A urinary assay for mutation and methylation biomarkers in the diagnosis and recurrence prediction of non-muscle invasive bladder cancer patients.

BACKGROUND: Currently, the clinical strategy for diagnosis of non-muscle invasive bladder cancer (NMIBC) such as cystoscopy and cytology are invasive and/or with limited accuracy. OncoUrine, a urinary assay for mutation and methylation biomarkers, have showed a high accuracy in the detection of upper tract urinary carcinoma (UTUC) patients with hematuria. The aim of this study is to evaluate the performance of OncoUrine in diagnosis of NMIBC patients. METHODS: In this multicenter prospective study, a total of 203 patients were enrolled, including 60 patients present with hematuria and 143 NMIBC patients under recurrence surveillance. Urine samples were collected before cystoscopy to undergo OncoUrine test. OncoUrine performance was calculated compared to clinical standard methods in hematuria cohort and recurrence surveillance cohort, respectively. Furthermore, NMIBC patients were followed up with a median time of 20.5 months (range 0.03 to 24.03 months) to assess the predictive value of OncoUrine during recurrence monitoring. RESULTS: For bladder cancer diagnosis, OncoUrine tested 47 samples and achieved a sensitivity/specificity/positive predictive value (PPV)/negative predictive value (NPV) of 80% (95% CI 44.2-96.5)/91.9% (95% CI 77.0-97.9)/72.7% (95% CI 39.3-92.7)/94.4% (95% CI 80.0-99.0) (kappa value 69.4%, 95% CI 44.4-94.3), indicating 72.3% of unnecessary cystoscopy. For recurrence diagnosis, OncoUrine tested 93 samples, and the sensitivity/specificity/PPV/NPV was 100% (95% CI 59.8-100.0)/68.2% (95% CI 57.1-77.7)/22.9% (95% CI 11.0-40.6)/100% (95% CI 92.3-100.0) (kappa value 27.0%, 95% CI 11.1-42.8), indicating 62.4% of spared cystoscopy. What is more, OncoUrine correctly predicted 80% (20/25) of final recurrence with 12/25 (48%) patients who were OncoUrine positive, but cystoscopy negative was followed with recurrence during follow-up. The test result of OncoUrine was also found significantly correlated with recurrence free survival (RFS) of NMIBC patients (median 34.4-month vs unreached; HR 6.0, 95% CI 2.7-13.5, P < 0.0001). CONCLUSIONS: OncoUrine showed potential value to reduce the frequency of unnecessary cystoscopy and the healthcare cost of bladder cancer patients. Patients with positive test results represented a population who were at high risk of recurrence and thus should be subject to frequent surveillance to ensure timely detection of any potential recurrence. This study has been registered in ClinicalTrials.gov with the number NCT04994197 posted on August 2021.

Genomic landscape of microsatellite instability in Chinese tumors: A comparison of Chinese and TCGA cohorts.

Microsatellite instability (MSI) is an important biomarker for predicting the response to immunotherapy and prognosis that mainly results from a defective DNA mismatch repair (MMR) system and strongly correlates with high tumor mutation burden (TMB). Herein, we developed a novel method that integrates MSI score, MMR mutation status and TMB level to identify MSI status from next-generation sequencing (NGS) data. The novel method displays a sensitivity of 96.80%, a specificity of 99.96% and an overall accuracy of 99.89%, compared to current standards. Using our novel method, we analyzed 11 395 Chinese patients across 30 cancer types. High microsatellite instability (MSI-H) was detected in 210 (1.84%) samples in 18 of 30 cancer types assessed. Mutations in ACVR2A (73%), KMT2D (68%), KMT2B (66%) and MMR-related genes (MLH1, MSH2, MSH6 and PMS2) were enriched in MSI-H samples. Furthermore, MSI-H samples were more likely to have high TMB (P < .01), high PD-L1 expression (P < .05) and more tumor-infiltrating immune cells than microsatellite-stable (MSS) samples. Compared to the TCGA patients, the prevalence of MSI-H in the Chinese cohort was significantly lower in colorectal, gastric and pancreatic cancer, while significantly higher in urinary and prostate cancer. Mutations in ACVR2A (73% vs 28%, P < .01) and MMR-related genes (51.4% vs 21.3%, P < .01) were significantly higher in the Chinese population. Thus, our study suggests the fraction of MSI-H attributable to MMR inactivation mutations were lower in European than in Chinese patients, while the proportion of MSI-H due to other events may be higher.

