Differential pre-malignant programs and microenvironment chart distinct paths to malignancy in human colorectal polyps.

Colorectal cancers (CRCs) arise from precursor polyps whose cellular origins, molecular heterogeneity, and immunogenic potential may reveal diagnostic and therapeutic insights when analyzed at high resolution. We present a single-cell transcriptomic and imaging atlas of the two most common human colorectal polyps, conventional adenomas and serrated polyps, and their resulting CRC counterparts. Integrative analysis of 128 datasets from 62 participants reveals adenomas arise from WNT-driven expansion of stem cells, while serrated polyps derive from differentiated cells through gastric metaplasia. Metaplasia-associated damage is coupled to a cytotoxic immune microenvironment preceding hypermutation, driven partly by antigen-presentation differences associated with tumor cell-differentiation status. Microsatellite unstable CRCs contain distinct non-metaplastic regions where tumor cells acquire stem cell properties and cytotoxic immune cells are depleted. Our multi-omic atlas provides insights into malignant progression of colorectal polyps and their microenvironment, serving as a framework for precision surveillance and prevention of CRC.

An Organoid Biobank of Neuroendocrine Neoplasms Enables Genotype-Phenotype Mapping.

Gastroenteropancreatic (GEP) neuroendocrine neoplasm (NEN) that consists of neuroendocrine tumor and neuroendocrine carcinoma (NEC) is a lethal but under-investigated disease owing to its rarity. To fill the scarcity of clinically relevant models of GEP-NEN, we here established 25 lines of NEN organoids and performed their comprehensive molecular characterization. GEP-NEN organoids recapitulated pathohistological and functional phenotypes of the original tumors. Whole-genome sequencing revealed frequent genetic alterations in TP53 and RB1 in GEP-NECs, and characteristic chromosome-wide loss of heterozygosity in GEP-NENs. Transcriptome analysis identified molecular subtypes that are distinguished by the expression of distinct transcription factors. GEP-NEN organoids gained independence from the stem cell niche irrespective of genetic mutations. Compound knockout of TP53 and RB1, together with overexpression of key transcription factors, conferred on the normal colonic epithelium phenotypes that are compatible with GEP-NEN biology. Altogether, our study not only provides genetic understanding of GEP-NEN, but also connects its genetics and biological phenotypes.

Divergent Routes toward Wnt and R-spondin Niche Independency during Human Gastric Carcinogenesis.

Recent sequencing analyses have shed light on heterogeneous patterns of genomic aberrations in human gastric cancers (GCs). To explore how individual genetic events translate into cancer phenotypes, we established a biological library consisting of genetically engineered gastric organoids carrying various GC mutations and 37 patient-derived organoid lines, including rare genomically stable GCs. Phenotype analyses of GC organoids revealed divergent genetic and epigenetic routes to gain Wnt and R-spondin niche independency. An unbiased phenotype-based genetic screening identified a significant association between CDH1/TP53 compound mutations and the R-spondin independency that was functionally validated by CRISPR-based knockout. Xenografting of GC organoids further established the feasibility of Wnt-targeting therapy for Wnt-dependent GCs. Our results collectively demonstrate that multifaceted genetic abnormalities render human GCs independent of the stem cell niche and highlight the validity of the genotype-phenotype screening strategy in gaining deeper understanding of human cancers.

Somatic inflammatory gene mutations in human ulcerative colitis epithelium.

With ageing, normal human tissues experience an expansion of somatic clones that carry cancer mutations1-7. However, whether such clonal expansion exists in the non-neoplastic intestine remains unknown. Here, using whole-exome sequencing data from 76 clonal human colon organoids, we identify a unique pattern of somatic mutagenesis in the inflamed epithelium of patients with ulcerative colitis. The affected epithelium accumulates somatic mutations in multiple genes that are related to IL-17 signalling-including NFKBIZ, ZC3H12A and PIGR, which are genes that are rarely affected in colon cancer. Targeted sequencing validates the pervasive spread of mutations that are related to IL-17 signalling. Unbiased CRISPR-based knockout screening in colon organoids reveals that the mutations confer resistance to the pro-apoptotic response that is induced by IL-17A. Some of these genetic mutations are known to exacerbate experimental colitis in mice8-11, and somatic mutagenesis in human colon epithelium may be causally linked to the inflammatory process. Our findings highlight a genetic landscape that adapts to a hostile microenvironment, and demonstrate its potential contribution to the pathogenesis of ulcerative colitis.

