Hypomagnesemia in Large B cell Lymphoma patients receiving CAR T cell therapy correlates with immune dysregulation and decreased PFS: results from the ZUMA-1 trial and Mayo Clinic cohort

doi:10.21203/rs.3.rs-4889200/v1

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Research Article

Hypomagnesemia in Large B cell Lymphoma patients receiving CAR T cell therapy correlates with immune dysregulation and decreased PFS: results from the ZUMA-1 trial and Mayo Clinic cohort

https://doi.org/10.21203/rs.3.rs-4889200/v1

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Background

Hypomagnesemia has been correlated with inferior outcomes in patients with large B cell lymphoma (LBCL) undergoing stem cell transplants. As T-cell and myeloid cell dysfunction have been associated with low magnesium conditions, we investigated whether serum magnesium (Mg) levels could predict clinical outcomes in LBCL patients who received chimeric antigen receptor T-cell therapy.

Methods

Patients with LBCL who received axi-cel under the ZUMA-1 trial or as FDA approved therapy at Mayo Clinic were examined. Serum samples were obtained at specified time points and cytokine analysis was performed. Single cell RNA sequencing was performed on peripheral blood mononuclear cells. The Student T-test, Kruskal Wallis, or Fisher's Exact Tests were used to compare differences in demographics across Mg levels. Survival curves were plotted using the Kaplan-Meier methodology and compared using the Wilcoxon test.

Results

We found that hypomagnesemia before lymphodepletion chemotherapy predicted for inferior progression-free and overall survival in the pivotal study ZUMA-1 (NCT02348216). These results were validated in an independent cohort of LBCL patients receiving axicabtagene ciloleucel (axi-cel) at Mayo Clinic. Hypomagnesemia correlated with increased inflammatory serum markers and cytokine levels including ferritin, IL-6, IL1Ra, IL-8, and MIP1a. scRNAseq analysis unveiled altered immune interactions between monocytes and T cells with a concordant immune suppressive transcriptome.

Conclusions

Hypomagnesemia at the time of CAR-T infusion associates with an unfavorable inflammatory profile and decreased response and survival in LBCL patients receiving axi-cel. These findings suggest a potentially actionable prognostic factor for patients with large cell lymphoma undergoing CAR-T.

Lymphoma

magnesium

cytokines

survival analysis

CAR-T

Magnesium (Mg) is essential for regulating cell proliferation and immunity (1, 2). Low serum Mg levels are common in hospitalized patients and associate with all-cause mortality(3). Although monitoring serum Mg is commonly performed in patients undergoing platinum-based chemotherapy, there are no established guidelines for the target Mg level in oncologic patients as there are for those with cardiac conditions (4). The relevance of Mg to lymphoma is derived from several lines of evidence. The discovery of X-linked immunodeficiency with Mg defect, EBV, and neoplasia (known as XMEN disease) suggests a role for Mg deficiency in the development of hematologic malignancies (5). Mg influx to the intracellular space is essential to coordinate T cell activation. In patients with XMEN, a transport channel mutation impairs Mg influx, negatively affecting cytolytic responses. Recent data demonstrate that a novel gene editing approach in XMEN can restore Mg transporter expression and, in turn, the function of CD8⁺ T cells and natural killer cells (6). Subsequent studies found that EBV viral loads were inversely related to plasma Mg levels suggesting that reduced Mg levels may impair T cell function, leading to uncontrolled viremia (7). There are also several studies supporting the prognostic role of hypomagnesemia. High posttransplant Mg levels have been associated with a lower incidence of relapse in patients with acute myeloid leukemia undergoing allogeneic stem cell transplant (SCT)(8). We have reported that serum Mg levels below normal and even in the low normal range (1.7–1.9 mg/dL) at the time of autologous SCT for patients with relapsed diffuse large B cell lymphoma (DLBCL) is associated with an inferior progression-free survival (PFS) and overall survival (OS) post-transplant (9). We also identified that hypomagnesemia at baseline is linked with inferior OS in patients with Burkitt Lymphoma (10).

