Standards, Policies, Protocols, and Regulations for Cell-Based Therapies

STANDARDS, POLICIES, PROTOCOLS, AND REGULATIONS FOR CELL-BASED THERAPIES a

Health Management Institute of Chinese PLA General Hospital, Beijing, People’s Republic of China; b Department of Transfusion Medicine, The Second Artillery General Hospital of Chinese People’s Liberation Army, Beijing, People’s Republic of China; cGeriatrics Institute of Xiyuan Hospital, Academy of Chinese Medical Sciences, Beijing, People’s Republic of China; d Department of Biotherapy of PLA 455 Hospital, Shanghai, People’s Republic of China; e Galactophore Department of the First Affiliated Hospital of Zhengzhou University, Henan, People’s Republic of China; fBeijing Key Laboratory of Normal Aging and Geriatrics, Geriatrics Institute of Chinese PLA General Hospital, Beijing, People’s Republic of China * †

Contributed equally. Contributed equally.

Correspondence: Qiang Zeng, M.D., Ph.D., Health Management Institute of Chinese PLA General Hospital, 28 Fuxing Road, Beijing 100853, People’s Republic of China. Telephone: 86 01068295521; E-Mail: zq301@ 126.com; or Xianyong Huang, M.D., Ph.D., Health Management Institute of Chinese PLA General Hospital, 28 Fuxing Road, Beijing 100853, People’s Republic of China. E-Mail: huangming301@ sina.cn; or Haixu Chen, Ph.D., Beijing Key Laboratory of Normal Aging and Geriatrics, Geriatrics Institute of Chinese PLA General Hospital, 28 Fuxing Road, Beijing 100853, People’s Republic of China. E-Mail: haixuchen@live. com Received July 8, 2015; accepted for publication November 23, 2015; published Online First on March 29, 2016. ©AlphaMed Press 1066-5099/2016/$20.00/0 http://dx.doi.org/ 10.5966/sctm.2015-0144

Residual b-Cell Function Predicts Clinical Response After Autologous Hematopoietic Stem Cell Transplantation HANG XIANG,a,* CHAO YANG,b,* TIANYUAN XIANG,c,* ZHENG WANG,d XIN GE,e FAN LI,f YUEHAN SU,d HAIXU CHEN,f,† XIANYONG HUANG,a,† QIANG ZENGa,† Key Words. Type 1 diabetes mellitus x Autologous hematopoietic stem cell transplantation x b-cell function x Predictive factors

ABSTRACT New strategies of autologous hematopoietic stem cell transplantation (auto-HSCT) have gained much interest for the treatment of type 1 diabetes mellitus. However, assessing the clinical response and residual b-cell function still has limitations. The aim of the study was to select the optimal quantitative index to assess pre-existing b-cell function and to explore its predictive function for clinical response after auto-HSCT therapy. In this study, all of the patients who had undergone auto-HSCT were clustered into a responder group (Db-score > 0) and a nonresponder group (Db-score £ 0). We compared their quantitative metabolic indexes at baseline and performed receiver-operating characteristic (ROC) analysis to analyze the correlations between the indexes and clinical response. KaplanMeier analysis was conducted to compare the cumulative response durations in each quartile of the selected indexes. In an average of 15.13 6 6.15 months of follow-up, 44 of 112 patients achieved a clinical response. The responder group had lower levels of fasting plasma glucose and quantitative insulin sensitivity check index (QUICKI) but higher levels of fasting C-peptide, fasting insulin, and homeostasis model assessments for insulin resistance (HOMA-IR). ROC analysis showed that HOMA-IR had the largest area under the curve (0.756), which was similar to that of QUICKI. Kaplan-Meier analysis further confirmed that the third quartile (1.3371–1.7018) of HOMA-IR or the second quartile (0.3523–0.3657) of QUICKI was preferential for a prolonged response. In conclusion, HOMA-IR and QUICKI could be optimal measurements for b-cell reserves, and they were predictive for the clinical response after auto-HSCT. STEM CELLS TRANSLATIONAL MEDICINE 2016;5:651–657

SIGNIFICANCE The b-score was comprehensive and reliable in evaluating clinical response after autologous hematopoietic stem cell transplantation (HSCT). The homeostasis model assessments for insulin resistance and the quantitative insulin sensitivity check index could serve as precise assessments for residual b-cell function and good predictors of clinical response. They might be used to select optimal clinical trial participants or predict the clinical response after auto-HSCT.

