Oral Cavity Squamous Cell Carcinoma Xenografts Retain Complex Genotypes and Intertumor Molecular Heterogeneity.

Herein, we report an oral cavity squamous cell carcinoma (OCSCC) patient-derived xenograft (PDX) platform, with genomic annotation useful for co-clinical trial and mechanistic studies. Genomic analysis included whole-exome sequencing (WES) and transcriptome sequencing (RNA-seq) on 16 tumors and matched PDXs and additional whole-genome sequencing (WGS) on 9 of these pairs as a representative subset of a larger OCSCC PDX repository (n = 63). In 12 models with high purity, more than 90% of variants detected in the tumor were retained in the matched PDX. The genomic landscape across these PDXs reflected OCSCC molecular heterogeneity, including previously described basal, mesenchymal, and classical molecular subtypes. To demonstrate the integration of PDXs into a clinical trial framework, we show that pharmacological intervention in PDXs parallels clinical response and extends patient data. Together, these data describe a repository of OCSCC-specific PDXs and illustrate conservation of primary tumor genotypes, intratumoral heterogeneity, and co-clinical trial application.

Biomarker and Tumor Responses of Oral Cavity Squamous Cell Carcinoma to Trametinib: A Phase II Neoadjuvant Window-of-Opportunity Clinical Trial.

Purpose: Ras/MEK/ERK pathway activation is common in oral cavity squamous cell carcinoma (OCSCC). We performed a neoadjuvant (preoperative) trial to determine the biomarker and tumor response of OCSCC to MEK inhibition with trametinib.Experimental Design: Patients with stage II-IV OCSCC received trametinib (2 mg/day, minimum 7 days) prior to surgery. Primary tumor specimens were obtained before and after trametinib to evaluate immunohistochemical staining for p-ERK1/2 and CD44, the primary endpoint. Secondary endpoints included changes in clinical tumor measurements and metabolic activity [maximum standardized uptake values (SUVmax) by F-18 fluorodeoxyglucose positron emission tomography/CT), and in tumor downstaging. Drug-related adverse events (AE) and surgical/wound complications were evaluated.Results: Of 20 enrolled patients, 17 (85%) completed the study. Three patients withdrew because of either trametinib-related (n = 2: nausea, duodenal perforation) or unrelated (n = 1: constipation) AEs. The most common AE was rash (9/20 patients, 45%). Seventeen patients underwent surgery. No unexpected surgical/wound complications occurred. Evaluable matched pre- and posttrametinib specimens were available in 15 (88%) of these patients. Reduction in p-ERK1/2 and CD44 expression occurred in 5 (33%) and 2 (13%) patients, respectively. Clinical tumor response by modified World Health Organization criteria was observed in 11 of 17 (65%) evaluable patients (median 46% decrease, range 14%-74%). Partial metabolic response (≥25% reduction in SUVmax) was observed in 6 of 13 (46%) evaluable patients (median 25% decrease, range 6%-52%). Clinical-to-pathologic tumor downstaging occurred in 9 of 17 (53%) evaluable patients.Conclusions: Trametinib resulted in significant reduction in Ras/MEK/ERK pathway activation and in clinical and metabolic tumor responses in patients with OCSCC. Clin Cancer Res; 23(9); 2186-94. ©2016 AACR.

A surprising cross-species conservation in the genomic landscape of mouse and human oral cancer identifies a transcriptional signature predicting metastatic disease.

PURPOSE: Improved understanding of the molecular basis underlying oral squamous cell carcinoma (OSCC) aggressive growth has significant clinical implications. Herein, cross-species genomic comparison of carcinogen-induced murine and human OSCCs with indolent or metastatic growth yielded results with surprising translational relevance. EXPERIMENTAL DESIGN: Murine OSCC cell lines were subjected to next-generation sequencing (NGS) to define their mutational landscape, to define novel candidate cancer genes, and to assess for parallels with known drivers in human OSCC. Expression arrays identified a mouse metastasis signature, and we assessed its representation in four independent human datasets comprising 324 patients using weighted voting and gene set enrichment analysis. Kaplan-Meier analysis and multivariate Cox proportional hazards modeling were used to stratify outcomes. A quantitative real-time PCR assay based on the mouse signature coupled to a machine-learning algorithm was developed and used to stratify an independent set of 31 patients with respect to metastatic lymphadenopathy. RESULTS: NGS revealed conservation of human driver pathway mutations in mouse OSCC, including in Trp53, mitogen-activated protein kinase, phosphoinositide 3-kinase, NOTCH, JAK/STAT, and Fat1-4. Moreover, comparative analysis between The Cancer Genome Atlas and mouse samples defined AKAP9, MED12L, and MYH6 as novel putative cancer genes. Expression analysis identified a transcriptional signature predicting aggressiveness and clinical outcomes, which were validated in four independent human OSCC datasets. Finally, we harnessed the translational potential of this signature by creating a clinically feasible assay that stratified patients with OSCC with a 93.5% accuracy. CONCLUSIONS: These data demonstrate surprising cross-species genomic conservation that has translational relevance for human oral squamous cell cancer. Clin Cancer Res; 20(11); 2873-84. ©2014 AACR.

Emerging insights into head and neck cancer metastasis.

