EMD were enrolled in clinical trials of anti-BCMA CAR-T therapy ( ChiCTR2000033925 ). Eight patients received anti-BCMA CAR-T therapy in combination with auto-HSCT and constituted the T-C group. A concurrent control group (C group) comprised ten additional R/R MM patients with EMD who received anti-BCMA CAR-T therapy alone, all were unable or did not receive sufficient stem cells for auto-HSCT. The enrolment period for this study concluded in December 2022, with a data cutoff date of December 2025. Anti-BCMA CAR-T cell therapy Peripheral blood mononuclear cells (PBMCs) were collected from patients with R/R MM in both the T-C and C groups and used for the manufacture of anti-BCMA CAR-T cells. The humanized anti-BCMA CAR construct, designated lenti-BCMA-2rd-CAR, was provided by Shanghai Genbase Biotechnology Co., Ltd. (Shanghai, China). Transduction efficiency was assessed by flow cytometry (FCM; BD Biosciences, San Jose, CA, USA) on the day of harvest following ex vivo expansion. All patients in C group received lymphodepleting chemotherapy with fludarabine (30 mg/m 2 ) and cyclophosphamide (400 mg/m 2 ) from day − 4 to day − 2. Autologous humanized anti-BCMA CAR-T cells (2×10 6 cells/kg) were infused on day 0 in C group or 2–4 days after the day of stem cell infusion in T-C group (Fig. 1 ). Auto-HSCT Before stem cell mobilization, all R/R MM patients with EMD in the T-C group achieved only minimal response (MR) or stable disease (SD) based on hematologic criteria. For mobilization, either cyclophosphamide (CTX, 3–5 g/m²) followed by granulocyte colony-stimulating factor (G-CSF, 300 µg/day, initiated 5–7 days after CTX and continued until stem cell collection) or G-CSF alone (300 µg/day on days 1–5) was administered. Peripheral blood stem cells were subsequently collected via leukapheresis. In the T-C group, the conditioning regimen was high-dose melphalan (200 mg/m²) [ 17 ]. The number of stem cells infused in T-C group was required as the following numbers: CD34 + cells > 2.0×10 6 /kg. Maintenance treatment after anti-BCMA CAR-T cell therapy All patients initiated lenalidomide or pomalidomide maintenance therapy 2–3 months following anti-BCMA CAR-T infusion, once hematologic toxicity had resolved. One patient in the C group subsequently underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT) three months after CAR-T cell therapy. Criteria for diagnosis and evaluation criteria for therapeutic efficacy The diagnosis of R/R MM, diagnosis of EMD, clinical response to the humanised anti-BCMA CAR-T cell therapy was assessed according to the International Myeloma Working Group Guidelines uniform response criteria for MM [ 18 ]. The proportion of MM cells was determined by bone marrow (BM) morphology and FCM. The M protein levels were detected by immunofixation electrophoresis. Assessable EMD was detected using CT, PET/CT or MRI. Therapeutic efficacy was assessed monthly during the first three months following anti-BCMA CAR-T cell infusion in all patients, including those in both the T-C and C groups. Thereafter, evaluations were conducted every 2 to 3 months. The clinical responses included stringent complete response (sCR), complete response (CR), very good partial response (VGPR), partial response (PR), MR, SD, and progressive disease (PD). We assessed the objective response rate (ORR) (including sCR, CR, VGPR, and PR), overall survival (OS), and progression-free survival (PFS). Adverse events (AEs) in the combination therapy The proportion of anti-BCMA CAR-T cells in peripheral blood was assessed by FCM on days 0, 4, 7, 14, and 28 following CAR-T cell infusion. Serum interleukin-6 (IL-6) concentrations were measured at the same timepoints (days 0, 7, 14, and 28) using enzyme-linked immunosorbent assay (ELISA). AEs associated with humanized anti-BCMA CAR-T therapy were systematically monitored. Cytokine release syndrome (CRS) and immune-effector cell-associated neurotoxicity syndrome (ICANS) were graded according to the consensus criteria established by the American Society for Transplantation and Cellular Therapy (ASTCT) [ 19 ], while hematologic toxicities were classified using the Common Terminology Criteria for Adverse Events (CTCAE), version 5.0 [ 20 ]. Statistical analysis Data are expressed as the mean ± SE. The probabilities of PFS and OS were estimated using the Kaplan-Meier method and compared with the log-rank test. All statistical analyses were performed using GraphPad Prism 7 and SPSS 17.0. Statistical significance was set at P < 0.05. Results R/R MM patient characteristics The baseline characteristics of the 18 patients with R/R MM enrolled in this clinical trial are summarized in Table 1 . All patients with R/R MM had at least one bone-unrelated EMD prior to enrolling in our clinical trial. No significant differences were observed between the T-C and C groups with respect to age, sex, high-risk cytogenetics, International Staging System (ISS) stage III, or median number of prior lines of therapy. All 18 patients were followed for more than three years. Table 1 Baseline characteristics of the R/R MM with EMD T-C group (n = 8) n% C group (n = 10) n% P values Sex: Male (%) 5 (62.5%) 4 (40.0%) P = 0.342 Age 57.4(48–71) 60.1(38–75) P = 0.210 KPS, > 80 8(100%) 10(100%) P = 1.000 Subtype, %k light chain 2(25.0%) 3(30.0%) P = 1.000 ISS stage, (I-II): III 2:6 3:7 P = 1.000 MM cells in BM, % 16.7 (0.9–55.6) 18.8 (0.4–86.8) P = 0.761 Serum M protein (g/L) 19.2 (4.6–49.7) 16.6 (2.4–56.6) P = 0.877 EMD number High-Risk Cytogenetics Numbers of prior therapy BCMA CAR-T therapy before Auto-HSCT before 1.75 (1–3) 2.25 (1–4) 4.63 (3–7) 1 (12.5%) 1 (12.5%) 2.13 (1–3) 2.38 (1–3) 5.10 (3–9) 2 (20.0%) 1 (10.0%) P = 0.300 P = 0.656 P = 0.651 P = 1.000 P = 1.000 Transduction and amplification efficiency, infusion dose of the humanized anti-BCMA CAR-T cells The mean anti-BCMA CAR transduction efficiency in the final products of T-C group and C group was 40.12 ± 6.92% and 37.41 ± 7.11%. On 2–4 days after stem cell infusion a dose of 2.09 ± 0.66×10 6 cells/kg anti-BCMA CAR-T cell was infused in T-C group, and on day 0 a dose of 2.12 ± 0.49×10 6 cells/kg anti-BCMA CAR-T cell was infused in C group. Stem cell infusion in T-C group The CD34 + cells infused in T-C group was 2.46 ± 0.72×10 6 cells/kg on day 0 during their auto-HSCT process. Clinical response to therapy After anti-BCMA CAR-T cell infusion, we evaluated the therapeutic effect through proportion of myeloma cells, M protein levels, and imaging examination of EMD. In T-C group, best hematological responses were evaluated: all the eight patients (8/8, 100.0%) obtained ORR two and three months after CAR-T cell infusion. Moreover, the EMD disappeared in all the eight patients in T-C group two month after CAR-T cell infusion. Therefore, regardless of whether combined with imaging examinations (Best Imaging EMD response) or not, the ORR of the T-C group was 100.0%. In C group, best hematological responses were evaluated: eight patients (8/10, 80.0%) obtained ORR three months after CAR-T cell infusion. But six patients (6/10, 60.0%) reached the best imaging EMD response in C group (Fig. 2 A-B). In T-C group, best hematological responses were evaluated two and three months after CAR-T cell infusion: seven patients (7/8, 87.5%) obtained sCR/CR, one patient (1/8, 12.5%) obtained VGPR. The best imaging EMD response was as same as the hematological responses two and three months after CAR-T cell infusion T-C group. In C group, best hematological responses were evaluated three months after CAR-T cell infusion: three patients (3/10, 30.0%) obtained sCR/CR, two patients (2/10, 20.0%) obtained VGPR, three patients (3/10, 30.0%) obtained PR, and two patients (2/10, 20.0%) obtained SD only. But the best imaging EMD response in this group, the EMD of six patients in C group disappeared three months after CAR-T cell therapy (Fig. 2 C-D). Re-progression of the disease and survival time Over a follow-up period exceeding three years, after the optimal response time without the combination of imaging examinations, two patients who obtained ORR in T-C group experienced BM relapse without EMD at 7 months and 19 months post CAR-T cell infusion. One of these two patients died from progressive MM, and the other succumbed to infection. The remaining six patients (6/8, 75.0%) remained alive in sCR or CR at the data cutoff. Of the patients in C group who obtained ORR, four patients (Pt 2, Pt 4, Pt 5, Pt 6) had disease progression again after anti-BCMA CAR-T cell therapy. All these four patients relapsed in BM, and at the same time, they had primary or new EMD. Only four patients (4/10, 40.0%) survived at CR/VGPR/PR state to the cutoff date in C group (including one patient who received allogeneic hematopoietic stem cell transplantation) (Fig. 3 A). The PFS and OS were higher in T-C group than that of in C group ( P PFS =0.0015 and P OS =0.0015). There were no differences of the PFS and OS between T-C group and C group at 6 months after therapy ( P PFS 6m =0.0500 and P OS 6m =0.1939). But the PFS and OS were higher in T-C group than that of in C group at 12 months ( P PFS 12m =0.0106 and P OS 12m =0.0106), and higher in T-C group than that of in C group at 24 months ( P PFS 24m =0.0037 and P OS 24m =0.0037) (Fig. 3 B, C). Expression of anti-BCMA CAR-T cells, T cells and IL-6 in peripheral blood In two groups, the proportion of anti-BCMA CAR-T cells was detected 0, 7, 14 and 28 days after CAR-T cell infusion. Peak of the anti-BCMA CAR-T cells in CD3 + T cells in peripheral blood was 45.24 (IQR 31.9, 63.4) % in T-C group, and 23.66 (IQR 1.6–46.7) % in C group. The peak of CAR-T cells was higher in T-C group than that of in C group ( P = 0.0040) (Fig. 4 A). The time to reach the peak of CAR-T cells was 15.25 (IQR 10, 21) days in T-C group, and 9.25 (IQR 4, 14) days in C group. The time to reach the peak of CAR-T cells was later in T-C group than that of in C group ( P = 0.0052) (Fig. 4 B). In two groups, level of IL-6 was detected 0, 7, 14 and 28 days after CAR-T cell infusion. Median peak of IL-6 in peripheral blood was 434.58 (IQR 86.3, 998.6) pg/mL in T-C group, and 104.44 (IQR 19.2, 506.7) pg/mL in C group. The median peak of IL-6 was higher in T-C group than that of in C group ( P = 0.0035) (Fig. 4 C). Although there was no difference of CD3 + CD4 + and CD3 + CD8 + T cell percentage in peripheral blood between the two groups ( P CD4 =0.0625 and P CD8 =0.3095), there were still differences between them. There was no difference of CD3 + CD4 + and CD3 + CD8 + T cell percentage between the two groups before condition, on day 0, on day 7 and on day 14. But the percentage of CD3 + CD4 + was higher in T-C group than that of in C group on day 28 after CAR-T cell infusion ( P = 0.0127). Moreover, the percentage of CD3 + CD8 + was higher in C group on day 28 after CAR-T cell infusion ( P = 0.0010) (Fig. 4 D). The correlation between the peak of CAR-T cell and IL-6 was not statistically significant in T-C group ( P = 0.1284). But the peak of CAR-T cell was correlated with the peak of IL-6 in C group ( P = 0.0188) (Fig. 5 A). The percentage of MM cells in BM was correlated with the peak of CAR-T cell in T-C group ( P = 0.0374), and correlated with the peak of IL-6 in C group ( P = 0.0013 But it was not correlated with the peak of CAR-T cell in C group ( P = 0.3253) and the peak of IL-6 in T-C group ( P = 0.5020) (Fig. 5 B). The level of serum M protein was correlated with the peak of IL-6 in C group ( P = 0.0072). But it was not correlated with the peak of CAR-T cell in two groups ( P = 0.0824 and 0.5122), and not correlated with the peak of IL-6 in T-C group ( P = 0.3043) (Fig. 5 C). Safety and adverse effects (AEs) In two groups, patients developed fever with or without chills, fatigue, headache, nausea, tachycardia, edema, and other symptoms 2 to 8 days after CAR-T cell infusion. The onset time of symptoms in C group was 2.68 ± 0.95 days, which was earlier than 4.85 ± 1.53 days in T-C group ( P = 0.0225). These symptoms lasted for 7 to 22 days and then subsided. The grade of CRS in T-C group was higher than that of in C group ( P = 0.0405), but there was no difference of the grade of ICANS between the two groups ( P = 0.5596) (Fig. 6 A). The ≥ grade 3 of CRS was found in three patients (3/8, 37.5%) in T-C group, and only one patient (1/10, 10.0%) in C group. The ≥ grade 1 of ICANS was found in two patients (2/8, 20.0%) in T-C group, and only 1 patient (1/10, 10.0%) in C group. The correlation between the grade of CRS and the grade of ICANS was not statistically significant in two groups ( P = 0.0784 and 0.0517) (Fig. 6 B). In our study, it was not statistically significant of the correlation between the grade of CRS and peak of CAR-T cell in two groups ( P = 0.1876 and 0.1855), and between the grade of CRS and peak of IL-6 in T-C group also ( P = 0.3891). But the grade of CRS was correlated with the peak of IL-6 in C group ( P = 0.0491) (Fig. 6CD). Then, the grade of ICANS was correlated with the peak of CAR-T cell in T-C group ( P = 0.0097), and the grade of ICANS was correlated with the peak of IL-6 in C group ( P < 0.0000). But it was not statistically significant of the correlation between the grade of ICANS and peak of CAR-T cell in C group ( P = 0.0539), between the grade of ICANS and peak of IL-6 in T-C group also ( P = 0.2683) (Fig. 6EF). Hematological toxicity in two groups and Stem cell implantation in T-C group Hematological toxicity was grade 1–4 in two groups. It occurred from 4 to 7 days and recovered 15–66 days post CAR-T cell infusion. The ≥ grade 3 of neutropenia, anemia and thrombocytopenia were found in 8 patients (8/8, 100.0%), 5 patients (5/8, 62.5%) and 7 patients (7/8, 87.5%) in T-C group, and 3 patients (3/10, 30.0%), 4 patients (4/10, 40.0%) and 2 patients (2/10, 20.0%) in C group (Fig. 7 A-C). Recovery time of neutropenia in T-C group was 34.63 ± 10.78 days, which was later than 16.88 ± 4.13 days in C group ( P = 0.0072). Recovery time of anemia was 36.00 ± 10.50 days in T-C group and 31.63 ± 5.22 days in C group. There was no difference of recovery time between the two groups ( P = 0.4117). Recovery time of thrombocytopenia was 29.38 ± 11.97 days in T-C group and 19.50 ± 4.38 days in C group. There was no difference of recovery time between the two groups ( P = 0.1241) (Fig. 7 D-E). Poor graft function (PGF) occurred in 3 patients (3/8, 37.5%) in T-C group. The three patients with PGF were as follows: one patient had a granulocyte implantation time of 61 days, another patient had a red blood cell implantation time of 63 days, and the third patient had a platelet implantation time of 66 days. None of the patients died of bacterial infections or invasive fungal diseases in these two groups in the course of their combination therapy or CAR-T therapy. Discussion Although MM remains an incurable malignancy, auto-HSCT is still recommended as standard therapy for eligible patients by international guidelines. Compared with treatment regimens based on PIs and IMiDs, auto-HSCT has been shown to significantly improve both PFS and OS [ 21 ]. While a subset of studies showed that auto-HSCT might cure a small number of patients, ranging from 6.3% to 31.3% [ 22 ], the survival benefits of auto-HSCT are limited [ 23 ]. EMD is recognized as a high-risk feature in MM. Although auto-HSCT may confer a survival benefit in MM patients with EMD, response rates to auto-HSCT in this subgroup remain suboptimal [ 24 ]. Moreover, among patients receiving auto-HSCT, compared with MM patients without EMD, the PFS and OS of MM patients with EMD were significantly shorter [ 25 , 26 ]. When malignant plasma cells acquire the ability to proliferate independently of the BM microenvironment and form EMD, they often exhibit enhanced invasiveness and resistance to therapy. These distinct biological features of EMD likely contribute to the significant clinical challenges associated with its treatment. The advent of novel therapeutic agents has led to substantial improvements in the treatment of MM. A study investigating cyclophosphamide, etoposide, and dexamethasone as salvage therapy in patients with R/R MM (including those with EMD) reported an ORR of 52%. Notably, 23% of these patients subsequently proceeded to auto-HSCT or CAR-T therapy, underscoring the potential role of this chemotherapy regimen as bridging therapy to auto-HSCT or CAR-T therapy in R/R MM and EMD [ 27 ]. For transplant-eligible patients with newly diagnosed multiple myeloma (NDMM), auto-HSCT remains the standard of care, as endorsed by international guidelines including those from the American Society of Clinical Oncology (ASCO) and the European Society for Medical Oncology (ESMO) [ 28 ]. Although auto-HSCT remains a cornerstone of therapy for eligible patients with MM, current evidence regarding its ability to mitigate the adverse prognostic impact of EMD remains conflicting [ 29 , 30 ]. In R/R MM patients with EMD, the role of double auto-HSCT in achieving deeper remission remains controversial. Current evidence suggests that double auto-HSCT offers only marginal improvements in PFS and OS compared with single auto-HSCT [ 31 ]. Furthermore, no survival benefit has been observed with double auto-HSCT in patients with ISS stage III disease or renal impairment, and it needs to be chosen with caution [ 32 ]. Collectively, these findings suggest that auto-HSCT, whether single or double, may not reliably improve outcomes in MM patients with EMD. BCMA CAR-T cell therapy has shown excellent and durable clinical efficacy in patients with R/R MM [ 33 – 35 ]. However, owing to the distinct biological features and adverse prognosis associated with EMD, the efficacy and optimal development strategies of CAR-T therapy in R/R MM patients with EMD have become the focus of attention [ 36 ]. In long-term follow-up studies, including our own previous results, the median PFS and median OS of R/R MM patients with EMD after anti-BCMA CAR-T cell therapy were significantly shorter than those of without EMD [ 16 , 37 – 39 ]. In this study, R/R MM patients with EMD in C group who achieved ORR in CAR-T cell therapy experienced rapid disease progression again. These patients remain an adverse prognostic factor for their CAR-T cell therapy, and the prognosis of CAR-T cell therapy is poor [ 39 , 40 ]. The efficacy and durability of CAR-T therapy in R/R MM patients with EMD remain limited and pose significant clinical challenges. The invasiveness and drug resistance of EMD are attributed to its distinctive tumor microenvironment and complex molecular and genetic alterations, however, the precise mechanisms underlying its evasion of immune surveillance and resistance to targeted therapies remain incompletely understood and warrant further investigation [ 41 ]. Optimizing combination therapeutic strategies for R/R MM patients with EMD represents an urgent clinical priority to enhance treatment efficacy and prolong remission duration. In a clinical trial of BCMA/G protein-coupled