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Clin Exp Med
Clin Exp Med
Clinical and Experimental Medicine
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10.1007/s10238-024-01477-y
Research
Enhanced platelet function through CAR-T cell therapy in relapsed/refractory multiple myeloma
Ma Ruixue 12
Zhang Qi 13
Liu Yang 13
Li Hujun 13
Chen Huimin 13
Zhang Qianqian 13
Qiao Jianlin 13
Qi Kunming 13
Shen Guifang 4
Sun Cai 13
Song Xuguang 13
Cao Jiang 13
Cheng Hai 13
Zhu Feng 13
Yan Zhiling 13
Sang Wei 13
Li Depeng 13
Sun Haiying 13
Zheng Junnian 56
Li Zhenyu lizhenyumd@163.com

13
Xu Kailin lihmd@163.com

13
http://orcid.org/0000-0001-5480-6577
Chen Wei feihu0808@163.com

137
1 grid.413389.4 0000 0004 1758 1622 Department of Hematology, The Affiliated Hospital of Xuzhou Medical University, No. 99 West Huaihai Road, Xuzhou, 221002 Jiangsu China
2 grid.24516.34 0000000123704535 Department of Hematology, Shanghai Fourth People’s Hospital, School of Medicine, Tongji University, 200434 Shanghai, China
3 https://ror.org/035y7a716 grid.413458.f 0000 0000 9330 9891 Blood Diseases Institute, Xuzhou Medical University, Xuzhou, Jiangsu China
4 grid.413389.4 0000 0004 1758 1622 Health Screening Center, The Affiliated Hospital of Xuzhou Medical University, Xuzhou, China
5 https://ror.org/035y7a716 grid.413458.f 0000 0000 9330 9891 Cancer Institute, Xuzhou Medical University, Xuzhou, Jiangsu China
6 grid.413389.4 0000 0004 1758 1622 Center of Clinical Oncology, The Affiliated Hospital of Xuzhou Medical University, Xuzhou, Jiangsu China
7 grid.89957.3a 0000 0000 9255 8984 The Affiliated Suqian First People’s Hospital of Nanjing Medical University, Suqian, Jiangsu China
4 9 2024
4 9 2024
2024
24 1 2107 7 2024
21 8 2024
© The Author(s) 2024
2024
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The influence of chimeric antigen receptor T (CAR-T) cell therapy on platelet function in relapsed/refractory (R/R) multiple myeloma (MM) has not been thoroughly investigated. Our cohort comprised fifty MM patients treated with CAR-T cells. The mean platelet closure time (PCT) induced by collagen/adenosine diphosphate (CADP) in peripheral blood was significantly prolonged before lymphodepletion (195.24 ± 11.740 s) and notably reduced post-CAR-T cell therapy (128.02 ± 5.60 s), with a statistically significant improvement (67.22, 95% CI 46.91–87.53, P < 0.001). This post-treatment PCT was not significantly different from that of healthy controls (10.64, 95% CI 1.11–22.40, P > 0.05). Furthermore, a pronounced enhancement in PCT was observed in patients with a response greater than partial remission (PR) following CAR-T cell infusion compared to pre-treatment values (P < 0.001). An extended PCT was also associated with a less favorable remission status. In patients with cytokine release syndrome (CRS) grades 0–2, those with a PCT over 240.5 s exhibited a shorter progression-free survival (PFS), with median PFS times of 10.2 months for the PCT > 240.5 s group versus 22.0 months for the PCT ≤ 240.5 s group. Multivariate analysis revealed that a PCT value exceeding 240.5 s is an independent prognostic factor for overall survival (OS) in R/R MM patients after CAR-T cell therapy. The study demonstrates that CAR-T cell therapy enhances platelet function in R/R MM patients, and PCT emerges as a potential prognostic biomarker for the efficacy of CAR-T cell therapy.

Supplementary Information

The online version contains supplementary material available at 10.1007/s10238-024-01477-y.

