Search

Fig. S2. (A) Metaphase spread of the cell line AG09391 hybridized with the NFIB(SG)/MTFR2(SR) FISH fusion probe set. The MTFR2 probe recognized homology with the chromosome 6q terminal band, compatible with mapping at 6q23.3; the NFIB probe recognized homology with the chromosome 9p terminal band, compatible with mapping at 9p22.3. Normal CUSG004 patient tissue hybridized with the NFIB(SG)/MTFR2(SR) FISH fusion probe set, showing negative pattern. CUSG004 patient tumor tissue (F0) and PDX tumor tissue (F1, F2, F5) hybridized with the NFIB(SG)/MTFR2(SR) FISH fusion probe set, with patient tumor and PDX tissue showing positive pattern. (B) CUSG005 patient (F0), CUSG005 PDX (F1), and CUSG006 PDX (F2) tumor tissue hybridized with the NFIB(SG)/MTFR2(SR) FISH fusion probe set, with CUSG005 tissue showing positive pattern and CUSG006 tissue a negative pattern. Our FISH probes also detected the MYB-NFIB fusion in the CUSG005 tumor and was likely due to the close proximity (

Excel file containing deferentially expressed genes (RNA-seq) for comparisons of CUSG006, -007, and -012 tumors.

Supplementary Methods, References, Figure Legends, and Tables.

Purpose: Salivary gland cancers (SGC) frequently present with distant metastases many years after diagnosis, suggesting a cancer stem cell (CSC) subpopulation that initiates late recurrences; however, current models are limited both in their availability and suitability to characterize these rare cells.Experimental Design: Patient-derived xenografts (PDX) were generated by engrafting patient tissue onto nude mice from one acinic cell carcinoma (AciCC), four adenoid cystic carcinoma (ACC), and three mucoepidermoid carcinoma (MEC) cases, which were derived from successive relapses from the same MEC patient. Patient and PDX samples were analyzed by RNA-seq and Exome-seq. Sphere formation potential and in vivo tumorigenicity was assessed by sorting for Aldefluor (ALDH) activity and CD44-expressing subpopulations.Results: For successive MEC relapses we found a time-dependent increase in CSCs (ALDH+CD44high), increasing from 0.2% to 4.5% (P=0.033), but more importantly we observed an increase in individual CSC sphere formation and tumorigenic potential. A 50% increase in mutational burden was documented in subsequent MEC tumors, and this was associated with increased expression of tumor-promoting genes (MT1E, LGR5, and LEF1), decreased expression of tumor-suppressor genes (CDKN2B, SIK1, and TP53), and higher expression of CSC-related proteins such as SOX2, MYC, and ALDH1A1. Finally, genomic analyses identified a novel NFIB–MTFR2 fusion in an ACC tumor and confirmed previously reported fusions (NTRK3–ETV6 and MYB–NFIB).Conclusions: Sequential MEC PDX models preserved key patient features and enabled the identification of genetic events putatively contributing to increases in both CSC proportion and intrinsic tumorigenicity, which mirrored the patient's clinical course. Clin Cancer Res; 24(12); 2935–43. ©2018 AACR.

PDF file - 1255K, Confirmation of GLI1 modulation by EGFR activation (S1); Epithelial gene expression profile and morphology predicts erlotinib sensitivity in HNSCC cell lines (S2); Zeb1 transcription factor is key driver of EGF induced EMT (S3); EGFR activation induces EMT in HNSCC cell lines characterized by expression of Vimentin (S4); Inter-pathway crosstalk as well as EGF induced EMT are blunted in erlotinib resistant cells (S5); Silencing of HhP transcription factor GLI1 increases EGFR driven EMT in Tu-167 cells (S6); MEK/ERK signaling is required for EMT-like state while both MEK/ERK and PI3K/AKT are required for invasion through Matrigel (S7); Proliferative inhibition of erltotinib (1μM and 2μM) plus IPI-926 (1μM) or SHH (250ng/ml) (S8); Cetuximab and IPI-926 modulate EGFR, HhP and EMT gene expression in vivo (S9).

PDF file - 113K

PDF file - 29K, EGFR dependent EMT quantified as percent Vimentin-positive cells in culture (S1); Combination therapy delays or blocks tumor re-growth in sensitive xenografts (S2).

Recurrence and metastasis are the major causes of mortality in head and neck squamous cell carcinoma (HNSCC). It is suggested that cancer stem cells (CSCs) play pivotal roles in recurrence and metastasis. Thus, a greater understanding of the mechanisms of CSC regulation may provide opportunities to develop novel therapies for improving survival by controlling recurrence or metastasis. Here, we report that overexpression of the gene encoding the catalytic subunit of PI3K (PIK3CA), the most frequently amplified oncogene in HNSCC, promotes epithelial‐to‐mesenchymal transition and enriches the CSC population. However, PIK3CA is not required to maintain these traits and inhibition of the phosphatidylinositol 3‐kinase (PI3K) signaling pathway paradoxically promotes CSC population. Molecular analysis revealed that overexpression of PIK3CA activates multiple receptor tyrosine kinases (RTKs), in which ephrin receptors (Ephs), tropomyosin receptor kinases (TRK) and mast/stem cell growth factor receptor (c‐Kit) contribute to maintain CSC population. Accordingly, simultaneous inhibition of these RTKs using a multi‐kinase inhibitor ponatinib has a superior effect at eliminating the CSC population and reduces metastasis of PIK3CA‐overexpressing HNSCC cells. Our result suggests that co‐targeting of Ephs, TRKs and the c‐Kit pathway may be effective at eliminating the PI3K‐independent CSC population, thereby providing potential targets for future development of a novel anti‐CSC therapeutic approach for HNSCC patients, particularly for patients with PIK3CA amplification., We report that overexpression of the gene encoding the catalytic subunit of PI3K (PIK3CA) promotes epithelial‐to‐mesenchymal transition and enriches the cancer stem cell (CSC) population. However, PIK3CA is not required to maintain these traits. Molecular analysis revealed that PIK3CA overexpression activates multiple receptor tyrosine kinases (RTK), in which tropomyosin receptor kinases, ephrin receptors, and mast/stem cell growth factor receptor (c‐Kit) contribute to maintain CSCs. Accordingly, co‐inhibition of these RTKs using a multi‐kinase inhibitor ponatinib eliminates CSCs and reduces metastasis of PIK3CA‐overexpressing head and neck squamous cell carcinoma cells.

