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Original article
Comparison of stem cell sources in the severity of dry eye after allogeneic haematopoietic stem cell transplantation
  1. Miki Uchino1,
  2. Yoko Ogawa1,
  3. Yuichi Uchino1,
  4. Takehiko Mori2,
  5. Shinichiro Okamoto2,
  6. Kazuo Tsubota1
  1. 1Department of Ophthalmology, Keio University School of Medicine, Tokyo, Japan
  2. 2Keio Bone Marrow Transplant Program, Division of Hematology, Department of Internal Medicine, Keio University School of Medicine, Tokyo, Japan
  1. Correspondence to Dr Yoko Ogawa, Department of Ophthalmology, Keio University School of Medicine, 35 Shinanomachi, Shinjyuku-ku, Tokyo, Japan; yoko{at}z7.keio.jp

Abstract

Aims To compare the incidence and severity of dry eye (DE) after allogeneic haematopoietic stem cell transplantation (HSCT) according to the stem cell source. The authors specifically focused on patients who received bone marrow transplantation (BMT), peripheral blood stem cell transplantation (PBSCT) and cord blood transplantation (CBT).

Methods Ninety-nine HSCT recipients who were prospectively followed-up for at least 100 days at Keio University Hospital were recruited. Ophthalmological examinations included evaluation of ocular surface findings and tear dynamics. The data on systemic graft-versus-host disease were collected by chart review.

Results Of the 99 patients (BMT, 67; PBSCT, 18; CBT, 14), 42 developed DE or showed worsened pre-existing DE after HSCT; 31 (46.3%) BMT group; 8 (44.0%) PBSCT group; and 3 (21.4%) CBT group (p=0.78). The median onset time of DE tended to be later in the PBSCT group (474 days, range 95–1559) than in the BMT (287 days, range 67–1216) or CBT (168 days, range 33–481) group, but the difference was not significant (p=0.23). However, the proportion of patients with severe DE was significantly higher in the PBSCT group (N=7, 87.5%) than in the BMT (N=12, 38.7%) or CBT (N=1, 33.3%) group (p=0.04) and CBT showed the lowest among all three stem cell sources.

Conclusion The data in this study suggested that the severity and onset time of DE were affected by the stem cell source. Close attention must be paid to the development of late-onset severe DE in PBSCT recipients.

  • Dry eye
  • graft-versus-host disease
  • haematopoietic stem cell transplantation
  • stem cell source
  • epidemiology
  • immunology
  • lacrimal gland
  • cornea
  • conjunctiva
  • tears
  • degeneration
  • treatment surgery
  • inflammation
  • medical education
  • wound healing
  • eye (tissue) banking
  • trauma
  • infection
  • optics and refraction
  • anterior chamber
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Introduction

Haematopoietic stem cell transplantation (HSCT) has become an established treatment modality for the management of haematological malignancies.1 In addition to bone marrow transplantation (BMT), both umbilical cord blood transplantation (CBT) and peripheral blood stem cell transplantation (PBSCT) have been increasingly used over the last 15 years,2 and several recent studies have suggested that CBT and PBSCT lead to outcomes similar to those observed with BMT in the setting of unrelated volunteer donor HSCT in adults.3 4

Despite improvements in post-transplantation immunosuppressive therapy, graft-versus-host disease (GVHD) remains a major complication that impedes the success of allogeneic HSCT.5 In the clinical setting, GVHD is divided into acute and chronic forms. Acute GVHD (aGVHD) usually occurs during the first 3 months following HSCT.5 T cells present in the donor's bone marrow at the time of transplant identify the HSCT patient as ‘non-self’ and attack the patient's skin, liver, stomach and/or intestines. Chronic GVHD (cGVHD) usually develops after the third month post-transplant5 and has features resembling scleroderma, exhibiting prominent fibrosis in skin lesions, pulmonary fibrosis and chronic immunodeficiency. The pathophysiology of cGVHD was originally considered to be a later phase of aGVHD, but several recent studies suggest that an autoimmune-like process induced by dysfunctional immunological recovery also plays some role. However, the exact mechanism of cGVHD, which is distinct from aGVHD, is still largely unknown and considered more complex, multistep and needs to be explored.

With the steady and significant increase in the number of long-term survivors after HSCT, the management of cGVHD has become increasingly important for improving their quality of life. The eye is one of the major target organs of cGVHD and dry eye (DE) is the major late ocular complication associated with this disease.6 7 Although there are other ocular complications as well and DE is not fatal, corneal epithelial defects can sometimes lead to blindness.8 Therefore, it is important to manage DE after HSCT properly for the recipient's quality of life and as a prophylaxis for blindness. In this prospective study, we compared the incidence and severity of DE after HSCT according to the stem cell source with the ultimate goal of improving the prophylaxis and treatment of this ophthalmic complication.

