Intraocular Biopsy Research Articles

Posterior uveal biopsy and the trans-scleral Essen forceps biopsy technique.

Differentiating neoplastic and non-neoplastic uveal tumours can present a diagnostic challenge; intra-ocular biopsy may be necessary. The novel trans-scleral Essen Forceps biopsy (TSEB) technique can improve diagnostic yield compared to fine needle aspiration biopsy (FNAB). We present a case demonstrating the technique and its added value. We also review the success rate of TSEB performed at two tertiary eye centres. Retrospective case report and consecutive case series from August 2021 to March 2023. Inclusion criteria were patients who underwent TSEB of posterior uveal lesions from Moorfields Eye Hospital and Sheffield Teaching Hospitals in the United Kingdom. The outcomes were biopsy success rate and complication rate RESULTS: Eleven biopsies met the inclusion criteria. Eight (73%) were successful, which comprised six uveal melanomas, one melanocytoma and one extranodal marginal zone (ENMZ) lymphoma. One TSEB did not yield tissue for histological examination because of perioperative sample handling. Two (18%) biopsies were histologically inconclusive; both were treated as uveal melanoma on clinical grounds or repeat biopsy. The only complication was vitreous loss and retinal hole without retinal detachment in one eye with a very posterior, shallow choroidal lesion. TSEB is an effective alternative to established biopsy techniques, yielding larger tissue samples than FNAB with intact tissue architecture. We recommend adding TSEB to the armamentarium of the ocular oncologist.

Gene Panels for Intraocular Lymphoma Can Overcome Diagnostic Difficulties and Enable Precision Medicine

Background: Definitive diagnosis of intraocular lymphoma (IOL) is often challenging, at least partially due to limited accessibility to the lesion and the rarity of tumor cells, with pathologically false-negative results reportedly occurring in approximately 30% of cases. Delayed diagnosis is often an important clinical issue, and the average time to diagnosis is approximately 1.5 years.Because of delayed diagnosis, IOLs are often found only after central nervous system (CNS) invasion has occurred, possibly leading to worse treatment outcomes. In this study, we tried to overcome diagnostic difficulties by using gene panel diagnostics of IOL. Furthermore, genetic analysis of IOLs may be able to predict the risk of recurrence.in Japan, a second-generation Bruton's tyrosine kinase (BTK) inhibitor, tirabrutinib, has been approved for the treatment of relapsed/refractory CNS lympoma. Herein, we present a case of IOL and CNS lesion with MYD88 mutation treated with tirabrutinib as part of precision medicine. Methods: We have developed a digital polymerase chain reaction-based system for multiplex gene panel testing of cell-free DNA (cfDNA) obtained from intraocular liquid biopsies (patent number 7149000, 2022/9/30). Our system can detect specific gene mutations in minute cfDNA by amplifying specific gene loci in bubbles, even from an extremely small number of cells. Using this multiplex gene panel testing, we analyzed genetic mutations in nine regions of four genes, MYD88, CD79B, BTG2, and PIM1, using cfDNA obtained from intraocular liquid biopsies. The diagnostic performance of the novel system was evaluated in 86 samlpes of patients with IOL. We also retrospectively analyzed the prognostic potential of the genetic mutations for IOL. Lastly, in the case of CNS lymphoma and IOL, we presented the MYD88 mutation treated with tirabrutinib. Results: Among the four disease-specific genes, the MYD88 mutation was present in 55 of 86 cases (64%), and frequencies of the CD79B, BTG2, and PIM1 mutations were 47%, 38%, and 29%, respectively. At least one of these mutations was found in 83 of the 86 IOL cases examined (97%), making this assay highly sensitive (Fig. 1). The MYD88 mutation was not associated with recurrence in patients with recurrent and refractory CNS lymphoma and IOL. Conversely,patients with the CD79B mutation had high rates of relapse for CNS lymphoma: the 4-year rate of recurrence-free survival was 35%, compared to 79% in patients without the CD79B mutation (p=0.012). Patients with the BTG2 mutation also had high rates of relapse (Fig. 2). Furthermore, we present a case of CNS lynphoma and IOL. By testing IOL gene panel, only MYD88 mutation was detected in the vitreous sample of IOL patient. We have experienced this case in which the second-generation BTK inhibitor,tirabrutinib, was effective not only for central but also for intraocular lesions. Conclusion: Our panel enables rapid and accurate diagnosis of IOL with only a small sample of intraocular liquid. We believe that this multiplex gene panel testing may aid in the diagnosis of IOL in difficult-to-diagnose patients, including those with chronic uveitis, by preventing CNS invasion due to diagnostic delay. Moreover, genetic profiling panels of IOL can help to stratify clinical outcomes.CD79B and BTG2 mutation is a potent predictive marker for relapse. In the clinical setting, we experienced a case of IOL with MYD88 mutation in which tirabrutinib was effective. Potentially, tirabrutinib also penetrates directly into the eye in intraocular lesions. Oral drug, tirabrutinib as eye dripping drug, might be effective as a therapeutic option for IOL and should be assessed in future clinical trials of precision medicine for IOL.

