Prevalence of Chronic Oral GVHD in a Cohort of Egyptian Pediatric Patients Following Allogenic Hemopoietic Stem Cell Transplantation

Introduction

  Hemopoietic stem cells transplantation (HSCT) refers to procedures by which haematopoietic stem cells of any source and from any donor type are donated to a recipient in order to help in repopulating and changing the haematopoietic system totally or partially . Bone marrow (BM), peripheral blood (PB), or cord blood (CB) are representing the sources of stem cells for HSCT Sureda et al. (2015).  HSCT is presently considered the standard management procedure for many hematological disorders Juric et al. (2016)

 According to data from the latest analysis of  the Center of International Blood and Marrow Transplant Research (CIBMTR), the estimated annual number of allogeneic transplant recipients  exceeded almost  8,000 cases per  year in the US in 2013 with 9,498 transplants estimated in 2019 Phelan et al. (2020). Whereas, in Egypt, for a period of 30 years of experience (from 1997 to 2017), 4256 transplants  were conducted  60% of which were allogenic Mahmoud et al. (2020).

In children and adolescents, allogeneic HSCT represents about 20% of all allogeneic HSCT practice , especially in rare and congenital diseases Sureda et al. (2015).

In 2020, the population of Egypt surpassed 100 million. There are fifteen transplant centers, where the transplant rate is 8.4 per million Mahmoud et al. (2020), this rate is considered higher than the rate previously reported in 2008, which was 2.8 per million  Mahmoud et al. (2008).  

Following allogenic HSCT, chronic graft-versus-host disease cGVHD is considered the most eminent non-relapse reason of morbidity and mortality (Grube et al.2016).  After allogenic HSCT, the incidence of cGVHD is almost 50% among all adult and pediatric patients who  received allogenic bone marrow transplant to manage acute leukemia, chronic leukemia and myelodysplastic syndrome  Arai et al. (2015).

Chronic GVHD persists for months or years. Eyes, oral cavity, GIT, liver, genitourinary tract, lungs and joints are the common sites of affection Schubert& Correa. (2008) and Fassil et al. (2012). Oral cGVHD  causes comorbidity manifested as pain and discomfort affecting general oral health status , functional abilities , and quality of  life in the patients Meier et al. (2011); Fassil et al. (2012) and Bassim et al. (2014).

 The clinical presentation of oral cGVHD  is variable. Oral manifestations occur at any site in the oral tissues. Oral mucosal lesions are present as erythema, ulcers, lichenoid lesions , and mucoceles in addition to xerostomia and sclerosis  Filipovich et al. (2005) and Pavletic et al. (2006). Oral affection affects quality of life of the patients as explained before . Therefore, the present study aims to detect the prevalence and predict risk factors for developing oral cGVHD after bone marrow transplant in a sample of Egyptian children.

Subjects and Methods

This is a cross - sectional study that  was conducted at Nasser Institute for Research and Treatment for pediatric patients undergoing allogeneic HSCT starting from June 2019 to December 2019. ClinicalTrials.gov Identifier: NCT03880214

1- Participants:

Children aged between 3 and 18 years old  who received  bone marrow transplantation at least 3 months  prior to conduction of the study , participants diagnosed with a neoplastic or non- neoplastic diseases can be included. The participants in the sample received cyclosporine (sandimmune) as immunomodulatory for prevention and treatment of GVHD, while patients (or their guardians) who do not approve to join the study or children receiving chemo or radiotherapy are excluded .

1.1 Ethical considerations:

This research had been approved by the ethical committee of the Faculty of Dentistry, Cairo University. Approval No. (11 7 18). Guardians of the children that met the inclusion criteria signed a consent form in Arabic approving their children to participate in the study. All the data needed were discussed.

2- Data collection

2.1 Study variables

-      Exposure: Exposure to hemopoietic stem cells transplantation.

-      Outcomes: Oral mucosal alteration, Salivary glands function and Maximal mouth opening.

-      Risk factors: Age, Gender, Type of stem cell, Previous acute attack of GVHD, conditioning regimen dose of CD34 and other organ involvement.

The data collected through:  

2.2 A standardized Transplant Database Questionnaire as mentioned in Hull et al. (2011)

2.3  Clinical examination:

2.3.1 Comprehensive intraoral clinical examination for the soft tissue under good lightening condition to detect any abnormalities. The oral lesions were recorded as oral mucosal alterations as listed by Treister et al. (2005) in the form of presence of variant oral lichen planus and oral lichenoid reactions, ulcers, erythema  or mucocele.

