CDH1 methylation analysis in invasive lobular breast carcinomas with and without gene mutation.

The proposed role of CDH1 (E-cadherin gene) methylation as a mechanism of gene inactivation in invasive lobular carcinoma (ILC) remains inconclusive. For many years, CDH1 promoter hypermethylation has been regarded as a mechanism for gene inactivation in ILC. However, this assumption has primarily relied on non-quantitative assays, which have reported CDH1 methylation frequencies ranging from 26 to 93% at CpG sites within the island region. Few studies employing quantitative methods and covering CpG island shores, regions of relatively low CpG density situated proximal to conventional promoter CpGs, have been conducted, revealing lower percentages of methylation ranging from 0 to 51%. Therefore, using the quantitative pyrosequencing method, we examined CDH1 methylation in the island region and shores in E-cadherin deficient ILC cases (15 with CDH1 mutation and 22 non-mutated), 19 cases of invasive breast carcinomas non-special type (IBC-NSTs), and five cases of usual ductal hyperplasia (UDH). Our analysis revealed CDH1 methylation frequencies ranging from 3 to 64%, with no significant increase in methylation levels in any group of ILCs (median = 12%) compared to IBC-NST (median = 15%). In addition, considering the poorly studied association between the number of tumor-infiltrating lymphocytes (TILs) and CDH1 methylation in breast cancer, we undertook a thorough analysis within our dataset. Our findings revealed a positive correlation between CDH1 methylation and the presence of TILs (r = 0.5; p-value < 0.05), shedding light on an aspect of breast cancer biology warranting further investigation. These findings challenge CDH1 methylation as a CDH1 inactivation mechanism in ILC and highlight TILs as a potential confounding factor in gene methylation.

The Prognostic Significance of Tumor-Infiltrating Lymphocytes, PD-L1, BRCA Mutation Status and Tumor Mutational Burden in Early-Stage High-Grade Serous Ovarian Carcinoma-A Study by the Spanish Group for Ovarian Cancer Research (GEICO).

Early stages are under-represented in studies on the molecular and immune features of high-grade serous ovarian carcinoma (HGSOC), and specific studies focused on early-stage HGSOC are required for a better prognostic stratification and to personalize chemotherapy. The aim of this study was to determine the prognostic significance of CD8+ and CD4+ tumor-infiltrating lymphocytes (TILs), tumoral cell PD-L1 expression, BRCA mutational status and tumor mutation burden (TMB) in early-stage HGSOC. A retrospective study was performed on stage I and II HGSOC from the Molecular Reclassification of Early Stages of Ovarian Cancer (RECLAMO) cohort from the Spanish Group of Ovarian Cancer Research (GEICO). Centralized histological typing was performed based on morphological and immunohistochemical features. Intraepithelial (i) and stromal (s) CD8+ and CD4+ T cells and PD-L1 were evaluated on tissue microarrays by immunohistochemistry. BRCA1 and BRCA2 mutation status and TMB were analyzed in tumor DNA using next-generation sequencing. The study included 124 tumors. High iCD8+ (>20 TILs/core), low/intermediate CD4+ (<20 TILs/core) and high CD8+/CD4+ ratio (>35/core) were associated with favorable outcomes. Tumor cell PD-L1 expression (TPS ≥ 1) was present in only 8% of tumors. In total, 11 (16%) and 6 (9%) out of 69 HGSOC tested carried pathogenic or likely pathogenic BRCA1 or BRCA2 mutations, respectively. Median TMB of 40 tumors analyzed was 5.04 mutations/Mb and only 6 tumors had 10 or more mutations/Mb. BRCA status and TMB were not associated with TILs or prognosis. When compared with studies on advanced HGSOC, our results suggested that prognostic variables differed according to stage and that more studies focused on early stages of HGSOC are needed to better stratify these tumors.

Evaluation of ERBB2 mRNA Expression in HER2-Equivocal (2+) Immunohistochemistry Cases.

