Molecular landscape of proliferative verrucous leukoplakia: a systematic review.

Proliferative verrucous leukoplakia (PVL) is a rare oral potentially malignant disorder characterised by multifocal origin and unpredictable long-term evolution to oral squamous cell carcinoma (OSCC) or oral verrucous carcinoma (OVC). Currently no predictive biomarkers are in clinical use. We aimed to explore the genomic profile of PVL. A total of 685 cases in 26 studies were included in this review. Genomic data were presented in 15% of studies and biomarker analysis was reported in 85% of studies. At first clinical presentation, PVL is characterised by a high loss of heterozygosity (LOH), similar to OSCC, and low copy number alterations (CNA). As these progress, more CNAs and mutations in CDKN2A and alterations to ELAVL1 expression are noted, but no TP53 mutations are identified. There is significantly lower LOH at 17p in early PVL compared with OSCC (p = 0.037). Deletions in chromosomal loci 17q12, 5q31.1 and amplifications in 7q11.2, 7q22 are shared between early lesions and OVC. PVL shows CNAs at 11q31. WNT signalling pathway genes (SUZ12, CTTN and FOLR3) are enriched in CN-altered regions. PVL stroma shows significantly lower α-SMA and higher CD34 expression than OVC and OSCC. The exact genomic landscape is currently unclear, and further studies are necessary to unravel this mystery.

Single-cell analysis reveals prognostic fibroblast subpopulations linked to molecular and immunological subtypes of lung cancer.

Fibroblasts are poorly characterised cells that variably impact tumour progression. Here, we use single cell RNA-sequencing, multiplexed immunohistochemistry and digital cytometry (CIBERSORTx) to identify and characterise three major fibroblast subpopulations in human non-small cell lung cancer: adventitial, alveolar and myofibroblasts. Alveolar and adventitial fibroblasts (enriched in control tissue samples) localise to discrete spatial niches in histologically normal lung tissue and indicate improved overall survival rates when present in lung adenocarcinomas (LUAD). Trajectory inference identifies three phases of control tissue fibroblast activation, leading to myofibroblast enrichment in tumour samples: initial upregulation of inflammatory cytokines, followed by stress-response signalling and ultimately increased expression of fibrillar collagens. Myofibroblasts correlate with poor overall survival rates in LUAD, associated with loss of epithelial differentiation, TP53 mutations, proximal molecular subtypes and myeloid cell recruitment. In squamous carcinomas myofibroblasts were not prognostic despite being transcriptomically equivalent. These findings have important implications for developing fibroblast-targeting strategies for cancer therapy.

The spatiotemporal dynamics of microglia across the human lifespan.

Microglia, the brain's resident macrophages, shape neural development and are key neuroimmune hubs in the pathological signatures of neurodevelopmental disorders. Despite the importance of microglia, their development has not been carefully examined in the human brain, and most of our knowledge derives from rodents. We aimed to address this gap in knowledge by establishing an extensive collection of 97 post-mortem tissues in order to enable quantitative, sex-matched, detailed analysis of microglia across the human lifespan. We identify the dynamics of these cells in the human telencephalon, describing waves in microglial density across gestation, infancy, and childhood, controlled by a balance of proliferation and apoptosis, which track key neurodevelopmental milestones. These profound changes in microglia are also observed in bulk RNA-seq and single-cell RNA-seq datasets. This study provides a detailed insight into the spatiotemporal dynamics of microglia across the human lifespan and serves as a foundation for elucidating how microglia contribute to shaping neurodevelopment in humans.

Phosphodiesterase type 5 inhibitors enhance chemotherapy in preclinical models of esophageal adenocarcinoma by targeting cancer-associated fibroblasts.

