Curtis R. Pickering

Exome Sequencing of Head and Neck Squamous Cell Carcinoma Reveals Inactivating Mutations in NOTCH1

The mutational profile of head and neck cancer is complex and may pose challenges to the development of targeted therapies. Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. To explore the genetic origins of this cancer, we used whole-exome sequencing and gene copy number analyses to study 32 primary tumors. Tumors from patients with a history of tobacco use had more mutations than did tumors from patients who did not use tobacco, and tumors that were negative for human papillomavirus (HPV) had more mutations than did HPV-positive tumors. Six of the genes that were mutated in multiple tumors were assessed in up to 88 additional HNSCCs. In addition to previously described mutations in TP53, CDKN2A, PIK3CA, and HRAS, we identified mutations in FBXW7 and NOTCH1. Nearly 40% of the 28 mutations identified in NOTCH1 were predicted to truncate the gene product, suggesting that NOTCH1 may function as a tumor suppressor gene rather than an oncogene in this tumor type.

Integrative Genomic Characterization of Oral Squamous Cell Carcinoma Identifies Frequent Somatic Drivers

The survival of patients with oral squamous cell carcinoma (OSCC) has not changed significantly in several decades, leading clinicians and investigators to search for promising molecular targets. To this end, we conducted comprehensive genomic analysis of gene expression, copy number, methylation, and point mutations in OSCC. Integrated analysis revealed more somatic events than previously reported, identifying four major driver pathways (mitogenic signaling, Notch, cell cycle, and TP53) and two additional key genes (FAT1, CASP8). The Notch pathway was defective in 66% of patients, and in follow-up studies of mechanism, functional NOTCH1 signaling inhibited proliferation of OSCC cell lines. Frequent mutation of caspase-8 (CASP8) defines a new molecular subtype of OSCC with few copy number changes. Although genomic alterations are dominated by loss of tumor suppressor genes, 80% of patients harbored at least one genomic alteration in a targetable gene, suggesting that novel approaches to treatment may be possible for this debilitating subset of head and neck cancers.

Assembly and Initial Characterization of a Panel of 85 Genomically Validated Cell Lines from Diverse Head and Neck Tumor Sites

Purpose: Human cell lines are useful for studying cancer biology and preclinically modeling cancer therapy, but can be misidentified and cross-contamination is unfortunately common. The purpose of this study was to develop a panel of validated head and neck cell lines representing the spectrum of tissue sites and histologies that could be used for studying the molecular, genetic, and phenotypic diversity of head and neck cancer. Methods: A panel of 122 clinically and phenotypically diverse head and neck cell lines from head and neck squamous cell carcinoma, thyroid cancer, cutaneous squamous cell carcinoma, adenoid cystic carcinoma, oral leukoplakia, immortalized primary keratinocytes, and normal epithelium was assembled from the collections of several individuals and institutions. Authenticity was verified by carrying out short tandem repeat analysis. Human papillomavirus (HPV) status and cell morphology were also determined. Results: Eighty-five of the 122 cell lines had unique genetic profiles. HPV-16 DNA was detected in 2 cell lines. These 85 cell lines included cell lines from the major head and neck primary tumor sites, and close examination shows a wide range of in vitro phenotypes. Conclusions: This panel of 85 genomically validated head and neck cell lines represents a valuable resource for the head and neck cancer research community that can help advance understanding of the disease by providing a standard reference for cell lines that can be used for biological as well as preclinical studies. Clin Cancer Res; 17(23); 7248–64. ©2011 AACR.