Xpert Breast Cancer STRAT4 is a RT-qPCR platform that studies the mRNA expression of ESR1, PGR, MKI67 and ERBB2, providing a positive or negative result for each of these breast cancer biomarkers. Its concordance with immunohistochemistry (IHC) and in situ hybridization (ISH) has been previously demonstrated, but none of the previous works was focused on HER2-equivocal (2+) cases identified by IHC. Thus, we studied the concordance between IHC/ISH and STRAT4 results for 112 HER2 2+ IBC samples, using 148 HER2 0+, 1+ and 3+ (no-HER2 2+) samples for comparison. We found 91.3% accuracy for the determination of HER2 status globally, 99.3% for no-HER2 2+ samples and 80.7% for HER2 2+ samples. Regarding the other biomarkers, we obtained 96.4% accuracy for estrogen receptor, 84.1% for progesterone receptor and 58.2% for Ki67. Our results suggest that the use of ERBB2 mRNA for the evaluation of HER2 2+ cases is not a reliable reflex method to assess the ERBB2 amplification status.

Progression to lung fibrosis in severe COVID-19 patients: A morphological and transcriptomic study in postmortem samples.

The development of lung fibrosis is a major concern in patients recovered from severe COVID-19 pneumonia. This study aimed to document the evolution of diffuse alveolar damage (DAD) to the fibrosing pattern and define the transcriptional programs involved. Morphological, immunohistochemical and transcriptional analysis were performed in lung samples obtained from autopsy of 33 severe COVID-19 patients (median illness duration: 36 days). Normal lung and idiopathic pulmonary fibrosis (IPF) were used for comparison. Twenty-seven patients with DAD and disease evolution of more than 2 weeks had fibrosis. Pathways and genes related with collagen biosynthesis and extracellular matrix (ECM) biosynthesis and degradation, myofibroblastic differentiation and epithelial to mesenchymal transition (EMT) were overexpressed in COVID-19. This pattern had similarities with that observed in IPF. By immunohistochemistry, pathological fibroblasts (pFBs), with CTHRC1 and SPARC expression, increased in areas of proliferative DAD and decreased in areas of mature fibrosis. Immunohistochemical analysis demonstrated constitutive expression of cadherin-11 in normal epithelial cells and a similar pattern of cadherin and catenin expression in epithelial cells from both normal and COVID-19 samples. Transcriptomic analysis revealed downregulation of the Hippo pathway, concordant with the observation of YAP overexpression in hyperplastic alveolar epithelial cells. Progression to fibrosis in severe COVID-19 is associated with overexpression of fibrogenic pathways and increased in CTHRC1- and SPARC-positive pFBs. Whereas the Hippo pathway seemed to be implicated in the response to epithelial cell damage, EMT was not a major process implicated in COVID-19 mediated lung fibrosis.

The Clonal Relationship Between the Ductal and Lobular Components of Mixed Ductal-Lobular Carcinomas Suggested a Ductal Origin in Most Tumors.

The relationship between the ductal and lobular components of invasive ductolobular carcinomas (IDLC) has not been fully elucidated. In this study, the molecular alterations of both components were analyzed in a series of 20 IDLC that were selected, not only by morphologic criteria, but also by the loss of E-cadherin expression in the lobular component. We found that 80% of tumors shared alterations of driver genes in both components, being PIK3CA the most common alteration. In addition, 45% of IDLC carried CDH1 mutations in their lobular component that were absent in the ductal component. Fluorescent in situ hybridization analysis of the CDH1 gene excluded homozygous CDH1 loss as a frequent cause of E-cadherin loss in tumors without CDH1 mutations. In addition, no pathogenic mutations of catenin genes were detected in this series of tumors. In 25% of tumors, actionable mutations in PIK3CA , AKT1 , and ERBB2 were found in only 1 component. Altogether, our results confirm that most IDLC derive from invasive carcinoma of no special type, in which a population of cells lose E-cadherin and acquire a lobular phenotype. The frequency of CDH1 mutations in IDLC appears to be lower than in conventional invasive lobular carcinomas, suggesting the implication of alternative mechanisms of E-cadherin loss. Moreover, molecular heterogeneity between ductal and lobular areas suggests the need for molecular characterization of both components to guide targeted therapies.

Neuropathological findings in fatal COVID-19 and their associated neurological clinical manifestations.