Identification of Quiescent LGR5+ Stem Cells in the Human Colon.

BACKGROUND & AIMS: In the mouse intestinal epithelium, Lgr5+ stem cells are vulnerable to injury, owing to their predominantly cycling nature, and their progenies de-differentiate to replenish the stem cell pool. However, how human colonic stem cells behave in homeostasis and during regeneration remains unknown. METHODS: Transcriptional heterogeneity among colonic epithelial cells was analyzed by means of single-cell RNA sequencing analysis of human and mouse colonic epithelial cells. To trace the fate of human colonic stem or differentiated cells, we generated LGR5-tdTomato, LGR5-iCasase9-tdTomato, LGR5-split-Cre, and KRT20-ERCreER knock-in human colon organoids via genome engineering. p27+ dormant cells were further visualized with the p27-mVenus reporter. To analyze the dynamics of human colonic stem cells in vivo, we orthotopically xenotransplanted fluorescence-labeled human colon organoids into immune-deficient mice. The cell cycle dynamics in xenograft cells were evaluated using 5-ethynyl-2'-deoxyuridine pulse-chase analysis. The clonogenic capacity of slow-cycling human stem cells or differentiated cells was analyzed in the context of homeostasis, LGR5 ablation, and 5-fluorouracil-induced mucosal injury. RESULTS: Single-cell RNA sequencing analysis illuminated the presence of nondividing LGR5+ stem cells in the human colon. Visualization and lineage tracing of slow-cycling LGR5+p27+ cells and orthotopic xenotransplantation validated their homeostatic lineage-forming capability in vivo, which was augmented by 5-FU-induced mucosal damage. Transforming growth factor-ß signaling regulated the quiescent state of LGR5+ cells. Despite the plasticity of differentiated KRT20+ cells, they did not display clonal growth after 5-FU-induced injury, suggesting that occupation of the niche environment by LGR5+p27+ cells prevented neighboring differentiated cells from de-differentiating. CONCLUSIONS: Our results highlight the quiescent nature of human LGR5+ colonic stem cells and their contribution to post-injury regeneration.

An Irf6-Esrp1/2 regulatory axis controls midface morphogenesis in vertebrates.

Irf6 and Esrp1 are important for palate development across vertebrates. In zebrafish, we found that irf6 regulates the expression of esrp1 We detailed overlapping Irf6 and Esrp1/2 expression in mouse orofacial epithelium. In zebrafish, irf6 and esrp1/2 share expression in periderm, frontonasal ectoderm and oral epithelium. Genetic disruption of irf6 and esrp1/2 in zebrafish resulted in cleft of the anterior neurocranium. The esrp1/2 mutant also developed cleft of the mouth opening. Lineage tracing of cranial neural crest cells revealed that the cleft resulted not from migration defect, but from impaired chondrogenesis. Analysis of aberrant cells within the cleft revealed expression of sox10, col1a1 and irf6, and these cells were adjacent to krt4+ and krt5+ cells. Breeding of mouse Irf6; Esrp1; Esrp2 compound mutants suggested genetic interaction, as the triple homozygote and the Irf6; Esrp1 double homozygote were not observed. Further, Irf6 heterozygosity reduced Esrp1/2 cleft severity. These studies highlight the complementary analysis of Irf6 and Esrp1/2 in mouse and zebrafish, and identify a unique aberrant cell population in zebrafish expressing sox10, col1a1 and irf6 Future work characterizing this cell population will yield additional insight into cleft pathogenesis.

Chromosome Engineering of Human Colon-Derived Organoids to Develop a Model of Traditional Serrated Adenoma.