Materials and methods

Patients

One hundred and forty-eight patients who underwent allogeneic HSCT (98 received BMT, 32 PBSCT and 18 CBT) from January 2000 to June 2004 at Keio University Hospital were prospectively followed by ophthalmologists before and after the transplant. We limited the study to those who underwent myeloablative HSCT and excluded reduced-intensity or mini-transplant recipients. Thirty-four patients who died and 15 who relapsed and needed to reduce or discontinue immunosuppressive drugs early in their course of treatment were also excluded from this study. Thus, 99 patients who survived at least 100 days with sustained engraftment were included in this study.

Of these 99 patients, 67 received BMT, 18 PBSCT and 14 CBT. All patients underwent standard clinical and ophthalmological examinations just before and 3, 6, 9, 12, 18 and 24 months after the transplantation and whenever indicated. cGVHD was diagnosed according to standard criteria.9 The research followed the tenets of the Declaration of Helsinki and informed consent was obtained from all patients.

Ophthalmological evaluation

Tear function tests and ocular surface evaluations

Ophthalmic examinations included the best corrected visual acuity for distance, assessment of conjunctival and corneal vital staining with rose bengal and fluorescein, tear film break up time (TBUT), Schirmer's test-I and lens and fundus examinations. The condition of the ocular surface was evaluated as reported previously.8 Briefly, the cornea was evaluated by the double vital staining method. Two microlitres of a preservative-free combination of 1% rose bengal and 1% fluorescein dye was instilled into the conjunctival sac by micropipette. For rose bengal score, the ocular surface was divided into three zones: nasal conjunctival, corneal and temporal. A score of 0–3 points was used for each zone with a minimum possible score of 0 and a maximum possible score of 9. Scarce punctate staining was given 1 point. Denser staining not covering the entire zone was given 2 points. Denser staining over the entire zone was given 3 points. For fluorescein staining, the cornea was divided into three equal zones—upper, middle and lower. Each zone has a staining score ranging from 0 to 3 points, as with the rose bengal stain and the minimum and maximum scores were 0 and 9, respectively. The presence of scarce staining in a zone was scored 1, frequent punctate staining not covering the entire zone was scored as 2 points and punctate staining covering the entire zone was scored as 3 points.

Tear dynamics were assessed by three different methods10: the TBUT, Schirmer's test-I, and Schirmer's test with nasal stimulation. To determine the TBUT, double vital staining was performed and the patients were requested to blink three times to ensure adequate mixing of the fluorescein dye in the tears. The time interval between the last complete blink and the appearance of the first corneal black spot was measured by a stopwatch and the mean of the three measurements was regarded as the TBUT in this study.

The Schirmer's test-I was performed without topical anaesthesia, after all the other examinations. Strips (Whatman No 41, Showa, Tokyo) were placed at the outer one-third of the temporal lower conjunctival fornix for 5 min. The strips were then removed and the length of wet filter paper (in mm) was recorded. Schirmer's test with nasal stimulation,10 which is considered to evoke maximal reflex tearing, was performed by applying a cotton swab to the nasal cavity.

Diagnostic criteria

DE was diagnosed as described previously.11 Briefly, any sign of tear film instability (TBUT <5 s, Schirmer's test-I <5 mm) with any abnormality of the ocular surface (rose bengal score >3 points and/or fluorescein score >1 point) was diagnosed as DE. We divided the cases of DE into two groups according to the degree of severity as severe dry eye (S-DE) and mild dry eye (M-DE). S-DE was defined as reduced reflex tearing in the Schirmer's test with nasal stimulation to less than 10 mm and abnormality of the ocular surface (rose bengal score ≥3 and/or fluorescein score ≥3). M-DE was defined as abnormality of the ocular surface (rose bengal score ≥3, fluorescein score ≥1) without reduced reflex tearing (Schirmer's test with nasal stimulation <10 mm).10 We also divided the DE cases into two groups according to the time of onset: within 100 days and after 100 days of HSCT.

Statistical analysis

The major endpoint of this study was to compare the incidence and severity of DE in patients who received HSCT with BMT, PBSCT or CBT. The analyses were performed with the StatView software (SAS Institute Inc, USA) for Windows 98/2000. The data are presented as medians with ranges. One-way analysis of variance was used to compare groups with respect to normally distributed continuous variables. The χ2 test was used to compare nominally scaled variables. Two-tailed p values of less than 0.05 were considered to indicate a statistically significant difference.