Consultations in Eye Pathology: Experience at the Ophthalmology Specialty Hospital.

Ophthalmic pathology is a discipline that relies heavily on a knowledge of clinical ophthalmology. The diagnosis of ocular and periocular lesions can be challenging because some lesions and diseases are unique to this region, whereas others may demonstrate site-specific differences from nonocular counterparts. Because of these challenges, ocular and periocular biopsies are frequently referred to specialized ophthalmic pathology centers for second opinion diagnoses. To analyze the referral patterns, diagnostic challenges, and diagnostic discrepancies for second opinion referrals at a dedicated ophthalmic pathology laboratory with an emphasis on lesions of special interest in ophthalmic pathology. Data sources included the pathology records of all slides and blocks received in consultation at the referral eye pathology center between December 1, 2015, and December 1, 2022, the personal experience of senior authors, and published peer-reviewed literature. Corneal, intraocular, and conjunctival biopsies are the most common types of cases received in consultation without the referring pathologist's diagnosis, likely reflecting diagnostic challenges. Degenerative intraocular processes occasionally raise concern for a neoplasm. Conjunctival melanocytic lesions are the most common conjunctival biopsies referred for second opinion diagnosis and require careful tissue sampling and clinical-pathologic correlation. Careful clinical-pathologic correlation, a high level of suspicion, and adequate sampling also are required for the accurate diagnosis of periocular sebaceous carcinoma. The diagnostic discrepancies involving uveal, retinal, conjunctival, eyelid, and temporal artery biopsies are most likely to adversely influence patient management and possible outcome. Such specimens may benefit from referral to specialized ophthalmic pathology laboratories.

Diagnostic Aqueous Humor Proteome Predicts Metastatic Potential in Uveal Melanoma.

Gene expression profiling (GEP) is clinically validated to stratify the risk of metastasis by assigning uveal melanoma (UM) patients to two highly prognostic molecular classes: class 1 (low metastatic risk) and class 2 (high metastatic risk). However, GEP requires intraocular tumor biopsy, which is limited by small tumor size and tumor heterogeneity; furthermore, there are small risks of retinal hemorrhage, bleeding, or tumor dissemination. Thus, ocular liquid biopsy has emerged as a less-invasive alternative. In this study, we seek to determine the aqueous humor (AH) proteome related to the advanced GEP class 2 using diagnostic AH liquid biopsy specimens. Twenty AH samples were collected from patients with UM, grouped by GEP classes. Protein expression levels of 1472 targets were analyzed, compared between GEP classes, and correlated with clinical features. Significant differentially expressed proteins (DEPs) were subjected to analysis for cellular pathway and upstream regulator identification. The results showed that 45 DEPs detected in the AH could differentiate GEP class 1 and 2 at diagnosis. IL1R and SPRY2 are potential upstream regulators for the 8/45 DEPs that contribute to metastasis-related pathways. AH liquid biopsy offers a new opportunity to determine metastatic potential for patients in the absence of tumor biopsy.

Abstract 5553: Diagnostic aqueous humor proteome predicts metastatic potential in uveal melanoma