BACKGROUND & AIMS: Traditional serrated adenomas (TSAs) are rare colorectal polyps with unique histologic features. Fusions in R-spondin genes have been found in TSAs, but it is not clear whether these are sufficient for TSA development, due to the lack of a chromosome engineering platform for human tissues. We studied the effects of fusions in R-spondin genes and other genetic alterations found in TSA using CRISPR-Cas9-mediated chromosome and genetic modification of human colonic organoids. METHODS: We introduced chromosome rearrangements that involve R-spondin genes into human colonic organoids, with or without disruption of TP53, using CRISPR-Cas9 (chromosome-engineered organoids). We then knocked a mutation into BRAF encoding the V600E substitution and overexpressed the GREM1 transgene; the organoids were transplanted into colons of NOG mice and growth of xenograft tumors was measured. Colon tissues were collected and analyzed by immunohistochemistry or in situ hybridization. We also established 2 patient-derived TSA organoid lines and characterized their genetic features and phenotypes. We inserted a bicistronic cassette expressing a dimerizer-inducible suicide gene and fluorescent marker downstream of the LGR5 gene in the chromosome-engineered organoids; addition of the dimerizer eradicates LGR5+ cells. Some tumor-bearing mice were given intraperitoneal injections of the dimerizer to remove LGR5-expressing cells. RESULTS: Chromosome engineering of organoids required disruption of TP53 or culture in medium containing IGF1 and FGF2. In colons of mice, organoids that expressed BRAFV600E and fusions in R-spondin genes formed flat serrated lesions. Patient-derived TSA organoids grew independent of exogenous R-spondin, and 1 line grew independent of Noggin. Organoids that overexpressed GREM1, in addition to BRAFV600E and fusions in R-spondin genes, formed polypoid tumors in mice that had histologic features similar to TSAs. Xenograft tumors persisted after loss of LGR5-expressing cells. CONCLUSIONS: We demonstrated efficient chromosomal engineering of human normal colon organoids. We introduced genetic and chromosome alterations into human colon organoids found in human TSAs; tumors grown from these organoids in mice had histopathology features of TSAs. This model might be used to study progression of human colorectal tumors with RSPO fusion gene and GREM1 overexpression.

Analysis of risk factors for immune-related adverse events in various solid tumors using real-world data.

The aim of this study was to determine the risk factors for immune-related adverse events (irAEs) induced by immune checkpoint inhibitors. The authors conducted a retrospective study in which patients with malignant melanoma, non-small-cell lung cancer, gastric cancer or renal cell carcinoma who received anti-PD-1/PD-L1 antibodies were included. Of 247 patients, 118 developed a total of 182 irAEs. In the multivariate Fine-Gray regression analysis, serum albumin level ≥3.6 g/dl (hazard ratio: 1.62; 95% CI: 1.10-2.39; p = 0.015) and history of Type I hypersensitivity reactions (hazard ratio: 1.48; 95% CI: 1.02-2.14; p = 0.037) were significantly associated with the development of irAEs. High serum albumin levels and history of Type I hypersensitivity reactions are risk factors for irAEs.

Post-progression survival following second-line chemotherapy in patients with advanced pancreatic cancer previously treated with gemcitabine: a meta-analysis.

Background Post-progression survival (PPS) could be a confounding element in interpreting data from clinical trials of second-line chemotherapy in patients with advanced pancreatic cancer (PC) previously treated with gemcitabine (GEM) because a recent meta-analysis of oxaliplatin combination therapy showed statistical heterogeneity for overall survival (OS) but not for progression-free survival (PFS). This study aimed to improve the understanding of the impact of PPS on OS in this setting. Methods Databases were searched to identify randomized controlled trials (RCTs) in the salvage setting. We evaluated relationships between OS and PFS, PPS, and other variables. Results Totally, 17 RCTs with 3253 patients were identified. Median OS was strongly and moderately associated with median PPS and PFS, respectively (r = 0.913; p < 0.001 and 0.780; p < 0.001, respectively). The proportion of patients with good performance status was significantly associated with both PPS and PFS (r = 0.574, p < 0.001 and 0.492, p < 0.001, respectively). The induction rate of subsequent chemotherapy was related to the duration of PPS and OS (r = 0.640, p < 0.001 and 0.647, p < 0.001, respectively). Median PPS and OS were significantly longer in recent trials than those in older trials (3.55 versus 2.78 months, p < 0.001 and 6.29 versus 5.02 months, p < 0.001). Conclusions Median PPS was strongly correlated with median OS. Given the recently increased opportunity for subsequent chemotherapy and supportive care, PPS may serve as an important element to clarify problems in this setting.

