Gastroenterology 2015;148:1362–1370
Combination of Granulocyte Colony-Stimulating Factor and Erythropoietin Improves Outcomes of Patients With Decompensated Cirrhosis Chandan Kumar Kedarisetty,1 Lovkesh Anand,1 Ankit Bhardwaj,2 Ajeet Singh Bhadoria,2 Guresh Kumar,2 Ashish Kumar Vyas,2 Paul David,2 Nirupama Trehanpati,2 Archana Rastogi,3 Chhagan Bihari,3 Rakhi Maiwall,1 Hitendra Kumar Garg,1 Chitranshu Vashishtha,1 Manoj Kumar,1 Vikram Bhatia,1 and Shiv Kumar Sarin1 CLINICAL LIVER
Departments of 1Hepatology, 2Biostatistics and Clinical Research, and 3Pathology, Institute of Liver and Biliary Sciences, New Delhi, India BACKGROUND & AIMS: Patients with decompensated cirrhosis have significantly reduced survival without liver transplantation. Granulocyte colony-stimulating factor (G-CSF) has been shown to increase survival in patients with acute-onchronic liver failure, and erythropoietin promoted hepatic regeneration in animal studies. We performed a double-blind, randomized, placebo-controlled trial to determine whether coadministration of these growth factors improved outcomes for patients with advanced cirrhosis. METHODS: In a prospective study, consecutive patients with decompensated cirrhosis seen at the Institute of Liver and Biliary Sciences, New Delhi (from May 2011 through June 2012) were randomly assigned to groups given subcutaneous G-CSF (5 mg/kg/d) for 5 days and then every third day (12 total doses), along with subcutaneous darbopoietin a(40 mcg/wk) for 4 weeks (GDP group, n ¼ 29), or only placebos (control group, n ¼ 26). All patients also received standard medical therapy and were followed for 12 months. Histology was performed on liver biopsies. The primary end point was survival at 12 months. RESULTS: Baseline characteristics of patients were comparable; alcohol intake was the most common etiology of cirrhosis. A higher proportion of patients in the GDP group than controls survived until 12 months (68.6% vs 26.9%; P ¼ .003). At 12 months, ChildTurcotte Pugh scores were reduced by 48.6% in the GDP group and 39.1% in the control group, from baseline (P ¼ .001); Model for End Stage Liver Disease scores were reduced by 40.4% and 33%, respectively (P ¼ .03). The need for large-volume paracentesis was significantly reduced in GDP group, compared with controls (P < .05). A lower proportion of patients in the GDP group developed septic shock (6.9%) during follow-up compared with controls (38.5%; P ¼ .005). No major adverse events were observed in either group. CONCLUSIONS: In a single-center randomized trial, a significantly larger proportion of patients with decompensated cirrhosis given a combination of G-CSF and darbopoietin a survived for 12 months more than patients given only placebo. The combination therapy also reduced liver severity scores and sepsis to a greater extent than placebo. Clinicaltrials.gov ID: NCT01384565.
Keywords: DPO; Liver Regeneration; Clinical Trial; Chronic Liver Disease.
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ost of the patients referred to a specialized hepatology center suffer from decompensation of endstage chronic liver disease. Decompensated cirrhosis is defined by the presence of ascites, variceal bleeding, encephalopathy, and/or jaundice.1 The only recommended treatment of choice is liver transplantation. A large proportion of cirrhotic patients succumb while waiting on the transplant list. Therefore, there is a large unmet need for studying newer treatment modalities, mainly focused on liver regeneration. During the last decade, the importance of bone marrow stem cell (BMSC) activation in liver disease has become apparent. CD34þ and CD133þ cells appear to be upregulated after liver resection or in diseased liver.2,3 BMSC are likely to differentiate into hepatocyte-like cells in vitro and in vivo.4 A potential approach to improve BMSC engraftment to the damaged liver could be their mobilization by using cytokine administration. Granulocyte colony-stimulating factor (G-CSF) is a 175-amino acid protein produced by recombinant DNA technology. G-CSF is the most potent cytokine currently available for mobilization of hematopoietic stem cells from the bone marrow. It is also widely used to stimulate and expand the bone marrow cells in ex vivo culture.5,6 The appearance of CD34þ cells during in vivo administration of G-CSF follows a distinct time course, with the maximal level of CD34þ cells occurring on day 5 after daily administration.7 In a recent phase II prospective trial, Gaia et al8 showed that multiple courses of G-CSF in decompensated cirrhotics were associated with BMSC mobilization. Spahr et al9 have reported that G-CSF mobilizes CD34þ cells, increases hepatocyte growth factor, and
Abbreviations used in this paper: ACLF, acute-on-chronic liver failure; AFP, a-fetoprotein; BMSC, bone marrow stem cell; CK, cytokeratin; CTP, Child-Turcotte Pugh; DPO, darbopoietin a; EPO, erythropoietin; G-CSF, granulocyte colony-stimulating factor; HCC, hepatocellular carcinoma; HVPG, hepatic venous pressure gradient; IHC, immunohistochemistry; LVP, large-volume paracentesis; MELD, Model for End Stage Liver Disease; TJLB, transjugular liver biopsy. © 2015 by the AGA Institute 0016-5085/$36.00 http://dx.doi.org/10.1053/j.gastro.2015.02.054
induces hepatic progenitor cells to proliferate within 7 days of administration in patients with alcoholic steatohepatitis. However, when they used G-CSF to mobilize BMSCs, which were subsequently taken from the bone marrow, concentrated, and then re-injected in the hepatic artery, they did not find reduction in the Model for End Stage Liver Disease (MELD) score.10 Acute-on-chronic liver failure (ACLF) is a distinct entity than decompensated cirrhosis of liver. It is characterized by progressive hepatic dysfunction after an acute insult on an underlying diagnosed or undiagnosed chronic liver disease, culminating in multi-organ failure and associated with high short-term mortality.11,12 For the first time in a randomized controlled trial, Garg et al13 have shown that the use of GCSF in patients with ACLF was associated with significantly improved survival of 69.6% compared with the 29% in the placebo group, along with increased recruitment of CD34þ cells on histology. Duan et al14 have shown that G-CSF improved survival in patients with hepatitis Brelated ACLF by increased neutrophil and more importantly CD34þ cell counts in peripheral circulation. However, there is limited information on the use of growth factors in patients with decompensated cirrhosis, a group distinct from ACLF. Erythropoietin (EPO), a pleiotropic cytokine known for its role in the stimulation of erythropoiesis,15 has emerged as a powerful clinical protective agent in a wide variety of tissues.16 We used darbopoietin a (DPO) due to better bioavailability, ease of administration as a once weekly injection and higher potency than recombinant human EPO.17 Greif et al18 reported similar results with intraperitoneal injection of recombinant human EPO (4 U/kg) 30 minutes before resection. Ben Ari et al19 recently showed a survival benefit of recombinant human EPO in a mouse model of fulminant hepatic failure induced by D-galactosamine/lipopolysaccharide. Based on these promising data, there is a potential for the use of EPO to stimulate hepatic regeneration and improve hepatic functions. EPO is often used to prevent or treat anemia in patients undergoing interferon and ribavirin therapy for the treatment of chronic hepatitis C, and has been found to be effective and safe. We hypothesized that the use of 2 growth factors (G-CSF and DPO), having different mechanisms of action and target cells, would lead to enhanced hepatic regeneration and improved survival in patients with decompensated liver disease.
Methods Patients with decompensated liver cirrhosis admitted to the Department of Hepatology, Institute of Liver and Biliary Sciences from May 2011 to June 2012 were consecutively screened, and those fulfilling the criteria were enrolled in 1:1 ratio into the randomized placebo controlled double-blind clinical trial. Patients aged younger than 18 years or older than 65 years, with evidence of alcoholic hepatitis or active alcohol abuse with last intake 1 month, with suspected autoimmune hepatitis (antinuclear antibody/ASMA-positive in titers 1:80 and/ or IgG 1.5 times upper limit of normal), hepatocellular carcinoma (HCC), any focus of sepsis as proven
G-CSF+ DPO Improve Survival in Cirrhosis 1363 by culture positivity or presence of spontaneous bacterial peritonitis, multi-organ dysfunction, grade 3 or 4 hepatic encephalopathy (as per West Haven criteria), human immunodeficiency virus seropositivity, pregnancy, uncontrolled hypertension, coronary artery disease, planned for liver transplantation and those refusing to participate in the study were excluded. Patients who were adequately treated for infection and became culture negative were also screened and, if found eligible, were enrolled in the study. After enrollment, patients underwent baseline etiological workup of cirrhosis (if not known previously), calculation of severity scores namely ChildTurcotte Pugh score (CTP), MELD, and MELD-Na scores and, wherever possible, a baseline transjugular liver biopsy (TJLB) and hepatic venous pressure gradient (HVPG) was done. Nearly all the patients were advised for liver transplantation due to the hepatic decompensation. Nonavailability of donors or other social issues were the main cause of their inability to get the liver transplantation and for opting for growth factor protocol. This single-center study protocol conformed to the Declaration of Helsinki and was approved by the Institutional Ethics Committee (approval file no: F. 8[10]05-06/AC/ILBS/1355). The study followed the CONSORT (Consolidated Standards of Reporting Trials) guidelines for randomized controlled trials and was registered at the ClinicalTrials.gov with full protocol access (identifier: NCT01384565). An informed written consent was taken from each patient at the time of enrollment. All authors had access to the study data and had reviewed and approved the final manuscript.
Randomization and Study Groups Randomization of the patients was done within 1 week of hospital admission after meeting all the inclusion and exclusion criteria. The block randomization was done with block size of 10. An independent statistician using computer-generated random number table performed sequence generation. Allocation concealment was done using sequentially numbered, opaque, sealed envelopes. Enrollment of participants, assessing eligibility and obtaining consent was carried out by 1 of the 2 authors (CKK or SKS). Randomization and administration of the drugs was done by another coauthor (AB). Both the study investigators and the patients were blinded to the study treatment through identical vials coded as A&B in the two groups, respectively. Standard medical therapy comprising albumin, diuretics, nutritional rehabilitation, b-blockers and treatment based on etiology, such as antivirals for hepatitis B, were continued in both groups. Patients with alcoholic liver disease were abstinent from alcohol throughout the study in both groups. These patients had undergone periodic psychosocial counseling in follow-up visits. Patients in the G-CSF and DPO protocol (GDP) group received G-CSF at a dose of 5 mg/kg subcutaneously at days 1, 2, 3, 4, 5 and then every third day until day 28 (total 12 doses), along with DPO 40 mg/wk subcutaneously for 4 weeks (total 4 doses). Patients in the control group received placebos in the same manner.
