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This diagnostic study describes the development of an assay for human papillomavirus–driven cancers of the oropharynx and the role viral integration could play in the process.

Background Head and neck squamous cell cancer (HNSCC) occurs at higher rates among persons with HIV (PWH). This study compares the impact of sociodemographic and clinicopathologic characteristics on outcomes among PWH-HNSCC compared with HNSCC patients without HIV. Methods Patient data from HNSCC individuals were collected at a single academic hospital center between 2002 and 2018. Forty-eight patients with HIV (HIV-HNSCC) and 2894 HNSCC patients without HIV were included. Multivariate analysis determined predictors of survival using Cox proportional hazards regression model. HIV-positive and -negative tumors were analyzed by quantitative immunofluorescence for expression of CD4, CD8, CD20 and PD-L1. Results HIV-HNSCC patients had a lower median overall survival than HNSCC patients without HIV (34 [18–84] vs 94 [86–103] months; P < .001). In multivariate analysis that included age, sex, race/ethnicity, stage, site, tobacco use, time to treatment initiation, and insurance status, HIV was an independent predictor of poorer survival, with a hazard ratio of 1.98 (95% CI: 1.32–2.97; P < .001). PWH with human papillomavirus (HPV)–positive oropharyngeal tumors also had worse prognosis than HPV-positive oropharyngeal tumors in the population without HIV (P < .001). The tumor microenvironment among HIV-HNSCC patients revealed lower intratumoral CD8 infiltration among HIV+ HPV+ tumors compared with HIV– HPV+ tumors (P = .04). Conclusions HIV-HNSCC patients had worse prognosis than the non-HIV population, with HIV being an independent predictor of poor clinical outcomes when accounting for important sociodemographic and clinicopathologic factors. Our findings highlight differences in tumor biology that require further detailed characterization in large cohorts and increased inclusion of PWH in immunotherapy trials.

Descriptive statistics for HNSC patients from the CHANCE study by p16 cytoplasmic/nuclear status.

Additional multiplex staining coexpression and ROI quantification.

Supplementary Figure from Comprehensive Viral Genotyping Reveals Prognostic Viral Phylogenetic Groups in HPV16-Associated Squamous Cell Carcinoma of the Oropharynx

Supplementary Table from Comprehensive Viral Genotyping Reveals Prognostic Viral Phylogenetic Groups in HPV16-Associated Squamous Cell Carcinoma of the Oropharynx

Human papillomavirus–positive (HPV+) squamous cell carcinoma of the oropharynx (OPSCC) is the most prevalent HPV-associated malignancy in the United States and is primarily caused by HPV subtype 16 (HPV16). Favorable treatment outcomes have led to increasing interest in treatment deescalation to reduce treatment-related morbidity. Prognostic biomarkers are needed to identify appropriately low-risk patients for reduced treatment intensity. Targeted DNA sequencing including all HPV16 open reading frames was performed on tumors from 104 patients with HPV16+ OPSCC treated at a single center. Genotypes closely related to the HPV16-A1 reference were associated with increased numbers of somatic copy-number variants in the human genome and poor recurrence-free survival (RFS). Genotypes divergent from HPV16-A1 were associated with favorable RFS. These findings were independent of tobacco smoke exposure. Total RNA sequencing was performed on a second independent cohort of 89 HPV16+ OPSCC cases. HPV16 genotypes divergent from HPV16-A1 were again validated in this independent cohort, to be prognostic of improved RFS in patients with moderate (less than 30 pack-years) or low (no more than 10 pack-years) of tobacco smoke exposure. In summary, we show in two independent cohorts that viral sequence divergence from the HPV16-A1 reference is correlated with improved RFS in patients with moderate or low tobacco smoke exposure.Implications:HPV16 genotype is a potential biomarker that could be easily adopted to guide therapeutic decision-making related to deescalation therapy.

Supplementary Figure S4 related to Figure 5D-F. (A) Individual tumor volume changes in Detroit 562-xenografted mice treated with vehicle, adavosertib (30 mg/kg, daily, p.o.), alisertib (30 mg/kg, daily, p.o.), or the combinations of alisertib with adavosertib. Tumor volume changes of all mice belonged to this study was shown in bottom panel (n = 6 per group). Significant differences between the combination and single drug alone are shown. (B) Body weight (g) of mice was monitored every other day. Graphs depict Mean {plus minus} SEM.