Results

The clinical profiles of the 99 patients are shown in table 1. There were no statistically significant differences in the median age (p=0.78) or sex (p=0.37) among the three groups.

Table 1

Characteristics of bone marrow, peripheral blood stem cell and cord blood transplants recipients

Forty-two patients (42.4%) developed DE or experienced a worsening of pre-existing DE after HSCT (table 2). DE was diagnosed in 31 patients (46.3%) in the BMT group, 8 (44.0%) in the PBSCT group and 3 (21.4%) in the CBT group (p=0.57). The median onset time of the DE was 287 days (range 67–1216) in the BMT group, 474 days (range 95–1559) in the PBSCT group, and 168 days (range 33–481) in the CBT group. Although it was not statistically significant, the CBT group showed an earlier onset of DE compared to the other groups (p=0.23). However, the incidence of DE onset within 100 days was significantly higher in patients who received CBT (2 patients, 66.7%) than BMT (4, 12.9%) or PBSCT (1, 12.5%) (p=0.05 for both; table 2). The incidence of DE did not correlate significantly with the recipients' age, sex or stem cell source.

Table 2

Characteristics of patients who developed dry eye after haematopoietic stem cell transplantation

Considering the severity of the DE, S-DE was observed in 12 patients (38.7%) in the BMT group, 7 (87.5%) in the PBSCT group and 1 (33.3%) in the CBT group. The incidence of S-DE was significantly higher in the PBSCT group than BMT or CBT group (p=0.04 for both; table 2). Following the onset of DE, the conjunctival and corneal findings of the S-DE group rapidly worsened despite treatment. For the M-DE group, the development of DE without reduced reflex tearing was observed in 19 patients (61.3%) in the BMT group, 1 patient (12.5%) in the PBSCT group and 2 patients (66.7%) in the CBT group. These patients' ocular surface findings were well controlled by applying commercially available artificial tears. None of the patients in this study experienced DE that led to corneal ulcer or blindness.

The relationship between the development of systemic GVHD and DE after HSCT is shown in table 3. We found that the patients with GVHD had a higher OR for DE in the BMT (OR 12.28, 95% CI 2.48 to 60.5) and CBT (OR 13.8, 95% CI 0.4 to 448.6) groups compared with the PBSCT group (OR 3, 95% CI 0.4 to 22.7).

Table 3

Relationship between dry eye and systemic graft-versus-host disease

Discussion

This is the first report to evaluate the incidence of GVHD-related DE according to the stem cell source, including bone marrow, peripheral blood and cord blood using detailed diagnostic techniques for DE. Unrelated CBT has been increasingly used as an alternative stem cell source for HSCT1; however, there have been no reports so far on the incidence and the severity of DE related to cGVHD after CBT in comparison with BMT or PBSCT. In this study, we found that the incidence of DE was comparable among the three stem cell recipient groups but the incidence of S-DE was significantly higher in the PBSCT recipients, while that in the BMT or CBT recipients was comparable. This finding is consistent with previous systemic cGVHD research examining the relationship between stem cell sources and the severity of systemic cGVHD.12 Our present results suggest that DE reflects the condition of systemic cGVHD well, and indicate that the careful evaluation and diagnosis of DE after HSCT can help predict the systemic condition.

The pathogenic process of DE associated with cGVHD is thought to involve T cells interacting with stromal fibroblasts in the cGVHD lacrimal gland. Because unmodified blood stem cells contain one log more T cells than unmodified bone marrow grafts,13 the number of donor T cells and fibrocytes and the timing of the infiltration may lead to the increased severity of DE in PBSCT recipients.

Studies comparing CBT, PBSCT or BMT published to date have not examined the histological events of ocular cGVHD with regard to the stem cell source and further studies will be required to elucidate them. Detailed studies assessing the time course and extent of the immune reconstitution after PBSCT compared with BMT or CBT are also needed.14

