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Supplementary Figure 1 from Sheddase Activity of Tumor Necrosis Factor-α Converting Enzyme Is Increased and Prognostically Valuable in Head and Neck Cancer

Tumor necrosis factor α converting enzyme (TACE) is a sheddase overexpressed in cancers that generates cancer cell growth and survival factors, and is implicated in carcinogenesis and tumor growth. This indicates that TACE could be a potentially important cancer biomarker. Unexpectedly, TACE expression in cancer tissues does not correlate with cancer stage or invasiveness. Although TACE sheddase activity is a more direct and potentially better indicator of TACE biology and might be a better cancer biomarker than TACE expression, it has not been studied in cancer tissues. In the present study, we developed a reliable specific assay for quantification of TACE sheddase activity, investigated TACE activity and TACE protein expression in head and neck cancer (HNC) tissues, and examined the correlation of the results with HNC clinical stages and likelihood to recur. We found that HNC cell lines and tissues contained remarkably higher quantities of TACE activity and TACE protein than normal keratinocytes or oral mucosa. siRNA silencing of TACE resulted in the inhibition of release of the tumorogenic factors amphiregulin and transforming growth factor α, and tumor protective factors tumor necrosis factor receptors from HNC cells. Importantly, TACE activity, but not TACE protein expression, was significantly higher in large, T3/T4, primary tumors relative to small, T1/T2, primary tumors, and especially in primary tumors likely to recur relative to those unlikely to recur. These data show that increased TACE activity in cancer is biologically and clinically relevant, and indicate that TACE activity could be a significant biomarker of cancer prognosis. (Cancer Epidemiol Biomarkers Prev 2009;18(11):2913–22)

Supplementary Figure Legends from Sheddase Activity of Tumor Necrosis Factor-α Converting Enzyme Is Increased and Prognostically Valuable in Head and Neck Cancer

Supplementary Figure 2 from Sheddase Activity of Tumor Necrosis Factor-α Converting Enzyme Is Increased and Prognostically Valuable in Head and Neck Cancer

Supplementary Figure 3 from Sheddase Activity of Tumor Necrosis Factor-α Converting Enzyme Is Increased and Prognostically Valuable in Head and Neck Cancer

PDF file - 109K, TMEM16A expression can be rescued by expression of cDNA that is resistant to the effects of anti-TMEM16A shRNA. Quantitative RT-PCR was used to demonstrate that the knock-down of endogenous TMEM16A induced by shRNA can be rescued by the expression of DNA engineered to be shRNA-resistant, labeled TMEM16A DNA* (mean � SEM; n=4, *P

PDF file - 72K, Patient survival is not impacted by TMEM16A amplification status. Disease-specific and overall survival of the cohort of SCCHN patients was not significantly correlated with TMEM16A amplification (A, B).

PDF file - 407K, TMEM16A overexpression promotes cell proliferation in HEK-293T cells. TMEM16A overexpression induced cellular proliferation (*P

PDF file - 109K, TMEM16A inhibition with T16A-inh01 (A01) alone or in combination with the MEK/ERK inhibitor UO126 leads to decreased tumor cell viability in vitro. UM-SCC1 and T24 cells were treated with increasing concentrations of A01. A dose dependent reduction in cell viability was observed (A, B *P

Based on the oncogenic role of phosphatidylinositol glycan (PIG) class U in human tumors, we explored the role of two additional subunits of the glycosylphosphatidylinositol (GPI) transamidase complex in human breast cancer. We found that PIG class T (PIG-T) and GPI anchor attachment 1 (GPAA1) were overexpressed in breast cancer cell lines and primary tumors. Forced expression of PIG-T and GPAA1 transformed NIH3T3 cells in vitro and increased tumorigenicity and invasion of these cells in vivo. Suppression of PIG-T expression in breast cancer cell lines led to inhibition of anchorage-independent growth. Moreover, we found that PIG-T and GPAA1 expression levels positively correlated with paxillin phosphorylation in invasive breast cancer cell lines. Furthermore, suppression of PIG-T and GPAA1 expression led to a decrease in paxillin phosphorylation with a concomitant decrease in invasion ability. These results suggest that the GPI transamidase complex is composed of a group of proto-oncogenes that individually or as a group contribute to breast cancer growth. This aberrant growth is mediated, at least partially, by phosphorylation of paxillin, contributing to invasion and progression of breast cancer. (Cancer Res 2006; 66(22): 9829-36) (Cancer Res 2006; 66(20): 9829-36)

PDF file - 101K, Cyclin D1 expression is reduced in the proliferating tissues derived from Tmem16a -/- mice when compared to wild-type littermates. Tissues harvested from Tmem16a -/- and wild-type mice were subjected to immunohistochemical staining for cyclin-D1. The proliferating mesoderm demonstrated intense nuclear staining of cyclin-D1 in wild-type mice, but not in knock-out littermates (A). Levels of cyclinD1 mRNA are correlated with TMEM16A expression in tissues derived from TMEM16A (-/-), (+/-) and (+/+) mice (B). TMEM16A overexpression did not induce activation of ERK5, another MAPK that can contribute to proliferation (C).

PDF file - 97K, TMEM16A manipulation via shRNA mediated knock-down or overexpression leads to a measurable change in chloride conductance. Quantitative PCR demonstrates a significant decrease (~75%) in TMEM16A levels after shRNA treatment (A; mean � SEM; n=3, ***P

PDF file - 139K, Anti-TMEM16A antibody recognizes endogeneous TMEM16A in immunohistochemistry. A rabbit polyclonal antibody was raised against a human TMEM16A epitope (as described in the Materials and Methods). This antibody was validated in immunohistochemistry using tissues obtained from Tmem16a -/- mice and wild-type littermates. Gastrointestinal stromal tumor (a tumor type that is known to overexpress TMEM16A) is used a positive control. The in-house antibody was compared against a commercial rabbit monoclonal DOG1 antibody (Thermo Fisher Scientific, Rockford, IL) which is a validated antibody that is used by our clinical Pathology laboratory for IHC (A). Surface biotinylation was used to demonstrate that TMEM16A is present on the cell membrane of T24 cells, and migrates at the expected molecular weight (~115kDa). Murine Tmem16a migrates at a higher molecular weight (~150 kDa), in accordance with previously published data (B).