INTRODUCTION Type 1 diabetes mellitus (T1DM), a common autoimmune disorder disease primarily affecting genetically susceptible individuals, is characterized by impairment of b-cell function and insulin production [1]. Although exogenous insulin can decrease or delay the occurrence of complications, it cannot halt the process of the disease. In recent years, hematopoietic stem cell (HSC) transplantation has been applied, with the aim of replicating pre-existing b cells or other progenitor cells by downregulating immunosuppression and inducing immunotolerance [2]. Among these methods, autologous HSC

transplantation (auto-HSCT) has been demonstrated to be safe and effective in improving b-cell function [3, 4]. However, there are still some concerns about the evaluation of the treatment. One concern was that clinical response should be evaluated by a more accurate and objective measurement. To date, this has been primarily defined by an increase of C-peptide and a decrease of glycated hemoglobin (HbA1c) or insulin use [5, 6]. These individual indicators cannot reflect the overall b-cell improvement because a patient might achieve remission in one or two indicators but not in the others [7]. Therefore, the b-score system was developed

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Table 1. Clinical characteristics of the responder and nonresponder groups at the baseline Variables

Responder group (Db-score > 0)

Nonresponder group (Db-score £ 0)

p value



Patients (n)

44

68

Follow-up (mo)

16.67 6 8.35

16.14 6 7.20

.73

Male patients, n (%)

21 (47.73)

33 (48.53)

.93

Age (yr)

14.70 6 4.83

16.46 6 5.72

.09

BMI (kg/m2)

19.66 6 5.62

20.13 6 4.12

.61

Insulin dose (IU/day)

14.50 6 3.63

16.16 6 4.97

.06

10 (14.70)

.62

Systemic hypertension, n (%)

8 (18.18)

Total cholesterol (mmol/l)

4.56 6 1.67

4.82 6 2.46

.54

LDL cholesterol (mmol/l)

2.22 6 1.34

2.37 6 1.06

.51

HDL cholesterol (mmol/l)

1.46 6 0.66

1.42 6 0.74

.77

Triglycerides (mmol/l)

1.42 6 1.03

1.85 6 1.24

.06

Leukocyte count before mobilization (3109 cells/l)

7.69 6 2.46

7.63 6 3.82

.93

Lymphocyte count before mobilization (3109 cells/l)

2.97 6 1.28

3.14 6 0.77

.38

CRP (mg/l)

4.54 6 2.53

5.45 6 1.73

.03a

IL-6 (pg/ml)

1.74 6 1.26

1.95 6 1.21

.38

IL-10 (mg/l)

31.21 6 13.62

34.66 6 16.24

.25

TNF-a (pg/ml)

3.52 6 1.75

4.87 6 1.36

.00a

IFN-g (pg/ml)

56.63 6 13.26

47.21 6 13.54

.00a

Anti-GAD antibody-positive, n (%)

23 (52.27)

35 (51.47)

.93

Anti-IA-2 antibody-positive, n (%)

15 (34.10)

21 (30.88)

.72

4 (9.09)

11 (16.17)

.28

Anti-insulin antibody-positive, n (%)

Values with a plus/minus sign are expressed as the mean 6 SD. a p , .05. Abbreviations: BMI, body mass index; CRP, C-reactive protein; GAD, glutamic acid decarboxylase; HDL, high-density lipoprotein; IA, islet antibody; IL, interleukin; LDL, low-density lipoprotein; TNF, tumor necrosis factor.

