ORAOV1, CCND1, and MIR548K Are the Driver Oncogenes of the 11q13 Amplicon in Squamous Cell Carcinoma.

11q13 amplification is a frequent event in human cancer and in particular in squamous cell carcinomas (SCC). Despite almost invariably spanning 10 genes, it is unclear which genetic components of the amplicon are the key driver events in SCC. A combination of computational, in vitro, ex vivo, and in vivo models leveraging efficient primary human keratinocyte genome editing by Cas9-RNP electroporation, identified ORAOV1, CCND1, and MIR548K as the critical drivers of the amplicon in head and neck SCC. CCND1 amplification drives the cell cycle in a CDK4/6/RB1-independent fashion and may confer a novel dependency on RRM2. MIR548K contributes to epithelial-mesenchymal transition. Finally, we identify ORAOV1 as an oncogene that acts likely via its ability to modulate reactive oxygen species. Thus, the 11q13 amplicon drives SCC through at least three independent genetic elements and suggests therapeutic targets for this morbid and lethal disease. IMPLICATIONS: This work demonstrates novel mechanisms and ways to target these mechanisms underlying the most common amplification in squamous cell carcinoma, one of the most prevalent and deadly forms of human cancer.

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.

Endoscopic Medial Reepithelization for Inflammatory Canal Stenosis.

OBJECTIVE: Inflammatory external auditory canal (EAC) Stenosis arises from infiltration of inflammatory cells, edema and eventual sclerosing of the medial EAC, leading to complete obstruction and conductive hearing loss. Current treatment includes surgical resection of the affected area with widening and reepithelization of the EAC via postauricular incision, but the condition is reported to recur with high frequency. Our aim was to assess the feasibility of endoscopic transcanal treatment as an alternative to postauricular canalplasty and understand its effect on recurrence rates. STUDY DESIGN: Retrospective case review. SETTING: Tertiary referral center. PATIENTS: Four patients were included who had bilateral conductive hearing loss and inflammatory canal stenosis, all with gross thickening of the tympanic membrane. INTERVENTIONS: Patients underwent endoscopic removal of obstructive tissue and reepithelization with split-thickness skin grafting. MAIN OUTCOME MEASURES: Postoperative air-bone gap (ABG), lack of recurrence, subjective reporting of hearing improvement, and lack of drainage. RESULTS: Eight of 8 ears (n = 4 patients) had significant improvement in hearing. No recurrence has been observed in any of the patients over a mean follow-up time of 90 months (range, 42-189 mo). Average reduction in ABG was 13.40 dB (SD = 9.0 dB) with a statistically significant difference between the pure tone average preoperative and postoperative ABG (p = 0.0008; n = 7). CONCLUSIONS: Endoscopic treatment of Inflammatory EAC stenosis obviates the need for postauricular incision and results in clinical improvement with a favorable recurrence rate.

A Rare Complication of Chronic Otitis Media: Central Skull Base Osteomyelitis Managed With Combined Endoscopic Transmastoid and Transsphenoidal Debridement.

OBJECTIVE: This report describes a case of otogenic central skull base osteomyelitis (CSBO) requiring complex surgical intervention and reviews the literature on management of this entity. PATIENT: A 76-year-old man presented with a nearly 20-year history of chronic otomastoiditis and cholesteatoma with ultimate progression to severe CSBO with involvement of the petrous apex, clivus, and craniocervical junction. INTERVENTIONS: CSBO was managed with culture-directed antibiotic therapy, hyperbaric oxygen, and surgical intervention including serial combined endoscopic transmastoid and transsphenoidal debridements. MAIN OUTCOME MEASURES: Symptom resolution, antibiotic holiday, and stable disease on surveillance imaging. RESULTS: With antibiotic treatment and surgical debridement including creation of a drainage pathway from the skull base to the sphenoid sinus, intermittent stretches of disease quiescence were realized over the course of nearly a decade. Despite extensive debridement and skull base reconstruction, the patient ultimately succumbed to the disease process. CONCLUSIONS: CSBO poses significant management challenges to the otologist. Herein, we present a rare case of CSBO managed over a prolonged period of time with antibiotics and combined anterior and lateral skull base debridement.

Human melanocyte development and melanoma dedifferentiation at single-cell resolution.

