Allogeneic hematopoietic stem cell transplantation (HCT) is a potentially curative treatment for malignant hematologic diseases and non-malignant diseases such as inborn errors of immunity (1). The graft-versus-leukemia (GVL) effect mediated by alloreactive donor T cells allows the treatment of hematologic malignancies with HCT. However, although natural killer cells only exert GVL effects, alloreactive donor T cells can also cause graft-versus-host disease (GVHD) (2). Despite advances in human leukocyte antigen typing and the use of immunosuppressants after transplantation, acute GVHD remains a leading cause of mortality and morbidity after allogeneic HCT (3).

After the skin, the gastrointestinal tract is the second most commonly affected organ, accounting for approximately 50% of all GVHD cases (4, 5). Approximately 25% of pediatric patients develop acute gastrointestinal GVHD after HCT (6). Gastrointestinal GVHD is commonly classified into upper and lower gastrointestinal GVHD based on clinical symptoms, and these two forms of GVHD have different prognoses. Upper gastrointestinal GVHD is associated with a better response to treatment with systemic steroids, and has a lower impact on overall survival (OS) and non-relapse mortality (NRM) than does lower gastrointestinal GVHD (7). The lower gastrointestinal tract is the most important organ in determining treatment response and outcome (8, 9). Among patients with lower gastrointestinal GVHD, those with clinical grades 3–4 have a higher risk of mortality than those with clinical grades 1–2 (4). Although the risk of severe gastrointestinal GVHD is known to be relatively lower in children than in adults, severe gastrointestinal GVHD has a 2-year mortality rate of 45% in pediatric patients treated with HCT (4).

Gastrointestinal GVHD is difficult to diagnose based on clinical manifestations alone due to non-specific symptoms and confounding factors such as chemoradiation toxicity, adverse drug reactions, and enteric infections (10, 11). Therefore, endoscopic and histologic findings play an important role in the diagnosis of gastrointestinal GVHD (12). However, previous studies have not clearly elucidated which of the clinical, endoscopic, and histologic grades correlates most strongly with the prognosis of GVHD. In addition, because endoscopy is an invasive procedure, it is not routinely performed in children, resulting in few studies reporting on endoscopic and histologic findings in pediatric patients. Therefore, in this study, we aimed to evaluate the outcomes according to the clinical, endoscopic, and histologic grades of gastrointestinal GVHD in pediatric patients treated with allogeneic HCT.

Although gastrointestinal GVHD is diagnosed on the basis of clinical manifestations, endoscopic and histologic examinations are required to exclude other conditions with similar symptoms (19, 20). Except for the purpose of diagnosis, the use of endoscopic and histologic grades of gastrointestinal GVHD is still controversial. In the current study, we demonstrated that the histologic severity of gastrointestinal GVHD was associated with both OS and NRM in pediatric patients treated with HCT. We also found that patients with mild-to-moderate histologic gastrointestinal GVHD, in addition to those with severe histologic gastrointestinal GVHD, had worse outcomes than patients without histologic gastrointestinal GVHD.

Endoscopic examinations can be difficult to perform in patients with a poor systemic status after HCT, especially in pediatric patients. Although the patient outcomes according to the clinical grade of gastrointestinal GVHD are well known, studies on prognostic differences according to endoscopic and histologic grades have been scarce, even in adult patients (21–26). In addition, although some studies have reported patient outcomes according to histologic grades, they mainly focused on grades 3–4 (4) and studies including all histologic grades are relatively rare. To our knowledge, this is the first study to address the significance of the histologic grades of gastrointestinal GVHD in predicting outcomes in pediatric patients treated with HCT.

The lower gastrointestinal tract is the organ most closely associated with mortality in patients with gastrointestinal GVHD (4, 27, 28). Although no standard method for the diagnosis of gastrointestinal GVHD has been established, the MAGIC guidelines identify histologic validation as the most definitive diagnostic test for patients with suspected gastrointestinal GVHD (5). Because the histologic findings of acute gastrointestinal GVHD can overlap with those of other disease entities, including infection and chemoradiation toxicity, the diagnosis is based on subtle histopathologic criteria.