to measure b-cell preservation, which was first applied for evaluation after islet transplantation [8]. The b-score system has been proved to be a more accurate assessment because it is a composite of several clinical and metabolic indicators in a gradient pattern. Another concern was that an optimal index that can precisely reflect the residual b-cell function should be explored because the latter was crucial for the outcome after auto-HSCT [9]. In previous studies, scientists have applied different indexes to assess the residual b-cell function, such as stimulated C-peptide [10], first-phase insulin response [11], intravenous glucose tolerance tests [11], and homeostasis model assessments of insulin resistance (HOMA-IR) [12]; however, it is difficult to determine which index is better in predicting the clinical response because these indexes have not been compared, and no correlation has been explored between those indexes and the clinical outcomes. In this retrospective study, we compared different quantitative indexes that indicate the residual b-cell function and explored their predictive function for the clinical response after auto-HSCT therapy.

MATERIAL AND METHODS Patients and Procedures This study was approved by the Board of Medical Ethics of PLA 455 Hospital (Shanghai, China), where it was conducted.

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Written informed consent in accordance with the Declaration of Helsinki was signed by all of the adult patients or by the parents of the adolescents. One hundred twenty-three patients (age # 30 years) were enrolled from January 2008 to July 2010; these patients were diagnosed with T1DM within 6 weeks by clinical findings and hyperglycemia, and the diagnosis was confirmed with positivity for antibodies against glutamic acid decarboxylase. The diagnosis of T1DM was based on "Diagnosis and Classification of Diabetes Mellitus," published by the American Diabetes Association in 2006 [13]. Patients with a history of ketoacidosis onset, presence of acute or chronic systemic infection, allergic disease, pregnancy, or any organ dysfunction were excluded from this study. Transplantation was performed after extensive physical and laboratory examination was completed. In brief, bone marrow HSCs were mobilized with granulocyte colony-stimulating factor (10 mg/kg per day) the day after administration of cyclophosphamide (2.0 g/m2). Then, HSCs (CD34+ cells) were collected from peripheral blood and separated by leukapheresis, with an ending cell count of no less than 3.0 3 106 cells/kg body weight. The stem cells were cryopreserved until use. The patients were conditioned with cyclophosphamide (200 mg/kg) and rabbit antithymocyte globulin (4.5 mg/kg) [3, 14, 15] before transplantation. Followup was performed at 1, 2, 3, 6, 9, 12, 18, and 24 months after the transplantation. S TEM C ELLS T RANSLATIONAL M EDICINE

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Data Collection and Assays The demographic and clinical data were recorded before transplantation. The meal tolerance test was performed after an overnight fast (.8 hours), with blood drawn for glucose, insulin, and C-peptide at baseline and then at 90 minutes after a standard oral glucose tolerance test (OGTT; 1.75 g/kg or a maximum of 75 g of glucose). Plasma glucose was measured by a modified hexokinase enzymatic method (Cobas c501 autoanalyzer, Roche Diagnostics, Berlin, Germany, http://www.roche.com). Serum insulin was measured by the electrochemiluminescence method (Elecsys 2010 system, Roche, Basel, Switzerland). HbA1c was measured by liquid chromatography (normal: 4.20%–6.50%); C-peptide was measured by chemiluminescence (normal: 0.90–7.10 ng/ml). Standard quality-control procedures were performed each day with standard samples (CV , 10%). Daily duplicate analyses were also conducted using samples collected from the participants (CV , 10%).

