STUB1 is an intracellular checkpoint for interferon gamma sensing.

Immune checkpoint blockade (ICB) leads to durable and complete tumour regression in some patients but in others gives temporary, partial or no response. Accordingly, significant efforts are underway to identify tumour-intrinsic mechanisms underlying ICB resistance. Results from a published CRISPR screen in a mouse model suggested that targeting STUB1, an E3 ligase involved in protein homeostasis, may overcome ICB resistance but the molecular basis of this effect remains unclear. Herein, we report an under-appreciated role of STUB1 to dampen the interferon gamma (IFNγ) response. Genetic deletion of STUB1 increased IFNGR1 abundance on the cell surface and thus enhanced the downstream IFNγ response as showed by multiple approaches including Western blotting, flow cytometry, qPCR, phospho-STAT1 assay, immunopeptidomics, proteomics, and gene expression profiling. Human prostate and breast cancer cells with STUB1 deletion were also susceptible to cytokine-induced growth inhibition. Furthermore, blockade of STUB1 protein function recapitulated the STUB1-null phenotypes. Despite these encouraging in vitro data and positive implications from clinical datasets, we did not observe in vivo benefits of inactivating Stub1 in mouse syngeneic tumour models-with or without combination with anti-PD-1 therapy. However, our findings elucidate STUB1 as a barrier to IFNγ sensing, prompting further investigations to assess if broader inactivation of human STUB1 in both tumors and immune cells could overcome ICB resistance.

Single-cell and bulk transcriptome sequencing identifies two epithelial tumor cell states and refines the consensus molecular classification of colorectal cancer.

The consensus molecular subtype (CMS) classification of colorectal cancer is based on bulk transcriptomics. The underlying epithelial cell diversity remains unclear. We analyzed 373,058 single-cell transcriptomes from 63 patients, focusing on 49,155 epithelial cells. We identified a pervasive genetic and transcriptomic dichotomy of malignant cells, based on distinct gene expression, DNA copy number and gene regulatory network. We recapitulated these subtypes in bulk transcriptomes from 3,614 patients. The two intrinsic subtypes, iCMS2 and iCMS3, refine CMS. iCMS3 comprises microsatellite unstable (MSI-H) cancers and one-third of microsatellite-stable (MSS) tumors. iCMS3 MSS cancers are transcriptomically more similar to MSI-H cancers than to other MSS cancers. CMS4 cancers had either iCMS2 or iCMS3 epithelium; the latter had the worst prognosis. We defined the intrinsic epithelial axis of colorectal cancer and propose a refined 'IMF' classification with five subtypes, combining intrinsic epithelial subtype (I), microsatellite instability status (M) and fibrosis (F).

Systematic evaluation of multiple qPCR platforms, NanoString and miRNA-Seq for microRNA biomarker discovery in human biofluids.

Aberrant miRNA expression has been associated with many diseases, and extracellular miRNAs that circulate in the bloodstream are remarkably stable. Recently, there has been growing interest in identifying cell-free circulating miRNAs that can serve as non-invasive biomarkers for early detection of disease or selection of treatment options. However, quantifying miRNA levels in biofluids is technically challenging due to their low abundance. Using reference samples, we performed a cross-platform evaluation in which miRNA profiling was performed on four different qPCR platforms (MiRXES, Qiagen, Applied Biosystems, Exiqon), nCounter technology (NanoString), and miRNA-Seq. Overall, our results suggest that using miRNA-Seq for discovery and targeted qPCR for validation is a rational strategy for miRNA biomarker development in clinical samples that involve limited amounts of biofluids.

A preliminary study for the assessment of PD-L1 and PD-L2 on circulating tumor cells by microfluidic-based chipcytometry.

AIM: Expression of PD-L1 in the tumor is associated with more favorable responses to anti-PD-1 therapy in multiple cancers. However, obtaining tumor biopsies for PD-L1 interrogation is an invasive procedure and challenging to assess repeatedly as the disease progresses. MATERIALS & METHODS: Here we assess an alternative, minimally invasive approach to analyze blood samples for circulating tumor cells (CTCs) that have broken away from the tumor and entered the periphery. Our approach uses sized-based microfluidic CTC enrichment and subsequent characterization with microfluidic-based cytometry (chipcytometry). CONCLUSION: We demonstrate tumor-cell detection and characterization for PD-L1, and other markers, in both spiked and patient samples. This preliminary communication is the first report using chipcytometry for the characterization of CTCs.

