and Graft-versus-Host Disease

Antonio Velasco Guardado,1 Lucı´aLopez-Corral,2 JoseA.Perez-Simon,2 Teresa Caballero-Velazquez,2 Teresa Flores Corral,3 Dolores Caballero Barrigon,2 Antonio Rodrı´guez Perez1

The gastrointestinal (GI) tract is the main target site of graft-versus-host disease (GVHD). Diagnosis is based on endoscopic and histological findings. Helicobacter pylori (HP) is a Gram-negative spiral bacterium linked to , , gastric mucosa–associated lymphoid tissue lymphoma, and adenocarcinoma and is frequently observed on in patients who have undergone transplantation. The role, if any, played by HP infection in the development of acute GVHD is unknown. We conducted a retrospective study between January 1, 1990, and December 31, 2008, of 338 upper GI (gastroscopies) performed on patients who underwent allogeneic stem cell transplantation with clinical suspicion of GVHD (198 patients). Acute and chronic GVHD were confirmed from histological features in 97 patients (51.3%) and 68 patients (36%), respectively. HP infection was detected in 69 patients (35%) and had a negative modulating effect on the development of acute GVHD (relative risk [RR], 0.60; 95% confidence interval, 0.46-0.79; P 5 .001) and chronic GVHD (RR, 0.75; 95% confidence interval, 0.61-0.92; P 5.016). Furthermore, the presence of HP was inversely correlated with the histological severity of GVHD (P 5.003). Our findings suggest that infection with HP may have a negative modulating effect on GVHD. Biol Blood Marrow Transplant 17: 765-769 (2011) Ó 2011 American Society for Blood and Marrow Transplantation KEY WORDS: Transplantation, Endoscopy, Bacterial colonization

INTRODUCTION stricture or concentric rings, and pancreatic exocrine insufficiency [3]. Allogeneic stem cell transplantation is the best Due to the frequency of GI symptoms during the first therapeutic option for many patients with hematologic 100 days after hematopoietic stem cell transplantation malignancies, although it is associated with significant (HSCT), more than 50% of patients are referred for an morbidity and mortality, due mostly to graft-versus- endoscopic evaluation of the enteric tract. Differential host disease (GVHD) [1]. Acute gastrointestinal (GI) diagnosis of GVHD and other conditions, such as toxic- GVHD presents as nausea, anorexia, vomiting, diar- ity from chemotherapy or radiotherapy, drug-related rhea, abdominal pain, hemorrhage, and ileus, and adverse effects, and bacterial, viral, parasitic, or fungal . carries a 80% risk of mortality in severe forms [2]. , should be performed [4,5]. Endoscopic The GI tract is also a target organ for chronic GVHD, evidence of GVHD in the and may clinical manifestations of which include anorexia, range from normal mucosa to nonspecific signs such nausea, vomiting, diarrhea, weight loss, esophageal as edema, erythema, frank ulcers, and erosion of the mucosa [6]. Helicobacter pylori (HP) is a Gram-negative bacillus that has been linked to several digestive disorders, in- From the 1Department of Gastroenterology; 2Hematology; and cluding gastric cancer and mucosa-associated lym- 3 Pathology, University Hospital of Salamanca, Salamanca, phoid tissue lymphoma. HP infection has coexisted Spain. Financial disclosure: See Acknowledgments on page 769. with the human species for more than 50,000 years Correspondence and reprint requests: Antonio Velasco Guardado, [7,8]. The infection is acquired during childhood and, Department of Gastroenterology, University Hospital of Sala- if untreated, remains present in the body throughout manca, Paseo San Vicente 50-180, 37007 Salamanca, Spain the lifespan, although it causes symptoms in only (e-mail: [email protected]). a subset of patients [9,10]. The prevalence of HP Received September 2, 2010; accepted November 10, 2010 Ó 2011 American Society for Blood and Marrow Transplantation infection is related to age, socioeconomic class, and 1083-8791/$36.00 country of origin, varying between 20% and 30% in doi:10.1016/j.bbmt.2010.11.014 developed countries and between 60% and 80% in