Measurement and Assessment The clinical response was evaluated by the b-score at the last visit. The b-score and the cutoff values were based on clinical judgment [8]. For this assessment, four components were considered and each item was awarded a score of 0–2. No points were awarded if fasting plasma glucose (FPG) was .7 mmol/l, HbA1c was .6.9%, C-peptide secretion was undetectable on stimulation, or daily insulin use was .0.24 units/kg; 1 point was awarded if FPG was in the range of 5.6–6.9 mmol/l, HbA1c was in the range of 6.2%–6.9%, stimulated C-peptide was in the range of 0.1–0.29 nmol/l, daily insulin use was in the range of 0.001–0.24 units/kg, or the patient was taking oral hypoglycemic agents (OHAs); 2 points were awarded if FPG was ,5.5 mmol/l, HbA1c was ,6.1%, stimulated and/or basal C-peptide was .0.3 nmol/l, or the patient was not receiving insulin or OHAs [8]. The surrogate indexes of insulin sensitivity and resistance were estimated with models as follows: HOMA-IR = fasting insulin concentration (mU/ml) 3 fasting glucose concentration (mg/dl)/ 405 [16]; quantitative insulin sensitivity check index (QUICKI) = 1/ [log fasting insulin concentration (mU/ml) + log fasting glucose concentration (mg/dl)] [16, 17].

Statistical Analysis Quantitative data are expressed as the mean 6 SD. Comparisons of the continuous variables between the two groups were performed by using the t test or Wilcoxon rank-sum test. A receiver-operating characteristic (ROC) analysis was performed to compare the predictive functions of the indexes. HOMA-IR and QUICKI were then stratified into quartiles for Kaplan-Meier estimates to determine the difference and linear trend for each level. The analyses were performed by using the SPSS 15.0 statistical package (IBM, Inc., Chicago, IL, http://www-01.ibm.com) and the SAS 8.0 package (SAS Institute, Inc., Cary, NC, http:/ www.sas.com). Any p values (two-tailed) ,.05 were considered to indicate statistical significance.

RESULTS

Figure 1. Comparison of the b-scores between the responder group and nonresponder group before and after auto-HSCT therapy. The data in the graph is expressed as median (bottom, middle, and upper lines in the box represent quartiles 1–3, respectively) and range (error bars) of the b-score values. Abbreviation: auto-HSCT, autologous hematopoietic stem cell transplantation.

experienced ketoacidosis 3 months after treatment, leaving 112 patients for this retrospective study. During an average of 16.34 6 7.64 months of follow-up (range, 1–24 months), 44 (39.29%) patients achieved a clinical response (Db-score . 0), but 68 (60.71%) patients did not achieve a clinical response (Db-score # 0). The average follow-up for the two groups was 16.67 6 8.35 months and 16.14 6 7.20 months, respectively (p . .05). The baseline characteristics of these two groups are shown in Table 1, and no significant difference between the groups was found except for C-reactive protein, tumor necrosis factor-a (TNF-a), and interferon-g (Table 1).

Improvement in b-Score and Its Components After Auto-HSCT The b-scores of the responder group and nonresponder group were significantly different at baseline (3.18 6 1.07 vs. 2.74 6 1.01) and after auto-HSCT (4.91 6 1.22 vs. 1.63 6 1.10) (Fig. 1). To explore the improvement in each component of the b-score, we analyzed components in the two groups and compared their response rates. The results showed that FPG and insulin/OHA decrease had higher response rates (48.21% and 55.36%, respectively); HbA1c and stimulated C-peptide had lower response rates (27.68% and 25.89%, respectively). The b-score showed an intermediary value of 39.29% (supplemental online Table 1). This result further confirmed that b-score was a comprehensive system to evaluate the improvement of b-cell function.

Clinical Characteristics at Baseline

Glucose Metabolic Indicators Before and After Auto-HSCT

One hundred twenty-three patients were enrolled in the study, excluding 10 patients lost to follow-up and 1 patient who

We compared the patients’ glucose metabolic indexes. At baseline, the results showed that the responder group had lower

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Table 2. Glucose metabolic indicators before and after autologous hematopoietic stem cell transplantation After auto-HSCTa

Baseline Responder group

Nonresponder group

p value

Responder group

Nonresponder group

p value

FCP (ng/ml)

2.19 6 0.79

1.88 6 0.65

.03b

2.38 6 1.24

1.96 6 1.17

.07

FSI (mU/ml)

