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CLINICAL—LIVER

GASTROENTEROLOGY 2012;142:505–512

CLINICAL LIVER

Granulocyte Colony–Stimulating Factor Mobilizes CD34 Cells and Improves Survival of Patients With Acute-on-Chronic Liver Failure

VISHAL GARG,* HITENDRA GARG, ARSHI KHAN, NIRUPAMA TREHANPATI, ASHISH KUMAR, BARJESH CHANDER SHARMA,* PUJA SAKHUJA, § and SHIV KUMAR SARIN* ,

Departments of *Gastroenterology and § Pathology, GB Pant Hospital, New Delhi; and Department of Hepatology, Institute of Liver and Biliary Sciences, New Delhi, India

Institute of Liver and Biliary Sciences, New Delhi, India BACKGROUND & AIMS: Acute-on-chronic liver failure

BACKGROUND & AIMS: Acute-on-chronic liver failure (ACLF) develops in patients with chronic liver disease and has high mortality. Mobilization of bone marrow– derived stem cells with granulocyte colony–stimulating factor (G- CSF) could promote hepatic regeneration. METHODS:

Consecutive patients with ACLF were randomly assigned to groups given 5 g/kg G-CSF subcutaneously (12 doses; group A, n 23) or placebo (group B, n 24) plus standard medical therapy. We assessed survival until day 60; Child– Turcotte–Pugh (CTP), Model for End-Stage Liver Disease (MELD), and Sequential Organ Failure Assessment (SOFA) scores; and the development of other related complications. RESULTS: After 1 week of treatment, group A had higher median leukocyte and neutrophil counts than group B (P .001). Sixteen patients in group A (69.6%) and 7 in group B (29%) survived; the actuarial probability of survival at day 60 was 66% versus 26%, respectively (P .001). Treatment with G-CSF also reduced CTP scores in group A by a median of 33.3% compared with an increase of 7.1% in group B (P .001), along with MELD (median reduction of 15.3% com- pared with an increase of 11.7% in group B; P .008) and SOFA scores (median reduction of 50% compared with an increase of 50% in group B; P .001). The percentages of patients who developed hepatorenal syndrome, hepatic en- cephalopathy, or sepsis were lower in group A than in group B (19% vs 71% [P .0002], 19% vs 66% [P .001], and 14% vs 41% [P .04], respectively). After 1 month of treatment, G-CSF increased the number of CD34 cells in the liver (by 45% compared with 27.5% in group B; P .01). CONCLU- SIONS: G-CSF therapy more than doubles the percent- age of patients with ACLF who survive for 2 months; it also significantly reduces CTP, MELD, and SOFA scores and prevents the development of sepsis, hepatorenal syndrome, and hepatic encephalopathy.

Keywords: Liver Regeneration; Liver Stem Cells; Growth Factor; Cirrhosis.

A cute-on-chronic liver failure (ACLF) is a serious acute insult of the liver on an underlying compensated

chronic liver disease. ACLF has been defined as an acute hepatic insult manifesting as jaundice (serum bilirubin level 5 mg/dL) and coagulopathy (international normal- ized ratio 1.5), complicated within 4 weeks by ascites and/or encephalopathy in a patient with previously diag- nosed or undiagnosed chronic liver disease (Asian Pacific Association for the Study of the Liver criteria). 1,2 ACLF is characterized by a high mortality rate caused by multi- organ failure. 2 Kjaergard et al reported a mortality rate of 51% in their systemic meta-analysis. 3 The short-term mor- tality may be as high as 65% at 3 months. 4 Available therapeutic options are limited. The Molecular Adsorbent Recirculating System is an option for patients with ACLF to give them additional time for recovery or to serve as a “bridge” to transplant. However, in a recent meta- analysis, 5 treatment with the Molecular Adsorbent Recircu- lating System did not appear to reduce mortality signifi- cantly compared with standard medical treatment. Liver transplant remains the only definitive therapy for patients with ACLF; however, the limited availability of do- nor organs, prohibitive costs, limited expertise, and lack of widespread availability limit its usefulness in the manage- ment of patients with ACLF. Furthermore, in patients with acute alcoholic hepatitis and ACLF, liver transplant cannot be considered because of a necessary requirement of absti- nence from alcohol use for 6 months. These patients are often quite sick and require repeated hospitalization with poor outcomes. Thus, there is an urgent need to evaluate new treatment options for patients with ACLF. The intriguing capability of blood-derived stem cells that differentiate into multiple cell lineages raises the exciting opportunity of using these cells for tissue repair when the pool of tissue-intrinsic stem cells is overwhelmed. 6