Neuroendocrine neoplasms of the lung and gastrointestinal system: convergent biology and a path to better therapies.

Neuroendocrine neoplasms (NENs) can develop in almost any organ and span a spectrum from well-differentiated and indolent neuroendocrine tumours (NETs) to poorly differentiated and highly aggressive neuroendocrine carcinomas (NECs), including small-cell lung cancer (SCLC). These neoplasms are thought to primarily derive from neuroendocrine precursor cells located throughout the body and can also arise through neuroendocrine transdifferentiation of organ-specific epithelial cell types. Hence, NENs constitute a group of tumour types that share key genomic and phenotypic characteristics irrespective of their site of origin, albeit with some organ-specific differences. The establishment of representative preclinical models for several of these disease entities together with analyses of human tumour specimens has provided important insights into crucial aspects of their biology with therapeutic implications. In this Review, we provide a comprehensive overview of the current understanding of NENs of the gastrointestinal system and lung from clinical and biological perspectives. Research on NENs has typically been siloed by the tumour site of origin, and a cross-cutting view might enable advances in one area to accelerate research in others. Therefore, we aim to emphasize that a better understanding of the commonalities and differences of NENs arising in different organs might more effectively inform clinical research to define therapeutic targets and ultimately optimize patient care.

dact1/2 modifies noncanonical Wnt signaling and calpain 8 expression to regulate convergent extension and craniofacial development.

Wnt signaling plays a crucial role in the early embryonic patterning and development, to regulate convergent extension during gastrulation and the establishment of the dorsal axis. Further, Wnt signaling is a crucial regulator of craniofacial morphogenesis. The adapter proteins Dact1 and Dact2 modulate the Wnt signaling pathway through binding to Disheveled, however, the distinct relative functions of Dact1 and Dact2 during embryogenesis remain unclear. We found that dact1 and dact2 genes have dynamic spatiotemporal expression domains that are reciprocal to one another and to wnt11f2l, that suggest distinct functions during zebrafish embryogenesis. We found that both dact1 and dact2 contribute to axis extension, with compound mutants exhibiting a similar convergent extension defect and craniofacial phenotype to the wnt11f2 mutant. Utilizing single-cell RNAseq and gpc4 mutant that disrupts noncanonical Wnt signaling, we identified dact1/2 specific roles during early development. Comparative whole transcriptome analysis between wildtype, gpc4 and dact1/2 mutants revealed a novel role for dact1/2 in regulating the mRNA expression of the classical calpain capn8. Over-expression of capn8 phenocopies dact1/2 craniofacial dysmorphology. These results identify a previously unappreciated role of capn8 and calcium-dependent proteolysis during embryogenesis. Taken together, our findings highlight the distinct and overlapping roles of dact1 and dact2 in embryonic craniofacial development, providing new insights into the multifaceted regulation of Wnt signaling.

Genotype-phenotype mapping of a patient-derived lung cancer organoid biobank identifies NKX2-1-defined Wnt dependency in lung adenocarcinoma.

Human lung cancer is a constellation of tumors with various histological and molecular properties. To build a preclinical platform that covers this broad disease spectrum, we obtained lung cancer specimens from multiple sources, including sputum and circulating tumor cells, and generated a living biobank consisting of 43 lines of patient-derived lung cancer organoids. The organoids recapitulated the histological and molecular hallmarks of the original tumors. Phenotypic screening of niche factor dependency revealed that EGFR mutations in lung adenocarcinoma are associated with the independence from Wnt ligands. Gene engineering of alveolar organoids reveals that constitutive activation of EGFR-RAS signaling provides Wnt independence. Loss of the alveolar identity gene NKX2-1 confers Wnt dependency, regardless of EGFR signal mutation. Sensitivity to Wnt-targeting therapy can be stratified by the expression status of NKX2-1. Our results highlight the potential of phenotype-driven organoid screening and engineering for the fabrication of therapeutic strategies to combat cancer.