Evasion of the host immune responses is critical for both persistent human papillomavirus (HPV) infection and associated cancer progression. We have previously shown that expression of the homeostatic chemokine CXCL14 is significantly downregulated by the HPV oncoprotein E7 during cancer progression. Restoration of CXCL14 expression in HPV-positive head and neck cancer (HNC) cells dramatically suppresses tumor growth and increases survival through an immune-dependent mechanism in mice. Although CXCL14 recruits natural killer (NK) and T cells to the tumor microenvironment, the mechanism by which CXCL14 mediates tumor suppression through NK and/or T cells remained undefined. Here we report that CD8+ T cells are required for CXCL14-mediated tumor suppression. Using a CD8+ T-cell receptor transgenic model, we show that the CXCL14-mediated antitumor CD8+ T-cell responses require antigen specificity. Interestingly, CXCL14 expression restores major histocompatibility complex class I (MHC-I) expression on HPV-positive HNC cells downregulated by HPV, and knockdown of MHC-I expression in HNC cells results in loss of tumor suppression even with CXCL14 expression. These results suggest that CXCL14 enacts antitumor immunity through restoration of MHC-I expression on tumor cells and promoting antigen-specific CD8+ T-cell responses to suppress HPV-positive HNC.

Hyoid bone elongation is an uncommon cause of stroke. Here, we report a case of hyoid bone elongation causing localized trauma to the internal carotid artery, resulting in multiple strokes. A 32-year-old woman presented with unilateral weakness and history of a recent stroke. Imaging revealed the greater horn of the hyoid bone extending between the external and internal carotid with associated thrombus at the carotid bifurcation and acute stroke. Carotid ultrasound demonstrated movement of the hyoid bone in and out of the space between the external carotid artery and internal carotid artery with neck rotation. Treatment involved anticoagulation and partial hyoid bone resection. After resection, the stroke symptoms had not recurred in the patient. Hyoid bone–related carotid injury is an infrequent etiology of stroke, with no established treatment guidelines. Partial hyoid bone resection and antithrombotic therapy are likely a reasonably safe and effective treatment.

Head and neck squamous cell carcinoma (HNSCC) is a biologically aggressive cancer in which high rates of recurrence (local, locoregional, or distant) contribute significantly to poor patient survival. One of the primary goals for current clinical practice of HNSCC surveillance is to detect recurrence at an early stage. However, current methods are either subjective, invasive, hard to access, not able to detect recurrence in a timely manner, or expensive. Saliva, given its directly adjacence to the anatomic location of HNSCC, may represent an opportunity to monitor HNSCC progression in a completely noninvasive, easy-to-access, and cost-effective method for HNSCC surveillance in a timely manner. We have developed a panel of seven biomarkers through screening of methylated microRNA loci (i.e., 9-1, 124-1, 124-2, 124-3, 129-2, 137, and 148) for HNSCC, and hereafter referred to as HNKlear. HNKlear demonstrated 92.1% sensitivity and 97.8% specificity in 189 HNSCC and 92 control tissue samples and 84.9% sensitivity and 95.4% in 86 pretreatment HNSCC and 108 control saliva samples. HNKlear is able to detect 93.5% T1 tumor and 91.2% stage I tumor in tissue, and 85.0% or 80.0% of HNSCC saliva when tumor is at T1 or stage I, respectively. In our prospective study, the value of HNKlear in patients’ saliva correlates with the tumor burden in 42 patients’ saliva before and after surgery within 3 months. HNKlear value is associated with progression-free survival in 57 primary tumor tissues and 38 treatment-naïve saliva. More importantly, HNKlear in saliva was shown to detect recurrence ranging from 90-337 days ahead of clinical detection of recurrence in the longitudinal follow-up of 43 HNSCC patients. Although these findings need to be validated in a large clinical trial, they suggest that analysis of certain methylated mciroRNA in saliva could have clinical significance for surveillance of HNSCC patients. Citation Format: Ling Lv, Steve Quattlebaum, Dexiang Gao, Yu Cao, Ben Milam, Francis Hall, Derek Smith, Elyse Handley, Sophia Bornstein, Neil Gross, John Song, Shi-Long Lu. Potential clinical value of methylated microRNAs in saliva-liquid biopsy for surveillance of head and neck squamous cell carcinoma [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B07.

Background To identify aberrant promoter methylation of genomic loci encoding microRNA (mgmiR) in head and neck squamous cell carcinoma (HNSCC) and to evaluate a biomarker panel of mgmiRs to improve the diagnostic accuracy of HNSCC in tissues and saliva. Methods Methylation of promoter regions of mgmiR candidates was initially screened using HNSCC and control cell lines and further selected using HNSCC and control tissues by quantitative methylation-specific PCR (qMS-PCR). We then examined a panel of seven mgmiRs for validation in an expanded cohort including 189 HNSCC and 92 non-HNSCC controls. Saliva from 86 pre-treatment HNSCC patients and 108 non-HNSCC controls was also examined using this panel of seven mgmiRs to assess the potentials of clinical utilization. Results Among the 315 screened mgmiRs, 12 mgmiRs were significantly increased in HNSCC cell lines compared to control cell lines. Seven out of the 12 mgmiRs, i.e., mgmiR9-1, mgmiR124-1, mgmiR124-2, mgmiR124-3, mgmiR129-2, mgmiR137, and mgmiR148a, were further found to significantly increase in HNSCC tumor tissues compared to control tissues. Using multivariable logistic regression with dichotomized variables, a combination of the seven mgmiRs had sensitivity and specificity of 92.6 and 92.4% in tissues and 76.7 and 86.1% in saliva, respectively. Area under the receiver operating curve for this panel was 0.97 in tissue and 0.93 in saliva. This model was validated by independent bootstrap validation and random forest analysis. Conclusions mgmiR biomarkers represent a novel and promising screening tool, and the seven-mgmiR panel is able to robustly detect HNSCC in both patient tissue and saliva. Electronic supplementary material The online version of this article (10.1186/s13148-018-0470-7) contains supplementary material, which is available to authorized users.

