Hereditary retinal diseases (RDs) are a group of diseases caused by monogenetic or multigenetic mutations in genes mostly expressed in the photoreceptors. RDs can eventually lead to severe vision impairment or blindness. Since the 1990s, ocular gene transfer mediated by adeno-associated virus (AAV)-derived vectors has been explored to treat hereditary ocular diseases via gene supplementation or gene editing, advancing gene therapy to the clinical trial stages and to one commercial product. By the end of 2024, 142 clinical trials had been initiated for different types of RDs. Immune responses remain a major concern in AAV-mediated gene therapy. Although the eye is considered as an immune-privileged organ, studies in animals and clinical evidence have demonstrated that ocular gene therapies mediated by AAV can trigger immune responses to the vector capsid and/or to the transgene genome and product. These immune responses may compromise the efficiency and safety of the therapy. In this review, we summarize clinical trials treating RDs with AAV and provide a comprehensive overview of reported immune responses, including local inflammation and systemic adaptive immune responses. Additionally, this review emphasizes that immunosuppression and immunomonitoring strategies are not yet standardized both for intraocular and systemic gene therapy clinical trials, and do not allow for accurate follow-up of side effects. It highlights the inter-individual variability among patients, reinforcing the need to evaluate multiple key immune parameters, including sensitive inflammation biomarkers, and to assess the impact of different immunosuppression regimens.

Adeno-associated viruses (AAVs) have been used in gene therapy, especially for inherited retinal diseases. Despite their effectiveness in gene transduction, immune responses to the AAV capsid and transgene products have been reported, which can compromise both the efficacy and the safety of AAV-mediated therapies. The eye is regarded as an immune-privileged organ where immune activity is constitutively suppressed. Here, we highlight that immunomonitoring in an ocular gene transfer reveals variable immune responses, whatever the species (human clinical trial, nonhuman primates, mice), the site of injection, the cassette, and the dose. We further explored factors contributing to this variability, investigating the potential correlation among immune parameters in a controlled experimental setting. In a syngeneic murine model after a subretinal injection of AAV, our results highlight an interindividual variability of immune parameters, emphasizing the importance of considering inherent variability among individuals when designing personalized therapies.

CRISPR-Cas9 is a powerful gene-editing tool with great potential for treating genetic diseases, including inherited retinal disorders. However, its bacterial origin can induce immune responses that may eliminate transduced cells, threatening editing efficiency. A deeper understanding of CRISPR-Cas9 immunogenicity is therefore needed. Previous studies have shown that systemic delivery via Cas9 induces an immune response, but the detailed inflammation and the impact of the vector remain unclear, especially in immune-privileged organs like the eye. In this study, we found that Cas9 delivered to the retina using adeno-associated virus (AAV) induced persistent inflammation, whereas delivery as naked ribonucleoprotein (RNP) complexes resulted in acute inflammation that faded three weeks post-injection. Inflammation was more severe in the rd10 mouse model of inherited retinal degeneration, which exhibits basal inflammation. These findings provide new insights into vector-dependent immune responses to Cas9 in the eye and highlight potential risks associated with its clinical application. ### Competing Interest Statement D.D. is a co-inventor on patent #9193956 (Adeno-associated virus virions with variant capsid and methods of use thereof), with royalties paid to Adverum Biotechnologies and on pending patent applications on noninvasive methods to target cone photoreceptors (EP17306429.6 and EP17306430.4) licensed to Gamut Tx now SparingVision. D.D. also has personal financial interests in Tenpoint Tx. and SparingVision, outside the scope of the submitted work. Fondation pour la Recherche Médicale, https://ror.org/04w6kn183, FRM SPF201909009287 Foundation Fighting Blindness, https://ror.org/011728q79, PPA-0922-0840-INSERM China Scholarship Council, https://ror.org/04atp4p48, 202108070132 LABEX LIFESENSES IHU FOReSIGHT Conseil Régional d'Île-de-France, https://ror.org/04ry1rt27, DIM C-Brains, DIM BioConvS