Exportin 1 inhibition prevents neuroendocrine transformation through SOX2 down-regulation in lung and prostate cancers.

In lung and prostate adenocarcinomas, neuroendocrine (NE) transformation to an aggressive derivative resembling small cell lung cancer (SCLC) is associated with poor prognosis. We previously described dependency of SCLC on the nuclear transporter exportin 1. Here, we explored the role of exportin 1 in NE transformation. We observed up-regulated exportin 1 in lung and prostate pretransformation adenocarcinomas. Exportin 1 was up-regulated after genetic inactivation of TP53 and RB1 in lung and prostate adenocarcinoma cell lines, accompanied by increased sensitivity to the exportin 1 inhibitor selinexor in vitro. Exportin 1 inhibition prevented NE transformation in different TP53/RB1-inactivated prostate adenocarcinoma xenograft models that acquire NE features upon treatment with the aromatase inhibitor enzalutamide and extended response to the EGFR inhibitor osimertinib in a lung cancer transformation patient-derived xenograft (PDX) model exhibiting combined adenocarcinoma/SCLC histology. Ectopic SOX2 expression restored the enzalutamide-promoted NE phenotype on adenocarcinoma-to-NE transformation xenograft models despite selinexor treatment. Selinexor sensitized NE-transformed lung and prostate small cell carcinoma PDXs to standard cytotoxics. Together, these data nominate exportin 1 inhibition as a potential therapeutic target to constrain lineage plasticity and prevent or treat NE transformation in lung and prostate adenocarcinoma.

Niacin restriction with NAMPT-inhibition is synthetic lethal to neuroendocrine carcinoma.

Nicotinamide phosphoribosyltransferase (NAMPT) plays a major role in NAD biosynthesis in many cancers and is an attractive potential cancer target. However, factors dictating therapeutic efficacy of NAMPT inhibitors (NAMPTi) are unclear. We report that neuroendocrine phenotypes predict lung and prostate carcinoma vulnerability to NAMPTi, and that NAMPTi therapy against those cancers is enhanced by dietary modification. Neuroendocrine differentiation of tumor cells is associated with down-regulation of genes relevant to quinolinate phosphoribosyltransferase-dependent de novo NAD synthesis, promoting NAMPTi susceptibility in vitro. We also report that circulating nicotinic acid riboside (NAR), a non-canonical niacin absent in culture media, antagonizes NAMPTi efficacy as it fuels NAMPT-independent but nicotinamide riboside kinase 1-dependent NAD synthesis in tumors. In mouse transplantation models, depleting blood NAR by nutritional or genetic manipulations is synthetic lethal to tumors when combined with NAMPTi. Our findings provide a rationale for simultaneous targeting of NAR metabolism and NAMPT therapeutically in neuroendocrine carcinoma.

Zebrafish models of alx-linked frontonasal dysplasia reveal a role for Alx1 and Alx3 in the anterior segment and vasculature of the developing eye.

The cellular and genetic mechanisms that coordinate formation of facial sensory structures with surrounding skeletal and soft tissue elements remain poorly understood. Alx1, a homeobox transcription factor, is a key regulator of midfacial morphogenesis. ALX1 mutations in humans are linked to severe congenital anomalies of the facial skeleton (frontonasal dysplasia, FND) with malformation or absence of eyes and orbital contents (micro- and anophthalmia). Zebrafish with loss-of-function alx1 mutations develop with craniofacial and ocular defects of variable penetrance, likely due to compensatory upregulation in expression of a paralogous gene, alx3. Here we show that zebrafish alx1;alx3 mutants develop with highly penetrant cranial and ocular defects that resemble human ALX1-linked FND. alx1 and alx3 are expressed in anterior cranial neural crest (aCNC), which gives rise to the anterior neurocranium (ANC), anterior segment structures of the eye and vascular pericytes. Consistent with a functional requirement for alx genes in aCNC, alx1; alx3 mutants develop with nearly absent ANC and grossly aberrant hyaloid vasculature and ocular anterior segment, but normal retina. In vivo lineage labeling identified a requirement for alx1 and alx3 during aCNC migration, and transcriptomic analysis suggested oxidative stress response as a key target mechanism of this function. Oxidative stress is a hallmark of fetal alcohol toxicity, and we found increased penetrance of facial and ocular malformations in alx1 mutants exposed to ethanol, consistent with a protective role for alx1 against ethanol toxicity. Collectively, these data demonstrate a conserved role for zebrafish alx genes in controlling ocular and facial development, and a novel role in protecting these key midfacial structures from ethanol toxicity during embryogenesis. These data also reveal novel roles for alx genes in ocular anterior segment formation and vascular development and suggest that retinal deficits in alx mutants may be secondary to aberrant ocular vascularization and anterior segment defects. This study establishes robust zebrafish models for interrogating conserved genetic mechanisms that coordinate facial and ocular development, and for exploring gene--environment interactions relevant to fetal alcohol syndrome.