4.15 6 0.60

3.18 6 1.00

b

4.21 6 1.02

3.48 6 1.53

.02b

FPG (mg/dl)

141.65 6 36.60

148.50 6 46.91

.03

124.62 6 52.57

138.16 6 72.26

.29

7.41 6 1.04

7.70 6 1.89

.43

7.35 6 1.24

7.63 6 1.36

.28

218.95 6 62.66

234.65 6 76.30

.08

213.63 6 73.88

223.42 6 84.61

.53

Variable

HbA1c (%) G2h (mg/dl)

.01

b

IN2h (mU/ml)

9.12 6 3.56

8.34 6 3.55

.51

9.25 6 4.47

9.02 6 3.61

.77

CP2h (ng/ml)

4.11 6 0.52

3.87 6 0.74

.06

4.36 6 1.48

4.13 6 0.58

.25

HOMA-IR

1.4377 6 0.4090

1.0866 6 0.2879

.00b

1.4225 6 0.4242

1.1336 6 0.2879

.00b

QUICKI

0.3637 6 0.0126

0.3813 6 0.0179

b

0.3574 6 0.0244

0.3738 6 0.0326

.01b

.00

Unless otherwise noted, values are expressed as the mean 6 SD. a The latest visit after autologous hematopoietic stem cell transplantation was defined as ending time. b p , .05. Abbreviations: CP2h, C-peptide 2 hours after glucose administration; FCP, fasting C-peptide; FPG, fasting plasm glucose; G2h, plasma glucose 2 hours after glucose administration; HbA1c, glycated hemoglobulin; HOMA-IR: homeostasis model assessments for insulin resistance; IN2h, serum insulin 2 hours after glucose administration; QUICKI, quantitative insulin sensitivity check index.

Table 3. Receiver-operating characteristic curve analysis of baseline glucose metabolic parameters for clinical response Variables

FCP FSI FPG

Cutoff

Sensitivity (%)

Specificity (%)

AUC (95% CI)

p value

1.56 mg/ml

64.4

47.6

0.575 (0.460–0.690)

.16

3.725 mU/ml 121.54 mg/dl

81.0

42.4

0.677 (0.571–0.784)

.04

85.7

49.2

0.521 (0.514–0.734)

.03

HbA1c

6.25%

85.7

42.4

0.598 (0.487–0.708)

.09

G2h

9.49 mg/dl

81.0

42.4

0.636 (0.524–0.748)

.06

IN2h

6.76 mU/ml

70.5

44.1

0.532 (0.416–0.648)

.59

0.462 (0.352–0.573)

.50

CP2h

3.71 ng/ml

54.5

41.2

HOMA-IR

1.2579

97.6

66.1

0.76 (0.659–0.853)a

.00

QUICKI

0.3603

71.2

59.5

0.76 (0.659–0.853)a

.00

a

HOMA-IR and QUICKI achieved the largest AUC for clinical response. Abbreviations: AUC, area under the receiver-operating characteristic curve; CP2h, C-peptide 2 hours after glucose administration; FCP, fasting C-peptide; FPG, fasting plasm glucose; G2h, plasma glucose 2 hours after glucose administration; HbA1c, glycated hemoglobulin; HOMA-IR: homeostasis model assessments for insulin resistance; IN2h, serum insulin 2 hours after glucose administration; QUICKI, quantitative insulin sensitivity check index.

levels of FPG and QUICKI (p , .05) but higher levels of fasting C-peptide (FCP), fasting serum insulin (FSI), and HOMA-IR (p , .05). However, no significant difference existed in HbA1c, plasma glucose (G2h), serum insulin, and C-peptide between the groups (p . .05). After auto-HSCT, significant difference still existed in FSI, HOMA-IR, and QUICKI, but not in the other indexes (Table 2).