Chimeric antigen receptor T-cell therapy (CAR-T) is a relatively new immune therapy where autologous T cells are engineered to express a chimeric antigen receptor targeting a cell surface antigen of interest (e.g., CD19)(11). Based on the role of Mg in T-cell functions and our findings in SCT, we investigated whether serum Mg of patients with large B cell lymphoma (LBCL) at the time of lymphodepletion for CAR-T cells could influence their clinical response and outcome. We also explored the impact of hypomagnesemia on the composition and function of peripheral immune cells, which may partially explain prognosis. Since Axicabtagene ciloleucel (axi-cel, Yescarta) was the first FDA-approved and most used CAR-T therapy, we focused on LBCL patients receiving axi-cel in the registrational study ZUMA-1 (NCT02348216) and an independent validation patient cohort in our standard of care (SOC) practice at Mayo Clinic.

Clinical data and sample collection

Baseline demographics, cytokine release syndrome, neurotoxicity, clinical response, EFS, OS, clinical labs, and correlative blood samples for chemokines and cytokines were collected from subjects enrolled in cohorts 1 and 2 of the ZUMA-1 study (12, 13). These patients are referred to as ZUMA-1 patients. Clinical data from the medical records of patients with LBCL who received SOC axi-cel between January 2018 and May 2020 at Mayo Clinic Rochester, MN, were reviewed after the Mayo Clinic Investigational Review Board (IRB) approval. They are referred to SOC patients. Serum Mg levels (normal range, 1.7–2.3 mg/dL) were collected on day − 5 before the start of LD chemotherapy. Baseline demographics, clinical response, disease progression date, and date of death were collected. Lugano lymphoma response criteria were used to assess clinical response (14). Additional blood was assayed for single-cell RNA sequencing for a subset of SOC subjects who consented to a Mayo IRB-approved biobank protocol.

Cytokine analysis from blood

Serum samples were obtained on Day − 5 (pre-lymphodepletion), Day 0 (post-conditioning and pre-axi-cel infusion) and at peak CAR-T expansion (typically day 7 or 14). A panel of analytes representing major categories of immune function were measured in serum by Meso Scale Discovery®. Spearman correlation analysis was performed to examine the relationship between cytokine levels at the indicated timepoints with baseline Mg level.

PBMNC isolation and scRNAseq

Blood samples from 13 sequentially relapsed/refractoryLBCL patient receiving CART cell therapy and enrolled to the biobank protocol which had either blood Mg level above 2.0 mg/dL and had durable complete remission for at least six months or more (Mg^high) or Mg level below 2.0 mg/dL and had progressive disease within six months (Mg^low) were used for scRNAseq. Peripheral blood mononuclear cells (PBMNC) were isolated from heparinized blood samples using Lymphoprep (Stemcell Technologies) density gradient centrifugation according to the manufacturer's instructions and cryopreserved in CryoStor CS10 (BioLife Solutions, Inc.). On the day of the single cell collection for RNA-sequencing, the cells were thawed for 1 min and washed with buffer (PBS with 2% BSA and 2 mM EDTA). PBMNC were counted and processed immediately for single-cell capture and 5' RNA-sequencing (10X Genomic). Gene expression libraries were prepared for each sample according to the manufacturer's protocol (10X Genomics). All libraries were sequenced using 10,000 cells to achieve a minimum of 5,000 cell reads per cell for gene expression.

scRNAseq analysis

Single-cell RNA sequencing (scRNA-Seq) data are aligned and quantified using 10X Genomics Cell Ranger Software Suite (v4.0) against the human reference genome (hg38). Supervised single-cell analysis was performed by mapping scRNA-seq datasets to the annotated human PBMC reference using the Seurat package (v4.0) (Hao YH, Hao S, et al., 2021). Differential gene expression analysis was performed between the CR High-Mg group and PD Low-Mg group at cluster level at different timepoints using the FindMarkers function by Seurat. The differentially expressed gene lists were used for GSEA (v4.2.3) analysis using -log10(p-value)*log2FC as the ranking score. CellChat (v1.5.0, Vu R et al., 2022) was used to perform cell-cell-interaction analysis separately for each condition at each timepoint by identifying over-expressed ligands or receptors among each cell subpopulation, followed by identifying over-expressed ligand-receptor interactions(15, 16). Biologically significant cell-cell communication was inferred by a probability value assigned to each interaction through integrating gene expression with CellChatDB, a manually curated database of known interactions between signaling ligands, receptors, and their cofactors. Cell-cell interactions between Mg^high and Mg^low groups were compared for each timepoint.

