Expression of p16 in conjunctival intraepithelial neoplasia does not correlate with HPV-infection.

The aim of our study was to identify the frequency of expression of p16(INK4a) (CDKN2A) and HPV (human papilloma virus) in different grades of conjunctival intraepithelial neoplasia (CIN).Twelve specimens including CIN I (2), II (3), III (5), and CIN with beginning invasion (2), as well as 15 control specimens, were stained with antibodies against p16(INK4a) and MIB1. The presence of HPV was examined by PCR.p16 as well as MIB1 were significantly elevated in CIN compared to control specimens (p<0.01) without correlation with the differentiation grade. Only two cases with CIN grade 3 contained HPV 16.As few control specimens also showed increased p16(INK4a) expression, p16(INK4a) seems not to be a very reliable marker for the exact determination of CIN. It could serve as an additional diagnostic tool besides the morphological characterization. Our study suggested that p16(INK4a) elevation is not associated with HPV infection.

Expression of p63 in conjunctival intraepithelial neoplasia and squamous cell carcinoma.

BACKGROUND: p63 is a homologue of the tumour suppressor gene p53, which is expressed in human basal squamous epithelium. Some investigators maintain that p63 plays a role in the development of squamous epithelium and, despite its homology to p53, it is considered to act as an oncogene. This study investigated the expression of p63 in conjunctival intraepithelial neoplasia of different grades, and conjunctival squamous cell carcinoma and its correlation to the proliferation marker MIB-1. MATERIAL AND METHODS: Seventeen conjunctival specimens excised with the suspicion of either conjunctival intraepithelial neoplasia (CIN) or squamous cell carcinoma were diagnosed histologically as follows: 2 squamous cell carcinomas of the conjunctiva, 2 CIN grade I, 3 CIN grade II, 7 CIN grade III, 2 CIN with beginning invasion and 1 normal conjunctiva with no dysplasia. Sixteen microscopically-normal postmortem conjunctival specimens and normal conjunctiva, CIN and carcinoma specimens were stained immunohistochemically with antibodies against p63 and MIB-1. At least 500 cells per specimen were counted and the percentage of positively-stained cells of each antibody was calculated. RESULTS: A mean of 80% (57-89%) of the dysplastic cells from the CIN specimens stained positively with antibodies against p63, especially in the lower two-thirds of the epithelium, statistically significantly more compared with the normal specimens (9-55%, mean 36%, p<0.001). Nevertheless, we did not find a correlation between the percentage of p63-positive cells and the differentiation grade of the malignant specimens. MIB-1 positivity was shown by 0-1% of the cells in the normal postmortem controls, by 3-30% (mean 12%) of the cells in the basal and occasionally in the middle layer of the CIN specimens, and 16-61% (mean 23%) in the carcinoma specimens. CONCLUSION: In conjunctival intraepithelial neoplasia and squamous cell carcinoma of the conjunctiva, p63 is preferentially expressed in the immature dysplastic epithelial cells. Its staining does not correlate with MIB-1-expression, and therefore does not appear to be linked to cell proliferation.

Penetrating limbo-keratoplasty for granular and lattice corneal dystrophy: survival of donor limbal stem cells and intermediate-term clinical results.

PURPOSE: To assess clinical follow-up data, and to identify donor epithelial cells after homologous penetrating central limbo-keratoplasty in patients with granular and lattice corneal dystrophies compared with patients who underwent conventional penetrating keratoplasty (PK). DESIGN: Mixed retrospective and prospective nonrandomized comparative case series. PARTICIPANTS AND CONTROLS: Twenty-six patients who underwent 33 limbo-keratoplasty procedures for granular or lattice corneal dystrophy since May 1995 and a historical control group of 24 patients who underwent 36 PK procedures between November 1986 and May 1995. METHODS: Postoperatively, all but 2 limbo-keratoplasty patients were treated with systemic immunosuppressants for 6 months. All patients received long-term topical immune prophylaxis with prednisolone-21-acetate 1% (2 drops per day). After obtaining informed consent, epithelial cells were harvested from 10 limbo-keratoplasty eyes of 8 patients with granular dystrophy and 7 limbo-keratoplasty eyes of 7 patients with lattice dystrophy. Conjunctival epithelium or buccal mucosal epithelium for recipient identification and corneal epithelial cells from 3 graft sites were harvested. Deep-frozen donor corneoscleral rims were analyzed to characterize donor features. Genetic analysis was performed by polymerase chain reaction of short tandem repeat (STR) loci. MAIN OUTCOME MEASURES: The ratio of dystrophy recurrences in the graft was clinically assessed. Donor features in epithelial cells were genetically established if at least 1 STR profile differed from that of the recipient. RESULTS: There were 5 recurrences in limbo-keratoplasty eyes with granular dystrophy and 2 recurrences in limbo-keratoplasty eyes with lattice dystrophy, compared with 15 and 6 recurrences in PK eyes, respectively. The differences between limbo-keratoplasty and PK were not statistically significant over time (log-rank test; P = 0.14 for granular dystrophy and P = 0.56 for lattice dystrophy; alpha error, 0.05). For genetic analysis, 12 of 17 samples were evaluated. Donor epithelial cells were detected in 5 of the 12 samples (42%). CONCLUSIONS: Limbo-keratoplasty tended to be associated with fewer recurrences of granular and lattice dystrophies. However, the difference was not yet statistically significant, probably due to the disappearance of the transplanted limbal stem cells over time. Genetic analysis confirmed the survival of transplanted limbal stem cells over several years in some limbo-keratoplasty eyes, which might correlate with less recurrence. Limbo-keratoplasty, therefore, is likely to represent a first step towards long-term recurrence-free survival of corneal grafts in patients with granular and lattice dystrophies.