Keywords

Relapsed/refractory
Multiple myeloma
Chimeric antigen receptor T cell
Platelet function
Prognosis
Suqian Key Laboratory of HematologyM202111 Chen Wei http://dx.doi.org/10.13039/501100004608 Natural Science Foundation of Jiangsu Province BK20221218 Chen Wei Development Fund of Affiliated Hospital of Xuzhou Medical UniversityXYFY2021001 Chen Wei issue-copyright-statement© Springer Nature Switzerland AG 2024
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pmcIntroduction

Anti-B-cell maturation antigen (BCMA) chimeric antigen receptor T (CAR-T) cell therapy is a new immune cell therapy that has achieved great efficacy in patients with relapse/refractory (R/R) multiple myeloma (MM) in recent years [1–3]. In our center, after infusion with the anti-BCMA or anti-GPRC5D CAR-T cells, patients achieved median progression-free survival (PFS) of 18.3 months and a manageable long-term safety profile [4, 5].

CAR-T therapy is usually associated with significant pancytopenia in recipients following lymphodepletion [6, 7]. Furthermore, thrombocytopenia is linked to poor prognosis in MM [8]. Patients received CAR-T therapy usually experienced delayed platelet recovery, as high as 36% patients of persistent grade ≥ 3 thrombocytopenia after day 21 has been reported by Li et al. [9]. Of patients experienced prolonged significant thrombocytopenia significantly impair patients’ quality of life, and had a shorter 1-year overall survival (OS) [10]. Moreover, platelet functions are affected in MM mainly by the inhibitory effect of paraprotein [11]. An increase in platelet reactivity was demonstrated post-lenalidomide treatment compared to pre-treatment [12]. On diagnosis, significantly prolonged median platelet closure time (PCT) in PFA-100 was seen in the MM patients, and therapy with thalidomide and dexamethasone caused marked shortening of PCT [13].

Platelet function analyzer simulates the process of platelet adhesion and aggregation after vascular injury in vivo according to the principle of external hemodynamics and reflects the platelet function using PCT [14, 15]. Previous study showed that patients with MM had a longer PCT than healthy controls [13]. In order to evaluate the effect of CAR-T cell therapy on PCT in patients with R/R MM, the present study examined the changes in PCT of patients with R/R MM before and after CAR-T cell therapy.

Patients and methods

Patients

We retrospectively collected the data on patients with R/R MM who received CAR-T cell therapy at the Hematology Department of Affiliated Hospital of Xuzhou Medical University from April 2019 to December 2021. All the enrolled patients received lymphodepletion with fludarabine (three daily doses of 30 mg/m2) and cyclophosphamide (one daily dose of 750 mg/m2) (FC) before CAR-T cell infusion. Patients received anti-BCMA CAR-T or G protein-coupled receptor class C group 5 member D (GPRC5D) CAR-T cell infusion. The details are provided in Table 1 and Supplemental Table 1). A total of 50 patients were enrolled in the healthy control group. The baseline data of the healthy control group and the group undergoing CAR-T cell therapy are shown in Supplemental Table 2. There was no statistically significant difference, and the two groups of patients were comparable. The study protocol was approved by the human studies review board at the Affiliated Hospital of Xuzhou Medical University (ClinicalTrials.gov # NCT02782351). Table 1 Characteristics, and clinical and laboratory data of patients with R/R MM before treatment

Characteristics	n %	
Sex	
 Male	31 (62)	
 Female	19 (38)	
MM type	
 IgG	24(48)	
 IgA	9 (18)	
 IgD	6 (12)	
Light chain type	10(20)	
 Non-secretory	1 (2)	
International Staging System (n)	
 I	13 (26)	
 II	21 (42)	
 III	16 (32)	
CAR-T cell constructs	
 Anti-CD19	2 (4)	
 Anti-BCMA	13 (26)	
 Anti-CD19 + Anti-BCMA	21 (42)	
 Anti-BCMA + Anti-CD138	3 (6)	
 Anti-GPRC5D	11 (22)	
Stem cell transplantation	14 (28)	
High-risk cytogenetics	17 (41)	
Daratumumab	5 (10)	
Proteasome inhibitor	50 (100)	
Immunomodulatory drugs	50 (100)	
The median lines of previous therapy	4 (3,5)	
R/R MM: relapse/refractory multiple myeloma; chimeric antigen receptor T: CAR-T; Anti-BCMA: anti–B-cell maturation antigen; Anti-CD19 + Anti-BCMA: the combination of anti–B-cell maturation antigen (BCMA) and anti-CD19; Anti-BCMA + Anti-CD138: the combination of anti–B-cell maturation antigen (BCMA) and anti-CD138; high-risk cytogenetics were defined as del(17p), t(14;16), t(14;20), or t(4;14)

