Edited by: Petros Christopoulos, Heidelberg University Hospital, Germany
Reviewed by: Andrea Aroldi, University of Milano Bicocca, Italy
Walter Hanel, The Ohio State University, United States
Yangmin Zhu, Guangdong Second Provincial General Hospital, China
*Correspondence: Raffaella Greco,
†These authors have contributed equally to this work
‡These authors have contributed equally to this work
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Hemophagocytic lymphohistiocytosis (HLH) is a hyper-inflammatory syndrome characterized by deficient NK-cell activity, cytokine storm and altered T-cell immunity, potentially sustained by multiple triggers. Recently, a hyperinflammatory condition resembling HLH has emerged as a potential complication of CAR T-cells. HLH represents a diagnostic conundrum due to its rarity, non-specific presentation and lack of validated biomarkers. We investigated a panel of serum cytokines which represent candidate markers to diagnose HLH after CAR-T. We analyzed 2 patients affected by B-cell lymphomas who received anti-CD19 CAR-T and developed HLH defined according to multiple diagnostic criteria. We identified four controls who did not develop HLH: one with CRS without cytopenia, one with cytopenia with CRS, one with both and one with none. We selected a set of acute-phase molecules including CD163, IL33R (quantitated through ELISA), CTLA-4, and CD80 (quantitated through Luminex) and studied their trend in frozen sera collected at parallel time-points. We studied case sera and compared them with controls. All patients developing CRS showed an early peak in IL2R, CD80, CTLA-4 and IL33R, followed by a second, more marked increase in case of HLH, absent in controls. The kinetics of the selected markers suggests that timing and extent of changes might help discriminating HLH from other causes of fever or cytopenia. Serum CD163, CTLA-4, CD80 and IL33R deserve prospective assessment as promising biomarkers to assist in the differential diagnosis between CRS, HLH and non-inflammatory cytopenias after CAR-T.
Chimeric antigen receptor T (CAR T)-cells have brought about a paradigm shift in the treatment of several lymphoid and plasma cell malignancies (
The mechanisms underlying CAR T-cell associated HLH remain poorly elucidated, and the presence of concomitant confounding factors (CRS, multi-factorial cytopenia) make its diagnosis elusive. Both the American Society for Transplant and Cell Therapy (ASTCT) and the European Bone Marrow Transplantation group (EBMT) have started cooperative efforts to characterize this disorder (
In our center, we reported 2 patients who received anti-CD19 CAR-T for B-cell lymphoma and developed IEC-HS. Based on literature data demonstrating their role in NK and T-cell mediated inflammation, we investigated a panel of serum markers as candidate adjuvants to diagnose CRS/MAS and IEC-HS, including soluble CD163, IL33R, CTLA-4, and CD80, and studied their trend in frozen sera collected at sequential time-points, before and after infusion. As control, we assessed CD25 levels (IL2R), a well-known HLH marker (
Summary of patient characteristics.
| Upn | Gender | Age | Diagnosis | Prior treatment lines | Product | CRS | ICANS | Treatment | HLH | Onset | HLH-treatment | Peak Ferritin (ng/mL) | Prolonged cytopenia (3 mo.) | Treatment (cytopenia) | Outcome |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 1 | M | 58 | MCL | R-CHOP, HDT+ASCT, Ibrutinib, AlloHCT | Brexu-ce | G1 | n.. | Tocilizumab (I); |
Yes | D11 | MPD, CsA, Anakinr | 6929 | No | n.. | HLH resolved; disease relapse at 4 mo. CR after salvag |
| 2 | M | 65 | DLBCL, NOS | R-CHOP, HDT+ASCT | Axi-ce | G3 | G3 | Tocilizumab (II); DEX; | Yes | D21 | MPD, CsA, Anakinra, VP16 | 5589 | n.. | n.. | Death due to HLH |