Abstract Introduction: Approximately half of patients with uveal melanoma (UM), a primary eye cancer, will develop metastatic disease. Gene expression profiling (GEP) is clinically validated to stratify risk of metastasis by assigning UM patients to two highly prognostic molecular classes: class 1 (low metastatic risk) and class 2 (high metastatic risk). However, GEP requires intraocular tumor biopsy which are limited by small tumor size and tumor heterogeneity; furthermore, there are small risks of retinal hemorrhage, bleeding or tumor dissemination. Thus, liquid biopsy has emerged as a significantly less invasive alternative. Blood biopsy has largely been unsuccessful for clinical use in UM due to low detection rates in the setting disease limited to the eye, however eye-specific aqueous humor (AH) liquid biopsy may be a better alternative. In this study, we seek to determine the AH proteome related to the advanced GEP class 2 using the diagnostic AH specimens. Method: Twenty UM treatment naive AH were collected before plaque brachytherapy. Patients were sub-grouped by GEP classes into GEP 1 (n=12) and GEP 2 (n=5). Three patients were classified as GEP unknown (GEP NA, n=3) due to the unavailability of tumor biopsy. Ten microliters of AH samples were analyzed by proximity extension assay-derived multiplexed Olink platform. Protein expression levels of 1472 targets were analyzed, compared between GEP classes and correlated with clinical features. Significant differentially expressed proteins (DEPs, fold-change (FC) > 2 or FC < 0.5, P < 0.01) were subjected to Qiagen ingenuity pathway analysis for cellular pathway and upstream regulator identification. Results: GEP 2 class was correlated with AJCC stages (P = 0.012), advanced clinical tumor stages (P = 0.007) and mutated BAP1 (P = 0.018). 45 DEPs were identified when comparing GEP classes. Among them, 31 are up-regulated DEPs [fold-change (FC) >2, P < 0.01] and 14 are down-regulated DEPs (FC < 0.5, P < 0.01) in GEP 2 compared to GEP1. The unsupervised clustering analysis showed that the 45 DEPs well-differentiate AH samples by GEP classes, and the 3 GEP NA samples were clustered with GEP1 class. Pathway analysis showed that 45 DEPs contribute to metastatic-related pathways including cellular movement, cellular proliferation and epithelial to mesenchymal transition. Two upstream regulators, IL1 receptor and SPRY2, were predicted to regulate 8 out of the 45 DEPs. IL1R2 (FC = 3.4, P = 0.021) and SPRY2 (FC = 0.6, P = 0.052) protein expression levels were found matched as prediction in our Olink dataset. Conclusions: 45 AH DEPs could differentiate GEP class 1 and 2 at the diagnostic stage and could be detected even when the tumor was too small to biopsy. IL1R and SPRY2 are potential upstream regulators for the 8/45 DEPs that contribute to metastasis-related pathways. AH liquid biopsy offers a new opportunity to determine metastatic potential for patients in the absence of tumor biopsy. Citation Format: Chen-Ching Peng, Liya Xu, Jesse Berry. Diagnostic aqueous humor proteome predicts metastatic potential in uveal melanoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5553.

Update in Molecular Testing for Intraocular Lymphoma.

Simple SummaryThe diagnosis of primary vitreoretinal lymphoma and central nervous system lymphoma is challenging. Intraocular biopsy and molecular testing are important for the diagnosis of cases with intraocular involvement. Intraocular biopsy does not always result in a tissue diagnosis. There are many new molecular tests that are currently being used to improve the yield of intraocular biopsy. This article will review the available molecular tests for intraocular lymphoma.The diagnosis of primary vitreoretinal lymphoma and central nervous system lymphoma is challenging. In cases with intraocular involvement, vitreous biopsy plays a pivotal role. Several diagnostic tests are employed to confirm a diagnosis and include cytologic evaluation, immunohistochemistry, flow cytometry, and cytokine analysis. The limitations of these conventional diagnostic tests stem from the often paucicellular nature of vitreous biopsy specimens and the fragility of malignant cells ex vivo. Several emerging molecular techniques show promise in improving the diagnostic yield of intraocular biopsy, possibly enabling more accurate and timely diagnoses. This article will review existing diagnostic modalities for intraocular lymphoma, with an emphasis on currently available molecular tests.

Challenges in the diagnosis and management of vitreoretinal lymphoma - Clinical and basic approaches.

Vitreoretinal lymphoma (VRL) is a subtype of diffuse large B-cell lymphoma and is sight- and life-threatening in the vast majority of patients. Lymphoma cells infiltrate the vitreous body and/or subretinal space and exhibit clinical signs of vitreous opacities and creamy white subretinal lesions. Although the intraocular signs can serve as clues to suspect VRL, they are nonspecific and may be misdiagnosed as uveitis. Histopathological evidence of malignant cells on vitreous biopsy, for instance, is the gold standard for diagnosis of VRL; however, cytological examination of the vitreous often results in a low success rate owing to the small quantity and poor quality of tissues and cells in the sample. Recent advancements in immunological, molecular, and gene analyses using intraocular samples have made it possible to accurately diagnose VRL. As for the management of VRL, local treatments with irradiation and/or intravitreal injections of anti-tumor agents (methotrexate or rituximab) are effective in suppressing intraocular VRL lesions. However, the effect of systemic chemotherapy, with or without brain irradiation, on preventing central nervous system involvements remains controversial. In this review article, we discuss the following concepts based on previous literature and our unpublished results: current ocular imaging examinations such as optical coherence tomography and fundus autofluorescence; immunological, molecular, and gene expression characterization of intraocular biopsies with special attention to flow cytometry; immunoglobulin gene rearrangement assays that use the polymerase chain reaction test; cytokine assays; gene mutations (MYD88, CD79B); and current local and systemic treatments of VRL.