In humans, epidermal melanocytes are responsible for skin pigmentation, defence against ultraviolet radiation and the deadliest common skin cancer, melanoma. Although there is substantial overlap in melanocyte development pathways between different model organisms, species-dependent differences are frequent and the conservation of these processes in human skin remains unresolved. Here, we used a single-cell enrichment and RNA-sequencing pipeline to study human epidermal melanocytes directly from the skin, capturing transcriptomes across different anatomical sites, developmental age, sexes and multiple skin tones. We uncovered subpopulations of melanocytes that exhibit anatomical site-specific enrichment that occurs during gestation and persists through adulthood. The transcriptional signature of the volar-enriched subpopulation is retained in acral melanomas. Furthermore, we identified human melanocyte differentiation transcriptional programs that are distinct from gene signatures generated from model systems. Finally, we used these programs to define patterns of dedifferentiation that are predictive of melanoma prognosis and response to immune checkpoint inhibitor therapy.

Transcriptional control of subtype switching ensures adaptation and growth of pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDA) is a heterogeneous disease comprised of a basal-like subtype with mesenchymal gene signatures, undifferentiated histopathology and worse prognosis compared to the classical subtype. Despite their prognostic and therapeutic value, the key drivers that establish and control subtype identity remain unknown. Here, we demonstrate that PDA subtypes are not permanently encoded, and identify the GLI2 transcription factor as a master regulator of subtype inter-conversion. GLI2 is elevated in basal-like PDA lines and patient specimens, and forced GLI2 activation is sufficient to convert classical PDA cells to basal-like. Mechanistically, GLI2 upregulates expression of the pro-tumorigenic secreted protein, Osteopontin (OPN), which is especially critical for metastatic growth in vivo and adaptation to oncogenic KRAS ablation. Accordingly, elevated GLI2 and OPN levels predict shortened overall survival of PDA patients. Thus, the GLI2-OPN circuit is a driver of PDA cell plasticity that establishes and maintains an aggressive variant of this disease.

Impact of pretreatment growth on Tumor control for vestibular schwannomas following gamma knife.

OBJECTIVES/HYPOTHESIS: To determine if volumetric growth prior to gamma knife (GK) radiosurgery predicts long-term tumor control. STUDY DESIGN: Retrospective cohort study. METHODS: Sporadic vestibular schwannomas (VS) treated with GK between 2002 and 2014 at a single tertiary care center were identified. Patients were included if they had over 6 months of pretreatment observation and over 1.5 years of posttreatment follow-up. Volumetric tumor analysis was performed on T1 postcontrast imaging. Pretreatment and posttreatment volume change was calculated. Tumors with over 20% volume increase were classified as growing. RESULTS: There were 62 patients included in this study; 48 had pretreatment growth and 14 had no pretreatment growth. Median tumor volume was 0.58 ± 1.8 cm3 and median follow-up was 3.3 ± 2.0 years. For tumors with and without pretreatment growth, salvage treatment rates were 2% and 7% (P = .35), and posttreatment radiologic stability rates were 73% and 86%, respectively (P = .33). Median pretreatment growth was 27 ± 33% per year for tumors with posttreatment radiographic growth and 18 ± 26% per year for tumors without posttreatment radiographic growth (P = .99). CONCLUSIONS: Pretreatment growth was not associated with increased salvage treatment or posttreatment radiographic progression rates in VS following GK. LEVEL OF EVIDENCE: 4 Laryngoscope, 129:743-747, 2019.

Oncogenic Signaling Pathways in The Cancer Genome Atlas.

Genetic alterations in signaling pathways that control cell-cycle progression, apoptosis, and cell growth are common hallmarks of cancer, but the extent, mechanisms, and co-occurrence of alterations in these pathways differ between individual tumors and tumor types. Using mutations, copy-number changes, mRNA expression, gene fusions and DNA methylation in 9,125 tumors profiled by The Cancer Genome Atlas (TCGA), we analyzed the mechanisms and patterns of somatic alterations in ten canonical pathways: cell cycle, Hippo, Myc, Notch, Nrf2, PI-3-Kinase/Akt, RTK-RAS, TGFß signaling, p53 and ß-catenin/Wnt. We charted the detailed landscape of pathway alterations in 33 cancer types, stratified into 64 subtypes, and identified patterns of co-occurrence and mutual exclusivity. Eighty-nine percent of tumors had at least one driver alteration in these pathways, and 57% percent of tumors had at least one alteration potentially targetable by currently available drugs. Thirty percent of tumors had multiple targetable alterations, indicating opportunities for combination therapy.

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.

Defining epithelial cell dynamics and lineage relationships in the developing lacrimal gland.