Follow-up Visits Follow-up was done at day 2, day 4, and at months 1, 3, 6, 9, and 12. During each follow-up visit, detailed clinical examination,
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1364 Kedarisetty et al complete blood counts, and kidney and liver function tests were done. a-Fetoprotein (AFP) was measured at baseline and at 1 month. Abdominal ultrasound was used to assess development of HCC at 3- to 6-month intervals throughout the study. The liver disease severity scores were calculated at each follow-up. Any adverse events were recorded during the entire period.
Assessment of Hepatic Regeneration
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Formalin-fixed, paraffin-embedded biopsy tissue sections obtained from TJLB, were cut at 3- to 4-mm thicknesses and were taken on poly-L-lysinecoated slides. These were deparaffinized by warming and Xylene. Immunohistochemistry (IHC) was done on 4 sections (of each biopsy) by using rabbit monoclonal antibody for markers of hematopoietic stem cells, namely CD34þ, CD133þ, and marker of proliferation, namely Ki67. There are currently a variety of established markers for human progenitor cells. These cells express biliary markers (such as biliary-type cytokeratins (oval cell marker 6, cytokeratin 7, and cytokeratin 19), and hematopoietic markers (such as the receptor for stem cell factor [c-kit], CD34þ). The expression of CD133þ is usually found in uncommitted progenitor cells.20 IHC staining was performed by peroxidase enzymelabeled streptavidin-biotin detection method using Polymer-HRP kit (Biogenex, San Ramon, CA) with diaminobenzidine as a chromogen and Meyer’s hematoxylin for contrasting. Both pathologists were blinded to the treatment protocol. Paired biopsies were studied for change in the markers on IHC. A minimum of 5 fields at 20 magnification was observed for CD34þ, CD133þ, and Ki67. CD34þ cells were expressed as mean percentage calculated in the 5 fields, CD133þ cells were expressed in form of grades namely grade 1: 70, and Ki67 was expressed as mean number detected in the 5 fields.
Primary and Secondary Outcomes The primary outcome was overall survival at 12 months. The time to event was calculated from the day of randomization into the study. The secondary outcomes were survival at 6 months; reduction in liver disease severity scores; reduction in need for large-volume paracentesis (LVP); development of new onset complications, such as acute kidney injury, sepsis, and variceal bleed; change in AFP levels at 1 month; hemodynamic improvement at 1 month; histological evidence of hepatic regeneration; and safety of treatment. Those patients lost to follow-up or who withdrew from the study were censored during the survival analysis. Septic shock was defined according to International Sepsis Definition21 and classified as culture positive or culture negative based on blood, urine, mini bronchoalveolar lavage in patients on mechanical ventilation, ascitic fluid, or any other sites.
Safety of the Therapy The patients were asked to report any adverse effects, such as pain abdomen, vomiting, altered behavior, diarrhea, jaundice, joint pains, or body rashes to the treating team. They were explained that fever and body aches could develop as part of common symptoms after the injections, but to record them and if these symptoms become worrisome, to report the hospital. They were also advised about the proper usage of analgesics and antipyretics. The patients kept a complete record of all
Gastroenterology Vol. 148, No. 7 symptoms and the nursing and clinical staff carefully monitored them.
Statistical Analyses The primary end point was to see the improvement in survival in treatment group as compared with placebo group. Assuming that the survival rate would be around 69% in the treatment group and 29% in the placebo group,13 with a ¼ 5% (2-sided) and power 80%, we needed to enroll a total of 52 cases in the trial (ie, 26 in each arm) based on log-rank test. It was possible to enroll 55 consecutive patients fulfilling the inclusion and exclusion criteria between May 2011 to June 2012 (29 in GDP and 26 in control group). The imbalance in allocation of patients is due to block randomization, with block size taken as 10 and defined enrollment period. Descriptive statistics were expressed as median (interquartile range [IQR]) or number (percentage). Comparison of continuous variables was done by Wilcoxon rank-sum test and categorical variables were compared by Fisher’s exact test or Pearson’s c2 test. The cumulative probability of survival was determined by Kaplan-Meier graph and compared by MantelCox log-rank test. We calculated the change in CTP; MELD and MELD-Na score from baseline to each follow-up as the %D change. Delta change was calculated as ([follow-up day score minus the baseline score]/[baseline score] 100). This delta change was compared between the 2 treatment groups at different time intervals using repeated measures analysis of variance test, followed by post-hoc comparison by Least Significant Difference method. However, the statistical significance for secondary end points should be interpreted cautiously from a false-positive perspective, as no adjustments for multiplicity were made. The intent of the study was to compare from a 2-sided perspective. All statistical tests were performed using SPSS for Windows version 15 (SPSS Inc., Chicago, IL).
Results A total of 750 patients with decompensated cirrhosis were screened during the enrollment period between May 2011 and June 2012 (Supplementary Figure 1) and followed up for 12 months. After careful assessment, 55 patients fulfilled the inclusion and exclusion criteria and were randomized; 29 patients were assigned to the GDP group and 26 patients to control group. The 2 study groups were well compared with respect to demographic characteristics, clinical and laboratory data, and activity scores (Table 1). Twenty-six patients (14 in GDP and 12 in control group) underwent baseline TJLB and HVPG. There was no bias in the selection of patients in the TJLB cohort as the baseline characteristics of TJLB and non-TJLB patients were comparable (Supplementary Table 1). Seven patients (5 in GDP and 2 in control group) got repeat TJLB and HVPG done at 1 month. The etiology of cirrhosis was comparable between the 2 groups (P ¼ .38) and was dominated by chronic alcohol abuse (Supplementary Figure 2). The predominant mode of hepatic decompensation was ascites in both the patient groups and was comparable (P ¼ .18). (Supplementary Figure 3)
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Male sex, n (%) Age, y, median (IQR) Jaundice, n (%) Moderate to severe ascites, n (%) Hepatic encephalopathy, n (%) Variceal bleed, n (%) Hemoglobin, g/dL, median (IQR) Platelet, 103/mm3, median (IQR) Sodium, mEq/dL, median (IQR) Total bilirubin, mg/dL, median (IQR) Albumin, g/dL, median (IQR) Grade of varices, median (IQR) HVPG in mm Hg (n ¼ 26), median (IQR) Ferritin, mg/dL, median (IQR) AFP, ng/mL, median (IQR) Need for LVP, n (%) CTP score, median (IQR) MELD score, median (IQR) MELD-Na score, median (IQR)
GDP group (n ¼ 29) 28 (97) 44 (3261) 23 (79) 26 (89.7) 2 (6.9) (grade 2) 8 (27.6) 9.1 (7.313.7) 99 (36172) 133 (121143) 4.8 (1.214.4) 2.6 (1.83.6) 2 (03) 16 (1125), n ¼ 14 350 (471830) 5.8 (3.28.7) 28 (96.6 ) 11 (913) 22 (1138) 24 (1039)
Control group (n ¼ 26) 25 46 19 23 0 5 8.5 107 130 5.5 2.9 2 15 426 4.3 26 11 20 25
(96) (2760) (73) (88.5) (0) (19.2) (6.812) (35187) (120136) (0.621.3) (1.73.7) (03) (1030), n ¼ 12 (261790) (2.86.3) ( 100) (713) (1030) (1035)
P value .71 .82 .89 .88 .17 .54 .26 .18 .19 .94 .74 .19 .22 .83 .14 .53 .63 .25 .93
*Continuous outcomes were compared using the Wilcoxon rank-sum test and categorical outcomes were compared using Fisher’s exact or Pearson’s c2 tests.
Primary Outcome The primary outcome of the study was whether the use of growth factors led to overall improved survival at 12 months. The difference was obvious from 1 month onward and remained so throughout the follow-up period. Using log-rank (Mantel-Cox) test, the cumulative probability of survival at 12 months was 68.6% (±8.7%) in the GDP group and 26.9% (±8.7%) in control group (P ¼ .003) (Figure 1).
(P ¼ .03). By using repeated measures analysis of variance test, we found that the comparison of %D CTP scores at different time points between the 2 groups showed a progressive reduction in CTP scores with a combination of growth factors that was statistically significant (P < .001) (Figure 2A). Similar results were seen in reference to MELD
Secondary Outcomes Overall survival at 6 months. One of the important secondary end points of the study was survival at 6 months with the use of growth factors. The cumulative probability of survival at 6 months was 82.7% (±7.2%) in the GDP group and 57.7% (±9.7%) in control group (P ¼ .03). Liver disease severity scores. The baseline CTP, MELD, and MELD-Na scores were comparable in both groups as shown in Table 1. Baseline CTP, MELD, and MELD-Na scores were predictive of mortality in the control group (P < .05), but not in the GDP group (P > .05) on subgroup analysis as shown in Supplementary Table 2. On multivariate analysis with adjustment for liver severity scores, we found that there was overall 77%, 73%, and 72% reduction in mortality by GDP group as compared with control group when adjusted for baseline CTP, MELD, and MELD-Na scores, respectively, as shown in Supplementary Table 3. Mean reduction in the severity scores was calculated as %D change at each follow-up from baseline in both the groups (%D change ¼ [follow-up day score minus the baseline score]/[baseline score] 100). Overall %D CTP at 12 months follow-up was 48.6% in GDP group and 39.1% in control group (P ¼ .001). The %D MELD score at 12 months was 40.4% in GDP group and 33% in control group
Figure 1. Kaplan-Meier curve showing the overall survival at 12 months in GDP group compared with the control group.