Abstract 1967: Potential of aqueous humor as a liquid biopsy for uveal melanoma

Abstract Introduction: Uveal melanoma (UM) is the most common primary intraocular tumor in adults and arises from the uvea (iris, ciliary body and choroid). Although there are prognostic biomarkers available, identifying them requires intraocular tumor biopsy. Blood biopsy has emerged as a significantly less invasive alternative that also offers the ability to longitudinally track disease. However, blood biopsy has been unsuccessful for clinical use in identifying prognostic biomarkers in UM due to low detection rates. Thus, eye-specific aqueous humor (AH) biopsy may be a better alternative. Methods: This case series includes 20 primary UM patients with 13 (65%) choroid and 7 (35%) ciliary body (CB) tumors. AH samples were collected prior to brachytherapy plaque placement (n = 17), after brachytherapy (n = 19) and at enucleation (n = 1). A tumor wash sample (n = 1) was collected after routine fine needle aspiration biopsy. Nucleic acids ([NA], dsDNA, RNA, ssDNA and miRNA) were quantitated by Qubit Assay Kits. All AH samples and cells from tumor washing underwent DNA isolation and was followed by shallow whole-genome sequencing for copy-number profiling. Results: Comparison of pre- and post-radiation samples revealed significantly higher concentration of ssDNA (n = 10 pairs, P = 0.039) and miRNA (n = 10 pairs, P = 0.008) in choroid post- samples and significantly higher concentration of dsDNA (n = 7 pairs, P = 0.031) and miRNA (n = 7 pairs, P = 0.016) in CB post- samples. Between choroid (n = 12) and CB (n = 7) post- samples, there was a significantly higher concentration of dsDNA (P = 0.022) and RNA (P = 0.009) in post- CB samples, suggesting NA content is released from the disturbance of tumor cells by radiation and due to the proximity of the tumor to the AH in CB. Highly recurrent UM somatic copy number alterations (SCNAs) of monosomy 3, 6p gain, 6q loss and 8q gain were identified in post- AH samples. There was a significant difference between altered SCNA profiles in CB and choroid (P= 0.038). Altered SCNA profiles were found in 1/12 (8.3%) and 4/7 (57.1%) post-radiation AH samples of choroid and CB, respectively. No SCNAs were found in the same eyes’ pre-radiation sample. A high concordance of DNA profiles between a tumor wash and post- AH sample was identified (r = 0.978) and UM mutations GNAQ and BAP1 were detected in 3/6 (50%) post- AH with 2/5 (40%) matching tumor samples, demonstrating AH dsDNA’s tumor-origin. Conclusions: The AH is a source of cfDNA, with a higher yield of NAs after radiation in UM. Given the significant increase in NA concentration after radiation and in CB compared to choroid tumors, we hypothesize that all detected NAs post-radiation are tumor-derived due to apoptosis of tumor cells and subsequent release of cfDNA and due to the proximity of the CB tumor to AH. These results represent the first time that UM SCNAs and mutations were identified in cfDNA isolated from the AH, and this suggests that the AH can serve as a liquid biopsy for UM, especially in CB tumors. Citation Format: Deborah H. Im, Chen-Ching Peng, Liya Xu, Mary E. Kim, Dejerianne Ostrow, Venkata Yellapantula, Moiz Bootwalla, Jaclyn A. Biegel, Xiaowu Gai, Peter Kuhn, James Hicks, Jesse L. Berry. Potential of aqueous humor as a liquid biopsy for uveal melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1967.

Deep Metagenomic Sequencing for Endophthalmitis Pathogen Detection Using a Nanopore Platform

To evaluate the utility of nanopore sequencing for identifying potential causative pathogens in endophthalmitis, comparing culture results against full-length 16S rRNA nanopore sequencing (16S Nanopore), whole genome nanopore sequencing (Nanopore WGS), and Illumina (Illumina WGS). Cross-sectional diagnostic comparison. Patients with clinically suspected endophthalmitis underwent intraocular vitreous biopsy as per standard care. Clinical samples were cultured by conventional methods, together with full-length 16S rRNA and WGS using nanopore and Illumina sequencing platforms. Of 23 patients (median age 68.5 years [range 47-88]; 14 males [61%]), 18 cases were culture-positive. Nanopore sequencing identified the same cultured organism in all of the culture-positive cases and identified potential pathogens in two culture-negative cases (40%). Nanopore WGS was able to additionally detect the presence of bacteriophages in three samples. The agreements at genus level between culture and 16S Nanopore, Nanopore WGS, and Illumina WGS were 75%, 100%, and 78%, respectively. Whole genome sequencing has higher sensitivity and provides a viable alternative to culture and 16S sequencing for detecting potential pathogens in endophthalmitis. Moreover, WGS has the ability to detect other potential pathogens in culture-negative cases. Whilst Nanopore and Illumina WGS provide comparable data, nanopore sequencing provides potential for cost-effective point-of-care diagnostics.