Statistical analysis

The Student T-test, Kruskal Wallis, or Fisher's Exact Tests were used to compare differences in demographics across Mg levels. To assess clinical outcomes, we used the following parameters: Progression-free survival (PFS) – defined from the time of CAR-T infusion to relapse, disease progression, or death of any cause; Event-free survival (EFS) - defined from the time of CAR-T infusion to relapse, disease progression, start of another lymphoma directed therapy or death of any cause, whichever occurred first; OS was defined from the time of CAR-T infusion to death of any cause. Survival curves were plotted using the Kaplan-Meier methodology and compared using the Wilcoxon test. Additional analysis between MG and CRP, Ferritin, and Cytokines were analyzed using Pearson Correlation Coefficient. A p-value < 0.05 was considered statistically significant (two-tailed testing). All analysis was performed with R (Version 3.6.3, R Foundation for Statistical Computing, Vienna, Austria).

Patient Characteristics

We included 108 patients from cohort 1 (enrolling DLBCL) and cohort 2 (enrolling primary mediastinal B cell lymphoma and transformed follicular lymphoma) of the ZUMA-1 clinical trial and 57 LBCL patients from an independent validation cohort who received axi-cel as SOC at Mayo Clinic. Demographics are shown in Table 1 and Table 2, respectively.

Table 1

Patient Characteristics stratified by magnesium level in ZUMA-1 Cohort
	Low Mg (< 1.7mg/dL) N = 23	Normal Mg (1.7 – <2.0 mg/dL) N = 48	High Mg (≥ 2.0) N = 37	Total N = 108	P value
Age Median (Q1, Q3) Range	56 (47, 67) 25–75	57 (51, 64) 23–76	59 (55, 64) 28–76	58 (51, 65) 23–76	0.59
Male Sex	15 (65%)	30 (63%)	28 (76%)	73 (68%)	0.42
Caucasian Race	19 (83%)	40 (83%)	33 (89%)	92 (85.2)	0.70
Diagnosis DLBCL HG PMBCL TFL	19 (83%) 0 (0%) 4 (17%)	33 (69%) 6 (13%) 9 (19%)	32 (87%) 2 (5%) 3 (8%)	84 (78%) 8 (7%) 16 (15%)	0.18
ECOG PS ≥ 1	16 (70%)	32 (67%)	14 (38%)	62 (57%)	0.01
Elevated LDH (> 190 U/L), no (%)	22 (96%)	39 (81%)	32 (87%)	93 (86%)	0.26
IPI ≥ 3, number (%)	12 (52.2%)	20 (41.7%)	17 (45.9%)	49 (45.4%)	0.71
Stage III/IV no (%)	19 (82.6%)	37 (77.1%)	34 (91.9%)	90 (83.3%)	0.19
Prior Lines of Therapy Median (Q1, Q3) Range	4 (2, 4) 1–10	3 (2, 4) 1–8	3 (2, 4) 2–6	3 (2, 4) 1–10	0.426
Prior ASCT	6 (26%)	13 (27%)	10 (27%)	29 (26.9%)	> 0.99
Abbreviations: Mg magnesium, DLBCL diffuse large B cell lymphoma, HG high-grade lymphoma, PMBCL primary mediastinal large B cell lymphoma, TFL transformed follicular lymphoma, ASCT autologous stem cell transplant, ECOG PS eastern cooperative oncology group performance status, LDH lactate dehydrogenase, IPI international prognostic index, LD chemo lymphodepleting chemotherapy

Table 2

Patient characteristics stratified by magnesium level in SOC Cohort
	Low Mg (< 1.7mg/dL) N = 12	Normal Mg (1.7 – <2.0 mg/dL) N = 30	High Mg (≥ 2.0) N = 15	Total N = 57	P value
Age Median (Q1, Q3) Range	61 (55, 65) 38–75	57 (41, 62) 27–77	60 (54, 64) 27–77	59 (44–64) 27–77	0.20
Male Sex	6 (50%)	18 (60%)	10 (67%)	34 (60%)	0.67
Caucasian Race	8 (67%)	26 (87%)	13 (87%)	47 (82%)	0.33
Diagnosis DLBCL HG PMBCL TFL	5 (42%) 4 (33%) 0 (0%) 3 (25%)	22 (73%) 2 (7%) 0 (0%) 6 (20%)	9 (60%) 1 (7%) 1 (7%) 4 (27%)	36 (63%) 7 (12%) 1 (2%) 13 (23%)	0.14
ECOG PS ≥ 1	6 (50%)	11 (37%)	6 (40%)	23 (40%)	0.72
Elevated LDH (> 220 U/L), no (%)	9 (75%)	19 (63%)	10 (67%)	38 (67%)	0.87
IPI ≥ 3, number (%)	9 (75%)	13 (43%)	9 (60%)	31 (54%)	0.18
Stage III/IV Yes (%)	12 (100%)	27 (90%)	15 (100%)	54 (95%)	0.41
Extranodal disease	11 (92%)	16 (53%)	10 (67%)	37 (65%)	0.07
Prior Lines of Therapy Median (Q1, Q3) Range	3.5 (3, 4) 3–5	3 (3, 4) 1–6	3.0 (2–4) 2–5	3.0 (3, 4) 1–6	0.25
Prior ASCT	6 (50%)	14 (47%)	5 (33%)	25 (44%)	0.68
Abbreviations: Mg magnesium, DLBCL diffuse large B cell lymphoma, HG high-grade lymphoma, PMBCL primary mediastinal large B cell lymphoma, TFL transformed follicular lymphoma, ASCT autologous stem cell transplant, ECOG PS eastern cooperative oncology group performance status, LDH lactate dehydrogenase, IPI international prognostic index, LD chemo lymphodepleting chemotherapy