Supplementary Data 1-9 from Overexpression of Glycosylphosphatidylinositol (GPI) Transamidase Subunits Phosphatidylinositol Glycan Class T and/or GPI Anchor Attachment 1 Induces Tumorigenesis and Contributes to Invasion in Human Breast Cancer

Prader-Willi syndrome (PWS) is a multisystem disorder with neurobehavioral, metabolic, and hormonal phenotypes, caused by loss of expression of a paternally-expressed imprinted gene cluster. Prior evidence from a PWS mouse model identified abnormal pancreatic islet development with retention of aged insulin and deficient insulin secretion. To determine the collective roles of PWS genes in β-cell biology, we used genome-editing to generate isogenic, clonal INS-1 insulinoma lines having 3.16 Mb deletions of the silent, maternal (control) or active, paternal PWS-alleles. PWS β-cells demonstrated a significant cell-autonomous reduction in basal and glucose-stimulated insulin secretion. Further, proteomic analyses revealed reduced levels of cellular and secreted hormones, including all insulin peptides and amylin, concomitant with reduction of at least ten endoplasmic reticulum (ER) chaperones, including GRP78 and GRP94. Critically, transcriptomic studies demonstrated that the broad reduction of ER chaperones originated from transcriptional downregulation without corresponding changes for Ins1 and Ins2. In contrast to the dosage compensation previously seen for ER chaperones in Grp78 or Grp94 gene knockouts or knockdown, compensation is precluded by the stress-independent deficiency of ER chaperones in PWS β-cells. Consistent with reduced ER chaperones levels, PWS INS-1 β-cells are more sensitive to ER stress, leading to earlier activation of all three arms of the unfolded protein response. These findings suggest that a chronic shortage of ER chaperones in PWS β-cells leads to a deficiency of protein folding and/or delay in ER transit of insulin and other cargo. In summary, our results illuminate the pathophysiological basis of pancreatic β-cell hormone deficits in PWS, with evolutionary implications for the multigenic PWS-domain, and indicate that PWS-imprinted genes coordinate concerted regulation of ER chaperone biosynthesis and β-cell secretory pathway function.

Phenylalanine hydroxylase–deficient (PAH-deficient) phenylketonuria (PKU) results in systemic hyperphenylalaninemia, leading to neurotoxicity with severe developmental disabilities. Dietary phenylalanine (Phe) restriction prevents the most deleterious effects of hyperphenylalaninemia, but adherence to diet is poor in adult and adolescent patients, resulting in characteristic neurobehavioral phenotypes. Thus, an urgent need exists for new treatments. Additionally, rodent models of PKU do not adequately reflect neurocognitive phenotypes, and thus there is a need for improved animal models. To this end, we have developed PAH-null pigs. After selection of optimal CRISPR/Cas9 genome-editing reagents by using an in vitro cell model, zygote injection of 2 sgRNAs and Cas9 mRNA demonstrated deletions in preimplantation embryos, with embryo transfer to a surrogate leading to 2 founder animals. One pig was heterozygous for a PAH exon 6 deletion allele, while the other was compound heterozygous for deletions of exon 6 and of exons 6–7. The affected pig exhibited hyperphenylalaninemia (2000–5000 μM) that was treatable by dietary Phe restriction, consistent with classical PKU, along with juvenile growth retardation, hypopigmentation, ventriculomegaly, and decreased brain gray matter volume. In conclusion, we have established a large-animal preclinical model of PKU to investigate pathophysiology and to assess new therapeutic interventions., A phenylalanine hydroxylase–deficient pig model of phenylketonuria is established and characterized.

The immunomodulatory drugs, lenalidomide and pomalidomide yield high response rates in multiple myeloma patients, but are associated with a high rate of thrombocytopenia and increased risk of secondary hematologic malignancies. Here, we demonstrate that the immunomodulatory drugs induce self-renewal of hematopoietic progenitors and upregulate megakaryocytic colonies by inhibiting apoptosis and increasing proliferation of early megakaryocytic progenitors via down-regulation of IKZF1. In this process, the immunomodulatory drugs degrade IKZF1 and subsequently down-regulate its binding partner, GATA1. This results in the decrease of GATA1 targets such as ZFPM1 and NFE2, leading to expansion of megakaryocytic progenitors with concomitant inhibition of maturation of megakaryocytes. The down-regulation of GATA1 further decreases CCND1 and increases CDKN2A expression. Overexpression of GATA1 abrogated the effects of the immunomodulatory drugs and restored maturation of megakaryocytic progenitors. Our data not only provide the mechanism for the immunomodulatory drugs induced thrombocytopenia but also help to explain the higher risk of secondary malignancies and long-term cytopenia induced by enhanced cell cycling and subsequent exhaustion of the stem cell pool.

Tumor genome sequencing has become an invaluable resource in determining targets for new therapies. In this report, we describe the case of a patient with metastatic urothelial carcinoma with sarcomatoid features. Sarcomatoid differentiation is a rare histologic subtype that confers a more aggressive course. The first-line treatment for patients with urothelial carcinoma is platinum-based chemotherapy. Next generation tumor sequencing performed using the FoundationOne assay revealed loss of one NF2 allele and an unbalanced der(22)t(10;22)(p11.22;q12.2) chromosomal rearrangement involving the other NF2 allele, resulting in truncation and predicted loss of function. Fluorescence in situ hybridization (FISH) analysis confirmed the presence of one NF2 signal. NF2 mutations have been found in a variety of cancers and result in activation of the mTOR pathway. As such, the use of mTOR inhibitors, such as everolimus are thought to be particularly effective in the case of NF2 loss. Our patient had a dramatic response to first-line chemotherapy, but unfortunately experienced subsequent progression of his cancer and could not tolerate everolimus. Although our patient's tumor demonstrated unique acquired genetic features including both loss of heterozygosity and truncation of the NF2 locus, he still achieved a meaningful response to platinum-based chemotherapy.

Background Human papillomavirus (HPV)-positive oropharyngeal cancer is generally associated with excellent response to therapy, but some HPV-positive tumors progress despite aggressive therapy. The purpose of this study was to evaluate viral oncogene expression and viral integration sites in HPV16- and HPV18-positive squamous cell carcinoma lines. Methods E6/E7 alternate transcripts were assessed by reverse transcriptase-polymerase chain reaction (RT-PCR). Detection of integrated papillomavirus sequences (DIPS-PCR) and sequencing identified viral insertion sites and affected host genes. Cellular gene expression was assessed across viral integration sites. Results All HPV-positive cell lines expressed alternate HPVE6/E7 splicing indicative of active viral oncogenesis. HPV integration occurred within cancer-related genes TP63, DCC, JAK1, TERT, ATR, ETV6, PGR, PTPRN2, and TMEM237 in 8 head and neck squamous cell carcinoma (HNSCC) lines but UM-SCC-105 and UM-GCC-1 had only intergenic integration. Conclusion HPV integration into cancer-related genes occurred in 7 of 9 HPV-positive cell lines and of these 6 were from tumors that progressed. HPV integration into cancer-related genes may be a secondary carcinogenic driver in HPV-driven tumors. © 2017 Wiley Periodicals, Inc. Head Neck 39: 840-852, 2017.