The tear-producing lacrimal gland is a tubular organ that protects and lubricates the ocular surface. The lacrimal gland possesses many features that make it an excellent model in which to investigate tubulogenesis, but the cell types and lineage relationships that drive lacrimal gland formation are unclear. Using single-cell sequencing and other molecular tools, we reveal novel cell identities and epithelial lineage dynamics that underlie lacrimal gland development. We show that the lacrimal gland from its earliest developmental stages is composed of multiple subpopulations of immune, epithelial and mesenchymal cell lineages. The epithelial lineage exhibits the most substantial cellular changes, transitioning through a series of unique transcriptional states to become terminally differentiated acinar, ductal and myoepithelial cells. Furthermore, lineage tracing in postnatal and adult glands provides the first direct evidence of unipotent KRT5+ epithelial cells in the lacrimal gland. Finally, we show conservation of developmental markers between the developing mouse and human lacrimal gland, supporting the use of mice to understand human development. Together, our data reveal crucial features of lacrimal gland development that have broad implications for understanding epithelial organogenesis.

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.

High intratumor genetic heterogeneity is related to worse outcome in patients with head and neck squamous cell carcinoma.

BACKGROUND: Although the presence of genetic heterogeneity within the tumors of individual patients is established, it is unclear whether greater heterogeneity predicts a worse outcome. A quantitative measure of genetic heterogeneity based on next-generation sequencing (NGS) data, mutant-allele tumor heterogeneity (MATH), was previously developed and applied to a data set on head and neck squamous cell carcinoma (HNSCC). Whether this measure correlates with clinical outcome was not previously assessed. METHODS: The authors examined the association between MATH and clinical, pathologic, and overall survival data for 74 patients with HNSCC for whom exome sequencing was completed. RESULTS: High MATH (a MATH value above the median) was found to be significantly associated with shorter overall survival (hazards ratio, 2.5; 95% confidence interval, 1.3-4.8). MATH was similarly found to be associated with adverse outcomes in clinically high-risk patients with an advanced stage of disease, and in those with tumors classified as high risk on the basis of validated biomarkers including those that were negative for human papillomavirus or having disruptive tumor protein p53 mutations. In patients who received chemotherapy, the hazards ratio for high MATH was 4.1 (95% confidence interval, 1.6-10.2). CONCLUSIONS: This novel measure of tumor genetic heterogeneity is significantly associated with tumor progression and adverse treatment outcomes, thereby supporting the hypothesis that higher genetic heterogeneity portends a worse clinical outcome in patients with HNSCC. The prognostic value of some known biomarkers may be the result of their association with high genetic heterogeneity. MATH provides a useful measure of that heterogeneity to be prospectively validated as NGS data from homogeneously treated patient cohorts become available.

The mutational landscape of head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is a common, morbid, and frequently lethal malignancy. To uncover its mutational spectrum, we analyzed whole-exome sequencing data from 74 tumor-normal pairs. The majority exhibited a mutational profile consistent with tobacco exposure; human papillomavirus was detectable by sequencing DNA from infected tumors. In addition to identifying previously known HNSCC genes (TP53, CDKN2A, PTEN, PIK3CA, and HRAS), our analysis revealed many genes not previously implicated in this malignancy. At least 30% of cases harbored mutations in genes that regulate squamous differentiation (for example, NOTCH1, IRF6, and TP63), implicating its dysregulation as a major driver of HNSCC carcinogenesis. More generally, the results indicate the ability of large-scale sequencing to reveal fundamental tumorigenic mechanisms.

Integration of genomic analysis and in vivo transfection to identify sprouty 2 as a candidate tumor suppressor in liver cancer.

UNLABELLED: Hepatocellular carcinoma (HCC) is 1 of the leading causes of cancer-related deaths worldwide, yet the molecular genetics underlying this malignancy are still poorly understood. In our study, we applied statistical methods to correlate human HCC gene expression data obtained from complementary DNA (cDNA) microarrays and corresponding DNA copy number variation data obtained from array-based comparative genomic hybridization. We have thus identified 76 genes that are up-regulated and show frequent DNA copy number gain, and 37 genes that are down-regulated and show frequent DNA copy loss in human HCC samples. Among these down-regulated genes is Sprouty2 (Spry2), a known inhibitor of receptor tyrosine kinases. We investigated the potential role of Spry2 in HCC by expressing dominant negative Spry2 (Spry2Y55F) and activated beta-catenin (DeltaN90-beta-catenin) in the mouse liver through hydrodynamic injection and sleeping beauty-mediated somatic integration. When stably expressed in mouse hepatocytes, Spry2Y55F cooperates with DeltaN90-beta-catenin to confer a neoplastic phenotype in mice. Tumor cells show high levels of expression of phospho-extracellular signal-regulated kinase (ERK), as well as deregulation of genes involved in cell proliferation, apoptosis, and angiogenesis. CONCLUSION: We identified a set of candidate oncogenes and tumor suppressor genes for human HCC. Our study provides evidence that inhibition of Spry activity cooperates with other oncogenes to promote liver cancer in mouse models, and Spry2 may function as a candidate tumor suppressor for HCC development in vivo. In addition, we demonstrate that the integration of genomic analysis and in vivo transfection is a powerful tool to identify genes that are important during hepatic carcinogenesis.