Single-virion sequencing of lamivudine-treated HBV populations reveal population evolution dynamics and demographic history.

BACKGROUND: Viral populations are complex, dynamic, and fast evolving. The evolution of groups of closely related viruses in a competitive environment is termed quasispecies. To fully understand the role that quasispecies play in viral evolution, characterizing the trajectories of viral genotypes in an evolving population is the key. In particular, long-range haplotype information for thousands of individual viruses is critical; yet generating this information is non-trivial. Popular deep sequencing methods generate relatively short reads that do not preserve linkage information, while third generation sequencing methods have higher error rates that make detection of low frequency mutations a bioinformatics challenge. Here we applied BAsE-Seq, an Illumina-based single-virion sequencing technology, to eight samples from four chronic hepatitis B (CHB) patients - once before antiviral treatment and once after viral rebound due to resistance. RESULTS: With single-virion sequencing, we obtained 248-8796 single-virion sequences per sample, which allowed us to find evidence for both hard and soft selective sweeps. We were able to reconstruct population demographic history that was independently verified by clinically collected data. We further verified four of the samples independently through PacBio SMRT and Illumina Pooled deep sequencing. CONCLUSIONS: Overall, we showed that single-virion sequencing yields insight into viral evolution and population dynamics in an efficient and high throughput manner. We believe that single-virion sequencing is widely applicable to the study of viral evolution in the context of drug resistance and host adaptation, allows differentiation between soft or hard selective sweeps, and may be useful in the reconstruction of intra-host viral population demographic history.

Pre-Analytical Considerations for Successful Next-Generation Sequencing (NGS): Challenges and Opportunities for Formalin-Fixed and Paraffin-Embedded Tumor Tissue (FFPE) Samples.

In cancer drug discovery, it is important to investigate the genetic determinants of response or resistance to cancer therapy as well as factors that contribute to adverse events in the course of clinical trials. Despite the emergence of new technologies and the ability to measure more diverse analytes (e.g., circulating tumor cell (CTC), circulating tumor DNA (ctDNA), etc.), tumor tissue is still the most common and reliable source for biomarker investigation. Because of its worldwide use and ability to preserve samples for many decades at ambient temperature, formalin-fixed, paraffin-embedded tumor tissue (FFPE) is likely to be the preferred choice for tissue preservation in clinical practice for the foreseeable future. Multiple analyses are routinely performed on the same FFPE samples (such as Immunohistochemistry (IHC), in situ hybridization, RNAseq, DNAseq, TILseq, Methyl-Seq, etc.). Thus, specimen prioritization and optimization of the isolation of analytes is critical to ensure successful completion of each assay. FFPE is notorious for producing suboptimal DNA quality and low DNA yield. However, commercial vendors tend to request higher DNA sample mass than what is actually required for downstream assays, which restricts the breadth of biomarker work that can be performed. We evaluated multiple genomics service laboratories to assess the current state of NGS pre-analytical processing of FFPE. Significant differences in pre-analytical capabilities were observed. Key aspects are highlighted and recommendations are made to improve the current practice in translational research.

Predictive Factors for BRCA1 and BRCA2 Genetic Testing in an Asian Clinic-Based Population.

PURPOSE: The National Comprehensive Cancer Network (NCCN) has proposed guidelines for the genetic testing of the BRCA1 and BRCA2 genes, based on studies in western populations. This current study assessed potential predictive factors for BRCA mutation probability, in an Asian population. METHODS: A total of 359 breast cancer patients, who presented with either a family history (FH) of breast and/or ovarian cancer or early onset breast cancer, were accrued at the National Cancer Center Singapore (NCCS). The relationships between clinico-pathological features and mutational status were calculated using the Chi-squared test and binary logistic regression analysis. RESULTS: Of 359 patients, 45 (12.5%) had deleterious or damaging missense mutations in BRCA1 and/or BRCA2. BRCA1 mutations were more likely to be found in ER-negative than ER-positive breast cancer patients (P=0.01). Moreover, ER-negative patients with BRCA mutations were diagnosed at an earlier age (40 vs. 48 years, P=0.008). Similarly, triple-negative breast cancer (TNBC) patients were more likely to have BRCA1 mutations (P=0.001) and that these patients were diagnosed at a relatively younger age than non-TNBC patients (38 vs. 46 years, P=0.028). Our analysis has confirmed that ER-negative status, TNBC status and a FH of hereditary breast and ovarian cancer (HBOC) are strong factors predicting the likelihood of having BRCA mutations. CONCLUSIONS: Our study provides evidence that TNBC or ER-negative patients may benefit from BRCA genetic testing, particularly younger patients (<40 years) or those with a strong FH of HBOC, in Asian patients.