The chemotherapy resistance of esophageal adenocarcinomas (EACs) is underpinned by cancer cell extrinsic mechanisms of the tumor microenvironment (TME). We demonstrate that, by targeting the tumor-promoting functions of the predominant TME cell type, cancer-associated fibroblasts (CAFs) with phosphodiesterase type 5 inhibitors (PDE5i), we can enhance the efficacy of standard-of-care chemotherapy. In ex vivo conditions, PDE5i prevent the transdifferentiation of normal fibroblasts to CAF and abolish the tumor-promoting function of established EAC CAFs. Using shotgun proteomics and single-cell RNA-seq, we reveal PDE5i-specific regulation of pathways related to fibroblast activation and tumor promotion. Finally, we confirm the efficacy of PDE5i in combination with chemotherapy in close-to-patient and in vivo PDX-based model systems. These findings demonstrate that CAFs drive chemotherapy resistance in EACs and can be targeted by repurposing PDE5i, a safe and well-tolerated class of drug administered to millions of patients world-wide to treat erectile dysfunction.

Comparison of optimized methodologies for isolating nuclei from esophageal tissue.

Single-nuclei RNA sequencing allows single cell-based analysis in frozen tissue, ameliorating cell recovery biases associated with enzymatic dissociation methods. The authors present two optimized methods for isolating and sequencing nuclei from esophageal tissue using a commercial EZ and citric acid (CA)-based method. Despite high endogenous RNase activity, these protocols produced libraries of expected fragment length (average length EZ: 745 bp; CA: 1232 bp) with comparable complexity (median Transcript/Gene number, EZ: 496/254; CA: 483/256). CA nuclei showed a higher proportion of ribosomal gene reads, potentially reflecting co-isolation of nuclei and adherent ribosomes. The authors identified 11 cell lineages in the combined datasets, with differences in cell type recovery between the two methods, providing utility dependent on experimental needs.

Single-nucleotide Fcγ receptor polymorphisms do not impact obinutuzumab/rituximab outcome in patients with lymphoma.

Single-nucleotide polymorphisms (SNPs) have been shown to influence Fcγ receptor (FcγR) affinity and activity, but their effect on treatment response is unclear. We assessed their importance in the efficacy of obinutuzumab or rituximab combined with chemotherapy in untreated advanced follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL) in the GALLIUM (www.clinicaltrials.gov #NCT01332968) and GOYA (#NCT01287741) trials, respectively. Genomic DNA was extracted from patients enrolled in GALLIUM (n = 1202) and GOYA (n = 1418). Key germline SNPs, FCGR2A R131H (rs1801274), FCGR3A F158V (rs396991), and FCGR2B I232T (rs1050501), were genotyped and assessed for their impact on investigator-assessed progression-free survival (PFS). In both cohorts there was no prognostic effect of FCGR2A or FCGR3A. In FL, FCGR2B was associated with favorable PFS in univariate and multivariate analyses comparing I232T with I232I, with a more modest association for rituximab-treated (univariate: hazard ratio [HR], 0.78; 95% confidence interval [CI], 0.54-1.14; P = .21) vs obinutuzumab-treated patients (HR, 0.56; 95% CI, 0.34-0.91; P = .02). Comparing T232T with I232I, an association was found for obinutuzumab (univariate: HR, 2.76; 95% CI, 1.02-7.5; P = .0459). Neither observation retained significance after multiple-test adjustment. FCGR2B was associated with poorer PFS in multivariate analyses comparing T232T with I232I in rituximab- but not obinutuzumab-treated patients with DLBCL (HR, 4.40; 95% CI, 1.71-11.32; P = .002; multiple-test-adjusted P = .03); however, this genotype was rare (n = 13). This study shows that FcγR genotype is not associated with response to rituximab/obinutuzumab plus chemotherapy in treatment-naive patients with advanced FL or DLBCL.

Prognostic significance of FCGR2B expression for the response of DLBCL patients to rituximab or obinutuzumab treatment.

