Hepatic Graft-versus-host Disease Research Articles

Serum Cytokeratin-18 Fragments as Quantitative Marker of Epithelial Apoptosis in Liver and Intestinal Graft-Versus-Host Disease.

Graft-versus-host disease (GvHD) is the main complication of allogeneic stem cell transplantation (SCT). However, diagnosis and differentiation of GvHD from infectious or toxic clinical conditions as well as evaluation of GvHD response to immunosuppressive treatment remains a challenge. Since apoptosis is a common histopathological finding in GvHD, we investigated whether the caspase-cleaved neo-epitope of cytokeratin 18 fragments (CK18F) might be a serum marker for ongoing GvHD-induced target organ inflammation.Longitudinal monitoring of serum CK18F kinetics by M30 antibody-based ELISA in 49 patients who fulfilled histopathological and/or clinical criteria diagnostic for GvHD following SCT showed that CK18F were highly elevated in hepatic and intestinal GvHD (6.3±3.8 fold and 4.6±0.8 fold, respectively, over baseline). Responses of GvHD to immunosuppressive therapy were paralleled by CK18F decreases. The extent of CK18F increases tended to correlate with serological severity of acute (r=0.96) and chronic (r=0.76) liver GvHD. Differentially relevant clinical conditions, such as non-complicated infection-related diarrhea and toxic hyperbilirubinemia were not associated with significant CK18F rises. In conclusion, CK18F monitoring provides a simple serum marker for quantitative assessment of GvHD-associated apoptotic activity in intestinal and hepatic GvHD and for distinguishing active GvHD from irreversible end organ damage. Prospective studies are warranted to demonstrate the significance of CK18F for diagnosis, differentiation, prognosis, and treatment guidance of GvHD.

Secondary Lymphoid Tissues Are Not Required for the Induction of GVHD Following Allogeneic BMT: A Shifting Paradigm for T Cell Allo-Activation.

Allogeneic (allo) bone marrow transplantation (BMT) is the only curative option for many patients with malignant and non-malignant diseases. Acute graft versus host disease (GVHD) is the major complication of allo-BMT and limits the utility of this treatment strategy. The induction of GVHD fundamentally depends upon the activation of donor T cells by host antigen presenting cells (APCs), and the prevailing hypothesis is that these critical interactions occur in secondary lymphoid organs, such as lymph nodes (LN) Peyer's patches (PP), and spleen (SP). We tested this hypothesis by using a well established, MHC disparate, murine SCT system (Balb/c → B6) and homozygous aly/aly (alymphoplasia) mice that are deficient in all LN and PP and heterozygous aly/+ littermate controls. Lethally irradiated, splenectomized, aly/aly mice (LN/PP/SP −/ −) and aly/+ sham mice (LN/PP/SP +/+) received BMT either from syngeneic (aly/aly) or allo (Balb/c) donors. In some experiments, wild-type B6 recipients of B6 or Balb/c BMT served as additional negative and positive GVHD controls respectively. The severity of GVHD was assessed by survival and well-described scoring systems of both clinical and target organ disease. As expected, greater than 95% of syngeneic (syn) BMT recipients survived and were indistinguishable from naïve, un-transplanted controls, whereas LN/PP/SP +/+ mice receiving allo-BMT showed significant signs of GVHD with ~40% mortality by day 49. All LN/PP/SP −/ − allo-BMT recipients also survived, but surprisingly, examination demonstrated that they too developed significant clinical GVHD compared to syn controls (score: 3.2 vs. 0.85) that was comparable in severity to LN/PP/SP +/+ mice (3.1). Moreover, histopathologic analysis demonstrated that LN/PP/SP −/ − allo-BMT recipients developed significantly greater GVHD target tissue damage in the liver, intestinal tract and skin compared to syn controls. In fact, LN/PP/SP −/ − allo-BMT recipients developed more severe hepatic GVHD compared to allo littermate (LN/PP/SP +/+) controls (30.8±1.9 vs. 20.7±2.2; p < 0.01). Similar differences in liver GVHD was also seen between allo groups as early as day 7 (16.0±2.2 vs. 7.3±0.9; p < 0.01). We next tested the ability of host aly/aly and aly/+ APCs to stimulate donor Balb/c T cells in vitro. No differences in proliferation, IFN γ production or CTL generation were detected, thus showing that the allo-stimulatory capacity of host APCs was not different between groups. In order to ascertain what extra-lymphoid host tissues might serve as initial sites for allo-antigen exposure, we examined donor T cell expansion (CD3+), activation (CD69+) and proliferation (CFSE) in the bone marrow compartment 3 days after BMT. We found that in each case, LN/PP/SP −/ − allo-BMT recipients had significantly higher numbers / divisions compared to allo, littermate, (LN/PP/SP +/+) controls. Collectively, these data challenge the paradigm that secondary lymphoid tissues are required for GVHD induction, and suggest that the bone marrow may represent an alternative site for allo-antigen recognition and donor T cell activation.