Cell cultures are valuable models of health and disease, especially in translational research. Cell culture is important for genome editing, stem cell technologies, cancer research, investigating the basic mechanisms involved in development and differentiation, elucidating the role of genetic alterations in normal and abnormal cellular behaviour and developing and testing therapeutics. The clinically important field of classical cytogenetics requires cell culture to obtain metaphase chromosomes from dividing cells at the mitotic stage of the cell cycle when the chromosomes are most compact and visible as distinct entities. Further, culture in vitro facilitates analysis of cells in a controlled environment; minimises morbidity and mortality of human, animal or plant subjects and enables replicate studies over long periods of time. Cell lines are essential models for testing therapeutic efficacy, including drug screening and sensitivity studies. The main difficulty with cell cultures in the clinical or research laboratory is their propensity to contamination, whether by microbes or stray cells from other cultures. It is imperative to verify the authenticity of (authenticate) cell lines and rule out microbial contamination of cell cultures before initiating a series of experiments, at regular intervals during each experimental study and at the end of the study to assure the quality and validity of experimental results. Key Concepts Cell cultures are valuable models in biomedical research. Cell cultures have a propensity to become contaminated by microbes. Intra- or interspecies cross-contamination of cell lines by mislabelling or stray cells from other cell lines results in misidentified cell cultures. High-quality research results rely on (1) cell line authentication to rule out cross-contamination and (2) testing cell cultures for and avoiding microbial contamination, at the beginning, at regular intervals during and at the end of a research study. Contaminated or misidentified cell cultures waste both time and money and produce invalid research results. Keywords: cell culture; tissue culture; aseptic technique; contamination; microbial; cross-contamination; authentication

Oral squamous cell carcinoma (OSCC) is a serious public health problem caused primarily by smoking and alcohol consumption or human papillomavirus. The cancer stem cell (CSC) theory posits that CSCs show unique characteristics, including self-renewal and therapeutic resistance. Examining biomarkers and other features of CSCs is critical to better understanding their biology. To this end, the results show that cellular SOX2 immunostaining correlates with other CSC biomarkers in OSCC cell lines and marks the rare CSC population. To assess whether CSC division patterns are symmetrical, resulting in two CSC, or asymmetrical, leading to one CSC and one cancer cell, cell size and fluorescence intensity of mitotic cells stained with SOX2 were analyzed. Asymmetrical SOX2 distribution in ≈25% of the mitoses analyzed was detected. Chromosomal instability, some of which is caused by chromosome segregation defects (CSDs), is a feature of cancer cells that leads to altered gene copy numbers. We compare chromosomal instability (as measured by CSDs) between CSCs (SOX2+) and non-CSCs (SOX2-) from the same OSCC cell lines. CSDs were more common in non-CSCs (SOX2-) than CSCs (SOX2+) and in symmetrical CSC (SOX2+) mitotic pairs than asymmetrical CSC (SOX2+/SOX2-) mitotic pairs. CSCs showed fewer and different types of CSDs after ionizing radiation treatment than non-CSCs. Overall, these data are the first to demonstrate both symmetrical and asymmetrical cell divisions with CSDs in OSCC CSC. Further, the results suggest that CSCs may undergo altered behavior, including therapeutic resistance as a result of chromosomal instability due to chromosome segregation defects. © 2016 Wiley Periodicals, Inc.

We present a comprehensive comparison of PAX5,IKZF1, and CDKN2A/B abnormalities in 21 B-cell precursor acute lymphoblastic leukemia (B-ALL) patients studied by aCGH and gene-specific FISH assays. In our cohort of B-ALL patients, alterations of IKZF1, PAX5, and CDKN2A/B were detected by aCGH analysis in 43, 52, and 57% of samples, respectively. Deletions of IKZF1 were present in 9 samples, including 5 cases positive for both PAX5 and IKZF1 deletions, implying digenic impairment. Furthermore, all cases with IKZF1 deletions also had additional genomic alterations, including BCR-ABL1 gene fusions, PAX5 deletions, CDKN2A/B deletions, and FLT3 amplification. Deletions of CDKN2A/B represented the most frequent abnormalities in our group of patients. Our study demonstrates the high incidence of PAX5, IKZF1, and CDKN2A/B alterations in B-ALL detected by aCGH analysis. Due to the small size and variability in the deletion breakpoints, FISH studies showed false-negative results in 10, 40, and 28% of the samples tested for the IKZF1,PAX5, and CDKN2A/B gene deletions, respectively. The PAX5 and IKZF1 abnormalities are highly specific to B-ALL and can be used as diagnostic markers. Moreover, IKZF1 alterations frequently coexist with a BCR-ABL gene fusion. Our study revealed multiple additional B-ALL-specific genomic alterations and showed that aCGH is a more sensitive method than FISH, allowing whole genome profiling and identification of aberrations of diagnostic and prognostic significance in patients with B-ALL.

We present a comprehensive comparison of PAX5,IKZF1, and CDKN2A/B abnormalities in 21 B-cell precursor acute lymphoblastic leukemia (B-ALL) patients studied by aCGH and gene-specific FISH assays. In our cohort of B-ALL patients, alterations of IKZF1, PAX5, and CDKN2A/B were detected by aCGH analysis in 43, 52, and 57% of samples, respectively. Deletions of IKZF1 were present in 9 samples, including 5 cases positive for both PAX5 and IKZF1 deletions, implying digenic impairment. Furthermore, all cases with IKZF1 deletions also had additional genomic alterations, including BCR-ABL1 gene fusions, PAX5 deletions, CDKN2A/B deletions, and FLT3 amplification. Deletions of CDKN2A/B represented the most frequent abnormalities in our group of patients. Our study demonstrates the high incidence of PAX5, IKZF1, and CDKN2A/B alterations in B-ALL detected by aCGH analysis. Due to the small size and variability in the deletion breakpoints, FISH studies showed false-negative results in 10, 40, and 28% of the samples tested for the IKZF1,PAX5, and CDKN2A/B gene deletions, respectively. The PAX5 and IKZF1 abnormalities are highly specific to B-ALL and can be used as diagnostic markers. Moreover, IKZF1 alterations frequently coexist with a BCR-ABL gene fusion. Our study revealed multiple additional B-ALL-specific genomic alterations and showed that aCGH is a more sensitive method than FISH, allowing whole genome profiling and identification of aberrations of diagnostic and prognostic significance in patients with B-ALL.

The landscape of human papillomavirus (HPV) infection in racial/ethnic subgroups of head and neck cancer (HNC) patients has not been evaluated carefully. In this study, a meta-analysis examined the prevalence of HPV in HNC patients of African ancestry. Additionally, a pooled analysis of subject-level data was also performed to investigate HPV prevalence and patterns of p16 (CDNK2A) expression amongst different racial groups. Eighteen publications (N = 798 Black HNC patients) were examined in the meta-analysis, and the pooled analysis included 29 datasets comprised of 3129 HNC patients of diverse racial/ethnic background. The meta-analysis revealed that the prevalence of HPV16 was higher among Blacks with oropharyngeal cancer than Blacks with non-oropharyngeal cancer. However, there was great heterogeneity observed among studies (Q test P < 0.0001). In the pooled analysis, after adjusting for each study, year of diagnosis, age, gender and smoking status, the prevalence of HPV16,18 in oropharyngeal cancer patients was highest in Whites (61.1%), followed by 58.0% in Blacks and 25.2% in Asians (P < 0.0001). There was no statistically significant difference in HPV16,18 prevalence in non-oropharyngeal cancer by race (P = 0.682). With regard to the pattern of HPV16,18 status and p16 expression, White patients had the highest proportion of HPV16,18+/p16+ oropharyngeal cancer (52.3%), while Asians and Blacks had significantly lower proportions (23.0 and 22.6%, respectively) [P < 0.0001]. Our findings suggest that the pattern of HPV16,18 status and p16 expression in oropharyngeal cancer appears to differ by race and this may contribute to survival disparities.