Purpose: Salivary gland cancers (SGC) frequently present with distant metastases many years after diagnosis, suggesting a cancer stem cell (CSC) subpopulation that initiates late recurrences; however, current models are limited both in their availability and suitability to characterize these rare cells. Experimental Design: Patient-derived xenografts (PDX) were generated by engrafting patient tissue onto nude mice from one acinic cell carcinoma (AciCC), four adenoid cystic carcinoma (ACC), and three mucoepidermoid carcinoma (MEC) cases, which were derived from successive relapses from the same MEC patient. Patient and PDX samples were analyzed by RNA-seq and Exome-seq. Sphere formation potential and in vivo tumorigenicity was assessed by sorting for Aldefluor (ALDH) activity and CD44-expressing subpopulations. Results: For successive MEC relapses we found a time-dependent increase in CSCs (ALDH+CD44high), increasing from 0.2% to 4.5% (P=0.033), but more importantly we observed an increase in individual CSC sphere formation and tumorigenic potential. A 50% increase in mutational burden was documented in subsequent MEC tumors, and this was associated with increased expression of tumor-promoting genes (MT1E, LGR5, and LEF1), decreased expression of tumor-suppressor genes (CDKN2B, SIK1, and TP53), and higher expression of CSC-related proteins such as SOX2, MYC, and ALDH1A1. Finally, genomic analyses identified a novel NFIB–MTFR2 fusion in an ACC tumor and confirmed previously reported fusions (NTRK3–ETV6 and MYB–NFIB). Conclusions: Sequential MEC PDX models preserved key patient features and enabled the identification of genetic events putatively contributing to increases in both CSC proportion and intrinsic tumorigenicity, which mirrored the patient's clinical course. Clin Cancer Res; 24(12); 2935–43. ©2018 AACR.

Salivary gland tumor (SGT) is a rare tumor type, which exhibits broad-spectrum phenotypic, biological, and clinical heterogeneity. Currently, the molecular mechanisms that cause SGT pathogenesis remain poorly understood. A lack of animal models that faithfully recapitulate the naturally occurring process of human SGTs has hampered research progress on this field. In this report, we developed an inducible keratin 5-driven conditional knockout mouse model to delete gene(s) of interest in murine salivary gland upon local RU486 delivery. We have deleted two major tumor suppressors, Pten, a negative regulator of the PI3K pathway, and Smad4, the central signaling mediator of TGFβ pathway, in the murine salivary gland. Our results have shown that deletion of either Pten or Smad4 in murine salivary gland resulted in pleomorphic adenomas, the most common tumor in human SGT patients. Deletion of both Pten and Smad4 in murine salivary gland developed several malignancies, with salivary adenoid cystic carcinoma (SACC) being the most frequently seen. Molecular characterization showed that SACC exhibited mTOR activation and TGFβ1 overexpression. Examination of human SGT clinical samples revealed that loss of Pten and Smad4 is common in human SACC samples, particularly in the most aggressive solid form, and is correlated with survival of SACC patients, highlighting the human relevance of the murine models. In summary, our results offer significant insight into synergistic role of Pten and Smad4 in SGT, providing a rationale for targeting mTOR and/or TGFβ signaling to control SGT formation and progression.

The limitations of cancer cell lines have led to the development of direct patient derived xenograft (PDX) models. However, the interplay between the implanted human cancer cells and recruited mouse stromal and immune cells alters the tumor microenvironment and limits the value of these models. To overcome these constraints, we have developed a technique to expand human hematopoietic stem and progenitor cells (HSPCs) and use them to reconstitute the radiation-depleted bone marrow of a NOD/SCID/IL2rg−/− (NSG) mouse on which a patient’s tumor is then transplanted (XactMice). The human HSPCs produce immune cells that home into the tumor and help replicate its natural microenvironment. Despite previous passage on nude mice, the expression of epithelial, stromal, and immune genes in XactMice tumors aligns more closely to that of the patient tumor than to those grown in non-humanized mice – an effect partially facilitated by human cytokines expressed by both the HSPC progeny and the tumor cells. The human immune and stromal cells produced in the XactMice can help recapitulate the microenvironment of an implanted xenograft, reverse the initial genetic drift seen after passage on non-humanized mice, and provide a more accurate tumor model to guide patient treatment.

This is a rare presentation of a morbidly obese male with a massive retropharyngeal goiter causing laryngeal compressive symptoms and unique airway management challenges. Flexible laryngoscopy revealed a retropharyngeal mass circumferentially compressing the oropharynx and supraglottis and preventing the visualization of the glottis. Awake tracheostomy was performed before total thyroidectomy. Airway compression from a goiter typically results from substernal extension, which usually does not cause difficulty with endotracheal intubation. Extensive retropharyngeal extension can cause supraglottic obstruction of the airway. In these cases, optimal management consists of a closely coordinated approach between anesthesia and surgery teams to establish a surgical airway.

BACKGROUND. EGFR and Src family kinases are upregulated in head and neck squamous cell carcinoma (HNSCC). EGFR interacts with Src to activate STAT3 signaling, and dual EGFR-Src targeting is synergistic in HNSCC preclinical models. pSrc overexpression predicted resistance to the EGFR inhibitor, erlotinib, in a prior window trial. We conducted a 4-arm window trial to identify biomarkers associated with response to EGFR and/or Src inhibition. METHODS. Patients with operable stage II-IVa HNSCC were randomized to 7-21 days of neoadjuvant erlotinib, the Src inhibitor dasatinib, the combination of both, or placebo. Paired tumor specimens were collected before and after treatment. Pharmacodynamic expression of EGFR and Src pathway components was evaluated by IHC of tissue microarrays and reverse-phase protein array of tissue lysates. Candidate biomarkers were assessed for correlation with change in tumor size. RESULTS. From April 2009 to December 2012, 58 patients were randomized and 55 were treated. There was a significant decrease in tumor size in both erlotinib arms (P = 0.0014); however, no effect was seen with dasatinib alone (P = 0.24). High baseline pMAPK expression was associated with response to erlotinib (P = 0.03). High baseline pSTAT3 was associated with resistance to dasatinib (P = 0.099). CONCLUSIONS. Brief exposure to erlotinib significantly decreased tumor size in operable HNSCC, with no additive effect from dasatinib. Baseline pMAPK expression warrants further study as a response biomarker for anti-EGFR therapy. Basal expression of pSTAT3 may be independent of Src, explain therapeutic resistance, and preclude development of dasatinib in biomarker-unselected cohorts. TRIAL REGISTRATION. NCT00779389. FUNDING. National Cancer Institute, American Cancer Society, Pennsylvania Department of Health, V Foundation for Cancer Research, Bristol-Myers Squibb, and Astellas Pharma.