Neuroendocrine neoplasms of gastrointestinal tract (GIT) and pancreas are heterogenous tumors. World Health Organization (WHO) 2019 classification introduced Grade (G)3 neuroendocrine tumor (NET) distinct from neuroendocrine carcinoma (NEC), based on molecular differences and to triage the patients for appropriate therapy. This distinction largely relies on morphology, which can be challenging at times. Genomic profiling has revealed TP53 and RB1 mutations in NECs, while death domain-associated protein 6 (DAXX) and alpha-thalassemia/mental retardation X-linked (ATRX), in G3NET. Their role as biological markers in differentiating these entities and their significance as prognostic markers are not yet established. This study aims at analyzing the diagnostic and prognostic role of p53 and ATRX in neuroendocrine neoplasms of GIT and pancreas. A single-centre, eight-year retrospective study of neuroendocrine neoplasm of GIT and pancreas comprised G2NET, G3NET and NEC. Tumor slides were stained by immunohistochemistry for p53 and ATRX. Strong nuclear staining of > 50

Purpose Neuroendocrine neoplasm of GIT and pancreas are heterogenous tumors with varying spectrum of clinicopathological characteristics. WHO 2019 classification, introduced Grade(G) 3 neuroendocrine tumor(NET) distinct from neuroendocrine carcinoma(NEC), to triage the patients for appropriate therapy. This distinction largely relies on morphology, which can be challenging at times. Genomic profiling has revealed TP53 and RB1 mutation in NECs while DAXX and ATRX in G3NET. Their role as biological markers in differentiating these entities and their significance as prognostic markers are not yet established. This study aims at analysing the diagnostic and prognostic role of p53 and ATRX in neuroendocrine neoplasms of GIT and Pancreas.Methodology: A single centre, ten year retrospective study. Tumour blocks of all G2, G3 NET and NEC were stained by immunohistochemistry for p53 and ATRX. Strong nuclear staining of > 50% of tumor cells for p53 was considered mutated. Nuclear staining of ATRX in < 5% of tumor cells was considered as ATRX loss.Results Mutated p53 was significantly associated with high grade tumors( NEC + G3NET) as compared to G2 tumors( p = 0.002). p53 mutation was frequent among NEC than G3 NET. Patients with mutated p53 had significantly lower survival irrespective of the grade(p = 0.001). There was no association of ATRX loss with grade or survival.Discussion G3NETs are genetically different from NECs. Molecular surrogates may play a role in accurately identifying these entities. Immunohistochemistry(IHC) for p53 has been used as surrogate for TP53 mutation in many tumors. Use of IHC for p53 in addition to histomorphology may facilitate accurate categorization NEC and G3 NET. Mutated p53 may also be used as an independent prognostic marker in Neuroendocrine tumors of GIT and Pancreas.

The anatomical differences between the retinas of humans and most animal models pose a challenge for testing novel therapies. Nonhuman primate (NHP) retina is anatomically closest to the human retina. However, there is a lack of relevant NHP models of retinal degeneration (RD) suitable for preclinical studies. To address this unmet need, we generated three distinct inducible cynomolgus macaque models of RD. We developed two genetically targeted strategies using optogenetics and CRISPR-Cas9 to ablate rods and mimic rod-cone dystrophy. In addition, we created an acute model by physical separation of the photoreceptors and retinal pigment epithelium using a polymer patch. Among the three models, the CRISPR-Cas9-based approach was the most advantageous model in view of recapitulating disease-specific features and its ease of implementation. The acute model, however, resulted in the fastest degeneration, making it the most relevant model for testing end-stage vision restoration therapies such as stem cell transplantation.