Impacts of ABO-Blood Type Incompatibility on Outcome of Unrelated Bone Marrow Transplantation through the Japan Marrow Donor Program.

ABO incompatibility between donor and recipient is not a barrier for successful allogeneic hematopoietic stem cell transplantation, but conflicting data still exist concerning its influence on transplant outcome, graft-versus-host disease (GVHD), relapse, and survival. We retrospectively analyzed the data of patients who underwent UR-BMT through the Japan Marrow Donor Program between January 1993 and September 2005, with complete data on ABO-blood group compatibility, age, and gender in donors and recipients.A total of 4,970 patients were transplanted with marrow from ABO-matched (M; n=2,513, 50.6%), major incompatible (MA; n=1,254, 25.2%), minor incompatible (MI; n=1,081, 21.8%), and bidirectional incompatible donors (IA; n=122, 2.5%), and were followed up over a median period of 325 days. Among these four groups, excluding age, there was no significant difference in the gender of patients and donors, number of transplantations, conditioning regimen, GVHD prophylaxis, and performance status before transplantation by the likelihood ratio test. The 5-year overall survival of any ABO-incompatible group was significantly lower compared to an identical group (Wilcoxon test, p<0.0001); the estimates for each group were 50.0% (M), 44.7% (MA), 46.7% (MI), and 41.3% (IA). Even in HLA-matched transplantation (n=2,608), a similar difference in overall survival was observed among the four groups (p=0.0124).In ABO-mismatched transplantation, the processing of bone marrow is necessary to prevent hemolysis of donor or recipient red blood cells as a result of the infusion of ABO-incompatible red blood cells or plasma contained within it. This procedure may reduce the number of hematopoietic stem cells. In fact, the mean number of total infused cells in each group was 3.10 (M), 1.52 (MA), 2.87 (MI), and 1.33 (IA) x108 per patient body weight (kg), with a significant difference in 4,210 patients in which data on the infused cell number were available (M; n=2,310, MA; n=996, MI; n=802, IA; n=102). To examine whether the difference in overall survival depended on the transplanted cell number, we used time-dependent Cox proportional hazards modeling to compare identical and major incompatible groups in terms of overall survival. Whereas the disease (standard and high-risk malignant disease, and benign disease; p=0.0000), patient age (p=0.0000), and ABO compatibility (p=0.0311) were elucidated to be significant risk factors, the number of infused cells was not (p=0.0603).Engraftment of red blood cells, white blood cells, and platelets were significantly delayed in major ABO mismatch in comparison with ABO identity (p<0.0001). Univariate analysis revealed a small but significant difference in the rate of grade III and IV GVHD among the four groups (p=0.0204). Patients with major and minor ABO incompatibility had a higher incidence of severe GVHD compared to ABO identity (21.9%, 20.4% vs 16.2%). There was no significant difference in GVHD of the skin and gut, but major and minor mismatch developed a higher incidence of moderate to severe hepatic GVHD compared to ABO match (p<0.0001, p=0.0010, respectively). ABO incompatibility had no significant effect on relapse, but the incidence of rejection was significantly higher with ABO-incompatible transplantation (p=0.0219).

Hepatitic pattern of graft versus host disease in children

Liver involvement by graft-versus-host disease (GVHD) is characterized histologically by bile duct damage, which may be severe. A different pattern, "hepatitic GVHD," has been described in adult patients. This pattern also shows marked lobular hepatitis and hepatocellular damage. We report the development of hepatitic GVHD in six pediatric patients. Clinical information and histologic features of liver biopsy samples were retrospectively reviewed. Patients' ages ranged from 3 to 11 years. Underlying diagnosis, pre-transplant conditioning and GVHD prophylaxis varied. Peripheral blood stem cells were the source of the allograft in four patients, matched sibling in one, and matched-unrelated donor in one. Hepatic GVHD was detected between 149 and 310 days post-transplant. Prior acute GVHD had developed in two patients, and involved the skin and/or gastrointestinal tract. No patients had significant ductopenia. Only one patient had significant lymphocytic infiltration of bile ducts (ductitis). Bile duct epithelial damage and significant portal/periportal inflammation were present in all patients. Lobular necro-inflammation was present in five patients. Five patients improved with immunosuppression and one died with progressive GVHD. This series focuses on hepatitic GVHD in pediatric patients. Clinical and histologic patterns are similar to what has been described in adults. Specific etiology and pathogenesis of this entity remain unclear.