Mutational Landscape of Aggressive Cutaneous Squamous Cell Carcinoma

Purpose: Aggressive cutaneous squamous cell carcinoma (cSCC) is often a disfiguring and lethal disease. Very little is currently known about the mutations that drive aggressive cSCC. Experimental Design: Whole-exome sequencing was performed on 39 cases of aggressive cSCC to identify driver genes and novel therapeutic targets. Significantly, mutated genes were identified with MutSig or complementary methods developed to specifically identify candidate tumor suppressors based upon their inactivating mutation bias. Results: Despite the very high-mutational background caused by UV exposure, 23 candidate drivers were identified, including the well-known cancer-associated genes TP53, CDKN2A, NOTCH1, AJUBA, HRAS, CASP8, FAT1, and KMT2C (MLL3). Three novel candidate tumor suppressors with putative links to cancer or differentiation, NOTCH2, PARD3, and RASA1, were also identified as possible drivers in cSCC. KMT2C mutations were associated with poor outcome and increased bone invasion. Conclusions: The mutational spectrum of cSCC is similar to that of head and neck squamous cell carcinoma and dominated by tumor-suppressor genes. These results improve the foundation for understanding this disease and should aid in identifying and treating aggressive cSCC. Clin Cancer Res; 20(24); 6582–92. ©2014 AACR.

Detection of somatic mutations and HPV in the saliva and plasma of patients with head and neck squamous cell carcinomas

Tumor DNA in saliva and plasma can provide a noninvasive biomarker for head and neck squamous cell carcinoma. A cancer test that’s worth a spit Head and neck squamous cell carcinoma is one of the most common cancers worldwide, and its incidence is increasing. This is a difficult-to-treat cancer for which few targeted agents are available, and there are no biomarkers for monitoring therapeutic progress. Wang et al. discovered that tumor DNA can be detected and analyzed in the blood of most patients with head and neck cancers, as well as in the saliva of those with cancers of the oral cavity. Moreover, they found preliminary evidence suggesting that tumor DNA may be detectable in saliva before clinical evidence of tumor recurrence, which may be useful for patient monitoring if this result is confirmed in larger studies. To explore the potential of tumor-specific DNA as a biomarker for head and neck squamous cell carcinomas (HNSCC), we queried DNA from saliva or plasma of 93 HNSCC patients. We searched for somatic mutations or human papillomavirus genes, collectively referred to as tumor DNA. When both plasma and saliva were tested, tumor DNA was detected in 96% of 47 patients. The fractions of patients with detectable tumor DNA in early- and late-stage disease were 100% (n = 10) and 95% (n = 37), respectively. When segregated by site, tumor DNA was detected in 100% (n = 15), 91% (n = 22), 100% (n = 7), and 100% (n = 3) of patients with tumors of the oral cavity, oropharynx, larynx, and hypopharynx, respectively. In saliva, tumor DNA was found in 100% of patients with oral cavity cancers and in 47 to 70% of patients with cancers of the other sites. In plasma, tumor DNA was found in 80% of patients with oral cavity cancers, and in 86 to 100% of patients with cancers of the other sites. Thus, saliva is preferentially enriched for tumor DNA from the oral cavity, whereas plasma is preferentially enriched for tumor DNA from the other sites. Tumor DNA in saliva was found postsurgically in three patients before clinical diagnosis of recurrence, but in none of the five patients without recurrence. Tumor DNA in the saliva and plasma appears to be a potentially valuable biomarker for detection of HNSCC.

Is p53 haploinsufficient for tumor suppression? Implications for the p53+/- mouse model in carcinogenicity testing.

The p53 tumor suppressor gene has been shown to be critical in preventing cancer in humans and mice. We have generated and extensively characterized p53-deficient mice lacking one (p53+/-) or both (p53-/-) p53 alleles. The p53-defi cient mice are much more susceptible to an array of different tumor types than their wild-type (p53+/+) littermates. The enhanced tumor susceptibility of the p53+/- mice has made them one of several transgenic mouse models that are being considered as substitutes for standard 2-year rodent carcinogenicity assays. In order to fully exploit this model, it will be important to understand some of the basic biological and molecular mechanisms that underlie its enhanced tumor susceptibility. With this in mind, we have explored the fate of the remaining wild-type p53 allele in spontaneously arising p53+/-tumors and have shown that over half of these tumors retain an intact, functional wild-type p53 allele. This suggests that p53 is haploinsufficient for tumor suppression and that mere reduction in p53 dosage is suffi cient to promote cancer formation. To support the idea that p53 is indeed a haploinsufficient tumor suppressor, we show here that normal p53+/-cells exhibit reduced parameters of growth control and stress response compared to their p53+/+ counterparts. We hypothesize that the reduced p53 dosage in the p53+/- cells provides an environment more conducive to the development of further oncogenic lesions and the initiation of a tumor. Finally, we have assessed p53 loss of heterozygosity (LOH) in carcinogen-induced p53+/- tumors and have found that some agents induce tumors that almost invariably exhibitp53 LOH, whereas other agents induce tumors that often retain the wild-type p53 allele. Our preliminary data suggest that LOH is dependent on both the mechanism of genotoxicity of the agent utilized and the tissue type targeted.