Owing to their small size and safety profiles, adeno-associated viruses (AAVs) have become the vector of choice for gene therapy applications in the retina. In addition to the naturally occurring AAVs, several engineered variants with enhanced properties are being developed for experimental and therapeutic applications. Nonetheless, there are still some challenges impeding successful application of AAVs for a broader range of retinal gene therapies. The small size of AAV particles ensures efficient tissue transduction but also limits the packaging capacity to a few kilobases. Further, AAV's ability to cross retinal barriers is still an obstacle to pan-retinal transduction of the outer retina with tolerable doses. Lastly, despite overall safety, there have been recent reports of immune responses to AAVs in the eye. Hence, evaluation and prediction of immune responses to AAVs has come to be considered an integral part of future clinical success. This review focuses on the use of AAV in clinical trials for retinal diseases, and discusses developments of variants and novel strategies to overcome immune responses to AAVs.

Gastric cancer is one of the most commonly occurring cancers worldwide, often presenting at an advanced stage. Combining targeted therapy with chemotherapeutic agents can enhance and extend the survival of these patients. This 4-year retrospective study aims to assess the prognostic role of Her2-Neu expression in gastric and gastroesophageal (GE) cancer. Clinicopathological features, histological type (Lauren classification) of adenocarcinoma, and Her2 immunohistochemical expression were correlated with disease-free and overall survival in 114 patients. A Her2 score of 0 and 1+ indicated negativity, while 3+ marked positivity. For cases with a 2+ score, fluorescent in situ hybridization (FISH) was conducted for definitive categorization. Statistical analysis employed IBM SPSS version 20.0 software. Among 114 patients, 13 displayed strong Her2-Neu immunopositivity (3 + ), 9 scored 2 + , and 92 were negative (0 [89] and 1+ [3]). FISH classified 4 and 5 cases as positive and negative, respectively. Most (64.7%) Her2-Neu-positive tumors occurred in the proximal stomach/GE junction (GEJ) and exhibited intestinal morphology (94.1%) with moderate differentiation (p-value < 0.05). Notably, 76.5% of Her2-Neu-positive patients exhibited advanced-stage disease with nodal/distant metastasis. The average disease-free survival was 15.4 months (standard error: 3.55) for positive Her2-Neu expression and 22.07 months (standard error: 1.364) for negative expression. The mean overall survival was 21.14 months (standard error: 3.702) for positive expression and 23.91 months (standard error: 1.474) for negative expression. Her2-Neu expression in gastric/GEJ adenocarcinomas correlates with reduced survival. Evaluating Her2-Neu in proximal gastric/GEJ cancers displaying low-grade intestinal morphology serves as both a predictive and prognostic indicator.

INTRODUCTION:Diaphragm disease, typically associated with long-term non-steroidal anti-inflammatory drug (NSAID) use, manifests as diaphragm-like small bowel strictures, often resulting in bowel obstruction.CASE DESCRIPTION:A 75-year-old male presented with features of recurrent subacute intestinal obstruction, later diagnosed with multiple small bowel strictures via CT imaging. Surgical intervention, including resection and anastomosis, was performed to alleviate the obstruction. Histopathological examination of the resected specimen confirmed diaphragm disease, challenging its traditional association with NSAID use.DISCUSSION:Diaphragm disease, characterized by mucosal and submucosal diaphragm-like strictures, is typically attributed to NSAID usage. However, this case underscores the possibility of diaphragm disease in the absence of NSAID exposure. Pathological findings supported the presence of diaphragm-like strictures, despite the patient's denial of NSAID use.CONCLUSION:This case emphasizes the importance of considering diaphragm disease as a differential diagnosis in patients with intermittent bowel obstruction, even in the absence of NSAID history.

