Probing mechanisms and improving management of glaucoma following Boston keratoprosthesis surgery.

Corneal blindness is a leading cause of visual impairment worldwide. The most common treatment is to replace the diseased cornea with standard corneal transplantation. In eyes at high risk of graft failure, the Boston keratoprosthesis type 1 (KPro) can be used to restore vision and is currently the most frequently used artificial cornea in the world. However, glaucoma is a well-known complication of KPro surgery and is the most important threat to vision in KPro-implanted eyes. This chronic disease is influenced by elevated intraocular pressure (IOP) and damages the optic nerve, leading to progressive vision loss. In KPro patients, glaucoma is highly prevalent and extremely challenging to manage, yet its exact cause remains unknown.

Probing mechanisms and improving management of glaucoma following Boston keratoprosthesis surgery.

ENGLISH SUMMARY: Corneal blindness is a leading cause of visual impairment worldwide. The most common treatment is to replace the diseased cornea by standard corneal transplantation. In eyes at high risk of graft failure, the Boston keratoprosthesis type 1 (KPro) can be used to restore vision and is currently the most frequently used artificial cornea in the world. However, glaucoma is a well-known complication of KPro surgery and is the most important threat to vision in KPro-implanted eyes (paper I). This chronic disease is influenced by elevated intraocular pressure (IOP) and damages the optic nerve, leading to progressive vision loss. In KPro patients, glaucoma is highly prevalent and extremely challenging to manage, yet its exact cause remains unknown. The overall purpose of this PhD Thesis (Geoffrion, 2021) was to better understand the mechanisms and how to improve management of glaucoma after KPro implantation. The approaches used in this thesis included investigating one of the largest KPro patient cohorts in North America, with a total of 157 operated patients at that time, as well as studying KPro surgery and outcomes in mice. The first objective was to identify risk factors for glaucoma development and progression after KPro implantation (paper II). Multivariate logistic regression revealed that high preoperative IOP signals a higher risk for both glaucoma development and progression. Stromal and endothelial corneal disorders were less associated with glaucoma progression, while autoimmune and ocular surface diseases precipitated glaucoma development. Second, there is no objective evidence that indicates the best order for glaucoma surgeries and KPro implantation. By comparing medical and surgical management in KPro eyes with either preexisting or de novo glaucoma (paper III), we showed that glaucoma surgery may be performed before or at the time of KPro in eyes with preexisting glaucoma to limit progression without increasing complications. In eyes with de novo glaucoma, glaucoma surgery did not increase complications compared with medications. Third, among glaucoma surgery interventions, the two most frequently implanted glaucoma drainage devices were compared in KPro patients (paper IV). Compared with the Ahmed glaucoma valve, the Baerveldt glaucoma implant was associated with lower failure rates, without increased postoperative complications. Fourth, even with aggressive management, many KPro patients suffer from progressive optic nerve damage, sometimes despite normal IOP. Inflammatory cytokines play an important role in glaucomatous optic neuropathy, but their role in KPro-associated glaucoma is still unknown. By analysing tear fluid of KPro patients by multiplex bead immunoassay (paper V), we identified that cytokines TNF-a, IL-1b, FGF-basic and IFN-g were elevated in KPro patients with glaucoma compared to those without. These cytokines correlated with optic nerve excavation and IOP. For the first time in humans, these results concorded with the elevations of TNF-a and IL-1b documented in the mouse KPro model. Ocular surface inflammation may thus reflect the inflammatory processes that perpetuate glaucoma damage years after KPro surgery. Fifth, we determined that miniaturized mouse KPro implantation requires extensive practice to be used as a reproducible model of glaucoma post-KPro (paper VI). KPro animal models with larger eyes and a full-thickness, 360-degree corneal excision should be prioritized to best validate human outcomes. In conclusion, glaucoma in KPro eyes is a long-lasting and multifactorial process. Most probable mechanisms combine IOP-independent inflammation mediated by TNF-a and IL-1b that prolong glaucoma damage, together with post-surgical angle closure elevating the IOP. Altogether, our results