receptor, class C group 5 member D (GPRC5D) dual-target CAR-T cell therapy in R/R MM patients with EMD, the ORR of 9 patients reached 100%, among which 44.4% achieved CR, and the 1-year OS rate was 60% [ 42 ]. However, its long-term efficacy still needs to be observed. Although the ORR and CR were satisfactory, more than half of the patients received anti-BCMA CAR-T cell therapy relapsed within one year, especially the R/R MM patients with EMD, whose prognosis was even worse [ 43 ]. Whether auto-HSCT and CAR-T cell therapy combine the advantages of both and is it feasible in terms of mechanism? Expansion of CD8 + T cells has been proven to be associated with a favorable prognosis of anti-BCMA CAR-T cell therapeutics [ 44 , 45 ]. Following auto-HSCT, the CD4⁺/CD8⁺ T-cell ratio remained inverted over an extended period, accompanied by rapid proliferation of CD8⁺ T cells [ 46 ]. In our study, we did not observe a sustained increase in proportion of CD8⁺ T cells in T-C group. Unexpectedly, however, the proportion of CD4⁺ T cells in T-C group was significantly elevated at day 28 post CAR-T cell infusion. Whether this shift in CD4⁺ T cell dynamics is associated with the durable clinical response observed in the T-C group warrants further investigation in a larger cohort with extended follow-up. In a study of CAR-T cell therapy for aggressive B-cell lymphoma, low CD4⁺ T cell counts one month after infusion predicted worse 12-month PFS [ 47 ]. This result might support our conclusion about the CD4⁺ T cell dynamics is associated with the durable clinical response. Previous studies have suggested that auto-HSCT might enhance the antitumor efficacy, expansion, and persistence of CAR-T cells [ 48 , 49 ]. Strategies to enhance the efficacy of CAR-T cell therapy include overcoming resistance mechanisms such as antigen escape and the immunosuppressive tumor microenvironment [ 50 ]. Moreover, the conditioning regimen associated with auto-HSCT may favorably modulate the tumor microenvironment, thereby creating a more conducive setting for subsequent immunotherapy and promoting the expansion of CAR-T cells, particularly in patients with EMD [ 49 ]. In our study, it was also observed that the peaks of CAR-T cells and IL-6 in the T-C group were higher. T cell dysfunction is commonly observed following auto-HSCT. Administering CAR-T cell therapy after auto-HSCT might facilitate the restoration of T lymphocyte function and thereby enhance both the intensity and durability of the antitumor response [ 51 ]. Therefore, the combination of auto-HSCT and CAR-T cell therapy demonstrates promising clinical efficacy and represents a feasible therapeutic approach for R/R MM patients with EMD, as supported by our findings. After a three-year follow-up, the T-C group demonstrated superior ORR, PFS, and OS compared with the C group. In theory, auto-HSCT prior to CAR-T cell infusion may reduce tumor burden and thereby mitigate the severity of CRS and ICANS. However, no similar results were observed in our cohort. Notably, the higher incidence of high-grade CRS in the T-C group might be attributable to a higher level of CAR-T cell expansion in this group. To date, data on the combination of auto-HSCT and CAR-T cell therapy in patients with R/R MM remain limited, with most published studies based on small sample sizes. Although patients in the T-C group experienced higher rates of AEs in our study, a three-year follow-up revealed that anti-BCMA CAR T-cell therapy combined with auto-HSCT was associated with improved PFS and OS in R/R MM patients with EMD. Moreover, CD4⁺ T cell dynamics might be associated with the durable clinical response observed in T-C group. Of course, the observed adverse events in T-C group might be attributable, at least in part, to auto-HSCT. However, it is difficult to distinguish whether the adverse effects are caused by auto-HSCT or CAR-T cell therapy. Future large-scale prospective clinical trials are warranted to clarify the etiology of these adverse effects. Declarations Acknowledgements We thank all our patients for their participation in our clinical trials. We thank the Shanghai Genbase Biotechnology Co., Ltd. for providing us with anti-BCMA CAR-T cells. Funding This work was financially supported by Tianjin Natural Science Foundation Project (25JCZDJC00830). Author Contributions Concept and design: DQ and LX Drafted or revised the manuscript: LX, LC and LR Acquisition of data: LX, NSY, GSQ, CR, WJ, QY Analysis and interpretation of data: LX, WJ, QY and LJY Study supervision: DQ Ethics approval and consent to participate This study was approved by the Medical Ethics Committee of the Department of Hematology, Tianjin First Center Hospital (Tianjin, China). (Approved No. of ethic committee: 2020N028KY). The Clinical trial in our study was registered at http://www.chictr.org.cn/index.aspx as ChiCTR2000033925 . The patient gave their written informed consent in accordance with the Declaration of Helsinki. The patients agreed to have their data used in our study. Conflict of Interest All authors have no conflict of interest to report. ORCID: Qi Deng https://orcid.org/0000-0002-3646-4953 References Binder M, Nandakumar B, Rajkumar SV, Kapoor P, Buadi FK, Dingli D, et al. 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San-Miguel J, Dhakal B, Yong K, Spencer A, Anguille S, Mateos MV, et al. Cilta-cel or standard care in lenalidomide-refractory multiple myeloma. N Engl J Med. 2023;389(4):335-347. Wang Y, Liu Y, Zhang X, Chen L, Li J, Zhao Y, et al. Real-world outcomes of extramedullary multiple myeloma in China: a multicenter retrospective study. Eur J Cancer. 2025; 220:115374. Jin C, Chen R, Fu S, Wang Y, Li J, Zhang Y, et al. Long-term follow-up of BCMA CAR-T cell therapy in patients with relapsed/refractory multiple myeloma. J Immunother Cancer. 2025;13(3): e009876. Abuhelwa Z, Almomen A, Alsharif F, Alzahrani M, Aljurf M. Outcomes of CAR T-cell therapy in high-risk multiple myeloma: a single-center experience. Front Oncol. 2025; 15:1663814. Pan D, Mouhieddine TH, Fu W, Lesokhin A, Mailankody S, Korde N, et al. Outcomes after CAR T cells in multiple myeloma patients with extramedullary and paramedullary disease. Blood. 2023;142(Suppl 1):1006. Lin Y, Raje N, Berdeja JG, Siegel DS, Madduri D, Liedtke M, et al. Idecabtagene vicleucel for relapsed and refractory multiple myeloma: post hoc 18-month follow-up of a phase 1 trial. Nat Med. 2023;29(9):2286-2294. Yao H, Li Y, Wang Q, Zhang L, Chen B, Zhou D, et al. Biomarkers predicting response to BCMA-targeted CAR T therapy in extramedullary multiple myeloma. J Hematol Oncol. 2025;18(1):56. Ho M, Zanwar S, Lin Y, Kapoor P, Binder M, Buadi FK, et al. Management of extramedullary disease in multiple myeloma: current challenges and future directions. Curr Oncol. 2025;32(3):456-468. Raje N, Berdeja J, Lin Y, Siegel D, Jagannath S, Madduri D, et al. Anti-BCMA CAR T-cell therapy bb2121 in relapsed or refractory multiple myeloma. N Engl J Med. 2019;380(18):1726-1737. Franssen LE, Stege CAM, Zweegman S, Mutis T, Groen RWJ. Resistance mechanisms towards CD38-directed antibody therapy in multiple myeloma. J Clin Med. 2020;9(4):1195. Bailur JK, McCachren SS, Doxie DB, Pendleton KM, Stefanek SC, Harville TO, et al. Early alterations in stem-like/resident T cells, innate and myeloid cells in the bone marrow in preneoplastic gammopathy. JCI Insight. 2020;5(5): e127807. Lagreca I, Riva G, Nasillo V, Dall’Olio FG, Potenza L, Fiorentini S, et al. The role of T cell immunity in monoclonal gammopathy and multiple myeloma: from immunopathogenesis to novel therapeutic approaches. Int J Mol Sci. 2022;23(9):5242. André Airosa Pardal, Ana Benzaquén, Pablo Granados, Paula Amat, Rafael Hernani, Aitana Balaguer-Roselló, et al. Patterns of Immune Recovery After Axicabtagene Ciloleucel for Aggressive B-Cell Lymphoma. HemaSphere. 2025; 9(10): e70229. Teoh PJ, Chng WJ. CAR T-cell therapy in multiple myeloma: more room for improvement. Blood Cancer J. 2021;11(5):84. Minnie SA, Hill GR. Autologous stem cell transplantation for myeloma: cytoreduction or an immunotherapy? Front Immunol. 2021; 12:651288. Choi T, Kang Y. Chimeric antigen receptor (CAR) T-cell therapy for multiple myeloma. Pharmacol Ther. 2022; 232:108007. Janakiram M, Arora N, Bachanova V, Callander NS, Costa LJ, Dhakal B, et al. Novel cell and immune engagers in optimizing tumor-specific immunity post-autologous transplantation in multiple myeloma. Transplant Cell Ther. 2022;28(2):61-69. Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 17 Jan, 2026 Reviewers invited by journal 15 Jan, 2026 Editor assigned by journal 14 Jan, 2026 First submitted to journal 12 Jan, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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04:01:10","extension":"xml","order_by":27,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":124121,"visible":true,"origin":"","legend":"","description":"","filename":"JTRMD26007450structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8582254/v1/32ccd3d7ade460770c077e60.xml"},{"id":100748025,"identity":"c4a35f33-395a-489c-99d3-3fe7b076c017","added_by":"auto","created_at":"2026-01-21 04:00:17","extension":"html","order_by":28,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":138265,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8582254/v1/85393d6ff8468811af32359f.html"},{"id":100748038,"identity":"d8f6a5dd-ce04-468c-929f-c9650a37ae3c","added_by":"auto","created_at":"2026-01-21 04:00:22","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":17994042,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe study design.