  2.3.2 Salivary glands function:

-      Assessed by measuring the resting salivary flow rate Walter et al. (2001), saliva collection was done while the patients were sitting down; their heads were tilted forward, they were instructed not to swallow or move their  tongues or lips, and their eyes were kept open. Any preexisting saliva was swallowed before the collection.

-      At the end of the collection time, Saliva was spitted in a container, then the volume was measured   and finally salivary flow rate was determined (in milliliters per minute). Children should be closely monitored to prevent them from swallowing and to prevent any  sample loss throughout the collection period. If this happened, the procedure is repeated at least 30 minutes after the first attempt.

-      We classified the examined children into normal and diminished unstimulated salivary flow rate according to findings by  by  Forcella et al. who reported that  unstimulated  salivary flow rate in healthy children ranged from (0.49, 1.05) ml/min  (Forcella et al. 2018)

-       

2.3.3 Maximal mouth opening estimation:

 The distance between upper and lower teeth was measured in millimeters from the incisal edge of the most vertically aligned maxillary central incisor to the labio-incisal edge of the opposing mandibular incisor using a caliber. The measurements taken were used to classify  subjects according to their degree of opening and their ages into ( normal or restricted ) according to the measurements reported in Müller et al. , they  plotted the average maximum mouth opening versus the age    (Müller et al. 2011)

3- Data management:

All  information obtained from the participants was stored in locked files in a limited access area. All data collection, process, and administrative forms were identified by a coded ID (Identification Number).

4- Sources of Bias:

-Selection bias was  minimized by enrolling the eligible participants in the study in a consecutive order of hospital visits. Concerning non-respondent bias, explaining the aim of the study and the importance to the participants helps to decrease it. For incomplete records, statistical analysis included them with reporting the cause of not completing the record.

5- Statistical methods:

Data were coded and entered using the statistical package for the Social Sciences (SPSS) version 26 (IBM Corp., Armonk, NY, USA).  Data were presented using mean, standard deviation, median, minimum and maximum in quantitative data and using frequency (count) and relative frequency (percentage) for categorical data. To compare  between quantitative variables the non-parametric Mann-Whitney test was used. To compare categorical data, Chi square (χ2) test was used. In case of presence of association, logistic regression was done to detect independent predictors of oral GVHD. P-values less than 0.05 were considered as statistically significant

Results

A total of 47 patients (31 male and 16 female) who underwent bone marrow transplantation were recruited for a 6 month duration from June 2019 to December 2019 .

1-      Demographic data and descriptive statistics for the study variables

The study participant's age distribution ranged from 3 to 17 years old with mean age of 9.38±4.2. Descriptive statistics for sex,   conditioning, exposure to acute GVHD, fate of acute GVHD, stem cells source (SC) and cGVHD were described in (Table 1).

Table (1): Descriptive statistics to study variables

RIC: Reduced Intensity Conditioning (uses less chemotherapy and radiation than standard myeloablative conditioning regimen)

MAC: myeloablative conditioning refers to (administration of total body irradiation)

PB: peripheral blood

BM: bone marrow

2-      Prevalence of chronic oral  graft versus disease

Regarding objective clinical findings, we observed the oral manifestation of GVHD (mucosal lesions, diminished salivary flow and restricted mouth opening). We found that the number of  patients with oral cGVHD (at least 1 oral manifestation)  in relation to the whole study population was 26 (55.3%), 15 patients clinically represented with chronic oral mucosal GVHD, 10 with diminished salivary flow and 22 with restricted mouth opening (Table 2). Combined oral lesions in the study group 14 patients (29.7%) have single oral manifestation, 8 patients (17.02%) have the three oral manifestations combined, while 4 patients (8.51%) have only two manifestations combined .

Table (2): Showing the distribution of site involvement in cGVHD

Regarding the site of mucosal affection, buccal mucosa was affected in 80% of patients with oral mucosal lesions. Other oral mucosal sites included tongue 4%, gingiva7% and labial mucosa 9%. Regarding the clinical presentation of the oral mucosal lesions, 9 patients had erythematous mucosa and white reticular lesion, 4 patients had ulcers and 2 patients had atrophic tongue. (Fig.1).

