Intra-tumor Genetic Heterogeneity and Mortality in Head and Neck Cancer: Analysis of Data from The Cancer Genome Atlas

Background: Although the involvement of intra-tumor genetic heterogeneity in tumor progression, treatment resistance, and metastasis is established, genetic heterogeneity is seldom examined in clinical trials or practice. Many studies of heterogeneity have had prespecified markers for tumor subpopulations, limiting their generalizability, or have involved massive efforts such as separate analysis of hundreds of individual cells, limiting their clinical use. We recently developed a general measure of intra-tumor genetic heterogeneity based on whole-exome sequencing (WES) of bulk tumor DNA, called mutant-allele tumor heterogeneity (MATH). Here, we examine data collected as part of a large, multi-institutional study to validate this measure and determine whether intra-tumor heterogeneity is itself related to mortality. Methods and Findings: Clinical and WES data were obtained from The Cancer Genome Atlas in October 2013 for 305 patients with head and neck squamous cell carcinoma (HNSCC), from 14 institutions. Initial pathologic diagnoses were between 1992 and 2011 (median, 2008). Median time to death for 131 deceased patients was 14 mo; median follow-up of living patients was 22 mo. Tumor MATH values were calculated from WES results. Despite the multiple head and neck tumor subsites and the variety of treatments, we found in this retrospective analysis a substantial relation of high MATH values to decreased overall survival (Cox proportional hazards analysis: hazard ratio for high/low heterogeneity, 2.2; 95% CI 1.4 to 3.3). This relation of intra-tumor heterogeneity to survival was not due to intra-tumor heterogeneity’s associations with other clinical or molecular characteristics, including age, human papillomavirus status, tumor grade and TP53 mutation, and N classification. MATH improved prognostication over that provided by traditional clinical and molecular characteristics, maintained a significant relation to survival in multivariate analyses, and distinguished outcomes among patients having oral-cavity or laryngeal cancers even when standard disease staging was taken into account. Prospective studies, however, will be required before MATH can be used prognostically in clinical trials or practice. Such studies will need to examine homogeneously treated HNSCC at specific head and neck subsites, and determine the influence of cancer therapy on MATH values. Analysis of MATH and outcome in human-papillomavirus-positive oropharyngeal squamous cell carcinoma is particularly needed. Conclusions: To our knowledge this study is the first to combine data from hundreds of patients, treated at multiple institutions, to document a relation between intra-tumor heterogeneity and overall survival in any type of cancer. We suggest applying the simply calculated MATH metric of heterogeneity to prospective studies of HNSCC and other tumor types

Lineage dynamics of murine pancreatic development at single-cell resolution.

Organogenesis requires the complex interactions of multiple cell lineages that coordinate their expansion, differentiation, and maturation over time. Here, we profile the cell types within the epithelial and mesenchymal compartments of the murine pancreas across developmental time using a combination of single-cell RNA sequencing, immunofluorescence, in situ hybridization, and genetic lineage tracing. We identify previously underappreciated cellular heterogeneity of the developing mesenchyme and reconstruct potential lineage relationships among the pancreatic mesothelium and mesenchymal cell types. Within the epithelium, we find a previously undescribed endocrine progenitor population, as well as an analogous population in both human fetal tissue and human embryonic stem cells differentiating toward a pancreatic beta cell fate. Further, we identify candidate transcriptional regulators along the differentiation trajectory of this population toward the alpha or beta cell lineages. This work establishes a roadmap of pancreatic development and demonstrates the broad utility of this approach for understanding lineage dynamics in developing organs

Inhibition of fatty acid oxidation as a therapy for MYC-overexpressing triple-negative breast cancer.

Expression of the oncogenic transcription factor MYC is disproportionately elevated in triple-negative breast cancer (TNBC), as compared to estrogen receptor-, progesterone receptor- or human epidermal growth factor 2 receptor-positive (RP) breast cancer. We and others have shown that MYC alters metabolism during tumorigenesis. However, the role of MYC in TNBC metabolism remains mostly unexplored. We hypothesized that MYC-dependent metabolic dysregulation is essential for the growth of MYC-overexpressing TNBC cells and may identify new therapeutic targets for this clinically challenging subset of breast cancer. Using a targeted metabolomics approach, we identified fatty acid oxidation (FAO) intermediates as being dramatically upregulated in a MYC-driven model of TNBC. We also identified a lipid metabolism gene signature in patients with TNBC that were identified from The Cancer Genome Atlas database and from multiple other clinical data sets, implicating FAO as a dysregulated pathway that is critical for TNBC cell metabolism. We found that pharmacologic inhibition of FAO catastrophically decreased energy metabolism in MYC-overexpressing TNBC cells and blocked tumor growth in a MYC-driven transgenic TNBC model and in a MYC-overexpressing TNBC patient-derived xenograft. These findings demonstrate that MYC-overexpressing TNBC shows an increased bioenergetic reliance on FAO and identify the inhibition of FAO as a potential therapeutic strategy for this subset of breast cancer