Distinct pathways of genomic progression to benign and malignant tumors of the liver.

We used several of the genetic lesions commonly associated with human liver tumors to reconstruct genetic progression to hepatocellular carcinoma and adenoma in mouse models. We initiated tumorigenesis with a transgene of the protooncogene MET or by hydrodynamic transfection of MET in combination with other genes into the livers of adult animals. Hepatocellular carcinoma in both instances arose from cooperation between MET and constitutively active versions of beta-catenin. In contrast, adenomas were produced by cooperation between MET and defective signaling through the transcription factor HNF1alpha. Prompted by these findings, we uncovered a coincidence between activation of the protein-tyrosine kinase encoded by MET and activating mutations of beta-catenin in a subset of human hepatocellular carcinomas. Inactivation of MET transgenes led to regression of hepatocellular carcinomas despite the persistence of activated beta-catenin. The tumors eventually recurred in the absence of MET expression, however, presumably after the occurrence of one or more events that cooperated with activated beta-catenin in lieu of MET. These results offer insight into hepatic tumorigenesis, provide mouse models that should be useful in the further study of hepatic tumorigenesis and for preclinical testing, and identify a subset of human hepatocellular carcinomas that may be susceptible to combination therapy directed against Met and the Wnt signaling pathway.

Inhibition of CDK1 as a potential therapy for tumors over-expressing MYC.

Tumor cells have a dysregulated cell cycle that may render their proliferation especially sensitive to the inhibition of cyclin-dependent kinases (CDKs), important regulators of cell cycle progression. We examined the effects of CDK1 inhibition in the context of different oncogenic signals. Cells transformed with MYC, but not cells transformed by a panel of other activated oncogenes, rapidly underwent apoptosis when treated with small-molecule CDK1 inhibitors. The inhibitor of apoptosis protein BIRC5 (survivin), a known CDK1 target, is required for the survival of cells overexpressing MYC. Inhibition of CDK1 rapidly downregulates survivin expression and induces MYC-dependent apoptosis. CDK1 inhibitor treatment of MYC-dependent mouse lymphoma and hepatoblastoma tumors decreased tumor growth and prolonged their survival. As there are no effective small-molecule inhibitors that selectively target the MYC pathway, we propose that CDK1 inhibition might therefore be useful in the treatment of human malignancies that overexpress MYC.

Bone morphogenetic protein antagonist gremlin 1 is widely expressed by cancer-associated stromal cells and can promote tumor cell proliferation.

Although tissue microenvironments play critical roles in epithelial development and tumorigenesis, the factors mediating these effects are poorly understood. In this work, we used a genomic approach to identify factors produced by cells in the microenvironment of basal cell carcinoma (BCC) of the skin, one of the most common human cancers. The global gene expression programs of stromal cell cultures derived from human BCCs showed consistent, systematic differences from those derived from nontumor skin. The gene most consistently expressed at a higher level in BCC tumor stromal cells compared with those from nontumor skin was GREMLIN 1, which encodes a secreted antagonist of the bone morphogenetic protein (BMP) pathway. BMPs and their antagonists are known to play a crucial role in stem and progenitor cell biology as regulators of the balance between expansion and differentiation. Consistent with the hypothesis that BMP antagonists might have a similar role in cancer, we found GREMLIN 1 expression in the stroma of human BCC tumors but not in normal skin in vivo. Furthermore, BMP 2 and 4 are expressed by BCC cells. Ex vivo, BMP inhibits, and Gremlin 1 promotes, proliferation of cultured BCC cells. We further found that GREMLIN 1 is expressed by stromal cells in many carcinomas but not in the corresponding normal tissue counterparts that we examined. Our data suggest that BMP antagonists may be important constituents of tumor stroma, providing a favorable microenvironment for cancer cell survival and expansion in many cancers.