| 3 | M | 41 | PMBCL | R-DA-EPOCH; HDT | Axi-ce | G1 | n.. | Tocilizumab (III); DEX; | No | n.. | n.. | 226 | No | n.. | Alive in CR |
| 4 | F | 69 | DLBCL, NOS | R-CHOP/MATRix, HDT+ASCT; | Axi-ce | G1. | n.. | Tocilizumab (I) | No | n.. | n.. | 345 | G3 neutropenia, G3 anemia, G4 thrombocytopeni | n.. | Prolonged cytopenia in FU |
| 5 | F | 70 | DHL | R-CHOP; MATRix | Axi-ce | G1 | G2 | Tocilizumab (I); DEX; | No | n.. | n.. | 2121 | G4 neutropenia, G3 anemia, G4 thrombocytopeni | Autologous CD34+ boost | Alive in CR |
| 6 | M | 52 | HGBCL, NOS | R-CODOX-M-IVAC; HDT+ASCT | Tisa-ce | n.. | n.. | n.. | No | n.. | n.. | 625 | No | n.. | Alive in CR |
AlloHSCT, allogeneic hematopoietic stem cell transplantation; ASCT, autologous stem cell transplantation; CR, complete remission; CRS, cytokine release syndrome; CsA, cyclosporin A; DLBCL, NOS, diffuse-large B cell lymphoma, not otherwise specified; DHL, double-hit lymphoma; FU, follow-up; HLH, hemophagocytic lymphohistiocytosis; HDT, high-dose chemotherapy; HGBCL, NOS, High-grade B cell lymphoma, not otherwise specified; ICANS, immune-effector cell associated cytotoxicity; MCL, mantle cell lymphoma; MPD, methylprednisolone; PMBCL, primary mediastinal B cell lymphoma; UPN, unified patient number; VP16, etoposide.
The first patient was a heavily pre-treated 58-year-old male affected by mantle cell lymphoma (MCL) who underwent brexucabtagene autoleucel (brexu-cel) for a relapse post-allogeneic stem cell transplant. After a G1 CRS on D+4, treated with a single tocilizumab dose, he developed a second febrile episode on D+11 with concomitant alteration in hepatic enzymes and hyperbilirubinemia, initially interpreted as a potential graft-versus-host disease (GvHD) flare and treated with steroids. Due to worsening cytopenia, hypofibrinogenemia, and liver and bone marrow bopsies consistent with hemophagocytosis (
The second patient was a 65-year-old male affected by refractory diffuse large B cell lymphoma (DLBCL) who received axicabtagene ciloleucel (axi-cel) and was admitted to the ICU on day +6 for a G3 CRS with concomitant G2 ICANS treated with inotropes, non-invasive ventilation, dexamethasone and broad-spectrum antibiotics due to severe neutropenia. Upon corticosteroid tapering, he developed pancytopenia, hepatocellular damage and inflammatory markers consistent with IEC-HS (elevated IL2R, triglycerides, ferritin). HLH diagnosis was made on day +21 when all major ASTCT criteria were fulfilled. The HScore was 152, indicating a 52% probability of IEC-HS, whereas sIL2R/Ferritin ratio was 0,55. Unfortunately, the patient had a poor response to high-dose steroids and anakinra and ultimately succumbed to multi-organ failure on day +42.
As controls, we identified four CAR T-cell recipients: one who developed CRS without significant cytopenia, one who developed long-term cytopenia without prior inflammatory issues, one with both and one with none of them. Three of the controls received axi-cel and one tisagenlecleucel (tisa-cel).
Samples were collected and banked in the context of the OSR CAR-T observational protocol. Time-points were pre-determined and included pre-lymphodepletion, infusion, D+3, D+14, D+21, D+28 and monthly thereafter. We analyzed soluble IL2R, CD163, CD80, CTLA4 and IL33R at each available time-point.
We assessed the serum concentration of our investigational markers by ELISA (see
Soluble IL2R, CTLA4, CD163, CD80 and IL33R trends for patients who did (red dots, green squares) and did not develop HLH (black triangles, plotted as aggregated data for simplicity) at parallel time-points. Blue band represents CRS clinical manifestations, whereas yellow band represents HLH. Patient 1 (MCL, Brexu-cel) and patient 2 (DLBCL, Axi-cel) showed remarkably higher serum levels than aggregated controls. LD, lymphodepletion.