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Figure 2. Change in liver severity scores. % Delta change (%D) was calculated as ([follow-up day score minus the baseline score]) / [baseline score] 100). (A) %D change in CTP scores from baseline to each follow-up. There was reduction in CTP scores in GDP group compared with control group with statistical significance. (B) %D change in MELD scores from baseline to each follow-up. There was reduction in MELD scores in GDP group compared with control group with statistical significance.
score (P < .001) (Figure 2B) and MELD-Na score (P < .001). This reduction was only noted in patients alive on follow up. Need for large-volume paracentesis. The need for LVP at baseline was 96.6% and 100% in GDP and control group, respectively (P ¼ .53); at 1 month, 41.4% and 76.9% (P ¼ .008); at 3 months, 22.2% and 52.6% (P ¼ .04) and at 6 months, none in the GDP group and 20% in the control group needed LVP (P ¼ .05). There was no difference seen at 9 and 12 months in both the groups (Supplementary Figure 4). Change in a-fetoprotein levels. AFP is an important tumor marker for early detection of HCC. However, it is also an equally important marker of liver regeneration. Therefore, AFP was measured at baseline and at 1 month. The baseline AFP levels were similar in both groups (Table 1). At
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1 month, AFP level was significantly higher in the GDP group compared with the control group (6.6 ± 3.6 ng/mL and 4.7 ± 2.7 ng/mL; P ¼ .018). This was suggestive of augmentation of hepatic regeneration with the use of growth factors. At 1 month, the median aspartate aminotransferase and alanine aminotransferase levels were 61 IU/ L (IQR, 3778 IU/L) and 36 IU/L (IQR, 2148 IU/L) in GDP group and 53 IU/L (IQR, 4183 IU/L) and 30 IU/L (IQR, 2149 IU/L) in control group, respectively. By using regression analyses, there was no interaction between aspartate aminotransferase or alanine aminotransferase and AFP at 1 month after completion of treatment protocol, therefore ruling out false positivity. Hemodynamic studies. In the 7 patients with baseline and repeat HVPG, the mean baseline (±SD) HVPG values in GDP and control groups were 19.7 ± 3.5 mm Hg and 19.8 ± 3.1 mm Hg, respectively. The repeat HVPG was 17 ± 9.8 mm Hg and 16 ± 2.8 mm Hg, respectively. The change in HVPG from baseline to 1 month was not statistically different between the 2 groups (P ¼ .19). This was observed probably due to insufficient power to detect an effect between the treatments with only 7 patients. Liver histology and immunohistochemistry. A total of 7 patients (5 in GDP group and 2 in control group) had undergone baseline and repeat TJLB. The pathologists were blinded to the treatment given to the patient before the reporting of the results. In the paired biopsies, percent of CD34þ cells increased from 37% to 53% in GDP group and 32.5% to 37.5% in control group (P ¼ .19). Figure 3 shows increase in the proportion of CD34þ cells and CD133þ cells in GDP group suggestive of augmented hepatic regeneration. Statistical significance was not observed, probably due to insufficient power to detect an effect between the treatments with only 7 patients (Supplementary Table 4). New onset complications. Variceal bleed was seen in 3 (10.3%) in the GDP group and 5 (19.2%) in the control group (P ¼ .43); spontaneous bacterial peritonitis in 2 (6.9%) and 2 (7.7%) (P ¼ .65); acute kidney injury in the form of hepatorenal syndrome in 3 (10.3%) and 3 (11.5%) (P ¼ .44). Three patients in the GDP group developed strangulation of umbilical hernia and underwent emergency surgery, but succumbed to the illness. One patient in the GDP group was detected to have a small HCC at 12 months of follow-up. However, this patient had a case of chronic hepatitis Brelated cirrhosis of liver on antiviral therapy (Supplementary Table 5). Sepsis. In the 12-month follow-up period, 12 patients with decompensated cirrhosis developed sepsis and presented with hypotension and shock. It was important to note that only 2 patients (6.9%) succumbed to the septic shock in the GDP group in comparison to 10 patients (38.5%) in the control group, the difference was statistically significant (P ¼ .005) (Figure 4). Three patients in the control group grew Enterococcus faecium; Klebsiella pneumoniae, Staphylococcus aureus in blood culture and one patient in Group B grew Stenotrophomonas maltophila in minibronchoalveolar lavage. Remaining patient samples were culture negative.
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Figure 3. Under 200 magnification in an area of 1.3 mm2 (5 fields), there was increased density of CD 34þ staining in lobular parenchyma (black arrow) and CD 133þ staining in ductules (black arrow) in paired liver biopsies in the GDP group.
There were a total of 28 deaths in the entire cohort. The etiologies were septic shock in 2 (6.9%) and 10 (38.5%) in GDP and control group, respectively, uncontrolled variceal bleed with shock in 1 (3.4%) and 3 (11.5%), postoperative complications after emergency umbilical hernia repair in 3 (10.3%) and 1 (3.8%), hepatorenal syndrome progressing to acute tubular necrosis on renal replacement therapy in 3 (10.3%) and 3 (11.5%) patients, respectively. In the 2 remaining cases, the cause of death could not be ascertained due to inaccessibility to information.
Safety of the Therapy G-CSF is known to cause leukocytosis.9 As shown in Supplementary Table 6, there was reactive leukocytosis in response to G-CSF on day 2, day 4, and at 1 month. By using repeated-measures analysis of variance test, we found that the total leukocyte counts between the 2 groups were statistically significant at different time points (P < .001). Malaise was seen as the predominant symptom in 37% patients, followed by generalized discomfort in 23% patients after G-CSF injection (Supplementary Figure 5A and B). Bodyache was the predominant
symptom seen in 45% patients, followed by myalgia in 30% patients and flu-like symptoms in 29% patients after DPO injection. None of the patients showed any evidence of portal vein or deep vein thrombosis on close Doppler ultrasound monitoring. One patient had noncardiac chest pain. There was no statistical difference in the symptomatology reported in the 2 groups. Although, none of the patients received both drugs on the same day, it was difficult to ascertain the adverse event to the same drug. All patients tolerated the treatment well and there was no treatment discontinuation noted.
Discussion The only proven treatment with survival benefit in decompensated cirrhotics is liver transplantation. Unfortunately, many patients succumb to their illness in the waiting list for a transplant or do not have available live donors. By treating the etiology of cirrhosis, for example, antivirals for chronic hepatitis B or chronic hepatitis C and alcohol abstinence in alcoholic liver disease, the disease progression can be retarded to some extent. However, it does not significantly reverse the advanced cirrhotic process.
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Figure 4. The Kaplan-Meier curve showing cumulative probability of septic shock was significantly lower in GDP group compared with the control group.
In this novel study, a combination of 2 growth factors, G-CSF and DPO targeting different cell lineages in the bone marrow, with different cytokine and chemokine cascades, showed that their use was able to achieve improved survival in these patients who ideally required liver transplantation. The baseline liver severity scores were not predictive of mortality in the GDP group, unlike the control group, probably due to altered natural history of cirrhosis in these patients. More importantly, there was a progressive reduction noted in severity scores on use of growth factors that explained the improved survival. The need for LVP had also significantly reduced in the GDP group. It was interesting to note an increase in the levels of AFP at 1 month in the intervention limb. This could be indicative of initiation of hepatic regeneration. In a seminal paper by Eleftheriou et al22 as early as 1975 and subsequently, the relevance of AFP levels in models on hepatic regeneration has been reported.23 One would, of course, need to be cautious of the initiation of dysplasia, followed by hepatocellular carcinoma, if the AFP levels continue to rise significantly. One of our important secondary study end points was induction of hepatic regeneration by the use of growth factors. Bone marrow and liver share an evolutionary conserved developmental relationship and are like 2 sides of the same coin. In fact, hematopoiesis is well known to occur in the fetal liver.24 The bone marrow acts as a reservoir for multiple stem cell populations, including hematopoietic stem cells, mesenchymal stem cells, and endothelial progenitor cells, which are mobilized into the peripheral circulation after
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injury.25 Systemic administration of G-CSF has been shown to mobilize hematopoietic stem cells, endothelial progenitor cells, and bone marrowderived mesenchymal stem cells.26,27 Similarly, EPO has been shown to mobilize endothelial progenitor cells and enhance their in vivo regenerative role via the phosphoinositide kinase-3Akt pathway.28 Patients who underwent repeat liver biopsy in GDP group showed an increased density of CD34þ, CD133þ, Ki67 cells at 1 month compared with control group, reiterating the concept of cross talk between liver and bone marrow to augment liver regeneration. Bacterial infections are much more common in patients with cirrhosis than in the general population.29 Arvaniti et al30 showed that cumulative mortality due to infection in decompensated cirrhosis was 43.5%, vs 13.5% with no infection. This stage classified as “critically ill cirrhotic” highlights the role of infection as a major player in the downhill course of decompensated cirrhosis. In the present study, the patients treated with growth factors had a significantly lower incidence of septic shock compared with those receiving placebo (6.9% vs 38.5%; P ¼ .005) during a follow-up of 12 months. This is an important observation in support of the use of growth factors, which, by modulation of the myeloid series in the bone marrow, are able to reduce the incidence of sepsis. We have recently shown that in ACLF, G-CSF recruits dendritic cells to the liver, which, in turn, reduce the intrahepatic CD8þ T cells and Th17 cells with reduced intrahepatic interferon gamma production, thereby restoring the immune disequilibrium.31 The safety of the combination therapy was also established in decompensated cirrhotics. None required discontinuation of the treatment. The strengths of the present study are that it was a double-blind randomized controlled trial showing clinical and survival benefits by the use of growth factors in decompensated cirrhotics. It could well be appreciated that because the study was invasive, as it required TJLB and HVPG measurement on 2 occasions and continuous monitoring, to recruit this many patients and complete follow-up were a major clinical responsibilities. The outcome of the study was quite gratifying, as the results strongly indicate that this protocol-based treatment can modulate the clinical course in a proportion of well-selected decompensated cirrhotic patients. The possible limitations of the study were a small number of repeat TJLBs at 1 month due to unwillingness of patients to undergo repeat procedure. It is unlikely that such psychological concerns could be easily overcome during the consent process until robust data on regenerative therapy are available in the field. Secondly, it would also have been more interesting to study the role of bone marrow as a source for recruitment of hematopoietic stem cells and macrophages.32 However, it needs to be assessed whether bone marrow biopsy can be done safely in these patients. Also, it would have been more educative to estimate CD34þ, CD133þ cells in peripheral and hepatic venous blood and to correlate these with IHC on liver biopsy or clinical parameters.
This is the first double-blind randomized controlled study (NCT01384565) showing clinical benefit and an overall improved survival using a combination of G-CSF and EPO in patients with decompensated liver disease. This is also the first human study using EPO in decompensated liver cirrhosis, demonstrating its potential in hepatic regeneration. The results of the present study would need to be confirmed in larger cohort of patients with decompensated liver disease.
Supplementary Material Note: To access the supplementary material accompanying this article, visit the online version of Gastroenterology at www.gastrojournal.org, and at http://dx.doi.org/10.1053/ j.gastro.2015.02.054.
References 1. D’Amico G, Garcia-Tsao G, Pagliaro L. Natural history and prognostic indicators of survival in cirrhosis: a systematic review of 118 studies. J Hepatol 2006; 44:217–231. 2. De Silvestro G, Vicarioto M, Donadel C, et al. Mobilization of peripheral blood hematopoietic stem cells following liver resection surgery. Hepatogastroenterology 2004; 51:805–810. 3. Lorenzini S, Isidori A, Catani L, et al. Stem cell mobilization and collection in patients with liver cirrhosis. Aliment Pharmacol Ther 2008;27:932–939. 4. Vassilopoulos G, Wang PR, Russell DW. Transplanted bone marrow regenerates liver by cell fusion. Nature 2003;422:901–904. 5. Pitchford SC, Furze RC, Jones CP, et al. Differential mobilization of subsets of progenitor cells from the bone marrow. Cell Stem Cell 2009;4:62–72. 6. Maiwall R, Kumar A, Sarin SK. Liver regeneration during acute-on-chronic liver failure using growth factors: in vivo or ex vivo indulgence of bone marrow? Gastroenterology 2013;145:901–904. 7. Hoffmann R. Hematology. Basic Principles and Practice. 5th ed. New York: Elsevier, 2009. 8. Gaia S, Olivero A, Smedile A, et al. Multiple courses of G-CSF in patients with decompensated cirrhosis: consistent mobilization of immature cells expressing hepatocyte markers and exploratory clinical evaluation. Hepatol Int 2013;7:1075–1083. 9. Spahr L, Lambert JF, Brandt LR, et al. Granulocyte-colony stimulating factor induces proliferation of hepatic progenitors in alcoholic steatohepatitis: a randomized trial. Hepatology 2008;48:221–229. 10. Spahr L, Chalandon Y, Terraz S, et al. Autologous bone marrow mononuclear cell transplantation in patients with decompensated alcoholic liver disease: a randomized controlled trial. PLoS One 2013;8:e53719. 11. Sarin SK, Kumar A, Almeida J, et al. Acute- on-chronic Liver Failure (ACLF): Consensus Recommendations of the Asian Pacific Association for the Study of the Liver (APASL). Hepatol Int 2009;3:269–282.
