Clinical and anatomic correlates of objectively measured zonular dehiscence in 427 postmortem pseudophakic eyes.

OBJECTIVE: This study aims to objectively measure the degree of zonular dehiscence in postmortem eyes and to assess for clinical and anatomic correlates. DESIGN: Cross-sectional study. MATERIALS: Four hundred and twenty-seven postmortem pseudophakic human eyes. METHODS: Eyes were obtained from the Lions Gift of Sight Eye Bank. Microscope photographs were taken of the eyes in Miyake-Apple view, and region-of-interest analysis was performed using ImageJ to measure the area, circumference, and diameter of the capsular bag, the ciliary ring, and the capsulorhexis. Clinical and anatomic parameters were assessed using simple linear regression analysis and one-way analysis of variance with post hoc Bonferroni testing. Zonular dehiscence was measured via 2 surrogates: capsule area over ciliary ring area ratio (CCR) and capsule-ciliary ring decentration (CCD). Low CCR and high CCD indicate more zonular dehiscence. RESULTS: CCR was significantly inversely correlated with smaller capsulorhexi (p = 0.012), lower intraocular lens power (p < 0.00001), younger age at death (p = 0.00002), and a longer cataract-to-death time (p = 0.00786). CCR also was significantly lower in glaucomatous cases (p = 0.0291). CCD was significantly correlated with longer cataract-to-death time (p = 0.000864), larger ciliary ring area (p = 0.001), more posterior capsule opacification (p = 0.0234), and higher Soemmering's ring opacity (p = 0.0003). There was also significantly more decentration in male eyes than in female eyes (p = 0.00852). CONCLUSIONS: CCR and CCD are novel measures of zonular dehiscence in postmortem eyes, with many interesting correlates. An enlarged ciliary ring area is possibly associated with zonular dehiscence in pseudophakic eyes and may be a quantifiable surrogate in vivo.

Histopathological analysis of residual lens cells in capsular opacities after cataract surgery using objective software.

Purpose: Remnant lens epithelial cells (LECs) within the capsular bag (CB) undergo epithelial-to-mesenchymal transition (EMT) and acquire a myofibroblast phenotype, depositing extracellular matrix (ECM) components, leading to posterior capsular opacification (PCO). This study histopathologically analyzes the LEC-to-myofibroblast transition and de novo ECM component deposition (i.e., smooth muscle actin (SMA) and fibronectin (FN) expression) and determines the intraocular lens (IOL) and patient factors associated with these changes. Methods: In total, 190 CBs with IOLs were removed from donor eyes. Digital images were obtained, and PCO was graded using published software (ADOS, Medical Parachute). Automated immunohistochemistry was performed using anti-SMA to detect EMT and anti-FN to document ECM remodeling. Slides were digitized and analyzed using the Positive Pixel Count v9 algorithm. Linear regression and Poisson regression were performed (P < 0.05). Results: SMA positive expression decreased as the time of IOL implantation increased (P < 0.0001). Positivity of SMA and FN demonstrated a positive correlation (P = 0.0002). Controlling for confounding factors in Poisson regression, hydrophobic and hydrophilic materials showed higher FN and SMA expression when compared to silicone material lenses (FN; P = 0.018; P < 0.0001, SMA; P = 0.001; P = 0.003, respectively). The square optic design had 29% higher SMA positivity compared to the opti-edge design (P = 0.042). One-piece haptic lenses had higher SMA expression compared to three-piece haptic (P = 0.042). A higher risk of expression of SMA and FN was seen in patients with a history of smoking, hypertension, and glaucoma (P < 0.05). Conclusion: This study demonstrated that SMA and FN expression is different according to IOL design and patient factors, thus indicating that LEC changes depend on lens biocompatibility. Therefore, by analyzing the histopathological composition of PCO by using LECs, further insight into the characteristics of IOLs that are important for biocompatibility can be ascertained.

Circulating tumor DNA tracking through driver mutations as a liquid biopsy-based biomarker for uveal melanoma.