Risk factors and treatment of hemorrhagic cystitis in children who underwent hematopoietic stem cell transplantation

A retrospective cohort of 163 children with 171 hematopoietic stem cell transplantation (HSCT) performed during Mar. 1992-Dec. 2005 were analyzed to evaluate the incidence, risk factors, management, and outcome of hemorrhagic cystitis (HC). Fourteen patients (8.2%) developed HC (6 boys, median age 6.6 years) at 0-166 days after HSCT (median 25 days), and lasted for 3-96 days (median 26 days). Older age at transplant (median 11.0 vs. 6.4 years, P = 0.013), allogeneic transplant (OR = 4.4, P = 0.02), cyclophosphamide-containing conditioning (OR = 4.87, P = 0.008), moderate-to-severe acute graft-versus-host disease (GVHD) (OR = 3.56, P = 0.025) and hepatic GVHD (OR = 3.62, P = 0.017) were associated with higher risks of HC in univariate but not multivariate analyses. While estrogen was ineffective in most patients, intravesical formalin, which was used in five patients, was found to be a very effective yet safe treatment for intractable HC. Patients with HC had longer hospital stay (median 175 vs. 88 days, P = 0.004). HC resolved after treatments in all cases but eight of the 14 patients subsequently died of other complications of HSCT. In conclusion, HC is a serious complication of allogeneic HSCT. Treatment with intravesical formalin appears effective and safe and can be considered early in severe HC to reduce the risk of morbidity and mortality.

Iron Overload Manifesting as Apparent Exacerbation of Hepatic Graft-versus-Host Disease after Allogeneic Hematopoietic Stem Cell Transplantation

Iron overload presenting as exacerbation of hepatic graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation has not been previously described. We report 6 patients with established hepatic GVHD in whom iron overload (median serum ferritin, 7231 μg/dL; median transferrin saturation, 77%) resulting from a lifetime median of 20 units of packed red blood cell transfusions was manifested by worsening of liver function. Liver biopsies performed in 4 patients confirmed severe iron overload and also hepatic GVHD. Analysis for the C282Y and H63D hemochromatosis gene mutation was negative for the homozygous state in all 6 patients. Erythropoietin-assisted phlebotomy resulted in normalization of liver function at a median of 7 months and of serum ferritin at a median of 11 months. Immunosuppressive therapy was successfully tapered in all 4 patients who completed the phlebotomy program, and this supported the impression that iron overload, rather than GVHD, was the principal cause of liver dysfunction. At a median follow-up of 50 months (range, 18-76 months) from the transplantation and 25 months (range, 5-36 months) from ferritin normalization, all 4 patients require maintenance phlebotomy. We conclude that iron overload can mimic GVHD exacerbation, thus resulting in unnecessary continuation or intensification of immunosuppressive therapy for GVHD, and that maintenance phlebotomy is necessary after successful iron-reduction therapy.

Living-Donor Liver Transplantation for Chronic Hepatic Graft-versus-Host Disease

To the Editor: In 2002, a four-year-old boy who had been diagnosed with acute lymphoblastic leukemia in 1999 received a bone marrow transplant from his mother, who had three HLA-antigen mismatches and an incompatible blood type. After engraftment, an erythematous rash, diarrhea, and hepatomegaly, accompanied by elevated liver-enzyme levels, developed in the boy. The findings were indicative of acute graft-versus-host disease (GVHD) and led to intensified immunosuppressive therapy with mycophenolate mofetil and corticosteroids. Although the gut and skin GVHD improved, the hepatic GVHD persisted and was confirmed as chronic hepatic GVHD on liver biopsy (Figure 1A and Figure 1B). The chronic GVHD . . .

Unusual extramedullary recurrences and breast relapse despite hepatic GVHD after allografting in Ph+-ALL

Extramedullary recurrences with or without bone marrow involvement are reported in up to a half of leukemic relapses after BMT. Our report describes a case of an extramedullary recurrence and breast relapse after second-allografting in a female patient with Ph+-acute lymphoblastic leukemia (ALL), occurring when there was active hepatic GHVD. This case illustrates the complex relationship between graft-versus-host disease (GVHD) and graft-versus-leukemia since she had no evidence of leukemia in her marrow demonstrating 100% full-donor chimerism while she had ALL relapse in her breast.