The two groups were similar regarding age, diagnosis, and sex. There were minor differences in the percent of prior autologous stem cell transplant (ASCT), performance status (PS), and abnormal LDH levels. The median serum Mg levels before lymphodepletion chemotherapy (LD) were similar between the two cohorts (1.9 mg/dL; range, 1.2–2.4 mg/dL in the ZUMA-1 cohorts and 1.9 mg/dL; range, 1.3–2.3 in the Mayo Clinic cohort). Mg levels from the start of LD chemotherapy through day 28 post-CAR-T infusion remained consistent (Supplemental Fig. 1).

Supplemental Fig. 1. Magnesium level over time. Magnesium levels from the start of LD chemotherapy through day 28 post-CAR-T infusion are shown for patients in the ZUMA-1 study (top panel) and the SOC cohort (bottom panel).

For analysis purpose, we divided patients in 3 groups based on Mg levels: Mg low (< 1.7 mg/dL [Mg^low]), Mg normal low (\(\:\ge\:\) 1.7 < 2.0 mg/dL [Mg^NL]) and Mg high (\(\:\ge\:\) 2mg/dL [Mg^high]). These cutoffs were chosen based on prognostic values identified on our prior work^9,10. In the ZUMA-1 cohorts, 21.3% (23/108) subjects were Mg^low, 44.4% (48/108) subjects Mg^NL, and 34.3% (34/108) subjects were in the Mg^high range. The Mayo Clinic cohort distribution was similar, with 21% (12/57) Mg^low, 53% (30/57) Mg^NL, and 26% (15/57) Mg^high. There was no difference in patient characteristics among the groups with different Mg levels except for a worse PS in those with Mg^low compared Mg^high in ZUMA-1 cohort (70% vs 38%, p = 0.01). However, this difference was not evident in the Mayo Clinic cohort (50% vs 40%, p = 0.72).

Magnesium level correlates with clinical response and survival

In the ZUMA-1 cohort, there was a lower overall response rate (ORR) in patients with hypomagnesemia (65%, 15/23) compared to those with normal (83%, 40/48) and high Mg levels (92%, 34/37; p = 0.03). This translated to an inferior PFS (p = 0.022; Fig. 1A) and OS (p = 0.0013; Fig. 1B).In the Mayo Clinic cohort, due to the smaller number of patients by tertile, we examined clinical response and survival using Mg < or ≥ 2.0 mg/dL grouping.

This is a commonly used clinical threshold for magnesium replacement in patients with cardiac issues.(17) Fitting a spline of the partial residuals for Mg at LD shows clinically significant benefit for Mg at 2.0 mg/dL and higher as a reasonable cutpoint (Supplemental Fig. 2C, D).

Supplemental Fig. 2. Survival by magnesium level for SOC cohort. A. Progression-free survival of patients with lymphoma in the SOC cohort undergoing CAR-T by day − 5 before the start of lymphodepleting chemotherapy serum magnesium level. p = 0.058. B. Overall survival of patients with lymphoma in the SOC cohort undergoing CAR-T by day − 5 before the start of lymphodepleting chemotherapy serum magnesium level. p = 0.032. The line color indicates magnesium level grouping. Spline plots for the relative hazard ratio are shown for EFS (C) and OS (D). The dotted lines correspond to a 95% confidence interval.