BAP1 Immunostain Status in Intraocular Biopsy Specimens for Uveal Melanoma Highly Correlates with Other Prognostic Markers

Introduction: Loss of BAP1 protein expression emerged as a negative prognostic marker in uveal melanoma (UM) and has primarily been studied in enucleations. Intraocular biopsy is frequently performed prior to UM globe-conserving therapy. Methods: We retrospectively evaluated BAP1 immunostaining of UM in 16 biopsies and 8 subsequent enucleations, and results were correlated with the UM-specific gene expression profile (GEP; n = 11), chromosome 3 status by FISH and/or chromosomal microarray (n = 12; 9 also had GEP), and clinical outcomes. Results: UM involved the choroid in 15 (of 16) cases. Biopsy was performed for prognostication (n = 12) or diagnosis (n = 4). Treatment included brachytherapy (n = 13; 5 followed by enucleation) or enucleation only (n = 3). BAP1 nuclear immunostaining was positive in 9, negative in 4, and equivocal in 3 biopsies. For the 3 equivocal biopsies, BAP1 immunostaining was positive in 2 (of 3) subsequent enucleations. BAP1 immunostaining was concordant between all 5 remaining biopsies and enucleations. BAP1-positive biopsies had disomy 3 (n = 6) or 3p loss (n = 1) and class 1 GEP (n = 6). BAP1-negative biopsies had monosomy 3 (n = 3) and class 2 GEP (n = 2). Median follow-up was 62.5 months (range, 17–150). For BAP1-positive UM patients, 8 were alive (7 without metastatic disease) and 3 had died (1 melanoma-related death). Among BAP1-negative UM patients, 2 were alive (1 with metastatic disease) and 3 had melanoma-related deaths. Conclusion: BAP1 immunostaining in biopsies highly correlates with results in subsequent enucleations and with well-established UM prognostic markers, representing a potential additional prognostic tool for UM biopsies.

Abstract 554: Aqueous humor as a liquid biopsy for uveal melanoma

Abstract Purpose: Metastatic Uveal Melanoma (UM) is usually fatal within one year of symptom onset. There are a variety of biomarkers that can stratify patients' metastatic risk, but identifying them requires intraocular tumor biopsy. Liquid biopsy offers a less invasive alterative without the risks of retinal detachment and intraocular hemorrhage. We investigated whether the aqueous humor (AH) has sufficient tumor-derived cell-free DNA (cfDNA) to function as a liquid biopsy for UM. Methods: This case series includes 12 AH samples from 7 eyes of 7 UM patients from whom AH was sampled during routine brachytherapy plaque placement and/or removal. Double stranded DNA (dsDNA), single stranded DNA (ssDNA), RNA, and microRNA (miRNA) were quantified with respective Qubit High Sensitivity Assay Kits (Thermo Fischer) for each sample. The lower limit of detection was 0.01 ng/µL for dsDNA, 0.05 ng/µL for ssDNA, 0.25 ng/µL for RNA, and 0.05 ng/µL for miRNA. CfDNA was then isolated and sequenced on an Illumina platform to assess genome-wide somatic copy number alterations (SCNAs). Results: Aqueous humor derived cfDNA was detected in all samples, ranging from 0.085-28.8 ng/µL (mean = 2.7 ng/µL). RNA was detected in 2 of 12 samples (mean = 1.6 ng/µL, range = 0.7-2.5 ng/µL), and miRNA was detected in 11 of 12 samples (mean = 1.7 ng/µL, range = 0.094-16.2 ng/µL). There was a significantly higher cfDNA concentration in post-radiation samples than pre-radiation samples, (median = 0.082 ng/µL in pre-radiation, 0.21 ng/µL in post-radiation, W = 1, z = -2.55, p < 0.05, Wilcoxon Signed-rank test). This finding was similar when comparing all nucleic acid concentrations (dsDNA, ssDNA, RNA, and miRNA) between pre- and post-radiation (median = 0.085 ng/µL in pre-radiation, 0.333 ng/µL in post-radiation, W = 1, z = -3.46, p = 0.00027). Confirmed UM genomic alterations of 6p gain, 6q loss, and 8q gain were identified in one post-radiation AH sample from an eye with an iridociliary body tumor. These SCNAs are characteristic of UM. No SCNAs were found in the AH from the pre-radiation sample in the same eye, or any of the other AH samples sequenced to date. Conclusions: The AH is a source of cfDNA in eyes with UM, with a higher yield of all nucleic acids detected after brachytherapy radiation treatment. UM associated SCNAs were identified in the AH of one eye with an iridociliary tumor after radiation only. We hypothesize this may be due to intratumoral necrosis from the radiation as well as the proximity of the primary tumor to the AH in the case where SCNAs were identified. These results represent the first time that UM SCNAs have been identified in cfDNA isolated from the AH, and this provides preliminary evidence that the AH can serve as a liquid biopsy for UM. Citation Format: Mary E. Kim, Liya Xu, Chen-Ching Peng, Peter Kuhn, James Hicks, Jesse L. Berry. Aqueous humor as a liquid biopsy for uveal melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 554.