The purpose of this review was to provide biological concepts of head and neck cancer metastasis. To attain this goal, we analyzed peer-reviewed articles related to head and neck cancer metastasis obtained though PubMed and archived articles. Articles related to the biologic principles of head and neck cancer metastasis were reviewed and summarized. As locoregional control has improved for patients with head and neck cancer, rates of distant metastasis have not decreased. As patients live longer, many will die of complications related to the development of disease at sites below the clavicles. Emerging evidence now suggests a more complicated framework of metastatic behavior for head and neck cancer. Here, we review the role of regional lymph nodes in containing advanced head and neck cancer, evidence for active as opposed to passive tumor cell metastasis, and clinical implications these concepts have on both treatment of head and neck cancer and future research.

ERK1/2 regulation of CD44 modulates oral cancer aggressiveness.

Carcinogen-induced oral cavity squamous cell carcinoma (OSCC) incurs significant morbidity and mortality and constitutes a global health challenge. To gain further insight into this disease, we generated cell line models from 7,12-dimethylbenz(a)anthracene-induced murine primary OSCC capable of tumor formation upon transplantation into immunocompetent wild-type mice. Whereas several cell lines grew rapidly and were capable of metastasis, some grew slowly and did not metastasize. Aggressively growing cell lines displayed ERK1/2 activation, which stimulated expression of CD44, a marker associated with epithelial to mesenchymal transition and putative cancer stem cells. MEK (MAP/ERK kinase) inhibition upstream of ERK1/2 decreased CD44 expression and promoter activity and reduced cell migration and invasion. Conversely, MEK1 activation enhanced CD44 expression and promoter activity, whereas CD44 attenuation reduced in vitro migration and in vivo tumor formation. Extending these findings to freshly resected human OSCC, we confirmed a strict relationship between ERK1/2 phosphorylation and CD44 expression. In summary, our findings identify CD44 as a critical target of ERK1/2 in promoting tumor aggressiveness and offer a preclinical proof-of-concept to target this pathway as a strategy to treat head and neck cancer.

Human-in-mouse modeling of primary head and neck squamous cell carcinoma.

OBJECTIVES/HYPOTHESIS: To develop a reliable modeling system for head and neck squamous cell carcinoma (HNSCC). STUDY DESIGN: Laboratory-based translational study. METHODS: HNSCC tissue was obtained from patients at biopsy/resection, cultured, and implanted into mice. In vivo, tumor growth, and survival was monitored by bioluminescence imaging. Histology and immunohistochemistry (IHC) were used to confirm HNSCC and human origin. RESULTS: Short-term culture techniques were optimized allowing survival of primary HNSCC cells more than 7 days in 76% of tumors. The size of the tumor biopsy collected did not correlate with the success of short-term culture or xenograft establishment. Xenograft modeling was attempted in primary HNSCCs from 12 patients with a success rate of 92%. Immunostaining confirmed human origin of epithelial tumor cells within the modeled tumor. Bioluminescence and Ki67 IHC suggested tumor proliferation within the model. Luciferase expression was maintained for as long as 100 days in modeled tumors. CONCLUSIONS: The techniques developed for short-term primary tumor culture followed by xenograft modeling provide a low-cost and tractable model for evaluation of HNSCC response to standard and novel therapies. The high success rate of human-in-mouse tumor formation from primary HNSCC suggests that selection pressures for tumor growth in this model may be less than those observed for establishment of cell lines. Bioluminescent imaging provides a useful tool for evaluating tumor growth and could be expanded to measure response of the modeled tumor to therapy. This model could be adapted for xenograft modeled growth of other primary tumor types.

Human papillomavirus in early laryngeal carcinoma.

OBJECTIVES/HYPOTHESIS: To examine the role of HPV status in the etiology, prognosis, and treatment of head and neck squamous cell carcinoma in early larynx malignancies. STUDY DESIGN: Retrospective. METHODS: Thirty-eight cases of T1 or carcinoma in situ (CIS) laryngeal lesions were examined for the presence of human papilloma virus (HPV) using an inclusive polymerase chain reaction (PCR)/hybridization technique capable of identifying 37 HPV subtypes. RESULTS: HPV DNA was detected in 6 (16%) of the 38 lesions, representing HPV types 16, 26, 31, 39, and 52, and p16 tumor suppressor protein expression was confirmed in 10 representative cases. This HPV prevalence is higher than that noted in many previous laryngeal cancer studies, possibly due to the relatively large panel of subtypes screened for in this study. Identification of HPV-26, which has been associated with uterine cervical cancer, in an early laryngeal cancer specimen represents the first evidence of this subtype in a laryngeal carcinoma. Consistent with reports focusing on head and neck squamous cell carcinoma (HNSCC) arising from other subsites within the upper aerodigestive tract, patients with HPV-positive laryngeal carcinomas were of younger age and were somewhat less likely to have a history of tobacco use, although the latter of the two findings did not reach statistical significance. CONCLUSIONS: Our findings emphasize the presence of a broad spectrum of HPV types in a relevant proportion of early laryngeal cancers, and together with evidence of an association of HPV tumor status with a more favorable clinical course, provide a rationale for the routine HPV testing of small larynx lesions.