Salivary glands regenerate after radiation injury through SOX2-mediated secretory cell replacement

Salivary gland acinar cells are routinely destroyed during radiation treatment for head and neck cancer that results in a lifetime of hyposalivation and co-morbidities. A potential regenerative strategy for replacing injured tissue is the reactivation of endogenous stem cells by targeted therapeutics. However, the identity of these cells, whether they are capable of regenerating the tissue, and the mechanisms by which they are regulated are unknown. Using in vivo and ex vivo models, in combination with genetic lineage tracing and human tissue, we discover a SOX2+ stem cell population essential to acinar cell maintenance that is capable of replenishing acini after radiation. Furthermore, we show that acinar cell replacement is nerve dependent and that addition of a muscarinic mimetic is sufficient to drive regeneration. Moreover, we show that SOX2 is diminished in irradiated human salivary gland, along with parasympathetic nerves, suggesting that tissue degeneration is due to loss of progenitors and their regulators. Thus, we establish a new paradigm that salivary glands can regenerate after genotoxic shock and do so through a SOX2 nerve-dependent mechanism

Specific Oral Microbial Differences in Proteobacteria and Bacteroidetes Are Associated with Distinct Sites When Moving from Healthy Mucosa to Oral Dysplasia-A Microbiome and Gene Profiling Study and Focused Review.

Oral potentially malignant disorders (OPMDs) are a group of conditions that carry a risk of oral squamous cell carcinoma (OSCC) development. Recent studies indicate that periodontal disease-associated pathogenic bacteria may play a role in the transition from healthy mucosa to dysplasia and to OSCC. Yet, the microbial signatures associated with the transition from healthy mucosa to dysplasia have not been established. To characterize oral microbial signatures at these different sites, we performed a 16S sequencing analysis of both oral swab and formalin-fixed, paraffin-embedded tissue (FFPE) samples. We collected oral swabs from healthy mucosa (from healthy patients), histologically normal mucosa adjacent to dysplasia, and low-grade oral dysplasia. Additionally, FFPE samples from histologically normal mucosa adjacent to OSCC, plus low grade and high-grade oral dysplasia samples were also collected. The collected data demonstrate significant differences in the alpha and beta microbial diversities of different sites in oral mucosa, dysplasia, and OSCC, as well as increased dissimilarities within these sites. We found that the Proteobacteria phyla abundance increased, concurrent with a progressive decrease in the Firmicutes phyla abundance, as well as altered levels of Enterococcus cecorum, Fusobacterium periodonticum, Prevotella melaninogenica, and Fusobacterium canifelinum when moving from healthy to diseased sites. Moreover, the swab sample analysis indicates that the oral microbiome may be altered in areas that are histologically normal, including in mucosa adjacent to dysplasia. Furthermore, trends in specific microbiome changes in oral swab samples preceded those in the tissues, signifying early detection opportunities for clinical diagnosis. In addition, we evaluated the gene expression profile of OSCC cells (HSC-3) infected with either P. gingivalis, T. denticola, F. nucelatum, or S. sanguinis and found that the three periodontopathogens enrich genetic processes related to cancer progression, including skin keratinization/cornification, while the commensal enriched processes related to RNA processing and adhesion. Finally, we reviewed the dysplasia microbiome literature and found a significant decrease in commensal bacteria, such as the Streptococci genus, and a simultaneous increase in pathogenic bacteria, mainly Bacteroidetes phyla and Fusobacterium genus. These findings suggest that features of the oral microbiome can serve as novel biomarkers for dysplasia and OSCC disease progression

Clinical and molecular characteristics, and their relations to overall survival.