Head and neck squamous cell carcinoma (HNSCC) affects 650,000 people worldwide and has a dismal 50% 5-year survival rate. Recurrence and metastasis are believed the two most important factors causing this high mortality. Understanding the biological process and the underlying mechanisms of recurrence and metastasis is critical to develop novel and effective treatment, which is expected to improve patients’ survival of HNSCC. MicroRNAs are small, non-coding nucleotides that regulate gene expression at the transcriptional and post-transcriptional level. Oncogenic and tumor-suppressive microRNAs have shown to regulate nearly every step of recurrence and metastasis, ranging from migration and invasion, epithelial-mesenchymal transition (EMT), anoikis, to gain of cancer stem cell property. This review encompasses an overview of microRNAs involved in these processes. The recent advances of utilizing microRNA as biomarkers and targets for treatment, particularly on controlling recurrence and metastasis are also reviewed.

Squamous cell carcinomas (SCCs) originate in stratified epithelia, with a small subset becoming metastatic. Epithelial stem cells are targets for driver mutations that give rise to SCCs, but it is unknown whether they contribute to oncogenic multipotency and metastasis. We developed a mouse model of SCC by targeting two frequent genetic mutations in human SCCs, oncogene Kras(G12D) activation and Smad4 deletion, to mouse keratin 15-expressing (K15+) stem cells. We show that transgenic mice developed multilineage tumors, including metastatic SCCs. Among cancer stem cell-enriched (CSC-enriched) populations, those with increased side population (SP) cells correlated with epithelial-mesenchymal transition (EMT) and lung metastasis. We show that microRNA-9 (miR-9) contributed to SP expansion and metastasis, and miR-9 inhibition reduced the number of SP cells and metastasis. Increased miR-9 was detected in metastatic human primary SCCs and SCC metastases, and miR-9-transduced human SCC cells exhibited increased invasion. We identified α-catenin as a predominant miR-9 target. Increased miR-9 in human SCC metastases correlated with α-catenin loss but not E-cadherin loss. Our results demonstrate that stem cells with Kras(G12D) activation and Smad4 depletion can produce tumors that are multipotent and susceptible to EMT and metastasis. Additionally, tumor initiation and metastatic properties of CSCs can be uncoupled, with miR-9 regulating the expansion of metastatic CSCs.

The EGF receptor (EGFR)-directed monoclonal antibody cetuximab is the only targeted therapy approved for the treatment of squamous cell carcinoma of the head and neck (HNSCC) but is only effective in a minority of patients. Epithelial-to-mesenchymal transition (EMT) has been implicated as a drug resistance mechanism in multiple cancers, and the EGFR and Hedgehog pathways (HhP) are relevant to this process, but the interplay between the two pathways has not been defined in HNSCC. Here, we show that HNSCC cells that were naturally sensitive to EGFR inhibition over time developed increased expression of the HhP transcription factor GLI1 as they became resistant after long-term EGFR inhibitor exposure. This robustly correlated with an increase in vimentin expression. Conversely, the HhP negatively regulated an EGFR-dependent, EMT-like state in HNSCC cells, and pharmacologic or genetic inhibition of HhP signaling pushed cells further into an EGFR-dependent phenotype, increasing expression of ZEB1 and VIM. In vivo treatment with cetuximab resulted in tumor shrinkage in four of six HNSCC patient-derived xenografts; however, they eventually regrew. Cetuximab in combination with the HhP inhibitor IPI-926 eliminated tumors in two cases and significantly delayed regrowth in the other two cases. Expression of EMT genes TWIST and ZEB2 was increased in sensitive xenografts, suggesting a possible resistant mesenchymal population. In summary, we report that EGFR-dependent HNSCC cells can undergo both EGFR-dependent and -independent EMT and HhP signaling is a regulator in both processes. Cetuximab plus IPI-926 forces tumor cells into an EGFR-dependent state, delaying or completely blocking tumor recurrence. Cancer Res; 73(11); 3381–92. ©2013 AACR.

Targeted therapy development in head and neck squamous cell carcinoma (HNSCC) is challenging given the rarity of activating mutations. Additionally, HNSCC incidence is increasing related to human papillomavirus (HPV). We sought to develop an in vivo model derived from patients reflecting the evolving HNSCC epidemiologic landscape, and use it to identify new therapies. Primary and relapsed tumors from HNSCC patients, both HPV+ and HPV-, were implanted on mice, giving rise to 25 strains. Resulting xenografts were characterized by detecting key mutations, measuring protein expression by IHC and gene expression/pathway analysis by mRNA-sequencing. Drug efficacy studies were run with representative xenografts using the approved drug cetuximab as well as the new PI3K inhibitor PX-866. Tumors maintained their original morphology, genetic profiles and drug susceptibilities through serial passaging. The genetic makeup of these tumors was consistent with known frequencies of TP53, PI3KCA, NOTCH1 and NOTCH2 mutations. Because the EGFR inhibitor cetuximab is a standard HNSCC therapy, we tested its efficacy and observed a wide spectrum of efficacy. Cetuximab-resistant strains had higher PI3K/Akt pathway gene expression and protein activation than cetuximab-sensitive strains. The PI3K inhibitor PX-866 had anti-tumor efficacy in HNSCC models with PIK3CA alterations. Finally, PI3K inhibition was effective in two cases with NOTCH1 inactivating mutations. In summary, we have developed an HNSCC model covering its clinical spectrum whose major genetic alterations and susceptibility to anticancer agents represent contemporary HNSCC. This model enables to prospectively test therapeutic-oriented hypotheses leading to personalized medicine.