In the liver regeneration process, together with hepato- cytes and intrahepatic stem cells, bone marrow– derived

Abbreviations used in this paper: ACLF, acute-on-chronic liver failure; CTP, Child–Turcotte–Pugh; G-CSF, granulocyte colony–stimulating fac- tor; HVPG, hepatic venous pressure gradient; MELD, Model for End- Stage Liver Disease; SOFA, Sequential Organ Failure Assessment. © 2012 by the AGA Institute

0016-5085/$36.00

doi:10.1053/j.gastro.2011.11.027

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stem cells may represent a third proliferative compart- ment. 7 Animal and human studies have suggested that such cells might contribute to the regeneration after dif- ferent kinds of liver injuries. 8 A potential approach to improve bone marrow– derived stem cell engraftment to the damaged liver could be their mobilization by using cytokine administration. 9 Granulo- cyte colony–stimulating factor (G-CSF) therapy has been widely studied in bone marrow transplant recipients and in the oncologic population. In an experimental rat model of fulminant hepatic failure and encephalopathy, rG-CSF not only ameliorated the histologically evident liver injury in a statistically significant manner but also enhanced the proliferative capacity of the hepatocytes. 10 In a recent study, mobiliza- tion of bone marrow– derived stem cells induced by G- CSF was observed in patients with severe cirrhosis. 11 In another study, it has been shown that G-CSF mobilizes CD34 cells, increases hepatocyte growth factor, and in- duces hepatic progenitor cells to proliferate within 7 days of administration in patients with alcoholic steatohepati- tis. 12 Mookerjee et al have shown that neutrophil dysfunc- tion in patients with alcoholic hepatitis superimposed on cirrhosis leads to the development of sepsis and further to the development of hepatorenal syndrome and hepatic encephalopathy in patients with alcoholic hepatitis super- imposed on cirrhosis. 13 G-CSF therapy, on the other hand, has been shown to improve neutrophil function in patients with glycogen storage disease type Ib. 14 There are no data whether G-CSF therapy prevents sepsis and im- proves survival in patients with ACLF. We undertook this study to evaluate the safety and efficacy of G-CSF therapy in reducing morbidity and mor- tality in patients with ACLF. We also investigated whether G-CSF therapy could improve the indices of severity of liver disease, such as Child–Turcotte–Pugh (CTP), Model for End-Stage Liver Disease (MELD), and Sequential Or- gan Failure Assessment (SOFA) scores. Sepsis, which sets in rather early in patients with ACLF, is partly related to neutrophil dysfunction. 13,15 The effect of G-CSF therapy on short-term (day 60) survival in patients with ACLF and prevention of new-onset hepatic encephalopathy and hepa- torenal syndrome was also studied. Whether CD34 cells are mobilized to the damaged liver after G-CSF administration in vivo by counting these cells in the peripheral venous blood and hepatic parenchyma was analyzed.

Patients and Methods Patients

Between December 2008 and August 2010, every consec- utive patient with ACLF (as defined by an acute hepatic insult manifesting as jaundice [serum bilirubin level 5 mg/dL] and coagulopathy [international normalized ratio 1.5), compli- cated within 4 weeks by ascites and/or encephalopathy in a patient with previously diagnosed or undiagnosed chronic liver disease [Asian Pacific Association for the Study of the Liver criteria]) 1 was enrolled and included in the study. The exclusion criteria included age younger than 12 and older than 75 years,

hepatocellular carcinoma or portal vein thrombosis, refusal to participate in the study, any concurrent evidence of sepsis, any significant comorbidities, multi-organ failure, grade 3 or 4 he- patic encephalopathy, HIV seropositivity, pregnancy, and any previous known hypersensitivity to G-CSF.