Comprehensive Genomic Profiling of Neuroendocrine Carcinomas of the Gastrointestinal System.

The neuroendocrine carcinoma of the gastrointestinal system (GIS-NEC) is a rare but highly malignant neoplasm. We analyzed 115 cases using whole-genome/exome sequencing, transcriptome sequencing, DNA methylation assays, and/or ATAC-seq and found GIS-NECs to be genetically distinct from neuroendocrine tumors (GIS-NET) in the same location. Clear genomic differences were also evident between pancreatic NECs (Panc-NEC) and nonpancreatic GIS-NECs (Nonpanc-NEC). Panc-NECs could be classified into two subgroups (i.e., "ductal-type" and "acinar-type") based on genomic features. Alterations in TP53 and RB1 proved common in GIS-NECs, and most Nonpanc-NECs with intact RB1 demonstrated mutually exclusive amplification of CCNE1 or MYC. Alterations of the Notch gene family were characteristic of Nonpanc-NECs. Transcription factors for neuroendocrine differentiation, especially the SOX2 gene, appeared overexpressed in most GIS-NECs due to hypermethylation of the promoter region. This first comprehensive study of genomic alterations in GIS-NECs uncovered several key biological processes underlying genesis of this very lethal form of cancer. SIGNIFICANCE: GIS-NECs are genetically distinct from GIS-NETs. GIS-NECs arising in different organs show similar histopathologic features and share some genomic features, but considerable differences exist between Panc-NECs and Nonpanc-NECs. In addition, Panc-NECs could be classified into two subgroups (i.e., "ductal-type" and "acinar-type") based on genomic and epigenomic features. This article is highlighted in the In This Issue feature, p. 587.

Concordance analysis of microsatellite instability status between polymerase chain reaction based testing and next generation sequencing for solid tumors.

Various malignancies exhibit high microsatellite instability (MSI-H) or mismatch repair deficiency (dMMR). The MSI-IVD kit, a polymerase chain reaction (PCR)-based method, was the first tumor-agnostic companion diagnostic to detect MSI status in MSI-H solid tumors. Recently, next-generation sequencing (NGS), which can also detect MSI-H/dMMR, has been made clinically available; however, its real-world concordance with PCR-based testing of MSI-H/dMMR remains to be investigated. The co-primary end points included the positive and negative predictive values of MSI-H/dMMR. A retrospective analysis of 80 patients who had undergone both MSI testing and NGS between July 2015 and March 2021 was conducted. Five patients were confirmed to have MSI-H in both examinations. Among the 75 patients diagnosed as microsatellite stable (MSS) by PCR-based testing, one with pancreatic cancer was diagnosed as having MSI-H after NGS. One patient with pancreatic cancer was diagnosed as having MSS in both tests was found to have a mutation in MLH1 by NGS, which was confirmed as dMMR by IHC staining. NGS had positive and negative predictive values of 100% (5/5) and 98.7% (74/75), respectively, for MSI-H. The concordance between NGS and PCR-based testing was 98.8% (79/80). Thus, NGS can be useful for evaluating MSI/MMR status in clinical practice and can be an important alternative method for detecting MSI-H/dMMR in the future.

The Entire Intestinal Tract Surveillance Using Capsule Endoscopy after Immune Checkpoint Inhibitor Administration: A Prospective Observational Study.