Table 2 Correlation analysis between platelet closure time and clinical data

Characteristics	CADP PCT	
r	P	
Age	 − 0.015	0.919	
Sex	 − 0.014	0.921	
PT	0.054	0.780	
APTT	0.157	0.276	
FIB	 − 0.218	0.129	
TT	0.336	0.027*	
IL-6	 − 0.132	0.380	
CRP	0.136	0.357	
Ferritin	 − 0.195	0.190	
Growth factor	 − 0.367	0.102	
(Exclude IgG type) IgG	0.172	0.434	
(Exclude IgA type) IgA	 − 0.394	0.017*	
(Exclude IgM type) IgM	 − 0.404	0.006*	
(Exclude κ-type) κ	0.059	0.822	
(Exclude λ-type) λ	 − 0.432	0.011*	
B microglobulin	 − 0.013	0.935	
Time from diagnosis to CAR- T cell infusion	 − 0.071	0.625	
CAR-T constructs	0.068	0.639	
Previous lines of therapy	 − 0.026	0.857	
Stem cell transplantation	 − 0.022	0.881	
CRS stage	0.289	0.042*	
ISS stage	0.360	0.010*	
PT prothrombin time, APTT activated partial thromboplastin time, FIB fibrinogen, TT thrombin time, IL-6 interleukin 6, CRP C-reactive protein, CAR-T chimeric antigen receptor T, DS stage Durie–Salmon stage, ISS International Staging System. Statistical analysis: Spearman correlation analysis

*P < 0.05

Test method and instruments to measure platelet closure time

A platelet function analyzer-200 (PFA-200), a Siemens Medical Diagnostic Products (Siemens, Shanghai, China)-approved device, was used to measure PCT. The PFA-200 system comprises a reservoir, capillary, and cartridges containing collagen/adenosine diphosphate (CADP). When the sample (blood) with platelets is added to the reservoir, the platelets are activated by CADP, and then adhere and aggregate to form a plug that can gradually obstruct the capillary hole; the closure time (CT) represents platelet function, which is measured in seconds. According to the manufacturer, the reference range of CADP-induced PCT is 68–121 s. We categorized patients as “prolonged CT” if PCT was longer than the reference ranges and as “normal PCT” if both were within the reference ranges. PCT was retested at 30 days after the CAR-T cells infusion, and patient efficacy was assessed at this time point.

Data observation

The clinical data collected included patients’ age, sex, platelet function closure time, white blood cell (WBC) count, hemoglobin concentration, red blood cell (RBC) distribution width (RDW), platelet count, platelet distribution width (PDW), prothrombin time (PT), activated partial clotting enzyme live time (APTT), fibrinogen (FIB), thrombin time (TT), Durie–Salmon (DS) staging, and International Staging System (ISS) staging. We acquired patient’s follow-up through reviewing in-patient medical records and making phone calls.

In this study, response to treatment was defined as a complete response (CR), a stringent complete response (sCR), a very good partial response (VGPR), and a partial response (PR). For patients with R/R MM, diagnosis, staging, and efficacy evaluation were based on the International Myeloma Working Group (IMWG) guidelines [16, 17]. Cytokine release syndrome (CRS) and the immune effector cell-associated neurotoxicity syndrome (ICANS) were graded according to criteria which were proposed by Lee et al. [18, 19]. Progression-free survival (PFS) is defined as the time from CAR-T cell infusion to disease progression or death from any cause; overall survival (OS) is defined as the time from CAR-T cell infusion to death from any cause.

