The aim of this study was to explore if measurement of pretransplant circulating CD161-expressing cells, in addition to clinical risk factors, could predict mucositis and infections in patients with multiple myeloma (MM) undergoing autologous stem cell transplantation (ASCT). To determine if CD161-expressing cells are likely to predict early complications, namely, mucositis (≥grade 3), infections, and cytomegalovirus (CMV) reactivation, we prospectively examined CD161-expressing cells (CD3+CD4+CD161+ and CD3+CD8+CD161+) in peripheral blood samples from 108 patients with MM undergoing ASCT. After adjusting for factors identified by univariate analysis that predicted mucositis (≥grade 3), infection before engraftment, and CMV reactivation, multivariate analyses revealed that the low proportion of CD3+CD4+CD161+ cells in peripheral blood was an independent predictor of mucositis (≥grade 3) (
High-dose therapy followed by autologous stem cell transplantation (ASCT) is the standard of care for transplant-eligible patients with multiple myeloma (MM) [
The innate immune system is a first line of defense against pathogens and sterile injury [
Although the influence of CD161-expressing T cells on early complications after allogeneic SCT has been examined [
A total of 108 consecutive patients with MM who underwent ASCT as part of front-line treatment at our institution between January 2012 and December 2015 were included in this analysis. To identify novel immune cell biomarkers predictive of early complications, we prospectively obtained peripheral blood samples before they underwent conditioning chemotherapy. In addition, various clinical and laboratory data that we hypothesized may contribute to the early development of complications following ASCT collected before the conditioning regimen was initiated. All patients received a pulmonary function test, echocardiography, liver Doppler ultrasound, and infection assessment prior to ASCT. The Institutional Review Board of the Catholic University of Korea approved the research protocol for all data analysis. This study was also conducted in accordance with the Declaration of Helsinki.
ASCT was performed after achieving a response greater than a partial response (PR). Some patients without progressive MM who were resistant to various agents (bortezomib and thalidomide) underwent ASCT. General ASCT procedures were performed as described previously [
Blood samples for cell population analysis were collected one day before conditioning chemotherapy. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood samples (10 mL) collected in EDTA-coated tubes by centrifugation in Ficoll-Paque Plus. PBMCs were processed immediately for analysis. Flow cytometry was used to evaluate the percentages of CD3+, CD4+CD161+, and CD8+CD161+ T cells; natural killer (NK) cells (CD16+CD56+); and myeloid-derived suppressor cells (MDSCs) [Lin-HLA-DR-CD11b+CD33+ (granulocytic) and HLA-DR-CD14+ (monocytic)]. Anti-CD3-allophycocyanin (APC), anti-CD4-fluorescein isothiocyanate (FITC), anti-CD8-phycoerythrin (PE), anti-CD161-PerCP-Cy5.5, anti-CD16-FITC, anti-CD56-PE, and anti-CD14-APC monoclonal antibodies (mAbs) were purchased from eBioscience (San Diego, CA, USA). Anti-Lineage cocktail 1 (Lin 1)-FITC, anti-HLA-DR-PerCP, rat anti-mouse CD11b-APC-Cy™7, and mouse anti-human CD33-V450 (BD BioSciences) mAbs were purchased from BD Biosciences (San Jose, CA). CD4+CD161+ and CD8+CD161+ T cells were gated on CD3+ cells and are expressed as percentages of lymphocytes. The frequencies of HLA-DR-Lin-CD11b+CD33+ and HLA-DR-CD14+ MDSCs are expressed as percentages of total PBMCs. Flow cytometry was performed using a FACS LSR Fortessa (BD Biosciences).
Toxicity was classified using the National Cancer Institute Common Toxicity Criteria grading scheme. Infections included microbially and clinically defined infections as proposed by the Immunocompromised Host Society [
Potential risk factors for the occurrence of early complications in patients with MM undergoing ASCT were assessed using logistic regression analysis. Optimal cutoffs for continuous variables were identified using receiver operating characteristic (ROC) curve analysis. To investigate whether the identified markers were independent predictors, covariates with a
A total of 108 patients with MM were analyzed, of whom 64 (59%) were male and 44 (41%) were female. The median age was 56 years (range, 32–67 years) and the median disease duration before ASCT was 6.8 months (range, 2.9–21.6 months). Demographic information for all patients is listed in Table
Patient characteristics.