The complete response (CR) rate trended higher in patients with Mg ≥ 2.0 mg/dL (80% v 52%; p-value = 0.07). Event free survival (EFS) was examined in the Mayo Clinic cohort due to some patients with stable disease or partial response receiving the next line of lymphoma-directed treatment before meeting Lugano's criteria for progressive disease. The patients with low Mg levels confirmed an inferior EFS (HR 0.41, 95% CI 0.18–0.93, p = 0.034; Fig. 1C), while the OS did not achieve significance (HR 0.49, 95% CI 0.17–1.28, p = 0.13; Fig. 1D) probably due to the low power and shorter follow-up. The EFS and OS by tertile grouping of Mg levels are shown in Supplemental Fig. 2.

Magnesium levels inversely correlate with inflammatory markers and immune effector cell-associated toxicities

In the ZUMA-1 cohort, Mg levels were inversely correlated with the abundance of interleukins such as IL1Ra, IL6 and MIP1a before LD and at CART cell peak (Fig. 2B), suggesting an impairment of immune effect.

Given the known contribution of myeloid cells to these cytokines and chemokines, we examined the monocyte transcriptomes at CAR-T peak expansion. We found that, CD14 and CD16 monocytes from patients with Mg^low levels were enriched for IL-6/STAT3 and TGFb gene pathways compared to those from patients with Mg^high levels (Fig. 2C). In line with this data, C-reactive protein (CRP) and ferritin levels, which are common inflammatory markers, were converse to Mg levels (Fig. 2A).

Accordingly, a lower number of patients with hypomagnesemia developed grade 3 or higher cytokine release syndrome (CRS) and neurologic events (Fig. 3A-B) as graded by Lee's criteria(18), which supports a lower immune reactivity in patients with low Mg levels. However, we did not observe a significant difference in the severity of CRS and Immune cell associated neurotoxicity syndrome (ICANS) among the Mg groups in the Mayo Clinic cohort (Fig. 3C-D).

Of note, during SOC practice CRS and neurotoxicity grading have been aligned with the American Society of Transplant and Cell Therapy (ASTCT) guideline(19). It is possible that the lack of correlation between Mg levels and immune effector cell-associated toxicity in the Mayo Clinic cohort depends on the recent improvement in the CRS management with tocilizumab and steroids, leading to an overall decrease of severe episodes in clinical practice.

Tight link between magnesium levels and immune cell interactions

To investigate whether the levels of Mg may impact systemic immune cells, we performed single-cell RNA-sequencing (scRNA-seq) of peripheral blood cells collected before LD chemotherapy (Pre-CART) and at peak CAR-T expansion (CART-Peak) of 13 LBCL patients from the Mayo Clinic cohort. Using CellChat analysis, we found that at Pre-CART, patients with Mg^high levels had a higher number of interactions between CD16 and CD14 monocytes and between these cells with CD4 and CD8 T cells compared to those with Mg^low levels (Fig. 4).

Similarly, CD8 T effector memory (T_EM) cells had an increased number of interactions with all other immune cells in Mg^high patients (Fig. 4), with the maximal interaction strength evident between CD8 T_EM cells and CD4 proliferating cells, followed by CD16 monocytes (Suppl Fig. 3A).

Supplemental Fig. 3. Immune cell interactions by magnesium levels. A. Heatmap showing differential interaction strength among the indicated immune cells before CAR-T infusion (left, Pre-CART) and at peak CAR-T expansion (right, CART-Peak) in patients with high magnesium (Mg^high, Mg > = 2.0 mg/dL) compared to those with low magnesium (Mg^low, Mg < 2.0 mg/dL) levels. Selected ligand-receptor interaction (y-axis) of immune cell populations in patients with Mg^High or Mg^Low levels as labeled (x-axis) against CD4 proliferating cells (B, top panel) and CD4 T effector memory (B, bottom panel) and CD8 T effector memory cells (C). P values are indicated by circle size. The means of the average expression level of interacting molecules are indicated by color.