Methods

The patients were evaluated in terms of their clinical pre- sentation and were investigated for the etiology of the liver disease. The diagnosis of the underlying chronic liver disease was based on clinical, radiologic, and endoscopic criteria. The presence of any of the following was taken as evidence of underlying chronic liver disease: high serum-ascites albumin gradient, grade 2 esophageal varices, hepatic venous pressure gradient (HVPG) 10 mm Hg, stage 2 fibrosis on histologic analysis, or portal vein 13 mm on ultrasonography. Wherever needed, HVPG measurement and a transjugular liver biopsy were performed to confirm the presence of underlying chronic liver disease. CD34 cell quantification. CD34 cells were assessed at baseline in the peripheral venous blood and the liver tissue. Briefly, peripheral blood mononuclear cells were isolated on the baseline and follow-up samples from peripheral blood by Ficoll– Hypaque density gradient centrifugation of fresh blood samples and were stored in freezing media containing 10% dimethyl sulfoxide and 90% fetal calf serum at 80°C for 24 hours and then were shifted to liquid nitrogen until used. The expression of CD34 on different cell types was assessed by using 3-color flow cytometry. The fluorochrome-labeled antibodies were used and single-color compensation was performed before acquisi- tion of samples. Lymphocytes were incubated with the antibod- ies for 30 minutes at 4°C and washed twice with fluorescence- activated cell sorter buffer containing 1 phosphate-buffered saline and 10% fetal calf serum. Cells were fixed with 4% para- formaldehyde. The surface expression of CD34 was measured on

a BD Calibur Flow cytometer (BD Biosciences, San Diego, CA)

and analyzed using CellQuest Pro software (BD Biosciences). Immunohistochemistry using antibody to CD34 (Spring Biosci- ences RTU) was performed on 4- m-thick sections of paraffin- embedded liver tissues using standard protocol. Antigen re-

trieval was performed in citrate buffer at pH 6 in a microwave for 2 minutes at half power and 2 minutes at full power. After the application of primary antibody, slides were incubated overnight in a humid chamber and subsequently washed in 3 changes of Tris buffer for 30 minutes. Biotinylated secondary antibodies were applied for 1 hour in the same chamber at room temper- ature followed by washing with Tris buffer. Next, streptavidin peroxidase reagent was applied and incubated in the same cham- ber for 1 hour at room temperature. Finally, reaction product was visualized by developing color using diaminobenzidine tet- rahydrochloride. Sections were counterstained with hematoxy- lin. CD34 scores were calculated by counting CD34-positive cells in sinusoids (arteries were not counted). Within 48 hours of admission, the patients were enrolled and randomized into 2 groups. A randomization code was generated. Randomization was performed with sequentially numbered en- velopes to either standard medical therapy with or without G-CSF, and the investigators as well as the patients were blinded to the treatment allotted. Patients in group A received G-CSF at

a dose of 5 g/kg subcutaneously, 12 doses over a period of 1

month (daily doses for the first 5 days and then every third day), along with the standard medical therapy, and patients in group

B received placebo (1 mL saline subcutaneously each time) along

with the standard medical therapy.

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Standard medical therapy included as per requirement lactu- lose, bowel wash, albumin, fresh frozen plasma, terlipressin, and antibiotics. Whenever needed, patients were supported with me- chanical ventilation, vasopressors, and renal replacement ther- apy. Patients with reactivation of hepatitis B and alcoholic hep- atitis were treated with tenofovir and pentoxifylline, respectively. Liver transplant and liver support devices could not be offered to the patients because they were not available at our center. During the treatment, daily physical examination was per- formed. White blood cell count, tests of liver function and kidney function, and estimation of prothrombin time and in- ternational normalized ratio were performed every alternate day during the first week of the treatment and weekly thereafter. The CTP and SOFA scores were used for assessing the severity of liver disease and were calculated at baseline and at days 7, 30, and 60. We calculated the change in CTP and SOFA score as the change, which was calculated as follows: (Score at Follow-up Day — Score at Baseline)/(Score at Baseline) %, %. Repeat estimation of CD34 cells in the peripheral venous blood and the liver tissue was performed after the completion of therapy at day 30, and repeat HVPG measurement and tran- sjugular liver biopsy were performed after the completion of therapy at day 30. The primary end point of the study was survival at day 60 as a result of the treatment used. The secondary end points of the study were (1) improvement in the CTP and SOFA scores and (2) development of new-onset hepatic encephalopathy, hepatorenal syndrome, and sepsis. The adverse effects of G-CSF therapy were carefully recorded throughout the study in each patient.