Statistical analysis

SPSS version 26.0 (IBM Corp., Armonk, NY, USA) and GraphPad Prism version 9 (GraphPad Inc., La Jolla, CA, USA) software were used to process the data. Continuous variables presenting normality were expressed as the mean ± SD (range), comparison of means between two groups was made using an independent t-test, and a paired t-test was used to assess the statistical significance of the difference between two population means in a study involving paired samples. Continuous variables presenting non-normality were expressed as the median (range), and stratified data were analyzed using the Cochran–Mantel–Haenszel test. Spearman correlation analysis was performed to test relationships between parameters. Follow-up time, overall survival (OS), and progression-free survival (PFS) were estimated using the Kaplan–Meier method. A receiver operating characteristic (ROC) curve was used to determine the optimal cutoff values for PCT. All tests were two-sided, and P < 0.05 was considered statistically significant.

Results

Patient characteristics

We conducted a retrospective analysis, compiling data on 81 individuals with R/R MM who received CAR-T cell therapy and 74 healthy controls over a period from January 2018 to February 2023. After applying the established inclusion and exclusion criteria, our final study group consisted of 50 R/R MM patients treated with CAR-T cell therapy and an equal number of healthy controls for comparison. Within the patient group, there were 31 males and 19 females, with a median age of 57.5 years (range: 41–72 years). The immunoglobulin types included 24 cases of IgG, 9 of IgA, 6 of IgD, 1 non-secreting type, and 10 light chain types, with further breakdown into 2 patients with λ-type light chain disease and 8 patients with κ-type light chain disease. A detailed overview of the patient characteristics is presented in Table 1.

CAR-T cell therapy improved PCT

The average PCT was notably longer in the pre-treatment for patients (195.24 ± 11.740 s) when juxtaposed with the healthy control group (117.38 ± 1.690 s) (95% CI 53.93–101.80, P < 0.001). A significant reduction in PCT post-treatment (128.02 ± 5.60 s) was observed (95% CI 46.91–87.53, P < 0.001). Post-treatment, PCT values among patients were not statistically different from those of the healthy control group (95% CI − 1.11 to 22.40, P > 0.05) (Fig. 1A).Fig. 1 Comparative analysis of platelet closure time (PCT) (A) and platelet count (B) in relapsed/refractory multiple myeloma (R/R MM) patients undergoing CAR-T cell therapy versus a control group. Statistically significant differences are denoted by P < 0.001; “ns” indicates no statistical significance. We utilized an independent t-test for the comparisons between each treatment group and the healthy control group. For the before and after treatment comparison, recognizing the paired nature of the samples, we employed a paired samples t-test

To assess the influence of platelet levels on PCT, we compared the average platelet count before and after CAR-T cell infusion. No significant difference was observed between the pre-infusion platelet levels of patients (145.41 ± 9.84 × 109/L) and the control group (129.70 ± 4.26 × 109/L) (P > 0.05). Similarly, no statistically significant change was noted in platelet levels between the pre-infusion (145.41 ± 9.84 × 109/L) and post-infusion (130.93 ± 9.99 × 109/L) (P > 0.05). Furthermore, no significant disparity in platelet levels was found between patients post-infusion and the healthy control group (P > 0.05) (Fig. 1B).

Correlation analysis of the relationship of PCT with clinical data

We explore the correlation between PCT and various clinical parameters. We identified subtypes, International Staging System (ISS) stage, thrombin time (TT), and CRS stage as factors associated with PCT. Negative correlations were observed between PCT and IgA (r = − 0.394, P = 0.017), or IgM (r = − 0.432, P = 0.011), while positive correlations were noted with ISS stage (r = 0.360, P = 0.010), (r = 0.336, P = 0.027), and CRS stage (r = 0.289, P = 0.042), as detailed in Table 2.

The PCT of patients with advanced ISS stage disease showed potential for improvement following CAR-T cell therapy. Prior to CAR-T cell infusion, the median PCT for stage III disease was notably higher at 230.0 s (interquartile range [IQR] 166.8, 291.8) compared to stage I disease at 125.5 s (IQR 114.3, 170.0) (P < 0.05). Post-infusion, the median PCT for stage III disease was reduced to 156.5 s (IQR 124.3, 235.3), yet it remained significantly longer than that of stage I at 116.0 s (IQR 95.8, 150.0) (P < 0.05) and stage II at 120.0 s (IQR 100.0, 147.0) (P < 0.05) (Supplemental Fig. 1).

