Stem cell transplantation Gastrointestinal graft-versus-host disease: from biomarkers to pathophysiology J. Ferrara ABSTRACT University of Michigan Hospitals and Acute graft-versus-host disease (GVHD) of the gastrointestinal tract (GI) affects up to 60% of Health Centers, MI, USA patients receiving allogeneic hematopoietic cell transplantation (HCT). It causes nausea, vomiting, anorexia, secretory diarrhea and, in severe cases, abdominal pain and/or hemorrhage. Acute GVHD is often clinically indistinguishable from other causes of GI dysfunction such as conditioning regimen Correspondence: toxicity, infection or medications. Endoscopic biopsy is often used to confirm the diagnosis, but histo- James Ferrara logical severity on biopsy does not consistently correlate with clinical outcome. Clinical stage II or E-mail: [email protected] greater (more than 1 L of diarrhea per day) is associated with reduced survival, but daily stool volume can vary considerably. Lower GI GVHD responds poorly to treatment compared to other target organs, and treatment with high-dose systemic steroid therapy carries significant risks, especially infectious Hematology Education: complications in profoundly immunosuppressed patients. the education program for the annual congress of the European Hematology Association Learning goals At the conclusion of this activity, participants should: 2013;7:347-350 - know the three parameters that define risk for death at the onset of gastrointestinal graft-versus- host disease; - know the functional importance of Paneth cells in the gastrointestinal tract; - know the relationship between Reg3 and the gastrointestinal microbiome. α versus Acute graft- -host disease (GVHD) of The research effort that identified REG3α the gastrointestinal tract (GI) affects up to 60% used a proteomics approach that analyzed of patients receiving allogeneic hematopoietic pooled plasma samples from 10 patients with cell transplantation (HCT).1,2 It causes nausea, biopsy-proven GI GVHD and 10 patients vomiting, anorexia, secretory diarrhea and, in without GVHD taken at similar times after severe cases, abdominal pain and/or hemor- HCT.12,13 We identified and quantified 562 pro- rhage.3 Acute GVHD is often clinically indis- teins, of which 74 were increased at least 2- tinguishable from other causes of GI dysfunc- fold in patients with GVHD. Of the 5 proteins tion such as conditioning regimen toxicity, preferentially expressed in the GI tract, only infection or medications. Endoscopic biopsy is one had commercially available antibodies often used to confirm the diagnosis,4 but histo- suitable for quantification of plasma concen- logical severity on biopsy does not consistent- trations by ELISA. This process identified ly correlate with clinical outcome.4-6 Clinical REG3α as the lead candidate for a biomarker stage II or greater (more than 1 L of diarrhea of GI GVHD. per day) is associated with reduced survival,1,2 We validated REG3α as a biomarker by but daily stool volume can vary considerably. measuring plasma concentration in samples Lower GI GVHD responds poorly to treatment from 850 allogeneic HCT recipients from the compared to other target organs, 2 and treat- University of Michigan, USA. Plasma REG3α ment with high-dose systemic steroid therapy concentrations were 3 times higher in patients carries significant risks, especially infectious at the onset of GI GVHD than in all other complications in profoundly immunosup- patients, including those with non-GVHD pressed patients.7,8 enteritis. Serum REG3α concentrations were The standard treatment of acute GVHD is also higher in GI GVHD in an independent higher dose systemic steroids, which has not validation set of 143 HCT patients from changed in 40 years. One reason for this lack Regensburg, Germany, and Kyushu, Japan, of progress is the lack of validated biomarkers although the absolute values were lower. This for acute GVHD. We have recently identified difference may be due to a center effect that and validated laboratory regenerating islet- depends on several factors, including varia- derived 3-alpha (REG3α), a C-type lectin tions in transplant conditioning regimens and secreted by Paneth cells,9,10 as a non-invasive, supportive care. For example, all patients in reliable blood biomarker specific for GVHD Regensburg and Kyushu received oral antibi- of the GI tract with diagnostic and prognostic otics as GVHD prophylaxis, whereas utility.11 Michigan patients did not, and thus increased Hematology Education: the education program for the annual congress of the European Hematology Association | 2013; 7(1) | 347 | 18th Congress of the European Hematology Association GI flora might account for greater REG3α secretion. number or pathological grade correlated better with clini- We next analyzed REG3α concentrations according to cal GVHD severity. Proportional odds logistical regres- diagnosis and type of GI symptom in all 1000 patients. In sion analysis demonstrated that Paneth cell numbers α patients with diarrhea caused by GVHD, REG3 concen- retained a strong correlation with GVHDP severity after