Cytogenetic alterations underlie the development of head and neck squamous cell carcinoma (HNSCC), whether tobacco and alcohol use, betel nut chewing, snuff or human papillomavirus (HPV) causes the disease. Many of the molecular genetic aberrations in HNSCC result from these cytogenetic alterations. This review presents a brief introduction to the epidemiology of HNSCC, and discusses the role of HPV in the disease, cytogenetic alterations and their frequencies in HNSCC, their molecular genetic and The Cancer Genome Atlas (TCGA) correlates, prognostic implications, and possible therapeutic considerations. The most frequent cytogenetic alterations in HNSCC are gains of 5p14-15, 8q11-12, and 20q12-13, gains or amplifications of 3q26, 7p11, 8q24, and 11q13, and losses of 3p, 4q35, 5q12, 8p23, 9p21-24, 11q14-23, 13q12-14, 18q23, and 21q22. To understand their effects on tumor cell biology and response to therapy, the cytogenetic findings in HNSCC are increasingly being examined in the context of the biochemical pathways they disrupt. The goal is to minimize morbidity and mortality from HNSCC using cytogenetic abnormalities to identify valuable diagnostic biomarkers for HNSCC, prognostic biomarkers of tumor behavior, recurrence risk, and outcome, and predictive biomarkers of therapeutic response to identify the most efficacious treatment for each individual patient's tumor, all based on a detailed understanding of the next generation biology of HNSCC.

HPV-related HNSCC generally have a better prognosis than HPV-negative HNSCC. However, a subgroup of HPV-positive tumors with poor prognosis has been recognized, particularly related to smoking, EGFR overexpression and chromosomal instability. Viral integration into the host genome might contribute to carcinogenesis, as is shown for cervical carcinomas. Therefore, all HPV16-positive HNSCC cell lines currently available have been carefully analyzed for viral and host genome parameters. The viral integration status, viral load, viral gene expression and the presence of aneusomies was evaluated in the cell lines UD-SCC-2, UM-SCC-047, UM-SCC-104, UPCI:SCC090, UPCI:SCC152, UPCI:SCC154 and 93VU147T. HPV integration was examined using FISH, APOT-PCR and DIPS-PCR. Viral load and the expression of the viral genes E2, E6 and E7 were determined via quantitative PCR. All cell lines showed integration-specific staining patterns and signals indicating transcriptional activity using FISH. APOT- and DIPS-PCR identified integration-derived fusion products in six cell lines and only episomal products for UM-SCC-104. Despite the observed differences in viral load and the number of viral integration sites, this did not relate to the identified viral oncogene expression. Furthermore, cell lines exhibited EGFR expression and aneusomy (except UPCI:SCC154). In conclusion, all HPV16-positive HNSCC cell lines showed integrated and/or episomal viral DNA that is transcriptionally active, although viral oncogene expression was independent of viral copy number and the number of viral integration sites. Because these cell lines also contain EGFR expression and aneusomy, which are parameters of poor prognosis, they should be considered suitable model systems for the development of new antiviral therapies.

As she said in writing about Theodore Puck in 2006, “it is impossible to write a comprehensive obituary of [Janet Rowley] in less than a dozen pages.1” Leading pioneer in cancer research and matriarch of cancer genetics and cytogenetics, Janet Davison Rowley, M.D. (Figure 1), died at her home from complications of ovarian cancer on December 17, 2013. How ironic that a woman whose numerous cancer research discoveries led to lifesaving treatments for so many cancer patients succumbed to cancer. Dr.

The Consortium for Mouse Cell Line Authentication was formed to validate Short Tandem Repeat (STR) markers for intraspecies identification of mouse cell lines. The STR profiling method is a multiplex polymerase chain reaction (PCR) assay comprised of primers targeting 19 mouse STR markers and two human STR markers (for interspecies contamination screening). The goals of the Consortium were to perform an interlaboratory study to-(1) validate the mouse STR markers to uniquely identify mouse cell lines (intraspecies identification), (2) to provide a public database of mouse cell lines with the National Institute of Standards and Technology (NIST)-validated mouse STR profiles, and (3) to publish the results of the interlaboratory study. The interlaboratory study was an international effort that consisted of 12 participating laboratories representing institutions from academia, industry, biological resource centers, and government. The study was based on 50 of the most commonly used mouse cell lines obtained from the American Type Culture Collection (ATCC). Of the 50 mouse cell lines, 18 had unique STR profiles that were 100% concordant (match) among all Consortium laboratory members, and the remaining 32 cell lines had discordance that was resolved readily and led to improvement of the assay. The discordance was due to low signal and interpretation issues involving artifacts and genotyping errors. Although the total number of discordant STR profiles was relatively high in this study, the percent of labs agreeing on allele calls among the discordant samples was above 92%. The STR profiles, including electropherogram images, for NIST-validated mouse cell lines will be published on the NCBI BioSample Database (https://www.ncbi.nlm.nih.gov/biosample/). Overall, the interlaboratory study showed that the multiplex PCR method using 18 of the 19 mouse STR markers is capable of discriminating at the intraspecies level between mouse cell lines. Further studies are ongoing to refine the assay including (1) development of an allelic ladder for improving the accuracy of allele calling and (2) integration of stutter filters to identify true stutter.

Oral squamous cell carcinoma (OSCC), a subset of head and neck squamous cell carcinoma (HNSCC), is the eighth most common cancer in the U.S.. Amplification of chromosomal band 11q13 and its association with poor prognosis has been well established in OSCC. The first step in the breakage-fusion-bridge (BFB) cycle leading to 11q13 amplification involves breakage and loss of distal 11q. Distal 11q loss marked by copy number loss of the ATM gene is observed in 25% of all Cancer Genome Atlas (TCGA) tumors, including 48% of HNSCC. We showed previously that copy number loss of distal 11q is associated with decreased sensitivity (increased resistance) to ionizing radiation (IR) in OSCC cell lines. We hypothesized that this radioresistance phenotype associated with ATM copy number loss results from upregulation of the compensatory ATR-CHEK1 pathway, and that knocking down the ATR-CHEK1 pathway increases the sensitivity to IR of OSCC cells with distal 11q loss. Clonogenic survival assays confirmed the association between reduced sensitivity to IR in OSCC cell lines and distal 11q loss. Gene and protein expression studies revealed upregulation of the ATR-CHEK1 pathway and flow cytometry showed G2 M checkpoint arrest after IR treatment of cell lines with distal 11q loss. Targeted knockdown of the ATR-CHEK1 pathway using CHEK1 or ATR siRNA or a CHEK1 small molecule inhibitor (SMI, PF-00477736) resulted in increased sensitivity of the tumor cells to IR. Our results suggest that distal 11q loss is a useful biomarker in OSCC for radioresistance that can be reversed by ATR-CHEK1 pathway inhibition.