Ethical Considerations

The study protocol was approved by the institutional ethics committee, and the study conformed to the Helsinki Declaration of 1977. All patients gave written informed consent to participate.

Statistical Analysis

This study was designed to compare the efficacy of G-CSF therapy as calculated by Fisher exact test in patients with ACLF as compared with placebo. The probability of death in the placebo group was assumed to be 65% at 2 months. 2 It was hypothesized that G-CSF therapy would decrease the risk to 25%. Using one-sided analysis with an value of 0.05 and power (1 — ) of 0.80, the required sample size was calculated to be 23 in each group. Descriptive statistics were expressed as median (range) or number (%). Comparison of continuous variables was performed by Mann–Whitney U test and categorical variables by Fisher exact test or Pearson 2 test. The actuarial probability of survival was calculated by Kaplan–Meier graph and compared by log- rank test. All statistical tests were performed using SPSS for Windows version 15 (SPSS Inc, Chicago, IL).

Results

Seventy consecutive patients with a clinical presen- tation of ACLF presenting to the gastroenterology service of our institute were screened from December 2008 to August 2010 (CONSORT Flow Diagram). Forty-seven pa- tients satisfying the inclusion criteria were included in the study: 23 patients randomized to G-CSF (group A) and 24 to placebo (group B). This was the first clinical presenta-

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tion as liver failure in these patients, and all the patients were diagnosed as having underlying chronic liver disease for the first time. The diagnostic workup of underlying chronic liver disease was therefore more elaborate and was based on a combination of imaging, HVPG measurement, upper gastrointestinal endoscopy, and/or a liver biopsy. Twenty-three patients could not be included for the fol- lowing reasons at the time of presentation: evidence of sepsis (n 7), hepatic encephalopathy grade 3 or 4 (n 7), acute liver failure (n 3), hepatocellular carcinoma (n 4), or refusal to participate in the study (n 2). The patients were randomized into either of the 2 treatment groups. The baseline parameters of the study popula- tion are summarized in Table 1. The etiologies of the acute event and the underlying chronic liver disease are summarized in Table 2.

Outcome

Forty-seven patients who fulfilled the inclusion and exclusion criteria were included in the study. The demographic profile was similar across the study groups. There were 20 male patients in group A (male/female,

20:3) and 21 male patients in group B (male/female, 7:1)

( P .71). The median age (40 years [range, 30 –65] in

group A and 40 years [range, 19 –55] in group B) ( P .70) was comparable in both groups. Alcohol-related liver disease was the most common etiology. Alcoholic hepatitis was seen in 27 patients

(57.4%). Ten patients (21.2%) had reactivation of hepatitis

B virus as the acute insult. Similarly, alcohol-related liver

disease was the most common underlying chronic liver disease, seen in 29 patients (61.7%). Eleven patients (23.4%) had hepatitis B virus–related chronic liver disease. Measurement of HVPG was performed in 21 patients at baseline (11 patients in group A and 10 patients in group B). The median HVPG was high and comparable in both

the groups at baseline (group A, 16 [13–28] mm Hg; group B, 19.25 [11–30] mm Hg; P .323). The HVPG value also helped us to confirm the presence of underlying chronic liver disease in these patients. There was no increase in spleen size during therapy, and no decrease was seen in the median platelet count during therapy. Baseline total leukocyte count was comparable in both groups (group A, 10.7 [3.9 –22.1] 10 3 /mm 3 ; group

B, 11.8 [3.8 –28.7] 10 3 /mm 3 ). G-CSF therapy resulted in

a significant increase in total leukocyte count at days 2, 4, 7, 14, 21, and 30. At days 42 and 60, the total leukocyte count was again comparable in both groups (Figure 1).