PDF file - 200KB, Immunoblotting of HPV(+) SCC090 cells stably transfected with either wild type or E545K mutant PIK3CA vectors with pS6, pS6K and pAKT antibodies. Actin antibody was used as a loading control.

qRT-PCR primers used in the study

Demographic and expression data

DAPT selectively increases cell death in CD133+ Accx11 cells as shown by PE-annexin and 7-AAD double staining. DMSO treatment serves as a control.

A. % line-1 methylation after 5-aza treatment in HPV+ (1-5) and HPV- tumors from the window clinical trial. B. % Line-1 methylation in HPV+ UMSCC47 cells before and after 5-aza treatment

PDF file - 37KB, Differentially expressed proteins in HPV(+) vs HPV(-) OPSCC (0.05

Human papilloma viruses (HPV) are linked to an epidemic increase in oropharyngeal head and neck squamous cell carcinomas (HNSCC), which display viral inactivation of tumor suppressors TP53 and RB1 and rapid regional spread. However, the role of genomic alterations in enabling the modulation of pathways that promote the aggressive phenotype of these cancers is unclear. Recently, a subset of HPV+ HNSCC has been shown to harbor novel genetic defects or decreased expression of TNF receptor–associated factor 3 (TRAF3). TRAF3 has been implicated as a negative regulator of alternative NF-κB pathway activation and activator of antiviral type I IFN response to other DNA viruses. How TRAF3 alterations affect pathogenesis of HPV+ HNSCC has not been extensively investigated. Here, we report that TRAF3-deficient HPV+ tumors and cell lines exhibit increased expression of alternative NF-κB pathway components and transcription factors NF-κB2/RELB. Overexpression of TRAF3 in HPV+ cell lines with decreased endogenous TRAF3 inhibited NF-κB2/RELB expression, nuclear localization, and NF-κB reporter activity, while increasing the expression of IFNA1 mRNA and protein and sensitizing cells to its growth inhibition. Overexpression of TRAF3 also enhanced TP53 and RB tumor suppressor proteins and decreased HPV E6 oncoprotein in HPV+ cells. Correspondingly, TRAF3 inhibited cell growth, colony formation, migration, and resistance to TNFα and cisplatin-induced cell death. Conversely, TRAF3 knockout enhanced colony formation and proliferation of an HPV+ HNSCC line expressing higher TRAF3 levels. Together, these findings support a functional role of TRAF3 as a tumor suppressor modulating established cancer hallmarks in HPV+ HNSCC.Significance: These findings report the functional role of TRAF3 as a tumor suppressor that modulates the malignant phenotype of HPV+ head and neck cancers. Cancer Res; 78(16); 4613–26. ©2018 AACR.

Figure S4. Body weight of MK-1775 treated mice

Supplementary figure legends

Supplementary Figure 1 from Gene Expression Differences Associated with Human Papillomavirus Status in Head and Neck Squamous Cell Carcinoma

Tables S18-20: Table S18. Secondary validation assay statistical analysis (absolute viability) Table S19. Differential viability and caspase 3/7 dependent apoptosis AUC of secondary validation assay Table S20. Statistical analysis summary differential viability and caspase 3/7 dependent apoptosis

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Validation of ACC cell cultures via microsatellite analysis.

Expression of SOX10, NOTCH1, FABP7, and CD133 in ACC tumor specimens from patients (P) and PDX (X). The comparison is based on previously produced Affymetrix data (6).