BACKGROUND: Uveal melanoma (UM) is the most common intraocular tumor in adults. Despite good primary tumor control, up to 50% of patients develop metastasis, which is lethal. UM often presents asymptomatically and is usually diagnosed by clinical examination and imaging, making it one of the few cancer types diagnosed without a biopsy. Hence, alternative diagnostic tools are needed. Circulating tumor DNA (ctDNA) has shown potential as a liquid biopsy target for cancer screening and monitoring. The aim of this study was to evaluate the feasibility and clinical utility of ctDNA detection in UM using specific UM gene mutations. METHODS: We used the highly sensitive digital droplet PCR (ddPCR) assay to quantify UM driver mutations (GNAQ, GNA11, PLCß4 and CYSTLR2) in cell-free DNA (cfDNA). cfDNA was analyzed in six well established human UM cell lines with known mutational status. cfDNA was analyzed in the blood and aqueous humor of an UM rabbit model and in the blood of patients. Rabbits were inoculated with human UM cells into the suprachoroidal space, and mutated ctDNA was quantified from longitudinal peripheral blood and aqueous humor draws. Blood clinical specimens were obtained from primary UM patients (n = 14), patients presenting with choroidal nevi (n = 16) and healthy individuals (n = 15). RESULTS: The in vitro model validated the specificity and accuracy of ddPCR to detect mutated cfDNA from UM cell supernatant. In the rabbit model, plasma and aqueous humor levels of ctDNA correlated with tumor growth. Notably, the detection of ctDNA preceded clinical detection of the intraocular tumor. In human specimens, while we did not detect any trace of ctDNA in healthy controls, we detected ctDNA in all UM patients. We observed that UM patients had significantly higher levels of ctDNA than patients with nevi, with a strong correlation between ctDNA levels and malignancy. Noteworthy, in patients with nevi, the levels of ctDNA highly correlated with the presence of clinical risk factors. CONCLUSIONS: We report, for the first time, compelling evidence from in vitro assays, and in vivo animal model and clinical specimens for the potential of mutated ctDNA as a biomarker of UM progression. These findings pave the way towards the implementation of a liquid biopsy to detect and monitor UM tumors.

Optical Coherence Tomography of Peri-Ocular Skin Cancers: An Optical Biopsy.

INTRODUCTION: Optical coherence tomography (OCT) imaging has been used as a diagnostic tool for retinal disease for several years, and OCT apparatuses are becoming increasingly powerful. However, OCT has yet to reach its full potential in ophthalmology clinics. Alike retinal layers, it has been shown that OCT is able to generate cross-sectional images of the skin and allows visualization of skin lesions in a histopathology-like manner. OBJECTIVE: We aim to validate OCT as an imaging modality for peri-ocular skin cancer. Through a series of cases, we highlight findings for 3 common eyelid malignancies: basal cell carcinoma, squamous cell carcinoma and sebaceous carcinoma. We propose an OCT image-based signature for basal cell carcinoma. METHODS: This is a prospective study. Fifty-eight lesions suspicious of malignancy from 57 patients were subjected to OCT imaging prior to the surgical excision of the lesion. OCT images were analysed and scored according to previously identified OCT features. Eight representative examples are presented, highlighting the OCT patterns for each malignancy side by side to its corresponding histopathological sections. RESULTS: Out of the 58 lesions analysed, 53 were malignant. A loss of the dermal-epidermal junction is observed in all malignant lesions. A strong link is observed between the presence of subepithelial hyporeflective nests on OCT and the diagnosis of basal cell carcinoma (present in 83% of cases). Conversely, lesions of epithelial origin such as squamous cell carcinoma are most often represented on OCT by acanthosis. Two supplementary cases, one basal cell carcinoma and one sebaceous carcinoma, are provided to illustrate how OCT imaging is a valuable tool in cases where clinical observations may be unusual. CONCLUSIONS: We provide evidence supporting the use of OCT for the evaluation of peri-ocular cancers. OCT enables visualization of the skin layers in vivo, before biopsy. Our results show that certain OCT features can contribute to include or exclude a diagnosis of basal cell carcinoma. By integrating this non-invasive imaging methodology into the routine assessment of peri-ocular skin lesions, especially in health care centres where access to specialists is limited, OCT imaging can increase clinical precision, reduce delays in patient referral and enhance patient care.

Histopathological changes in the anterior segment with anterior and posterior chamber intraocular lens.