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8582254/v1/96784b41cb35e832984c689b.png"},{"id":100748146,"identity":"51137947-8457-47e0-9167-f1049cb542c5","added_by":"auto","created_at":"2026-01-21 04:00:54","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":23695380,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eClinical response to therapy (A) \u003c/strong\u003eBest hematological responses: all the eight patients (8/8, 100.0%) obtained ORR in T-C group and 8 patients (8/10, 80.0%) obtained ORR in C group.\u003cstrong\u003e (B) \u003c/strong\u003eBest Imaging EMD response:\u003cstrong\u003e \u003c/strong\u003ethe EMD disappeared in all the 8 patients in T-C group and 6 patients (6/10, 60.0%) in C group.\u003cstrong\u003e(C)\u003c/strong\u003e Best hematological responses: 7 patients (7/8, 87.5%) obtained sCR/CR, 1 patient (1/8, 12.5%) obtained VGPR in T-C group. 3 patients (3/10, 30.0%) obtained sCR/CR, 2 patients (2/10, 20.0%) obtained VGPR, 3 patients (3/10, 30.0%) obtained PR, and 2 patients (2/10, 20.0%) obtained SD in C group. \u003cstrong\u003e(D) \u003c/strong\u003eBest Imaging EMD response: it was as same as the hematological responses in T-C group. The EMD of 6 patients in C group disappeared after CAR-T cell therapy.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8582254/v1/e16880aeffcc9d55f9ede836.png"},{"id":100748075,"identity":"cc624914-8a03-4c41-bc1f-d9309698b95e","added_by":"auto","created_at":"2026-01-21 04:00:35","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":39291242,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRe-progression of the disease and survival time (A) \u003c/strong\u003eSix patients (6/8, 75.0%) survived at CR state to cutoff date in T-C group. Four patients (4/10, 40.0%) survived at CR/VGPR/PR state to the cutoff date in C group.\u003cstrong\u003e (B)\u003c/strong\u003e The PFS and OS were higher in T-C group than that of in C group. But it was no differences between the two groups at 6 months.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8582254/v1/b66dfb0f586408b8920820d6.png"},{"id":100748157,"identity":"58b3d32b-9d9e-4a85-93df-b005e229b438","added_by":"auto","created_at":"2026-01-21 04:00:58","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":11965995,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression of anti-BCMA CAR-T cells, T cells and IL-6 (A) \u003c/strong\u003eThe peak of CAR-T cells was higher in T-C group than that of in C group.\u003cstrong\u003e (B)\u003c/strong\u003e The time to reach the peak of CAR-T cells was later in T-C group than that of in C group.\u003cstrong\u003e(C) \u003c/strong\u003eThe median peak of IL-6 was higher in T-C group than that of in C group.\u003cstrong\u003e (D)\u003c/strong\u003e The percentage of CD3+CD4+ was higher in T-C group, and the percentage of CD3+CD8+ was higher in C group on day 28 after CAR-T cell infusion.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8582254/v1/a37f7afd90dc8f599e348281.png"},{"id":100748016,"identity":"fd7378fa-9ebc-4efa-aea6-bdeb0fa9e222","added_by":"auto","created_at":"2026-01-21 04:00:15","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":30776830,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe related factors of the peak of CAR-T cell and the peak of IL-6 (A)\u003c/strong\u003e The peak of CAR-T cell was correlated with the peak of IL-6 in C group. \u003cstrong\u003e(B)\u003c/strong\u003e The percentage of MM cells in BM was correlated with the peak of CAR-T cell in T-C group, and correlated with the peak of IL-6 in C group. \u003cstrong\u003e(C)\u003c/strong\u003e The level of serum M protein was correlated with the peak of IL-6 in C group.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8582254/v1/a053f18e60b18c09db13ddc6.png"},{"id":100748031,"identity":"b9376983-08e4-448d-8c74-131d65fd2411","added_by":"auto","created_at":"2026-01-21 04:00:19","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":34127421,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSafety and adverse effects (A)\u003c/strong\u003e The grade of CRS in T-C group was higher. It was no difference of grade of ICANS between the two groups. \u003cstrong\u003e(B) \u003c/strong\u003eThere was no correlation between the grade of CRS and the grade of ICANS in two groups.\u003cstrong\u003e(CD) \u003c/strong\u003eThere was no correlation between the grade of CRS and peak of CAR-T cell in two groups, no correlation between the grade of CRS and peak of IL-6 in T-C group. It has correlation between the grade of CRS and peak of IL-6 in C group.\u003cstrong\u003e(EF) \u003c/strong\u003eThere was no correlation between the grade of ICANS and peak of CAR-T cell in C group, between the grade of ICANS and peak of IL-6 in T-C group.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-8582254/v1/29feabf7aa99441ffda19ac6.png"},{"id":100748140,"identity":"76f19535-bea7-4a8d-8850-c373014d2462","added_by":"auto","created_at":"2026-01-21 04:00:51","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":43620977,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHematological toxicity (A-C) \u003c/strong\u003eThe ≥ grade 3 of neutropenia, anemia and thrombocytopenia in two groups.\u003cstrong\u003e(D-E)\u003c/strong\u003e Recovery time of neutropenia in T-C group was later than thatin C group. There was no difference of anemia and thrombocytopenia recovery time between the two groups.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-8582254/v1/ab050479c31e647672195288.png"},{"id":100747974,"identity":"01c1014d-6491-43ff-8cfb-e2738672ff20","added_by":"auto","created_at":"2026-01-21 03:59:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":887714,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8582254/v1/f5f27ed1-c9de-45ef-be2d-0d8d009c8d02.pdf"}],"financialInterests":"","formattedTitle":"Long-term follow-up results of anti-BCMA CAR-T cell therapy combined with autologous hematopoietic stem cell transplantation in relapsed/refractory multiple myeloma with extramedullary disease","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMultiple myeloma (MM), a malignant neoplasm of plasma cells, accounts for approximately 10% of all hematologic malignancies and has an annual incidence of 6.6 cases per 100,000 individuals [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In recent years, the introduction of novel therapeutic agents, including proteasome inhibitors (PIs), immunomodulatory drugs (IMiDs), monoclonal antibodies, autologous stem cell transplantation (auto-HSCT), and chimeric antigen receptor T-cell (CAR-T) therapy, has substantially improved survival outcomes in patients with multiple myeloma (MM) [\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Despite these advances, multiple myeloma is still considered incurable, with recurrent relapses representing the major therapeutic hurdle [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Extramedullary disease (EMD) is a frequent complication in relapsed/refractory (R/R) MM and is consistently associated with inferior survival outcomes [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. At initial diagnosis, EMD is present in approximately 2% to 5% of MM patients, whereas its prevalence rises to 20% to 40% in the R/R MM patients [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. MM patients with EMD have a significantly poorer prognosis compared to those without EMD, with shorter median progression-free survival (PFS) and overall survival (OS) [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Especially for EMD not related to bones, the prognosis is even worse [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. MM patients with EMD still have a poor response to conventional therapies, including PIs, chemotherapy and ASCT, resulting in poor PFS and OS [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In CAR-T cell therapy for R/R MM, EMD is also one of the adverse factors affecting the prognosis of this therapy, especially for MM patients with non-bone-related extramedullary diseases, the prognosis is the worst [\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Effective strategies to improve prognosis and prolong survival in MM patients with EMD remain an unmet clinical need. To address this challenge, we developed a combined therapeutic approach integrating anti-BCMA CAR-T cell therapy with auto-HSCT. To date, long-term outcomes of combining CAR-T therapy with ASCT in MM patients with EMD remain unreported. Our study represents the first clinical observation with three-year follow-up, offering insights into the sustained efficacy and safety of this strategy.Preliminary results indicate that this strategy enhances both PFS and OS in patients with MM with EMD.\u003c/p\u003e"},{"header":"Patients and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePatients enrolled in the study\u003c/h2\u003e \u003cp\u003eBetween June 2020 and December 2022, 18 R/R MM patients with at least one site of EMD were enrolled in clinical trials of anti-BCMA CAR-T therapy (\u003cem\u003eChiCTR2000033925\u003c/em\u003e). Eight patients received anti-BCMA CAR-T therapy in combination with auto-HSCT and constituted the T-C group. A concurrent control group (C group) comprised ten additional R/R MM patients with EMD who received anti-BCMA CAR-T therapy alone, all were unable or did not receive sufficient stem cells for auto-HSCT. The enrolment period for this study concluded in December 2022, with a data cutoff date of December 2025.