Absence of β7 integrin results in less graft-versus-host disease because of decreased homing of alloreactive T cells to intestine

The alpha4beta7 integrin plays a central role in the homing of T cells to the gut. We hypothesized that absence of the beta7 subunit would result in a reduction of intestinal graft-versus-host disease (GVHD) and an improvement in overall GVHD morbidity and mortality in recipients of hematopoietic stem cell transplantation (HSCT). Analysis of alloreactive beta7-/- T cells showed intact activation, proliferation, cytokine production, and cytotoxicity. However, recipients of beta7-/- donor T cells in murine HSCT models experienced less GVHD morbidity and mortality than recipients of wild-type (WT) T cells, associated with a decrease in donor T-cell infiltration of the liver and intestine and with an overall significant decrease in hepatic and intestinal GVHD. In graft-versus-tumor (GVT) experiments, we demonstrated intact or even enhanced GVT activity of beta7-/- donor T cells. In conclusion, beta7-/- donor T cells caused less GVHD morbidity and mortality than WT donor T cells because of selectively decreased T-cell infiltration of the liver and intestines. Our data suggest that strategies to target the beta7 integrin have the clinical potential to alleviate or prevent GVHD while sparing or potentiating GVT activity.

Liver biopsy in the management of liver dysfunction after allogeneic hematopoietic stem-cell transplantation

To investigate the pathologic and clinical characteristics of patients with liver dysfunction after allogeneic hemopoietic stem cell transplantation (Allo-HSCT), and evaluate the role of liver biopsy in diagnosis and management of liver dysfunction post Allo-HSCT. Ten patients with hematological diseases, 5 males and 5 females, aged 34.5 (15-56), underwent Allo-HSCT, 6 receiving Allo-HSCT from their HLA-matched siblings, one receiving haploidentical transplant, two receiving cord blood transplantation from sibling, and one receiving unrelated BMT. The conditioning regimens included Cy/TBI (in 2 cases), and non-TBI regimen (in 8 cases). All the patients received cyclosporine A, short term MTX and MMF for the prophylaxis of graft-versus-host disease (GVHD). Test of liver function and examination of HBV and HCV related serologic markers were performed for the patients and recipients before transplantation and for those who had liver dysfunction after transplantation. Percutaneous liver biopsy was performed for these 10 patients. Biopsy specimens were examined histologically by hematoxylin-eosin staining, Masson staining and immunohistochemical stating of related viruses. All 10 patients showed liver dysfunction on average 3.5 months (2-5 months) after the transplantation. Seven patients positive in HBV and HCV serologic markers before HSCT remained positive after the transplantation. Three patients negative in HBV/HCV markers before and after Allo-HSCT were diagnosed as with GVHD clinically. Eight patients had other manifestations of chronic GVHD. Eight patients received liver biopsy 5.5 months (4-12 months) because of poor improvement in the initial treatment stage. The result of biopsy showed hepatic GVHD in 3 patients and hepatic GVHD concomitant with HBV or HCV hepatitis reactivation in 7 patients. The liver function of 3 patients diagnosed as with hepatic GVHD returned to normal after the use of the second-line anti-GVHD therapy. The other 7 patients used anti-virus agents combined with immunosuppressive agents with the result that 2 patients remained alive with normal liver function and 5 patients died due to pulmonary complication (2 cases) or hepatic failure and hepatic coma (3 cases). Liver biopsy is of distinguished diagnostic value for patients with refractory hepatic GVHD after Allo-HSCT. The prognosis is poor for patients having hepatic GVHD concomitant with active hepatic virus infection. At this time the role of liver biopsy is limited and management should be made according to the clinical features and liver pathology.