Severe cases of Coronavirus Disease 2019 (COVID-19) can present with multiple neurological symptoms. The available neuropathological studies have described different lesions; the most frequent was the presence of neuroinflammation and vascular-related lesions. The objective of this study was to report the neuropathological studies performed in a medical institution, with abundant long intensive care unit stays, and their associated clinical manifestations. This is a retrospective monocentric case series study based on the neuropathological reports of 13 autopsies with a wide range of illness duration (13-108 days). A neuroinflammatory score was calculated based on the quantification of CD8- and CD68-positive cells in representative areas of the central nervous system. This score was correlated afterwards with illness duration and parameters related to systemic inflammation. Widespread microglial and cytotoxic T-cell activation was found in all patients. There was no correlation between the neuroinflammatory score and the duration of the illness; nor with parameters of systemic inflammation such as the peak of IL-6 or the HScore (a parameter of systemic macrophage activation syndrome). Two patients had global hypoxic ischaemic damage and five patients had subacute infarcts. One patient had many more brain vascular microthrombi compared to the others and multiple subacute pituitary infarcts. SARS-CoV-2 RNA was not detected with qRT-PCR. The proportion of brain lesions in severe COVID-19 patients could be related to illness duration. In our series, with abundant long hospitalisation stays, neuroinflammation was present in all patients and was more prominent between day 34 and day 45 after onset of symptoms. Clinical correlation showed that two patients with the highest neuroinflammatory scores had severe encephalopathies that were not attributable to any other cause. The second most frequent lesions were related to vascular pathology.

Impact of the COVID-19 pandemic on pathology training: a survey among Spanish residents.

This study aimed to assess the impact of the COVID-19 pandemic on the training of Spanish pathology residents by conducting an online survey. It was distributed among the pathologists who had been residents during the pandemic and consisted of 31 questions about the demographics and the impact of the pandemic on their pathology training. In total, 114 residents completed the survey; they reported a decrease in the number of biopsies, cytology samples, autopsies, research activities, sessions, case corrections, and courses, as well as the detrimental effect this had on their training. Half of the residents had to cancel rotations and 32% were redeployed to other departments. The COVID-19 pandemic has affected pathology training, and our results can be used to understand the challenges residents have faced and mitigate their impact.

Cytological and molecular characterization of secretory breast carcinoma.

Secretory breast carcinoma is a rare neoplasm, histologically well-characterized, and secondary to ETV6-NTRK3 gene fusion, whose cytological features are scarcely described in the literature. We report the case of a woman with a history of secretory breast carcinoma 8 years before, who presented a periareolar nodule. A recurrence was diagnosed by fine-needle aspiration based on the cytomorphological features and pan-TRK immunocytochemistry on the cell block, and the patient underwent a mastectomy. The histology and molecular studies performed on the surgical specimen (immunohistochemistry, FISH and NGS) confirmed the diagnosis. Cytological smears showed abundant epithelial cellularity, in groups and single cells. These cells showed moderate atypia, with abundant cytoplasm. We observed intracytoplasmic inclusions and extracellular metachromatic globules. Immunocytochemical and immunohistochemical studies showed a triple negative breast tumour. NTRK overexpression was demonstrated with immunocytochemistry against pan-TRK on the cell block, as well as with immunohistochemistry in the surgical specimen. NTRK3 rearrangement was proved by FISH. In the primary tumour and in the recurrence, we demonstrated ETV6-NTRK3 fusion by NGS. After conducting a literature review, we have found 26 articles describing the cytological features of secretory breast carcinoma in 33 patients. The smears were described as groups of epithelial cells with vacuolated cytoplasm, single signet ring cells and a globular extracellular secretion. In only two cases molecular confirmation of the diagnosis with ETV6-NTRK3 fusion was proven, although not in the cytological specimen, but in the subsequent biopsy. The distinct cytological features of secretory breast carcinoma can help in its diagnosis, thus guiding the molecular studies. This is the first reported case that proves TRK overexpression, as a fusion surrogate, in the cytological sample.

RNA SARS-CoV-2 Persistence in the Lung of Severe COVID-19 Patients: A Case Series of Autopsies.