High-risk human papillomaviruses (HPVs) are causally associated with multiple human cancers. Previous studies have shown that the HPV oncoprotein E7 induces immune suppression; however, the underlying mechanisms remain unknown. To understand the mechanisms by which HPV deregulates host immune responses in the tumor microenvironment, we analyzed gene expression changes of all known chemokines and their receptors using our global gene expression data sets from human HPV-positive and -negative head/neck cancer and cervical tissue specimens in different disease stages. We report that, while many proinflammatory chemokines increase expression throughout cancer progression, CXCL14 is dramatically downregulated in HPV-positive cancers. HPV suppression of CXCL14 is dependent on E7 and associated with DNA hypermethylation in the CXCL14 promoter. Using in vivo mouse models, we revealed that restoration of Cxcl14 expression in HPV-positive mouse oropharyngeal carcinoma cells clears tumors in immunocompetent syngeneic mice, but not in Rag1-deficient mice. Further, Cxcl14 reexpression significantly increases natural killer (NK), CD4+ T, and CD8+ T cell infiltration into the tumor-draining lymph nodes in vivo. In vitro transwell migration assays show that Cxcl14 reexpression induces chemotaxis of NK, CD4+ T, and CD8+ T cells. These results suggest that CXCL14 downregulation by HPV plays an important role in suppression of antitumor immune responses. Our findings provide a new mechanistic understanding of virus-induced immune evasion that contributes to cancer progression., IMPORTANCE Human papillomaviruses (HPVs) are causally associated with more than 5% of all human cancers. During decades of cancer progression, HPV persists, evading host surveillance. However, little is known about the immune evasion mechanisms driven by HPV. Here we report that the chemokine CXCL14 is significantly downregulated in HPV-positive head/neck and cervical cancers. Using patient tissue specimens and cultured keratinocytes, we found that CXCL14 downregulation is linked to CXCL14 promoter hypermethylation induced by the HPV oncoprotein E7. Restoration of Cxcl14 expression in HPV-positive cancer cells clears tumors in immunocompetent syngeneic mice, but not in immunodeficient mice. Mice with Cxcl14 reexpression show dramatically increased natural killer and T cells in the tumor-draining lymph nodes. These results suggest that epigenetic downregulation of CXCL14 by HPV plays an important role in suppressing antitumor immune responses. Our findings may offer novel insights to develop preventive and therapeutic tools for restoring antitumor immune responses in HPV-infected individuals.

The purpose of this study was to investigate the incidence of symptomatic and asymptomatic deep venous thrombosis in patients undergoing harvest of a free flap from the lower extremity who were receiving standard chemoprophylaxis while hospitalized.A retrospective review of 65 consecutive patients undergoing surgery between 2011 and 2013 was performed to determine the incidence of symptomatic deep venous thrombosis. These patients were screened for deep venous thrombosis based on development of symptoms. Prospective evaluation of a similar consecutive population of 37 patients between 2014 and 2015 was then performed to determine the incidence of asymptomatic deep venous thrombosis. These patients underwent routine duplex ultrasonography of both legs at postoperative weeks 1 and 4.Symptomatic deep venous thrombosis occurred in 2.9 percent of all patients. In the prospective cohort, 8.1 percent of the patients were found to have an acute deep venous thrombosis by postoperative week 1. At postoperative week 4, 16.7 percent of the patients developed a new, acute deep venous thrombosis. The estimated costs of screening and treating deep venous thrombosis in the retrospective group and the prospective group were $222 and $2259, respectively. The cost of routine chemoprophylaxis without duplex screening for an additional 14 days after discharge was $125 per patient.The rate of asymptomatic deep venous thrombosis may be much higher than previously appreciated in this population of very high-risk patients, especially during the 2 weeks after discharge. Extending the duration of chemoprophylaxis to 4 weeks after surgery may be warranted.Therapeutic, IV.

Detection of DNA methylation has produced promising results as biomarkers for head and neck squamous cell carcinoma (HNSCC). However, current panels are limited by an insufficient number of sensitive and specific tumor markers. MicroRNAs (miR) play an important role in tumorigenesis, and may represent a novel panel of molecules for the development of cancer biomarkers. We investigated methylation of three miRNA promoter sites of miR-9 (miR-9-1, miR-9-2, miR-9-3) in 107 human head and neck tissue samples and controls. We found methylations of miR-9-1 and miR-9-3 were higher in oral and oropharyngeal carcinomas than that in laryngeal carcinoma, achieving a combined sensitivity of 63% and 56%, respectively, for these two tumor types, compared to 21% for the laryngeal carcinoma. Quantitative PCR of miR-9 showed reduced expression associated with methylation of miR-9 in tumor tissues. To investigate the functional consequences of miR-9 methylation, we found that miR-9 methylation is correlated with miR-9 expression level in human HNSCC cell lines. Demethylation treatment using 5-aza-deoxycytidine restored its expression in a miR-9 methylated human HNSCC cell line UM-SCC22A. Furthermore, cell proliferation and viability was significantly inhibited, while PTEN expression was elevated after transfection of miR-9 into the UM-SCC22A cell line. In summary, our results suggest that methylations of miR-9-1 and miR-9-3 are sensitive and specific biomarkers for HNSCC, particularly for oral and oropharyngeal squamous cell carcinomas. In addition, miR-9 may function as a tumor suppressor in HNSCC through inhibition of cell proliferation and elevation of tumor suppressor PTEN.

Objectives: To compare the toxicity and outcomes of three radiotherapy techniques—three-dimensional conformal (3D-RT), accelerated fractionation with concomitant boost (AFxCB), and intensity modulated radiotherapy (IMRT)—in the combined modality treatment of stage III–IV squamous cell carcinoma (SCC) of the oropharynx. Study Design: Retrospective review. Methods: Between 1998 and 2007, a total of 87 patients were treated; 23 were treated with 3D-RT, 32 with AFxCB, and 32 with IMRT. Systemic therapy consisted of platinum-based chemotherapy in 81 and anti-epidermal growth factor receptor (anti-EGFR)-targeted therapy in 6 cases. Median radiotherapy doses were 70Gy with 3D-RT, 72Gy with AFxCB, and 69.3Gy with IMRT. Locoregional control, survival outcomes, and feeding tube (PEG) dependence were compared using log-rank method. The incidence of acute mucositis and skin reaction, and grade ≥2 xerostomia at 6, 12, and 18 months after radiotherapy was compared using Fisher's exact test. Results: Median follow-up was 24 months (range 3 to 103 months) for living patients. Two-year overall survival (OS), disease-free survival (DFS), and locoregional control (LRC) were 77.3%, 69.5%, and 86.4%, respectively. There was a trend toward improvement in LRC in patients treated with IMRT. Acute grade ≥3 skin and mucosal toxicity were significantly lower with IMRT compared to AFxCB (P < .001). Grade ≥2 xerostomia was significantly reduced with IMRT compared to AFxCB and 3D-RT (P < .001). There was no difference in the actuarial rate of PEG dependence (P = .96). Conclusions: Compared to AFxCB and 3D-RT, IMRT confers an improvement in toxicity and appears to have similar efficacy in patients with SCC of the oropharynx.