The ATR-CHEK1 pathway is upregulated and overactivated in Ataxia Telangiectasia (AT) cells, which lack functional ATM protein. Loss of ATM in AT confers radiosensitivity, although ATR-CHEK1 pathway overactivation compensates, leads to prolonged G(2) arrest after treatment with ionizing radiation (IR), and partially reverses the radiosensitivity. We observed similar upregulation of the ATR-CHEK1 pathway in a subset of oral squamous cell carcinoma (OSCC) cell lines with ATM loss. In the present study, we report copy number gain, amplification, or translocation of the ATR gene in 8 of 20 OSCC cell lines by FISH; whereas the CHEK1 gene showed copy number loss in 12 of 20 cell lines by FISH. Quantitative PCR showed overexpression of both ATR and CHEK1 in 7 of 11 representative OSCC cell lines. Inhibition of ATR or CHEK1 with their respective siRNAs resulted in increased sensitivity of OSCC cell lines to IR by the colony survival assay. siRNA-mediated ATR or CHEK1 knockdown led to loss of G(2) cell cycle accumulation and an increased sub-G(0) apoptotic cell population by flow cytometric analysis. In conclusion, the ATR-CHEK1 pathway is upregulated in a subset of OSCC with distal 11q loss and loss of the G(1) phase cell cycle checkpoint. The upregulated ATR-CHEK1 pathway appears to protect OSCC cells from mitotic catastrophe by enhancing the G(2) checkpoint. Knockdown of ATR and/or CHEK1 increases the sensitivity of OSCC cells to IR. These findings suggest that inhibition of the upregulated ATR-CHEK1 pathway may enhance the efficacy of ionizing radiation treatment of OSCC.

Lenalidomide (LEN) treatment in multiple myeloma (MM) results in a superior outcome. However, there is concern for increased myelodysplastic syndrome/acute myeloid leukemia (MDS/AML) associated with LEN. Thus, bone marrow morphology and cytogenetics studies from 40 patients were evaluated for early signs of MDS prior to therapy, during therapy and at follow-up. Newly diagnosed patients with MM treated with LEN and dexamethasone (LD) alone or followed by autologous stem cell transplant (LD/ASCT), or patients with relapsed/refractory MM treated with LEN, bendamustine and dexamethasone (BLD) were included. One patient developed MDS. Baseline prevalence of mild morphologic myelodysplasia was highest in pretreated patients with MM (BLD, 71%), but was also seen in newly diagnosed patients (LD and LD/ASCT, 17%). The prevalence of myelodysplasia did not increase over time. Thus, this study did not reveal rapidly emerging MDS in 39 of 40 patients with MM treated with LEN. The development of MDS in one patient suggests that longer follow-up is needed for all.

Head and neck squamous cell carcinoma (HNSCC) is a major public health concern. Recent data indicate the presence of cancer stem cells (CSC) in many solid tumors, including HNSCC. Here, we assessed the stem cell (SC) characteristics, including cell surface markers, radioresistance, chromosomal instability, and in vivo tumorigenic capacity of CSC isolated from HNSCC patient specimens. We show that spheroid enrichment of CSC from early and short-term HNSCC cell cultures was associated with increased expression of CD44, CD133, SOX2 and BMI1 compared with normal oral epithelial cells. On immunophenotyping, five of 12 SC/CSC markers were homogenously expressed in all tumor cultures, while one of 12 was negative, four of 12 showed variable expression, and two of the 12 were expressed heterogeneously. We showed that irradiated CSCs survived and retained their self-renewal capacity across different ionizing radiation (IR) regimens. Fluorescence in situ hybridization (FISH) analyses of parental and clonally-derived tumor cells revealed different chromosome copy numbers from cell to cell, suggesting the presence of chromosomal instability in HNSCC CSC. Further, our in vitro and in vivo mouse engraftment studies suggest that CD44+/CD66− is a promising, consistent biomarker combination for HNSCC CSC. Overall, our findings add further evidence to the proposed role of HNSCC CSCs in therapeutic resistance.

Human papillomavirus (HPV)-positive oropharyngeal cancer is generally associated with excellent response to therapy, but some HPV-positive tumors progress despite aggressive therapy. The purpose of this study was to evaluate viral oncogene expression and viral integration sites in HPV16- and HPV18-positive squamous cell carcinoma lines.E6/E7 alternate transcripts were assessed by reverse transcriptase-polymerase chain reaction (RT-PCR). Detection of integrated papillomavirus sequences (DIPS-PCR) and sequencing identified viral insertion sites and affected host genes. Cellular gene expression was assessed across viral integration sites.All HPV-positive cell lines expressed alternate HPVE6/E7 splicing indicative of active viral oncogenesis. HPV integration occurred within cancer-related genes TP63, DCC, JAK1, TERT, ATR, ETV6, PGR, PTPRN2, and TMEM237 in 8 head and neck squamous cell carcinoma (HNSCC) lines but UM-SCC-105 and UM-GCC-1 had only intergenic integration.HPV integration into cancer-related genes occurred in 7 of 9 HPV-positive cell lines and of these 6 were from tumors that progressed. HPV integration into cancer-related genes may be a secondary carcinogenic driver in HPV-driven tumors. © 2017 Wiley Periodicals, Inc. Head Neck 39: 840-852, 2017.

Sarcomas are heterogeneous, and their treatment and prognosis are driven by the morphologic subtype and the clinical stage. Classic cytogenetics and fluorescence in situ hybridization (FISH) analysis play an important role in their diagnostic work up.Forty-six cases of soft-tissue sarcoma were reviewed that underwent karyotyping and simultaneous FISH analysis at initial diagnosis. They included 10 dedifferentiated liposarcomas, 10 myxoid liposarcomas, and 14 synovial sarcomas. Six tumors were investigated for EWSR1 rearrangement. Six high-grade miscellaneous sarcomas were also examined.The dedifferentiated liposarcoma had complex karyotypes and MDM2 amplification by FISH, and of these, 5 tumors with myxoid changes also had complex signals for DDIT3. All but 4 myxoid liposarcomas had complex karyotypes, in addition to the characteristic translocation. FISH analysis displayed DD1T3 rearrangement. All synovial sarcomas except 1 recurrence had a t(X;18) translocation by karyotyping and FISH. The EWSR1 rearrangement was present in all extraskeletal myxoid chondrosarcomas, angiomatoid fibrous histiocytoma, atypical Ewing sarcoma, and a clear-cell sarcoma, all of which had characteristic karyotypes. Seven high-grade sarcomas had no specific karyotype or rearrangements for DDIT3, SS18, and EWSR1 by FISH.There is good correlation between karyotyping and FISH. Complex FISH signals found in dedifferentiated liposarcomas may be related to an increased chromosome 12 copy number and ploidy. Karyotyping is an important baseline standard for the quality assurance of newly developed FISH probes. It also provides a global view of chromosomal changes and the opportunity to investigate the role of other genetic alterations and potential therapeutic targets.