Absence of inducible costimulator on alloreactive T cells reduces graft versus host disease and induces Th2 deviation

AbstractInducible costimulator (ICOS) is expressed on activated and memory T cells and is involved in the regulation of cytokine production. We studied the role of ICOS on alloreactive T cells in graft versus host disease (GVHD) and determined that ICOS expression was up-regulated on alloreactive T cells in recipients of an allogeneic hematopoietic stem cell transplantation (allo-HSCT) with GVHD. We compared ICOS-/- T cells with wild-type (WT) T cells in 2 GVHD models. In both models, recipients of ICOS-/- T cells demonstrated significantly less GVHD morbidity and mortality, which was associated with less intestinal and hepatic GVHD but increased cutaneous GVHD. In addition, recipients of ICOS-/- donor T cells displayed a slight decrease in graft versus leukemia (GVL) activity. Further analysis of alloreactive ICOS-/- T cells showed no defect in activation, proliferation, cytotoxicity, and target organ infiltration. Recipients of ICOS-/- T cells had decreased serum levels of interferon-γ (IFN-γ), while interleukin-4 (IL-4) and IL-10 levels were increased, suggesting that alloreactive ICOS-/- T cells are skewed toward T helper-2 (Th2) differentiation. These data suggest a novel role for ICOS in the regulation of Th1/Th2 development of activated T cells. In conclusion, alloreactive ICOS-/- donor T cells induce less GVHD due to a Th2 immune deviation while GVL activity is slightly diminished.

The role of TNF in hepatic histopathological manifestations and hepatic CD8+ T cell alloresponses in murine MHC class I disparate GVHD

Transfer of B6 T cells to major histocompatibility complex (MHC) class I disparate bm1 x B6 F1 mice leads to the development of hepatic graft-versus-host disease (GVHD) characterized by an active hepatitis with portal and lobular inflammation as well as bile duct inflammation and venulitis. The present studies determined the role of tumor necrosis factor (TNF) in hepatic GVHD. B6 responder cells were cultured with irradiated MHC class I disparate bm1 or syngeneic spleen cells (SpC) in the presence or absence of TNF receptor inhibitor [TNFR-immunoglobulin (Ig)]. Recipient bm1 x B6 F1 mice were irradiated (600 cGy) and reconstituted with 5 x 10(6) T cell-depleted B6 bone marrow cells and 1 x 10(7) B6 SpC. Mice were injected with an adenovirus encoding TNFR-Ig [TNF inhibitor-encoding adenovirus (Adv-TNFi)] or beta-galactosidase (Adv-betagal). Severity of liver GVHD was assessed by a composite histopathological score consisting of the sum of scores for venulitis, lobular hepatitis, and bile duct inflammation. Addition of TNFR-Ig reduced cell proliferation in mixed lymphocyte cultures using B6 responder SpC by 71% +/- 12.8% and interferon-gamma responses by 78% +/- 18%. GVHD-induced "wasting disease" was reduced in Adv-TNFi recipients [4.4%+/-5.2% weight loss (n=11)] compared with Adv-betagal recipients [16.1%+/-7.6% weight loss (n=11; P=0.0004)] 9 days post-transplant. Composite histopathological scores and individual venulitis scores were reduced with the addition of Adv-TNFi. Hepatic CD8+ T cells in the recipients of Adv-TNFi were reduced as compared with recipients of Adv-betagal. In conclusion, Adv-TNFi reduces MHC class I disparate alloproliferative responses and hepatic GVHD.

GI complications in pediatric patients post-BMT

This retrospective study comprehensively examined hepatic and gastrointestinal complications post-bone marrow transplant (BMT) in a heterogeneous group of 132 pediatric patients that underwent 142 transplants. Hyperbilirubinemia occurred in 28% of this population with clinically evident jaundice in 16%. Acute graft-versus-host disease (GVHD) occurred in 46% of the population, with liver involvement in 39% and intestinal involvement in 60% of those with acute GVHD. Veno-occlusive disease (VOD) occurred in 18% of the population. A greater increase in hepatic transaminases was noted in GVHD and VOD than nonspecific liver injury. Serum bilirubin may help to differentiate between VOD and hepatic GVHD. Biliary sludging occurred in 20% of patients and was associated with increased morbidity. Common post transplant gastrointestinal complications included mucositis in 90%, vomiting in 85% and abdominal pain in 71%. TPN support post transplant was required in 91%. Diarrhea occurred in 67% with the most common identified etiologies reported as GVHD (27%), viral (6%), Clostridium difficile (8%) infections and unknown (28%). Typhilitis developed in 3.5%. Melena or hematochezia occurred in 11 patients (8%). However, gastrointestinal bleeding was disproportionately represented in intensive care unit admissions (5/27) and 100 day mortality (5/21). Gastrointestinal and hepatic complications represent a major cause of morbidity and mortality in pediatric BMT recipients.