Fc γ receptor IIB (FcγRIIB) is an inhibitory molecule capable of reducing antibody immunotherapy efficacy. We hypothesized its expression could confer resistance in patients with diffuse large B-cell lymphoma (DLBCL) treated with anti-CD20 monoclonal antibody (mAb) chemoimmunotherapy, with outcomes varying depending on mAb (rituximab [R]/obinutuzumab [G]) because of different mechanisms of action. We evaluated correlates between FCGR2B messenger RNA and/or FcγRIIB protein expression and outcomes in 3 de novo DLBCL discovery cohorts treated with R plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) reported by Arthur, Schmitz, and Reddy, and R-CHOP/G-CHOP-treated patients in the GOYA trial (NCT01287741). In the discovery cohorts, higher FCGR2B expression was associated with significantly shorter progression-free survival (PFS; Arthur: hazard ratio [HR], 1.09; 95% confidence interval [CI], 1.01-1.19; P = .0360; Schmitz: HR, 1.13; 95% CI, 1.02-1.26; P = .0243). Similar results were observed in GOYA with R-CHOP (HR, 1.26; 95% CI, 1.00-1.58; P = .0455), but not G-CHOP (HR, 0.91; 95% CI, 0.69-1.20; P = .50). A nonsignificant trend that high FCGR2B expression favored G-CHOP over R-CHOP was observed (HR, 0.67; 95% CI, 0.44-1.02; P = .0622); however, low FCGR2B expression favored R-CHOP (HR, 1.58; 95% CI, 1.00-2.50; P = .0503). In Arthur and GOYA, FCGR2B expression was associated with tumor FcγRIIB expression; correlating with shorter PFS for R-CHOP (HR, 2.17; 95% CI, 1.04-4.50; P = .0378), but not G-CHOP (HR, 1.37; 95% CI, 0.66-2.87; P = .3997). This effect was independent of established prognostic biomarkers. High FcγRIIB/FCGR2B expression has prognostic value in R-treated patients with DLBCL and may confer differential responsiveness to R-CHOP/G-CHOP.

Rapid microfluidic isolation of virally infected primary bronchial epithelial cells for single-cell RNA sequencing.

Single-cell RNA sequencing (scRNA-seq) of the bronchial epithelium enables examination of cellular subtypes and their responses to viral infections. Here, an optimized method for the isolation of virally infected primary bronchial epithelial cells using a commercially available microfluidic device is presented. Using this method single cells can be rapidly isolated with minimal equipment available in most laboratories. Isolation can be carried out inside biological safety cabinets, permitting the use of virally infected cells. Both cell-line and primary cells isolated using the device retained sufficient RNA integrity for the generation of short-read sequencing-compatible cDNA libraries to facilitate scRNA-seq.

Genomic Analysis of Response to Neoadjuvant Chemotherapy in Esophageal Adenocarcinoma.

Neoadjuvant therapy followed by surgery is the standard of care for locally advanced esophageal adenocarcinoma (EAC). Unfortunately, response to neoadjuvant chemotherapy (NAC) is poor (20-37%), as is the overall survival benefit at five years (9%). The EAC genome is complex and heterogeneous between patients, and it is not yet understood whether specific mutational patterns may result in chemotherapy sensitivity or resistance. To identify associations between genomic events and response to NAC in EAC, a comparative genomic analysis was performed in 65 patients with extensive clinical and pathological annotation using whole-genome sequencing (WGS). We defined response using Mandard Tumor Regression Grade (TRG), with responders classified as TRG1-2 (n = 27) and non-responders classified as TRG4-5 (n =38). We report a higher non-synonymous mutation burden in responders (median 2.08/Mb vs. 1.70/Mb, p = 0.036) and elevated copy number variation in non-responders (282 vs. 136/patient, p < 0.001). We identified copy number variants unique to each group in our cohort, with cell cycle (CDKN2A, CCND1), c-Myc (MYC), RTK/PIK3 (KRAS, EGFR) and gastrointestinal differentiation (GATA6) pathway genes being specifically altered in non-responders. Of note, NAV3 mutations were exclusively present in the non-responder group with a frequency of 22%. Thus, lower mutation burden, higher chromosomal instability and specific copy number alterations are associated with resistance to NAC.

Dual dean entrainment with volume ratio modulation for efficient droplet co-encapsulation: extreme single-cell indexing.