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Purpose:Human papillomavirus (HPV)-negative head and neck squamous cell carcinomas (HNSCC) commonly bear disruptive mutations in TP53, resulting in treatment resistance. In these patients, direct targeting of p53 has not been successful, but synthetic lethal approaches have promise. Although Aurora A kinase (AURKA) is overexpressed and an oncogenic driver, its inhibition has only modest clinical effects in HPV-negative HNSCC. We explored a novel combination of AURKA and WEE1 inhibition to overcome intrinsic resistance to AURKA inhibition.Experimental Design: AURKA protein expression was determined by fluorescence-based automated quantitative analysis of patient specimens and correlated with survival. We evaluated treatment with the AURKA inhibitor alisertib (MLN8237) and the WEE1 inhibitor adavosertib (AZD1775), alone or in combination, using in vitro and in vivo HNSCC models.Results:Elevated nuclear AURKA correlated with worse survival among patients with p16(−) HNSCC. Alisertib caused spindle defects, G2–M arrest and inhibitory CDK1 phosphorylation, and cytostasis in TP53 mutant HNSCC FaDu and UNC7 cells. Addition of adavosertib to alisertib instead triggered mitotic entry and mitotic catastrophe. Moreover, in FaDu and Detroit 562 xenografts, this combination demonstrated synergistic effects on tumor growth and extended overall survival compared with either vehicle or single-agent treatment.Conclusions:Combinatorial treatment with adavosertib and alisertib leads to synergistic antitumor effects in in vitro and in vivo HNSCC models. These findings suggest a novel rational combination, providing a promising therapeutic avenue for TP53-mutated cancers.

Human papillomavirus (HPV) is associated with a subset of head and neck squamous cell carcinoma (HNSCC). Between 15% and 35% of HNSCCs harbor HPV DNA. Demographic and exposure differences between HPV-positive (HPV+) and negative (HPV−) HNSCCs suggest that HPV+ tumors may constitute a subclass with different biology, whereas clinical differences have also been observed. Gene expression profiles of HPV+ and HPV− tumors were compared with further exploration of the biological effect of HPV in HNSCC. Thirty-six HNSCC tumors were analyzed using Affymetrix Human 133U Plus 2.0 GeneChip and for HPV by PCR and real-time PCR. Eight of 36 (22%) tumors were positive for HPV subtype 16. Statistical analysis using Significance Analysis of Microarrays based on HPV status as a supervising variable resulted in a list of 91 genes that were differentially expressed with statistical significance. Results for a subset of these genes were verified by real-time PCR. Genes highly expressed in HPV+ samples included cell cycle regulators (p16INK4A, p18, and CDC7) and transcription factors (TAF7L, RFC4, RPA2, and TFDP2). The microarray data were also investigated by mapping genes by chromosomal location (DIGMAP). A large number of genes on chromosome 3q24-qter had high levels of expression in HPV+ tumors. Further investigation of differentially expressed genes may reveal the unique pathways in HPV+ tumors that may explain the different natural history and biological properties of these tumors. These properties may be exploited as a target of novel therapeutic agents in HNSCC treatment.

PDF file - 157KB, UMSCC-47 parental and stably overexpressing mutant or wild-type PI3KCA cells were treated with increasing concentrations of AKT (MK-2206) or dual PI3KCA/mTOR (BEZ-235) inhibitor, and cells survival was assessed after 7 days of treatment with trypan blue exclusion. Concentrations inhibiting growth by 50% are presented.

Isolation and characterization of CD133 fractions from ACC xenografts. (A) Variation in the percentage of CD133+ cells across ACC xenografts. (B) qRT-PCR analysis of CD133 expression confirms adequate separation of CD133+ and CD133- cells isolated from ACC xenografts. (C-E) Selectivity of SOX10, NOTCH1, and FABP7 to CD133+ cells isolated directly from ACC xenografts as confirmed by qRT-PCR. (F, G) Selectivity of JAG1 and NR2F1 to CD133- cells isolated directly from ACC xenografts as confirmed by qRT-PCR.

Purpose: To identify novel therapeutic drug targets for p53-mutant head and neck squamous cell carcinoma (HNSCC).Experimental Design: RNAi kinome viability screens were performed on HNSCC cells, including autologous pairs from primary tumor and recurrent/metastatic lesions, and in parallel on murine squamous cell carcinoma (MSCC) cells derived from tumors of inbred mice bearing germline mutations in Trp53, and p53 regulatory genes: Atm, Prkdc, and p19Arf. Cross-species analysis of cell lines stratified by p53 mutational status and metastatic phenotype was used to select 38 kinase targets. Both primary and secondary RNAi validation assays were performed on additional HNSCC cell lines to credential these kinase targets using multiple phenotypic endpoints. Kinase targets were also examined via chemical inhibition using a panel of kinase inhibitors. A preclinical study was conducted on the WEE1 kinase inhibitor, MK-1775.Results: Our functional kinomics approach identified novel survival kinases in HNSCC involved in G2–M cell-cycle checkpoint, SFK, PI3K, and FAK pathways. RNAi-mediated knockdown and chemical inhibition of the WEE1 kinase with a specific inhibitor, MK-1775, had a significant effect on both viability and apoptosis. Sensitivity to the MK-1775 kinase inhibitor is in part determined by p53 mutational status, and due to unscheduled mitotic entry. MK-1775 displays single-agent activity and potentiates the efficacy of cisplatin in a p53-mutant HNSCC xenograft model.Conclusions: WEE1 kinase is a potential therapeutic drug target for HNSCC. This study supports the application of a functional kinomics strategy to identify novel therapeutic targets for cancer. Clin Cancer Res; 20(16); 4274–88. ©2014 AACR.