ROC Analysis of Various Indexes for Remission After Auto-HSCT ROC analysis was performed to compare the predictive function of these indicators. The area under the ROC curve (AUC) of QUICKI was 0.76 (95% confidence interval, 0.659–0.853; p = .00), at the cutoff value of 0.3603, with a sensitivity and specificity of 71.2% and 59.5%, respectively. HOMA-IR had an AUC similar to that of QUICKI, but its sensitivity (97.6%) and specificity (66.1%) were higher than those of QUICKI at the cutoff value of 1.2579. The AUCs of FSI, FPG, and PG2h were 0.677, 0.521, and 0.636, respectively, which were smaller than those of QUICKI and HOMA-IR. The smallest AUCs were those for HbA1c, FCP,

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and serum insulin 2 hours after glucose administration: 0.598, 0.575, and 0.532, respectively (Table 3).

Comparison of Stratified Indexes With Kaplan-Meier Estimates For each quartile of HOMA-IR, the response duration was significantly different (chi-square = 32.18; p = .00) (Table 4). Among those, the patients in the third quartile (1.3371–1.7018) had the longest response duration (22.93 6 0.53 months). This was followed by patients in the fourth (.1.7018) and second (1.1277–1.3372) quartiles, with median response durations of 16.50 6 0.75 months and 14.07 6 1.54 months, respectively. The shortest response duration, at 10.56 6 1.51 months, was for patients in the first quartile (,1.1277) (Fig. 2). As a counterpart, each quartile of QUICKI also had a significant difference in response durations (chi-square = 30.64; p = .00) (Table 4). Patients in the second quartile (.0.3759) of QUICKI had the longest response duration (22.93 6 0.53 months). In contrast, patients in the fourth quartile had the shortest response duration (10.56 6 1.51 months), notably shorter than duration among those in the first and third quartiles (16.50 6 1.14 months and 14.25 6 1.14 months, respectively) (Fig. 2). S TEM C ELLS T RANSLATIONAL M EDICINE

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Table 4. Comparison of cumulative response duration of homeostasis model assessments for insulin resistance and quantitative insulin sensitivity check index Variables

Range

Mean duration

SE

95% CI

10.56

1.51

7.60–13.51

1.54 11.05–17.09

HOMA-IR Quartile 1

,1.1277

Quartile 2

1.1277–1.3372

14.07

Quartile 3a

1.3371–1.7018

22.93

0.53

16.50

0.75 14.57–17.52

Quartile 4

.1.7018

21.89–23.97

Log-rank (Mantel-Cox): chi-square = 32.18, p = .00 QUICKI Quartile 1 Quartile 2a Quartile 3 Quartile 4

,0.3523

16.50

1.14

14.27–18.73

0.3523–0.3657

22.93

0.53

21.89–23.97

0.3657–0.3759

14.25

1.59 11.14–17.36

10.50

1.45

.0.3759

7.65–13.35

Log-rank (Mantel-Cox): chi-square = 30.64, p = .00 Values are expressed as months. a The third quartile of HOMA-IR and the second quartile of QUICKI predict the longest response. Abbreviations: CI, confidence interval; HOMA-IR: homeostasis model assessments for insulin resistance; QUICKI, quantitative insulin sensitivity check index.