Remarkably, at CART-Peak, the same interactions decreased dramatically in patients with Mg^high (Fig. 4A and Suppl Fig. 3A). In particular, there was a profound decrease in the overall interaction strength of all CD8 T cell subsets, including CD8 naïve, proliferating, central memory (T_CM), and T_EM cells in the Mg^high group (Suppl Fig. 3A). In contrast, we observed a substantial upregulation of the CD4 T_EM cells, suggesting the presence of a sustained antigen-restricted immune response (Fig. 4B). A transcription factors enrichment analysis identified significant enrichment for SPI1 (also known as PU.1) in Mg^low patients compared to those Mg^high. The transcription factor SPI1 plays an essential role in monocyte differentiation(20, 21) and reprograms macrophages towards suppressive M2 subtype(22). There was also an enrichment of KMT2A (also known as MLL1), a histone methyltransferase that regulates mono- and dimethylation of histone 3 at lysine 4 (H3K4me1/2) primarily at gene enhancers. This epigenetic change may alter the chromatin accessibility to allow transcription factor binding (e.g., SPI1) and gene activation. Along the same line, JMJD1C (also known as KDM3C) was significantly increased in Mg^low patients who progressed. KDM3C belongs to an enzymatic family that specifically demethylates H3K4me1/2 resulting in suppression(23) of the NF-kB-mediated immune response.(24) Moreover, Mg^low non-responders showed an upregulation of the proto-oncogene MYC, which favors stemness and cell growth as well as orchestrates changes in the TME, including immune suppression(25) (Fig. 4C). Finally, we performed a ligand-receptor analyses to identify potentially unique inhibitory cell-cell communication mechanisms in each immune subset at CART cell peak. Interestingly, we found a significant downregulation of multiple HLA subtypes linked to CD8 T cells (naïve, proliferating, T_CM, and T_EM) interacting with CD14 and CD16 monocytes (Fig. 4D), CD4 proliferating cells, CD4 and CD8 T_EM cells in Mg^high responders (Suppl Fig. 3B-C). By contrast, the same interactions were significantly increased in the Mg^low progressors (Fig. 4D and Suppl Fig. 3B-C). The results of colocalized ligand-receptor pairs and cell-specific markers infer a signaling network facilitating contact-dependent communication and immune response. Altogether, these data underscore the relevance of Mg in modulating the intercellular interaction for a successful CART cell function.

Magnesium plays a critical role in modulating immune surveillance against pathogens and cancer cells. Previous studies have shown a faster rate of tumor growth and a lower infiltration of T cells in vivo models of hypomagnesemia. Additionally, the relevance of Mg levels has shown remarkable implication in immunotherapeutic strategies in B cell lymphoma including checkpoint inhibitors and autologous SCT, however the impact of Mg on CART cell therapy in humans remains unexplored. To answer this question, we leveraged two independent patient cohorts including the ZUMA-1 trial and our Mayo Clinic SOC cohort. Our data demonstrate the prognostic impact of Mg in relapsed/refractory LBCL patients receiving axi-cel CAR-T therapy and uncovered its functional implication in immune response.

Interestingly, we found that low levels of Mg prior to the start of lymphodepletion chemotherapy were associated with increased expression of inflammatory markers such as CRP and ferritin. These inflammatory markers are also known to be prognostic for poor response and decreased survival(26). In addition, hypomagnesemia is associated with decreased presence of T and NK cells and increased levels of myeloid cell-associated inflammatory cytokines and chemokines. Myeloid-derived cytokines are a driver of CAR-T-associated toxicities, CRS, and ICANS. Correspondingly, patients in the ZUMA-1 study with more severe CRS and ICANS have been found to display hypomagnesemia at the time of lymphodepletion. However, this correlation was not confirmed in the Mayo Clinic cohort likely due to changes in CRS management and the lower incidence of severe CRS in SOC practice. (27) (28)

There is minimal data on serum Mg levels status in lymphoma correlated with clinical outcomes in general and in the setting of CAR-T cell therapy specifically. Previous CAR-T trials have documented that 19% of patients in the phase 1 axi-cel trial(13) experienced hypomagnesemia, and all but 1 were grade 1. The study with lisocabtagene maraleucel(29) also reported 19% hypomagnesemia, with all being ≤ grade 2. Neither of these studies reported outcomes by Mg level status. Of note, grade 1 hypomagnesemia (levels between 1.2–1.6 mg/dL) may be considered a "mild toxicity" but was associated with poor outcomes in our study. We previously reported in a much larger study of relapsed DLBCL patients undergoing a standard autologous SCT that serum Mg levels < 2.0 mg/dL predicted an inferior outcome (9). Based on our studies of Mg in relapsed DLBCL patients undergoing autologous SCT, and the previous studies that hypomagnesemia leads to impaired T cell responses (30–32), we hypothesized that patients with hypomagnesemia undergoing CAR-T cell therapy would have inferior outcomes. Taken together, our data in the registration ZUMA-1 study and our SOC patients confirm that hypomagnesemia is a prognostic factor and predictor of inferior PFS/EFS compared to patients with high normal Mg levels at this timepoint.