Frequent gene amplification of the receptor-activated calcium-dependent chloride channel TMEM16A (TAOS2 or ANO1) has been reported in several malignancies. However, its involvement in human tumorigenesis has not been previously studied. Here, we show a functional role for TMEM16A in tumor growth. We found TMEM16A overexpression in 80% of head and neck squamous cell carcinoma (SCCHN), which correlated with decreased overall survival in patients with SCCHN. TMEM16A overexpression significantly promoted anchorage-independent growth in vitro, and loss of TMEM16A resulted in inhibition of tumor growth both in vitro and in vivo. Mechanistically, TMEM16A-induced cancer cell proliferation and tumor growth were accompanied by an increase in extracellular signal–regulated kinase (ERK)1/2 activation and cyclin D1 induction. Pharmacologic inhibition of MEK/ERK and genetic inactivation of ERK1/2 (using siRNA and dominant-negative constructs) abrogated the growth effect of TMEM16A, indicating a role for mitogen-activated protein kinase (MAPK) activation in TMEM16A-mediated proliferation. In addition, a developmental small-molecule inhibitor of TMEM16A, T16A-inh01 (A01), abrogated tumor cell proliferation in vitro. Together, our findings provide a mechanistic analysis of the tumorigenic properties of TMEM16A, which represents a potentially novel therapeutic target. The development of small-molecule inhibitors against TMEM16A may be clinically relevant for treatment of human cancers, including SCCHN. Cancer Res; 72(13); 3270–81. ©2012 AACR.

Human cancer cell lines have been important tools for biological and preclinical investigation, and establishing their relationship to genomic alterations and subtypes found in tumors could further accelerate functional and therapeutic discoveries. We conducted an integrated analysis of the genomic and transcriptomic profiles of 15 human papillomavirus negative (HPV−) and 11 HPV(+) head and neck squamous cell carcinoma (HNSCC) lines, to identify models of important molecular subtypes uncovered among 279 HNSCC tumors from The Cancer Genome Atlas (TCGA). We developed analytic approaches to detect copy number alterations (CNAs) driving altered expression and predict deleterious mutations in HNSCC lines for comparison with TCGA. Strikingly, HNSCC lines displayed recurrent amplifications on chromosomes 3q22-29, 5p15, 11q13/22, and 8p11 that drive increased expression of a cluster of ~100 mRNAs encoding multiple known and understudied candidate oncogenes. These CNAs, together with loss and under-expression or mutations of putative tumor suppressor genes, recapitulate genomic alterations found in more aggressive HNSCC tumor subtypes. Among these, concurrent 3q26.3 amplification and TP53 mutation in HPV(−) HNSCCs is associated with worse overall survival. Common and distinctive CNAs, transcriptome, and mutations converge on biologically and therapeutically important signal pathways and functions in HNSCCs differing in HPV status, including PI3K, Hippo, TGF-β, Wnt/β-catenin, TP63, TP53/death, cell cycle/mitosis, stemness/differentiation, chromatin remodelling/DNA replication, post-transcriptional regulation, and mitochondrial biosynthesis. Our findings elucidate and validate genomic alterations underpinning numerous discoveries made with historic and recently derived HNSCC lines, and provide valuable models for future preclinical and therapeutic studies in subsets of HNSCCs with worse prognosis. Supported by NIDCD/NIH intramural projects ZIA-DC-000073, ZIA-DC-000074. This work utilized the computational resources of the NIH HPC Biowulf cluster. (http://hpc.nih.gov) Citation Format: Hui Cheng, Xinping Yang, Han Si, Anthony Saleh, Wenming Xiao, Jamie Coupar, Susanne M. Gollin, Robert L. Ferris, Natalia Issaeva, Wendell G. Yarbrough, Mark E. Prince, Thomas E. Carey, Carter Van Waes, Zhong Chen. Recurrent genomic copy alterations deregulate the transcriptome and key signaling networks in head and neck cancer cell lines and tumors with worse prognosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5346.

Background: Polymorphisms in DNA repair genes have been reported contributing factors in head and neck cancer risk but studies have shown conflicting results.Objective: To clarify the impact of DNA repair gene polymorphisms in head and neck cancer risk.Method: A meta-analysis including 30 case–control studies was performed.Results: Marginally statistically significant association was found for XRCC1 codon 399 (for Caucasians only), XPD Asp312Asn and XRCC1 codon 194 variants and head and neck cancer.Conclusion: Assessments of the effects of smoking, alcohol, human papillomavirus and race/ethnicity on the association between DNA repair gene polymorphisms and head and neck cancer are needed.

SUMMARY Cell lines are important tools for biological and preclinical investigation, and establishing their relationship to genomic alterations in tumors could accelerate functional and therapeutic discoveries. We conducted integrated analyses of genomic and transcriptomic profiles of 15 human papillomavirus (HPV)-negative and 11 HPV-positive head and neck squamous cell carcinoma (HNSCC) lines to compare with 279 tumors from The Cancer Genome Atlas (TCGA). We identified recurrent amplifications on chromosomes 3q22–29, 5p15, 11q13/22, and 8p11 that drive increased expression of more than 100 genes in cell lines and tumors. These alterations, together with loss or mutations of tumor suppressor genes, converge on important signaling pathways, recapitulating the genomic landscape of aggressive HNSCCs. Among these, concurrent 3q26.3 amplification and TP53 mutation in most HPV(–) cell lines reflect tumors with worse survival. Our findings elucidate and validate genomic alterations underpinning numerous discoveries made with HNSCC lines and provide valuable models for future studies., Graphical Abstract, In Brief Cheng et al. reveal that head and neck squamous cell cancer cell lines display recurrent chromosome copy number alterations and mutations found in tumors with worse prognosis. The copy alterations correlate with expression of multiple genes implicated in squamous cell signaling pathways and programs that provide candidates for mechanistic studies and targeted therapy.

Lymphoplasmacytic lymphoma (LPL) is a small B-cell lymphoma with plasmacytic differentiation that does not fulfill the criteria for any other small B-cell lymphoma. Cytogenetic characterization of nodal LPL is limited and the distinction from marginal zone lymphomas with plasmacytic differentiation can be problematic. Thus, 17 cases of lymph node-based LPL were studied with fluorescence immunophenotypic and interphase cytogenetics for the investigation of neoplasia (FICTION) using a CD79a antibody and probes to detect trisomies of chromosomes 3 (15 cases), 12 (16 cases), and 18 (17 cases); rearrangements (R) of IgH (10 cases), BCL6 (6 cases), PAX5 (7 cases), and MALT1 (16 cases); and deletion 6q21 (7 cases). Cases with IgH R were further studied with an IgH/BCL2 probe. In cases without FICTION studies, previously reported fluorescence in situ hybridization results for IgH, PAX5, and deletion 6q21 were available from prior studies. The histopathology, immunophenotype, and available clinical data were also reviewed. Three pathologic categories were recognized: 5 classic LPL, 5 vaguely nodular polymorphous (VN-P), and 7 other. Among the classic LPL, 4/4 had an IgM paraproteinemia, 5/5 had bone marrow involvement (BM+), and 1/5 had +MALT1. One of one VN-P LPL had an IgM paraprotein, 2/4 were IgM+, 2/4 IgG+, 1/3 had BM+, and 1/5 had an IgH R. Among the other cases, 2/3 had a paraprotein, 2/7 were IgM+, 5/7 IgG+, and 0/3 had BM+. Of these cases, 1 showed +12, 1 +18, and 1 IgH/BCL2 rearrangement plus +18. None of the 17 cases had a 6q21 deletion or +3. Therefore, with rare exception, lymph node-based LPL with classic or more varied histopathologic features does not have the cytogenetic abnormalities frequently associated with bone marrow-based LPL/Waldenstrom macroglobulinemia or many of the marginal zone lymphomas. The search for better objective inclusionary criteria for LPL must continue.