DISCUSSION Insulin production is greatly reduced in patients with T1DM because the pancreatic islet b cells are severely impaired by the autoimmune antibodies. This progression can be halted or reversed by early interruption of stem cell transplantation before all the b cells are lost [18, 19]. In recent years, different types of stem cell transplantation have been applied to treat T1DM [2]. One of the strategies is b-cell replacement, which aims to preserve residual b cells and restore insulin-producing b cells through the transplantation of insulinproducing cells, such as embryonic stem cells [20], induced pluripotent stem cells [21], mesenchymal stromal cells [22, 23], and islet transplantation [24]. This strategy is severely encumbered by ethical issues, donor scarcity, and graft rejection [25]. Another strategy is b-cell regeneration, which aims to rebuild immunosuppression and induce immunotolerance [2]. For this strategy, allogenic HSC transplantation or auto-HSC transplantation has been performed for clinical trials. The safety and effectiveness of auto-HSCT in treating patients with new-onset T1DM have been demonstrated [3, 4]; this therapy can modulate the immune system [26] and reverse b-cell destruction [14], without associated graft-versus-host disease. However, although most studies have defined insulin requirement, C-peptide, or HbA1c changes as clinical response [27, 28], these indicators could not reflect the overall improvement of b-cell function [29]. For this reason, a composite index, the b-score, which is composed of the major aspects of function, has been suggested [8]. It was first applied to evaluate the success of islet transplantation and proved to be a better assessment because of its soundness and simplicity [8]. In this study, we introduced the b-score to evaluate the clinical response, and we found that the response rate of the b-score was between the higher rates of FPG and insulin/OHA reduction and the lower rates of HbA1c and stimulated C-peptide. Previous studies have applied one of these indicators to evaluate the success of auto-HSCT, but the effectiveness might be overestimated or underestimated by using just one of these indicators. However, the b-score, which

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contains subjective and objective responses, could provide a more sound and reliable assessment of clinical improvement. In the past few years, residual b-cell function was closely correlated with clinical outcomes [9]. The Diabetes Control and Complications Trial reported that residual b-cell function was correlated with the incidence of diabetic complications [30] and that continuing pancreatic secretion function was important in avoiding hypoglycemia during treatment [30]. Currently, the many methods to assess b-cell function include insulin sensitivity and resistance [31]. One method is direct measurement, including the hyperinsulinemic-euglycemic glucose clamp and insulin suppression test, which are very precise but not suitable for clinical study because they are labor-intensive and time-consuming [32]. Another method is indirect measurement, such as the intravenous glucose measurement of the OGTT, which is dynamic and less complex [33]. In addition, there are surrogate measurements that are composite indicators, such as HOMA-IR and QUICKI [16, 17]. For this study, we selected several indexes from all three measurements to analyze residual b-cell function, and we explored their association with clinical response. Our results showed that most of the indexes were closely correlated with clinical response, except the dynamic glucose indicators. This could be interpreted as indicating that the static metabolism at baseline was more crucial for the recovery of b-cell function. When this is combined with the result of the higher response rate of FPG and the lower response rate of stimulated C-peptide, we speculate that basic static metabolism might be recovered in the early phase, but maturation might be required for the regenerative cells to function in glucose stimulation. Among the indexes evaluated, HOMA-IR showed a stronger correlation with clinical response than the other indexes. HOMA-IR is an index for steady insulin resistance, which reflects a feedback loop between the liver and b cell. It describes the glucose-insulin homeostasis by a set of nonlinear equations, which consist of fasting glucose and insulin or C-peptide [34]. A previous study suggested that during the progression of the autoimmune process, T1DM was affected not only by the decreased b-cell secretion function but also by the increased insulin resistance [35]. HOMA-IR can effectively estimate insulin resistance and b-cell deficiency and performs well in accordance with the euglycemic clamp, the gold standard of insulin resistance [36]. Siljander et al. revealed that HOMA-IR was important for the stratification of the disease risk in prediabetic children [11]. Another report suggested that HOMA-IR could serve as a prognostic marker for the development of T1DM [37]. In this study, we found that HOMA-IR could well predict clinical response after auto-HSCT, with a sensitivity of 97.6% and a specificity of 66.1%. Further investigation of Kaplan-Meier estimates identified a predictive function of HOMA-IR for clinical remission duration, which showed the third quartile (1.3371–1.7018) for the longest remission. In contrast with our findings, Caumo et al. thought that HOMA-IR was unsuitable for the evaluation of b-cell function after islet transplantation [12]. This disagreement might have resulted from their small number of patients. Considering for this, HOMA-IR should be applied cautiously to evaluate patients with severely impaired b-cell function or absent insulin secretion [31]. Another index, the QUICKI, is a reliable and reproducible method that can accurately predict insulin sensitivity in a wide range of insulin-resistant states [38, 39]. It is among the most thoroughly evaluated and validated surrogate indexes of insulin sensitivity [40]. Studies showed that QUICKI is in line with the