G-CSF+ DPO Improve Survival in Cirrhosis 1369 12. Moreau R, Jalan R, Gines P, et al. Acute-on-chronic liver failure is a distinct syndrome that develops in patients with acute decompensation of cirrhosis. Gastroenterology 2013;144:1426–1437. 13. Garg V, Garg HK, Khan A, et al. Granulocyte-colony stimulating factor (G- CSF) therapy mobilizes CD34 cells and improves survival in patients with acute on chronic liver failure. Gastroenterology 2012;142:505–512. 14. Duan XZ, Liu FF, Tong JJ, et al. Granulocyte colony stimulating factor therapy improves survival in patients with hepatitis B-associated acute-on-chronic liver failure. World J Gastroenterol 2013;19:1104–1110. 15. Fischer JW. Landmark advances in development of erythropoietin. Exp Biol Med 2010;235:1398–1411. 16. Lewis LD. Preclinical and clinical studies: a preview of potential future applications of erythropoietin agents. Semin Hematol 2004;41:17–25. 17. Egrie JC, Browne JK. Development and characterization of erythropoiesis stimulating protein (NESP). Br J Cancer 2001;84:3–10. 18. Greif F, Ben Ari Z, Taya R, et al. Dual effect of EPO on liver protection and regeneration after subtotal hepatectomy in rats. Liver Transplant 2010;16:631–638. 19. Ben Ari Z, Zilbermints V, Pappo O, et al. Erythropoetin increases survival and attenuates fulminant hepatic failure induced by D-galactosamine/ lipopolysaccharide in mice. Transplantation 2011;92:18–24. 20. Spee B, Carpino G, Schotanus BA, et al. Characterization of the liver progenitor cell niche in liver diseases: potential involvement of Wnt and Notch signalling. Gut 2010;59:247–257. 21. Levy MM, Fink MP, Marshall JC, et al. 2001 SCCM/ ESICM/ACCP/ATS/SIS International Sepsis Definitions Conference. Crit Care Med 2003;31:1250–1256. 22. Eleftheriou N, Thomas H, Heathcote J, et al. Proceedings: serum alpha-fetoprotein levels in liver disease: relation to hepatocellular regeneration and development of hepatoma. Gut 1975;16:835. 23. Fausto N. Messenger RNA in regenerating liver: implications for understanding of regulated growth. Mol Cell Biochem 1984;59:131–147. 24. Martin MA, Bhatia M. Analysis of the human fetal liver hematopoietic microenvironment. Stem Cells Dev 2005; 14:493–504. 25. Chen Y, Xiang LX, Shao JZ, et al. Recruitment of endogenous bone marrow mesenchymal stem cells towards injured liver. J Cell Mol Med 2010;14:1494–1508. 26. Liu F, Pan X, Chen G, et al. Hematopoietic stem cells mobilized by granulocyte colony-stimulating factor partly contribute to liver graft regeneration after partial orthotopic liver transplantation. Liver Transpl 2006; 12:1129–1137. 27. Takamiya M, Okigaki M, Jin D, et al. Granulocyte colonystimulating factor-mobilized circulating c-Kitþ/Flk-1þ progenitor cells regenerate endothelium and inhibit neointimal hyperplasia after vascular injury. Arterioscler Thromb Vasc Biol 2006;26:751–757. 28. Urao N, Okigaki M, Yamada H, et al. Erythropoietinmobilized endothelial progenitors enhance reendothelialization via Akt-endothelial nitric oxide synthase
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29.
30.
31.
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activation and prevent neointimal hyperplasia. Circ Res 2006;98:1405–1413. Bruns T, Zimmermann HW, Stallmach A. Risk factors and outcome of bacterial infections in cirrhosis. World J Gastroenterol 2014;20:2542–2554. Arvaniti V, D’Amico G, Fede G, et al. Infections in patients with cirrhosis increase mortality four- fold and should be used in determining prognosis. Gastroenterology 2010; 139:1246–1256. Khanam A, Trehanpati N, Garg V, et al. Altered frequencies of dendritic cells and IFN-g secreting T cells with granulocyte colony stimulating factor (GCSF) therapy in acute-on-chronic liver failure. Liver Int 2014; 34:505–513. Thomas AJ, Pope C, Wojtacha D, et al. Macrophage therapy for murine liver fibrosis recruits host effector
Gastroenterology Vol. 148, No. 7 cells improving fibrosis, regeneration, and function. Hepatology 2011;53:2003–2015.
Received May 11, 2014. Accepted February 28, 2015. Reprint requests Address requests for reprints to: Shiv Kumar Sarin, MD, DM, FNA, FNASc, Department of Hepatology, Institute of Liver and Biliary Sciences, New Delhi 110 070, India. e-mail: [email protected]; fax: þ91 (011) 26123504. Acknowledgments The authors would like to thank their staffs, Ms Anila, and Ms Madhu for data compilation. The generic drugs G-CSF, DPO, and placebos were provided by Dr. Reddys’ Laboratories, Hyderabad, India. They provided no financial assistance of any kind. Conflicts of interest The authors disclose no conflicts.
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Supplementary Figure 1. Study design (screening, randomization, and follow-up of subjects).
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Supplementary Figure 2. Etiology of liver disease.
Supplementary Figure 3. Mode of decompensation.
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Supplementary Figure 4. Need for large volume paracentesis at baseline and at each follow up in both groups.
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Supplementary Figure 5. Adverse events of the drugs. (A) Graphical presentation of % patients adverse events reported after administration of G-CSF in GPD group or G-CSF matched placebo in control group. (B) Graphical presentation of % patients adverse events reported after administration of DPO in GPD group or DPO matched placebo in control group.
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Supplementary Table 1.Baseline Characteristics of Patients in Transjugular Liver Biopsy and NonTransjugular Liver Biopsy Cohort Parameter
TJLB cohort (n ¼ 26)
Non-TJLB cohort (n ¼ 29)
P value
Male sex, n (%) Moderate to severe ascites, n (%) Hepatic encephalopathy, n (%) Hemoglobin, g/dL Platelet, 103/mm3 Urea, mg/dL Creatinine, mg/dL Sodium, mEq/dL Total bilirubin, mg/dL Albumin, g/dL INR AFP, ng/mL Need for LVP, n (%) CTP score MELD score MELD-Na score
25 (96.2) 24 (92.3) 5 (19.2) 9.3 ± 1.5 109.7 ± 39.7 33.4 ± 22.5 0.74 ± 0.3 131.4 ± 4.6 6.8 ± 4.9 2.8 ± 0.4 2.5 ± 2.1 5.8 ± 3.4 25 (96.2) 10.9 ± 1.5 21.8 ± 6.3 25.5 ± 5.7
28 (96.6) 25 (86.2) 4 (13.8) 9.2 ± 1.5 94.1 ± 35.3 32.1 ± 26.8 0.79 ± 0.3 131.2 ± 5.9 4.7 ± 2.7 2.7 ± 0.6 2.1 ± 0.6 7.2 ± 7.3 27 (93.1) 10.6 ± 1.5 19.6 ± 5.1 24.1 ± 5.2
.74 .67 .72 .81 .13 .85 .54 .92 .05 .3 .58 .41 .96 .36 .17 .34
NOTE. Data are presented as mean ± SD, except where indicated. INR, international normalized ratio.
Supplementary Table 2.Baseline Liver Severity Scores and Prediction of Mortality in Each Treatment Group Liver severity score
Treatment group
CTP baseline
GDP Placebo GDP Placebo GDP Placebo
MELD baseline MELD-Na baseline
Hazard ratio
Upper limit
Lower limit
P value
1.42 2.05 1.14 1.13 1.17 1.17
2.53 3.44 1.35 1.25 1.37 1.33
0.80 1.22 0.97 1.02 1.00 1.04
.23 .01 .12 .02 .06 .01
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Supplementary Table 3.Impact of Treatment Group on Outcomes When Adjusted for Baseline Liver Severity Score 95% CI
Treatment group MELD baseline Treatment group MELD-Na baseline Treatment group CTP baseline
P value
HR
Lower
Upper
0.00 0.01 0.00 0.00 0.00 0.00
0.27 1.13 0.28 1.17 0.23 1.78
0.12 1.04 0.13 1.06 0.10 1.22
0.61 1.24 0.64 1.29 0.54 2.58
CI, confidence interval; HR, hazard ratio.
Supplementary Table 4.Immunohistochemistry Markers for Liver Regeneration in Liver Biopsies CD34þ cells (%) Treatment group GDP
Control
a
CD133þ cells (gradea)
Ki67 (n)
Serial no.
Baseline
1 month
Baseline
1 month
Baseline
1 month
1 2 3 4 5 1 2
35 30 40 30 50 40 25
60 40 50 60 55 45 30
1 1 2 3 2 1 0
3 1 2 3 3 1 1
1 0 1 1 1 1 0
2 0 1 2 3 2 1
CD133þ cells were labeled in form of grades. Grade 1: 70.
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Supplementary Table 5.Incidence of New Onset Complications in the 2 Treatment Groups New onset complication
GDP group (n ¼ 29), n (%)
UGI bleed Acute kidney injury SBP SBE/pneumonia Hepatic encephalopathy Septic shock Umbilical hernia strangulation Spontaneous hematoma HCC
3 3 2 2 2 2 3 2 1
Control group (n ¼ 26), n (%)
(10.3) (10.3) (6.9) (6.9) (6.9) (6.9) (10.3) (6.9) (3.4)
P value
5 3 2 1
(19.2) (11.5) (7.7) (3.8) 0 10 (38.5) 1 (3.8) 0 0
.43 .45 .65 .54 .14 .005 .35 .14 .53
SBE, spontaneous bacterial empyema; SBP, spontaneous bacterial peritonitis; UGI, upper gastrointestinal.