Correlation between PCT improvement and CAR-T cell infusion effectiveness

Patients in the remission > PR group showed a median PCT of 158.50 s (115.50, 202.25) prior to lymphodepletion (pre-LD), whereas the remission ≤ PR group exhibited a higher median PCT of 220.00 s (146.50, 300.00), indicating a significant statistical difference (P = 0.028). One month post-CAR-T cell infusion, a reassessment revealed a median PCT of 108.50 s (95.50, 127.50) for the > PR group and 155.00 s (122.00, 197.50) for the ≤ PR group (Fig. 2). A significant decrease in PCT was observed in the > PR group after treatment compared to pre-treatment levels (P < 0.001) (Fig. 2). Similarly, for the ≤ PR group, a notable reduction in PCT post-treatment was observed in comparison to pre-treatment values (P < 0.01) (Fig. 2).Fig. 2 Assessment of CADP-induced platelet closure times (CADP PCTs) in patients exhibiting varied levels of remission. The significance of differences is indicated by *P < 0.05; **P < 0.01; ***P < 0.001. For the statistical analysis, we employed an independent samples t-test to compare the platelet counts between the groups with remission greater than partial response (> PR) and those with remission less than or equal to partial response (≤ PR), both before and after treatment initiation. Additionally, to evaluate the changes within each group from before to after treatment, we utilized a paired samples t-test on the before and after treatment samples

Association of PCT improvement and favorable survival outcomes

As of the cutoff date (February 28, 2023), the median follow-up duration was 23.0 months (95% CI, 15.2–0.8). The median PFS and OS for all participants were 17.5 months (95% CI 13.3–21.7) and not reached, respectively (Fig. 3). A receiver operating characteristic (ROC) curve was utilized to determine the optimal cutoff value for survival distributions based on PCT, identified as 240.5 s (AUC 0.713, 95% CI 0.510–0.915, P = 0.039). Patients with a PCT exceeding 240.5 s experienced inferior PFS compared to those with PCT at or below 240.5 s, with medians of 12.0 months versus 18.5 months, respectively (P = 0.013). In terms of OS, patients with higher PCT also had a worse prognosis compared to those with lower PCT, with medians of 34.0 months versus not reached (P = 0.001) (Fig. 4). Univariate Cox analysis indicated an increased risk of death for high PCT versus low PCT (HR 6.282, 95% CI 1.266–31.179, P = 0.025), along with other factors such as hemoglobin levels, activated partial thromboplastin time (APTT), and history of autologous hematopoietic stem cell transplantation. Multivariate analysis confirmed that a PCT value above 240.5 s (HR 13.417, 95% CI 1.545–116.495, P = 0.019) is an independent risk factor for OS in R/R MM patients treated with CAR-T cells. A summary of the univariate and multivariable Cox analysis results is found in Table 3.Fig. 3 Examination of progression-free survival (A) and overall survival (B) in the R/R MM patient cohort. The 95% CI represents the 95% confidence interval. Survival curves were constructed using the Kaplan–Meier method, with log-rank tests employed to assess differences in survival probabilities between groups

Fig. 4 Patient prognosis post-CAR-T cell therapy. A Progression-free survival (PFS), B overall survival (OS) for the entire patient cohort, stratified by CADP-induced platelet closure time (PCT) levels prior to CAR-T cell infusion. Survival curves were constructed using the Kaplan–Meier method, with log-rank tests employed to assess differences in survival probabilities between groups