Severity Indices

At baseline, the CTP, MELD, and SOFA scores were comparable in both groups. G-CSF therapy resulted in significant improvement in CTP, MELD, and SOFA scores at days 7, 30, and 60. There was a significant and

progressive improvement in the indices of severity of liver failure, as evident in the reduction in the CTP, MELD, and SOFA scores. The median change in CTP score at days

7, 30, and 60 was 15.38%, 25%, and 33.33% in group

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Table 1. Baseline Characteristics of the Study Population

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Parameters

Group A (n 23)

Group B (n 24)

P value

Male/female Age ( y ) Ascites Total leukocyte count ( 10 3 /mm 3 )

20/3

21/3

.71

40 (30–65) 23 (100) 10.7 (3.9–22.1) 8.3 (2.4–19.1) 128 (50–265) 131 (124–138) 0.8 (0.5–3.7) 25.6 (9.0–43.5) 2.20 (1.66–3.92) 65 (21–250) 2.6 (1.8–3.5) 5 (10.6) 2 (1–2) 2 (0–3) (n 22) 15 (65.2) 4 (8.5) 5.34 (5.04–6.60) (n 4) 16 (13–28) (n 11) 4 (0–5) (n 10) 12 (11–14) 29 (21–40) 5 (4–9)

40 (19–55) 24 (100) 11.8 (3.8–28.7) 8.7 (3.1–26.6) 143 (75–186) 130 (115–146) 1.0 (0.3–4.9) 23.9 (6.2–36.1) 2.71 (1.70–4.53) 86 (34–247) 2.5 (2.0–3.8) 8 (17) 2 (1–2) 2 (0–4) (n 20) 17 (70.8) 5 (10.6) 5.50 (4.76–7.93) (n 7) 19.25 (11–30) (n 10) 4 (0–4) (n 8) 12 (10–14) 31.5 (20–40) 6 (4–10)

.70

1

.34

Absolute neutrophil count ( 10 3 /mm 3 ) Platelets ( 10 3 /mm 3 ) Sodium ( mEq/dL ) Creatinine (mg/dL ) Bilirubin ( mg/dL ) International normalized ratio Alanine aminotransferase ( IU/L ) Albumin ( g/dL ) Encephalopathy

.43

.90

.19

.06

.53

.12

.11

.27

.51

Grade

of

encephalopathy varix (n 42)

.28

Grade

of

.32

Grade of varices 2 Hepatorenal syndrome HBV DNA log 10 ( IU/mL) (n 11) HVPG ( mm Hg ) (n 21) Fibrosis score (modified Ishak) (n 18) CTP score MELD score SOFA score

.76

1

.91

.32

.237

.91

.069

.40

NOTE. All values are expressed as median (range) or number (%).

A and 0%, 0%, and 7.14% in group B, respectively. Me-

dian change in MELD score at days 7, 30, and 60 was 7.41%, 18.23%, and 15.34% in group A and 3.33%, 6.25%, and 11.76% in group B, respectively. Median change in SOFA score at days 7, 30, and 60 was 20%, 25%, and 50% in group A and 20%, 0%, and 50% in group B, respectively.

Development of Complications

Four patients in group A and 17 patients in group

B developed hepatorenal syndrome during follow-up ( P

.002). Four patients in group A and 16 patients in group

B developed new-onset hepatic encephalopathy on fol-

low-up ( P .001). Three patients in group A and 10 patients in group B developed clinically evident sepsis during the follow-up period ( P .04).

Table 2. Etiology of the Acute Event and the Underlying Chronic Liver Disease

Acute event

Group A

Group B

Alcoholic hepatitis Reactivation of hepatitis B virus Antitubercular therapy Hepatitis E virus infection Cryptogenic Underlying chronic liver disease Alcoholic liver disease Hepatitis B Cryptogenic Wilson’s disease

15 (65)

12 (50)

4 (17)

6 (25)

2 (9)

1 (4)

1 (4)

2 (8)

1 (4)

3 (12)

17 (74)

12 (50)

4 (17)

7 (30)

2 (9)

4 (16)

1 (4)

NOTE. All values are expressed as n (%). There was no significant difference between groups A and B.

The probability of development of hepatorenal syn- drome, new-onset hepatic encephalopathy, and sepsis was significantly lower in patients in group A compared with patients in group B (0.19 vs 0.71 [ P .0002], 0.19 vs 0.66 [ P .001], and 0.14 vs 0.41 [ P .04]), respectively.

CD34 Cell Mobilization

At baseline, CD34 cells were comparable in the peripheral blood and liver tissue. G-CSF therapy led to a significant increase in the CD34 cells in the liver tissue at follow-up (day 30) (Table 3 and Figure 2).