Epibulbar Seeding of Uveal MALT Lymphoma Following Vitrectomy

Purpose: One of the most feared complications of intraocular tumor biopsy with fine needle aspiration biopsy (FNAB) or vitrectomy-assisted biopsy is seeding of the tumor cells directly into the needle tract. We present a case of epibulbar seeding of uveal MALT lymphoma following vitrectomy. Observations: A 67-year-old male presented with painless loss of vision of the left eye and creamy yellow infiltrates at the level of the choroid. A diagnostic 25-gauge pars plana vitrectomy was performed using a standard 3-port approach. Choroidal biopsy of the lesion demonstrated the presence of Mucosa-Associated Lymphoid Tissue (MALT) lymphoma. Three weeks following vitrectomy, the patient developed a large fleshy subconjunctival mass centered over the prior superonasal sclerotomy site. The lesion was completely excised and histopathology of the lesion demonstrated MALT lymphoma with an identical immunohistochemical staining pattern to that of the choroidal lesion. Conclusions and Importance: Transvitreal biopsy with fine needle aspiration or vitrectomy is an increasingly common and safe procedure for clinical diagnosis of intraocular tumors. However, there is a risk of tumor seeding of the needle tract and precautions to reduce this risk are warranted.

Uveal Melanoma Biopsy. A Review

In intraocular tumors, diagnosis is usually based on clinical examination and imaging without the need for invasive surgery or tissue sampling. The diagnosis can be confirmed by biopsy, however, in the case of intraocular malignancy, the biopsy is considered controversial. Due to the development of uveal melanoma cytogenetic prognostics and the progression in generalised uveal melanoma treatment, intraocular melanoma biopsy is becoming increasingly important. Diagnostic biopsy of intraocular tumors is indicated in cases of diagnostic uncertainty for findings with conflicting non-invasive test results and for small melanocyte lesions. Tumor prognostic biopsy is performed to obtain a tissue sample for tumor cytogenetic testing, which can help to determine the prognosis and specific metastatic risk of the patient. For anterior segment tumors, anterior chamber fluid sampling, thin-needle iris biopsy, punch biopsy, surgical biopsy or biopsy using vitrectomy may be used. For posterior segment tumors, procedures include transscleral or transretinal thin-needle biopsy, vitrectomy-assisted biopsy, punch biopsy, endoresection or transscleral exoresection. Complications of intraocular melanoma biopsy include too small or non-valuable sample collection, intra-tumoral heterogeneity, intra-ocular trauma and induction of intraocular or extraocular tumor dissemination.

Cell-free DNA profiling in retinoblastoma patients with advanced intraocular disease: An MSKCC experience.

PurposeThe enucleation rate for retinoblastoma has dropped from over 95% to under 10% in the past 10 years as a result of improvements in therapy. This reduces access to tumor tissue for molecular profiling, especially in unilateral retinoblastoma, and hinders the confirmation of somatic RB1 mutations necessary for genetic counseling. Plasma cell‐free DNA (cfDNA) has provided a platform for noninvasive molecular profiling in cancer, but its applicability in low tumor burden retinoblastoma has not been shown. We analyzed cfDNA collected from 10 patients with available tumor tissue to determine whether sufficient tumorderived cfDNA is shed in plasma from retinoblastoma tumors to enable noninvasive RB1 mutation detection.MethodsTumor tissue was collected from eye enucleations in 10 patients diagnosed with advanced intra‐ocular unilateral retinoblastoma, three of which went on to develop metastatic disease. Tumor RB1 mutation status was determined using an FDA‐cleared tumor sequencing assay, MSK‐IMPACT. Plasma samples were collected before eye enucleation and analyzed with a customized panel targeting all exons of RB1.ResultsTumor‐guided genotyping detected 10 of the 13 expected somatic RB1 mutations in plasma cfDNA in 8 of 10 patients (average variant allele frequency 3.78%). Without referring to RB1 status in the tumor, de novo mutation calling identified 7 of the 13 expected RB1 mutations (in 6 of 10 patients) with high confidence.ConclusionPlasma cfDNA can detect somatic RB1 mutations in patients with unilateral retinoblastoma. Since intraocular biopsies are avoided in these patients because of concern about spreading tumor, cfDNA can potentially offer a noninvasive platform to guide clinical decisions about treatment, follow‐up schemes, and risk of metastasis.