Supplementary Table 6.Total Leukocyte Counts (Millions/mm3) at Different Follow-Up Intervals Until 3 Months in the 2 Treatment Groups Time point Baseline Day 2 Day 4 1 month 3 months
GDP group (n ¼ 29) 7.5 19.3 22.5 13.4 7.5
± ± ± ± ±
NOTE. Data are presented as mean ± SD.
4 11 12.3 8.4 4.2
Control group (n ¼ 26)
P value
± ± ± ± ±
.20
Combination of Granulocyte Colony-Stimulating Factor and Erythropoietin Improves Outcomes of Patients With Decompensated Cirrhosis Chandan Kumar Kedarisetty,1 Lovkesh Anand,1 Ankit Bhardwaj,2 Ajeet Singh Bhadoria,2 Guresh Kumar,2 Ashish Kumar Vyas,2 Paul David,2 Nirupama Trehanpati,2 Archana Rastogi,3 Chhagan Bihari,3 Rakhi Maiwall,1 Hitendra Kumar Garg,1 Chitranshu Vashishtha,1 Manoj Kumar,1 Vikram Bhatia,1 and Shiv Kumar Sarin1 CLINICAL LIVER
Departments of 1Hepatology, 2Biostatistics and Clinical Research, and 3Pathology, Institute of Liver and Biliary Sciences, New Delhi, India BACKGROUND & AIMS: Patients with decompensated cirrhosis have significantly reduced survival without liver transplantation. Granulocyte colony-stimulating factor (G-CSF) has been shown to increase survival in patients with acute-onchronic liver failure, and erythropoietin promoted hepatic regeneration in animal studies. We performed a double-blind, randomized, placebo-controlled trial to determine whether coadministration of these growth factors improved outcomes for patients with advanced cirrhosis. METHODS: In a prospective study, consecutive patients with decompensated cirrhosis seen at the Institute of Liver and Biliary Sciences, New Delhi (from May 2011 through June 2012) were randomly assigned to groups given subcutaneous G-CSF (5 mg/kg/d) for 5 days and then every third day (12 total doses), along with subcutaneous darbopoietin a(40 mcg/wk) for 4 weeks (GDP group, n ¼ 29), or only placebos (control group, n ¼ 26). All patients also received standard medical therapy and were followed for 12 months. Histology was performed on liver biopsies. The primary end point was survival at 12 months. RESULTS: Baseline characteristics of patients were comparable; alcohol intake was the most common etiology of cirrhosis. A higher proportion of patients in the GDP group than controls survived until 12 months (68.6% vs 26.9%; P ¼ .003). At 12 months, ChildTurcotte Pugh scores were reduced by 48.6% in the GDP group and 39.1% in the control group, from baseline (P ¼ .001); Model for End Stage Liver Disease scores were reduced by 40.4% and 33%, respectively (P ¼ .03). The need for large-volume paracentesis was significantly reduced in GDP group, compared with controls (P < .05). A lower proportion of patients in the GDP group developed septic shock (6.9%) during follow-up compared with controls (38.5%; P ¼ .005). No major adverse events were observed in either group. CONCLUSIONS: In a single-center randomized trial, a significantly larger proportion of patients with decompensated cirrhosis given a combination of G-CSF and darbopoietin a survived for 12 months more than patients given only placebo. The combination therapy also reduced liver severity scores and sepsis to a greater extent than placebo. Clinicaltrials.gov ID: NCT01384565.
Keywords: DPO; Liver Regeneration; Clinical Trial; Chronic Liver Disease.
M
ost of the patients referred to a specialized hepatology center suffer from decompensation of endstage chronic liver disease. Decompensated cirrhosis is defined by the presence of ascites, variceal bleeding, encephalopathy, and/or jaundice.1 The only recommended treatment of choice is liver transplantation. A large proportion of cirrhotic patients succumb while waiting on the transplant list. Therefore, there is a large unmet need for studying newer treatment modalities, mainly focused on liver regeneration. During the last decade, the importance of bone marrow stem cell (BMSC) activation in liver disease has become apparent. CD34þ and CD133þ cells appear to be upregulated after liver resection or in diseased liver.2,3 BMSC are likely to differentiate into hepatocyte-like cells in vitro and in vivo.4 A potential approach to improve BMSC engraftment to the damaged liver could be their mobilization by using cytokine administration. Granulocyte colony-stimulating factor (G-CSF) is a 175-amino acid protein produced by recombinant DNA technology. G-CSF is the most potent cytokine currently available for mobilization of hematopoietic stem cells from the bone marrow. It is also widely used to stimulate and expand the bone marrow cells in ex vivo culture.5,6 The appearance of CD34þ cells during in vivo administration of G-CSF follows a distinct time course, with the maximal level of CD34þ cells occurring on day 5 after daily administration.7 In a recent phase II prospective trial, Gaia et al8 showed that multiple courses of G-CSF in decompensated cirrhotics were associated with BMSC mobilization. Spahr et al9 have reported that G-CSF mobilizes CD34þ cells, increases hepatocyte growth factor, and
Abbreviations used in this paper: ACLF, acute-on-chronic liver failure; AFP, a-fetoprotein; BMSC, bone marrow stem cell; CK, cytokeratin; CTP, Child-Turcotte Pugh; DPO, darbopoietin a; EPO, erythropoietin; G-CSF, granulocyte colony-stimulating factor; HCC, hepatocellular carcinoma; HVPG, hepatic venous pressure gradient; IHC, immunohistochemistry; LVP, large-volume paracentesis; MELD, Model for End Stage Liver Disease; TJLB, transjugular liver biopsy. © 2015 by the AGA Institute 0016-5085/$36.00 http://dx.doi.org/10.1053/j.gastro.2015.02.054
induces hepatic progenitor cells to proliferate within 7 days of administration in patients with alcoholic steatohepatitis. However, when they used G-CSF to mobilize BMSCs, which were subsequently taken from the bone marrow, concentrated, and then re-injected in the hepatic artery, they did not find reduction in the Model for End Stage Liver Disease (MELD) score.10 Acute-on-chronic liver failure (ACLF) is a distinct entity than decompensated cirrhosis of liver. It is characterized by progressive hepatic dysfunction after an acute insult on an underlying diagnosed or undiagnosed chronic liver disease, culminating in multi-organ failure and associated with high short-term mortality.11,12 For the first time in a randomized controlled trial, Garg et al13 have shown that the use of GCSF in patients with ACLF was associated with significantly improved survival of 69.6% compared with the 29% in the placebo group, along with increased recruitment of CD34þ cells on histology. Duan et al14 have shown that G-CSF improved survival in patients with hepatitis Brelated ACLF by increased neutrophil and more importantly CD34þ cell counts in peripheral circulation. However, there is limited information on the use of growth factors in patients with decompensated cirrhosis, a group distinct from ACLF. Erythropoietin (EPO), a pleiotropic cytokine known for its role in the stimulation of erythropoiesis,15 has emerged as a powerful clinical protective agent in a wide variety of tissues.16 We used darbopoietin a (DPO) due to better bioavailability, ease of administration as a once weekly injection and higher potency than recombinant human EPO.17 Greif et al18 reported similar results with intraperitoneal injection of recombinant human EPO (4 U/kg) 30 minutes before resection. Ben Ari et al19 recently showed a survival benefit of recombinant human EPO in a mouse model of fulminant hepatic failure induced by D-galactosamine/lipopolysaccharide. Based on these promising data, there is a potential for the use of EPO to stimulate hepatic regeneration and improve hepatic functions. EPO is often used to prevent or treat anemia in patients undergoing interferon and ribavirin therapy for the treatment of chronic hepatitis C, and has been found to be effective and safe. We hypothesized that the use of 2 growth factors (G-CSF and DPO), having different mechanisms of action and target cells, would lead to enhanced hepatic regeneration and improved survival in patients with decompensated liver disease.
Methods Patients with decompensated liver cirrhosis admitted to the Department of Hepatology, Institute of Liver and Biliary Sciences from May 2011 to June 2012 were consecutively screened, and those fulfilling the criteria were enrolled in 1:1 ratio into the randomized placebo controlled double-blind clinical trial. Patients aged younger than 18 years or older than 65 years, with evidence of alcoholic hepatitis or active alcohol abuse with last intake 1 month, with suspected autoimmune hepatitis (antinuclear antibody/ASMA-positive in titers 1:80 and/ or IgG 1.5 times upper limit of normal), hepatocellular carcinoma (HCC), any focus of sepsis as proven
G-CSF+ DPO Improve Survival in Cirrhosis 1363 by culture positivity or presence of spontaneous bacterial peritonitis, multi-organ dysfunction, grade 3 or 4 hepatic encephalopathy (as per West Haven criteria), human immunodeficiency virus seropositivity, pregnancy, uncontrolled hypertension, coronary artery disease, planned for liver transplantation and those refusing to participate in the study were excluded. Patients who were adequately treated for infection and became culture negative were also screened and, if found eligible, were enrolled in the study. After enrollment, patients underwent baseline etiological workup of cirrhosis (if not known previously), calculation of severity scores namely ChildTurcotte Pugh score (CTP), MELD, and MELD-Na scores and, wherever possible, a baseline transjugular liver biopsy (TJLB) and hepatic venous pressure gradient (HVPG) was done. Nearly all the patients were advised for liver transplantation due to the hepatic decompensation. Nonavailability of donors or other social issues were the main cause of their inability to get the liver transplantation and for opting for growth factor protocol. This single-center study protocol conformed to the Declaration of Helsinki and was approved by the Institutional Ethics Committee (approval file no: F. 8[10]05-06/AC/ILBS/1355). The study followed the CONSORT (Consolidated Standards of Reporting Trials) guidelines for randomized controlled trials and was registered at the ClinicalTrials.gov with full protocol access (identifier: NCT01384565). An informed written consent was taken from each patient at the time of enrollment. All authors had access to the study data and had reviewed and approved the final manuscript.
Randomization and Study Groups Randomization of the patients was done within 1 week of hospital admission after meeting all the inclusion and exclusion criteria. The block randomization was done with block size of 10. An independent statistician using computer-generated random number table performed sequence generation. Allocation concealment was done using sequentially numbered, opaque, sealed envelopes. Enrollment of participants, assessing eligibility and obtaining consent was carried out by 1 of the 2 authors (CKK or SKS). Randomization and administration of the drugs was done by another coauthor (AB). Both the study investigators and the patients were blinded to the study treatment through identical vials coded as A&B in the two groups, respectively. Standard medical therapy comprising albumin, diuretics, nutritional rehabilitation, b-blockers and treatment based on etiology, such as antivirals for hepatitis B, were continued in both groups. Patients with alcoholic liver disease were abstinent from alcohol throughout the study in both groups. These patients had undergone periodic psychosocial counseling in follow-up visits. Patients in the G-CSF and DPO protocol (GDP) group received G-CSF at a dose of 5 mg/kg subcutaneously at days 1, 2, 3, 4, 5 and then every third day until day 28 (total 12 doses), along with DPO 40 mg/wk subcutaneously for 4 weeks (total 4 doses). Patients in the control group received placebos in the same manner.