Table 3 Univariate and multivariate Cox regression analyses for OS

Characteristics	Univariate	Multivariate	
HR (95% CI)	P-value	HR (95% CI)	P-value	
Sex (female vs. male)	0.853 (0.212–3.435)	0.823			
Age (> 65y vs. ≤ 65y)	0.043 (0.000–1335.577)	0.550			
CADP CT (> 240 s vs. ≤ 240 s)	8.955 (1.892–42.377)	0.006	13.417 (1.545–116.495)	0.019	
HGB(≥ 100 g/L vs. < 100 g/L)	5.108 (1.061–24.589)	0.042	2.221 (0.403–12.243)	0.360	
PLT (≥ 50 × 109 g/L vs. < 50 × 109 g/L)	1.280 (0.159–10.287)	0.816			
PT (≥ 12.5 s vs. < 12.5 s)	9.460 (1.052–85.108)	0.045	17.377 (0.825–365.997)	0.066	
APTT (≥ 36.5 s vs. < 36.5 s)	2.485 (0.599–10.317)	0.210			
Albumin (≥ 40 g/L vs. < 40 g/L)	1.211 (0.310–4.731)	0.783			
LDH (≥ 240 U/L vs. < 240 U/L)	0.270 (0.033–2.176)	0.219			
Calcium (≥ 10.2 mg/dL vs. < 10.2 mg/dL)					
ISS stage	Reference				
 I	0.996 (0.166–5.974)	0.997			
 II	2.742 (0.531–14.151)	0.228			
 III					
CAR-T cell constructs	Reference				
 Anti-BCMA	0.000 (0.000-Inf)	0.971			
 Anti-CD19	1.796 (0.460–7.007)	0.399			
 Anti-CD19 + Anti-BCMA	0.000 (0.000-Inf)	0.978			
 Anti-BCMA + Anti-CD138	0.000 (0.000-Inf)	0.975			
 Anti-GPRC5D					
Chromosomal abnormality	Reference				
 High risk	0.000 (0.000-Inf)	0.989			
 Standard risk	1.929 (0.243–15.292)	0.534			
 Non-assessable	1.630 (0.338–7.861)	0.543			
Previous therapy lines(≥ 3 vs. < 3)	3.894 (1.112–13.635)	0.033	3.266 (0.807–13.216)	0.097	
Previous stem cell transplantation (yes vs. no)	0.853 (0.212–3.435)	0.823			
Characteristics of patients were collected before CAR-T cell therapy. OS overall survival, HR hazard ratio, 95% CI 95% confidence interval, HBG hemoglobin, PLT platelet, PT prothrombin time, APTT activated partial thromboplastin time, LDH lactic dehydrogenase. CAR-T: chimeric antigen receptor T; Inf: infinity; Anti-BCMA: anti–B-cell maturation antigen; Anti-CD19 + Anti-BCMA: the combination of anti–B-cell maturation antigen (BCMA) and anti-CD19; Anti-BCMA + Anti-CD138: the combination of anti–B-cell maturation antigen (BCMA) and anti-CD138

Subgroup analysis revealed that the incidence of hemoglobin levels ≥ 100 g/L at disease onset and ISS stage I was significantly lower in patients with PCT above 240.5 s compared to those with PCT at or below 240.5 s, with respective differences being statistically significant (P = 0.026 and P = 0.049). Conversely, the incidence of lactate dehydrogenase levels ≥ 240 U/L was higher in the PCT > 240.5 s group, although this difference did not reach statistical significance (Table 4). Table 4 Comparison of clinical indicators in Pre-LD CAR-T R/R MM patients with different PCT levels

Characteristics	CADP PCT > 240.5 s	CADP PCT ≤ 240.5 s	P-value	
Sex, n (%)			0.960	
 Female	6 (37.5)	13 (38.2)		
 Male	10 (62.5)	21 (61.8)		
Age, n (%)			0.695	
 > 65y	1 (6.2)	5 (14.7)		
 ≤ 65y	15 (93.8)	29 (85.3)		
HB, n (%)			0.026*	
 ≥ 100 g/L	4 (25.0)	20 (58.8)		
 < 100 g/L	12 (75.0)	14 (41.2)		
PT, n (%)			1.000	
 < 12.5 s	14 (87.5)	31 (91.2)		
 ≥ 12.5 s	2 (12.5)	3 (8.8)		
APTT, n (%)			1.000	
 < 36.5 s	16 (100.0)	33 (97.1)		
 ≥ 36.5 s	0 (0.0)	1 (2.9)		
TT, n (%)			0.217	
 ≥ 16.6 s	8 (61.5)	11 (40.7)		
 < 16.6 s	5 (38.5)	16 (59.3)		
ALB, n (%)			0.525	
 ≥ 40 g/L	6 (37.5)	16 (47.1)		
 < 40 g/L	10 (62.5)	18 (52.9)		
LDH, n (%)			0.226	
 ≥ 240 U/L	6 (46.2)	4 (20.0)		
 < 240 U/L	7 (53.8)	16 (80.0)		
Ca, n (%)			0.963	
 < 10.2 mg/dL	11 (73.3)	26 (78.8)		
 ≥ 10.2 mg/dL	4 (26.7)	7 (21.2)		
ISS stage, n (%)			0.049*	
 I	1 (6.3)	12 (35.3)		
 II	7 (43.8)	14 (41.2)		
 III	8 (50.0)	8 (23.5)		
Chi-square test.