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAnti-BCMA CAR-T cell therapy\u003c/h3\u003e\n\u003cp\u003ePeripheral blood mononuclear cells (PBMCs) were collected from patients with R/R MM in both the T-C and C groups and used for the manufacture of anti-BCMA CAR-T cells. The humanized anti-BCMA CAR construct, designated lenti-BCMA-2rd-CAR, was provided by Shanghai Genbase Biotechnology Co., Ltd. (Shanghai, China). Transduction efficiency was assessed by flow cytometry (FCM; BD Biosciences, San Jose, CA, USA) on the day of harvest following ex vivo expansion.\u003c/p\u003e \u003cp\u003eAll patients in C group received lymphodepleting chemotherapy with fludarabine (30 mg/m\u003csup\u003e2\u003c/sup\u003e) and cyclophosphamide (400 mg/m\u003csup\u003e2\u003c/sup\u003e) from day \u0026minus;\u0026thinsp;4 to day \u0026minus;\u0026thinsp;2. Autologous humanized anti-BCMA CAR-T cells (2\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells/kg) were infused on day 0 in C group or 2\u0026ndash;4 days after the day of stem cell infusion in T-C group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eAuto-HSCT\u003c/h3\u003e\n\u003cp\u003eBefore stem cell mobilization, all R/R MM patients with EMD in the T-C group achieved only minimal response (MR) or stable disease (SD) based on hematologic criteria. For mobilization, either cyclophosphamide (CTX, 3\u0026ndash;5 g/m\u0026sup2;) followed by granulocyte colony-stimulating factor (G-CSF, 300 \u0026micro;g/day, initiated 5\u0026ndash;7 days after CTX and continued until stem cell collection) or G-CSF alone (300 \u0026micro;g/day on days 1\u0026ndash;5) was administered. Peripheral blood stem cells were subsequently collected via leukapheresis. In the T-C group, the conditioning regimen was high-dose melphalan (200 mg/m\u0026sup2;) [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The number of stem cells infused in T-C group was required as the following numbers: CD34\u0026thinsp;+\u0026thinsp;cells\u0026thinsp;\u0026gt;\u0026thinsp;2.0\u0026times;10\u003csup\u003e6\u003c/sup\u003e/kg.\u003c/p\u003e\n\u003ch3\u003eMaintenance treatment after anti-BCMA CAR-T cell therapy\u003c/h3\u003e\n\u003cp\u003eAll patients initiated lenalidomide or pomalidomide maintenance therapy 2\u0026ndash;3 months following anti-BCMA CAR-T infusion, once hematologic toxicity had resolved. One patient in the C group subsequently underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT) three months after CAR-T cell therapy.\u003c/p\u003e\n\u003ch3\u003eCriteria for diagnosis and evaluation criteria for therapeutic efficacy\u003c/h3\u003e\n\u003cp\u003eThe diagnosis of R/R MM, diagnosis of EMD, clinical response to the humanised anti-BCMA CAR-T cell therapy was assessed according to the International Myeloma Working Group Guidelines uniform response criteria for MM [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The proportion of MM cells was determined by bone marrow (BM) morphology and FCM. The M protein levels were detected by immunofixation electrophoresis. Assessable EMD was detected using CT, PET/CT or MRI. Therapeutic efficacy was assessed monthly during the first three months following anti-BCMA CAR-T cell infusion in all patients, including those in both the T-C and C groups. Thereafter, evaluations were conducted every 2 to 3 months. The clinical responses included stringent complete response (sCR), complete response (CR), very good partial response (VGPR), partial response (PR), MR, SD, and progressive disease (PD). We assessed the objective response rate (ORR) (including sCR, CR, VGPR, and PR), overall survival (OS), and progression-free survival (PFS).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eAdverse events (AEs) in the combination therapy\u003c/h2\u003e \u003cp\u003eThe proportion of anti-BCMA CAR-T cells in peripheral blood was assessed by FCM on days 0, 4, 7, 14, and 28 following CAR-T cell infusion. Serum interleukin-6 (IL-6) concentrations were measured at the same timepoints (days 0, 7, 14, and 28) using enzyme-linked immunosorbent assay (ELISA). AEs associated with humanized anti-BCMA CAR-T therapy were systematically monitored. Cytokine release syndrome (CRS) and immune-effector cell-associated neurotoxicity syndrome (ICANS) were graded according to the consensus criteria established by the American Society for Transplantation and Cellular Therapy (ASTCT) [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], while hematologic toxicities were classified using the Common Terminology Criteria for Adverse Events (CTCAE), version 5.0 [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SE. The probabilities of PFS and OS were estimated using the Kaplan-Meier method and compared with the log-rank test. All statistical analyses were performed using GraphPad Prism 7 and SPSS 17.0. Statistical significance was set at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eR/R MM patient characteristics\u003c/h2\u003e \u003cp\u003eThe baseline characteristics of the 18 patients with R/R MM enrolled in this clinical trial are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. All patients with R/R MM had at least one bone-unrelated EMD prior to enrolling in our clinical trial. No significant differences were observed between the T-C and C groups with respect to age, sex, high-risk cytogenetics, International Staging System (ISS) stage III, or median number of prior lines of therapy. All 18 patients were followed for more than three years.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eBaseline characteristics of the R/R MM with EMD\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eT-C group (n\u0026thinsp;=\u0026thinsp;8) n%\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC group (n\u0026thinsp;=\u0026thinsp;10) n%\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eP values\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSex: Male (%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5 (62.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4 (40.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.342\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAge\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e57.4(48\u0026ndash;71)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e60.1(38\u0026ndash;75)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.210\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eKPS, \u0026gt;\u0026thinsp;80\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8(100%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10(100%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSubtype, %k light chain\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2(25.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3(30.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eISS stage, (I-II): III\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2:6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3:7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMM cells in BM, %\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16.7 (0.9\u0026ndash;55.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18.8 (0.4\u0026ndash;86.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.761\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSerum M protein (g/L)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e19.2 (4.6\u0026ndash;49.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16.6 (2.4\u0026ndash;56.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.877\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eEMD number\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003eHigh-Risk Cytogenetics\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003eNumbers of prior therapy\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003eBCMA CAR-T therapy before\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003eAuto-HSCT before\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.75 (1\u0026ndash;3)\u003c/p\u003e \u003cp\u003e2.25 (1\u0026ndash;4)\u003c/p\u003e \u003cp\u003e4.63 (3\u0026ndash;7)\u003c/p\u003e \u003cp\u003e1 (12.5%)\u003c/p\u003e \u003cp\u003e1 (12.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.13 (1\u0026ndash;3)\u003c/p\u003e \u003cp\u003e2.38 (1\u0026ndash;3)\u003c/p\u003e \u003cp\u003e5.10 (3\u0026ndash;9)\u003c/p\u003e \u003cp\u003e2 (20.0%)\u003c/p\u003e \u003cp\u003e1 (10.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.300\u003c/p\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.656\u003c/p\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.651\u003c/p\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.000\u003c/p\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eTransduction and amplification efficiency, infusion dose of the humanized anti-BCMA CAR-T cells\u003c/h2\u003e \u003cp\u003eThe mean anti-BCMA CAR transduction efficiency in the final products of T-C group and C group was 40.12\u0026thinsp;\u0026plusmn;\u0026thinsp;6.92% and 37.41\u0026thinsp;\u0026plusmn;\u0026thinsp;7.11%. On 2\u0026ndash;4 days after stem cell infusion a dose of 2.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.66\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells/kg anti-BCMA CAR-T cell was infused in T-C group, and on day 0 a dose of 2.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells/kg anti-BCMA CAR-T cell was infused in C group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStem cell infusion in T-C group\u003c/h2\u003e \u003cp\u003eThe CD34\u0026thinsp;+\u0026thinsp;cells infused in T-C group was 2.