The exact role of viral replication in patients with severe COVID-19 has not been extensively studied, and it has only been possible to demonstrate the presence of replicative virus for more than 3 months in a few cases using different techniques. Our objective was to study the presence of RNA SARS-CoV-2 in autopsy samples of patients who died from COVID-19 long after the onset of symptoms. Secondary superimposed pulmonary infections present in these patients were also studied. We present an autopsy series of 27 COVID-19 patients with long disease duration, where pulmonary and extrapulmonary samples were obtained. In addition to histopathological analysis, viral genomic RNA (gRNA) and viral subgenomic RNA (sgRNA) were detected using RT-PCR and in situ hybridization, and viral protein was detected using immunohistochemistry. This series includes 26 adults with a median duration of 39 days from onset of symptoms to death (ranging 9-108 days), 92% of them subjected to immunomodulatory therapy, and an infant patient. We detected gRNA in the lung of all but one patient, including those with longer disease duration. SgRNA was detected in 11 out of 17 patients (64.7%) with illness duration up to 6 weeks and in 3 out of 9 patients (33.3%) with more than 6 weeks of disease progression. Viral protein was detected using immunohistochemistry and viral mRNA was detected using in situ hybridization in 3 out of 4 adult patients with illness duration of <2 weeks, but in none of the 23 adult patients with an illness duration of >2 weeks. A remarkable result was the detection of viral protein, gRNA and sgRNA in the lung cells of the pediatric patient after 95 days of illness. Additional pulmonary infections included: 9 acute bronchopneumonia, 2 aspergillosis, 2 cytomegalovirus, and 1 BK virus infection. These results suggest that in severe COVID-19, SARS-CoV-2 could persist for longer periods than expected, especially in immunocompromised populations, contributing to the persistence of chronic lung lesions. Additional infections contribute to the fatal course of the disease.

Comparison Between Real-time Ammonium and pH Measurement, Immunohistochemistry, and Histochemistry for the Diagnosis of Helicobacter pylori.

BACKGROUND: Helicobacter pylori (HP) infection has been implicated in several malignant and nonmalignant conditions. The confirmatory diagnosis of HP requires an endoscopic biopsy, followed by a rapid urease test, culture, and/or histopathologic examination using hemotoxylin and eosin, histochemical stains, or immunohistochemistry against HP. EndoFaster is a novel device that can perform real-time ammonium and pH measurements in gastric juice, allowing a diagnosis of HP during gastroduodenal endoscopy. GOAL: This study aimed to validate the accuracy of EndoFaster and to compare different histochemical and immunohistochemical techniques for the diagnosis of HP infection. STUDY: Consecutive patients who underwent upper endoscopy at our center were prospectively enrolled. During the endoscopy procedure, gastric juice was aspirated to perform an automatic analysis by EndoFaster and gastric biopsies were taken. Histologic sections were reviewed to assess the histopathologic features. The sensitivity, specificity, positive predictive value, and negative predictive value were calculated for the EndoFaster test, Diff-Quick (DQ), and immunohistochemistry against HP (anti-HP) using Warthin-Starry as the gold standard for HP detection. RESULTS: Overall, 80 patients were enrolled. In 19 cases (23,75%), histology was normal. In the remaining cases, varying degrees of inflammation were found. The sensitivity, specificity, positive predictive value, and negative predictive value were 73.33%, 86.00%, 75.86%, and 84.31% for the EndoFaster test using 67 ppm/mL as the positive threshold; 73.33%, 100%, 100%, and 86.21% for DQ; and 79.31%, 88.00%, 79.31%, and 88.00% for anti-HP, respectively. CONCLUSIONS: The EndoFaster test has good sensitivity and specificity for the diagnosis of HP during the gastroscopy procedure. DQ and anti-HP are excellent alternatives to Warthin-Starry for the detection of HP.

Immune-related gene expression signatures: a step forward in the stratification of patients with ovarian clear cell carcinoma†.

Clear cell carcinoma of the ovary (CCC) is one of the five major histological types of ovarian cancer and presents specific clinicopathological features, such as a higher prevalence in the Asian population and a poor response to conventional chemotherapy. In a recent publication in The Journal of Pathology, Heong, Tan, Miwa et al demonstrated the heterogeneity of the immune landscape of CCC. They reported the immune signatures observed in a large cohort of CCC, including tumours from both Asian and Caucasian women. The authors analysed three cohorts from Europe, Japan, and Singapore, with a total of 246 tumours, and evaluated 730 immune-related genes using NanoString technology. The study revealed four main transcriptional subtypes characterised by the expression of specific sets of genes: PD1-high (11%), CTLA4-high (29%), antigen-presentation (42%), and a pro-angiogenic subtype (18%). The two main conclusions of the study were: (1) that CCCs in women of Asian and Caucasian descent share significant molecular similarities, since all four molecular signatures are present in all the cohorts analysed, without any evident differences in frequency; and (2) that the PD1-high and CTLA4-high subtypes were associated with worse clinical outcomes and may be useful when stratifying treatment in early-stage tumours. The immune signature could represent a promising biomarker of immunotherapy response if future prospective studies confirm it. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Primary epithelioid hemangioendothelioma of the penis: a case report and literature review.