Follow-up Visits Follow-up was done at day 2, day 4, and at months 1, 3, 6, 9, and 12. During each follow-up visit, detailed clinical examination,
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1364 Kedarisetty et al complete blood counts, and kidney and liver function tests were done. a-Fetoprotein (AFP) was measured at baseline and at 1 month. Abdominal ultrasound was used to assess development of HCC at 3- to 6-month intervals throughout the study. The liver disease severity scores were calculated at each follow-up. Any adverse events were recorded during the entire period.
Assessment of Hepatic Regeneration
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Formalin-fixed, paraffin-embedded biopsy tissue sections obtained from TJLB, were cut at 3- to 4-mm thicknesses and were taken on poly-L-lysinecoated slides. These were deparaffinized by warming and Xylene. Immunohistochemistry (IHC) was done on 4 sections (of each biopsy) by using rabbit monoclonal antibody for markers of hematopoietic stem cells, namely CD34þ, CD133þ, and marker of proliferation, namely Ki67. There are currently a variety of established markers for human progenitor cells. These cells express biliary markers (such as biliary-type cytokeratins (oval cell marker 6, cytokeratin 7, and cytokeratin 19), and hematopoietic markers (such as the receptor for stem cell factor [c-kit], CD34þ). The expression of CD133þ is usually found in uncommitted progenitor cells.20 IHC staining was performed by peroxidase enzymelabeled streptavidin-biotin detection method using Polymer-HRP kit (Biogenex, San Ramon, CA) with diaminobenzidine as a chromogen and Meyer’s hematoxylin for contrasting. Both pathologists were blinded to the treatment protocol. Paired biopsies were studied for change in the markers on IHC. A minimum of 5 fields at 20 magnification was observed for CD34þ, CD133þ, and Ki67. CD34þ cells were expressed as mean percentage calculated in the 5 fields, CD133þ cells were expressed in form of grades namely grade 1: 70, and Ki67 was expressed as mean number detected in the 5 fields.
Primary and Secondary Outcomes The primary outcome was overall survival at 12 months. The time to event was calculated from the day of randomization into the study. The secondary outcomes were survival at 6 months; reduction in liver disease severity scores; reduction in need for large-volume paracentesis (LVP); development of new onset complications, such as acute kidney injury, sepsis, and variceal bleed; change in AFP levels at 1 month; hemodynamic improvement at 1 month; histological evidence of hepatic regeneration; and safety of treatment. Those patients lost to follow-up or who withdrew from the study were censored during the survival analysis. Septic shock was defined according to International Sepsis Definition21 and classified as culture positive or culture negative based on blood, urine, mini bronchoalveolar lavage in patients on mechanical ventilation, ascitic fluid, or any other sites.
Safety of the Therapy The patients were asked to report any adverse effects, such as pain abdomen, vomiting, altered behavior, diarrhea, jaundice, joint pains, or body rashes to the treating team. They were explained that fever and body aches could develop as part of common symptoms after the injections, but to record them and if these symptoms become worrisome, to report the hospital. They were also advised about the proper usage of analgesics and antipyretics. The patients kept a complete record of all
Gastroenterology Vol. 148, No. 7 symptoms and the nursing and clinical staff carefully monitored them.
Statistical Analyses The primary end point was to see the improvement in survival in treatment group as compared with placebo group. Assuming that the survival rate would be around 69% in the treatment group and 29% in the placebo group,13 with a ¼ 5% (2-sided) and power 80%, we needed to enroll a total of 52 cases in the trial (ie, 26 in each arm) based on log-rank test. It was possible to enroll 55 consecutive patients fulfilling the inclusion and exclusion criteria between May 2011 to June 2012 (29 in GDP and 26 in control group). The imbalance in allocation of patients is due to block randomization, with block size taken as 10 and defined enrollment period. Descriptive statistics were expressed as median (interquartile range [IQR]) or number (percentage). Comparison of continuous variables was done by Wilcoxon rank-sum test and categorical variables were compared by Fisher’s exact test or Pearson’s c2 test. The cumulative probability of survival was determined by Kaplan-Meier graph and compared by MantelCox log-rank test. We calculated the change in CTP; MELD and MELD-Na score from baseline to each follow-up as the %D change. Delta change was calculated as ([follow-up day score minus the baseline score]/[baseline score] 100). This delta change was compared between the 2 treatment groups at different time intervals using repeated measures analysis of variance test, followed by post-hoc comparison by Least Significant Difference method. However, the statistical significance for secondary end points should be interpreted cautiously from a false-positive perspective, as no adjustments for multiplicity were made. The intent of the study was to compare from a 2-sided perspective. All statistical tests were performed using SPSS for Windows version 15 (SPSS Inc., Chicago, IL).
Results A total of 750 patients with decompensated cirrhosis were screened during the enrollment period between May 2011 and June 2012 (Supplementary Figure 1) and followed up for 12 months. After careful assessment, 55 patients fulfilled the inclusion and exclusion criteria and were randomized; 29 patients were assigned to the GDP group and 26 patients to control group. The 2 study groups were well compared with respect to demographic characteristics, clinical and laboratory data, and activity scores (Table 1). Twenty-six patients (14 in GDP and 12 in control group) underwent baseline TJLB and HVPG. There was no bias in the selection of patients in the TJLB cohort as the baseline characteristics of TJLB and non-TJLB patients were comparable (Supplementary Table 1). Seven patients (5 in GDP and 2 in control group) got repeat TJLB and HVPG done at 1 month. The etiology of cirrhosis was comparable between the 2 groups (P ¼ .38) and was dominated by chronic alcohol abuse (Supplementary Figure 2). The predominant mode of hepatic decompensation was ascites in both the patient groups and was comparable (P ¼ .18). (Supplementary Figure 3)
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Male sex, n (%) Age, y, median (IQR) Jaundice, n (%) Moderate to severe ascites, n (%) Hepatic encephalopathy, n (%) Variceal bleed, n (%) Hemoglobin, g/dL, median (IQR) Platelet, 103/mm3, median (IQR) Sodium, mEq/dL, median (IQR) Total bilirubin, mg/dL, median (IQR) Albumin, g/dL, median (IQR) Grade of varices, median (IQR) HVPG in mm Hg (n ¼ 26), median (IQR) Ferritin, mg/dL, median (IQR) AFP, ng/mL, median (IQR) Need for LVP, n (%) CTP score, median (IQR) MELD score, median (IQR) MELD-Na score, median (IQR)
GDP group (n ¼ 29) 28 (97) 44 (3261) 23 (79) 26 (89.7) 2 (6.9) (grade 2) 8 (27.6) 9.1 (7.313.7) 99 (36172) 133 (121143) 4.8 (1.214.4) 2.6 (1.83.6) 2 (03) 16 (1125), n ¼ 14 350 (471830) 5.8 (3.28.7) 28 (96.6 ) 11 (913) 22 (1138) 24 (1039)
Control group (n ¼ 26) 25 46 19 23 0 5 8.5 107 130 5.5 2.9 2 15 426 4.3 26 11 20 25
(96) (2760) (73) (88.5) (0) (19.2) (6.812) (35187) (120136) (0.621.3) (1.73.7) (03) (1030), n ¼ 12 (261790) (2.86.3) ( 100) (713) (1030) (1035)
P value .71 .82 .89 .88 .17 .54 .26 .18 .19 .94 .74 .19 .22 .83 .14 .53 .63 .25 .93
*Continuous outcomes were compared using the Wilcoxon rank-sum test and categorical outcomes were compared using Fisher’s exact or Pearson’s c2 tests.
Primary Outcome The primary outcome of the study was whether the use of growth factors led to overall improved survival at 12 months. The difference was obvious from 1 month onward and remained so throughout the follow-up period. Using log-rank (Mantel-Cox) test, the cumulative probability of survival at 12 months was 68.6% (±8.7%) in the GDP group and 26.9% (±8.7%) in control group (P ¼ .003) (Figure 1).
(P ¼ .03). By using repeated measures analysis of variance test, we found that the comparison of %D CTP scores at different time points between the 2 groups showed a progressive reduction in CTP scores with a combination of growth factors that was statistically significant (P < .001) (Figure 2A). Similar results were seen in reference to MELD
Secondary Outcomes Overall survival at 6 months. One of the important secondary end points of the study was survival at 6 months with the use of growth factors. The cumulative probability of survival at 6 months was 82.7% (±7.2%) in the GDP group and 57.7% (±9.7%) in control group (P ¼ .03). Liver disease severity scores. The baseline CTP, MELD, and MELD-Na scores were comparable in both groups as shown in Table 1. Baseline CTP, MELD, and MELD-Na scores were predictive of mortality in the control group (P < .05), but not in the GDP group (P > .05) on subgroup analysis as shown in Supplementary Table 2. On multivariate analysis with adjustment for liver severity scores, we found that there was overall 77%, 73%, and 72% reduction in mortality by GDP group as compared with control group when adjusted for baseline CTP, MELD, and MELD-Na scores, respectively, as shown in Supplementary Table 3. Mean reduction in the severity scores was calculated as %D change at each follow-up from baseline in both the groups (%D change ¼ [follow-up day score minus the baseline score]/[baseline score] 100). Overall %D CTP at 12 months follow-up was 48.6% in GDP group and 39.1% in control group (P ¼ .001). The %D MELD score at 12 months was 40.4% in GDP group and 33% in control group
Figure 1. Kaplan-Meier curve showing the overall survival at 12 months in GDP group compared with the control group.