inform glaucoma risk profiling of transplant recipients, improvement of surgical management of KPro patients with glaucoma and development of targeted treatments to minimize glaucomatous damage after KPro. Ultimately, this work has the potential to preserve the vision of thousands of patients who undergo KPro surgery every year worldwide and to provide insight for the role of inflammation in other diseases involving neuronal damage. RÉSUMÉ (FRENCH SUMMARY): La cécité cornéenne est l'une des causes les plus importantes de déficience visuelle dans le monde. Le traitement usuel est de remplacer la cornée malade par une greffe de cornée traditionnelle. Dans les yeux à haut risque d'échec de greffe, la kératoprothèse de Boston de type 1 (KPro) peut rétablir la vision et est la cornée artificielle la plus utilisée au monde. Cependant, le glaucome est une complication importante de la KPro (papier I). Cette maladie chronique est influencée par une pression intraoculaire (PIO) élevée et endommage le nerf optique, menant à une perte de vision. Chez les patients avec KPro, le glaucome est fréquent et difficile à contrôler, mais sa cause exacte demeure inconnue. L'objectif principal de cette thèse est de découvrir les mécanismes et d'optimiser la prise en charge du glaucome après l'implantation de la KPro. Pour ce faire, nous avons investigué l'une des plus grandes cohortes de patients KPro en Amérique du Nord avec un total de 157 patients, ainsi qu'un groupe de souris ayant reçu une implantation de kératoprothèse. Le premier but était d'identifier les facteurs de risque pour le développement et la progression du glaucome après la KPro (papier II). Par régression logistique multivariée, nous avons démontré qu'une PIO préopératoire élevée mène à un plus grand risque de développement et de progression du glaucome. Les maladies cornéennes stromales ou endothéliales sont moins associées à une progression, alors que les maladies autoimmunes ou de la surface oculaire précipitent le développement du glaucome. Deuxièmement, il n'existe aucune donnée objective pour indiquer le meilleur ordre des chirurgies de glaucome et de KPro. En comparant les traitements médicaux et chirurgicaux des yeux KPro avec glaucome (papier III), nous avons démontré que les chirurgies de glaucome peuvent limiter la progression en étant effectuées avant ou pendant l'implantation de KPro dans les yeux avec glaucome préexistant, sans augmenter les complications. Dans le glaucome de novo, les chirurgies de glaucome n'augmentent pas les complications en comparaison aux médicaments. Troisièmement, les deux implants de glaucome les plus communs ont été étudiés chez les patients KPro (papier IV). Comparé à la valve Ahmed, l'implant Baerveldt est associé à des taux d'échec plus bas, sans augmentation des complications. Quatrièmement, même avec une prise en charge agressive, plusieurs patients KPro souffrent de glaucome qui progresse, parfois sans PIO élevée. Les cytokines inflammatoires jouent un rôle dans la pathophysiologie du glaucome, mais leur rôle dans le glaucome associé à la KPro est inconnu. En analysant les larmes de patients KPro (papier V), nous avons identifié que les cytokines TNF-a, IL-1b, FGF-basic et IFN-g sont élevées chez les patients KPro avec glaucome comparé à ceux sans glaucome. Ces cytokines corrèlent avec l'excavation du nerf optique et la PIO. Pour la première fois chez les humains, ces résultats concordent avec les niveaux élevés de TNF-a et IL-1b documentés dans le modèle murin de KPro. L'inflammation de la surface oculaire pourrait donc refléter les processus inflammatoires qui perpétuent le dommage glaucomateux. Cinquièmement, nous avons déterminé que l'implantation de la KPro miniature chez la souris requiert beaucoup de pratique pour être utilisé comme modèle de glaucome post-KPro (papier VI). Des modèles animaux avec des yeux plus larges et une excision cornéenne de pleine épaisseur sur 360 degrés devraient être priorisés pour valider les résultats chez l'humain. En conclusion, le glaucome associé à la KPro est un processus multifactoriel qui persiste à long terme. Les mécanismes probables combinent l'inflammation médiée par TNF-a et IL-1b et une fermeture de l'angle qui augmente la PIO. Nos résultats contribuent à établir les facteurs de risque de glaucome pour les receveurs de KPro, à améliorer leur prise en charge et à développer des thérapies ciblées. Ce travail a le potentiel de préserver la vision de milliers de patients recevant une KPro chaque année dans le monde et d'aider à mieux comprendre le rôle de l'inflammation dans d'autres maladies avec atteinte neuronale.

Assessment of the Role and Timing of Glaucoma Surgery in Boston Keratoprosthesis Type 1 Patients.