The future of single cell diversity screens involves ever-larger sample sizes, dictating the need for higher throughput methods with low analytical noise to accurately describe the nature of the cellular system. Current approaches are limited by the Poisson statistic, requiring dilute cell suspensions and associated losses in throughput. In this contribution, we apply Dean entrainment to both cell and bead inputs, defining different volume packets to effect efficient co-encapsulation. Volume ratio scaling was explored to identify optimal conditions. This enabled the co-encapsulation of single cells with reporter beads at rates of ∼1 million cells per hour, while increasing assay signal-to-noise with cell multiplet rates of ∼2.5% and capturing ∼70% of cells. The method, called Pirouette coupling, extends our capacity to investigate biological systems.

V-ATPase Inhibition Decreases Mutant Androgen Receptor Activity in Castrate-resistant Prostate Cancer.

Prostate cancer is critically dependent on androgen receptor (AR) signaling. Despite initial responsiveness to androgen deprivation, most patients with advanced prostate cancer subsequently progress to a clinically aggressive castrate-resistant prostate cancer (CRPC) phenotype, typically associated with expression of splice-variant or mutant AR forms. Although current evidence suggests that the vacuolar-ATPase (V-ATPase), a multiprotein complex that catalyzes proton transport across intracellular and plasma membranes, influences wild-type AR function, the effect of V-ATPase inhibition on variant AR function is unknown.Inhibition of V-ATPase reduced AR function in wild-type and mutant AR luciferase reporter models. In hormone-sensitive prostate cancer cell lines (LNCaP, DuCaP) and mutant AR CRPC cell lines (22Rv1, LNCaP-F877L/T878A), V-ATPase inhibition using bafilomycin-A1 and concanamycin-A reduced AR expression, and expression of AR target genes, at mRNA and protein levels. Furthermore, combining chemical V-ATPase inhibition with the AR antagonist enzalutamide resulted in a greater reduction in AR downstream target expression than enzalutamide alone in LNCaP cells. To investigate the role of individual subunit isoforms, siRNA and CRISPR-Cas9 were used to target the V1C1 subunit in 22Rv1 cells. Whereas transfection with ATP6V1C1-targeted siRNA significantly reduced AR protein levels and function, CRISPR-Cas9-mediated V1C1 knockout showed no substantial change in AR expression, but a compensatory increase in protein levels of the alternate V1C2 isoform.Overall, these results indicate that V-ATPase dysregulation is directly linked to both hormone-responsive prostate cancer and CRPC via impact on AR function. In particular, V-ATPase inhibition can reduce AR signaling regardless of mutant AR expression.

CD8+CD103+ tissue-resident memory T cells convey reduced protective immunity in cutaneous squamous cell carcinoma.

BACKGROUND: Tumor infiltrating lymphocytes play a key role in antitumor responses; however, while several memory T-cell subtypes have been reported in inflammatory and neoplastic conditions, the proportional representation of the different subsets of memory T cells and their functional significance in cancer is unclear. Keratinocyte skin cancer is one of the most common cancers globally, with cutaneous squamous cell cancer (cSCC) among the most frequent malignancies capable of metastasis. METHODS: Memory T-cell subsets were delineated in human cSCCs and, for comparison, in non-lesional skin and blood using flow cytometry. Immunohistochemistry was conducted to quantify CD103+ cells in primary human cSCCs which had metastasized (P-M) and primary cSCCs which had not metastasized (P-NM). TIMER2.0 (timer.cistrome.org) was used to analyze TCGA cancer survival data based on ITGAE expression. Immunofluorescence microscopy was performed to determine frequencies of CD8+CD103+ cells in P-M and P-NM cSCCs. RESULTS: Despite intertumoral heterogeneity, most cSCC T cells were CCR7-/CD45RA- effector/resident memory (TRM) lymphocytes, with naive, CD45RA+/CCR7- effector memory re-expressing CD45RA, CCR7+/L-selectin+ central memory and CCR7+/L-selectin- migratory memory lymphocytes accounting for smaller T-cell subsets. The cSCC CD8+ T-cell population contained a higher proportion of CD69+/CD103+ TRMs than that in non-lesional skin and blood. These cSCC CD69+/CD103+ TRMs exhibited increased IL-10 production, and higher CD39, CTLA-4 and PD-1 expression compared with CD103- TRMs in the tumor. CD103+ cells were more frequent in P-M than P-NM cSCCs. Analysis of TCGA data demonstrated that high expression of ITGAE (encoding CD103) was associated with reduced survival in primary cutaneous melanoma, breast carcinoma, renal cell carcinoma, kidney chromophobe cancer, adrenocortical carcinoma and lower grade glioma. Immunofluorescence microscopy showed that the majority of CD103 was present on CD8+ T cells and that CD8+CD103+ cells were significantly more frequent in P-M than P-NM cSCCs. CONCLUSION: These results highlight CD8+CD103+ TRMs as an important functional T-cell subset associated with poorer clinical outcome in this cancer.