Many investigators have attempted to develop histologic scoring systems for GVHD. Lerner et al., Melson et al., and Sale et al. proposed histologic criteria that have been widely accepted and used by clinicians and pathologists (18, 23, 29). The method that is most consistent with clinical data regarding diagnostic accuracy, treatment response, and outcomes is known as the Lerner method, which assigns four grades according to the degree of epithelial cell damage (30). In this scoring system for histologic severity, cases in which only the apoptotic bodies are visible are classified as grade 1, and cases with other additional histologic findings related to epithelial damage are systematically classified as grades 2–4. Because mild epithelial apoptosis can also be seen in other conditions such as inflammation, infection, and drug reactions (e.g., mycophenolate mofetil), the absent and mild-to-moderate categories of histologic gastrointestinal GVHD (grades 0–2) have received less attention than the severe category, and relatively few attempts have been made to analyze their association with patient outcomes.

False-positive results due to conditioning regimens have been reported to typically occur within 20 days of myeloablation (31). In this study, the median duration of endoscopy after HCT was 52 days, with no significant difference between groups based on histologic severity (p = 0.082). Furthermore, the proportions of patients receiving the conditioning regimens were not statistically different when the groups were stratified by histologic severity (p = 0.180). Therefore, the probability of a false-positive for gastrointestinal GVHD due to conditioning therapy in this study is considered low.

The histologic grades of gastrointestinal GVHD correlate with the clinical grades (23, 24, 32). Melson et al. reported that gastrointestinal crypt loss was highly correlated with the clinical grade of gastrointestinal GVHD based on the amount of diarrhea (23). In addition, Narkhede et al. found that the Spearman’s correlation coefficient between the histologic and clinical grades of gastrointestinal GVHD was 0.6, indicating a moderate association between the two components (24). Our study also revealed a strong correlation (ρ = 0.7, p = 0.0052) between the clinical and histologic grades of lower gastrointestinal GVHD, which is consistent with the results of previous studies. In addition, when the histologic grades were divided into severity categories, early onset of gastrointestinal GVHD symptoms, hematochezia, and severe clinical gastrointestinal GVHD were significantly more prevalent in patients with severe histologic gastrointestinal GVHD than in those with absent or mild-to-moderate histologic gastrointestinal GVHD. However, there was no additional association between the histologic severity of gastrointestinal GVHD and the prevalence of GVHD in other organs or overall GVHD grade.

The correlation between the histologic and endoscopic grades is controversial. Cruz-Correa et al. found a positive association between the endoscopic and histologic grades of gastrointestinal GVHD (odds ratio 12.0, 95% CI 3.9–37.2) (16). Meanwhile, Cheung et al. reported that endoscopic evaluation had low sensitivity and specificity for the diagnosis of gastrointestinal GVHD (33). In our study, the correlation between clinical and endoscopic grades was low (ρ = 0.3, p = 0.048) and that between endoscopic and histologic grades was moderate (ρ = 0.5, p = 0.001).

Several studies have shown that early onset of acute gastrointestinal GVHD, severe clinical gastrointestinal GVHD, hematochezia, mismatched donor type, and low albumin and high serum bilirubin levels are risk factors for mortality in patients with gastrointestinal GVHD (4, 34–36). Furthermore, in a study of 23 adult patients with gastrointestinal GVHD, a severe histologic grade was highly correlated with clinical grades 2–4, and patients with severe histologic gastrointestinal GVHD had a statistically significantly higher mortality rate than those with absent or mild-to-moderate histologic gastrointestinal GVHD (23). In addition, a study of 231 adult patients with GVHD confirmed that histologic grade was an important factor in mortality; however, this study included both skin and gastrointestinal GVHD cases (24).