PURPOSE: To determine the role and optimal timing of glaucoma surgery in relation to Boston keratoprosthesis type 1 (KPro) implantation. DESIGN: Retrospective, comparative, nonrandomized clinical study. METHODS: Single-center study of a total of 100 eyes (100 patients) implanted with a KPro between 2008 and 2017, and diagnosed with glaucoma before or after KPro. Patients were separated into 2 groups: those with preexisting glaucoma and those who developed de novo glaucoma after KPro. Groups were then divided based on whether patients were medically or surgically managed. Glaucoma surgery included glaucoma drainage device (GDD) implantation, trabeculectomy, and cyclophotocoagulation (CPC). Primary outcomes included best-corrected visual acuity (BCVA), glaucoma progression, and complications. Differences in outcomes were compared using parametric and nonparametric tests, as well as log-rank test to compare time-to-outcome events. RESULTS: Among 72 eyes with preexisting glaucoma, 27 (38%) had glaucoma surgery before KPro (18 GDD), whereas 45 (62%) were medically managed only. Among the latter, 19 (42%) needed glaucoma surgery post-KPro (16 GDD). Among 28 eyes with de novo glaucoma, 12 (43%) had glaucoma surgery post-KPro (9 GDD). For eyes with preexisting glaucoma, glaucoma progression was greater with glaucoma surgery performed post-KPro (100%) compared with pre-KPro (74%, P = .016) and to medical management (54%, P = .002). No increase in complications were observed with glaucoma surgery compared to medications only (P > .05), whereas fewer eyes maintained a BCVA of 20/200 or better over time with medical management (P = .013). Eyes with de novo glaucoma had similar progression, BCVA, and complications between medical and surgical care (P > .05). CONCLUSIONS: Glaucoma surgery should be performed before or at the same time as KPro implantation in eyes with preexisting glaucoma. Complication rates are not increased when glaucoma surgery is performed in KPro eyes with either preexisting or de novo glaucoma. To ensure optimal glaucoma control, glaucoma surgery should be performed as early as possible in KPro eyes with good visual potential.

Perspectives for preclinical mouse models of glaucoma after Boston keratoprosthesis type 1.

Animal models of the Boston keratoprosthesis type 1 (KPro) are needed to study glaucoma damage after KPro implantation to control for confounding comorbidities common in human KPro recipients. The purpose of this study was to determine the feasibility of establishing a reproducible mouse model of glaucoma after KPro surgery, specifically that of a miniaturized mouse model of KPro (mKPro). In the present study, a total of 20 corneas of donor C57BL/6 mice (n = 10) were implanted in one eye of each recipient BALB/C mouse (n = 20), assembled as part of the mKPro, either with or without intraoperative lensectomy. Main feasibility outcomes consisted in incidence rates of loss of tone, capsule nicking, and lens extrusion, as well as acquisition of posterior segment optical coherence tomography (OCT) images. With lensectomy (n = 10), loss of ocular tone and retinal detachment occurred in 100% of mice. Without lensectomy (n = 10), capsule nicking and opening, as well as lens extrusion, occurred in 80% of mice. Causes of these complications included the large proportion of intraocular volume occupied by the lens, the shallow anterior chamber, and thus the lack of available intraocular volume to implant the KPro if the lens remains present. Successful mouse KPro surgery may require a great deal of practice to be useful as a reproducible model. Animal KPro models ought to be pursued further by research teams in future studies.

Tear Film Cytokine Profile of Patients With the Boston Keratoprosthesis Type 1: Comparing Patients With and Without Glaucoma.

Purpose: Inflammatory cytokines are involved in glaucoma pathogenesis. The purpose is to compare cytokine levels in the tear film of Boston keratoprosthesis (KPro) patients with and without glaucoma, relative to controls, and correlate levels with clinical parameters. Methods: This cross-sectional study enrolled 58 eyes (58 patients): 41 KPro eyes with glaucoma, 7 KPro eyes without glaucoma, and 10 healthy controls. Twenty-seven cytokines were measured by multiplex bead immunoassay. Intraocular pressure (IOP), cup-to-disk ratio (CDR), retinal nerve fiber layer, visual acuity, topical medications, and angle closure were assessed in all KPro eyes. Cytokine levels between groups were analyzed by nonparametric tests, and correlations with clinical parameters by Spearman's test. Results: Levels of TNF-ɑ, IL-1ß, FGF-basic, and IFN-É£ were significantly higher in KPro with glaucoma compared to KPro without (P = 0.020; 0.008; 0.043; 0.018, respectively). KPro groups had similar characteristics and topical antibiotic/steroid regimen. Levels of IL-1Ra, IL-15, VEGF, and RANTES were significantly higher in KPro with glaucoma compared to controls (P < 0.001; = 0.034; < 0.001; = 0.001, respectively). IL-1ß and IFN-É£ levels were positively correlated with CDR (r = 0.309, P = 0.039 and r = 0.452, P = 0.006, respectively) and IOP (r = 0.292, P = 0.047 and r = 0.368, P = 0.023, respectively). TNF-α and FGF-basic levels were positively correlated with CDR (r = 0.348, P = 0.022 and r = 0.344, P = 0.021, respectively). Conclusions: TNF-α, IL-1ß, FGF-basic, IFN-É£ are elevated in tears of KPro patients with glaucoma and correlate with CDR and IOP. These results show, for the first time in humans, concordance with documented elevations of TNF-α and IL-1ß in the murine KPro model. Ocular surface inflammation may reflect inflammatory processes of KPro glaucoma.