Purpose To establish the safety and toxicity profile of daily gefitinib with radiation alone or with concurrent chemoradiotherapy in previously untreated patients with locally advanced squamous cell head and neck cancer (LAHNC). Patients and Methods Patients with intermediate-stage LAHNC were treated with concomitant boost radiation (RT) alone with escalating doses of daily gefitinib (250 or 500 mg; cohort I). Once a safety profile was determined with RT alone, patients with high-risk disease were then treated with daily gefitinib (250 or 500 mg), weekly cisplatin (CDDP; 30 mg/m2), and once-daily RT (cohort II). Patients also received post-RT gefitinib at 250 mg daily for a period of up to 2 years. Results Twenty-three patients were enrolled and assessable for toxicity. No dose-limiting toxicities (DLTs) were observed in patients treated in cohort I at either 250 or 500 mg of gefitinib daily with concomitant boost RT to 72 Gy. In patients receiving chemoradiotherapy and gefitinib (cohort II), DLTs included one grade 4 diarrhea and one grade 4 neutropenic fever. Fifteen patients started maintenance gefitinib, and eight (53%) experienced grade 1 to 2 acne-like skin rash and diarrhea, but no grade 3 or 4 toxicity occurred. Conclusion Gefitinib (250 or 500 mg daily) was well tolerated with concomitant boost RT or concurrent chemoradiotherapy with weekly CDDP. Protracted administration of gefitinib for up to 2 years at 250 mg daily was also tolerated well.

Immediate reconstruction of composite head and neck defects using free tissue transfer is an accepted treatment standard. There remains, however, ongoing debate on whether the costs associated with this reconstructive approach merit its selection, especially considering poor patient prognoses and the high cost of care.A retrospective review of the last 100 consecutive patients undergoing microsurgical reconstruction for head and neck cancer by the two senior surgeons was performed to determine whether microsurgical complications or postoperative medical complications had the more profound influence on morbidity and mortality outcomes and the true costs of these reconstructions.Two patients required re-exploration of the microsurgical anastomoses, for a re-exploration rate of 2 percent, and one flap failed, for a flap success rate of 99 percent. The major surgical complication rate requiring a second operative procedure was 6 percent. Sixteen percent had minor surgical complications related to the donor site. Major medical complications, defined as a significant risk to the patient's life, occurred in 5 percent of the patients, but there was a 37 percent incidence of "minor" medical complications primarily caused by pulmonary problems and alcohol withdrawal. Postsurgical complications almost doubled the average hospital stay from 13.5 days for those patients without complications to 24 days for patients with complications. Thirty-six percent of the true cost of microsurgical reconstruction of head and neck cancer was due to the intensive care unit and hospital room costs, and 24 percent was due to operating room costs. Postsurgical complications resulted in a 70.7 percent increase in true costs, reflecting a prolonged stay in the intensive care unit and not an increase in operating room costs or regular hospital room costs.Postoperative medical complications in these elderly, debilitated patients related to pulmonary problems and alcohol withdrawal were statistically far more important in negatively affecting the outcomes and true costs of microsurgical reconstruction.

The anterolateral thigh (ALT) free flap has become the most widely used fasciocutaneous free flap for head and neck reconstruction.1 However, there has been no report demonstrating the indications or options for increasing arterial inflow and venous outflow to an ALT flap based on separate vascular pedicles. The overall size of the ALT flap is dependent on the number and location of perforators. Large series of ALT free flaps report flaps with average dimensions of approximately 8 × 23 cm.2 What remains uncertain is the maximum dimensions of an ALT flap that can be harvested based on a single perforator. Moreover, given that the perforators of the ALT flap sequentially arise from the descending branch of the circumflex artery as it descends down the thigh, it is likely that a long ALT flap can be more reliably harvested than a wide ALT flap. Multiple skin islands can also be created from an ALT flap either with a strip of de-epithelialization between the 2 skin islands or with complete division of the skin paddle if 2 separate perforators exist, each supplying a separate island of skin. The purpose of this article is to introduce the options and indications for supercharging and venous augmenting an ALT flap when confronted with an extensive head and neck defect that may not reliably be reconstructed with a routine ALT flap based on a single perforator.

The upper aerodigestive tract is predisposed to the formation of multiple primary tumors due to field cancerization. TGF-alpha/EGFR autocrine signaling appears to play an important role in squamous cell carcinoma of the head and neck (SCCHN) and upregulation of TGF-alpha and EGFR is an early event in SCCHN carcinogenesis. STAT proteins, including Stat3, are activated by TGF-alpha and EGFR and strategies that downmodulate TGF-alpha or EGFR inhibit SCCHN cell proliferation and abrogate Stat3 activation. Targeting Stat3 leads to SCCHN growth inhibition, increases apoptosis and a downmodulation of Bcl-xL expression in head and neck tumors. These studies support the role of Stat3 as an oncogene, which is activated early in SCCHN carcinogenesis, and efforts to understand EGFR-mediated Stat3 signaling could facilitate novel strategies that will interfere with this growth promoting pathway. Oncogene (2000).