Gene therapy is a potential cure for several inherited retinal dystrophies and adeno-associated virus (AAV) has emerged as a vector of choice for therapeutic gene delivery to the retina. However, prior exposure to AAVs can cause a humoral immune response resulting in the presence of antibodies in the serum, which can subsequently interfere with the AAV-mediated gene therapy. The antibodies bind specifically to a serotype, but often display broad cross-reactivity. A subset of these antibodies called Neutralizing Antibodies (NABs) can render the AAV inactive thereby reducing the efficacy of the therapy. The pre-existing NAB levels against different serotypes vary by species, and these variations need to be considered while designing studies. Since large animals often serve as preclinical models to test gene therapies, in this review we compile studies reporting pre-existing NABs against commonly used AAV serotypes in humans and large animal models and discuss strategies to deal with NABs.

Inherited retinal diseases (IRDs) are a leading cause of blindness in industrialized countries, and gene therapy is quickly becoming a viable option to treat this group of diseases. Gene replacement using a viral vector has been successfully applied and advanced to commercial use for a rare group of diseases. This, and the advances in gene editing, are paving the way for the emergence of a new generation of therapies that use CRISPR–Cas9 to edit mutated genes in situ. These CRISPR-based agents can be delivered to the retina as transgenes in a viral vector, unpackaged transgenes or as proteins or messenger RNA using non-viral vectors. Although the eye is considered to be an immune-privileged organ, studies in animals, as well as evidence from clinics, have concluded that ocular gene therapies elicit an immune response that can under certain circumstances result in inflammation. In this review, we evaluate studies that have reported on pre-existing immunity, and discuss both innate and adaptive immune responses with a specific focus on immune responses to gene editing, both with non-viral and viral delivery in the ocular space. Lastly, we discuss approaches to prevent and manage the immune responses to ensure safe and efficient gene editing in the retina.

The quest for neuroprotective factors that can prevent or slow down the progression of retinal degeneration is still ongoing. Acute hypoxic stress has been shown to provide transient protection against subsequent damage in the retina. Stanniocalcins - STC1 and STC2 - are secreted glycoproteins that are hypoxia-regulated and were shown to be cytoprotective in various in vitro studies. Hence, we investigated the expression of stanniocalcins in the normal, degenerating and hypoxic retina. We show that the expression of Stc1 and Stc2 in the retina was detectable as early as postnatal day 10 and persisted during aging. Retinal expression of Stc2, but not Stc1, was induced in mice in an in vivo model of acute hypoxia and a genetic model of chronic hypoxia. Furthermore, we show that HIF1, not HIF2, is responsible for regulating Stc2 in cells with the molecular response to hypoxia activated due to the absence of von Hippel Lindau protein. Surprisingly, Stc2 was not normally expressed in photoreceptors but in the inner retina, as shown by laser capture microdissection and immunofluorescence data. The expression of both Stc1 and Stc2 remained unchanged in the degenerative retina with an almost complete loss of photoreceptors, confirming their expression in the inner retina. However, the absence of either Stc1 or Stc2 had no effect on retinal architecture, as was evident from retinal morphology of the respective knockout mice. Taken together our data provides evidence for the differential regulation of STC1 and STC2 in the retina and the prospect of investigating STC2 as a retinal neuroprotective factor.

Positive clinical outcomes in adeno-associated virus (AAV)-mediated retinal gene therapy have often been attributed to the low immunogenicity of AAVs and immune privilege of the eye. However, several recent studies have shown potential for inflammatory responses. The current understanding of the factors contributing to inflammation, such as the pre-existence of serum antibodies against AAVs and their contribution to increases in antibody levels post-injection, is incomplete. The parameters that regulate the generation of new antibodies in response to the AAV capsid or transgene after intraocular injections are also insufficiently described. This study is a retrospective analysis of the pre-existing serum antibodies in correlation with changes in antibody levels after intraocular injections of AAV in non-human primates (NHPs) of the species Macaca fascicularis. In NHP serums, we analyzed the binding antibody (BAB) levels and a subset of these called neutralizing antibodies (NABs) that impede AAV transduction. We observed significantly higher pre-existing serum BABs against AAV8 compared with other serotypes and a dose-dependent increase in BABs and NABs in the serums collected post-injection, irrespective of the serotype or the mode of injection. Lastly, we were able to demonstrate a correlation between the serum BAB levels with clinical grading of inflammation and levels of transgene expression.