Cancer is one of the five leading causes of death in the Caribbean. Viral infections have been associated with cancer development and propagation, but the prevalence of such infections in the Caribbean is unknown. This review of the published literature shows that in 161,196 subjects from 14 Caribbean Islands, the adjusted prevalence of HHV8 infection is 4.5%, HTLV-1: 1.0%, HPV: 57.5%, HCV: 0.4%, HBV: 9.4%, and EBV: 92.2%. With the exception of HCV, the prevalence was significantly higher in the Caribbean than in the United States. These findings may significantly contribute to the high incidence of cancer observed in the Caribbean.

About 45% of head and neck squamous cell carcinomas (HNSCC) are characterized by amplification of chromosomal band 11q13. This amplification occurs by a breakage-fusion-bridge (BFB) cycle mechanism. The first step in the BFB cycle involves breakage and loss of distal 11q, from FRA11F (11q14.2) to 11qter. Consequently, numerous genes, including three critical genes involved in the DNA damage response pathway, MRE11A, ATM, and H2AFX are lost in the step preceding 11q13 amplification. We hypothesized that this partial loss of genes on distal 11q may lead to a diminished DNA damage response in HNSCC. Characterization of HNSCC using fluorescence in situ hybridization (FISH) revealed concurrent partial loss of MRE11A, ATM, and H2AFX in all four cell lines with 11q13 amplification and in four of seven cell lines without 11q13 amplification. Quantitative microsatellite analysis and loss of heterozygosity studies confirmed the distal 11q loss. FISH evaluation of a small series of HNSCC, ovarian, and breast cancers confirmed the presence of 11q loss in at least 60% of these tumors. All cell lines with distal 11q loss exhibited a diminished DNA damage response, as measured by a decrease in the size and number of γ-H2AX foci and increased chromosomal instability following treatment with ionizing radiation. In conclusion, loss of distal 11q results in a defective DNA damage response in HNSCC. Distal 11q loss was also unexpectedly associated with reduced sensitivity to ionizing radiation. Although the literature attributes the poor prognosis in HNSCC to 11q13 gene amplification, our results suggest that distal 11q deletions may be an equally significant factor. © 2007 Wiley-Liss, Inc.

Nonrandom, reciprocal translocations between nonhomologous chromosomes are critical cellular events that lead to malignant transformation. Therefore, understanding the mechanisms involved in these chromosomal rearrangements is essential for understanding the process of carcinogenesis. There has been substantial discussion in the literature over the past ten years about mechanisms involved in constitutional chromosomal rearrangements, including deletions, duplications, and translocations. Yet our understanding of the mechanisms of chromosomal rearrangements in cancer is still developing. This review presents what is known about the mechanisms involved in selected nonrandom chromosomal translocations in leukemia.

Background. Head and neck squamous cell carci- noma (HNSCC) cell lines are important preclinical models in the search for novel and targeted therapies to treat head and neck cancer. Unlike many other cancer types, a wide variety of pri- mary and metastatic HNSCC cell lines are available. An easily accessible guide that organizes important characteristics of HNSCC cell lines would be valuable for the selection of appro- priate HNSCC cell lines for in vitro or in vivo studies. Methods. A literature search was performed. Results. Cell growth and culture parameters from HNSCC cell lines were catalogued into tables or lists of selected charac- teristics. Methods for establishing cancer cell lines and basic cell culture maintenance techniques were reviewed. Conclusions. A compendium of HNSCC cell line characteris- tics is useful for organizing the accumulating information regard- ing cell line characteristics to assist investigators with the devel

Human papillomavirus (HPV), a sexually transmitted virus causes cervical carcinomas, and is associated with approximately 36% of oropharyngeal tumours where HPV16 is the predominant genotype. The cervical cancer incidence rate in Trinidad and Tobago is about two times higher than the worldwide rate. We have for the first time determined the prevalence and type distribution of cervical HPV infections among cancer-free Afro-Caribbean women from Tobago, and compared it with the HPV subtypes observed in their oral cavity. Thirty-five per cent of the women were cervical HPV positive. The most common high-risk type detected in the cervix was HPV45 rather than HPV16 and 18. The prevalence of HPV infection in the oral mucosa was 6.6%. The distribution of HPV genotypes in healthy Tobagonian women is different from that reported in studies conducted in European and North American populations. This may have important implications for vaccine introduction in this and other Afro-Caribbean countries.