BAsE-Seq: a method for obtaining long viral haplotypes from short sequence reads.

We present a method for obtaining long haplotypes, of over 3 kb in length, using a short-read sequencer, Barcode-directed Assembly for Extra-long Sequences (BAsE-Seq). BAsE-Seq relies on transposing a template-specific barcode onto random segments of the template molecule and assembling the barcoded short reads into complete haplotypes. We applied BAsE-Seq on mixed clones of hepatitis B virus and accurately identified haplotypes occurring at frequencies greater than or equal to 0.4%, with >99.9% specificity. Applying BAsE-Seq to a clinical sample, we obtained over 9,000 viral haplotypes, which provided an unprecedented view of hepatitis B virus population structure during chronic infection. BAsE-Seq is readily applicable for monitoring quasispecies evolution in viral diseases.

Specifying and sustaining pigmentation patterns in domestic and wild cats.

Color markings among felid species display both a remarkable diversity and a common underlying periodicity. A similar range of patterns in domestic cats suggests a conserved mechanism whose appearance can be altered by selection. We identified the gene responsible for tabby pattern variation in domestic cats as Transmembrane aminopeptidase Q (Taqpep), which encodes a membrane-bound metalloprotease. Analyzing 31 other felid species, we identified Taqpep as the cause of the rare king cheetah phenotype, in which spots coalesce into blotches and stripes. Histologic, genomic expression, and transgenic mouse studies indicate that paracrine expression of Endothelin3 (Edn3) coordinates localized color differences. We propose a two-stage model in which Taqpep helps to establish a periodic pre-pattern during skin development that is later implemented by differential expression of Edn3.

Digital gene expression for non-model organisms.

Next-generation sequencing technologies offer new approaches for global measurements of gene expression but are mostly limited to organisms for which a high-quality assembled reference genome sequence is available. We present a method for gene expression profiling called EDGE, or EcoP15I-tagged Digital Gene Expression, based on ultra-high-throughput sequencing of 27-bp cDNA fragments that uniquely tag the corresponding gene, thereby allowing direct quantification of transcript abundance. We show that EDGE is capable of assaying for expression in >99% of genes in the genome and achieves saturation after 6-8 million reads. EDGE exhibits very little technical noise, reveals a large (10(6)) dynamic range of gene expression, and is particularly suited for quantification of transcript abundance in non-model organisms where a high-quality annotated genome is not available. In a direct comparison with RNA-seq, both methods provide similar assessments of relative transcript abundance, but EDGE does better at detecting gene expression differences for poorly expressed genes and does not exhibit transcript length bias. Applying EDGE to laboratory mice, we show that a loss-of-function mutation in the melanocortin 1 receptor (Mc1r), recognized as a Mendelian determinant of yellow hair color in many different mammals, also causes reduced expression of genes involved in the interferon response. To illustrate the application of EDGE to a non-model organism, we examine skin biopsy samples from a cheetah (Acinonyx jubatus) and identify genes likely to control differences in the color of spotted versus non-spotted regions.

Genetics of Sex-linked yellow in the Syrian hamster.

Alternating patches of black and yellow pigment are a ubiquitous feature of mammalian color variation that contributes to camouflage, species recognition, and morphologic diversity. X-linked determinants of this pattern--recognized by variegation in females but not in males--have been described in the domestic cat as Orange, and in the Syrian hamster as Sex-linked yellow (Sly), but are curiously absent from other vertebrate species. Using a comparative genomic approach, we develop molecular markers and a linkage map for the euchromatic region of the Syrian hamster X chromosome that places Sly in a region homologous to the centromere-proximal region of human Xp. Comparison to analogous work carried out for Orange in domestic cats indicates, surprisingly, that the cat and hamster mutations lie in nonhomologous regions of the X chromosome. We also identify the molecular cause of recessively inherited black coat color in hamsters (historically referred to as nonagouti) as a Cys115Tyr mutation in the Agouti gene. Animals doubly mutant for Sly and nonagouti exhibit a Sly phenotype. Our results indicate that Sly represents a melanocortin pathway component that acts similarly to, but is genetically distinct from, Mc1r and that has implications for understanding both the evolutionary history and the mutational mechanisms of pigment-type switching.