Survival

G-CSF therapy resulted in significantly improved 60-day survival as compared with placebo in patients with

60-day survival as compared with placebo in patients with Figure 1. Median total leukocyte count in

Figure 1. Median total leukocyte count in the 2 groups of patients.

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Table 3. CD34 Cell Profile in the Peripheral Blood and Liver in the Study Groups

Group A

Group B

P value

Peripheral

vein

CD34

cells ( % , baseline) (n 47)

0.72 (0–3.77) (n 23)

0.54 (0.08–67.44) (n 24)

.502

Peripheral

vein

CD34

cells ( % , day 30) (n 25)

0.43 (0.02–3.51) (n 15)

0.41 (0.1–2.15) (n 10)

.803

P

value

.02

.11

Hepatic

Hepatic

CD34

CD34

cells ( % , baseline) (n 18)

27.5 (20–70) (n 10)

30 (15–60) (n 8)

.583

cells ( % , day 30) (n 18)

45 (30–70) (n 10)

20 (10–30) (n 8)

.001

P value

 

.01

.03

NOTE. All values are expressed as median (range) or number (%).

ACLF. Sixteen patients (69%) in group A and 7 patients (29%) in group B survived ( P .01, with the actuarial probability of survival [day 60] of 66% vs 26% [ P .001]) (Figure 3). Seven patients died in group A (progressive liver failure, 2; multi-organ failure, 3; variceal bleed, 2) and 17 patients died in group B (progressive liver failure, 2; multi-organ failure, 12; variceal bleed, 2; intracranial bleed, 1). Forty-two percent of patients (3 of 7) in group A and 66% of patients (12 of 18) in group B died because of multi-organ failure. Thus, G-CSF therapy tended to prevent the develop- ment of multi-organ failure and subsequent death.

Adverse Events

G-CSF therapy was very well tolerated in our pa- tient group. One patient had a transient rash (improved with omission of one dose), one patient had herpes zoster at the end of treatment (treated with acyclovir), and in one patient one dose of G-CSF was omitted because of high fever. Other patients completed therapy without any ma- jor adverse events.

Discussion

This is the first large randomized clinical trial that has shown clinical benefit with G-CSF therapy in patients with ACLF. Patients treated with G-CSF showed a signifi- cantly better 60-day survival compared with placebo (P

.001). Moreover, G-CSF therapy resulted in a significant improvement in the median CTP, MELD, and SOFA scores (P .001). The probability of development of hepatorenal syndrome, new-onset hepatic encephalopathy, and sepsis was also significantly lower in the treated group. ACLF is a recently defined clinical entity. 1,2 It connotes a serious acute insult of the liver associated with a very high short-term mortality rate. Approximately two-thirds of the patients may die in the absence of early liver transplant, which is often not readily available. 4 Most patients in the western series already have multi-organ failure at presenta- tion, which has a rapid downhill course leading to mortality. G-CSF has been used in patients with chronic liver disease as well as in animal models of acute-and-chronic liver injury with promising results. 10,11,16 19 These encour- aging results support our data on the benefits in patients with ACLF. More than 95% of the patients with ACLF in our study presented with acute-onset jaundice and ascites. The jaundice was severe in these patients, with a serum bilirubin value 20 mg/dL. Patients with any evidence of sepsis were excluded at baseline. In our study, the most common etiology of the underlying chronic liver disease was alcohol, followed by hepatitis B. Alcoholic hepatitis, sepsis, and upper gastrointestinal bleed- ing have been reported to be the most common causes of ACLF in studies from Western countries. 2026 In the present study, ACLF was defined according to the Asian Pacific

Figure 2. CD34 immunostain- ing in sinusoidal cells in pre- therapy and posttherapy biopsy specimens shows an increase in the cells in the posttherapy bi- opsy specimen (original magnifi- cation 200 ).

biopsy specimens shows an increase in the cells in the posttherapy bi- opsy specimen (original magnifi-