In this study, severe clinical and histologic grades were associated with mortality, which is also consistent with the findings in adult studies (Table 3). Therefore, histologic findings are not only important clues for the diagnosis of gastrointestinal GVHD but also factors related to outcomes. As shown in Figure 2, histologic grade 3—4 is strongly associated with clinical grade 3–4, but lower histologic grades are distributed without significant association with clinical grade severity. Histologic grade was lower than the clinical grade in 68.6% of the patients, and patients with absent or mild-to-moderate histologic GVHD accounted for 78.5% of the study population. Therefore, low histologic grade does not equate to “mild” GVHD and it can be present even in patients with advanced clinical grade. Since the clinical and histologic severity of GVHD were found to influence the outcomes in the current study, we evaluated the cumulative incidence of NRM by combining these two factors (Figure 5). We observed that even among patients with the same clinical severity of gastrointestinal GVHD, the outcome was worse in those with higher histologic grades. In addition, patients with mild-to-moderate histologic gastrointestinal GVHD also had worse outcomes than those without histologic GVHD. This finding highlights that not only severe histologic grade but also mild-to-moderate histologic grade is a risk factor for worse outcomes (grade 1–2 vs. 0: HR 1.67, 95% CI 1.07–3.21, p = 0.0315). Therefore, histologic evaluation should be performed in addition to endoscopic evaluation in patients suspected of having acute gastrointestinal GVHD, not only for diagnostic purpose but also for prognostic evaluation.

The current grading system of histologic gastrointestinal GVHD reflect only short-term ongoing damage, and it is not immunologically proven why mild-to-moderate histologic GVHD is associated with worse outcomes. Recently, attempts have been made to overcome this limitation and to elucidate the relationship between the lower grades of the Lerner’s grading system and prognosis. Myerson et al. categorized low-level Lerner grade I into four activity grade categories based on the average frequency of apoptotic bodies (apoptotic index), which associated with therapeutic intervention (37). In the future, histologic grading of acute gastrointestinal GVHD, including immunologic features, should be investigated not only for the diagnosis of GVHD, but also for guiding treatment decisions and predicting outcomes.

The present study had several limitations. First, this was a retrospective study with certain disadvantages compared with prospective studies. However, all patients were hospitalized or attended the outpatient clinic at regular intervals during study period, allowing for consistent clinical evaluation and monitoring of acute gastrointestinal GVHD. Second, selection bias may have occurred by excluding patients with inadequate endoscopic and histologic evaluation. Most patients who were not included in this study were patients with mild gastrointestinal GVHD who responded well to glucocorticoids, the first-line therapy for GVHD, and were not candidates for endoscopy based on risk–benefit considerations at the time of treatment. Well-designed prospective studies of gastrointestinal GVHD in pediatric patients are needed to address the limitations of this study.

In conclusion, higher clinical and histologic grades of gastrointestinal GVHD are risk factors for NRM in pediatric patients treated with HCT. Furthermore, the histologic severity of gastrointestinal GVHD is a relevant factor affecting OS and NRM in these patients, with patients with mild-to-moderate or severe histologic gastrointestinal GVHD having worse outcomes than patients without histologic GVHD. These findings support the importance of assessing the histologic grade in the diagnostic evaluation of patients with clinical gastrointestinal GVHD. In addition, our results emphasize the prognostic value of the histologic grade of gastrointestinal GVHD.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving human participants were reviewed and approved by Institutional Review Board of Samsung Medical Center (file no. 2021–12-129). Written informed consent from the participants’ legal guardian/next of kin was not required to participate in this study in accordance with the national legislation and the institutional requirements.

ESK, MJK, and KHY contributed to the conception and design of the study, interpreted the data, drafted the original manuscript, and revised the manuscript critically for important intellectual content. ESK and YK supported the data acquisition. ESK analyzed the data. All authors approved the final version to be published.

This study was supported by a National Research Foundation of Korea (NRF) grant funded by the Ministry of Science and ICT (MSIT) of Korea (no. 2022R1F1A1073691).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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