We decided to investigate the value of soluble CD80 and CTLA4 and their relationship with post-CAR-T cell HLH. Both these molecules are T-cell regulators. Soluble CD80 is release by monocytes and B cells, whereas sCTLA-4 is released by different cell types, including regulatory T cells and monocytes. Both molecules can exert alternate stimulatory or inhibitory functions on T cells (
Compared to the control group, patient 1 showed similar baseline levels of sIL33R and increased levels of sCTLA-4, sCD163 and sCD80 before cell infusion. Two weeks after treatment, at HLH onset, we observed a marked increase of all the evaluated markers compared to the controls, concomitantly with the appearance of the second febrile episode and the worsening clinical conditions. Subsequently, sCD80, sCD163 and sCTLA-4 plummeted, in line with the clinical improvement, whereas sIL33R displayed an initial reduction, followed by a second peak at day +28. As expected, sIL2R peaked 2 weeks after CAR-T cell infusion, compatible with the diagnosis of IEC-HS, followed by a gradual decrease in line with the clinical improvement. At day +100, after HLH resolution, the level of all the markers except sCD163 was comparable with that of the control group.
Patient 2 showed a rapid increase in sIL2R, sCD80, sCTLA-4 and sIL33R immediately after CAR-T cell infusion, compared to the control group. The examined markers underwent a gradual decrease at subsequent timepoints, reaching levels comparable with that of the control group by day 21. Later on, in line with further clinical deterioration, they underwent a second peak (D+28). Conversely, sCD163 showed a slower but progressive increase, reaching the peak of concentration 4 weeks after CAR-T cell infusion.
Unfortunately, no data were available concerning CAR T-cell kinetics in peripheral blood or in tissue biopsies at the time.
In conclusion, CRS/MAS and IEC-HS are elusive entities, requiring further clinical and translational investigations. Prompt diagnosis and treatment are hampered by the difficulties in ruling out differentials. We investigated a panel of candidate markers which might represent plausible co-adjuvants in the distinction between HLH and other CAR-associated hyperinflammatory syndromes. Soluble CD163, CTLA4, CD80 and IL33R showed a more marked increase in patients with CRS/MAS and IEC-HS compared to CRS alone or negative controls, thus representing promising biomarkers to distinguish CRS/MAS and IEC-HS from CRS and non-inflammatory new-onset cytopenia. So far, these markers chiefly reflect the severity of inflammation and shall be not used as diagnostic tools. However, we believe that broader prospective studies are warranted to study their kinetics across timepoints and potentially validate their role to discriminate distinct clinical events such as CRS and IEC-HS at relevant timepoints.
The original contributions presented in the study are included in the article/
The studies involving humans were approved by IRCCS Ospedale San Raffaele Ethical Review Board. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.
EC: Writing – review & editing, Writing – original draft, Conceptualization, Data curation. PA: Writing – original draft, Writing – review & editing. MD: Investigation, Methodology, Conceptualization, Supervision, Writing – review & editing, Writing – original draft. AU: Formal Analysis, Methodology, Writing – review & editing, Investigation, Writing – original draft. ML: Writing – original draft, Writing – review & editing. MN: Writing – original draft, Writing – review & editing. ET: Writing – original draft, Writing – review & editing. FP: Writing – review & editing, Writing – original draft. GB: Writing – original draft, Writing – review & editing. LL: Writing – review & editing, Writing – original draft. AB: Writing – review & editing, Writing – original draft. ED: Writing – review & editing, Writing – original draft. MP: Writing – review & editing, Writing – original draft. CC: Writing – original draft, Writing – review & editing. JP: Writing – original draft, Writing – review & editing. AR: Writing – review & editing, Writing – original draft. AF: Writing – review & editing, Writing – original draft. CB: Writing – review & editing, Writing – original draft. FC: Writing – review & editing, Writing – original draft. MC: Writing – original draft, Investigation, Writing – review & editing, Supervision, Conceptualization. RG: Data curation, Methodology, Writing – review & editing, Conceptualization, Supervision, Writing – original draft.
The author(s) declare that no financial support was received for the research and/or publication of this article.
We acknowledge all the patients, family members, and staff who participated in the study.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.
The author(s) declare that no Generative AI was used in the creation of this manuscript.
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Liver biopsy samples collected from patient 1 via transjugular biopsy.
panel displaying trends in common biomarkers of IEC-HS (and HLH in general) for patient 1 (red circles) and patient 2 (green squares) over time after CAR T-cell product infusion.