Stem cell (SC) research holds the promise of controlling and/or curing a variety of diseases. SC applications include cell therapy, disease modeling, and developmental biology. This commentary focuses on an important study that examined Notch signaling in murine satellite muscle SCs (Lin et al., 2013). The results highlight the possible serious side effects of NOTCH inhibitors, a class of targeted drugs that are being examined for treating a variety of cancers (Krop et al., 2012). Skeletal muscles, unlike many other tissues, possess a unique regenerative capacity (Shi and Garry, 2006). Many researchers believe that this unique feature may be the key to treating myopathies, other degenerative diseases, and cancer (Jackson et al., 2010). Much of the regenerative potential of muscle depends on a population of SCs called satellite cells (Le Grand and Rudnicki, 2007). The maintenance of stemness in adult SC is regulated by two main factors: the “SC niche” and the stemness regulatory pathways. Stemness regulatory pathways regulate stemness via SC genes and regulatory transcription factors, including the NOTCH pathway proteins. Changes in expression of these transcription factors regulate the balance between self-renewal and differentiation, vital for tissue regeneration. NOTCH is a crucial signaling pathway that regulates stemness during both embryogenesis and adulthood (Le Grand and Rudnicki, 2007). In humans, the NOTCH signaling pathway involves four receptors and five ligands, is extremely complex, and is controlled by interconnected positive and negative regulators (Fouillade et al., 2012). To add further complexity, NOTCH family members have been shown to carry out different and sometimes opposing functions in the same tissue and/or cell type (Yin et al., 2010). The role of the Notch pathway was first identified in satellite cells from murine muscle, which were shown to have reduced regenerative potential (Le Grand and Rudnicki, 2007). Satellite cells rely on the NOTCH pathway for maintenance of quiescence (Mourikis et al., 2012), which is characterized by reversible mitotic arrest and reduced metabolic activity (Le Grand and Rudnicki, 2007; Bjornson et al., 2012). Inactivation of the NOTCH pathway is crucial for the process of differentiation (Bjornson et al., 2012). To understand the role of the Notch protein in regulating SC quiescence and muscle regeneration, Lin et al. (2013) developed a unique conditional knock-out mouse model (Pax7-Cre+/dnMaml1+ mutant mice) in which Notch signaling is specifically blocked in the satellite SC using the dominant negative pan-Notch inhibitor, dnMaml1. The Pax7-Cre+/dnMaml1+ mutant mice showed myopathy manifested as muscle damage and weakness as early as 6 months of age and a shortened life span of about 8 months. The satellite SC pool was totally depleted in the mutant mice due to skewing of the balance between self-renewal and differentiation to differentiation rather than self-renewal. Further, the progeny of myoblasts showed reduced proliferation compared to controls. These experiments and others post cardiotoxin (CTX)-induced injury provided evidence that Notch pathway blockade leads to loss of quiescence and depletion of the satellite SC pool. These results are concordant with reports of other studies that blocked murine Notch pathway signaling by selectively inhibiting the recombining binding protein-Jj (RBP-Jj), a nuclear factor involved in the canonical Notch signaling pathway (Bjornson et al., 2012; Mourikis et al., 2012). In addition to its important role in muscle development, regeneration and repair, the NOTCH pathway is dysfunctional commonly in several developmental, vascular and cardiac diseases, including leukoencephalopathy and pulmonary arterial hypertension (Fouillade et al., 2012; Groth and Fortini, 2012). NOTCH is also dysregulated in a variety of human tumors, including cancers of the breast, lung, pancreas, colon, and brain (Groth and Fortini, 2012), and in head and neck squamous cell cancer (HNSCC) (Stransky et al., 2011). NOTCH gene mutations have been found in approximately 15% of HNSCC (Gaykalova et al., 2014), and are usually associated with loss-of-function (Stransky et al., 2011), gain-of-function, or both (Gaykalova et al., 2014). Functional studies suggest that NOTCH1 may act as a tumor suppressor gene in HNSCC (Pickering et al., 2013). The Lin et al. (2013) paper is pertinent to the clinical application of NOTCH inhibitors, which primarily target the presenilin/gamma-secretase aspartyl protease cleavage that releases the NOTCH intracellular domain (NICD) from the membrane (Fouillade et al., 2012). Marked toxicity affecting the gastrointestinal tract and immune systems has been demonstrated in clinical and preclinical trials, but muscle side effects have not yet been noted (Groth and Fortini, 2012). However, in combination with satellite SC exhaustion associated with aging, the side effects of NOTCH inhibitors in older cancer patients might be substantial (Carlson et al., 2009). Muscle side effects were not demonstrated in clinical trials most likely due to early termination of most trials to date (e.g., NCT01088763), in which acute side effects affected rapidly proliferating cellular compartments. Further, the role of the NOTCH pathway in regulating squamous epithelial differentiation is consistent with reports of cutaneous disorders and malignancies as side effects of NOTCH inhibitors in an Alzheimer's disease clinical trial (NCT00762411) and in Notch1 knockout mice (Nicolas et al., 2003). These results clearly show that more research concerning the effects and side effects of inhibiting the NOTCH pathway in normal and tumor cells is crucial before targeted therapeutic approaches against the NOTCH pathway are implemented further. In addition, to avoid possibly severe and life-threatening skin or muscle-related side effects, alternative therapeutic approaches should be examined, including intra-tumoral administration of Notch inhibitors in preclinical models, as suggested by Pickering et al. (2013). Another approach may be to develop alternative small molecule inhibitors or antibodies to inhibit specific NOTCH receptors, their activating ligands, or other components of the NOTCH pathway in tumor cells (Fouillade et al., 2012). For example, Wu et al. generated antibodies that specifically target murine and human Notch receptors; short-term administration (2–3 weeks) showed fewer and less severe side effects compared to other Notch inhibitors (Wu et al., 2010). Targeted therapies against stemness regulatory pathways like NOTCH are promising in a variety of disorders. Owing to the high possibility of muscle side effects after long-term administration, we recommend following patients by physical exam for muscle wasting and avoiding concomitant use of steroids, which can lead to myopathies. The study by Lin and colleagues (2013) highlights the critical gap in SC targeting, which is a better understanding of SCs and their complex regulatory pathways. A detailed understanding of regulatory mechanisms in SCs is essential before we can attempt to manipulate them in beneficial ways to eradicate disorders of regeneration like muscular dystrophy, and SC-driven resistance to cancer therapies, while avoiding dysregulation of normal SC processes and cellular proliferation.

Oropharyngeal squamous cell carcinoma (OPSCC) arises from squamous epithelial cells that line mucosal surfaces in the oropharynx. This region includes the middle segment of the pharynx, that is, the base of the tongue, soft palate, tonsils, and side and back walls of the throat. The majority of OPSCC cases (~62 %) in the United States are caused by human papillomavirus (HPV), which is the most common sexually transmitted infection worldwide. HPV not only causes OPSCC, but cervical, vaginal, vulvar, anal, penile, and laryngeal cancers, genital warts, and respiratory papillomas. There is no cure for HPV, although prophylactic vaccination is available, safe, and highly effective if the three injection series is completed prior to infection. As a result of widespread vaccination of both males and females, the frequency of HPV-induced tumors is expected to decrease significantly. However, in the United States, HPV vaccination is not widespread; only ~57 % of girls ages 13–17 and ~35 % of teen boys have been vaccinated with at least one injection. In developing countries, such as Rwanda with government-implemented, school-based HPV vaccination, three-dose vaccination completion rates exceed 90 %. This chapter reviews the biology, prevalence, and transmission of HPV infection, the incidence, risk factors for and features of HPV-associated OPSCC, and strategies for preventing HPV infection and HPV-associated cancers.

Two distinct etiologies of head and neck squamous cell carcinoma (HNSCC) have been proposed, DNA damage owing to tobacco and alcohol exposure and human papillomavirus (HPV) oncogene-mediated transformation. Common genetic alterations in HNSCC include TP53 mutations, 11q13 amplification (amp) and CDKN2A/p16 mutations or promoter methlyation. However, in HPV+ HNSCC it is frequent to observe wild-type TP53 and expression of p16. The relationship of this unusual pattern with 11q13 amp has not been tested. In a retrospective study on 125 HNSCC patients, only 17% (five out of 30) of HPV+ vs 44% (39 out of 89) of HPV − tumours expressed 11q13 amp (adjusted odds ratio (OR)=0.2, 95% confidence interval (CI)=0.1–0.6). A subpopulation of tumours (n=69) were classified according to the three molecular markers, TP53, p16 and 11q13 amp. In addition to wild-type TP53, and p16 expression, HPV+ tumours were more likely not to be amplified at 11q13 (OR=6.5, 95% CI=1.8–23.9). As HPV+ HNSCC lack the genetic alterations which are common in other tumours, we hypothesise that HPV infection may represent an early event in the HNSCC carcinogenic process, thus suggesting a distinct molecular pathway.