Our observation that DE was diagnosed later in PBSCT than in BMT and CBT recipients remains to be elucidated. One possible explanation is that the early development of DE may have been prevented by the earlier use of immunosuppressants in the PBSCT recipients. Systemic immune suppressants are often applied early in PBSCT because PBSCT can result in more severe systemic GVHD than CBT or BMT.15 16 Studies have shown the duration of systemic immunosuppressant treatment was longer in peripheral blood stem cell recipients than in the bone marrow recipients, suggesting that cGVHD after PBSCT is more protracted than BMT.16 17 In peripheral blood stem cell recipients, cGVHD continue to occur over 6 months after transplantation. This type of cGVHD is defined as late onset of cGVHD.15 17 Our results suggest that, when the immunosuppressants were being tapered off in the PBSCT recipients, late-onset DE related to cGVHD appear and rapidly progress, leading to S-DE. We also found that the patients with systemic GVHD had a higher OR for DE in the BMT and CBT groups than in the PBSCT group. These results also suggested that the prolonged use of systemic immunosuppressants in PBSCT patients masked their DE when systemic GVHD occurred. In contrast, once systemic GVHD occurred in the BMT and CBT recipients, mild DE, but not severe DE might have developed due to earlier taper of systemic immunosuppressants than PBSCT. These findings suggest that we may be able to start using topical immunosuppressants such as ciclosporin or FK506 before systemic immunosuppressants are tapered off to prevent or slow down the onset or progression of DE related to cGVHD. Close communication between ophthalmologists and internists would be important to determine the timing of commencement or cessation and the dose of systemic and topical immunosuppressants.

The patients with haematological malignancies receiving CBT from unrelated donors with partially mismatched human leucocyte antigen (HLA) had a lower risk of S-DE and earlier onset of DE than the PBSCT or BMT recipients. The earlier onset of DE after CBT compared with BMT or PBSCT validated the specificity of our results, because cord blood cells proliferate more rapidly and generate a larger number of progeny compared with bone marrow cells.18 In CBT, the incidence of aGVHD is lower than that expected based on the degree of HLA mismatch and the time for haematopoietic recovery is consistently longer than that for other haematopoietic stem cell sources; furthermore, the dosage of both nucleated cells and CD34 cells influences the success of HCST.19 20 The main differences between CBT and BMT are reported to be the number of nucleated cells in the graft and HLA compatibility.21

Cord blood has unique immunological features.18 22 The reason for the low immunological reaction in cord blood cells has been attributed to the significant decrease in transforming growth factor β-1 and macrophage chemoattractant protein-122 and the increase in anti-inflammatory cytokine interleukin-10, which probably results in downmodulation of GVHD.23 It has been reported that cord blood contains a high proportion of ‘naive’ phenotype T cells expressing the CD45RA/CD45RO, CD62L.24 These properties may influence and reduce the ocular surface and lacrimal gland immune response, leading to the low incidence of S-DE after CBT.

In particular, in terms of the DE associated with cGVHD, the clinical results reported in this study suggest that cord blood from unrelated donors could be a safe and effective stem cell source. However, there is a report of a rapidly worsening case of severe DE with pseudomembrane after CBT.25

Taken together, our study suggests that the following series of events occur in ocular cGVHD depending on the stem cell sources. First, breakdown of the blood vessel results in the invasion of donor T cells and fibroblasts or fibrocytes into the ocular tissues, perhaps due to homing signals released as part of an alloimmune response.26–28 The donor cells from various stem cell sources interact with recipient residual inflammatory cells, then activate and migrate to the ocular surface and lacrimal gland microenvionment. These activated fibroblasts exacerbate the production of excessive extracellular matrix with the end effect being impaired ocular surface and lacrimal gland function, resulting in S-DE. Peripheral blood stem cells contain a large number of mature T cells, and some donor fibrocytes29 may have the potential to activate these inflammatory cascades, leading to S-DE than BMT or CBT. Therefore, PBSCT recipients require more dose of immune suppressive treatment. In contrast, cord blood has various anti-inflammatory properties23 facilitating the inhibition of developing S-DE. Further studies investigating the immune process depending on the stem cell sources in the ocular cGVHD would be useful for clarifying the complex pathogenesis of cGVHD as well as for the development of therapeutic strategies for this disease.

In conclusion, we found that the incidence and severity of DE differ with the stem cell source used. Improvements in the early diagnosis of DE and therapeutic strategies for treating it may lead to a substantial decrease in the morbidity associated with DE. Studies aimed at improving the diagnosis and treatment of cGVHD after HSCT should be continued, with a focus on the effects of stem cell sources and the importance of DE.

References

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Footnotes

  • Funding The study was supported by two grants from the Japanese Ministry of Education, Science, Sports and Culture (No. 23592590).

  • Competing interests None.

  • Ethics approval The examination procedure in the outpatient clinic and the examination of documents are within the routine work for patients in the outpatient clinic. The examination procedure for the patients and examination of the patients' documents were approved for dry eye patients by the Institutional Review Board of Keio University. However, no specific approval was obtained for this study.

  • Provenance and peer review Not commissioned; externally peer reviewed.

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