trations at the onset of GVHD were 5 times higher than in accounting for pathological grade ( =0.008), but patho- α patients with diarrhea from other causes. REG3 concen- logical grade did not correlate with GVHDP severity after trations at the onset of symptoms continued to distinguish Paneth cell numbers were accounted for ( =0.27). Thus between GVHD and non-GVHD etiologies in patients Paneth cell number in the duodenum correlates better with with both small and large volumes of diarrhea. clinical GI GVHD severity than histological grade. The clinical utility of any biomarker increases if it pro- Patients with a complete response (CR) to therapy also vides prognostic information regarding the future status of had significantly more Paneth cells than patients with a a disease and/or patient, e.g. the likelihood of response to partial response (PR); patients with no response (NR) had treatment. We, therefore, evaluated the prognostic signifi- the fewest Paneth cells corresponding to a highly signifi- α cance of REG3 plasma levels in more than 160 patients cantP difference among the three treatment responses taken at the time of diagnosis of lower GI GVHD. Four ( <0.0001) (Figure 2). The number of Paneth cells also weeks is a common time point at which to evaluate stronglydata not correlatedshown with maximum GI GVHD severity response.14 REG3α concentrations were 3-fold lower at ( ). We found that patients whose duodenal biopsies con- the timeversus of GVHD diagnosis in patients who did not respond those in patients who experienced a com- tained a mean of 4 or fewer Paneth cells at the onset of vs. GVHD were more than twice as likely to die from GVHD- plete or partial responseP to therapy (635±132 ng/mL 240±61 ng/mL; <0.001). Patients responding to therapy related causes as comparedvs. Pto patients with 5 or more α Paneth cells (55% 23%; <0.0001). This large differ- still exhibited REG3 concentrations more thanP 3 times that of non-GVHD controls (77±22 ng/mL; <0.001). ence in GVHD-related mortality translated into a signifi- Because the response to treatment at four weeks strongly cant difference in overall vs.survival atP six months from the correlates with non-relapse mortality (NRM), we hypoth- onset of GI GVHD (16% 42%, =0.01). esized that the REG3α concentration at GVHD diagnosis The current GI GVHD pathological grading system has would also correlate with NRM. We, therefore, divided the major limitations. First, the correlation between severity patients into two equal groups based upon the median of colonic changes and clinical GVHD severity is poor REG3α concentration. NRM was twice as high in patients and so many pathologists do not report pathological grade with high REG3α concentrations, and this difference in order to avoid confusion by clinicians. Second, the remained significant after adjusting for known risk factors grading system has not been standardized. Third, there is of donor type, degree of HLA match, conditioning intensi- no definition as to how to resolve the variable severity within biopsy sections from the same patient. These find- ty, age,vs. and baseline disease severity:P 59% (95%CI: 4%- 69%) 34% (95%CI: 2%-46%); <0.001). ings demonstrate that a straightforward quantification of Data regarding the clinical stage, histological grade, Paneth cell number can help establish the diagnosis of GI REG3α concentration, and level of GVHD at onset were GVHD and has prognostic importance. Recent research supports the correlation between Paneth available in 140vs patients. The plasma concentration of REG3α (above . below the median), the clinical severity cells and GI GVHD, and our observations provide insights vs into physiological mechanisms and suggest Paneth cells of GVHD (stage 1vs . stage 2-4), and the histological α severity (stage 1-3 . stage 4) at the time of GVHD diag- secrete α-defensins and REG3 , which are antimicrobial nosis independently predicted both lack of response to peptides. These peptides function to regulate the microbio- GVHD therapy and 1-year NRM after adjustment for the ta of the intestine through selective activity against non- commensal bacteria, while generally sparing commensal aforementioned risk factors. When lack of response to therapy and NRM were modeled simultaneously on all four parameters, all the three parameters remained statisti- cally significant. The inclusion of all three characteristics that remained statistically significant on simultaneous modeling demonstrated that patients with increasing num- bers of risk factors present at onset had increasing risk for NRM. Paneth cells are major producers of REG3α. Given the importance of these biomarkers, we evaluated 118 patients who had duodenal biopsies obtained at the time of GVHD- related diarrhea, and 15 patients who had minimal or no GI symptoms but had small duodenal biopsies available. We quantitated the number of Paneth cells per high pow- ered field (HPF) and found a very strong statistical corre- lation between the number of Paneth cells per HPF and the clinicalP severity of GI GVHD at the time of biopsy ( <0.0001) (Figure 1).