*P < 0.05

Subgroup survival analysis among patients with varying degrees of CRS indicated that in the CRS 0–2 grade group, patients with PCT above 240.5 s had a worse prognosis for PFS and OS compared to those with PCT at or below 240.5 s, with medians of 10.2 months versus 22.0 months for PFS (P = 0.002) and 16.2 months versus not reached for OS (P < 0.001) (Fig. 5A, B). In contrast, for patients with CRS grades 3–5, no significant differences in PFS and OS were observed between those with PCT above 240.5 s and those with PCT at or below 240.5 s, with medians of 12.0 months versus 14.4 months for PFS (P = 0.954) and 19.2 months versus not reached for OS (P = 0.829) (Fig. 5C, D).Fig. 5 Impact of CADP-induced platelet closure time (PCT) prior to CAR-T cell infusion on patient survival outcomes. A Progression-free survival (PFS) rates among patients with cytokine release syndrome (CRS) grades 0–2, stratified by CADP-induced PCT levels prior to CAR-T cell therapy initiation. B Overall survival (OS) rates among patients with CRS grades 0–2, categorized by CADP-induced PCT levels before the commencement of CAR-T cell treatment. C PFS rates for patients experiencing CRS grades 3–5, differentiated by the CADP-induced PCT levels recorded before CAR-T cell infusion. D OS rates for patients with CRS at grades 3–5, with stratification based on the CADP-induced PCT levels measured prior to undergoing CAR-T cell therapy. Survival curves were constructed using the Kaplan–Meier method, with log-rank tests employed to assess differences in survival probabilities between groups

Discussion

In our retrospective analysis, we examined the platelet function in patients with R/R MM. We specifically evaluated the PCT induced by CADP and discovered that patients with R/R MM exhibited a significantly longer PCT compared to a healthy control group prior to CAR-T cell infusion. Notably, the PCT was observed to improve following CAR-T cell infusion, suggesting a potential link between PCT improvement and factors such as immunoglobulin type, TT, ISS stage, and CRS grade. Furthermore, our findings indicate that PCT may serve as an indicator of the effectiveness of CAR-T cell therapy.

Previous research has established that platelet function is compromised in the context of myocardial infarction and can act as an independent predictor of the condition’s severity, contributing to the risk stratification of patients with myocardial infarction [20]. In our study, paired-sample t-tests were utilized to compare PCT levels before and after treatment, revealing a significant reduction post-treatment. Robak et al. [13] reported longer PCT in MM patients treated with the thalidomide/dexamethasone regimen compared to healthy controls, while they observed a significant decrease in PCT levels 4 weeks post-chemotherapy. Our results corroborate the notion that CAR-T cell therapy can improve PCT. Additionally, we found no significant difference in PCT between patients post-CAR-T cell therapy and healthy controls, nor in platelet counts before and after treatment compared to controls, which may imply a significant post-therapy improvement in PCT. The underlying mechanism might involve the sequestration of abnormal immunoglobulins secreted by MM patients on platelet surfaces, leading to diminished platelet adhesion and aggregation [21, 22]. We consider both the reduction in tumor burden and the CAR-T therapy’s effect on the surrounding environment to be significant factors contributing to PCT improvement post-CAR-T infusion. The sequestration of abnormal immunoglobulins on platelet surfaces could be mitigated by the decrease in these immunoglobulins, thus contributing to the observed PCT improvement. Additionally, CAR-T therapy may influence the synthesis of platelet-activating factors.

Our study further revealed a correlation between improved PCT levels in patients with remissions > PR. Spearman correlation analysis demonstrated a positive association between PCT and TT, where TT represents the time required for the conversion of fibrinogen to fibrin. An elongated TT signifies an increased risk of bleeding for the patient [23]. The reduction in PCT can substantially mitigate the bleeding risk, offering clinical treatment guidance. We have previously reported a high incidence of coagulation disorders post-CAR-T cell infusion, including prolonged activated partial thromboplastin time (APTT), which is indicative of a poor prognosis and suggests that elevated APTT levels are linked to an increased risk of mortality [24].