BACKGROUND: Despite the proven efficacy of immune checkpoint inhibitors (ICIs) against various types of malignancies, they have been found to induce immune-related adverse events, such as enterocolitis; however, the clinical features of ICI-induced enterocolitis remain to be sufficiently elucidated, which is significant, considering the importance of early detection in the appropriate management and treatment of ICI-induced enterocolitis. Therefore, the current study aimed to determine the utility of capsule endoscopy as a screening tool for ICI-induced enterocolitis. METHODS: This single-center, prospective, observational study was conducted on patients with malignancy who received any ICI between April 2016 and July 2020 at Keio University Hospital. Next, second-generation capsule endoscopy (CCE-2) was performed on day 60 after ICI initiation to explore the entire gastrointestinal tract. RESULTS: Among the 30 patients enrolled herein, 23 underwent CCE-2. Accordingly, a total of 23 findings were observed in 14 (60.8%) patients at any portion of the gastrointestinal tract (7 patients in the colon, 4 patients in the small intestine, 2 patients in both the colon and the small intestine, and 1 patient in the stomach). After capsule endoscopy, 2 patients (8.7%) developed ICI-induced enterocolitis: both had significantly higher Capsule Scoring of Ulcerative Colitis than those who had not developed ICI-induced enterocolitis (p = 0.0455). No adverse events related to CCE-2 were observed. CONCLUSIONS: CCE-2 might be a safe and useful entire intestinal tract screening method for the early detection of ICI-induced enterocolitis in patients with malignancies.

Synergistic roles of Wnt modulators R-spondin2 and R-spondin3 in craniofacial morphogenesis and dental development.

Wnt signaling plays a critical role in craniofacial patterning, as well as tooth and bone development. Rspo2 and Rspo3 are key regulators of Wnt signaling. However, their coordinated function and relative requirement in craniofacial development and odontogensis are poorly understood. We showed that in zebrafish rspo2 and rspo3 are both expressed in osteoprogenitors in the embryonic craniofacial skeleton. This is in contrast to mouse development, where Rspo3 is expressed in osteoprogenitors while Rspo2 expression is not observed. In zebrafish, rspo2 and rspo3 are broadly expressed in the pulp, odontoblasts and epithelial crypts. However, in the developing molars of the mouse, Rspo3 is largely expressed in the dental follicle and alveolar mesenchyme while Rspo2 expression is restricted to the tooth germ. While Rspo3 ablation in the mouse is embryonic lethal, zebrafish rspo3-/- mutants are viable with modest decrease in Meckel's cartilage rostral length. However, compound disruption of rspo3 and rspo2 revealed synergistic roles of these genes in cartilage morphogenesis, fin development, and pharyngeal tooth development. Adult rspo3-/- zebrafish mutants exhibit a dysmorphic cranial skeleton and decreased average tooth number. This study highlights the differential functions of Rspo2 and Rspo3 in dentocranial morphogenesis in zebrafish and in mouse.

ALX1-related frontonasal dysplasia results from defective neural crest cell development and migration.

A pedigree of subjects presented with frontonasal dysplasia (FND). Genome sequencing and analysis identified a p.L165F missense variant in the homeodomain of the transcription factor ALX1 which was imputed to be pathogenic. Induced pluripotent stem cells (iPSC) were derived from the subjects and differentiated to neural crest cells (NCC). NCC derived from ALX1L165F/L165F iPSC were more sensitive to apoptosis, showed an elevated expression of several neural crest progenitor state markers, and exhibited impaired migration compared to wild-type controls. NCC migration was evaluated in vivo using lineage tracing in a zebrafish model, which revealed defective migration of the anterior NCC stream that contributes to the median portion of the anterior neurocranium, phenocopying the clinical presentation. Analysis of human NCC culture media revealed a change in the level of bone morphogenic proteins (BMP), with a low level of BMP2 and a high level of BMP9. Soluble BMP2 and BMP9 antagonist treatments were able to rescue the defective migration phenotype. Taken together, these results demonstrate a mechanistic requirement of ALX1 in NCC development and migration.