Epithelioid hemangioendothelioma (EHE) is a rare vascular tumour with an intermediate behaviour between benign hemangioma and malignant angiosarcoma. There is scarce data on the penile EHE's management and its natural history, as our knowledge is based on few reported cases with a short follow-up period. We present a case report and conducted a literature review, including 17 cases. The relevance of this case report derives from the need for better clinical characterization of patients with penile EHE and the importance of defining the outcomes. We report the case of a 53-year-old male with a 1-year history of sleep-related painful erections. Imaging techniques showed a well-defined hypoechoic and hypervascular solid nodule on the dorsal aspect of the penis. It was surgically removed, and the histopathological study revealed a low-risk EHE of the penis. Follow-up magnetic resonance imaging (MRI) and computed tomography did not demonstrate local recurrence nor metastases. According to the literature review, most of the patients were in their fifth and sixth decades of life at the time of diagnosis and lesions were usually located in the glans. The most common clinical presentation was as a painful mass. Follow-up period ranged from 2 months to 5 years. Three patients showed systemic metastases, two of which died due to cancer. The conclusions from the literature review are limited by the reduced number of cases and the short follow-up. This case report highlights the importance of understanding the diagnosis and treatment of this type of rare non-squamous malignant tumours of the penis. Penile EHE is a malignant vascular tumour that is very rare in this location. The best treatment is local excision, with re-excision or intraoperative margins assessment. Occasionally, systemic chemotherapy and radiation therapy can be useful. There is consensus on the importance of very strict follow-up of these patients.

Late Cardiac Pathology in Severe Covid-19. A Postmortem Series of 30 Patients.

The role of SARS-CoV-2 as a direct cause in the cardiac lesions in patients with severe COVID-19 remains to be established. Our objective is to report the pathological findings in cardiac samples of 30 patients who died after a prolonged hospital stay due to Sars-Cov-2 infection. We performed macroscopic, histological and immunohistochemical analysis of the hearts of 30 patients; and detected Sars-Cov-2 RNA by RT-PCR in the cardiac tissue samples. The median age of our cohort was 69.5 years and 76.6% were male. The median time between symptoms onset and death was 36.5 days. The main comorbidities were arterial hypertension (13 patients, 43.3%), dyslipidemia (11 patients, 36.7%), cardiovascular conditions (8 patients, 26.7%), and obesity (8 patients, 26.7%). Cardiovascular conditions included ischemic cardiopathy in 4 patients (13.3%), hypertrophic cardiomyopathy in 2 patients (6.7%) and valve replacement and chronic heart failure in one patient each (3.3%). At autopsy, the most frequent histopathological findings were coronary artery atherosclerosis (8 patients, 26.7%), left ventricular hypertrophy (4 patients, 13.3%), chronic epicardial inflammation (3 patients, 10%) and adipose metaplasia (2 patients, 6.7%). Two patients showed focal myocarditis, one due to invasive aspergillosis. One additional patient showed senile amyloidosis. Sars-Cov-2 RNA was detected in the heart of only one out of 30 patients, who had the shortest disease evolution of the series (9 days). However, no relevant cardiac histological alterations were identified. In present series, cardiac pathology was only modest in most patients with severe COVID-19. At present, the contribution of a direct effect of SARS-CoV-2 on cardiac lesions remains to be established.

Pulmonary vascular proliferation in patients with severe COVID-19: an autopsy study.

Diffuse alveolar damage and thrombi are the most common lung histopathological lesions reported in patients with severe COVID-19. Although some studies have suggested increased pulmonary angiogenesis, the presence of vascular proliferation in COVID-19 lungs has not been well characterised. Glomeruloid-like microscopic foci and/or coalescent vascular proliferations measuring up to 2 cm were present in the lung of 14 out of 16 autopsied patients. These lesions expressed CD31, CD34 and vascular endothelial cadherin. Platelet-derived growth factor receptor-ß immunohistochemistry and dual immunostaining for CD34/smooth muscle actin demonstrated the presence of pericytes. These vascular alterations may contribute to the severe and refractory hypoxaemia that is common in patients with severe COVID-19.