p53 is regulated by aerobic glycolysis in cancer cells by the CtBP family of NADH-dependent transcriptional regulators.

High rates of glycolysis in cancer cells are a well-established characteristic of many human tumors, providing rapidly proliferating cancer cells with metabolites that can be used as precursors for anabolic pathways. Maintenance of high glycolytic rates depends on the lactate dehydrogenase-catalyzed regeneration of NAD+ from GAPDH-generated NADH because an increased NADH:NAD+ ratio inhibits GAPDH. Here, using human breast cancer cell models, we identified a pathway in which changes in the extramitochondrial-free NADH:NAD+ ratio signaled through the CtBP family of NADH-sensitive transcriptional regulators to control the abundance and activity of p53. NADH-free forms of CtBPs cooperated with the p53-binding partner HDM2 to suppress p53 function, and loss of these forms in highly glycolytic cells resulted in p53 accumulation. We propose that this pathway represents a "glycolytic stress response" in which the initiation of a protective p53 response by an increased NADH:NAD+ ratio enables cells to avoid cellular damage caused by mismatches between metabolic supply and demand.

Clinical significance of TP53, BIRC3, ATM and MAPK-ERK genes in chronic lymphocytic leukaemia: data from the randomised UK LRF CLL4 trial.

Despite advances in chronic lymphocytic leukaemia (CLL) treatment, globally chemotherapy remains a central treatment modality, with chemotherapy trials representing an invaluable resource to explore disease-related/genetic features contributing to long-term outcomes. In 499 LRF CLL4 cases, a trial with >12 years follow-up, we employed targeted resequencing of 22 genes, identifying 623 mutations. After background mutation rate correction, 11/22 genes were recurrently mutated at frequencies between 3.6% (NFKBIE) and 24% (SF3B1). Mutations beyond Sanger resolution (<12% VAF) were observed in all genes, with KRAS mutations principally composed of these low VAF variants. Firstly, employing orthogonal approaches to confirm <12% VAF TP53 mutations, we assessed the clinical impact of TP53 clonal architecture. Whilst ≥ 12% VAF TP53mut cases were associated with reduced PFS and OS, we could not demonstrate a difference between <12% VAF TP53 mutations and either wild type or ≥12% VAF TP53mut cases. Secondly, we identified biallelic BIRC3 lesions (mutation and deletion) as an independent marker of inferior PFS and OS. Finally, we observed that mutated MAPK-ERK genes were independent markers of poor OS in multivariate survival analysis. In conclusion, our study supports using targeted resequencing of expanded gene panels to elucidate the prognostic impact of gene mutations.

Single-cell analysis based dissection of clonality in myelofibrosis.

Cancer development is an evolutionary genomic process with parallels to Darwinian selection. It requires acquisition of multiple somatic mutations that collectively cause a malignant phenotype and continuous clonal evolution is often linked to tumor progression. Here, we show the clonal evolution structure in 15 myelofibrosis (MF) patients while receiving treatment with JAK inhibitors (mean follow-up 3.9 years). Whole-exome sequencing at multiple time points reveal acquisition of somatic mutations and copy number aberrations over time. While JAK inhibition therapy does not seem to create a clear evolutionary bottleneck, we observe a more complex clonal architecture over time, and appearance of unrelated clones. Disease progression associates with increased genetic heterogeneity and gain of RAS/RTK pathway mutations. Clonal diversity results in clone-specific expansion within different myeloid cell lineages. Single-cell genotyping of circulating CD34 + progenitor cells allows the reconstruction of MF phylogeny demonstrating loss of heterozygosity and parallel evolution as recurrent events.

