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Prostaglandin Analogue CLINICAL SCIENCES A Study of Histopathological Features of Latanoprost-Treated Irides With or Without Darkening Compared With Non–Latanoprost-Treated Irides Daniel M. Albert, MD, MS; Ronald E. Gangnon, PhD; Hans E. Grossniklaus, MD; W. Richard Green, MD; Soesiawati Darjatmoko, BS; Amol D. Kulkarni, MD Objectives: To study the histopathological features of and P=.005, respectively). The average thickness and pig- latanoprost-treated irides with or without darkening, com- mentation of the anterior border layer was greater in the pared with non–latanoprost-treated irides. latanoprost-treateddarkenediridesthanintheother2groups (P=.03 and P=.02, respectively). The latanoprost-treated Methods: Iridectomy specimens and patient history darkened irides had increased pigmentation of the stroma forms were independently examined by 3 ophthalmic pa- (PϽ.001), stromal fibroblasts (PϽ.001), melanocytes thologists in a masked fashion. Specimens were evalu- (P=.005), vascular endothelium (P=.02), and adventitia ated for premalignant changes and for differences in level (PϽ.001) relative to the other 2 groups. of pigmentation and degrees of cellularity, inflamma- tion, and vascular abnormalities. Conclusions: There is no histopathological evidence of premalignant changes in latanoprost-treated darkened iri- Results: The specimens consisted of 22 latanoprost-treated des. The latanoprost-induced iris color changes are due darkened irides, 35 latanoprost-treated irides without dark- to a thickening of the anterior border layer and an in- ening, and 35 non–latanoprost-treated irides. There was a creased amount of melanin in the anterior border layer statistically significant decrease in the number of nuclear in- and within the stromal melanocytes. vaginations and prominent nucleoli in latanoprost-treated darkened irides compared with the other 2 groups (P=.004 Arch Ophthalmol. 2008;126(5):626-631 ROSTAGLANDIN ANALOGUES sociatedwithanincreaseinmelanocytenum- constitute one of the most ef- ber. That study found no evidence of adverse fective classes of antiglaucoma histopathological effects in latanoprost- drugsandincludevariouscon- treated irides compared with controls. geners such as latanoprost, A recent study by Arranz-Marquez et al4 unoprostone isopropyl, travoprost, and bi- compared iridectomy specimens from pa- P1 matoprost. In various clinical studies, these tients with latanoprost-induced darkening drugs often have been demonstrated to cause (n=22) and those from untreated controls increased pigmentation of the iris.2 A small (n=8). The authors found that latanoprost- number of studies have consisted of histo- induced darkened irides had an increased pathological evaluations to determine the number of melanocytes with intranuclear in- mechanism of iris darkening.3-5 In 2004, we clusions, nuclear invaginations, prominent performed the first large study to evaluate, nucleoli, and mitotic figures suggestive of Author Affiliations: atypia. Furthermore, they found more gran- Departments of Ophthalmology in a systematic manner, key histopathologi- and Visual Sciences (Drs Albert cal findings in iris specimens obtained from ules of melanin in the vascular walls and an and Kulkarni and patients with (n=449) or without (n=142) increased number of melanocytes and free Mr Darjatmoko) and a history of latanoprost therapy.3 That study melanin granules in the stroma compared Biostatistics and Medical included latanoprost-treated and control or with controls. To evaluate these findings, we Informatics (Dr Gangnon), nontreatediridesbutdidnotspecificallylook repeatedthestudyandcomparedlatanoprost- University of Wisconsin School at irides with darkening as a separate group. treated irides with darkening, latanoprost- of Medicine and Public Health, The study investigated the safety aspects of treated irides without darkening, and non– Madison; and Departments of treatment, including evidence of malignant latanoprost-treated control irides. Ophthalmology, Emory change or cell proliferation, such as the pres- University School of Medicine, Atlanta, Georgia ence of melanoma, the presence of mitoses, METHODS (Dr Grossniklaus), and Wilmer and an increased number of cells. The inves- Eye Institute, The Johns tigatorsconcludedthatlatanoprosttreatment Iris specimens were obtained at the time of iri- Hopkins University, Baltimore, caused increased amounts of melanin within dectomy during glaucoma surgery. The source Maryland (Dr Green). the iris stromal melanocytes but was not as- of the specimens has been previously cited.3 The (REPRINTED) ARCH OPHTHALMOL / VOL 126 (NO. 5), MAY 2008 WWW.ARCHOPHTHALMOL.COM 626 ©2008 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/29/2021 Table 1. Melanocytic Cellular Atypia Mean (SD) No. of Features Latanoprost-Treated Latanoprost-Treated Irides With No P Atypia Irides With Darkening Darkening Control Irides Valuea Intranuclear inclusions 1.3 (1.6) 1.1 (0.9) 1.3 (1.1) .67 Nuclear invaginations 2.5 (1.7) 4.0 (1.6) 3.4 (1.4) .004 Prominent nucleoli 1.9 (1.3) 3.1 (1.5) 2.7 (1.1) .005 a Based on the F test from analysis of variance models for continuous and ordinal variables and Pearson ␹2 test for categorical variables. tissue was received in 10% neutral-buffered formalin at the La- received institutional review board approval from the University tanoprost Pathology Center (LPC), University of Wisconsin, of Wisconsin Health Sciences Human Subjects Committee. Madison, and labeled by the LPC coordinator with a unique Comparisons of groups were based on the F test from analy- study accession number. The formalin-fixed tissue specimens sis of variance models for continuous and ordinal variables and were processed overnight on an automated tissue processor, Pearson ␹2 test for categorical variables. Ordinal variables were embedded in paraffin, serially sectioned at 5 µm, and mounted converted to integer equivalents for analysis. Statistical signifi- on treated slides. The first 3 slides were stained with hema- cance was set at PϽ.05. No formal adjustment for multiple com- toxylin-eosin, the next 3 slides were bleached with potassium parisons was used. permanganate before staining with hematoxylin-eosin, and the next 7 slides were left unstained. All slides were labeled with the unique study accession number assigned to the specimen. RESULTS If any tissue remained after sectioning, the paraffin block was archived. Also included were the slides of 11 of the 22 cases From May 1, 2005, through March 31, 2007, 92 iris speci- with latanoprost-induced darkening from the study by Arranz- mens from 92 unique patients were evaluated by the 3 Marquez et al.4 Sets of microscopic slides (1 hematoxylin-eosin–stained slide, reviewing pathologists. The specimens consisted of 22 1 bleached hematoxylin-eosin–stained slide, and 1 unstained slide) latanoprost-treated darkened irides, 35 latanoprost- were sent to the 3 reviewing pathologists (D.M.A., H.E.G., and treated irides without darkening, and 35 non–latanoprost- W.R.G.), who examined the sets independently and with no treated irides. The 22 latanoprost-treated darkened iri- knowledge of therapy history. They completed the accompany- des included 11 cases obtained from Arranz-Marquez et ing pathology grading form, which included an assessment of the al4 and 11 other cases from the LPC. Evaluation of the quality of the specimens, their location, and their orientation. The composite pathology grading forms indicated that the la- specimens were graded by level of pigmentation as none, mild, tanoprost-treated groups with or without darkening and moderate, and heavy. The thickness of the anterior border layer the control group were comparable in terms of quality, was ascertained by measuring the average number of flattened location, and orientation of the iridectomy specimens. cells on the iris surface, oriented parallel to the iris surface, and overlying the loose connective tissue of the stroma proper. These All specimens were obtained from the peripheral iris. were counted in 3 separate areas of the anterior border layer, and No melanomas were reported on the pathology grad- the average was determined by each of the 3 examiners. The num- ing form in either of the latanoprost-treated groups or ber given represents the further average of these 3 determina- in the control group. The iris specimens in all of the groups tions. These forms were returned to the LPC coordinator, who were evaluated for melanocytic atypia (Table 1). The completed a composite grading form using specific rules for com- various features suggestive of melanocytic atypia in- bining data from the 3 grading forms. Specimens were adjudi- cluded intranuclear inclusions, nuclear invaginations, cated using a protocol formulated by the 3 pathologists before prominent nucleoli, and mitotic figures. No mitotic fig- the beginning of the study. ures were noted in any of the iris specimens. There were The patient history form that accompanied each specimen no statistically significant differences in the number of included iris color (blue, hazel, or brown); type of glaucoma; ocular history; type of surgery; treatment history, including the intranuclear inclusions among the 3 groups. There was start of medical treatment; medication history, including spe- a statistically significant decrease in the number of nuclear cific medications used; duration of latanoprost therapy (Ͻ3 invaginations and prominent nucleoli in latanoprost- months, 3 months to 3 years, or Ͼ3 years); other glaucoma treated darkened
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