Uveitis Masquerade Syndrome: Typical Symptoms and Presentations

The uveitis masquerade syndrome (UMS) describes a series of malignant diseases that mimic inflammatory conditions without belonging to the group of immune-mediated uveitis entities. The correct diagnosis is often difficult to establish, despite a detailed ophthalmological and general medical history using all imaging modalities and interdisciplinary cooperation. In the case of recurrent inflammation, it is very important to consider an UMS, since any delay in proving the underlying diagnosis and initiating the appropriate therapy may be associated with the risk of systemic spread and consecutive deterioration of the prognosis. Often, after all conservative options have been exhausted, the only remaining option to confirm the correct diagnosis might be an intraocular biopsy. The present work focuses on the leading symptoms, differential diagnoses and clinical signs of UMS.

Targeted Next-Generation Sequencing of 117 Routine Clinical Samples Provides Further Insights into the Molecular Landscape of Uveal Melanoma.

Uveal melanoma (UM) has well-characterised somatic copy number alterations (SCNA) in chromosomes 1, 3, 6 and 8, in addition to mutations in GNAQ, GNA11, CYSLTR2, PLCB4, BAP1, SF3B1 and EIF1AX, most being linked to metastatic-risk. To gain further insight into the molecular landscape of UM, we designed a targeted next-generation sequencing (NGS) panel to detect SCNA and mutations in routine clinical UM samples. We compared hybrid-capture and amplicon-based target enrichment methods and tested a larger cohort of primary UM samples on the best performing panel. UM clinical samples processed either as fresh-frozen, formalin-fixed paraffin embedded (FFPE), small intraocular biopsies or following irradiation were successfully profiled using NGS, with hybrid capture outperforming the PCR-based enrichment methodology. We identified monosomy 3 (M3)-UM that were wild-type for BAP1 but harbored SF3B1 mutations, novel frameshift deletions in SF3B1 and EIF1AX, as well as a PLCB4 mutation outside of the hotspot on exon 20 coinciding with a GNAQ mutation in some UM. We observed samples that harboured mutations in both BAP1 and SF3B1, and SF3B1 and EIF1AX, respectively. Novel mutations were also identified in TTC28, KTN1, CSMD1 and TP53BP1. NGS can simultaneously assess SCNA and mutation data in UM, in a reliable and reproducible way, irrespective of sample type or previous processing. BAP1 and SF3B1 mutations, in addition to 8q copy number, are of added importance when determining UM patient outcome.

Prognostic Utility of Whole-Genome Sequencing and Polymerase Chain Reaction Tests of Ocular Fluids in Postprocedural Endophthalmitis

To associate detection of potential pathogen DNA in endophthalmitis with clinical outcomes. Prospective cohort study. Patients in whom endophthalmitis was diagnosed following an intraocular procedure were recruited. Clinical outcome data from baseline, week-1, month-1, and month-3 visits were collected. Intraocular biopsy samples were cultured by standard methods. Quantitative polymerase chain reaction (qPCR) was performed for specific pathogens and whole-genome sequencing (WGS). A total of 50 patients (mean age 72 years old; 52% male) were enrolled. Twenty-four cases were culture-positive and 26 were culture-negative. WGS identified the cultured organism in 76% of culture-positive cases and identified potential pathogens in 33% of culture-negative cases. Month-1 and -3 visual acuities did not vary by pathogen-positive versus pathogen-negative cases as detected by either culture or WGS. Visual outcomes of Staphylococcus epidermidis endophthalmitis were no different than those of pathogen-negative cases, whereas the patients infected with other pathogens showed worse outcome. Higher baseline bacterial DNA loads of bacteria other than those of S epidermidis detected by WGS were associated with worse month-1 and -3 visual acuity, whereas the S epidermidis loads did not appear to influence outcomes. Torque teno virus (TTV) and Merkel cell polyomavirus (MCV) were detected by qPCR in 49% and 19% of cases, respectively. Presence of TTV at presentation was associated with a higher rate of secondary pars plana vitrectomy (P= .009) and retinal detachment (P= .022). The presence and higher load of bacteria other than S epidermidis detected by WGS orDNAfrom TTV by qPCR in ocular fluids is associatedwith worse outcomes in post-procedure endophthalmitis.