Neuroendocrine neoplasm of GIT (gastrointestinal tract) and pancreas is heterogenous with variable clinical features and disease outcomes. Despite multiple attempts of risk stratification by grading and staging, some have unpredictable clinical courses. Well-differentiated grade 3 neuroendocrine tumour (G3NET) is a recent subcategory introduced in the 2019 WHO classification based on morphology, molecular profile and prognosis distinguishing it from neuroendocrine carcinoma(NEC). This study aimed at describing the spectrum of NENs encountered in a tertiary centre with focus on reclassifying previously reported G3 tumours into G3 NET and NEC and comparing the survival between them. This is an 8-year retrospective study of all gastro-entero-pancreatic neuroendocrine neoplasms reclassified according to the 2019 WHO classification based on morphology and Ki-67 index with analysis of the survival rates between the categories. Minimum follow-up period was 20 months. Eighty-six patients with NENs of gastro-entero-pancreas were included, with median age group of 40–60 years (age range 9 to 79 years) and male:female ratio of 1.7:1. The tumour grade correlated with the TNM staging and most of the syndromic NETs were low grade. Eleven percent of the tumours were reclassified as well-differentiated G3NETs. The survival of G3 NETs was higher than NEC. Grading of NEN is vital for therapeutic decisions and for prognostication. Currently, morphology is the key to recognise the well-differentiated G3 NETs, but can be subject to interobserver variability. Molecular surrogates may play a role in accurately identifying these entities, the validity of which is warranted.

Introduction: Kayexalate (Sodium Polystyrene Sulfonate/SPS) and K-bind (Calcium Polystyrene Sulfonate/CPS) are cation exchange resins, commonly used for treatment of hyperkalaemia. SPS/CPS induced injury of the gastrointestinal tract(GIT) is rare, can be potentially life threatening but is under-recognized. This study aims to increase awareness of pathologists and clinicians of this under-reported complication of a drug commonly used to treat hyperkalaemia. Materials: Study population comprised patients with SPS/CPS (Kayexalate or its analogues) crystals identified in gastrointestinal specimens from 2017-2019 at a tertiary care centre. Clinical details, relevant investigations, imaging and endoscopic findings, patient follow up details were obtained from the hospital electronic information system. Results: A total of 10 patients with SPS/ CPS crystals in the GIT were encountered over 2 years. Male to female ratio was 9:1, with mean age 66.5years (range 52-82 years). Eight cases were mucosal biopsies and 2 were resection specimens. Additional pathology (tumours, colonic perforation) was present in 80% of patients. The characteristic morphological appearance of the CPS/SPS crystals on H&E stains were supported by special stains -Periodic acid Schiff(PAS) and Acid fast Bacilli(AFB). In all cases, the treatment history with SPS/CPS for hyperkalaemia was obtained only after the histological examination. Most common etiology of hyperkalaemia encountered was chronic kidney disease(CKD)/ Acute on chronic kidney disease. Conclusion: It is important for pathologists to recognise the presence of these crystals especially in small biopsies as early feedback to clinicians can help in appropriate management and avoidance of more serious adverse outcome. To the best of our knowledge, this is the first series of 10 consecutive cases of SPS/CPS crystals encountered in gastrointestinal tract to be reported from India.