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.72\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells/kg on day 0 during their auto-HSCT process.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eClinical response to therapy\u003c/h2\u003e \u003cp\u003eAfter anti-BCMA CAR-T cell infusion, we evaluated the therapeutic effect through proportion of myeloma cells, M protein levels, and imaging examination of EMD. In T-C group, best hematological responses were evaluated: all the eight patients (8/8, 100.0%) obtained ORR two and three months after CAR-T cell infusion. Moreover, the EMD disappeared in all the eight patients in T-C group two month after CAR-T cell infusion. Therefore, regardless of whether combined with imaging examinations (Best Imaging EMD response) or not, the ORR of the T-C group was 100.0%. In C group, best hematological responses were evaluated: eight patients (8/10, 80.0%) obtained ORR three months after CAR-T cell infusion. But six patients (6/10, 60.0%) reached the best imaging EMD response in C group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn T-C group, best hematological responses were evaluated two and three months after CAR-T cell infusion: seven patients (7/8, 87.5%) obtained sCR/CR, one patient (1/8, 12.5%) obtained VGPR. The best imaging EMD response was as same as the hematological responses two and three months after CAR-T cell infusion T-C group. In C group, best hematological responses were evaluated three months after CAR-T cell infusion: three patients (3/10, 30.0%) obtained sCR/CR, two patients (2/10, 20.0%) obtained VGPR, three patients (3/10, 30.0%) obtained PR, and two patients (2/10, 20.0%) obtained SD only. But the best imaging EMD response in this group, the EMD of six patients in C group disappeared three months after CAR-T cell therapy (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-D).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eRe-progression of the disease and survival time\u003c/h2\u003e \u003cp\u003eOver a follow-up period exceeding three years, after the optimal response time without the combination of imaging examinations, two patients who obtained ORR in T-C group experienced BM relapse without EMD at 7 months and 19 months post CAR-T cell infusion. One of these two patients died from progressive MM, and the other succumbed to infection. The remaining six patients (6/8, 75.0%) remained alive in sCR or CR at the data cutoff. Of the patients in C group who obtained ORR, four patients (Pt 2, Pt 4, Pt 5, Pt 6) had disease progression again after anti-BCMA CAR-T cell therapy. All these four patients relapsed in BM, and at the same time, they had primary or new EMD. Only four patients (4/10, 40.0%) survived at CR/VGPR/PR state to the cutoff date in C group (including one patient who received allogeneic hematopoietic stem cell transplantation) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe PFS and OS were higher in T-C group than that of in C group (\u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003ePFS\u003c/em\u003e\u003c/sub\u003e=0.0015 and \u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eOS\u003c/em\u003e\u003c/sub\u003e=0.0015). There were no differences of the PFS and OS between T-C group and C group at 6 months after therapy (\u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003ePFS 6m\u003c/em\u003e\u003c/sub\u003e=0.0500 and \u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eOS 6m\u003c/em\u003e\u003c/sub\u003e=0.1939). But the PFS and OS were higher in T-C group than that of in C group at 12 months (\u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003ePFS 12m\u003c/em\u003e\u003c/sub\u003e=0.0106 and \u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eOS 12m\u003c/em\u003e\u003c/sub\u003e=0.0106), and higher in T-C group than that of in C group at 24 months (\u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003ePFS 24m\u003c/em\u003e\u003c/sub\u003e=0.0037 and \u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eOS 24m\u003c/em\u003e\u003c/sub\u003e=0.0037) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, C).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eExpression of anti-BCMA CAR-T cells, T cells and IL-6 in peripheral blood\u003c/h2\u003e \u003cp\u003eIn two groups, the proportion of anti-BCMA CAR-T cells was detected 0, 7, 14 and 28 days after CAR-T cell infusion. Peak of the anti-BCMA CAR-T cells in CD3\u0026thinsp;+\u0026thinsp;T cells in peripheral blood was 45.24 (IQR 31.9, 63.4) % in T-C group, and 23.66 (IQR 1.6\u0026ndash;46.7) % in C group. The peak of CAR-T cells was higher in T-C group than that of in C group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0040) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The time to reach the peak of CAR-T cells was 15.25 (IQR 10, 21) days in T-C group, and 9.25 (IQR 4, 14) days in C group. The time to reach the peak of CAR-T cells was later in T-C group than that of in C group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0052) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). In two groups, level of IL-6 was detected 0, 7, 14 and 28 days after CAR-T cell infusion. Median peak of IL-6 in peripheral blood was 434.58 (IQR 86.3, 998.6) pg/mL in T-C group, and 104.44 (IQR 19.2, 506.7) pg/mL in C group. The median peak of IL-6 was higher in T-C group than that of in C group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0035) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAlthough there was no difference of CD3\u0026thinsp;+\u0026thinsp;CD4\u0026thinsp;+\u0026thinsp;and CD3\u0026thinsp;+\u0026thinsp;CD8\u0026thinsp;+\u0026thinsp;T cell percentage in peripheral blood between the two groups (\u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eCD4\u003c/em\u003e\u003c/sub\u003e=0.0625 and \u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eCD8\u003c/em\u003e\u003c/sub\u003e=0.3095), there were still differences between them. There was no difference of CD3\u0026thinsp;+\u0026thinsp;CD4\u0026thinsp;+\u0026thinsp;and CD3\u0026thinsp;+\u0026thinsp;CD8\u0026thinsp;+\u0026thinsp;T cell percentage between the two groups before condition, on day 0, on day 7 and on day 14. But the percentage of CD3\u0026thinsp;+\u0026thinsp;CD4\u0026thinsp;+\u0026thinsp;was higher in T-C group than that of in C group on day 28 after CAR-T cell infusion (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0127). Moreover, the percentage of CD3\u0026thinsp;+\u0026thinsp;CD8\u0026thinsp;+\u0026thinsp;was higher in C group on day 28 after CAR-T cell infusion (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0010) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eThe correlation between the peak of CAR-T cell and IL-6 was not statistically significant in T-C group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.1284). But the peak of CAR-T cell was correlated with the peak of IL-6 in C group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0188) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). The percentage of MM cells in BM was correlated with the peak of CAR-T cell in T-C group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0374), and correlated with the peak of IL-6 in C group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0013 But it was not correlated with the peak of CAR-T cell in C group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.3253) and the peak of IL-6 in T-C group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.5020) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). The level of serum M protein was correlated with the peak of IL-6 in C group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0072). But it was not correlated with the peak of CAR-T cell in two groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0824 and 0.5122), and not correlated with the peak of IL-6 in T-C group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.3043) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eSafety and adverse effects (AEs)\u003c/h2\u003e \u003cp\u003eIn two groups, patients developed fever with or without chills, fatigue, headache, nausea, tachycardia, edema, and other symptoms 2 to 8 days after CAR-T cell infusion. The onset time of symptoms in C group was 2.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.95 days, which was earlier than 4.85\u0026thinsp;\u0026plusmn;\u0026thinsp;1.53 days in T-C group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0225). These symptoms lasted for 7 to 22 days and then subsided.\u003c/p\u003e \u003cp\u003eThe grade of CRS in T-C group was higher than that of in C group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0405), but there was no difference of the grade of ICANS between the two groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.5596) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). The \u0026ge;\u0026thinsp;grade 3 of CRS was found in three patients (3/8, 37.5%) in T-C group, and only one patient (1/10, 10.0%) in C group. The \u0026ge;\u0026thinsp;grade 1 of ICANS was found in two patients (2/8, 20.0%) in T-C group, and only 1 patient (1/10, 10.0%) in C group. The correlation between the grade of CRS and the grade of ICANS was not statistically significant in two groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0784 and 0.0517) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn our study, it was not statistically significant of the correlation between the grade of CRS and peak of CAR-T cell in two groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.1876 and 0.1855), and between the grade of CRS and peak of IL-6 in T-C group also (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.3891). But the grade of CRS was correlated with the peak of IL-6 in C group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0491) (Fig.