Transcriptome-based molecular staging of human stem cell-derived retinal organoids uncovers accelerated photoreceptor differentiation by 9-cis retinal

ABSTRACTRetinal organoids generated from human pluripotent stem cells exhibit considerable variability in temporal dynamics of differentiation. To assess the maturity of neural retina in vitro, we performed transcriptome analyses of developing organoids from human embryonic and induced pluripotent stem cell lines. We show that the developmental variability in organoids was reflected in gene expression profiles and could be evaluated by molecular staging with the human fetal and adult retinal transcriptome data. We also demonstrated that addition of 9-cis retinal, instead of widely-used all-trans retinoic acid, accelerated rod photoreceptor differentiation in organoid cultures, with higher rhodopsin expression and more mature mitochondrial morphology evident by day 120. Our studies thus provide an objective transcriptome-based modality for determining the differentiation state of retinal organoids, which should facilitate disease modeling and evaluation of therapies in vitro.Summary StatementThree-dimensional organoids derived from human pluripotent stem cells have been extensively applied for investigating organogenesis, modeling diseases and development of therapies. However, substantial variations within organoids pose challenges for comparison among different cultures and studies. We generated transcriptomes of multiple distinct retinal organoids and compared these to human fetal and adult retina gene profiles for molecular staging of differentiation state of the cultures. Our analysis revealed the advantage of using 9-cis retinal, instead of the widely-used all-trans retinoic acid, in facilitating rod photoreceptor differentiation. Thus, a transcriptome-based comparison can provide an objective method to uncover the maturity of organoid cultures across different lines and in various study platforms.

Molecular Staging of Patients with Colon Cancer. The C-Closer-II Study: A Multicentre Study in Portugal

Introduction: Approximately 20% - 30% of histological lymph node-negative patients with colorectal cancer relapse at five years after surgical treatment. This recurrence is likely due to occult nodal disease undetected by standard histopathological practice which has implications in terms of the clinical management of patients.Material and Methods: Lymph nodes were collected from colectomy specimens. A central section from each lymph node was histologically examined following haematoxylin-eosin staining and the remaining tissue was subjected to OSNA - one step nucleic acid amplification analysis.Results: A total of 1046 lymph nodes from 59 pN0 patients were assessed. Of these, 753 lymph nodes were examined by both methods. The median number of lymph nodes assessed with OSNA - one step nucleic acid amplification was 12 (IQR: 7;16). Among pN0 patients, 17 had OSNA - one step nucleic acid amplification-positive lymph nodes, resulting in a positive molecular staging rate of 28.8% (95% CI: 17.8 - 42.1). Among these patients, 12 (70.59%) were molecular-staged as pN1 and 5 (29.41%) were molecular staged as pN2. The tumour burden of lymph nodes assessed with OSNA - one step nucleic acid amplification ranged from 270 to 17 000 cytokeratin 19 mRNA copies/μL. Most of these patients (88.2%) were found to have lymph nodes with micrometastases only (250 - 4999 copies/μL).Discussion: We provide the results from the first study of the use of the OSNA - one step nucleic acid amplification assay in colorectal cancer patients in Portugal. Our results are in-line with other international studies, showing the improvement on patients’ staging by molecular examination of lymph nodes.Conclusion: In our study, 28.8% of patients with histologically negative lymph nodes were found to have metastatic lymph nodes using OSNA - one step nucleic acid molecular assessment. OSNA - one step nucleic acid assay allows a more accurate staging of patients with colorectal cancer and standardizes lymph node assessment.

The 46th David A. Karnofsky Memorial Award Lecture: Oligometastasis—From Conception to Treatment

Metastasis from most adult solid tumors generally has been considered to be widespread and incurable. Here, I present clinical and molecular data to support the hypothesis that some metastases are limited in number and pace and are curable with ablative therapies. I advance the hypothesis that immunotherapy combined with radiotherapy may be a general strategy to increase the number of patients with metastatic cancer amenable to cure. I further suggest that, in the context of ablative radiotherapy, the potential synergies between immunotherapy and radiotherapy are principally within the local tumor microenvironment and require treatment of all or most sites of metastatic disease. Improvements in the molecular staging of metastasis, immunotherapy strategies, and radiotherapy delivery are likely to improve outcomes for patients with metastatic cancer.

Molecular staging of gastric cancer with a novel panel of prognostic microRNAs.

52 Background: Molecular heterogeneity of gastric cancer discerns patient survival in ways that clinically-based prediction models cannot. To date, individual microRNAs(miRs) expressed by gastric tumors have been associated with survival. We offer a unique molecular-based method of prognosticating the survival of gastric cancer patients with an efficient panel of miRs. Methods: From The Cancer Genome Atlas(TCGA), we studied subjects with gastric adenocarcinoma, who had undergone R0-R1 resection and had data on clinical characteristics, overall survival (OS), and miR expression. Of miRs quantified by TCGA, miRs expressed by at least 15% of subjects’ tumors were evaluated as continuous variables. From 10 replicate samples, each containing 80% of current subjects, miRs were selected using proportional hazards regression adjusted for age with stepwise selection. Cross-validated miRs were retained for the panel if they optimized an accelerated failure-time model of OS using the full cohort. Results: Among subjects (n=254, age 64.7+10.9 years), median OS was 2.38 (95% Confidence Interval: 2.09-3.85) years. Of the 913 evaluable miRs, 10 distinct miRs (miR-373, miR-548ay, miR-659, miR-891a, miR-1243, miR- 4685, miR-6718, miR-6733, miR-6808, and miR-8072) were cross-validated as being prognostic and retained for the age-adjusted panel. Panel-predicted survival estimates, grouped into tertiles, distinguished progressively worse survival at 2 years, OS was 81.0%+5.1% for upper tertile, 61.5%+6.3% for middle tertile, and 29.6%+6.3% for lower tertile. As shown in Table 1, each tertile included subjects at all levels of TNM stage, and tertile was not significantly associated with stage (chi-square test, p=0.26). Conclusions: This unique miRs panel represents a molecular staging system for gastric cancer that has the potential to prognosticate patients' survival more accurately than existing clinical systems can. [Table: see text]