Graft-Versus-Host-Disease and Health-Related Quality of Life in Young Long-term Survivors of Cancer and Allogeneic Hematopoietic Stem Cell Transplantation.

Purpose: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an established treatment predominantly for malignancies. Chronic graft-versus-host disease (cGVHD) is the leading long-term complication after allo-HSCT, but knowledge on cGVHD and health-related quality of life (HRQOL) in long-term survivors of allo-HSCT performed in childhood, adolescence, and young adulthood (CAYA) is scarce. Therefore, we aimed to (1) assess prevalence and risk factors of active cGVHD using the 2014 National Institutes of Health-Consensus criteria, (2) investigate associations between cGVHD severity, patient-reported symptom burden, and HRQOL, and (3) compare HRQOL of survivors to population norms. Methods: We conducted a nationwide cross-sectional study in long-term survivors of CAYA allo-HSCT combining clinical examinations and patient-reported outcome measures. Results: We included 103 survivors, 55 (53%) females, median age of 19.6 years [range 0.3-29.9] at HSCT, 16.8 years [6.0-32.0] from HSCT, and 77 (75%) with underlying malignancy. Overall, 32 (31%) survivors were diagnosed with active cGVHD. The risk of active cGVHD was increased with prior acute GVHD and reduced with in vivo T cell depletion. cGVHD severity was associated with increased symptom burden, but not with adverse HRQOL. Compared to Norwegian population norms, allo-HSCT survivors reported significantly lower HRQOL. Conclusion: These results indicate a high prevalence of cGVHD in long-term survivors of CAYA allo-HSCT. Although we did not find an association between cGVHD severity and HRQOL, survivors reported significantly poorer HRQOL compared to population norms. Knowledge on the long-term consequences of cGVHD will be important for optimizing treatment and long-term follow-up care after CAYA allo-HSCT.

Corneal endothelial cell loss following cataract surgery in patients with pseudoexfoliation syndrome: a 2-year prospective comparative study.

PURPOSE: To compare the corneal endothelial cell density (ECD) before and after cataract surgery in patients with and without pseudoexfoliation syndrome (PEX). METHODS: In this prospective study, we compared the ECD in 62 PEX patients with 62 patients without PEX (controls). The mean age was 78.3 ± 6.2 years and 77.2 ± 5.9 years, respectively. Patients were examined before and at 6 months, 1 year and 2 years after cataract surgery. The corneal endothelium was examined with confocal microscopy, and the ECD was counted both automatically and semi-manually. Nine patients in the PEX group (15%) and 11 patients in the control group (18%) were lost to follow-up in the 2-year period. Within the PEX and the control groups, we also compared the ECD between patients with and without glaucoma. RESULTS: Before surgery, the ECD (semi-manual counting) was 2258 ± 342 cells/mm2 in the PEX group and 2322 ± 321 cells/mm2 in the control group (p = 0.29). There were no significant differences in postoperative ECD between these groups at any visit. After 2 years, the ECD had changed by -679 ± 337 cells/mm2 and -704 ± 484 cells/mm2 , respectively (p = 0.78). The preoperative ECD was lower in eyes with glaucoma compared to eyes without glaucoma, both within the PEX group (p = 0.05) and the control group (p = 0.03). After surgery, there were no differences between eyes with or without glaucoma. CONCLUSION: The ECD was not significantly different in PEX eyes compared with control eyes, neither before nor after cataract surgery. However, there seemed to be a lower ECD in eyes with glaucoma before surgery.

Ocular findings and ocular graft-versus-host disease after allogeneic stem cell transplantation without total body irradiation.

Patients treated with allogeneic stem cell transplantation (allo-SCT) often develop ocular complications. To investigate the ocular findings in young long-term survivors after allo-SCT without TBI, we examined 96 patients more than 5 years after transplantation. All patients were under 30 years of age at transplantation. The mean follow-up time was 16.8 years (range 6.0-26.1 years). The study was a part of the Norwegian Allo Survivorship Study investigating health impairments in young survivors after allo-SCT. Ophthalmological examination included visual acuity, tear break-up time, corneal fluorescein staining, Schirmer I test, tear film osmolarity, biomicroscopy and dilated ophthalmoscopy. In patients with known systemic chronic GVHD (cGVHD), ocular GVHD (oGVHD) diagnosed by clinical examination was compared with diagnosis using National Institutes of Health (NIH) or International Chronic Ocular Graft-vs-Host-Disease (ICCGVHD) Consensus Group criteria. We diagnosed dry eye disease (DED) in 52 patients (54%), cataract in 3 patients (3%) and retinopathy in 1 patient (1%). Systemic cGVHD was a risk factor for DED (OR 4.40, CI 1.33-14.56, p = 0.02). Comparison of diagnostic criteria suggests that the more stringent ICCGVHD criteria can better differentiate DED from oGVHD after allo-SCT as compared with the NIH criteria.

The effect of culture medium and carrier on explant culture of human limbal epithelium: A comparison of ultrastructure, keratin profile and gene expression.