Genomic programming of IRF4-expressing human Langerhans cells.

Langerhans cells (LC) can prime tolerogenic as well as immunogenic responses in skin, but the genomic states and transcription factors (TF) regulating these context-specific responses are unclear. Bulk and single-cell transcriptional profiling demonstrates that human migratory LCs are robustly programmed for MHC-I and MHC-II antigen presentation. Chromatin analysis reveals enrichment of ETS-IRF and AP1-IRF composite regulatory elements in antigen-presentation genes, coinciding with expression of the TFs, PU.1, IRF4 and BATF3 but not IRF8. Migration of LCs from the epidermis is accompanied by upregulation of IRF4, antigen processing components and co-stimulatory molecules. TNF stimulation augments LC cross-presentation while attenuating IRF4 expression. CRISPR-mediated editing reveals IRF4 to positively regulate the LC activation programme, but repress NF2EL2 and NF-kB pathway genes that promote responsiveness to oxidative stress and inflammatory cytokines. Thus, IRF4-dependent genomic programming of human migratory LCs appears to enable LC maturation while attenuating excessive inflammatory and immunogenic responses in the epidermis.

Clinical significance of DNA methylation in chronic lymphocytic leukemia patients: results from 3 UK clinical trials.

Chronic lymphocytic leukemia patients with mutated immunoglobulin heavy-chain genes (IGHV-M), particularly those lacking poor-risk genomic lesions, often respond well to chemoimmunotherapy (CIT). DNA methylation profiling can subdivide early-stage patients into naive B-cell-like CLL (n-CLL), memory B-cell-like CLL (m-CLL), and intermediate CLL (i-CLL), with differing times to first treatment and overall survival. However, whether DNA methylation can identify patients destined to respond favorably to CIT has not been ascertained. We classified treatment-naive patients (n = 605) from 3 UK chemo and CIT clinical trials into the 3 epigenetic subgroups, using pyrosequencing and microarray analysis, and performed expansive survival analysis. The n-CLL, i-CLL, and m-CLL signatures were found in 80% (n = 245/305), 17% (53/305), and 2% (7/305) of IGHV-unmutated (IGHV-U) cases, respectively, and in 9%, (19/216), 50% (108/216), and 41% (89/216) of IGHV-M cases, respectively. Multivariate Cox proportional analysis identified m-CLL as an independent prognostic factor for overall survival (hazard ratio [HR], 0.46; 95% confidence interval [CI], 0.24-0.87; P = .018) in CLL4, and for progression-free survival (HR, 0.25; 95% CI, 0.10-0.57; P = .002) in ARCTIC and ADMIRE patients. The analysis of epigenetic subgroups in patients entered into 3 first-line UK CLL trials identifies m-CLL as an independent marker of prolonged survival and may aid in the identification of patients destined to demonstrate prolonged survival after CIT.

An optimised tissue disaggregation and data processing pipeline for characterising fibroblast phenotypes using single-cell RNA sequencing.

Single-cell RNA sequencing (scRNA-Seq) provides a valuable platform for characterising multicellular ecosystems. Fibroblasts are a heterogeneous cell type involved in many physiological and pathological processes, but remain poorly-characterised. Analysis of fibroblasts is challenging: these cells are difficult to isolate from tissues, and are therefore commonly under-represented in scRNA-seq datasets. Here, we describe an optimised approach for fibroblast isolation from human lung tissues. We demonstrate the potential for this procedure in characterising stromal cell phenotypes using scRNA-Seq, analyse the effect of tissue disaggregation on gene expression, and optimise data processing to improve clustering quality. We also assess the impact of in vitro culture conditions on stromal cell gene expression and proliferation, showing that altering these conditions can skew phenotypes.

Author Correction: Authentication and characterisation of a new oesophageal adenocarcinoma cell line: MFD-1.

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