Characteristic | Total |
---|---|
Age, years, median (range) | 56 (32–67) |
Sex (M/F) | 64 (59)/44 (41) |
BMI (kg/m2), median (range) | 24.6 (17.6–33.0) |
Serum M-protein | |
IgG, kappa | 28 (26) |
IgG, lambda | 31 (29) |
IgA, kappa | 5 (5) |
IgA, lambda | 9 (8) |
Light chain, kappa | 11 (10) |
Light chain, lambda | 17 (16) |
Other | 7 (6) |
Durie-Salmon stage | |
I-II | 14 (13) |
IIIA/B | 73 (68)/21 (19) |
Cytogenetics |
|
Standard risk/high risk/NA | 38 (35)/46 (43)/24 (22) |
Myeloma bone disease on plain radiographs, yes/no | 64 (59)/44 (41) |
Creatinine at diagnosis, mg/dL, median (range) | 1.02 (0.55–11.61) |
|
3.46 (0.2–44.0) |
Duration from diagnosis to ASCT, months, median (range) | 6.8 (2.9–21.6) |
|
|
Creatinine, mg/dL, median (range) | 0.70 (0.34–10.28) |
GFR |
104.62 (5.11–208.22) |
Diabetes | 13 (12) |
Arrhythmia/cardiac/heart valve disease | 2 (2)/5 (5)/0 (0) |
Pulmonary | |
Mild/moderate/severe | 17 (16)/45 (42)/25 (23) |
Hepatic | |
Mild/moderate-severe | 15 (14)/0 (0) |
HCT-CI | |
0–4/≥4 | 63 (58)/45 (42) |
ASCT, autologous stem cell transplantation; BMI, body mass index; GFR, glomerular filtration rate; HCT-CI, hematopoietic cell transplantation comorbidity index; NA, not available.
Among the 108 patients, 54 patients (50%) developed mucositis, including 25 with grade 2 and 29 with ≥grade 3. Analysis of risk factors was performed for mucositis (≥grade 3), because mucositis (≥grade 3) had a substantial effect on infection risk. The incidence of infection before engraftment was 52%. Twenty-nine patients (27%) developed CMV reactivation, nine of whom received ganciclovir therapy. One patient died due to severe sepsis within 100 days.
The median MNC count was 1.2 × 106 cells/mL (range, 0.2–3.4 × 106 cells/mL), and the mean (±standard error) proportions of CD3+CD4+CD161+ T cells and CD3+CD8+CD161+ T cells were
Proportions of pretransplant CD3+CD4+CD161+ cells according to (a) the occurrence of mucositis (≥grade 3), (b) infection before engraftment, and (c) CMV reactivation.
In addition, although we compared the proportions of NK cells and MDSCs according to the occurrence of mucositis (≥grade 3), infection before engraftment, and CMV reactivation in 59 patients with available data, there were no significant associations with early complications (Supplementary Table
The results of univariate analysis for potential risk factors predicting the development of mucositis (≥grade 3) following ASCT are listed in Table
Univariate analyses of risk factors for the occurrence of mucositis, infection before engraftment, and CMV reactivation.
Variable | Mucositis (≥grade 3) | Infection before engraftment (CDI + MDI) | CMV reactivation | |||
---|---|---|---|---|---|---|
RR (95% CI) |
|
RR (95% CI) |
|
RR (95% CI) |
| |
Age (years), continuous | 1.03 (0.98–1.09) | 0.272 | 1.00 (0.95–1.04) | 0.846 | 1.04 (0.99–1.11) | 0.149 |
Sex (female versus male) | 2.73 (1.14–6.53) | 0.024 | 2.25 (1.02–4.95) | 0.044 | 0.57 (0.23–1.40) | 0.216 |
BMI (kg/m2), continuous | 0.84 (0.72–0.99) | 0.037 | 0.86 (0.75–0.99) | 0.039 | 0.87 (0.75–1.02) | 0.088 |
Diabetes (yes versus no) | 0.46 (0.10–2.20) | 0.330 | 1.10 (0.34–3.50) | 0.878 | 1.85 (0.55–6.20) | 0.319 |
Cr (mg/dl), continuous | 1.10 (0.77–1.56) | 0.617 | 1.59 (0.81–3.10) | 0.178 | 1.15 (0.81–1.65) | 0.433 |
GFR |
0.98 (0.97–0.99) | 0.006 | 0.99 (0.98–1.00) | 0.096 | 1.00 (0.99–1.01) | 0.450 |
Pulmonary | ||||||
None-mild | 1 | 1 | 1 | |||
Moderate | 2.21 (0.79–6.19) | 0.130 | 0.89 (0.37–2.12) | 0.790 | 1.52 (0.55–4.19) | 0.415 |
Severe | 1.72 (0.52–5.71) | 0.374 | 2.86 (0.97–8.42) | 0.057 | 1.77 (0.56–5.55) | 0.332 |
Hepatic | ||||||
None | 1 | 1 | 1 | |||
Mild | 2.03 (0.65–6.32) | 0.222 | 0.41 (0.13–1.30) | 0.130 | 0.64 (0.17–2.47) | 0.521 |
Moderate-severe | - | - | - | - | - | - |
HCT-CI | ||||||
0–4 | 1 | 1 | 1 | |||
≥4 | 0.89 (0.29–2.71) | 0.836 | 3.44 (1.15–10.28) | 0.027 | 0.89 (0.29–2.71) | 0.836 |
Proportion of CD3+CD4+CD161+ cells (%), continuous | 0.88 (0.76–1.02) | 0.098 | 0.90 (0.80–1.00) | 0.055 | 0.86 (0.73–1.00) | 0.047 |
Proportion of CD3+CD8+CD161+ cells (%), continuous | 0.99 (0.93–1.06) | 0.781 | 0.97 (0.92–1.03) | 0.352 | 0.94 (0.88–1.02) | 0.129 |
BMI, body mass index; CDI, clinically defined infection; CI, confidence interval; CMV, cytomegalovirus; GFR, glomerular filtration rate; HCT-CI, hematopoietic cell transplantation comorbidity index; MDI, microbially defined infection; RR, relative risk.