\u0026nbsp;6CD). Then, the grade of ICANS was correlated with the peak of CAR-T cell in T-C group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0097), and the grade of ICANS was correlated with the peak of IL-6 in C group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0000). But it was not statistically significant of the correlation between the grade of ICANS and peak of CAR-T cell in C group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0539), between the grade of ICANS and peak of IL-6 in T-C group also (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.2683) (Fig.\u0026nbsp;6EF).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eHematological toxicity in two groups and Stem cell implantation in T-C group\u003c/h2\u003e \u003cp\u003eHematological toxicity was grade 1\u0026ndash;4 in two groups. It occurred from 4 to 7 days and recovered 15\u0026ndash;66 days post CAR-T cell infusion. The \u0026ge;\u0026thinsp;grade 3 of neutropenia, anemia and thrombocytopenia were found in 8 patients (8/8, 100.0%), 5 patients (5/8, 62.5%) and 7 patients (7/8, 87.5%) in T-C group, and 3 patients (3/10, 30.0%), 4 patients (4/10, 40.0%) and 2 patients (2/10, 20.0%) in C group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eRecovery time of neutropenia in T-C group was 34.63\u0026thinsp;\u0026plusmn;\u0026thinsp;10.78 days, which was later than 16.88\u0026thinsp;\u0026plusmn;\u0026thinsp;4.13 days in C group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0072). Recovery time of anemia was 36.00\u0026thinsp;\u0026plusmn;\u0026thinsp;10.50 days in T-C group and 31.63\u0026thinsp;\u0026plusmn;\u0026thinsp;5.22 days in C group. There was no difference of recovery time between the two groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.4117). Recovery time of thrombocytopenia was 29.38\u0026thinsp;\u0026plusmn;\u0026thinsp;11.97 days in T-C group and 19.50\u0026thinsp;\u0026plusmn;\u0026thinsp;4.38 days in C group. There was no difference of recovery time between the two groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.1241) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD-E).\u003c/p\u003e \u003cp\u003ePoor graft function (PGF) occurred in 3 patients (3/8, 37.5%) in T-C group. The three patients with PGF were as follows: one patient had a granulocyte implantation time of 61 days, another patient had a red blood cell implantation time of 63 days, and the third patient had a platelet implantation time of 66 days. None of the patients died of bacterial infections or invasive fungal diseases in these two groups in the course of their combination therapy or CAR-T therapy.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003e Although MM remains an incurable malignancy, auto-HSCT is still recommended as standard therapy for eligible patients by international guidelines. Compared with treatment regimens based on PIs and IMiDs, auto-HSCT has been shown to significantly improve both PFS and OS [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. While a subset of studies showed that auto-HSCT might cure a small number of patients, ranging from 6.3% to 31.3% [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], the survival benefits of auto-HSCT are limited [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eEMD is recognized as a high-risk feature in MM. Although auto-HSCT may confer a survival benefit in MM patients with EMD, response rates to auto-HSCT in this subgroup remain suboptimal [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Moreover, among patients receiving auto-HSCT, compared with MM patients without EMD, the PFS and OS of MM patients with EMD were significantly shorter [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. When malignant plasma cells acquire the ability to proliferate independently of the BM microenvironment and form EMD, they often exhibit enhanced invasiveness and resistance to therapy. These distinct biological features of EMD likely contribute to the significant clinical challenges associated with its treatment. The advent of novel therapeutic agents has led to substantial improvements in the treatment of MM. A study investigating cyclophosphamide, etoposide, and dexamethasone as salvage therapy in patients with R/R MM (including those with EMD) reported an ORR of 52%. Notably, 23% of these patients subsequently proceeded to auto-HSCT or CAR-T therapy, underscoring the potential role of this chemotherapy regimen as bridging therapy to auto-HSCT or CAR-T therapy in R/R MM and EMD [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. For transplant-eligible patients with newly diagnosed multiple myeloma (NDMM), auto-HSCT remains the standard of care, as endorsed by international guidelines including those from the American Society of Clinical Oncology (ASCO) and the European Society for Medical Oncology (ESMO) [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Although auto-HSCT remains a cornerstone of therapy for eligible patients with MM, current evidence regarding its ability to mitigate the adverse prognostic impact of EMD remains conflicting [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In R/R MM patients with EMD, the role of double auto-HSCT in achieving deeper remission remains controversial. Current evidence suggests that double auto-HSCT offers only marginal improvements in PFS and OS compared with single auto-HSCT [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Furthermore, no survival benefit has been observed with double auto-HSCT in patients with ISS stage III disease or renal impairment, and it needs to be chosen with caution [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Collectively, these findings suggest that auto-HSCT, whether single or double, may not reliably improve outcomes in MM patients with EMD.\u003c/p\u003e \u003cp\u003eBCMA CAR-T cell therapy has shown excellent and durable clinical efficacy in patients with R/R MM [\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. However, owing to the distinct biological features and adverse prognosis associated with EMD, the efficacy and optimal development strategies of CAR-T therapy in R/R MM patients with EMD have become the focus of attention [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In long-term follow-up studies, including our own previous results, the median PFS and median OS of R/R MM patients with EMD after anti-BCMA CAR-T cell therapy were significantly shorter than those of without EMD [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. In this study, R/R MM patients with EMD in C group who achieved ORR in CAR-T cell therapy experienced rapid disease progression again. These patients remain an adverse prognostic factor for their CAR-T cell therapy, and the prognosis of CAR-T cell therapy is poor [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. The efficacy and durability of CAR-T therapy in R/R MM patients with EMD remain limited and pose significant clinical challenges. The invasiveness and drug resistance of EMD are attributed to its distinctive tumor microenvironment and complex molecular and genetic alterations, however, the precise mechanisms underlying its evasion of immune surveillance and resistance to targeted therapies remain incompletely understood and warrant further investigation [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Optimizing combination therapeutic strategies for R/R MM patients with EMD represents an urgent clinical priority to enhance treatment efficacy and prolong remission duration. In a clinical trial of BCMA/G protein-coupled receptor, class C group 5 member D (GPRC5D) dual-target CAR-T cell therapy in R/R MM patients with EMD, the ORR of 9 patients reached 100%, among which 44.4% achieved CR, and the 1-year OS rate was 60% [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. However, its long-term efficacy still needs to be observed.\u003c/p\u003e \u003cp\u003eAlthough the ORR and CR were satisfactory, more than half of the patients received anti-BCMA CAR-T cell therapy relapsed within one year, especially the R/R MM patients with EMD, whose prognosis was even worse [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Whether auto-HSCT and CAR-T cell therapy combine the advantages of both and is it feasible in terms of mechanism? Expansion of CD8\u0026thinsp;+\u0026thinsp;T cells has been proven to be associated with a favorable prognosis of anti-BCMA CAR-T cell therapeutics [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Following auto-HSCT, the CD4⁺/CD8⁺ T-cell ratio remained inverted over an extended period, accompanied by rapid proliferation of CD8⁺ T cells [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. In our study, we did not observe a sustained increase in proportion of CD8⁺ T cells in T-C group. Unexpectedly, however, the proportion of CD4⁺ T cells in T-C group was significantly elevated at day 28 post CAR-T cell infusion. Whether this shift in CD4⁺ T cell dynamics is associated with the durable clinical response observed in the T-C group warrants further investigation in a larger cohort with extended follow-up. In a study of CAR-T cell therapy for aggressive B-cell lymphoma, low CD4⁺ T cell counts one month after infusion predicted worse 12-month PFS [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. This result might support our conclusion about the CD4⁺ T cell dynamics is associated with the durable clinical response.