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Figure 2. Change in liver severity scores. % Delta change (%D) was calculated as ([follow-up day score minus the baseline score]) / [baseline score] 100). (A) %D change in CTP scores from baseline to each follow-up. There was reduction in CTP scores in GDP group compared with control group with statistical significance. (B) %D change in MELD scores from baseline to each follow-up. There was reduction in MELD scores in GDP group compared with control group with statistical significance.
score (P < .001) (Figure 2B) and MELD-Na score (P < .001). This reduction was only noted in patients alive on follow up. Need for large-volume paracentesis. The need for LVP at baseline was 96.6% and 100% in GDP and control group, respectively (P ¼ .53); at 1 month, 41.4% and 76.9% (P ¼ .008); at 3 months, 22.2% and 52.6% (P ¼ .04) and at 6 months, none in the GDP group and 20% in the control group needed LVP (P ¼ .05). There was no difference seen at 9 and 12 months in both the groups (Supplementary Figure 4). Change in a-fetoprotein levels. AFP is an important tumor marker for early detection of HCC. However, it is also an equally important marker of liver regeneration. Therefore, AFP was measured at baseline and at 1 month. The baseline AFP levels were similar in both groups (Table 1). At
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1 month, AFP level was significantly higher in the GDP group compared with the control group (6.6 ± 3.6 ng/mL and 4.7 ± 2.7 ng/mL; P ¼ .018). This was suggestive of augmentation of hepatic regeneration with the use of growth factors. At 1 month, the median aspartate aminotransferase and alanine aminotransferase levels were 61 IU/ L (IQR, 3778 IU/L) and 36 IU/L (IQR, 2148 IU/L) in GDP group and 53 IU/L (IQR, 4183 IU/L) and 30 IU/L (IQR, 2149 IU/L) in control group, respectively. By using regression analyses, there was no interaction between aspartate aminotransferase or alanine aminotransferase and AFP at 1 month after completion of treatment protocol, therefore ruling out false positivity. Hemodynamic studies. In the 7 patients with baseline and repeat HVPG, the mean baseline (±SD) HVPG values in GDP and control groups were 19.7 ± 3.5 mm Hg and 19.8 ± 3.1 mm Hg, respectively. The repeat HVPG was 17 ± 9.8 mm Hg and 16 ± 2.8 mm Hg, respectively. The change in HVPG from baseline to 1 month was not statistically different between the 2 groups (P ¼ .19). This was observed probably due to insufficient power to detect an effect between the treatments with only 7 patients. Liver histology and immunohistochemistry. A total of 7 patients (5 in GDP group and 2 in control group) had undergone baseline and repeat TJLB. The pathologists were blinded to the treatment given to the patient before the reporting of the results. In the paired biopsies, percent of CD34þ cells increased from 37% to 53% in GDP group and 32.5% to 37.5% in control group (P ¼ .19). Figure 3 shows increase in the proportion of CD34þ cells and CD133þ cells in GDP group suggestive of augmented hepatic regeneration. Statistical significance was not observed, probably due to insufficient power to detect an effect between the treatments with only 7 patients (Supplementary Table 4). New onset complications. Variceal bleed was seen in 3 (10.3%) in the GDP group and 5 (19.2%) in the control group (P ¼ .43); spontaneous bacterial peritonitis in 2 (6.9%) and 2 (7.7%) (P ¼ .65); acute kidney injury in the form of hepatorenal syndrome in 3 (10.3%) and 3 (11.5%) (P ¼ .44). Three patients in the GDP group developed strangulation of umbilical hernia and underwent emergency surgery, but succumbed to the illness. One patient in the GDP group was detected to have a small HCC at 12 months of follow-up. However, this patient had a case of chronic hepatitis Brelated cirrhosis of liver on antiviral therapy (Supplementary Table 5). Sepsis. In the 12-month follow-up period, 12 patients with decompensated cirrhosis developed sepsis and presented with hypotension and shock. It was important to note that only 2 patients (6.9%) succumbed to the septic shock in the GDP group in comparison to 10 patients (38.5%) in the control group, the difference was statistically significant (P ¼ .005) (Figure 4). Three patients in the control group grew Enterococcus faecium; Klebsiella pneumoniae, Staphylococcus aureus in blood culture and one patient in Group B grew Stenotrophomonas maltophila in minibronchoalveolar lavage. Remaining patient samples were culture negative.
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Figure 3. Under 200 magnification in an area of 1.3 mm2 (5 fields), there was increased density of CD 34þ staining in lobular parenchyma (black arrow) and CD 133þ staining in ductules (black arrow) in paired liver biopsies in the GDP group.
There were a total of 28 deaths in the entire cohort. The etiologies were septic shock in 2 (6.9%) and 10 (38.5%) in GDP and control group, respectively, uncontrolled variceal bleed with shock in 1 (3.4%) and 3 (11.5%), postoperative complications after emergency umbilical hernia repair in 3 (10.3%) and 1 (3.8%), hepatorenal syndrome progressing to acute tubular necrosis on renal replacement therapy in 3 (10.3%) and 3 (11.5%) patients, respectively. In the 2 remaining cases, the cause of death could not be ascertained due to inaccessibility to information.
Safety of the Therapy G-CSF is known to cause leukocytosis.9 As shown in Supplementary Table 6, there was reactive leukocytosis in response to G-CSF on day 2, day 4, and at 1 month. By using repeated-measures analysis of variance test, we found that the total leukocyte counts between the 2 groups were statistically significant at different time points (P < .001). Malaise was seen as the predominant symptom in 37% patients, followed by generalized discomfort in 23% patients after G-CSF injection (Supplementary Figure 5A and B). Bodyache was the predominant
symptom seen in 45% patients, followed by myalgia in 30% patients and flu-like symptoms in 29% patients after DPO injection. None of the patients showed any evidence of portal vein or deep vein thrombosis on close Doppler ultrasound monitoring. One patient had noncardiac chest pain. There was no statistical difference in the symptomatology reported in the 2 groups. Although, none of the patients received both drugs on the same day, it was difficult to ascertain the adverse event to the same drug. All patients tolerated the treatment well and there was no treatment discontinuation noted.
Discussion The only proven treatment with survival benefit in decompensated cirrhotics is liver transplantation. Unfortunately, many patients succumb to their illness in the waiting list for a transplant or do not have available live donors. By treating the etiology of cirrhosis, for example, antivirals for chronic hepatitis B or chronic hepatitis C and alcohol abstinence in alcoholic liver disease, the disease progression can be retarded to some extent. However, it does not significantly reverse the advanced cirrhotic process.
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Figure 4. The Kaplan-Meier curve showing cumulative probability of septic shock was significantly lower in GDP group compared with the control group.
In this novel study, a combination of 2 growth factors, G-CSF and DPO targeting different cell lineages in the bone marrow, with different cytokine and chemokine cascades, showed that their use was able to achieve improved survival in these patients who ideally required liver transplantation. The baseline liver severity scores were not predictive of mortality in the GDP group, unlike the control group, probably due to altered natural history of cirrhosis in these patients. More importantly, there was a progressive reduction noted in severity scores on use of growth factors that explained the improved survival. The need for LVP had also significantly reduced in the GDP group. It was interesting to note an increase in the levels of AFP at 1 month in the intervention limb. This could be indicative of initiation of hepatic regeneration. In a seminal paper by Eleftheriou et al22 as early as 1975 and subsequently, the relevance of AFP levels in models on hepatic regeneration has been reported.23 One would, of course, need to be cautious of the initiation of dysplasia, followed by hepatocellular carcinoma, if the AFP levels continue to rise significantly. One of our important secondary study end points was induction of hepatic regeneration by the use of growth factors. Bone marrow and liver share an evolutionary conserved developmental relationship and are like 2 sides of the same coin. In fact, hematopoiesis is well known to occur in the fetal liver.24 The bone marrow acts as a reservoir for multiple stem cell populations, including hematopoietic stem cells, mesenchymal stem cells, and endothelial progenitor cells, which are mobilized into the peripheral circulation after
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injury.25 Systemic administration of G-CSF has been shown to mobilize hematopoietic stem cells, endothelial progenitor cells, and bone marrowderived mesenchymal stem cells.26,27 Similarly, EPO has been shown to mobilize endothelial progenitor cells and enhance their in vivo regenerative role via the phosphoinositide kinase-3Akt pathway.28 Patients who underwent repeat liver biopsy in GDP group showed an increased density of CD34þ, CD133þ, Ki67 cells at 1 month compared with control group, reiterating the concept of cross talk between liver and bone marrow to augment liver regeneration. Bacterial infections are much more common in patients with cirrhosis than in the general population.29 Arvaniti et al30 showed that cumulative mortality due to infection in decompensated cirrhosis was 43.5%, vs 13.5% with no infection. This stage classified as “critically ill cirrhotic” highlights the role of infection as a major player in the downhill course of decompensated cirrhosis. In the present study, the patients treated with growth factors had a significantly lower incidence of septic shock compared with those receiving placebo (6.9% vs 38.5%; P ¼ .005) during a follow-up of 12 months. This is an important observation in support of the use of growth factors, which, by modulation of the myeloid series in the bone marrow, are able to reduce the incidence of sepsis. We have recently shown that in ACLF, G-CSF recruits dendritic cells to the liver, which, in turn, reduce the intrahepatic CD8þ T cells and Th17 cells with reduced intrahepatic interferon gamma production, thereby restoring the immune disequilibrium.31 The safety of the combination therapy was also established in decompensated cirrhotics. None required discontinuation of the treatment. The strengths of the present study are that it was a double-blind randomized controlled trial showing clinical and survival benefits by the use of growth factors in decompensated cirrhotics. It could well be appreciated that because the study was invasive, as it required TJLB and HVPG measurement on 2 occasions and continuous monitoring, to recruit this many patients and complete follow-up were a major clinical responsibilities. The outcome of the study was quite gratifying, as the results strongly indicate that this protocol-based treatment can modulate the clinical course in a proportion of well-selected decompensated cirrhotic patients. The possible limitations of the study were a small number of repeat TJLBs at 1 month due to unwillingness of patients to undergo repeat procedure. It is unlikely that such psychological concerns could be easily overcome during the consent process until robust data on regenerative therapy are available in the field. Secondly, it would also have been more interesting to study the role of bone marrow as a source for recruitment of hematopoietic stem cells and macrophages.32 However, it needs to be assessed whether bone marrow biopsy can be done safely in these patients. Also, it would have been more educative to estimate CD34þ, CD133þ cells in peripheral and hepatic venous blood and to correlate these with IHC on liver biopsy or clinical parameters.
This is the first double-blind randomized controlled study (NCT01384565) showing clinical benefit and an overall improved survival using a combination of G-CSF and EPO in patients with decompensated liver disease. This is also the first human study using EPO in decompensated liver cirrhosis, demonstrating its potential in hepatic regeneration. The results of the present study would need to be confirmed in larger cohort of patients with decompensated liver disease.
Supplementary Material Note: To access the supplementary material accompanying this article, visit the online version of Gastroenterology at www.gastrojournal.org, and at http://dx.doi.org/10.1053/ j.gastro.2015.02.054.