765 766 A. Velasco Guardado et al. Biol Blood Marrow Transplant 17:765-769, 2011 some ethnic groups and developing countries [11]. proposed by Cruz-Correa et al. [16]: stage 0, normal; Humans are the primary reservoir, and several trans- stage I, loss of vascular pattern and/or focal moderate mission methods have been proposed, including erythema; stage II, edema and/or diffuse moderate oral-oral, waterborne transmission, fecal-oral, and erythema; stage III, edema, erythema, erosions, and/ gastro-oral [12]. Chronic HP infection can produce en- or bleeding; stage IV, ulceration, exudates, and/or doscopic findings similar to those seen in stage I and II bleeding. GVHD, and histological analysis may reveal apoptotic In the histological analysis, the presence of apopto- bodies [13,14]. Mucosal and histological tic bodies was considered ‘‘compatible with GVHD.’’ examination of specimens for the presence of HP is The histological findings were staged according to the diagnostic method of choice for patients in whom the classification system proposed by McDonald and an endoscopy is clinically indicated, with a sensitivity Sale [17] in 1984: grade 0, normal; grade I, single cell of 100%, a specificity of 66%, and positive and necrosis (apoptosis) noted under medium power; grade negative predictive values of 84% and 100%, II, evidence of epithelial damage by crypt/gland ab- respectively [15]. scesses, epithelial flattening, or crypt/gland dilations; We conducted a retrospective study in patients grade III, dropout of one or more crypt/gland; grade with GI symptoms after allogeneic HSCT referred IV, total epithelial denudation. for an endoscopy to the Gastroenterology Department A diagnosis of HP infection was confirmed if the to establish the diagnosis of GVHD and assess the bacterium was detected in the biopsy specimen from presence of HP infection, as well as any association the gastric antrum. of this infection with the development and severity of In the statistical analyses, performed with SPSS GVHD. version 15.0 (SPSS, Chicago, IL), percentages were used for categorical variables, and measures of central MATERIALS AND METHODS tendency (mean and/or median) and dispersion (stan- dard deviation) were used for continuous variables. We performed a retrospective study on all of the All dichotomous variables were analyzed with c2 patients who underwent allogeneic HSCT at the Uni- contingency tests to compare the frequencies of of versity Hospital of Salamanca between January 1, categorical variables or, where appropriate, Fisher’s 1990, and December 31, 2008, and who were referred exact test and Monte Carlo simulations, and 95% to our department for an upper GI endoscopy due to confidence intervals (CIs) for odds ratios (ORs) were suspected GVHD. Patient and donor characteristics derived. Continuous and categorical variables were are summarized in Table 1. The graft sources were analyzed by analysis of variance. Multivariate logistic not manipulated, and there were no mismatched do- regression was used to analyze confounding factors. nors. All patients received bacterial prophylaxis with Statistical significance was set at P \ .05. meropenem during the period of neutropenia, and omeprazole was administered until engraftment and RESULTS achievement of adequate oral intake. The endoscopic findings from these explorations, histological findings Baseline characteristics of the study group are of the (a minimum of 3 biopsies) performed summarized in Tables 1 and 2. HP infection was during the procedure, and the presence of HP were detected in 69 patients (35%). The incidence of HP retrospectively compiled. One pathologist involved was significantly lower in patients with acute GVHD in allogeneic HSCT for 15 years reviewed all of the than in those without acute GVHD (15.5% vs 38%; samples. Standard hematoxylin and eosin was the stain relative risk [RR], 0.58; 95% CI, 0.44-0.76; P \. 001) used in all cases. The presence of apoptotic bodies in (Figure 1). The association was confirmed by logistic the biopsy specimen (minimum criterion), crypt ab- regression adjusted for sex, age, donor type, progenitor scesses, and/or destruction of glands were considered cell source, sex mismatch, and type of conditioning criteria for a diagnosis of acute or chronic GVHD, af- regimen (OR, 2.87; 95% CI, 1.33-6.16; P 5 .007), as ter ruling out other causes that also could induce these well as for GVHD prophylaxis (OR, 2.64; 95% CI, findings, such as drug-related toxicity or infections by 1.25-5.55; P 5 .010). blood and/or stool cultures, cytomegalovirus infection Similarly, the incidence of HP was lower in pa- by antigenemia assay, or a reduced dose of the suspect tients with histologically confirmed chronic GVHD drug. than in those without it (16.2% vs 32.2%; RR, 0.75; Twenty-six patients required two endoscopies to 95% CI, 0.61-0.92; P 5 .016) (Figure 2). These results confirm or rule out acute GVHD in the first 100 were confirmed using logistic regression adjusted by days post-HSCT. For these patients, we included the sex, age, donor type, progenitor cell source, sex mis- endoscopy in which the endoscopic and histological match, and type of conditioning regimen (OR, 2.59; features were of a higher grade. Endoscopic findings 95% CI, 1.12-5.96; P 5 .025), but not when adjusted were staged according to the classification system by GVHD prophylaxis. Biol Blood Marrow Transplant 17:765-769, 2011 H pylori Infection and GVHD 767