It has long been known that effective T-cell function requires Mg(33, 34). Specifically, the lymphocyte function-associated antigen 1 (LFA-1), a T cell surface protein required for the formation of the immune synapse, necessitates Mg for its conformation and ability to activate T cells(35). This was further validated recently by Lötscher et al. (36) who demonstrated the importance of extracellular Mg to CD8 T-cell function via LFA-1 signaling. They showed that hypomagnesemia impaired response to checkpoint inhibitor therapy in lung cancer patients. They also retrospectively reviewed 96 patients treated with axi-cel in the SOC practice and found that those with hypomagnesemia around the time of CAR-T treatment (using a level < 1.7 mg/dL) had an inferior outcome. Our data further identifies altered monocyte transcriptomes, including enrichment of cytokine pathways such as IL-6 and TGFb, which are known to be involved in unfavorable inflammation(37) and immune suppression(38, 39). In addition, altered monocyte interaction with T cells may further contribute to the correlation of hypomagnesemia with decreased PFS. Our findings come from a larger cohort of patients in both the registration clinical trial and SOC practice. In addition, we focused our analysis using Mg at a defined timepoint immediately before LD chemotherapy, a more useful timepoint to facilitate identifying patients at increased risk for potential intervention.

Mg replacement could be a readily available intervention to facilitate a favorable environment for the activities of the infused CAR-T cells. However, replacing Mg in ill patients can be difficult as these patients typically do not eat well and may waste Mg through the GI tract (diarrhea) and kidney (due to prior platinum-based chemotherapy(40) and anti-fungals such as amphotericin(41)). Laboratory serum Mg deficiency at our institution is defined as a Mg level < 1.7 mg/dL. However, this study found a cut-off of < 2.0 mg/dL was associated with poor response rate and EFS. This is the same level we found to be prognostic in the DLBCL SCT study (9). In addition, values less than 2.0 mg/dL are associated with an increased risk of congestive heart disease mortality(42) and sudden cardiac death(43) in patients with cardiac pathologies. In patients with a risk of cardiac arrhythmia and other events, it is common practice to keep the Mg level at ≥ 2.0 mg/dL. Future investigation is needed to see if replacing Mg to this level will also benefit immunotherapy.

This study provides insight into the clinical relevance of hypomagnesemia in patients with lymphoma undergoing CAR-T therapy. Our analysis has similar demographics and percentages of patients with hypomagnesemia compared to other published clinical trials on CAR-T (13) (29). However, we acknowledge the limitation of our study concerning its relatively small sample size and retrospective design. We anticipate that prospective studies evaluating the immune cell dysfunctions associated with hypomagnesemia will shed light into intervention strategies that can improve the outcome of patients treated with cellular therapies.

Large B cell lymphoma – LBCL

Magnesium – Mg

Axicabtagene ciloleucel – Axi-cel

X-linked immunodeficiency with Mg defect, EBV and neoplasia – XMEN

Stem cell transplant – SCT

Progression-free survival – PFS

Overall survival – OS

Chimeric antigen receptor T-cell – CAR-T

Standard of care – SOC

Investigonal review board – IRB

Event free survival – EFS

Autologous stem cell transplant – ASCT

Performance status – PS

Overall response rate – ORR

Lymphodepletion chemotherapy – LD

Complete response – CR

C-reactive protein – CRP

Cytokine release syndrome – CRS

Immune cell associated neurotoxicity syndrome – ICANS

American society of transplant cell therapy – ASTCT

Single-cell RNA-sequencing – scRNA-seq

Lymphocyte function-associated antigen 1 – LFA-1

Ethics approval and consent to participate - All study protocols were approved by the Mayo Clinic Investigational Review Board.

Consent for publication – Not applicable.