Liver transplantation for severe hepatic graft-versus-host disease: An analysis of aggregate survival data

Graft-versus-host disease (GVHD) often occurs after bone marrow transplantation (BMT). GVHD may lead to cirrhosis or complete destruction of the bile ducts, and few effective treatment options exist for such cases. Orthotopic liver transplantation (OLT) has been described as an option, but to date the patient survival, graft survival, and GVHD recurrence rates after OLT have been unknown. Cases of OLT for GVHD were accumulated from several sources: (1) cases of OLT performed at a single institution, (2) the English-language medical literature, and (3) the United Network for Organ Sharing (UNOS) liver transplant registry. Descriptive data were derived from pre- and post-OLT information; survival analysis was performed using the Kaplan-Meier method. One case of OLT for GVHD after BMT was found at our institution, and another 6 cases were previously reported in the literature. Extrahepatic GVHD recurred in 2 cases, but no recurrence of hepatic GVHD was reported. The UNOS registry contained an additional 73 patients who underwent OLT for hepatic GVHD. The 1- and 5-year actuarial patient survival rates were 72.4% and 62.9%, respectively. Although 4 patients required retransplantation, no deaths or retransplants were attributed to the recurrence of hepatic GVHD. OLT is an effective treatment for hepatic GVHD after BMT or non-liver organ transplant. Long-term disease-free survival is obtainable in these cases, and recurrence of hepatic GVHD has not been reported. These findings suggest that OLT should be considered as an effective treatment option for cases of hepatic GVHD recalcitrant to medical treatment.

Liver disease is a major cause of mortality following allogeneic bone-marrow transplantation

Liver disease is an important cause of morbidity and mortality among recipients of bone-marrow transplantation (BMT). The aim of this retrospective study was to determine the incidence, risk factors and clinical evolution of liver disease following allogeneic BMT. A total of 103 patients (mean age 22.8 years (SD 10.9); 31.1% aged < 18 years; 66% males) transplanted in a single institution were enrolled. Data on donors and recipients were collected, including hematological disease, alanine transaminase, alkaline phosphatase, bilirubin, hepatitis B virus (HBV) and hepatitis C virus (HCV) markers (including HBV-DNA and HCV-RNA). Fifty six of 103 patients died, with liver disease the main cause of death (27 of 56, 48%). Overall the incidence of liver failure attributed to hepatic graft-versus-host-disease (GVHD) was 22.3% (23 of 103; 74% HBV/HCV infected) and veno-occlusive disease (VOD) was 9.7% (10 of 103; 80% HBV/HCV infected). Fourteen patients had hepatitis reactivations (four hepatic GVHD and three VOD). Donors' HCV-RNA status and serum bilirubin above 2 mg/dl were predictive of hepatic GVHD [adjusted odds ratio (AOR) 11.1, 95% confidence interval (CI) 0.99-33.12; AOR 3.93, 95% CI 1.09-14.62; P < 0.05, respectively] and an abnormal alkaline phosphatase could predict severe liver disease (AOR 2.78, 95% CI 1.01-7.54; P < 0.05). Development of severe liver disease (hepatic GVHD or VOD) was a significant predictor of mortality (AOR 4.57, 95% CI 1.09-20.32; P < 0.05) with a low probability of survival (19.3%, SD 7.9%) compared with those without liver disease (52.1%, SD 7.6%; log-rank P = 0.0003). Hepatic GVHD is a common complication following BMT and an important cause of liver-related mortality. The high prevalence of HCV and HBV may have contributed to the outcome of hepatic GVHD and VOD. Therefore, antiviral therapy should be considered early to prevent relentless progression of liver disease.

Dueling Lymphocytes: Picking the Winner

Dueling Lymphocytes: Picking the Winner

Pathologic Features May Predict the Clinical Outcome of Hepatic Graft Versus Host Disease (H-GVHD).