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Figure 3. Kaplan–Meier graph. The actuarial probability of survival (at day 60) was higher in
Figure 3. Kaplan–Meier graph. The actuarial probability of survival (at
day 60) was higher in group A than group B.
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Association for the Study of the Liver criteria, in which overt sepsis is not included in the definition of ACLF. This defi- nition allows screening of a much larger population of patients and an early identification of potentially treatable patients before the onset of overt sepsis (Figure 4). In addition to its well-characterized ability to induce neutrophil production, G-CSF is a potent stimulus for inducing neutrophil release from the bone marrow. 2730 It has been seen that in mice, a single injection of G-CSF leads to a 5-fold increase in circulating neutrophils that begins within 30 minutes and peaks at 12 hours after injection. 27 Similar results have been reported in human

studies with G-CSF. 28 In our study, G-CSF therapy led to a significant increase in total leukocyte count across the total duration of therapy and the counts again became comparable in both the groups at 2 months. The exact mechanism of the benefits of G-CSF therapy is not clear. Several studies in vitro and in vivo have documented the neutrophil-activating effect of G-CSF, indicating that it should be considered a potent immedi- ate activator of mature circulating cells capable of prim- ing respiratory burst, inducing the release of secretory vesicles and cytoplasmic granules, and modulating expres- sion of surface adhesion molecules. The polymorphonu- clear surface antigen CD11b/CD18 expression and the plasma elastase- 1AT complex levels are increased follow- ing G-CSF administration. 31,32 Although we did not study the neutrophil functions, it is possible that some of the preceding mechanisms could have resulted in the preven- tion of sepsis, multi-organ failure, and improved survival in our study group. A recent experimental study has shown that the use of colony-stimulating factor 1 pro- vides bone marrow– derived macrophages, which, on en- graftment in the liver, help in reducing the hepatic fibro- sis and support hepatic regeneration. 33 We observed that G-CSF therapy also led to a signifi- cant increase in the CD34 cell population in the liver tissue after 4 weeks of G-CSF administration protocol. This important observation gives credence to the role of recruitment of bone marrow– derived cells after G-CSF stimulation in patients with ACLF. There was a significant decrease in the peripheral blood CD34 cell population after 4 weeks, perhaps because of increased migration and settlement of the CD34 cells in the liver attributable to G-CSF therapy. It cannot be said with certainty whether a mere increase in the frequency of these cells in the hepatic

mere increase in the frequency of these cells in the hepatic Figure 4. Role of neutrophil

Figure 4. Role of neutrophil dysfunction in the pathogenesis of ACLF and the possible role of G-CSF therapy in preventing the development of sepsis and re- lated complications.

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tissue translated into enhanced regeneration and improved functional outcomes in patients with ACLF. However, it can be argued that this approach of prolonged 30-day adminis- tration of G-CSF is probably more beneficial than the pro- tocol of ex vivo, 1- to 2-week expansion of bone marrow– derived cells for allogeneic transplantation through the hepatic artery in such sick patients. 34,35 Further, clinical studies may address the in vivo versus ex vivo approach of bone marrow–derived cells in hepatic regeneration. Our results are at variance with those of an earlier study by Di Campli et al, who used G-CSF in patients with ACLF without any demonstrable biochemical or clinical benefit. 28 These contrasting results could be attributable to a very short duration (5 days) of G-CSF therapy used in their study and partly because of the differences in the patient popula- tion, especially in relation to the presence of sepsis and inclusion of patients with decompensated liver disease in the form of ascites, variceal bleeding, and encephalopathy. On the other hand, our patient group had an acute hepatic insult (defined by the presence of jaundice and encephalop- athy) over an underlying compensated or undiagnosed chronic liver disease without any overt sepsis. We have shown that reversal to the prior compensated state is possi- ble by timely and prolonged administration of G-CSF. An important observation of our study was that the G-CSF therapy tended to prevent the development of multi-organ failure. This could be explained by prevention of sepsis in these patients with the drug. Neutrophil dysfunction has been shown to cause sepsis and further to the development of hepatorenal syndrome and hepatic encephalopathy in patients with ACLF. G-CSF therapy further showed an improvement in the CTP, MELD, and SOFA scores and significantly improved survival. Our data show that patients with ACLF had about 60% incidence of multi-organ failure (multi-organ failure was defined as 2 or more organ failure [apart from liver failure], with organ failure defined as SOFA score 3 for the respective organ system). Similar observations have been previously reported as cause of death in patients with cirrhosis. 36 The patients with ACLF in our study who survived the initial 60 days were carefully followed up for assessing long- term survival and development of morbidities. It was impor- tant to note that these patients returned slowly to compen- sated stage and did not require an emergency liver transplant or die subsequently. These observations support the conten- tion that in the patients with ACLF who have not previously been symptomatic because of compensated chronic liver disease, if the acute hepatic insult can be overcome by ag- gressive clinical management supplemented by the use of G-CSF therapy, long-term survival is improved. This could possibly be related to initiation of hepatic regeneration by G-CSF therapy. These benefits were not seen in the patients receiving placebo (Figures 3 and 4). By giving G-CSF before the onset of sepsis, we have been able to achieve significant improvement in patient survival and reduction in the development of multi-organ failure. The aim of defining any disease entity is not only to char- acterize clearly the subset of patients, but also to specify