Objectives:Early detection of head and neck squamous cell carcinoma (HNSCC) improves long-term survival. More than half of patients with HNSCC present with locoregional or metastatic disease at the time of diagnosis. MicroRNAs are promising markers for early detection, as they are stable, tissue-specific, and known to be involved in processes critical for tumor development and progression. Methylated miRNA sequences have already been studied in early detection of lung and gastrointestinal (GI) cancers.Methods:Genomic DNA was extracted from 30 SCC samples, 24 samples of grossly normal adjacent mucosa from those tumor patients, and 8 samples from normal controls (no cancer). The gDNA was treated with bisulfate to identify methylation patterns, and quantitative polymerase chain reaction was run for 6 miRNA markers (9-1, 9-3, 124-1, 124-2, 124-3, 137) to determine the level of expression in each sample.Results:Overall expression of each marker was significantly higher in the tumor group when compared to the c...

Purpose To present the first report of a Phase I trial evaluating concurrent and maintenance erlotinib and reirradiation in patients with recurrent or secondary primary head-and-neck cancer (HNC). Methods and Materials Patients with recurrent or new primary HNC with an interval of at least 6 months since prior radiation were eligible. Patients were treated in 3 sequential cohorts: Cohort I, 100 mg of erlotinib daily with reirradiation at 61.6 Gy in 28 fractions; Cohort II, 150 mg of erlotinib with 61.6 Gy in 28 fractions; and Cohort III, 150 mg of erlotinib with 66 Gy in 30 fractions. Maintenance erlotinib started immediately after reirradiation at 150 mg daily and was continued for 2 years or until disease progression or dose-limiting toxicity. Dose-limiting toxicities were defined as any Grade 4 or 5 toxicity or a toxicity-related delay in radiation therapy of greater than 7 days. Results Fourteen patients were accrued, 3 to Cohort I, 4 to Cohort II, and 7 to Cohort III. Thirteen patients were evaluable for toxicity. Median follow-up was 8.4 months overall and 15.1 months for surviving patients. One patient had a dose-limiting toxicity in Cohort III. This patient declined initial percutaneous endoscopic gastrostomy tube placement, was hospitalized with Grade 3 dysphagia and aspiration, and required a delay in radiation therapy of greater than 7 days. No Grade 4 acute toxicity was observed. Acute Grade 3 toxicity occurred in 9 of 13 patients. No erlotinib-related toxicity of Grade 3 or greater was observed during maintenance therapy. One patient had Grade 5 carotid hemorrhage 6 months after reirradiation, and another patient had Grade 3 osteoradionecrosis. Conclusions Reirradiation (66 Gy in 2.2 Gy fractions) with concurrent and maintenance erlotinib (150 mg daily) for recurrent or new primary HNC is feasible.

Purpose To evaluate the survival and patterns of relapse for patients with squamous cell carcinoma (SCC) of the oral tongue. Patients and Methods Between 1999 and 2007, 50 patients with SCC of the oral tongue were treated at the University of Colorado Denver. Of the 50 patients, 38 had newly diagnosed SCC of the oral tongue (13 with stage I-II and 25 with stage III-IV disease), and 12 presented with locally recurrent SCC. Of the 50 patients, 49 were treated with initial surgery and 1 with definitive chemoradiotherapy. Adjuvant radiotherapy or chemoradiotherapy was administered to 42 patients after surgery. Of the 13 patients with newly diagnosed stage I-II disease, 7 did not receive adjuvant therapy. The actuarial locoregional control, freedom from distant relapse, and survival were determined using the Kaplan-Meier method, and comparisons were made using the log-rank test. Results The median follow-up was 29 months (range 4 to 95) for living patients. The 2-year locoregional control and freedom from distant relapse rate was 58% and 83%, respectively. Locoregional control was particularly low among patients with stage I-II disease, for whom the 2-year locoregional control rate was only 35%. The median survival time and 2-year survival rate for all patients was 42 months and 65%, respectively. The 2-year survival rate for patients with stage I-II oral tongue cancer was 77% compared with 52% for patients with stage III-IV disease ( P = .04). Conclusions Despite aggressive therapy, patients with SCC of the oral tongue have a low rate of local tumor control and survival, particularly among those with stage I-II disease. These patients should be considered for inclusion in clinical trials evaluating novel postoperative therapies.

Objective: To evaluate the outcomes of patients with locally advanced head and neck squamous cell carcinoma with N3 neck nodes treated with definitive chemoradiation. Study Design: Retrospective review. Methods: Thirty-two patients with nonmetastatic locally advanced head and neck squamous cell carcinoma and N3 neck disease treated with concurrent chemoradiation therapy were evaluated. Overall survival, disease- free survival, locoregional control, and distant control were recorded. Results: Median follow-up for surviving patients was 25 (range, 3–93) months. Seventeen of 32 (53%) patients failed, 13 in distant sites only, 2 in the neck only, 1 in the neck and a distant site, and 1 in the neck and primary site. The absolute rates of locoregional control and distant control were 88% and 56%, respectively. Actuarial overall survival and disease-free survival at 2 years were 51% and 29%, respectively. Conclusion: Patients with N3 neck disease treated with chemoradiation experience a very high rate of distant failure. Future studies investigating the role of additional systemic therapy in these patients are warranted.

To determine efficacy and toxicities of postoperative concurrent chemoradiation using docetaxel in high-risk head and neck cancer.High-risk patients were enrolled 2-8 weeks after surgery. Treatment included 60 Gy for 6 weeks with weekly docetaxel 25 mg/m(2) and erythropoietin alpha 40,000 U for hemoglobinor =12 g/dL. Primary endpoints included locoregional control (LC), disease-free survival (DFS), and patterns of failure (POF). Secondary endpoints were toxicity and quality of life.Eighteen patients were enrolled (14 male, 4 female), aged 24-70 years (median, 55 years). Primary site included oropharynx = 7, oral cavity = 8, hypopharynx = 1, and larynx = 2. Pathologic American Joint Committee on Cancer Stage was III = 3 patients, IV = 15 patients. High-risk eligibility includedor =2 positive lymph nodes = 13, extracapsular extension = 10, positive margins = 8 (11 patients with two or more risk factors). Docetaxel was reduced to 20 mg/m(2)/week after 5 patients had prolonged Grade 3 or higher mucositis. Overall, number of doses delivered was 2 of 6 = 1, 3 of 6 = 2, 4 of 6 = 2, 5 of 6 = 4, 6 of 6 = 9 patients. With median follow-up of 30 months (range, 5-66), 10 (56%) patients are alive and have no evidence of disease (NED); POF: three local recurrences (two with distant) and 1 distant only. One-year survival was 76%, median PFS and DFS had not been reached. Three-year LC was 82%. No Grade 3 or higher late toxicities were observed, although a few cases of prolonged mucositis and taste loss (3 months) were seen, particularly at 25 mg/m(2)/week.Postoperative radiation therapy with weekly docetaxel 20 or 25 mg/m(2)/week for high-risk postoperative head and neck cancer caused intolerable mucosal toxicity, prompting early study termination. Further studies should consider 15 mg/m(2). Actuarial 3-year LC is 82%, similar to cisplatin-based chemoradiation regimens. Distant metastasis remains an important issue requiring additional systemic interventions.