<p>Clinical and molecular characteristics, and their relations to overall survival.</p

Salivary glands regenerate after radiation injury through SOX2-mediated secretory cell replacement.

Salivary gland acinar cells are routinely destroyed during radiation treatment for head and neck cancer that results in a lifetime of hyposalivation and co-morbidities. A potential regenerative strategy for replacing injured tissue is the reactivation of endogenous stem cells by targeted therapeutics. However, the identity of these cells, whether they are capable of regenerating the tissue, and the mechanisms by which they are regulated are unknown. Using in&nbsp;vivo and ex&nbsp;vivo models, in combination with genetic lineage tracing and human tissue, we discover a SOX2+ stem cell population essential to acinar cell maintenance that is capable of replenishing acini after radiation. Furthermore, we show that acinar cell replacement is nerve dependent and that addition of a muscarinic mimetic is sufficient to drive regeneration. Moreover, we show that SOX2 is diminished in irradiated human salivary gland, along with parasympathetic nerves, suggesting that tissue degeneration is due to loss of progenitors and their regulators. Thus, we establish a new paradigm that salivary glands can regenerate after genotoxic shock and do so through a SOX2 nerve-dependent mechanism

Specific Oral Microbial Differences in Proteobacteria and Bacteroidetes Are Associated with Distinct Sites When Moving from Healthy Mucosa to Oral Dysplasia—A Microbiome and Gene Profiling Study and Focused Review

Oral potentially malignant disorders (OPMDs) are a group of conditions that carry a risk of oral squamous cell carcinoma (OSCC) development. Recent studies indicate that periodontal disease-associated pathogenic bacteria may play a role in the transition from healthy mucosa to dysplasia and to OSCC. Yet, the microbial signatures associated with the transition from healthy mucosa to dysplasia have not been established. To characterize oral microbial signatures at these different sites, we performed a 16S sequencing analysis of both oral swab and formalin-fixed, paraffin-embedded tissue (FFPE) samples. We collected oral swabs from healthy mucosa (from healthy patients), histologically normal mucosa adjacent to dysplasia, and low-grade oral dysplasia. Additionally, FFPE samples from histologically normal mucosa adjacent to OSCC, plus low grade and high-grade oral dysplasia samples were also collected. The collected data demonstrate significant differences in the alpha and beta microbial diversities of different sites in oral mucosa, dysplasia, and OSCC, as well as increased dissimilarities within these sites. We found that the Proteobacteria phyla abundance increased, concurrent with a progressive decrease in the Firmicutes phyla abundance, as well as altered levels of Enterococcus cecorum, Fusobacterium periodonticum, Prevotella melaninogenica, and Fusobacterium canifelinum when moving from healthy to diseased sites. Moreover, the swab sample analysis indicates that the oral microbiome may be altered in areas that are histologically normal, including in mucosa adjacent to dysplasia. Furthermore, trends in specific microbiome changes in oral swab samples preceded those in the tissues, signifying early detection opportunities for clinical diagnosis. In addition, we evaluated the gene expression profile of OSCC cells (HSC-3) infected with either P. gingivalis, T. denticola, F. nucelatum, or S. sanguinis and found that the three periodontopathogens enrich genetic processes related to cancer progression, including skin keratinization/cornification, while the commensal enriched processes related to RNA processing and adhesion. Finally, we reviewed the dysplasia microbiome literature and found a significant decrease in commensal bacteria, such as the Streptococci genus, and a simultaneous increase in pathogenic bacteria, mainly Bacteroidetes phyla and Fusobacterium genus. These findings suggest that features of the oral microbiome can serve as novel biomarkers for dysplasia and OSCC disease progression

A single-cell atlas and lineage analysis of the adult Drosophila ovary.

The Drosophila ovary is a widely used model for germ cell and somatic tissue biology. Here we use single-cell RNA-sequencing (scRNA-seq) to build a comprehensive cell atlas of the adult Drosophila ovary that contains transcriptional profiles for every major cell type in the ovary, including the germline stem cells and their niche cells, follicle stem cells, and previously undescribed subpopulations of escort cells. In addition, we identify Gal4 lines with specific expression patterns and perform lineage tracing of subpopulations of escort cells and follicle cells. We discover that a distinct subpopulation of escort cells is able to convert to follicle stem cells in response to starvation or upon genetic manipulation, including knockdown of escargot, or overactivation of mTor or Toll signalling