OBJECTIVE: Patients have shown a lowering of intraocular pressure (IOP) after cataract surgery. Histopathology studies have reported trabecular meshwork (TM) changes in pseudophakic eyes with posterior chamber intraocular lens (PCIOL) and have eluded to the mechanisms for IOP decrease. Unlike PCIOLs, TM histopathology changes after implantation of an anterior chamber intraocular lens (ACIOL) have not been studied, to our knowledge. Therefore, this study aims to examine the histopathological changes in both the TM and corneal endothelium among donor eyes with ACIOL, PCIOL, and phakic eyes. METHODS: Forty fixed postmortem donor eyes were obtained, sectioned, and embedded. Slides were stained with Masson's trichrome and CD31 vascular endothelial antibody, and further digitalized. Customized Medical Parachute TMAN software quantified the cellular components, the trabecular extracellular matrix (ECM), ECM fibrosis, and trabecular area. Schlemm's canal and corneal endothelium were quantified across the ACIOL, PCIOL, and phakic groups. RESULTS: Cellular area component of the TM was lower in the ACIOLs and PCIOLs than in phakic eyes, but statistically significant only between PCIOL and phakic eyes (p = 0.0023). ECM area component, TM fibrosis score and TM lamellae area, ciliary process fibrosis, and CD31 expression in Schlemm's canal showed no differences (p = 0.40, 0.99, 0.10, 0.83, 0.45). Significantly lower corneal endothelial cells were seen in ACIOLs compared with both PCIOLs and phakic eyes (p = 0.0002). CONCLUSIONS: ACIOLs and PCIOLs in our sample group showed that there is loss of cellular components in the TM compared with the phakic eyes, with PCIOLs displaying the least amount of TM cells statistically, in this cohort. The ACIOLs led to a greater loss of corneal endothelial cells than both PCIOLs and phakic eyes after cataract surgery. The endothelial cells in Schlemm's canal did not seem to be affected by the IOL placements. Therefore, this study illustrates that there are histopathological differences seen with the placements of ACIOLs in both TM and cornea.

The Use of Digital Microscopy to Compare the Thicknesses of Normal Corneas and Ex Vivo Rejected Corneal Grafts with a Focus on the Descemet's Membrane.

OBJECTIVE: To compare the thickness of corneal layers, specifically the Descemet's membrane (DM), in normal corneas and in failed grafts due to rejection (FGRs) using the digital histopathology and to propose a model for the measurement of corneal layers using this method. METHODS: This is a prospective, cross-sectional study performed at the MUHC-McGill University Ocular Pathology & Translational Research Laboratory (McGill University, Montreal, Canada). Histopathological sections of 25 normal human corneas and 40 FGRs were fully digitalized and examined. Inclusion criteria: samples diagnosed as normal corneas or FGRs, from patients older than 18 years of age. Exclusion criteria: histopathological sections without adequate tissue or missing epidemiological information. For each sample, the thicknesses of the epithelium, stroma, and DM were acquired. From a perpendicular plane of reference, two central measurements and two nasal and two temporal peripheral measurements were obtained. RESULTS: There were differences between the normal and FGR groups in the mean central thickness of the epithelium (p < 0.001), the nasal and temporal stromal regions (p < 0.001), and of the DM in the nasal and temporal regions (p < 0.001). Compared with the extremities of the sample (nasal and temporal), the mean thickness of the DM in normal corneas was lower in the central region (p < 0.001), and this difference was not found in the FGR group. CONCLUSIONS: Normal corneas have a thinner epithelium in the central region than the FGR group. In addition, the stroma and DM thicknesses of the nasal and temporal periphery were significantly higher in normal corneas than in those from the FGR group. The digital microscopy protocol applied in this study may be useful for further research studies regarding cornea and other tissues.

Novel application of anterior segment optical coherence tomography for periocular imaging.