\u003c/p\u003e \u003cp\u003ePrevious studies have suggested that auto-HSCT might enhance the antitumor efficacy, expansion, and persistence of CAR-T cells [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Strategies to enhance the efficacy of CAR-T cell therapy include overcoming resistance mechanisms such as antigen escape and the immunosuppressive tumor microenvironment [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Moreover, the conditioning regimen associated with auto-HSCT may favorably modulate the tumor microenvironment, thereby creating a more conducive setting for subsequent immunotherapy and promoting the expansion of CAR-T cells, particularly in patients with EMD [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. In our study, it was also observed that the peaks of CAR-T cells and IL-6 in the T-C group were higher. T cell dysfunction is commonly observed following auto-HSCT. Administering CAR-T cell therapy after auto-HSCT might facilitate the restoration of T lymphocyte function and thereby enhance both the intensity and durability of the antitumor response [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Therefore, the combination of auto-HSCT and CAR-T cell therapy demonstrates promising clinical efficacy and represents a feasible therapeutic approach for R/R MM patients with EMD, as supported by our findings. After a three-year follow-up, the T-C group demonstrated superior ORR, PFS, and OS compared with the C group. In theory, auto-HSCT prior to CAR-T cell infusion may reduce tumor burden and thereby mitigate the severity of CRS and ICANS. However, no similar results were observed in our cohort. Notably, the higher incidence of high-grade CRS in the T-C group might be attributable to a higher level of CAR-T cell expansion in this group.\u003c/p\u003e \u003cp\u003eTo date, data on the combination of auto-HSCT and CAR-T cell therapy in patients with R/R MM remain limited, with most published studies based on small sample sizes. Although patients in the T-C group experienced higher rates of AEs in our study, a three-year follow-up revealed that anti-BCMA CAR T-cell therapy combined with auto-HSCT was associated with improved PFS and OS in R/R MM patients with EMD. Moreover, CD4⁺ T cell dynamics might be associated with the durable clinical response observed in T-C group. Of course, the observed adverse events in T-C group might be attributable, at least in part, to auto-HSCT. However, it is difficult to distinguish whether the adverse effects are caused by auto-HSCT or CAR-T cell therapy. Future large-scale prospective clinical trials are warranted to clarify the etiology of these adverse effects.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank all our patients for their participation in our clinical trials.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe thank the Shanghai Genbase Biotechnology Co., Ltd. for providing us with anti-BCMA CAR-T cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was financially supported by Tianjin Natural Science Foundation Project (25JCZDJC00830).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConcept and design: DQ and LX\u003c/p\u003e\n\u003cp\u003eDrafted or revised the manuscript: LX, LC and LR\u003c/p\u003e\n\u003cp\u003eAcquisition of data: LX, NSY, GSQ, CR, WJ, QY\u003c/p\u003e\n\u003cp\u003eAnalysis and interpretation of data: LX, WJ, QY and LJY\u003c/p\u003e\n\u003cp\u003eStudy supervision: DQ\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Medical Ethics Committee of the Department of Hematology, Tianjin First Center Hospital (Tianjin, China). (Approved No. of ethic committee: 2020N028KY). The Clinical trial in our study was registered at http://www.chictr.org.cn/index.aspx as \u003cem\u003eChiCTR2000033925\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eThe patient gave their written informed consent in accordance with the Declaration of Helsinki. The patients agreed to have their data used in our study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors have no conflict of interest to report.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eORCID:\u0026nbsp;\u003c/strong\u003e Qi Deng \u0026nbsp; https://orcid.org/0000-0002-3646-4953\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eBinder M, Nandakumar B, Rajkumar SV, Kapoor P, Buadi FK, Dingli D, et al. Mortality trends in multiple myeloma after the introduction of novel therapies in the United States. 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Novel cell and immune engagers in optimizing tumor-specific immunity post-autologous transplantation in multiple myeloma. Transplant Cell Ther. 2022;28(2):61-69.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-translational-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jtrm","sideBox":"Learn more about [Journal of Translational Medicine](http://translational-medicine.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/jtrm/default.aspx","title":"Journal of Translational Medicine","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Relapsed, Refractory, Multiple myeloma, Extramedullary disease, Anti-BCMA CAR-T, Autologous hematopoietic stem cell transplantation","lastPublishedDoi":"10.21203/rs.3.rs-8582254/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8582254/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eRelapsed/refractory multiple myeloma (R/R MM) with extramedullary disease (EMD) carries a poor prognosis. Responses to current therapies, including autologous stem cell transplantation (auto-HSCT) and chimeric antigen receptor T-cell (CAR-T) therapy, remains unsatisfactory, or with frequent early progression despite initial response.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eEighteen patients with R/R MM with EMD were enrolled in clinical trials evaluating anti-BCMA CAR-T therapy. Of these, eight patients were treated with ASCT in combination (T-C group), while the remaining ten underwent CAR-T therapy alone (C group). We systematically compared clinical responses, CAR-T cell expansion kinetics, T-cell subset profiles, serum interleukin-6 (IL-6) levels, treatment-related toxicities, and long-term outcomes between the two cohorts.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eIn the T-C group, all 8 patients achieved an overall response (ORR) based on combined hematologic and imaging assessments of EMD. In contrast, among the 10 patients in the C group, 8 met hematologic criteria for ORR, but only 6 demonstrated radiographic response in EMD lesions. Progression-free survival (PFS) and overall survival (OS) were markedly improved in the T-C group. This cohort also exhibited higher peak levels of both CAR-T cells and interleukin-6 (IL-6). On day 28 post-infusion, the proportion of CD3⁺CD4⁺ T cells were significantly greater in the T-C group. While cytokine release syndrome (CRS) tended to be more severe in this group, the incidence and severity of immune effector cell-associated neurotoxicity syndrome (ICANS) were comparable between groups. Hematologic recovery was delayed in the T-C group, and three of eight patients developed poor graft function.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eWhen followed up for more than 3 years, the combination of anti-BCMA CAR-T cell therapy and auto-HSCT was associated with improved PFS and OS in R/R MM patient with EMD. Moreover, CD4⁺ T cell dynamics might be associated with the durable clinical response observed in the T-C group. Although the T-C group experienced higher-grade CRS and more prolonged hematologic toxicity, no treatment-related deaths due to treatment-related toxicities were observed. (Trial registration: \u003cem\u003eChiCTR2000033925\u003c/em\u003e)\u003c/p\u003e","manuscriptTitle":"Long-term follow-up results of anti-BCMA CAR-T cell therapy combined with autologous hematopoietic stem cell transplantation in relapsed/refractory multiple myeloma with extramedullary disease","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-21 03:58:43","doi":"10.21203/rs.3.rs-8582254/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2026-01-17T15:05:51+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-15T14:01:13+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-14T13:20:37+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Translational Medicine","date":"2026-01-12T08:26:09+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-translational-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jtrm","sideBox":"Learn more about [Journal of Translational Medicine](http://translational-medicine.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/jtrm/default.aspx","title":"Journal of Translational Medicine","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"512a3cd0-9646-4501-be57-1be3b3326b19","owner":[],"postedDate":"January 21st, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-11T17:14:05+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-21 03:58:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8582254","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8582254","identity":"rs-8582254","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}