In multivariate analysis (Table
Multivariate analyses of risk factors for the occurrence of mucositis, infection before engraftment, and CMV reactivation.
Variable | Mucositis (≥grade 3) | Infection before engraftment (CDI + MDI) | CMV reactivation | |||
---|---|---|---|---|---|---|
RR (95% CI) |
|
RR (95% CI) |
|
RR (95% CI) |
| |
CD3+CD4+CD161+ cells (%) (≤3.72 versus >3.72) | 0.19 (0.04–0.73) | 0.020 | 0.20 (0.06–0.73) | 0.014 | 0.25 (0.09–0.72) | 0.010 |
Sex (female versus male) | 6.39 (1.74–29.71) | 0.009 | 6.87 (2.05–23.04) | 0.020 | - | - |
BMI (kg/m2), continuous | 0.82 (0.64–1.03) | 0.094 | 0.87 (0.71–1.08) | 0.213 | 0.95 (0.78–1.16) | 0.595 |
GFR |
0.98 (0.97–1.00) | 0.020 | 1.00 (0.98–1.01) | 0.502 | - | - |
Pulmonary | ||||||
None/mild | - | - | 1 | - | - | |
Moderate | - | - | 1.29 (0.40–4.12) | 0.671 | - | - |
Severe | - | - | 5.54 (1.10–27.85) | 0.038 | - | - |
BMI, body mass index; CDI, clinically defined infection; CI, confidence interval; CMV, cytomegalovirus; GFR, glomerular filtration rate; MDI, microbially defined infection; RR, relative risk.
Multivariate analyses including clinical risk factors showing the predictive role of the proportion of pretransplant CD3+CD4+CD161+ cells for (a) the occurrence of mucositis (≥grade 3) and (b) infection before engraftment.
Female sex, decreased BMI, decreased GFR, and severe pulmonary comorbidity predicted the occurrence of infection before engraftment. In addition, decreased BMI predicted the occurrence of CMV reactivation (Table
Myeloablative high-dose chemotherapy followed by ASCT prolongs survival for patients with MM [
CD161 is a type II transmembrane glycoprotein with characteristics of the C-type lectin superfamily. This receptor is expressed on NK cells, 25% of all adult peripheral T cells, more than 90% of all peripheral blood monocytes, and on
In this study, we sought to identify predictors of mucositis in a homogeneous group of patients with MM undergoing ASCT following high-dose melphalan treatment, because mucositis risk is believed to vary according to the conditioning regimen intensity, SCT type (autologous versus allogeneic), and patient-related factors. Regarding SCT-associated mucositis, previous studies have reported that significant oral mucositis occurs in about 75% of all patients undergoing SCT [
CMV reactivation has been demonstrated to be relatively common after ASCT for MM (48.5%), especially when tandem transplantation is performed using combination chemotherapy with high-dose melphalan [
We identified that circulating CD3+CD4+CD161+ T cells, in addition to clinical risk factors, were associated with the occurrence of early complications in the setting of ASCT in patients with MM. Pretransplant predictive immune markers that can predict the occurrence of early complications will help assess transplant eligibility and identify those patients who require dose modification of conditioning chemotherapy and intensified prophylactic strategies.
The authors declare that they have no conflicts of interest.
This research was supported by a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (no. HI16C0047).
Supplementary Table 1: pretransplant cell populations according to the occurrence of mucositis, infection, and CMV reactivation.