OBJECTIVE: This study aims to assess the use of optical coherence tomography (OCT) imaging for periocular skin lesions and to determine which characteristic features of these images can be correlated to histopathology. DESIGN: This is an ongoing prospective study with Research Ethics Board approval. PARTICIPANTS: Fifty patients over 18 years old with lesions clinically suspicious of nonmelanoma skin cancer on the periocular region were included in this study. METHODS: After consent was obtained, clinical photographs and dermatoscopic images were obtained (DermLite II Hybrid M) from the lesion and its contralateral side. Subsequently, the patient was subjected to OCT imaging using the anterior segment module of a spectral domain OCT (Optovue Avanti) and images of the contralateral skin were also obtained. Surgical excision of the lesion was performed and sent for histopathological examination as per routine treatment. OCT images were then correlated to their matching digitalized histopathology section (Philips Ultra Fast Scanner 1.6 RA). RESULTS: Based on the OCT images acquired from 50 patients, 8 predominant architectural features have been correlated to histopathology: hyperkeratosis, acanthosis, loss of dermal-epidermal junction delineation, hyporeflective tumour nests, cystic structures, "bunch of grapes" nodules, hyperreflective nests, and ulcerations. Results observed from 45 malignant lesions (basal cell carcinoma, squamous cell carcinoma, and sebaceous gland carcinoma) suggest that groups of features and their layout within the same OCT image may be associated to specific tumour characteristics. CONCLUSIONS: Current data suggest that anterior-segment OCT imaging is a noninvasive imaging modality for periocular lesions and may be a valuable tool to help differentiate between some tumour types before a biopsy is performed.

Optimizing optical coherence tomography and histopathology correlation in retinal imaging.

OBJECTIVE: To develop a methodology to correlate optical coherence tomography (OCT) images and histopathological sections from the same eye. Part 1: To determine the best fixative for optimal OCT and histopathological analysis in post-mortem eyes. Part 2: A protocol is proposed to correlate histopathological features and OCT scans from the same post-mortem eyes. DESIGN: Experimental study. PARTICIPANTS: Part 1: Twenty-three rabbit eyes and 14 post-mortem human eyes. Part 2: Nineteen post-mortem human eyes. METHODS: Part 1: Six different fixatives were tested, and specimens were evaluated on 4 criteria: globe shape, structure opacification, retinal detachment, and nuclear details. Part 2: Based on the findings from Part 1, fixed human eyes were imaged using OCT. Orientation-controlled histopathological processing was performed to obtain serial tissue sections from paraffin embedded tissue, which were matched to corresponding OCT images. RESULTS: Part 1: Of the 6 fixatives, 2% glutaraldehyde and Davidson's solution met the proposed criteria in rabbit eyes. Of these, glutaraldehyde showed similar results in human eyes and was selected for Part 2. Part 2: Using anatomical landmarks, cross-sectional histopathological sections in the same orientation as the OCT images were correlated to their corresponding OCT images. Retinal lesions such as a macular hole, an epiretinal membrane, and the presence of drusen were easily correlated, proving the reliability of our methodology. Moreover, the photoreceptor's inner/outer junction was correlated to a hyperreflective band on OCT. CONCLUSIONS: A standardized protocol was developed to correlate OCT images and histopathological findings by generating serial cross-sections of the retina, which can be used to better understand otherwise ambiguous OCT findings.

Removing Subjective Post-Mortem Grading from Posterior Capsular Opacification: A New Automated Detector Opacification Software, ADOS.

Purpose/aim: Posterior capsular opacification (PCO) is the leading complication following cataract surgery. However, PCO grading methods vary between studies and rely on subjective scoring methods; hence, they are prone to error. Therefore, software that reduces subjectivity and enhances reproducibility is needed. The aim of the study was to evaluate the reproducibility and objectivity of a novel automated custom-designed PCO detection software. MATERIALS AND METHODS: In total, 165 fixed eyes with intraocular lenses were sectioned to obtain a Miyake Apple view (MAV) image. The capsular bag (CB) with the in situ IOL was removed and imaged using the Olympus DSX110 stereoscope. Central, peripheral, and Soemmering's ring area (SRA) and intensity (SRI) were graded by two ophthalmologists using a scale of 0-4. Software was developed to detect PCO, and these values were correlated with subjective scoring. RESULTS: Intra-observer agreement for MAV and CB images ranged from poor to moderate, with very good agreement for SRI (k = 0.88, 0.86, respectively). Agreement between graders was fair to good (k = 0.31-0.77). Miyake Apple view and capsular bag images showed good correlation; central PCO showed the least correlation for both evaluators (k = 0.29, 0.72). Subjective average grading versus software correlation demonstrated very good correlation for intensity and good for area (r = 0.85, 0.61). Reproducibility of the methodology resulted in good to very good correlation. CONCLUSIONS: Software correlates with previous scoring methods and is a reliable and reproducible system. Moreover, the capsular bag view, as opposed to the Miyake Apple view, allows visualization of the capsular bag directly and avoids overlapping structures (vitreous, cornea) that may interfere with proper PCO quantification.