Long-term results of allogeneic penetrating limbo-keratoplasty in total limbal stem cell deficiency.

OBJECTIVE: To determine the prognosis of allogeneic penetrating limbo-keratoplasty in patients with total limbal stem cell deficiency and to find out if donor limbal stem cells survive in the long run. DESIGN: Noncomparative prospective case series. PARTICIPANTS: Forty-eight patients with total limbal stem cell deficiency. INTERVENTION: Allogeneic penetrating limbo-keratoplasty. All patients received systemic cyclosporin A and/or mycophenolate mofetil in the postoperative course. Thirteen patients received grafts with 0 to 1 HLA mismatches in the HLA-A, HLA-B, and HLA-DR loci; 13 patients received grafts with 2 to 6 mismatches; and 22 patients received untyped grafts. MAIN OUTCOME MEASURES: Long-term clear graft survival and survival of donor limbal stem cells. RESULTS: Five years postoperatively, 65% of the grafts with 0 to 1 mismatches, 41% of the grafts with 2 to 6 mismatches, and 14% of the untyped grafts were clear centrally (estimation according to Kaplan-Meier log rank test, P = 0.03). Immunogenetic analysis of epithelial cells from the surface of the graft could be performed successfully in 7 of 9 patients and revealed donor DNA in the epithelium of 5 of these 7 patients up to 56 months postoperatively. CONCLUSIONS: Long-term survival of donor epithelium could be demonstrated immunogenetically in patients undergoing allogeneic penetrating limbo-keratoplasty. Human leukocyte antigen-matched grafts seem to deliver better results than untyped grafts. Progress with matching and immunosuppressive strategies may further improve current results.

[Epithelial damage of corneal grafts after prolonged storage in dextran-containing organ culture medium - a prospective study]. / Hornhautepithelschädigung bei langer Transplantatverweildauer in dextranhaltigem Organkulturmedium - eine prospektive Studie.

BACKGROUND: High-molecular dextran added to organ culture medium is used for deswelling of corneal grafts before surgery. As we had the impression to have more often epithelial desquamation if storage time in dextran-containing medium was longer than 1 day, we investigated this problem in a pilot study. METHODS: We examined prospectively the effect of the storage period on the graft epithelium one day after penetrating keratoplasty in 137 corneal grafts which were stored in dextran-containing storage medium (dextran T500 6 %). 88 corneal grafts were stored for 1 - 2 days (12 - 48 hours) and 49 corneal grafts were stored for 3 - 4 days (60 - 96 hours). Before deswelling, all 137 grafts had been stored 10 - 14 days in dextran-free organ culture medium. Postoperative epithelial defects observed 1 day after surgery were classified as margin and central erosions of the graft. RESULTS: With a storage period in dextran-containing organ culture medium of 1 - 2 days statistically significantly less epithelial defects were observed in comparison to a longer storage period of 3 - 4 days (33 % vs. 57 %, p = 0,005). We found a statistically high significant correlation between storage time in dextran-containing organ culture medium and central erosions (p = 0,001), whereas margin erosions were observed after 1 - 2 days as well as after 3-4 days (p = 0,2). CONCLUSION: Our data show that early postoperative epithelial stability of corneal grafts depends on the storage period in dextran-containing organ culture medium.

Influencing factors on chronic endothelial cell loss characterised in a homogeneous group of patients.

BACKGROUND/AIM: Advanced donor age, long death to excision time interval, and factors related to organ culture can trigger unfavourable intracellular processes in the graft endothelium and contribute to chronic endothelial cell loss after penetrating keratoplasty. The aim of this study was to investigate factors influencing chronic endothelial cell loss in a homogeneous group of patients. METHODS: 177 patients after first normal risk keratoplasties for keratoconus were retrospectively selected from the quality control database of our clinic. For 71 of them at least four central endothelial cell density values were documented in follow up. From these patients, only those 53 without any further intraocular procedures, without glaucoma, and without graft rejection were considered. A scatter plot of logarithmised endothelial cell density values against postoperative time was drawn for each patient. The slope of the regression line then equals the constant of decay in central endothelial cell density. The influence of donor age and storage time in organ culture on this index value of cell loss was investigated by means of linear regression analysis. RESULTS: Mean loss of central endothelial cell density was 16.7% per year. Regression analysis revealed a statistically significant negative linear effect of both postmortem time (beta = -0.324; p = 0.014) and donor age (beta = -0.282; p = 0.036) and a trend for storage time in organ culture (beta = -0.195; p = 0.142) in a combined linear regression model. CONCLUSION: Increased postmortem time and advanced donor age exert a significant negative effect on chronic endothelial cell loss. Storage time in organ culture seems to be third influencing factor. These negative influences may be reduced by compensating advanced donor age with minimised postmortem and storage time.