 Fig 1: Clinical photos of a 6 year male patient, (A) and (B) showing the lichenoid lesions (reticulation and atrophy) in the buccal mucosa bilaterally. (C) showing another male 4 years old patients with the upper and lower lips showing lichenoid changes at the junction of labial mucosa and vermillion border. While , (D) showing the limitation of mouth opening. (E) , (F) showing erythematous lesions and oral ulcers bilaterally on the buccal mucosa of 7 years female patient. (G ) is showing erythema and psudomembrane in the left buccal mucosa of 6 years old male patient. (H) showing lichenoid changes in the vermillion of 12 y male patient and areas of erythema and ulceration are appearing in left buccal mucosa and the tongue in addition to sever limitation of mouth opening ,(I) is showing the skin affection in the same patient.

3-      Relation between oral cGVHD and systemic affection

Out of 32 cGVHD patients, 17 patients (53.1%) had combined oral and systemic cGVHD,

6 patients (18.7%) had only systemic involvement, while 9 patients (28.1%) had only oral involvement (table 3). There is a statistically significant association between oral involvement and systemic involvement in patients with cGVHD where P value < 0.05 (Table 3).

We observed that 11 patients out of 26 patients with oral cGVHD (42.3%) had skin manifestations.

Table (3): Results chi- square test comparing appearance of oral cGVHD and systemic involvement

 *P-value significant at < 0.05

4-      Association of the oral manifestations of cGVHD and the study variables

No association was detected between oral manifestations and age, sex, transplant type, source of stem cells or occurrence of acute GVHD.  However, association was significant between the trio oral manifestations (mucosal ,diminished salivary flow and restricted mouth opening) where the combined lesions out of the total patients with oral cGVHD was 12 patients (46.1%)(Tables 4,5,6,7).

Table (4): Results of Mann-Whitney test for comparing appearance of oral cGVHD manifestations and the age of  the child

 *P-value significant at < 0.05

Table (5): Results of chi- square test comparing appearance of oral mucosal cGVHD and qualitative study variables

*P-value significant at < 0.05

Table (6): Results chi- square test comparing change in salivary flow rate and qualitative study variables.

*P-value significant at < 0.05

Table (7): Results chi- square test comparing change in length of mouth opening and qualitative study variables

*P-value significant at < 0.05

Discussion

The lack of studies discussing the prevalence of oral GVHD in Egypt is quite evident that to our knowledge, our study is the first study that investigates the prevalence of oral chronic GVHD in a population of Egyptian pediatric patients after allogeneic HSCT.

NIH consensus project (2005) defined the diagnostic criteria of oral cGVHD , criteria include oral mucosal cGVHD, diminished salivary flow and fibrosis of the peri-oral tissues additionally some other associated lesions for instance mucoceles, candidiasis and  pseudo membranes formation Filipovich (2005).

The study population consisted of 47 patients who underwent allogeneic HSCT 31 males and 16 females, the vast majority of these patients received peripheral blood stem cells (PBSCT) (83.0%).

The age distribution of the participants of the study ranged from 3 to 17 years old with mean age of 9.38y ±4.2y. We observed that in the literature there are few studies that tackled the oral cGVHD in children Berkowitz (1987), Dahllof et al. (1988), Dahllöf et al. (1989) ,Nicolatou-Galitis et al. (2001), Treister et al. (2005). In agreement with our finding on pediatric patients is Berkowitz (1987) who found that mean age in the study group was 9.3 (7 mo -10y). Similarly, Dahllof et al. (1988) and Dahllöf et al. (1989) found that mean age in the study group was 7y (1-12). Also, Nicolatou-Galitis et al. (2001) found that mean age in the study group was 10.7 y (1.5-15).

 Furthernore, in the present study the prevalence of oral GVHD related to sex distribution was more common in males (66.0%) than females (34.0%), which seems to be in agreement with other studies that investigated the oral cGVHD in pediatric patients establishing that male predominance was detected Berkowitz et al. (1987) Dahllof et al. (1988) Nicolatou-Galitis et al. (2001).

Our study revealed that chronic GVHD was developed by 68.08% of patients  after transplantation with 55.3% of them  having oral involvement.

This is in accordance with Hull et al. (2011). The study done at Westmead Hospital BMTU in Australia, sample consisted of 88 adults who underwent allogeneic HSCT assessing the prevalence of oral long-term complications. Chronic GVHD was detected in 72% of the subjects after transplantation with 56% of the subjects suffered from oral manifestations Hull et al. (2011).

Similarly, our results are consistent with Imanguli et al. (2006) who found that the oral cavity is affected in 70% of those who have undergone hematopoietic stem cell transplantation.