Table 1. Baseline Characteristics of the Study Group Number of patients 197 Sex, n (%) Men 114 (57.9) Women 83 (42.1) Age, years, mean (SD) 45.3 (14.0) Type of donor, n (%) Related 130 (69.5) Unrelated 51 (27.3) Others 6 (3.2) Source of progenitor cells, n (%) Peripheral blood 155 (83.3) Bone marrow 31 (16.7) Type of conditioning, n (%) Reduced intensity 118 (61.5) Myeloablative 74 (38.5) ATG 5 (2.5) Campath 2 (1) TBI (myeloablative) 17(23) Donor–recipient sex mismatch, n (%) 72 (42.4) GVHD prophylaxis, n (%) CSA/tacrolimus + MTX 142 (80.2) CSA/tacrolimus + MMF 22 (12.4) Others 13 (7.4) Endoscopic studies, n 379 Gastroscopy, n (%) 338 (89.2) First 100 days, n (%) 223 (66.0) Days after transplantation, mean (SD) 41 (24.0) Figure 1. Incidence of HP infection among patients with and without After 100 days, n (%) 115 (34.0) acute GVHD. Days after transplantation, mean (SD) 269 (210.0) ATG indicates antithymocyte globulin; CSA, cyclosporine; MMF, myco- Furthermore, HP infection was associated with phenolate mofetil; MTX, methotrexate; SD, standard deviation; TBI, significantly lower histological severity (11.1% severe total body irradiation. forms in HP-positive cases vs 88.9% in HP-negative cases; P 5 .003), as indicated by a negative Pearson cor- Interestingly, in the patients without acute relation coefficient (r 520.26; P \ .001) (Table 3). In GVHD, the detection of HP in the biopsy specimens contrast, we found no significant correlation with endo- obtained within the first 100 days post-HSCT was scopic severity according to the classification system of related to a significantly lower risk for the subsequent Cruz-Correa et al. [16]. development of chronic GVHD. In this regard, we The overall incidence rate of GVHD was 92.4% found that 21.7% of the patients with HP infection de- (n 5 182), and HP infection did not affect this rate veloped chronic GVHD, compared with 52.2% of HP-negative patients (RR, 0.73; 95% CI, 0.58-0.93; P 5 .011). Multivariate logistic regression analysis confirmed this association when adjusted by sex, age, donor type, progenitor cell source, and type of condi- tioning regimen (OR, 3.87; 95% CI, 1.25-11.94; P 5 .018) and by GVHD prophylaxis (OR, 3.45; 95% CI, 1.10-10.83; P 5 .034).

Table 2. Frequency and Severity of GVHD Overall GVHD, n (%) 182 (92.4) Acute GVHD, n (%) 137 (75.2) GI GVHD 97 (70.8) Grade I 23 (23.7) Grade II 56 (57.7) Grade III 15 (15.4) Grade IV 3 (3.1) Cutaneous GVHD 52 (38) GVHD 30 (21.9) Chronic GVHD, n (%) 120 (65.9) GI GVHD 68 (56.6) Mild 20 (29.4) Moderate 36 (53) Severe 12 (17.6) Cutaneous GVHD 60 (50.0) Hepatic GVHD 37 (30.8) Figure 2. Incidence of HP infection among patients with and without chronic GVHD. 768 A. Velasco Guardado et al. Biol Blood Marrow Transplant 17:765-769, 2011

Table 3. Relationship between Histological Grade of GI GVHD According to the McDonald and Sales Classification System [17] and HP Infection

Not Compatible Grade I Grade II Grade III Grade IV

HP-positive, n (% of histological grade) 16 (41.0) 26 (31.0) 4 (9.1) 2 (11.1) 0 HP-negative, n (% of histological grade) 23 (59.0) 58 (69.0) 40 (90.9) 16 (88.9) 0