The Hox gene Antennapedia is essential for wing development in insects.

A long-standing view in the field of evo-devo is that insect forewings develop without any Hox gene input. The Hox gene Antennapedia (Antp), despite being expressed in the thoracic segments of insects, has no effect on wing development. This view has been obtained from studies in two main model species: Drosophila and Tribolium. Here, we show that partial loss of function of Antp resulted in reduced and malformed adult wings in Bombyx, Drosophila and Tribolium. Antp mediates wing growth in Bombyx by directly regulating the ecdysteriod biosynthesis enzyme gene (shade) in the wing tissue, which leads to local production of the growth hormone 20-hydroxyecdysone. Additional targets of Antp are wing cuticular protein genes CPG24, CPH28 and CPG9, which are essential for wing development. We propose, therefore, that insect wing development occurs in an Antp-dependent manner. This article has an associated 'The people behind the papers' interview.

Regulatory encoding of quantitative variation in spatial activity of a Drosophila enhancer.

Developmental enhancers control the expression of genes prefiguring morphological patterns. The activity of an enhancer varies among cells of a tissue, but collectively, expression levels in individual cells constitute a spatial pattern of gene expression. How the spatial and quantitative regulatory information is encoded in an enhancer sequence is elusive. To link spatial pattern and activity levels of an enhancer, we used systematic mutations of the yellow spot enhancer, active in developing Drosophila wings, and tested their effect in a reporter assay. Moreover, we developed an analytic framework based on the comprehensive quantification of spatial reporter activity. We show that the quantitative enhancer activity results from densely packed regulatory information along the sequence, and that a complex interplay between activators and multiple tiers of repressors carves the spatial pattern. Our results shed light on how an enhancer reads and integrates trans-regulatory landscape information to encode a spatial quantitative pattern.

Enhancer evolutionary co-option through shared chromatin accessibility input.

The diversity of forms in multicellular organisms originates largely from the spatial redeployment of developmental genes [S. B. Carroll, Cell 134, 25-36 (2008)]. Several scenarios can explain the emergence of cis-regulatory elements that govern novel aspects of a gene expression pattern [M. Rebeiz, M. Tsiantis, Curr. Opin. Genet. Dev. 45, 115-123 (2017)]. One scenario, enhancer co-option, holds that a DNA sequence producing an ancestral regulatory activity also becomes the template for a new regulatory activity, sharing regulatory information. While enhancer co-option might fuel morphological diversification, it has rarely been documented [W. J. Glassford et al., Dev. Cell 34, 520-531 (2015)]. Moreover, if two regulatory activities are borne from the same sequence, their modularity, considered a defining feature of enhancers [J. Banerji, L. Olson, W. Schaffner, Cell 33, 729-740 (1983)], might be affected by pleiotropy. Sequence overlap may thereby play a determinant role in enhancer function and evolution. Here, we investigated this problem with two regulatory activities of the Drosophila gene yellow, the novel spot enhancer and the ancestral wing blade enhancer. We used precise and comprehensive quantification of each activity in Drosophila wings to systematically map their sequences along the locus. We show that the spot enhancer has co-opted the sequences of the wing blade enhancer. We also identified a pleiotropic site necessary for DNA accessibility of a shared regulatory region. While the evolutionary steps leading to the derived activity are still unknown, such pleiotropy suggests that enhancer accessibility could be one of the molecular mechanisms seeding evolutionary co-option.