References 1. D’Amico G, Garcia-Tsao G, Pagliaro L. Natural history and prognostic indicators of survival in cirrhosis: a systematic review of 118 studies. J Hepatol 2006; 44:217–231. 2. De Silvestro G, Vicarioto M, Donadel C, et al. Mobilization of peripheral blood hematopoietic stem cells following liver resection surgery. Hepatogastroenterology 2004; 51:805–810. 3. Lorenzini S, Isidori A, Catani L, et al. Stem cell mobilization and collection in patients with liver cirrhosis. Aliment Pharmacol Ther 2008;27:932–939. 4. Vassilopoulos G, Wang PR, Russell DW. Transplanted bone marrow regenerates liver by cell fusion. Nature 2003;422:901–904. 5. Pitchford SC, Furze RC, Jones CP, et al. Differential mobilization of subsets of progenitor cells from the bone marrow. Cell Stem Cell 2009;4:62–72. 6. Maiwall R, Kumar A, Sarin SK. Liver regeneration during acute-on-chronic liver failure using growth factors: in vivo or ex vivo indulgence of bone marrow? Gastroenterology 2013;145:901–904. 7. Hoffmann R. Hematology. Basic Principles and Practice. 5th ed. New York: Elsevier, 2009. 8. Gaia S, Olivero A, Smedile A, et al. Multiple courses of G-CSF in patients with decompensated cirrhosis: consistent mobilization of immature cells expressing hepatocyte markers and exploratory clinical evaluation. Hepatol Int 2013;7:1075–1083. 9. Spahr L, Lambert JF, Brandt LR, et al. Granulocyte-colony stimulating factor induces proliferation of hepatic progenitors in alcoholic steatohepatitis: a randomized trial. Hepatology 2008;48:221–229. 10. Spahr L, Chalandon Y, Terraz S, et al. Autologous bone marrow mononuclear cell transplantation in patients with decompensated alcoholic liver disease: a randomized controlled trial. PLoS One 2013;8:e53719. 11. Sarin SK, Kumar A, Almeida J, et al. Acute- on-chronic Liver Failure (ACLF): Consensus Recommendations of the Asian Pacific Association for the Study of the Liver (APASL). Hepatol Int 2009;3:269–282.
G-CSF+ DPO Improve Survival in Cirrhosis 1369 12. Moreau R, Jalan R, Gines P, et al. Acute-on-chronic liver failure is a distinct syndrome that develops in patients with acute decompensation of cirrhosis. Gastroenterology 2013;144:1426–1437. 13. Garg V, Garg HK, Khan A, et al. Granulocyte-colony stimulating factor (G- CSF) therapy mobilizes CD34 cells and improves survival in patients with acute on chronic liver failure. Gastroenterology 2012;142:505–512. 14. Duan XZ, Liu FF, Tong JJ, et al. Granulocyte colony stimulating factor therapy improves survival in patients with hepatitis B-associated acute-on-chronic liver failure. World J Gastroenterol 2013;19:1104–1110. 15. Fischer JW. Landmark advances in development of erythropoietin. Exp Biol Med 2010;235:1398–1411. 16. Lewis LD. Preclinical and clinical studies: a preview of potential future applications of erythropoietin agents. Semin Hematol 2004;41:17–25. 17. Egrie JC, Browne JK. Development and characterization of erythropoiesis stimulating protein (NESP). Br J Cancer 2001;84:3–10. 18. Greif F, Ben Ari Z, Taya R, et al. Dual effect of EPO on liver protection and regeneration after subtotal hepatectomy in rats. Liver Transplant 2010;16:631–638. 19. Ben Ari Z, Zilbermints V, Pappo O, et al. Erythropoetin increases survival and attenuates fulminant hepatic failure induced by D-galactosamine/ lipopolysaccharide in mice. Transplantation 2011;92:18–24. 20. Spee B, Carpino G, Schotanus BA, et al. Characterization of the liver progenitor cell niche in liver diseases: potential involvement of Wnt and Notch signalling. Gut 2010;59:247–257. 21. Levy MM, Fink MP, Marshall JC, et al. 2001 SCCM/ ESICM/ACCP/ATS/SIS International Sepsis Definitions Conference. Crit Care Med 2003;31:1250–1256. 22. Eleftheriou N, Thomas H, Heathcote J, et al. Proceedings: serum alpha-fetoprotein levels in liver disease: relation to hepatocellular regeneration and development of hepatoma. Gut 1975;16:835. 23. Fausto N. Messenger RNA in regenerating liver: implications for understanding of regulated growth. Mol Cell Biochem 1984;59:131–147. 24. Martin MA, Bhatia M. Analysis of the human fetal liver hematopoietic microenvironment. Stem Cells Dev 2005; 14:493–504. 25. Chen Y, Xiang LX, Shao JZ, et al. Recruitment of endogenous bone marrow mesenchymal stem cells towards injured liver. J Cell Mol Med 2010;14:1494–1508. 26. Liu F, Pan X, Chen G, et al. Hematopoietic stem cells mobilized by granulocyte colony-stimulating factor partly contribute to liver graft regeneration after partial orthotopic liver transplantation. Liver Transpl 2006; 12:1129–1137. 27. Takamiya M, Okigaki M, Jin D, et al. Granulocyte colonystimulating factor-mobilized circulating c-Kitþ/Flk-1þ progenitor cells regenerate endothelium and inhibit neointimal hyperplasia after vascular injury. Arterioscler Thromb Vasc Biol 2006;26:751–757. 28. Urao N, Okigaki M, Yamada H, et al. Erythropoietinmobilized endothelial progenitors enhance reendothelialization via Akt-endothelial nitric oxide synthase
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Gastroenterology Vol. 148, No. 7 cells improving fibrosis, regeneration, and function. Hepatology 2011;53:2003–2015.
Received May 11, 2014. Accepted February 28, 2015. Reprint requests Address requests for reprints to: Shiv Kumar Sarin, MD, DM, FNA, FNASc, Department of Hepatology, Institute of Liver and Biliary Sciences, New Delhi 110 070, India. e-mail: [email protected]; fax: þ91 (011) 26123504. Acknowledgments The authors would like to thank their staffs, Ms Anila, and Ms Madhu for data compilation. The generic drugs G-CSF, DPO, and placebos were provided by Dr. Reddys’ Laboratories, Hyderabad, India. They provided no financial assistance of any kind. Conflicts of interest The authors disclose no conflicts.
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Supplementary Figure 1. Study design (screening, randomization, and follow-up of subjects).
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Supplementary Figure 2. Etiology of liver disease.
Supplementary Figure 3. Mode of decompensation.
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Supplementary Figure 4. Need for large volume paracentesis at baseline and at each follow up in both groups.
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Supplementary Figure 5. Adverse events of the drugs. (A) Graphical presentation of % patients adverse events reported after administration of G-CSF in GPD group or G-CSF matched placebo in control group. (B) Graphical presentation of % patients adverse events reported after administration of DPO in GPD group or DPO matched placebo in control group.
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Supplementary Table 1.Baseline Characteristics of Patients in Transjugular Liver Biopsy and NonTransjugular Liver Biopsy Cohort Parameter
TJLB cohort (n ¼ 26)
Non-TJLB cohort (n ¼ 29)
P value
Male sex, n (%) Moderate to severe ascites, n (%) Hepatic encephalopathy, n (%) Hemoglobin, g/dL Platelet, 103/mm3 Urea, mg/dL Creatinine, mg/dL Sodium, mEq/dL Total bilirubin, mg/dL Albumin, g/dL INR AFP, ng/mL Need for LVP, n (%) CTP score MELD score MELD-Na score
25 (96.2) 24 (92.3) 5 (19.2) 9.3 ± 1.5 109.7 ± 39.7 33.4 ± 22.5 0.74 ± 0.3 131.4 ± 4.6 6.8 ± 4.9 2.8 ± 0.4 2.5 ± 2.1 5.8 ± 3.4 25 (96.2) 10.9 ± 1.5 21.8 ± 6.3 25.5 ± 5.7
28 (96.6) 25 (86.2) 4 (13.8) 9.2 ± 1.5 94.1 ± 35.3 32.1 ± 26.8 0.79 ± 0.3 131.2 ± 5.9 4.7 ± 2.7 2.7 ± 0.6 2.1 ± 0.6 7.2 ± 7.3 27 (93.1) 10.6 ± 1.5 19.6 ± 5.1 24.1 ± 5.2
.74 .67 .72 .81 .13 .85 .54 .92 .05 .3 .58 .41 .96 .36 .17 .34
NOTE. Data are presented as mean ± SD, except where indicated. INR, international normalized ratio.
Supplementary Table 2.Baseline Liver Severity Scores and Prediction of Mortality in Each Treatment Group Liver severity score
Treatment group
CTP baseline
GDP Placebo GDP Placebo GDP Placebo
MELD baseline MELD-Na baseline
Hazard ratio
Upper limit
Lower limit
P value
1.42 2.05 1.14 1.13 1.17 1.17
2.53 3.44 1.35 1.25 1.37 1.33
0.80 1.22 0.97 1.02 1.00 1.04
.23 .01 .12 .02 .06 .01
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Supplementary Table 3.Impact of Treatment Group on Outcomes When Adjusted for Baseline Liver Severity Score 95% CI
Treatment group MELD baseline Treatment group MELD-Na baseline Treatment group CTP baseline
P value
HR
Lower
Upper
0.00 0.01 0.00 0.00 0.00 0.00
0.27 1.13 0.28 1.17 0.23 1.78
0.12 1.04 0.13 1.06 0.10 1.22
0.61 1.24 0.64 1.29 0.54 2.58
CI, confidence interval; HR, hazard ratio.
Supplementary Table 4.Immunohistochemistry Markers for Liver Regeneration in Liver Biopsies CD34þ cells (%) Treatment group GDP
Control
a
CD133þ cells (gradea)
Ki67 (n)
Serial no.
Baseline
1 month
Baseline
1 month
Baseline
1 month
1 2 3 4 5 1 2
35 30 40 30 50 40 25
60 40 50 60 55 45 30
1 1 2 3 2 1 0
3 1 2 3 3 1 1
1 0 1 1 1 1 0
2 0 1 2 3 2 1
CD133þ cells were labeled in form of grades. Grade 1: 70.
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Supplementary Table 5.Incidence of New Onset Complications in the 2 Treatment Groups New onset complication
GDP group (n ¼ 29), n (%)
UGI bleed Acute kidney injury SBP SBE/pneumonia Hepatic encephalopathy Septic shock Umbilical hernia strangulation Spontaneous hematoma HCC
3 3 2 2 2 2 3 2 1
Control group (n ¼ 26), n (%)
(10.3) (10.3) (6.9) (6.9) (6.9) (6.9) (10.3) (6.9) (3.4)
P value
5 3 2 1
(19.2) (11.5) (7.7) (3.8) 0 10 (38.5) 1 (3.8) 0 0
.43 .45 .65 .54 .14 .005 .35 .14 .53
SBE, spontaneous bacterial empyema; SBP, spontaneous bacterial peritonitis; UGI, upper gastrointestinal.
Supplementary Table 6.Total Leukocyte Counts (Millions/mm3) at Different Follow-Up Intervals Until 3 Months in the 2 Treatment Groups Time point Baseline Day 2 Day 4 1 month 3 months
GDP group (n ¼ 29) 7.5 19.3 22.5 13.4 7.5
± ± ± ± ±
NOTE. Data are presented as mean ± SD.
4 11 12.3 8.4 4.2
Control group (n ¼ 26)
P value
± ± ± ± ±
.20