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

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.

Glucose, not Glutamine is the Dominant Energy Source Required for Proliferation and Survival of Head and Neck Squamous Carcinoma Cells

Tumor metabolism is an essential contributor to disease progression and response to treatment. An understanding of the metabolic phenotype of head and neck squamous cell carcinoma (HNSCC) will allow the development of appropriate antimetabolic strategies for this tumor type.

p38 Regulates Cyclooxygenase-2 in Human Mammary Epithelial Cells and Is Activated in Premalignant Tissue

The immediate-early gene, cyclooxygenase-2 (COX-2), is induced in a variety of inflammatory and neoplastic processes and is believed to play an important role in tumorigenesis. In this study, we identify an important upstream regulatory pathway of COX-2 expression in variant human mammary epithelial cells (vHMEC), which has been shown to exhibit phenotypes important for malignancy. We find that the stress-activated kinase, p38, is phosphorylated and activated in vHMEC compared with HMEC and is responsible for the expression of COX-2 in vHMEC as cells grow in culture. Furthermore in this capacity, p38 acts to stabilize the COX-2 transcript rather than activate COX-2 transcription. Inhibition of p38 kinase, using a chemical inhibitor, down-regulates COX-2 and decreases cell survival. Examination of archived tissue from women with ductal carcinoma in situ reveals epithelial cells that not only overexpress COX-2 but also have an abundance of activated phospho-p38 in the nucleus and cytoplasm, mirroring the expression observed in vitro. These epithelial cells are found within premalignant lesions as well as in fields of morphologically normal tissue that surround the lesions. In contrast, low phospho-p38 staining was observed in the majority of normal tissue obtained from reduction mammoplasty. These data help define the regulation of COX-2 expression in early carcinogenesis and provide alternative candidates for targeted prevention of COX-2-induced phenotypes and breast cancer.

Chk1/2 Inhibition Overcomes the Cisplatin Resistance of Head and Neck Cancer Cells Secondary to the Loss of Functional p53

Despite the use of multimodality therapy using cisplatin to treat patients with advanced stage squamous cell carcinoma of the head and neck (HNSCC), there is an unacceptably high rate of treatment failure. TP53 is the most commonly mutated gene in HNSCC, and the impact of p53 mutation on response to cisplatin treatment is poorly understood. Here, we show unambiguously that wild-type TP53 (wtp53) is associated with sensitivity of HNSCC cells to cisplatin treatment, whereas mutation or loss of TP53 is associated with cisplatin resistance. We also show that senescence is the major cellular response to cisplatin in wtp53 HNSCC cells and that cisplatin resistance in p53-null or -mutant TP53 cells is due to their lack of senescence. Given the dependence on checkpoint kinase (Chk)1/2 kinases to mediate the DNA damage response in p53-deficient cells, there is potential to exploit this to therapeutic advantage through targeted inhibition of the Chk1/2 kinases. Treatment of p53-deficient HNSCC cells with the Chk inhibitor AZD7762 sensitizes them to cisplatin through induction of mitotic cell death. This is the first report showing the ability of a Chk kinase inhibitor to sensitize TP53-deficient HNSCC to cisplatin in a synthetic lethal manner, which has significance given the frequency of TP53 mutations in this disease and because cisplatin has become part of standard therapy for aggressive HNSCC tumors. These preclinical data provide evidence that a personalized approach to the treatment of HNSCC based on Chk inhibition in p53-mutant tumors may be feasible. Mol Cancer Ther; 12(9); 1860–73. ©2013 AACR.