BACKGROUND: Microsatellite instability (MSI) accounts for 15-20% of colorectal cancer (CRC) and is considered to have favorable stage-adjusted prognosis compared to Microsatellite stable (MSS) CRCs. Determination of MSI in stage II CRC is important for management decisions regarding adjuvant chemotherapy administration. The aim of this study was to determine the prognostic and predictive significance of MSI in stage 2 CRC in the Indian scenario. MATERIALS AND METHODS: A total of 195 patients who underwent curative surgery for stage II CRC from 2010 to 2017 were included. MSI testing by immunohistochemistry (DNA MisMatch Repair proteins) was performed in all. Various clinicopathological factors and disease-free survival and overall survival were assessed between MSI and MSS groups. The effect of treatment in terms of survival benefits with adjuvant therapy in the MSI group was also assessed. RESULTS: 27.1% of the CRCs' showed MSI. Younger age (<50 years), family history of cancer, synchronous/metachronous malignancies, proximal (right sided) location, poor morphological tumour differentiation, mucin production, and presence of peritumoral (Crohn's-like) lymphocytic response showed statistically significant association with MSI. Majority (56%) of our patients showed combined loss of MLH1 and PMS2. Overall, survival among the MSI patients was significantly higher (76.6 +/- 4.149 months) than the MSS patients (65.05 +/- 3.555) P = 0.04. MSI patients did not show any differences in survival with or without treatment. CONCLUSION: This study highlights the distinct clinicopathological features of MSI-related CRC and the relevance of MSI testing of stage II CRC for management decisions and prognostication.

Contrasting with fish or amphibian, retinal regeneration from Müller glial cells is largely limited in mammals. In our quest towards the identification of molecular cues that may boost their stemness potential, we investigated the involvement of the Hippo pathway effector YAP, which we previously found to be upregulated in Müller cells following retinal injury. We report that conditional Yap deletion in Müller cells prevents the upregulation of cell cycle genes that normally accompanies reactive gliosis upon photoreceptor cell death. This occurs as a consequence of defective EGFR signaling. Consistent with a function of YAP in triggering Müller glia cell cycle re-entry, we further show that in Xenopus , a species endowed with efficient regenerative capacity, YAP is required for their injury-dependent proliferative response. Finally, and noteworthy, we reveal that YAP overactivation in mouse Müller cells is sufficient to induce their reprogramming into highly proliferative cells. Overall, we unravel a pivotal role for YAP in tuning Müller cell response to injury and highlight a novel YAP-EGFR axis by which Müller cells exit their quiescence state, a critical step towards regeneration.

Introduction10%–15% of colorectal cancer (CRC) is due to Microsatellite Instability(MSI). The aim of the study was to determine the prognostic significance of detecting MSI in Stage II CRC and to understand the demographic and pathological characteristics of the disease in the Indian scenario.MethodsThis was an 8 year retrospective study (2010 to 2017) of 195 patients with Stage II colorectal adenocarcinoma who were categorised into Microsatellite stable (MSS) or MSI based on immunohistochemical expression of the DNA Mismatch Repair(dMMR) proteins. Various clinical and pathological factors as per the Revised Bethseda criteria were compared and analysed between the MSI and MSS groups by Chi- square test and T Test. Kaplan- Meier method was used to calculate the Disease Free Survival (DFS) and Overall-survival (OS) for the 2 groups. Log Rank test was applied to know the strength of association between the DFS and OS with each of the parameters. The data was analysed using IBM SPSS version 20 software.ResultsThere were 53 (27%) patients in the MSI group. Younger age, and presence of synchronous or metachronous malignancies, right sided location of tumour, poorly differentiated adenocarcinoma, mucin production and presence of peritumoral Crohn’s like lymphocytic response showed statistically significant association with MSI. A definite relationship of MSI status with family history could not be established. The mean DFS in MSI group was 74.7±3.496 months as compared to 69.2±3.631 months in MSS group. Disease related death was seen in 2.8% and 15.7% of patients in MSI and MSS group respectively, p=0.042. Overall survival among the MSI patients was significantly higher (76.6±4.149 months) than the MSS patients (65.05±3.555) p=0.04. MSI patients did not show improved survival with adjuvant therapy.ConclusionsEarly stage MSI related CRC has good prognosis even without adjuvant chemotherapy. Knowledge of the MSI status in CRC is useful in management decisions and prognostication. In addition it can help to detect those with Lynch Syndrome who may not fulfil the Revised Bethesda criteria.