(P 5 .14). The median follow-up duration was 64.5 saliva) and the development of GVHD in 46 patients months (range, 31.2-108.7 months). A total of 47 who underwent allogeneic HSCT. The second, by patients (23.8%) have died due to nonrelapse mortal- Au et al. [24], examined the association between HP ity, with a GVHD-related mortality rate of 40.4% infection before transplantation and the subsequent (n 5 19). The rate of response to immunosuppressive development of mucositis (drug-related toxicity) and therapy was 77.7% (n 5 153). acute GVHD in 128 patients. No increased risk of mu- cositis was found in patients with HP colonization; however, these patients exhibited a significant reduc- DISCUSSION tion in the risk of developing acute GI GVHD (RR, 0.36; 95% CI, 0.13-0.98; P 5 .04). On multivariate HP stimulates the release of a variety of inflamma- analysis, the absence of HP infection and the presence tory mediators, both directly by bacterial products of mucositis remained independent predictive factors and indirectly as a result of interaction with gastric ep- of the development of GI GVHD. ithelial cells. These factors allow HP to thrive in In the present study, we found that HP may protect a niche that is inhospitable to almost every other en- against the development of GVHD, and this effect teric organism [18]. Recent epidemiologic studies remains when we consider patients who have not sus- have suggested that colonization with HP might tained acute GVHD. Accordingly, HP-positive pa- protect against the development of certain autoim- tients had a lower risk of acute and chronic GVHD. mune disorders, such as asthma [19,20].Anegative It could be argued that gastroscopy was performed association between inflammatory bowel disease only in patients with GI symptoms, so that in the ab- (IBD) and HP infection has been established in sence of acute GVHD, we might have selected patients numerous studies. A recent meta-analysis of 23 stud- with other concomitant comorbidities, such as HP in- ies with a total of 5903 patients evaluated the relation- fection, which could account for these symptoms. ship between HP infection and IBD [21]. Thirteen of Nevertheless, when we specifically analyzed patients the studies noted a significant RR (\1), and no study without acute GVHD, the detection of HP on endos- found an RR of .1. Some 27% of the patients with copy performed during the first 100 days post-HSCT IBD carried an HP infection, compared with an HP significantly decreased the risk of subsequent chronic infection rate of 40.9% in the control group. The GVHD. These results are consistent with the negative authors concluded that these results suggest that HP correlation observed between HP infection and histo- infection might have a beneficial effect on the devel- logical severity. Type of conditioning and donor, pro- opment of IBD. Further studies are needed to confirm genitor cell source, and type of GVHD prophylaxis this conclusion. were not confounding variables in the association On the other hand, recent laboratory tests have as- between GVHD and HP. cribed a certain modulating capacity of the immune re- This study has some limitations, including its ret- sponse to HP, with an increased number of regulatory rospective nature and the fact that HP infection was di- T cells of the gastric mucosa that might modify the im- agnosed after the onset of symptoms. The standard mune response and prevent a Th1/Th17 response hematoxylin and eosin stain is excellent for identifying [13,14]. Chen and Blaser [22] suggested that the inten- histologically chronic or chronic active inflammation sity of the host response against especially aggressive and demonstrates HP when large numbers of HP strands (with the cagA gene) might alter the pattern are present. Silver staining is better at detecting the of the Th1/Th2 immune response, with the subse- organism when few bacteria are present, but does not quent induction of immunoregulating lymphocytes optimally demonstrate tissue histology. The best ap- that might prevent immune hyperreactivity responses, proach is a combination of hematoxylin and eosin such as asthma or allergy, in cases where the Th2 and Diff-Quick (Newark, Delaware) stain; thus, we pattern is predominant. likely underestimated the incidence of HP [25]. Two prospective studies of the relationship be- To the best of our knowledge, this is the first study tween GVHD and HP have been published to date. to suggest that HP infection might have a protective The first, by Correia-Silva et al. [23], found no effect against acute and chronic GVHD. Further relationship between HP infection (established on prospective studies to evaluate the presence of HP the basis of polymerase chain reaction analysis of before transplantation ( breath test) and the Biol Blood Marrow Transplant 17:765-769, 2011 H pylori Infection and GVHD 769 relationship between CagA strands with the subse- 11. Go MF. Review: natural history and epidemiology of Helicobacter quent development of GVHD are merited. pylori infection. Aliment Pharmacol Ther. 2002;16(Suppl 1):3-15. 12. Graham DY, Sung J. Helicobacter pylori. In: Feldman M, Friedman LS, Brandt LJ, editors. Sleisenger & Fordtran’s Gastroin- testinal and Liver Disease: Pathophysiology, Diagnosis, and Manage- ment. 8th ed, Vol. 1. Philadelphia: Elsevier; 2006, p. 1049-1066. ACKNOWLEDGMENTS 13. Jones NL, Shannon PT, Cutz E, et al. 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