Apoptotic Blocks in Primary Non-Hodgkin B Cell Lymphomas Identified by BH3 Profiling.

To determine causes of apoptotic resistance, we analyzed 124 primary B cell NHL samples using BH3 profiling, a technique that measures the mitochondrial permeabilization upon exposure to synthetic BH3 peptides. Our cohort included samples from chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), high-grade B cell lymphoma with translocations in MYC and BCL2 (HGBL-DH), mantle cell lymphoma (MCL) and marginal zone lymphoma (MZL). While a large number of our samples displayed appropriate responses to apoptosis-inducing peptides, pro-apoptotic functional defects, implicating BAX, BAK, BIM or BID, were seen in 32.4% of high-grade NHLs (12/37) and in 3.4% of low-grade NHLs (3/87, p < 0.0001). The inhibition of single anti-apoptotic proteins induced apoptosis in only a few samples, however, the dual inhibition of BCL2 and MCL1 was effective in 83% of samples, indicating MCL1 was the most common cause of lack of response to the BCL2 inhibitor, venetoclax. We then profiled Toledo and OCI-Ly8 high-grade lymphoma cell lines to determine which drugs could reduce MCL1 expression and potentiate venetoclax responses. Doxorubicin and vincristine decreased levels of MCL1 and increased venetoclax-induced apoptosis (all p < 0.05). Overall, in primary NHLs expressing BCL2 that have no defects in pro-apoptotic signaling, a poor response to venetoclax is primarily due to the presence of MCL1, which may be overcome by combining venetoclax with doxorubicin and vincristine-based chemotherapy or with other anti-microtubule inhibitors.

Glaucoma Risk Factors and Outcomes Following Boston Keratoprosthesis Type 1 Surgery.

PURPOSE: To evaluate glaucoma risk factors and associated outcomes of the Boston keratoprosthesis type I (KPro). DESIGN: Clinical case-control study. METHODS: This is a single-center study of 140 eyes of 118 patients who underwent KPro surgery between October 2008 and March 2017 by a single surgeon. A total of 118 eyes of 118 patients with at least 6 months of follow-up were analyzed to account for intereye correlation. Patients without glaucoma were compared to those diagnosed with glaucoma, which included treatment with intraocular pressure (IOP)-lowering medications or glaucoma surgery. A subgroup analysis compared eyes with pre-KPro glaucoma with those with post-KPro glaucoma. Statistical analysis was performed using univariate and multivariate analyses and Kaplan-Meier survival curves. Main outcome measures were glaucoma diagnosis and progression. Other outcomes included demographics, preoperative diagnosis, best-corrected visual acuity, IOP, cup-to-disc ratio progression and postoperative complications. RESULTS: The mean age at surgery was 60.7 ± 16.7 years, with a follow-up of 6.9 ± 3.2 years. De novo KPro glaucoma incidence was 24% (n = 28/118), equivalent to 3.4 cases per 100 eye-years, with onset at 2.1 ± 2.2 postoperative years. A total of 17 of 118 eyes (14%) did not have glaucoma. Multiple logistic regression showed that high preoperative IOP was a predictor of higher rates of glaucoma development (odds ratio [OR] = 1.538, 95% confidence interval [CI] = 1.030-2.297, P = .035) and progression (OR = 1.450, 95% CI = 1.084-1.937, P = .012). Stromal and endothelial disorders were protective preoperative diagnoses for glaucoma progression after KPro (OR = 0.002, 95% CI = 0.000-0.227, P = .010). A greater proportion of eyes with autoimmune and ocular surface diseases developed de novo glaucoma after KPro compared with other preoperative diagnoses (P < .05). A total of 45% of glaucomatous KPro eyes suffered postoperative glaucoma progression. The mean final best-corrected visual acuity of the cohort was 1.76 ± 1.0, with no difference between eyes with and without glaucoma (P > .05). The rate of serious vision-threatening complications was higher in KPro eyes without glaucoma (77%) than in those with glaucoma (41%, P = .006). CONCLUSIONS: High preoperative IOP signals a higher risk for glaucoma development and progression after KPro surgery. Autoimmune diseases and ocular surface diseases precipitate de novo glaucoma, whereas stromal and endothelial disorders protect against glaucoma progression after KPro. The minority of KPro eyes without glaucoma remain at high risk of complications that can hinder promising visual outcomes. Despite all available treatments and surgical interventions, a majority of eyes will suffer from glaucoma progression, even later during follow-up.

