Inflammatory monocytes promote pre-engraftment syndrome and tocilizumab can therapeutically limit pathology in patients

Unrelated cord blood transplantation (UCBT) is an effective treatment for hematopoietic disorders. However, this attractive approach is frequently accompanied by pre-engraftment syndrome (PES), severe cases of PES are associated with enhanced mortality and morbidity, but the pathogenesis of PES remains unclear. Here we show that GM-CSF produced by cord blood-derived inflammatory monocytes drives PES pathology, and that monocytes are the main source of IL-6 during PES. Further, we report the outcome of a single arm, single-center clinical study of tocilizumab in the treatment of steroid-refractory severe PES patients (www.chictr.org.cn ChiCTR1800015472). The study met the primary outcome measure since none of the patients was nonrelapse death during the 100 days follow-up. The study also met key secondary outcomes measures of neutrophil engraftment and hematopoiesis. These findings offer a therapeutic strategy with which to tackle PES and improve nonrelapse mortality.

Source data is provided as a Source Data file. Percentage of monocytes in GM-CSF + cells obtained from peripheral blood stem cells and cord blood mononuclear cells. Each data point represents a biologically-independent sample. n = 6 and 12, respectively. Mann-Whitney test (two-sided) (p=0.0241). (c) ELISA of GM-CSF in supernatants from peripheral blood stem cells or cord blood mononuclear cells and cultured for 6 h. n = 6 and 11, respectively. Mean difference=23.99; 95% CI: 2.158-45.82; Unpaired t-test with Welch's correction (two-sided) (p=0.0335). PBSC, peripheral blood stem cells. CB, cord blood mononuclear cells. Each data point represents a biologically-independent sample. Data in (b) and (c) are presented as mean ± SEM. * p < 0.05. Source data is provided as a Source Data file. Representative histograms of GM-CSFRα in monocytes, T cells, NK cells, and B cells from peripheral blood stem cells or cord blood; n=9 for peripheral blood stem cells; n=11 for cord blood.
Source data is provided as a Source Data file. secretion. Subsequently, these cells undergo rapid expansion in the recipient. Levels of both GM-CSF and IL-6 were increased in the sera of PES patients. Intervention with tocilizumab (TCZ), the monoclonal antibody that targets the IL-6 receptor, is an effective treatment for patients with refractory PES. We created this schematic diagram using Adobe Illustrator CS6.

Official title
A single arm, single-center clinical study of tocilizumab in the treatment of corticosteroids unresponsive pre-engraftment syndrome patients after unrelated cord blood transplantation

Participating Centers
Study participants were recruited from the First Affiliated Hospital of the University of Science and Technology in China (Anhui Provincial Hospital), Hefei, China.

Brief summary
Pre-engraftment syndrome (PES) is common following UCBT and is characterized by non-infectious high-grade fever, skin rash, diarrhea and other clinical findings. At present, steroids treatment is the first-line therapy for PES. However, some patients are steroid-refractory. In our study, we found that IL-6 is a signature cytokine of PES, and to test the efficacy of this finding into a clinical setting, we conducted a single-arm trial to treat the patients who suffered steroid-refractory severe PES after a single-unit UCBT as a first HSCT. Eligible patients were treated with the anti-IL-6 receptor monoclonal antibody-tocilizumab. The study will evaluate if tocilizumab help to ameliorate PES, and also study the safety of treatment with tocilizumab.

Estimated enrollment
10 participants, if necessary, the sample size can be further expanded. (2) Myeloablative conditioning regimens, GVHD prophylaxis without ATG;

Study execution time
(3) Any age, any sex, any race; (4) Must be met the diagnostic criteria of PES, if symptoms were not relieved after 3 days of methylprednisolone (2mg/kg/d) treatment, and the patient had a fever in excess of 38.3C for three consecutive days; (5) Patients and their families voluntarily participate in this research and sign informed consent.

Discontinuation of subjects from treatment
The treatment must be stopped for any one of the following reasons: (1) Severe organ dysfunction during the research course; (2) Withdrawal of informed consent for any reason;

Primary Objective
Non-relapse mortality

Recruitment of subjects
(1) All patients meeting the inclusion criteria were recruited.
(2) All patients included in our study signed informed consent.
(3)Ineligible patients were given other treatment under the guidance of a doctor.

Treatment protocols
All patients were treated following the standard UCBT procedures in our center [Zhu X, Huang L, Zheng C, et al: European group for blood and marrow transplantation risk score predicts the outcome of patients with acute leukemia receiving single umbilical cord blood transplantation. Biol Blood Marrow Transplant 23:2118-2126, 2017].

Cord blood selection
Cord blood and recipient HLA typing were determined using molecular techniques, with a minimum antigen split-level resolution for HLA-A and -B and allele-level resolution at DRB1. Cord blood units are from Chinese cord blood banks serologically matched for ≥4 of 6 HLA antigens and containing at least 2.5×10 7 TNC/kg and 1.2×10 5 CD34 + cells/kg of recipient body weight before freezing were chosen for transplantation. Complete information regarding allele-level HLA matching for HLA-A, -B, -Cw, -DRB1 and -DQB1 loci was obtained from the Chinese cord blood banks.