Histopathological trabecular meshwork remodeling after cataract surgery detected with an advanced image analyzer.

PURPOSE: To compare the histopathological morphometry of the trabecular meshwork and ciliary processes in pseudophakic eyes and phakic eyes using advanced image analyzer technology. SETTING: McGill University, Montreal, Quebec, Canada. DESIGN: Retrospective case series. METHODS: Thirty-five pseudophakic eyes and 25 phakic eyes were sectioned and converted into digital slides. The total trabecular meshwork area and the ciliary body stroma were demarcated. The area of the trabecular meshwork, cellular and noncellular trabecular meshwork compartments, trabecular space, distance from scleral spur to inner uveal trabecular portion, and degree of fibrosis of the ciliary processes were evaluated. RESULTS: The trabecular meshwork area was larger in the pseudophakic group than the phakic group (P = .03). Furthermore, a trend of larger trabecular space recorded was seen in the pseudophakic group than the phakic group (P = .14). No differences in the proportion of cellular (P = .88) and noncellular trabecular meshwork compartments (P = .4) were seen between groups. The scleral spur to inner uveal trabecular portion distance was longer in the pseudophakic group than the phakic group (P = .008) and correlate with the trabecular meshwork area (P = .0001, r = 0.56). In the ciliary processes, a higher degree of fibrosis was measured in the pseudophakic group than the phakic group (P = .02). CONCLUSIONS: There were significant histopathological changes in the trabecular meshwork and higher fibrosis in the ciliary processes in pseudophakic eyes compared with phakic eyes. These findings support the hypothesis that trabecular meshwork remodeling after cataract surgery is involved in lowering intraocular pressure.

Evaluation of nicotinamide as an anti-inflammatory and anti-angiogenic agent in uveal melanoma cell lines.

PURPOSE:: To investigate the effect of nicotinamide on the secretion of pro-an giogenic and pro-inflammatory cytokines in uveal melanoma cell lines. METHODS:: Two human uveal melanoma cell lines (92.1 and OCM-1) were treated with nicotinamide (10 mmol/L) or control media for 48 hours in culture. The su perna tant from each culture was used in sandwich enzyme-linked immuno sorbent assay-based angiogenesis and inflammation arrays to evaluate the effects of exogenously administered nicotinamide on the secretion of a total of 20 pro-an gio genic and pro-inflammatory proteins. RESULTS:: Seven pro-angiogenic cytokines were detected under control conditions for both uveal melanoma cell lines. Treatment with nicotinamide resulted in a significant decrease in secretion of the following pro-angiogenic cytokines: angiogenin, angiopoietin-2, epidermal growth factor, and vascular epithelial growth factor-A in the 92.1 cells; basic fibroblast growth factor in the OCM-1 cells; and placenta growth factor in both cell lines. Among the pro-inflammatory proteins, monocyte chemotactic protein-1 and interleukin-8 were expressed in both untreated cell lines and both were significantly reduced when treated with nicotinamide. CONCLUSIONS:: Results from this in vitro model suggest that nicotinamide may have anti-inflammatory and anti-angiogenic properties, which may open the possibility of using it as a chemopreventive agent for uveal melanoma; however, further studies including animal models are warranted.

Evaluation of nicotinamide as an anti-inflammatory and anti-angiogenic agent in uveal melanoma cell lines / Avaliação da nicotinamida como agente anti-inflamatório e anti-angiogênico em linhas celulares de melanoma uveal

ABSTRACT Purpose: To investigate the effect of nicotinamide on the secretion of pro-an giogenic and pro-inflammatory cytokines in uveal melanoma cell lines. Methods: Two human uveal melanoma cell lines (92.1 and OCM-1) were treated with nicotinamide (10 mmol/L) or control media for 48 hours in culture. The su perna tant from each culture was used in sandwich enzyme-linked immuno sorbent assay-based angiogenesis and inflammation arrays to evaluate the effects of exogenously administered nicotinamide on the secretion of a total of 20 pro-an gio genic and pro-inflammatory proteins. Results: Seven pro-angiogenic cytokines were detected under control conditions for both uveal melanoma cell lines. Treatment with nicotinamide resulted in a significant decrease in secretion of the following pro-angiogenic cytokines: angiogenin, angiopoietin-2, epidermal growth factor, and vascular epithelial growth factor-A in the 92.1 cells; basic fibroblast growth factor in the OCM-1 cells; and placenta growth factor in both cell lines. Among the pro-inflammatory proteins, monocyte chemotactic protein-1 and interleukin-8 were expressed in both untreated cell lines and both were significantly reduced when treated with nicotinamide. Conclusions: Results from this in vitro model suggest that nicotinamide may have anti-inflammatory and anti-angiogenic properties, which may open the possibility of using it as a chemopreventive agent for uveal melanoma; however, further studies including animal models are warranted.