Revisiting the developmental and cellular role of the pigmentation gene yellow in Drosophila using a tagged allele.

Pigmentation is a diverse and ecologically relevant trait in insects. Pigment formation has been studied extensively at the genetic and biochemical levels. The temporality of pigment formation during animal development, however, is more elusive. Here, we examine this temporality, focusing on yellow, a gene involved in the formation of black melanin. We generated a protein-tagged yellow allele in the fruit fly Drosophila melanogaster, which allowed us to precisely describe Yellow expression pattern at the tissue and cellular levels throughout development. We found Yellow expressed in the pupal epidermis in patterns prefiguring black pigmentation. We also found Yellow expressed in a few central neurons from the second larval instar to adult stages, including a subset of neurons adjacent to the clock neurons marked by the gene Pdf. We then specifically examined the dynamics of Yellow expression domain and subcellular localization in relationship to pigment formation. In particular, we showed how a late step of re-internalization is regulated by the large low-density lipoprotein receptor-related protein Megalin. Finally we suggest a new function for Yellow in the establishment of sharp pigmentation pattern boundaries, whereby this protein may assume a structural role, anchoring pigment deposits or pigmentation enzymes in the cuticle.

Genome-wide identification and characterization of Fox genes in the silkworm, Bombyx mori.

The forkhead box (Fox) transcription factor family has a characteristic of forkhead domain, a winged DNA-binding domain. The Fox genes have been classified into 23 subfamilies, designated FoxA to FoxS, of which the FoxR and FoxS subfamilies are specific to vertebrates. In this review, using whole-genome scanning, we identified 17 distinct Fox genes distributed on 13 chromosomes of the silkworm, Bombyx mori. A phylogenetic tree showed that the silkworm Fox genes could be classified into 13 subfamilies. The FoxK subfamily is specifically absent from the silkworm, although it is present in other lepidopteran insects, including Danaus plexippus and Heliconius melpomene. Microarray data revealed that the Fox genes have distinct expression patterns in the tissues on day 3 of the 5th instar larva. A Gene Ontology analysis suggested that the Fox genes have roles in cellular components, molecular functions, and biological processes, except in pore complex biogenesis. An analysis of the selective pressure on the proteins indicated that most of the amino acid sites in the Fox proteins are undergoing strong purifying selection. Here, we summarize the general characteristics of the Fox genes in the silkworm, which should support further functional studies of the silkworm Fox proteins.

Mutation of a cuticular protein, BmorCPR2, alters larval body shape and adaptability in silkworm, Bombyx mori.

Cuticular proteins (CPs) are crucial components of the insect cuticle. Although numerous genes encoding cuticular proteins have been identified in known insect genomes to date, their functions in maintaining insect body shape and adaptability remain largely unknown. In the current study, positional cloning led to the identification of a gene encoding an RR1-type cuticular protein, BmorCPR2, highly expressed in larval chitin-rich tissues and at the mulberry leaf-eating stages, which is responsible for the silkworm stony mutant. In the Dazao-stony strain, the BmorCPR2 allele is a deletion mutation with significantly lower expression, compared to the wild-type Dazao strain. Dysfunctional BmorCPR2 in the stony mutant lost chitin binding ability, leading to reduced chitin content in larval cuticle, limitation of cuticle extension, abatement of cuticle tensile properties, and aberrant ratio between internodes and intersegmental folds. These variations induce a significant decrease in cuticle capacity to hold the growing internal organs in the larval development process, resulting in whole-body stiffness, tightness, and hardness, bulging intersegmental folds, and serious defects in larval adaptability. To our knowledge, this is the first study to report the corresponding phenotype of stony in insects caused by mutation of RR1-type cuticular protein. Our findings collectively shed light on the specific role of cuticular proteins in maintaining normal larval body shape and will aid in the development of pest control strategies for the management of Lepidoptera.