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potential therapeutic options. Identification of patients with ACLF before the onset of sepsis gives a window of opportu- nity of about 7–14 days for intervention. Our data show that introduction of therapies such as G-CSF during this “golden period” can reduce the development of sepsis and multi- organ failure and improve survival in patients with ACLF. Because the most common etiology of acute hepatic insult in our study was alcoholic hepatitis (57.4%), larger clinical trials using G-CSF therapy should be undertaken in patients with ACLF of different etiologies. G-CSF therapy was very well tolerated in our patient group. G-CSF has been used previously in patients with ACLF as well as in patients with alcoholic hepatitis. In summary, the results of this novel randomized con- trolled study clearly show distinct clinical benefits with prolonged G-CSF therapy for patients with ACLF. With its ease of administration and potential benefits of hepatic regeneration, in vivo G-CSF therapy merits further evalu- ation in patients with ACLF.

Supplementary Material

Note: To access the supplementary material accompanying this article, visit the online version of Gastroenterology at www.gastrojournal.org, and at doi:

10.1053/j.gastro.2011.11.027.

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893.

Received March 28, 2011. Accepted November 4, 2011.

Reprint requests Address requests for reprints to: Shiv Kumar Sarin, MD, Department of Hepatology, Institute of Liver & Biliary Sciences, New Delhi 110 070, India. e-mail: shivsarin@gmail.com.

Acknowledgments This trial is registered with ClincalTrials.gov (NCT01036932). Presented as an oral presentation at the 2010 annual meeting of the American Association for the Study of Liver Diseases in Boston, Massachusetts.

Conflicts of interest The authors disclose no conflicts.

March 2012

G–CSF AND SURVIVAL OF PATIENTS WITH LIVER FAILURE

2012 G–CSF AND SURVIVAL OF PATIENTS WITH LIVER FAILURE Assessed for eligibility (n=70) Randomized (n=47) Excluded
2012 G–CSF AND SURVIVAL OF PATIENTS WITH LIVER FAILURE Assessed for eligibility (n=70) Randomized (n=47) Excluded

Assessed for eligibility (n=70)

Randomized (n=47)
Randomized (n=47)

Excluded (n=23) Not meeting inclusion criteria (n=21) Declined to participate (n=2) Other reasons (n=0)

Allocated to intervention (Group A) (n=23)

Received allocated intervention (n=23)Allocated to intervention (Group A) (n=23) Did not receive allocated intervention (give reasons) (n=0)

Did not receive allocated intervention (give reasons) (n=0)Allocated to intervention (Group A) (n=23) Received allocated intervention (n=23)

Lost to follow-up (give reasons) (n=0)

Discontinued intervention (give reasons) (n=0)

Analysed (n=23)

Excluded from analysis (give reasons) (n=0)Analysed (n=23)

Allocated to intervention (Group B) (n=24)

Received allocated intervention (n=24)Allocated to intervention (Group B) (n=24) Did not receive allocated intervention (give reasons) (n=0)

Did not receive allocated intervention (give reasons) (n=0)Allocated to intervention (Group B) (n=24) Received allocated intervention (n=24)

Lost to follow-up (give reasons) (n=0)

Discontinued intervention (give reasons) (n=0)

Analysed (n=24)

Excluded from analysis (give reasons) (n=0)Analysed (n=24)

p
p
Analy
Analy

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