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Figure 2. Kaplan-Meier curves for clinical response for each quartile of HOMA-IR (A) and QUICKI (B). Abbreviations: Cum, cumulative; HOMAIR, homeostasis model assessments for insulin resistance; QUICKI, quantitative insulin sensitivity check index.

standard glucose clamp in evaluating b-cell secretion and insulin resistance [41]. QUICKI has a positive predictive power for the development of diabetes [42]. Furthermore, QUICKI is correlated with HOMA-IR in a mathematical fashion (i.e., QUICKI is proportional to 1/log [HOMA-IR]) [43]. In this study, we observed that QUICKI had a similar efficiency in estimating clinical response as HOMA-IR, but a reverse linear trend in predicting response duration, stemming from their intrinsic characteristics [39]. Several key points should be noted. One key point is that insulin/OHAs use might influence the other components of the b-score (e.g., FPG). However, the factors involved in the b-score interact with each other. For example, the dose of insulin/OHAs was coordinated with controlled HbA1c, which was determined by the glucose metabolism in the past few weeks. Therefore, the b-score system was a composite system that consisted of the previous (HbA1c) and the present (FPG) glucose metabolism, the static (FPG) and the dynamic (stimulated C-peptide) glucose metabolism, and insulin or OHAs use. It was an all-around assessment of b-cell improvement. Another key point is that static metabolism indexes of HOMA-IR and QUICKI were more effective predictors than the individual indicators, such as FCP, FSI, and FPG, possibly because the composite indexes were more likely to predict a clinical response with high efficiency [7, 44]. This study had some limitations. First, the indication of residual b-cell function by HOMA-IR and QUICKI should be confirmed by the golden criteria of the hyperinsulinemic-euglycemic glucose clamp. Although a good congruence has been found in many insulin-resistant states, further validation would be helpful to confirm this agreement in T1DM. Second, the relationship between the indexes and the response should be corroborated by a long-term follow-up because a subset of patients might relapse after a transient remission [45]. Third, the mechanism of regeneration remains unclear, although clinical response has been demonstrated by several clinical trials [3, 46, 47]. Several pathogenic factors are involved in this disease, such as autoreactive T-cell count, antibodies, and cytokines, but we found that no factor

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was correlated with or predictive for the clinical response except for TNF-a [45]. Thus, further study is still needed to elucidate the mechanism of b-cell restoration and to explore more useful biomarkers to evaluate the success of auto-HSCT.

CONCLUSION The b-score was comprehensive and reliable in evaluating clinical response after auto-HSCT. The indexes of HOMA-IR and QUICKI could serve as precise assessments for residual b-cell function and good predictors for the clinical response. They might be applied for the selection of optimal clinical trial participants or used as the predictor for the clinical response after auto-HSCT treatment.

ACKNOWLEDGMENTS This study was supported by the State Science and Technology Support Program (No. 2012BAI37B04), Military Eleventh Five Year Health Promotion Program (No. 10BJZ18), National Nature Science Foundation of China (No. 31440039), and Beijing National Science Foundation of China (No. 5154035).

AUTHOR CONTRIBUTIONS H.X.: study design, drafting of the manuscript; C.Y.: data analysis and interpretation; T.X.: statistical analysis, data collection; Z.W.: provision of study material or patients; X.G.: clinical support; F.L.: technical and material support; Y.S: collection of data; H.C.: proofreading and revision; X.H.: administrative support; Q.Z.: study concept and design, obtaining of funding, final approval of manuscript.

DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST The authors indicated no potential conflicts of interest.

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Residual β-Cell Function Predicts Clinical Response After Autologous Hematopoietic Stem Cell Transplantation.

New strategies of autologous hematopoietic stem cell transplantation (auto-HSCT) have gained much interest for the treatment of type 1 diabetes mellit...
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