Liver histology is regarded as inadequate for grading the severity of H-GVHD. Thus, liver biopsies (LB) are only performed to rule out other causes of liver injury when the results may lead to a radical therapeutic change. In this study we reviewed the pathologic features in LB from 33 consecutive evaluable patients with at least one LB, confirmed diagnosis of H-GVHD and no other concomitant causes of liver dysfunction. We asked whether the pathologic features in the first LB may predict their clinical outcome. Underlying diseases were AML (9), ALL (8), CML (7), MM (4), MDS (3), NHL (1) and AA (1). H-GVHD developed after BM (15) or PB (11) allo-HSCT, or DLI (7). Twenty-eight donors were related and 5 unrelated, 30 HLA-matched and 3 mismatched. All patients received myeloablative TBI-based (31) or chemotherapy only (2) conditioning regimens, and GVHD prophylaxis with cyclosporine-methotrexate (21) or T-cell depletion (12). The onset was acute in 22 (median day 35, range 5–86) and chronic in 11 (median day 139, range 103–545). The median overall survival (OS) from the onset of liver dysfunction was 6.2 months, with a non-relapse mortality (NRM) of 73.2% at 1 year (CI95=50.2–96.2). The causes of NRM were infection (8), liver failure (3), hemorrhage (3) and others (4). The first LB was performed at a median of 14 days (4–68) from the onset of abnormal liver function tests. The commonest pathologic finding in this series was bile duct damage, but ductopenia was uncommon (4). Neither the presence nor the degree of bile duct damage associated with the clinical outcome. Lobular inflammation (LI) was present in 25 cases (grade 1: 16; grade 2: 6; grade 3: 3). Higher degree of LI in the first LB associated with a reduced NRM (P=.001) and improved OS (P<.001). Patients with LI grades 2/3 had higher median OS than those with grades 0/1 (not-reached vs 4.3 months; P=.012), and were more likely to respond to primary treatment (RPT) with steroids (P=.044), the most important clinical prognostic factor for OS in our series (HR=4.3; P=.001). Presentation of H-GVHD as hepatitis, with markedly increased aminotransferases levels, has been reported, in particular after DLI. In our group, there was no association between DLI-induced H-GVHD and LI, or between LI and aminotrasferases peak or biopsy-time levels. No study to date has shown that LI in the LB associated with clinical outcome in H-GVHD. In liver transplantation however, a histologically documented lobular hepatitic phase has been shown to anticipate ductopenia in chronic rejection, and associate with good response to treatment. In addition to LI, hepatocyte ballooning (HB) was associated with NRM (P=.003) and OS (P=.018) in our series. Patients with HB grades 0/1 had higher median OS than those with grades 2/3 (6.5 months vs 1.3 months; P=.004). HB did not associate with RPT. In multivariate analysis the effects of LI and HB on NRM (HR=5.1, P=.033; and HR=5.5, P=. 018, respectively) and OS (HR=4.0, P=.032; and HR=4.2, P=.037, respectively) remained significant. All other pathologic features examined had no association with patient clinical outcome. Our results suggest that LI and HB in the first diagnostic LB from patients with H-GVHD may predict the probability of RPT, NRM and OS. These results should be considered in the indications of LB and the selection of candidate patients with H-GVHD for new experimental therapies and in the design of future trials. Prospective validation of our findings is under way.

B-Cell Depletion and Prevention of Hemolysis in Allogeneic Peripheral Blood Stem Cell Transplantation with Minor ABO Incompatibility.

Immune hemolysis is a severe complication of allogeneic peripheral blood stem cell (PBSC) transplantation with minor ABO-incompatibility, principally observed after conventional allogeneic PBSCT. The B-cell depletion of the PBSC graft is a prophylactic intervention which may decrease incidence and severity of this event. Eight patients who underwent allogeneic PBSCT in our institution between August 1999 and April 2004 were analysed. They all received a B-cell depleted transplantation performed because of minor ABO-incompatibility. There were 5 males and 3 females and the median age was 42.5 years (19–65). The pre-transplant diagnosis were solid tumours for 2 patients and haematological malignancies for 6. All received allogeneic PBSC recruited by G-CSF from 3 HLA identical unrelated and 5 HLA identical sibling donors. The median number of nucleated cells and CD34+ cells harvested were 7.75x108 /Kg (3.9–11) and 6x106/kg (4.3–8.7) and the median number of infused cells were 5.5x108 /Kg (3.4–8.71) and 5x 106/kg (3.1–8.33) respectively. Two patients received myeloablative and 6 patients reduced intensity conditioning regimens (RIC). As graft-versus-host-disease (GVHD) prophylaxis, cyclosporin with methotrexate was given after myeloablative conditioning. After RIC, cyclosporin alone (n=4) or cyclosporin associated to methotrexate (n=1) or cyclosporin associated to mycophenolate mofetil (n=1) were given. The ex-vivo manipulation consisted of a negative CD19 immuno-selection using the Isolex 300 I Baxter procedure. The median number of CD19+ cells (x106/kg) before and after ex-vivo manipulation was respectively 73 and 0.05, which represented a B-cell log depletion of 3.16. The ex-vivo manipulation nevertheless induced a nucleated cell depletion of 20% (from 7.75 x108 cells/kg to 5.52x108 cells/kg after B-cell depletion) and a CD34+ cell loss of 18.5% (from 6x106 cells/kg to 5x106 cells/kg). Engrafment was observed for all patients and median time to neutrophiles (>0.5 G/l) and platelets (>50 G/l) recovery were respectively 19 and 12 days. Acute GVHD occurred for 5 patients whereas 3 patients developed chronic GVHD. At last follow-up, 4 patients died, 3 due to progressive disease, one due to hepatic GVHD. None of these 8 patients developed any clinical severe hemolysis, except one patient who presented a biological hemolysis with the development of a positive antiglobulin test 3 weeks after transplantation, without any consequences. Three months after transplant and at the last follow-up, the 4 long-term alive patients showed a stable erythropoietic reconstitution with an ABO-RhD donor determination. These data indicate that B-cell depletion is an efficient and safe method which could be used in case of minor ABO incompatibility.