In another study, lima et al. (2012), cGVHD was observed in 32.5% (13/40) of the patients undergoing allogeneic bone marrow transplantation  and about  50%of all the patients showed oral involvement  (25/51; 49%). Some patients had manifested combined oral lesions.

In addition, a study done by Treister et al. (2005) in a pediatric cohort, for each child subjective and objective assessments of mucosal, salivary gland function, and oral sclerosis were done.  Oral mucosal cGVHD was diagnosed in 22 (45%) of 49 patients. Only 4 (8%) of 49 patients reported painful mouth. However, in contrast to our findings, subjective and objective salivary gland and sclerotic diseases were observed far less often.

In contrast to our results, Berkowitz et al. (1987) found that prevalence of oral cGVHD was (20%) 8/41 pediatric patients who underwent HSCT. Similarly,  the study conducted by Dahllof et al. (1988, 1989) showed the prevalence of oral cGVHD (20%) 9/45 pediatric patients. Also, Nicolatou-Galitis et al. (2001) detected (10%) only 9 patients with oral cGVHD out of 90 patients screened for cGVHD. These studies diagnosed the oral cGVHD based on having mucosal oral lesions and xerostomia defined either as diminished salivary flow Dahllof et al. (1988, 1989) or with clinical signs of oral dryness as in Berkowitz et al. (1987) and Nicolatou-Galitis et al. (2001). All of them did not consider the limitation of mouth opening in diagnosing the oral cGVHD explaining the difference in our results.

Diagnosis of oral signs and symptoms in children is more difficult than in adults, as clinical examinations can be challenging in some patients Treister et al. (2005). As seen in  our study  we missed the salivary flow measurement of 7 patients as they could not follow the instruction of saliva collection.   

Regarding the mucosal clinical  presentation, we seem to be in agreement with Schubert et al. (2008) who studied patients in age range of  3 to 41 years and found that the most common clinical findings were erythema, atrophy or lichenoid lesions appearing on  the buccal and labial  mucosae. Also, clinical presentations and site distribution detected in many studies Filipovich et al. (2005) and Pavletic et al. (2006) are in agreement with our findings.

In contrast to our finding in predicting risk factors, an association with transplant type have been previously proposed, where a higher degree of oral involvement (70%) seen in peripheral blood stem cell transplant relative to bone marrow transplant (53%)  Pavletic et al. (2005). In addition, considering the age as risk factor  Storb et al. (1983) and Carlens et al. (1998) found that younger patients are considered to have a decreased risk of chronic GVHD.

Concerning the dose of CD34, human hematopoietic stem cells expressing CD34 have the ability to bind  E-selectin guiding  the migration to specialized the bone marrow vascular bed  expressing the  vascular selectins , maximizing the good results after transplantation. Zaucha et al. (2001)

There is a controversy concerning whether to restrict the dose of infused CD34 cells/kg in recipients of both related and unrelated donor grafts. The effect of higher CD34 cell dose in the graft on the risk of cGVHD is debatable Zaucha et al. (2001). In adults, as some studies have revealed , there are only diverse associations Przepiorka et al.(2001) or none at all  Cao et al , (2005) and Pulsipher et al. (2009). Increasing the risk of GVHD is considered  the major concern when higher doses of CD34 cells present in the graft.  In children, consistent with our findings is Kałwakw et al, (2010) who have found absolutely no correlation between the CD34 cell dose and the incidence of severe aGVHD and cGVHD.

Regarding  statistical association between the trio oral manifestation (mucosal lesion, diminished salivary flow and restricted mouth opening) that was detected in our study, it is considered consistent with findings of  Bassim et al. (2014) and  confirming the diagnostic criteria of oral cGVHD established in 2005 at National Institutes of Health Consensus about diagnosis and staging of cGVHD Filipovich et al. (2005).

Unfortunately, the small sample size is considered a limitation in our study; therefore, further studies are recommended on a larger sample size.

Finally, The high prevalence of oral cGVHD among the children underwent bone marrow transplantation in Egypt suggests that oral medicine specialists should have an active participation  in the long-term follow-up of these patients for  proper management of oral cGVHD.

Conclusion

In a sample of Egyptian pediatric patients who underwent bone marrow transplantation, the prevalence of cGVHD is 68.08%, patients with oral cGVHD is 55.3%. No association was detected between oral manifestations and age, sex, transplant type, source of stem cells or occurrence of acute GVHD. There was significant association between systemic and oral manifestations mainly skin, conditioning regimen and diminished salivary flow and between the trio oral manifestations where the combined lesions out of the total patients with oral cGVHD was 12 patients (46.1%).