Availability of data and materials - The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests - SMA: Research funding (to institution) for clinical trials from Bristol Myers Squibb, Takeda, Pfizer, Affimed, AstraZeneca, Regeneron and ADC Therapeutics. MKD: Research funding (to institution) from Bristol Myers Squibb, Novartis and Pharmacyclics and lecture/honararia from Kite Pharma. JM: Consulting for Pharmacyclics/Abbvie, Bayer, Gilead/Kite, Beigene, Pfizer, Janssen, Celgene/BMS, Kyowa, Alexion, Fosunkite, Seattle Genetics, Karyopharm, Aurobindo, Verastem, Genmab, Genzyme, Genentech/Roche, ADC Therapeutics, Epizyme, Beigene, Novartis, Morphosys/Incyte, MEI, TG Therapeutics, AstraZeneca, Eli Lilly; Research funding (to institution) – Bayer, Gilead/Kite, Celgene, Merck, Portola, Incyte, Genentech, Pharmacyclics, Seattle Genetics, Janssen, Millennium, Novartis, Beigene. Honoraria - Targeted Oncology, OncView, Curio, and Physicians' Education Resource. YL: Janssen, Sanofi, NexImmune, Caribou, BMS, Pfizer, Regeneron, Genentech. No disclosures were reported by the other authors.

Funding – Supported in part by the University of Iowa/Mayo Clinic Lymphoma SPORE CA97274; Mayo Clinic Center for Individualized Medicine, Bernard E. and Edith B. Waterman, Henry J. Predolin Foundation, and other generous benefactors of Mayo Clinic; Mayo Clinic Department of Medicine Innovation Acceleration Award; R01CA219960; R01CA203836.

Authors' contributions – JJG, PM, YL, TEW – Conceptualization, writing original draft. JJG, PM, MAH, YL, TEW – data curation, statistical analysis. JJG, PM, ZW, YL, RB, SG, HZ, EB, KM, GM, ZS, RK, MAH, PW, JW, ASAS, GR, SMA, NNB, PBJ, JP, JCVB, AK, UD, YW, PJH, AR, JM, EM, JEC, HSM, MKD, SSK, JJK, RS, MM, YL, TEW – Critical appraisal and writing/editing the manuscript. PM, PW – Performed single cell analysis. ZW, JJK, RS, MM – Interleukin levels analysis. All authors read and approved the final manuscript.

Acknowledgements – This work ws supported in part by the NIH/NCI University of Iowa, Mayo Clinic Lymphoma SPORE (CA97274); Mayo Clinic Department of Medicine Acceleration Award; Mayo Clinic Center for Individualized Medicine, Bernard E. and Edith B. Waterman, Henry J. Predolin Foundation, and other generous benefactors of Mayo Clinic; Mayo Clinic Comprehensive Cancer Center Novel Biotherapeutics Data Science Workstream. We thank our study coordinators, patients and family.

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Competing interest reported. SMA: Research funding (to institution) for clinical trials from Bristol Myers Squibb, Takeda, Pfizer, Affimed, AstraZeneca, Regeneron and ADC Therapeutics. MKD: Research funding (to institution) from Bristol Myers Squibb, Novartis and Pharmacyclics and lecture/honararia from Kite Pharma. JM: Consulting for Pharmacyclics/Abbvie, Bayer, Gilead/Kite, Beigene, Pfizer, Janssen, Celgene/BMS, Kyowa, Alexion, Fosunkite, Seattle Genetics, Karyopharm, Aurobindo, Verastem, Genmab, Genzyme, Genentech/Roche, ADC Therapeutics, Epizyme, Beigene, Novartis, Morphosys/Incyte, MEI, TG Therapeutics, AstraZeneca, Eli Lilly; Research funding (to institution) – Bayer, Gilead/Kite, Celgene, Merck, Portola, Incyte, Genentech, Pharmacyclics, Seattle Genetics, Janssen, Millennium, Novartis, Beigene. Honoraria - Targeted Oncology, OncView, Curio, and Physicians' Education Resource. YL: Janssen, Sanofi, NexImmune, Caribou, BMS, Pfizer, Regeneron, Genentech. No disclosures were reported by the other authors.

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Hypomagnesemia in Large B cell Lymphoma patients receiving CAR T cell therapy correlates with immune dysregulation and decreased PFS: results from the ZUMA-1 trial and Mayo Clinic cohort

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Abstract

Background

Methods

Results

Conclusions

Figures

Background

Methods

Clinical data and sample collection

Cytokine analysis from blood

PBMNC isolation and scRNAseq

scRNAseq analysis

Statistical analysis

Results

Patient Characteristics

Magnesium level correlates with clinical response and survival

Magnesium levels inversely correlate with inflammatory markers and immune effector cell-associated toxicities

Tight link between magnesium levels and immune cell interactions

Discussion

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

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Version 1