Allogeneic peripheral blood stem cell transplantation in patients with haematological malignancies.

To report the initial data on allogeneic peripheral blood stem cell transplantation for haematological malignancies in Pakistan. A single centre descriptive study. Bismillah Taqee Institute of Health Sciences and Blood Diseases Centre from September 1999 to June 2004. Patients with haematological malignancies were included who had received allogeneic PBSC transplantation of Filgrastim (rhG-CSF) mobilized peripheral blood stem cells from HLA-identical siblings (except one 5/6 antigen sibling) with Busulphan and Cyclophosphamide standard conditioning therapy in all patients. No patient received antibiotics for gut decontamination. Empirical antibiotics included Ceftriaxone and Amikacin for febrile neutropenia, oral Itraconazole for antifungal prophylaxis while oral acyclovir was used for antiviral prophylaxis. All donors and recipients were CMV IgG positive Cyclosporin A / Methotrexate were given for graft versus host disease (GvHD) prophylaxis. Stem cells were harvested using Haemonetics MCS+ cell separator. All patients received G-CSF starting from day +4 until their neutrophil count rose to normal. There were 21 patients with age range of 8-38 years and male to female ratio of 2:1. Engraftment was achieved in all patients; median time to absolute neutrophil count of > 0.5 x 10(9)/l was 10 days (range 8 - 12 days) and platelet count of > 20 x 10(9)/l was 14 days (12-17 days). Acute graft versus host disease ( aGvHD) was seen in 7 patients; one patient had grade IV skin and hepatic GvHD; another patient had grade III gut GvHD, grade II GvHD was seen in 3 patients while grade I skin aGvHD was seen in 2 patients. Median hospital stay was 34 days. Treatment related mortality was seen in 3 patients (18%). Chronic GvHD was seen in 5 patients. Four more patients died during the follow-up period. Malaria was seen in 2 while tuberculosis developed in one case. Relapse was seen in 2 patients. The estimated probability of survival at one hundred day, at one year and five years was 82, 47 and 40 percent respectively. Haematopoietic stem cell transplant programme can be developed in a developed country setting. Post transplant complications are similar to what have been reported in the developed countries. In endemic areas malaria could prove to be fatal if not recognised and treated early.

Non-HLA immunogenetic polymorphisms and the risk of complications after allogeneic hemopoietic stem-cell transplantation.

Existing data indicate that non-human leukocyte antigen (HLA) immunogenetic polymorphisms influence the risk of complications after allogeneic hemopoietic stem-cell transplantation. However, prior studies have been limited by small sample size and limited genotyping. We examined 22 polymorphisms in 11 immunoregulatory genes including cytokines, mediators of apoptosis, and host-defense molecules by polymerase chain reaction using sequence-specific primers in 160 related myeloablative transplants. Associations were confirmed in two independent cohorts. An intronic polymorphism in the tumor necrosis factor gene (TNF 488A) was associated with the risk of acute graft-versus-host disease (GVHD) (odds ratio [OR] 16.9), grades II to IV acute GVHD (OR 3.3), chronic GVHD (OR 12.5), and early death posttransplant (OR 3.4). Recipient Fas -670G and donor interleukin (IL)-6 -174G were independent risk factors for acute GVHD. Recipient IL-10 ATA and Fas -670 genotype were independent risk factors for chronic GVHD. Recipient IL-1beta +3953T was associated with hepatic acute GVHD, and Fas -670G was associated with major infection. These results highlight the potential importance of cytokine and apoptosis gene polymorphisms in stem-cell transplantation, and indicate that non-HLA genotyping may be useful to identify individuals at the highest risk of complications and new targets for therapeutic intervention.