The use of retinoids to prevent and/or treat cancers, including head and neck squamous cell carcinoma (HNSCC), has been limited by the development of resistance and unwanted side effects. Receptor-selective retinoids are potentially less toxic than nonselective compounds. The present investigation was undertaken to determine the mechanism of responsiveness to an retinoic acid receptor-selective retinoid (LGD1550) that has shown antitumor efficacy in a xenograft model of HNSCC.A series of HNSCC cell lines were characterized with respect to proliferation and apoptosis after LGD1550 treatment. Relative responsiveness to LGD1550 was examined with respect to modulation of epidermal growth factor receptor (EGFR) signaling pathways.Cells were either growth inhibited and underwent apoptosis or were resistant to treatment with this compound. Retinoids have been shown to decrease the gene transcription rates of transforming growth factor (TGF)-alpha and EGFR in HNSCC. LGD1550 responsiveness was accompanied by decreased expression of TGF-alpha, EGFR, and modulation of EGFR signaling pathways, including signal transducers and activators of transcriptions and mitogen-activated protein kinase. In contrast, EGFR autocrine signaling pathways were not altered in HNSCC cells that were resistant to the growth inhibitory effects of LGD1550.These results suggest that there is a correlation between the efficacy of receptor-selective retinoids and modulation of TGF-alpha/EGFR signaling in HNSCC. Therefore, alterations of these signaling pathways may serve as a biomarker of clinical response.

6033 Background: The epidermal growth factor receptor (EGFR) and Src family kinases are upregulated in HNSCC. EGFR interacts with cSrc to activate oncogenic STAT3 signaling; dual targeting is syner...

Purpose: Data suggest that human papilloma virus (HPV)-positive head and neck squamous cell carcinomas (HNSCC) are distinct from HPV negative tumors. We have generated a direct patient tumor xenograft model (DPTM) of HNSCC. DPTM maintain tumor histology better than cell lines and are optimally positioned to investigate in vivo tumor sensitivity and changes in tumor initiating cell (TIC) biology in response to therapy based upon HPV status. Phosphatidyl inositol 3 kinase (PI3K) is a downstream signal of epidermal growth factor receptor (EGFR) that may play a critical role in TIC biology and resistance to chemotherapy or anti-EGFR monoclonal antibodies in HNSCC. PX-866 is an irreversible pan-isoform inhibitor of class 1 PI3K currently in early clinical trials. We aimed to test the in vivo efficacy of PX-866, cetuximab, docetaxel, or a combination of PX-866 and cetuximab or docetaxel. Methods: DPTM were left untreated or exposed to PX-866, docexatel, cetuximab, or a combination of PX-866 plus cetuximab or docetaxel for 25-29 days. Tumor measurements were taken twice weekly during therapy and thereafter until study conclusion. TIC population were determined by flow cytometry. Results: We have treated three DPTM (CUHN015, CUHN022, CUHN027) with PX-866, cetuximab, or PX-866 plus cetuximab, and three DPTM (CUHN004, CUHN011, CUHN015) with PX-866, docetaxel, or the combination of PX-866 plus docetaxel. Neither combination had increased toxicity compared with the single agents. All six untreated DPTM had rapid tumor growth. In two DPTM (CUHN015 and CUHN022) cetuximab was similar to no treatment, whereas single agent PX-866 was slightly more effective than no treatment in CUHN022 and significantly more effective in CUHN015. In CUHN027 both PX-866 and cetuximab arrested growth but the combination caused greater than 50% tumor reduction. The combination of cetuximab and PX-866 was more effective than cetuximab alone in all DPTM. Two xenografts treated with PX-866, docetaxel, or the combination showed decreased tumor growth with PX-866 compared to either docetaxel or no treatment (CUHN004 and CUHN015). In all three DTPM the combination of PX-866 and docetaxel was more effective than either single agent. In the HPV-positive xenograft (CUHN022) cetuximab had no effect. Treatment with chemotherapeutic agents induced TIC accumulation that was partially reversed by combination therapy. Conclusions: PX-866 was equal or superior to cetuximab or docetaxel at slowing tumor growth in 5 of 6 DPTM of HNSCC. The combination of PX-866 plus another agent was superior to single agent therapy in 5 of 6 DPTM. Cetuximab was more effective in HPV-negative cases. Correlations with pathway gene mutations and pharmacodynamic events, including pathway inhibition and TIC subpopulation dynamics, will be presented. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 652. doi:10.1158/1538-7445.AM2011-652

5552 Background: Postoperative radiation with concurrent cisplatin has shown improved efficacy over radiation alone in randomized trials for patients with HRPHNC: ≥2 involved nodes (N), extra-capsular nodal disease (EC), +margins (+M), N3 disease (N3). This phase II trial in HRPHNC evaluates the efficacy and toxicities of postop CR using weekly docetaxel, and with weekly Epo in patients with suboptimal Hgb (≤12 gm/dl). Methods: HRPHNC patients were enrolled 2–8 weeks after surgery with PS 0–1, no distant metastases, and no prior regional radiation or chemotherapy. Treatment consisted of conformal radiation (60Gy in 6 weeks) with weekly Doc 25mg/m2 and Epo 40,000U. Primary endpoints include local/regional control, DFS, and patterns of failure. Secondary endpoints include toxicity and QOL. Results: 15 HRPHNC (11 male, 4 female) ages 22–69 years (median 51) were enrolled. Characteristics include: Oropharynx=6, Oral Cavity=6, Hypopharynx=1, Larynx=1; AJCC stage III=2 pts, IV=13 pts. HRPHNC eligibility include...