We observed an elongation of PCT in correlation with the progression of ISS stages. This prolongation might be attributed to the aberrant functionality of platelets in malignant blood disorders, potentially linked to the altered membrane receptors and the heightened synthesis and release of factors pivotal to platelet function [25]. These intricate interactions suggest a role for platelets in the pathogenesis of MM, indicating a close association with the disease’s status [14, 26].

In our investigation, we observed no substantial correlation between the duration of cytopenias and CRS. Our findings indicate that patients with an elongated PCT following CAR-T cell therapy experienced a diminished PFS and OS when juxtaposed with those who exhibited a reduced PCT. This observation suggests that PCT may function as a prognostic biomarker, insinuating that individuals with an extended PCT are potentially at a higher risk of unfavorable outcomes. It accentuates the significance of vigilant PCT level surveillance preceding the administration of CAR-T cell therapy.

Furthermore, survival analysis indicated that within the group of patients experiencing mild-to-moderate CRS (grades 0–2), those with a PCT exceeding 240.5 s had inferior PFS and OS. This suggests that PCT might be instrumental in identifying patients at risk of a poor prognosis, particularly among those with mild-to-moderate CRS. The interplay between CRS, an immune-mediated condition characterized by inflammation, and platelets, which play a significant role in inflammatory processes, could be a contributing factor [27]. Cytokines can disrupt platelet function, and CAR-T therapy might affect the synthesis of platelet-activating factors in MM patients, leading to PCT prolongation [28, 29]. This is further supported by the finding that PCT was significantly longer in patients with severe CRS reactions (P < 0.05). Additionally, the activation of abnormal immunoglobulins, alongside the suppression of normal bone marrow plasma cells and humoral immune deficiencies, could also result in PCT prolongation.

The study's limitations include its retrospective nature, single-center design, and limited sample size. Future research involving larger cohorts is warranted to validate these findings.

In summary, our study suggests that CAR-T cell therapy can enhance platelet function, and PCT can serve as a valuable parameter for evaluating the efficacy of CAR-T cell infusion.

Supplementary Information

Below is the link to the electronic supplementary material.Supplementary file1 (DOC 130 KB)

Acknowledgements

We thank numerous individuals who participated in this study.

Author contributions

Wei Chen, Kailin Xu, and Zhenyu Li designed the research. Ruixue Ma, Qianqian Zhang, and Huimin Chen collected the data. Wei Chen and Ruixue Ma designed the figures, analyzed and interrupted the results, wrote the first draft of manuscript. Jianlin Qiao wrote the revised manuscript. Jiang Cao, Hai Cheng, Feng Zhu, Zhiling Yan, Wei Sang, Depeng Li, Haiying Sun provided the detailed information of patients. Qi Zhang, Yang Liu, Kunming Qi, Cai Sun, Xuguang Song, and Junnian Zheng provided the blood sample of patients. Guifang Shen provided the blood sample of healthy controls. All authors were involved at each stage of manuscript preparation and approved the final version.

Funding

This study was supported by grants from Suqian Key Laboratory of Hematology (M202111). Natural Science Foundation of Jiangsu Province (BK20161177, BK20221218), Project of the Jiangsu Provincial Health and Family Planning Commission/ international (JSH-2017-008), China Postdoctoral Science Foundation project (2016M590507, 2018T110557), Xuzhou clinical Backbone Training Project (2018GG006), Development Fund of Affiliated Hospital of Xuzhou Medical University (XYFY2021001).

Data availability statement

No datasets were generated or analyzed during the current study.

Declarations

Conflict of interest

The authors declare no competing interests.

Ethics statement

The study protocol was approved by the human studies review board at the Affiliated Hospital of Xuzhou Medical University (ClinicalTrials.gov # NCT02782351). All patients provided written informed consent before received CAR-T cell therapy. The clinical investigation was conducted according to the principles of the Declaration of Helsinki.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Ruixue Ma, Qi Zhang and Yang Liu have contributed equally to this work.
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