RESUMO Objetivo: Acredita-se que a nicotinamida (NIC) seja capaz de diminuir a angiogênese induzida pelo fator de crescimento endotelial vascular (VEGF). Investigar os efeitos da nicotinamida sobre a secreção de citocinas pró-angiogênicas e pró-inflamatórias em linhagens de células de melanoma uveal humano (UM). Métodos: Duas linhagens de células humanas de UM (92,1 e OCM-1) foram tratadas com NIC (10 mmol/L) ou apenas com meio de cultura por 48 horas. O sobrenadante das culturas obtido após a administração de nicotinamida foi comparado com o sobrenadante das culturas controle quanto à expressão de 20 fatores pró-angiogênicos e pró-inflamatórios, pela técnica de enzyme-linked immunosorbent assay (ELISA). Resultados: Sete citocinas pró-angiogênicas foram detectadas nas condições de controle em ambas as linhagens de células de UM. O tratamento com nicotinamida promoveu uma redução significativa da secreção das seguintes citocinas angiogênicas: Angiogenina, ANG2, EGF e VEGF-A em células 92.1; bFGF em células OCM-1; PIGF em ambas as linhagens celulares. Quanto às proteínas pró-inflamatórias, a expressão de MCP-1 e IL-8 foi significativamente reduzida com a administração de nicotinamida em relação às culturas de células que não receberam o tratamento. Conclusões: Nicotinamida apresenta propriedades anti-inflamatórias e anti-angiogênicas em modelo experimental in vitro. Tais efeitos sugerem a possibilidade de utilizar esta substância na quimioprevenção do UM. Entretanto, estudos com modelos experimentais in vivo são necessários para melhor avaliar o benefício do tratamento do UM com nicotinamida.

Pericyte Status in Routinely Discarded Vitrectomy Samples May Be an Early Marker of Diabetic Retinopathy.

BACKGROUND: Diabetic retinopathy (DR) is the leading cause of blindness among the working-age population. The earliest morphological manifestation of the disease is pericyte loss, as shown by animal models. AIMS: The purpose of this study was to evaluate the presence of pericytes in vitreous samples (VS) from diabetic and nondiabetic patients. METHODS: VS from 125 patients with and without diabetes were analyzed. Thirty-three of the VS contained blood vessels and were therefore included in further analysis. Pericyte status was evaluated using α-smooth muscle actin and quantified using the following scoring system: total loss (3), >50% loss (2), <50% loss (1), and no loss (0). RESULTS: Of the 33 VS, 29 samples were from patients with diabetes and 4 from nondiabetic patients. Six diabetic cases had a score of 1, 8 diabetic cases had a score of 2, and 15 cases had a score of 3. A positive correlation between glycemia levels and pericyte loss was observed (p = 0.0016; Spearman's r = 0.61). Moreover, all nondiabetic cases had a score of 0 (sensitivity and specificity = 100%). CONCLUSION: Pericyte loss in VS might be a sensitive and specific marker of DR that correlates with glycemia levels. Furthermore, VS, which are currently discarded, may contain valuable information for diabetic management.

Epithelial Inclusion Cyst in Conjunctival Melanoma.

Conjunctival melanoma is the second most common conjunctival malignancy. Its differential diagnosis with other conjunctival melanocytic neoplasms is inherently difficult. The presence of epithelial cysts is a useful feature in conjunctival tumors and favors a benign lesion. Herein 2 cases of conjunctival melanoma with cysts are presented. To the best of our knowledge, this is the first series of conjunctival melanoma with epithelial inclusion cysts. This series emphasizes the importance of considering several malignant features when reviewing conjunctival melanocytic lesions, as malignancy can exist even in the presence of epithelial inclusion cysts.