Multiple DICER1-related tumors in a child with a large interstitial 14q32 deletion.

Germ-line interstitial deletions involving the 14q32 chromosomal region, resulting in 14q32 deletion syndrome, are rare. DICER1 is a recently described cancer-predisposition gene located at 14q32.13. We report the case of a male child with a ∼5.8 Mbp 14q32.13q32.2 germ-line deletion, which included the full DICER1 locus. We reviewed available clinical and pathological material, and conducted genetic analyses. In addition to having congenital dysmorphic features, the child developed multiple DICER1 syndrome-related tumors before age 5 y: a pediatric cystic nephroma (pCN), a ciliary body medulloepithelioma (CBME), and a small lung cyst (consistent with occult pleuropulmonary blastoma Type I/Ir cysts seen in DICER1 mutation carriers). He also developed a cerebral spindle-cell sarcoma with myogenous differentiation. Our investigations revealed that the deletion encompassed 31 protein-coding genes. In addition to the germ-line DICER1 deletion, somatic DICER1 RNase IIIb mutations were found in the CBME (c.5437G > A, p.E1813K), pCN (c.5425G > A, p.G1809R), and sarcoma (c.5125G > A, p.D1709N). The sarcoma also harbored a somatic TP53 mutation: c.844C > T, p.R282W. Additional copy number alterations were identified in the CBME and sarcoma using an OncoScan array. Among the 8 cases with molecularly-defined 14q32 deletions involving DICER1 and for whom phenotypic information is available, our patient and one other developed DICER1-related tumors. Biallelic DICER1 mutations have not previously been reported to cause cerebral sarcoma, which now may be considered a rare manifestation of the DICER1 syndrome. Our study shows that DICER1-related tumors can occur in children with 14q32 deletions and suggests surveillance for such tumors may be warranted.

The genomic landscape of two Burkitt lymphoma cases and derived cell lines: comparison between primary and relapse samples.

Relapse occurs in 10-40% of Burkitt lymphoma (BL) patients that have completed intensive chemotherapy regimens and is typically fatal. While treatment-naive BL has been characterized, the genomic landscape of BL at the time of relapse (rBL) has never been reported. Here, we present a genomic characterization of two rBL patients. The diagnostic samples had mutations common in BL, including MYC and CCND3. Additional mutations were detected at relapse, affecting important pathways such as NFκB (IKBKB) and MEK/ERK (NRAS) signaling, glutamine metabolism (SIRT4), and RNA processing (ZFP36L2). Genes implicated in drug resistance were also mutated at relapse (TP53, BAX, ALDH3A1, APAF1, FANCI). This concurrent genomic profiling of samples obtained at diagnosis and relapse has revealed mutations not previously reported in this disease. The patient-derived cell lines will be made available and, along with their detailed genetics, will be a valuable resource to examine the role of specific mutations in therapeutic resistance.

High-throughput screening in niche-based assay identifies compounds to target preleukemic stem cells.

Current chemotherapies for T cell acute lymphoblastic leukemia (T-ALL) efficiently reduce tumor mass. Nonetheless, disease relapse attributed to survival of preleukemic stem cells (pre-LSCs) is associated with poor prognosis. Herein, we provide direct evidence that pre-LSCs are much less chemosensitive to existing chemotherapy drugs than leukemic blasts because of a distinctive lower proliferative state. Improving therapies for T-ALL requires the development of strategies to target pre-LSCs that are absolutely dependent on their microenvironment. Therefore, we designed a robust protocol for high-throughput screening of compounds that target primary pre-LSCs maintained in a niche-like environment, on stromal cells that were engineered for optimal NOTCH1 activation. The multiparametric readout takes into account the intrinsic complexity of primary cells in order to specifically monitor pre-LSCs, which were induced here by the SCL/TAL1 and LMO1 oncogenes. We screened a targeted library of compounds and determined that the estrogen derivative 2-methoxyestradiol (2-ME2) disrupted both cell-autonomous and non-cell-autonomous pathways. Specifically, 2-ME2 abrogated pre-LSC viability and self-renewal activity in vivo by inhibiting translation of MYC, a downstream effector of NOTCH1, and preventing SCL/TAL1 activity. In contrast, normal hematopoietic stem/progenitor cells remained functional. These results illustrate how recapitulating tissue-like properties of primary cells in high-throughput screening is a promising avenue for innovation in cancer chemotherapy.