Conditioning regimens
The myeloablative conditioning regimens were based on a full dose of busulfan (BU, total 12.8 mg/kg, 0.8 mg/kg every 6 hours for 4 days), cyclophosphamide (60 mg/kg daily for 2 days) plus fludarabine (30 mg/m 2 daily for 4 days) or total body irradiation (total 12 Gy, 3 cGy twice a day for two days) and cyclophosphamide (a total of 120 mg/kg administered as 60 mg/kg daily for 2 days) plus cytarabine (2 g/m2 twice a day for 2 days).

GVHD prophylaxis
All patients received a combination of cyclosporine (CsA) and mycophenolate mofetil (MMF) without antithymocyte globulin (ATG) as GVHD prophylaxis.

Cytokine supportive care
Granulocyte colony-stimulating factor (G-CSF; 5 to 7 g/kg per day) was added on Day 6 after UCBT to stimulate neutrophil recovery.

Tocilizumab treatment
All recruited patients were given a comprehensive workup, including urine and blood cultures through both peripheral and central lines, and none of the patients responded to empirical antibiotic therapy. Consequently, these patients were treated with methylprednisolone (2mg/kg/d), if symptoms were not relieved after 3 days of methylprednisolone treatment, and the patient had a fever in excess of 38.3C for three consecutive days, then tocilizumab was administered.

Definitions and evaluation
(1) The date of cord blood infusion was defined as day 0.
(2) The date of neutrophil engraftment was defined as the first day of a neutrophil count ≥0.5 ×10 9 /L for three consecutive days.
(3) The date of platelet engraftment was defined as the first day of platelet recovery to ≥20×10 9 /L without transfusion support for seven consecutive days.
(4) Acute GVHD was defined and scored from 0 to IV, according to the type and severity of organ involvement. [Przepiorka D, Weisdorf D, Martin P, et al: 1994 Consensus . According to these criteria, mild cGVHD reflects one or two organs involved with no more than score 1 plus lung score 0; moderate cGVHD involves three or more organs involved with no more than score 1 or at least one organ (not lung) with a score of 2 or lung score 1; and extensive cGVHD is diagnosed when at least one organ with a score of 3 or lung score of 2 or 3. The diagnosis was mainly based on clinical manifestations.
(6) Relapse was defined by the morphological evidence of disease in the peripheral blood, BM or extramedullary sites. Time to relapse was defined from the date of transplantation to the date of disease recurrence. Patients exhibiting minimal residual disease (for example, the presence of BCR/ABL RNA transcripts by PCR) were not classified as having relapsed.
(7) Overall survival was calculated from day 0 to the time of death or last follow-up.
(8) Disease-free survival (DFS) was defined as the time from day 0 to the date of disease progression or death from any cause.
(9) Transplant-related mortality was defined as all causes of death other than those related directly to malignant disease itself, occurring at any time after transplantation.

Safety profile
Safety analyses were performed based on the incidences of adverse events, transplantrelated complications.

Adverse events
(1) Definition: Adverse events refer to an unforeseeable medical condition during or after treatment or an event worsening health conditions that does not necessarily have a causal relationship with the treatment regimen used. Adverse events include symptoms, signs and abnormal laboratory results.
(2) Recording and grading: Adverse events were recorded and graded according to the Common Terminology Criteria for Adverse Events, Version 4.0.
(3) Monitoring: All patients were monitored by daily physical examination and routine blood tests prior to neutrophil. Liver and kidney function tests were performed at least twice a week within 30 days after UCBT. After neutrophil engraftment, serum cytomegalovirus (CMV)-DNA was also detected once a week until day 90.

Transplant-related complications
(1) Bleeding events were the major transplant-related complication after UCBT.
(2) Grading: The assessment of bleeding events, with the exception of mild petechiae, was Bleeding severity was graded as minor, major-nonlife-threatening and major-life-threatening. Major bleeding was defined if it induced a reduction in the hemoglobin level of at least 20 g/L, transfusion of at least two blood-pack units, or symptomatic bleeding in a critical area or organ. Transfusion events due to aplasia post chemotherapy were not included. Major bleeding was considered to be life-threatening if it resulted in fatality, symptomatic intracranial or pulmonary bleeding, bleeding with a decrease in the hemoglobin level of at least 50 g/L, bleeding requiring the transfusion of at least four red blood-cell units or inotropic agents, or surgery. All other bleeding was considered minor.

Statistical analysis
Numerical variables were described as medians and compared using the Mann-Whitney U test, and Pearson's chi-square test was used for categorical variables.
Cumulative incidence curves were used in a competing-risks setting, to calculate the probability of neutrophil engraftment, platelet engraftment and recovery, acute GVHD, TRM, and relapse. The occurrence of death prior to neutrophil engraftment, platelet engraftment and platelet recovery was considered a competing risk. Competing risks for the occurrence of GVHD were death without GVHD and relapse without GVHD.
The competing risk for TRM was death with relapse. When relapse of leukemia was the outcome of interest among patients with this disease, death without relapse was a competing risk event. The probabilities of OS and DFS were estimated using the Kaplan-Meier method.
All statistical analyses were performed using R software package (version 3.5.0), Prism