Patients with limbal stem cell deficiency (LSCD) often experience pain and photophobia due to recurrent epithelial defects and chronic inflammation of the cornea. Successfully restoring a healthy corneal surface in these patients by transplantation of ex vivo expanded human limbal epithelial cells (LECs) may alleviate these symptoms and significantly improve their quality of life. The clinical outcome of transplantation is known to be influenced by the quality of transplanted cells. Presently, several different protocols for cultivation and transplantation of LECs are in use. However, no consensus on an optimal protocol exists. The aim of this study was to examine the effect of culture medium and carrier on the morphology, staining of selected keratins and global gene expression in ex vivo cultured LECs. Limbal biopsies from cadaveric donors were cultured for three weeks on human amniotic membrane (HAM) or on tissue culture coated plastic (PL) in either a complex medium (COM), containing recombinant growth factors, hormones, cholera toxin and fetal bovine serum, or in medium supplemented only with human serum (HS). The expanded LECs were examined by light microscopy (LM), transmission electron microscopy (TEM), immunohistochemistry (IHC) for keratins K3, K7, K8, K12, K13, K14, K15 and K19, as well as microarray and qRT-PCR analysis. The cultured LECs exhibited similar morphology and keratin staining on LM, TEM and IHC examination, regardless of the culture condition. The epithelium was multilayered, with cuboidal basal cells and flattened superficial cells. Cells were attached to each other by desmosomes. Adhesion complexes were observed between basal cells and the underlying carrier in LECs cultured on HAM, but not in LECs cultured on PL. GeneChip Human Gene 2.0 ST microarray (Affymetrix) analysis revealed that 18,653 transcripts were ≥2 fold up or downregulated (p ≤ 0.05). Cells cultured in the same medium (COM or HS) showed more similarities in gene expression than cells cultured on the same carrier (HAM or PL). When each condition was compared to HAM/COM, no statistical difference was found in the transcription level of the selected genes associated with keratin expression, stemness, proliferation, differentiation, apoptosis, corneal wound healing or autophagy. In conclusion, the results indicate that ex vivo cultures of LECs on HAM and PL, using culture media supplemented with COM or HS, yield tissues with similar morphology and keratin staining. The gene expression appears to be more similar in cells cultured in the same medium (COM or HS) compared to cells cultured on the same carrier (HAM or PL).

Clinical transplantation of ex vivo expanded autologous limbal epithelial cells using a culture medium with human serum as single supplement: a retrospective case series.

PURPOSE: Presently, our clinic is the only centre in Scandinavia that offers patients with corneal surface pathology including limbal stem cell deficiency (LSCD) transplantation of ex vivo expanded limbal epithelial cells (LECs). We here present clinical data of the first nine patients with LSCD who were transplanted with autologous LECs expanded in medium completely free of any animal-derived products and non-human/recombinant growth factors (including Cholera Toxin), and with autologous human serum as the only growth supplement. METHODS: We conducted a noncomparative retrospective study of patients with LSCD at our centre between 2009 and 2011. The diagnosis was based on history and clinical signs. A biopsy was taken from healthy limbus, and the epithelium was expanded on amniotic membrane (AM) in medium containing autologous serum and subsequently transplanted to the affected eye. RESULTS: Successful outcome was defined as relief of pain and photophobia and/or improved best corrected visual acuity (BCVA) and/or reestablishment of a stable corneal epithelium and regression of corneal vascularization. Five of the nine transplanted patients (55.6%) had an improvement in either subjective symptoms or objective findings (11- to 28-month follow-up). CONCLUSIONS: Our clinical study shows that patients with LSCD can be treated successfully with transplantation of LECs expanded ex vivo in a medium with autologous serum as the only growth supplement. The use of this novel culture system, which is devoid of animal-derived products and non-human/recombinant growth factors (including Cholera Toxin), reduces the risks of inter-species disease transmission and host immune responses to xenogenic proteins, both obvious advantages for the patient.

Ex vivo expanded autologous limbal epithelial cells on amniotic membrane using a culture medium with human serum as single supplement.

In patients with limbal stem cell deficiency (LSCD), transplantation of ex vivo expanded human limbal epithelial cells (HLECs) can restore the structural and functional integrity of the corneal surface. However, the protocol for cultivation and transplantation of HLECs differ significantly, and in most protocols growth additives such as cholera toxins, exogenous growth factors, hormones and fetal calf serum are used. In the present article, we compare for the first time human limbal epithelial cells (HLECs) cultivated on human amniotic membrane (HAM) in a complex medium (COM) including fetal bovine serum to a medium with human serum as single growth supplement (HSM), and report on our first examinations of HLECs expanded in autologous HSM and used for transplant procedures in patients with LSCD. Expanded HLECs were examined by genome-wide microarray, RT-PCR, Western blotting, and for cell viability, morphology, expression of immunohistochemical markers and colony forming efficiency. Cultivation of HLECs in HSM produced a multilayered epithelium where cells with markers associated with LESCs were detected in the basal layers. There were few transcriptional differences and comparable cell viability between cells cultivated in HSM and COM. The p63 gene associated with LESCs were expressed 3.5 fold more in HSM compared to COM, and Western blotting confirmed a stronger p63α band in HSM cultures. The cornea-specific keratin CK12 was equally found in both culture conditions, while there were significantly more CK3 positive cells in HSM. Cells in epithelial sheets on HAM remaining after transplant surgery of patients with LSCD expressed central epithelial characteristics, and dissociated cells cultured at low density on growth-arrested fibroblasts produced clones containing 21 ± 12% cells positive for p63α (n = 3). In conclusion, a culture medium without growth additives derived from animals or from animal cell cultures and with human serum as single growth supplement may serve as an equivalent replacement for the commonly used complex medium for ex vivo expansion of HLECs on HAM.