Molecular Features of Metaplastic Breast Carcinoma: An Infrequent Subtype of Triple Negative Breast Carcinoma.

Metaplastic breast carcinoma (MBC) is a heterogeneous group of infrequent invasive carcinomas that display differentiation of the neoplastic epithelium towards squamous cells and/or mesenchymal-type elements. Most MBC have a triple negative phenotype and poor prognosis. Thus, MBC have worse survival rates than other invasive breast carcinomas, including other triple negative breast carcinomas (TNBC). In this study, we reviewed the molecular features of MBC, pointing out the differences among subtypes. The most frequently mutated genes in MBC were TP53 and PIK3CA. Additionally, mutations in the other genes of the PI3K/AKT pathway indicated its importance in the pathogenesis of MBC. Regarding copy number variations (CNVs), MYC was the most frequently amplified gene, and the most frequent gene loss affected the CDKN2A/CDKN2B locus. Furthermore, the pattern of mutations and CNVs of MBC differed from those reported in other TNBC. However, the molecular profile of MBC was not homogeneous among histological subtypes, being the alterations in the PI3K pathway most frequent in spindle cell carcinomas. Transcriptomic studies have demonstrated an epithelial to mesenchymal program activation and the enrichment of stemness genes in most MBC. In addition, current studies are attempting to define the immune microenvironment of these tumors. In conclusion, due to specific molecular features, MBC have a different clinical behavior from other types of TNBC, being more resistant to standard chemotherapy. For this reason, new therapeutic approaches based on tumor molecular characteristics are needed to treat MBC.

Low Prevalence of HER2-Positive Breast Carcinomas among Screening Detected Breast Cancers.

Conflicting results have been reported regarding the prevalence of screen-detected human epidermal growth factor receptor 2 (HER2)-positive breast carcinomas and non-screen detected HER2-positive breast carcinomas. To address this issue, we evaluated the prevalence of HER2-positive breast carcinomas in two independent regional screening programs in Spain. The clinicopathologic and immunohistochemical characteristics of 479 (306 and 173) screen-detected breast carcinomas and 819 (479 and 340) non-screen-detected breast carcinomas diagnosed in women between 50 and 69-year-olds were compared. The prevalence of HER2-positive breast carcinomas was 8.8% and 6.4% in the two series of screen-detected tumors, compared with 16.4% and 13% in non-screen-detected carcinomas. These differences were statistically significant. This lower prevalence of HER2-positive in-screen-detected breast carcinomas was observed in both hormone receptor positive (luminal HER2) and hormone-receptor-negative (HER2 enriched) tumors. In addition, a lower prevalence of triple-negative and a higher prevalence of luminal-A breast carcinomas was observed in screen-detected tumors. Moreover, a literature review pointed out important differences in subrogate molecular types in screen-detected breast carcinomas among reported series, mainly due to study design, technical issues and racial differences.

Mesenchymal stem cell therapy in retinal and optic nerve diseases: An update of clinical trials.

Retinal and optic nerve diseases are degenerative ocular pathologies which lead to irreversible visual loss. Since the advanced therapies availability, cell-based therapies offer a new all-encompassing approach. Advances in the knowledge of neuroprotection, immunomodulation and regenerative properties of mesenchymal stem cells (MSCs) have been obtained by several preclinical studies of various neurodegenerative diseases. It has provided the opportunity to perform the translation of this knowledge to prospective treatment approaches for clinical practice. Since 2008, several first steps projecting new treatment approaches, have been taken regarding the use of cell therapy in patients with neurodegenerative pathologies of optic nerve and retina. Most of the clinical trials using MSCs are in I/II phase, recruiting patients or ongoing, and they have as main objective the safety assessment of MSCs using various routes of administration. However, it is important to recognize that, there is still a long way to go to reach clinical trials phase III-IV. Hence, it is necessary to continue preclinical and clinical studies to improve this new therapeutic tool. This paper reviews the latest progress of MSCs in human clinical trials for retinal and optic nerve diseases.