Intraocular biopsy in uveitis.

An intraocular biopsy is performed for diagnostic, prognostic and investigational purposes. Biopsies help to confirm or exclude malignancies and differentiate inflammatory from infectious processes. Histopathological analysis is the final verdict in unresponsive uveitis, atypical inflammation, metastases and masquerade syndromes. Advances and refinement of techniques in cytopathology, immunohistochemistry, microbiological and molecular biologic study offer much more than just diagnosis. They provide prognosis based on cell characteristics and are helpful in planning treatment and intervention. Many biopsy procedures have evolved to provide more safety and minimise complications thus improving the quality of specimens or samples available for analysis. The type of biopsy and technique adopted varies based on the clinical suspicion, size and location of lesions. In uveitis, a working diagnosis of intraocular inflammation is made on clinical examination and laboratory investigations and ancillary tests. Malignancy and uveitis is interlinked and masquerade syndromes are among the commonest indications for biopsy and analysis of specimen. The various types of intraocular biopsies include aqueous tap, fine needle aspiration biopsy, vitreous biopsy, iris and ciliary body, and retinochoroidal biopsy. They will be reviewed in this article with respect to current perspective

Multi-center analysis of intraocular biopsy technique and outcomes for uveal melanoma: Ocular Oncology Study Consortium report 4.

To investigate the relationship between surgical approach for intraocular tumor biopsy of uveal melanoma and tumor morphologic features such as size and intraocular location and the effect of these variables on diagnostic yield and biopsy outcome. Consecutive patients from nine Ocular Oncology centers with uveal melanoma (UM) undergoing tumor biopsy immediately preceding I125 plaque brachytherapy with tissue sent for gene expression profiling (GEP) testing were reviewed retrospectively. Three hundred sixty patients were included (50% men, mean age 60.2years). Overall biopsy yield was 99% and 83% for GEP and cytopathology, respectively. Surgeon choice of biopsy approach (trans-vitreal vs. trans-scleral) was found to associate with both tumor location and tumor thickness. A trans-scleral rather than trans-vitreal approach was used more commonly for anteriorly located tumors (92% vs. 38% of posterior tumors, p< 0.001) and thicker tumors (86% vs. 55% of thin tumors, p< 0.001). When performing trans-vitreal biopsies, ocular oncologists with previous vitreoretinal surgery fellowship training were more likely to use wide-field surgical viewing systems, compared with indirect ophthalmoscopy (82.6% vs. 20.6%, p< 0.001). Surgical complications were rare and occurred more frequently with trans-vitreal biopsies (3.6% vs. 0.46%, p= 0.046). In this multi-center analysis of UM tumor biopsy, surgical yield was high for obtaining tumor tissue for GEP and cytopathology analysis with both trans-scleral and trans-vitreal techniques. Fellowship-trained ocular oncologists' preferred intraocular biopsy techniques associated strongly with tumor location, tumor thickness, and fellowship training of the surgeon. Short-term complication rates were low.

Intraocular Biopsy and ImmunoMolecular Pathology for "Unmasking" Intraocular Inflammatory Diseases.

Intraocular inflammation can hide a variety of eye pathologies. In 33% of cases, to obtain a correct diagnosis, investigation of the intraocular sample is necessary. The combined analyses of the intraocular biopsy, using immuno-pathology and molecular biology, point to resolve the diagnostic dilemmas in those cases where history, clinical tests, and ophthalmic and systemic examinations are inconclusive. In such situations, the teamwork between the ophthalmologist and the molecular pathologist is critically important to discriminate between autoimmune diseases, infections, and intraocular tumors, including lymphoma and metastases, especially in those clinical settings known as masquerade syndromes. This comprehensive review focuses on the diagnostic use of intraocular biopsy and highlights its potential to enhance research in the field. It describes the different surgical techniques of obtaining the biopsy, risks, and complication rates. The review is organized according to the anatomical site of the sample: I. anterior chamber containing aqueous humor, II. iris and ciliary body, III. vitreous, and IV. choroid and retina. We have excluded the literature concerning biopsy for choroidal melanoma and retinoblastoma, as this is a specialized area more relevant to ocular oncology.