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Supplementary Methods, related references, and Figures: S1, 2 Effects TRAF3 on alternate pathway proteins; S2, Effects TRAF3 on alternate pathway proteins; S3, Effects of TNF/LTB on NF-kB reporter activity and TRAF3 siRNA on alternate pathway; S4, Effects TRAF3 on cisplatin and anti-apoptotic genes; S5, TRAF3 loss with passage; S6-8, TRAF3 effects on IRF expression; S9, TRAF3 effects on TP53, RB, p21; S10, TRAF3 effects on E6; S11, Diagram illustrating the genetic and cellular mechanisms linked to deficient TRAF3 in HPV+ HNSCC.

Tables S9-17: Table S9. HT RNAi primary validation screen with siRNA pool deconvolution Table S10. Differential analysis of primary validation screen (CTG) N=12 Table S11. Differential analysis of primary validation screen (CTG) N=3 Pooled Table S12. Statistical analysis summary of differential viability (CTG) Table S13. Statistical analysis (absolute viability) of primary validation screen (CTG) Table S14. HT RNAi primary validation screen summary of hits (CTG) Table S15. HT caspase 3/7-dependent apoptosis AUC analysis (Apotox) N=12 Table S16. HT caspase 3/7-dependent apoptosis AUC analysis (Apotox) N=3 pooled Table S17. Statistical analysis summary caspase 3/7 dependent apoptosis (Apotox)

Supplementary Table Legends

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Supplementary Figures legends

Co-occurence of TRAF3 and NF-kappaB pathway alterations

Genes and pathways suppressed by FABP7 knockdown in Accx11 cells.

Tables S1-8:Table S1. Characteristics of the HNSCC cell lines Table S2. Characteristics of the MSCC cell lines with germline mutations in p53 pathway genes Table S3. Human kinome (713 kinases) Sigma Table S4. Murine kinome (572 kinases) Ambion Table S5. Interspecies kinome (508 kinases) Table S6. Primary validation siRNAs Table S7. Secondary validation siRNAs Table S8. Interspecies kinase targets selected for validation

Purpose: Human papillomavirus (HPV) is linked with a subset of head and neck squamous cell carcinomas (HNSCC). HPV-positive HNSCCs show a better prognosis than HPV-negative HNSCCs, which may be explained by sensitivity of the HPV-positive HNSCCs to ionizing radiation (IR). Although the molecular mechanism behind sensitivity to IR in HPV-positive HNSCCs is unresolved, DNA damage response (DDR) might be a significant determinant of IR sensitivity. An important player in the DDR, SMG-1 (suppressor with morphogenetic effect on genitalia), is a potential tumor suppressor and may therefore be deregulated in cancer. No studies have yet been conducted linking defects in SMG-1 expression with cancer. We investigated whether deregulation of SMG-1 could be responsible for defects in the DDR in oropharyngeal HNSCC.Experimental Design: Expression and promoter methylation status of SMG-1 were investigated in HNSCCs. To identify a functional link between HPV infection and SMG-1, we transfected the HPV-negative cells with an E6/E7 expression construct. SMG-1 short hairpin RNAs were expressed in HPV-negative cells to estimate survival upon IR.Results: Forced E6/E7 expression in HPV-negative cells resulted in SMG-1 promoter hypermethylation and decreased SMG-1 expression. Due to promoter hypermethylation, HPV-positive HNSCC cells and tumors express SMG-1 at lower levels than HPV-negative SCCs. Depletion of SMG-1 in HPV-negative HNSCC cells resulted in increased radiation sensitivity, whereas SMG-1 overexpression protected HPV-positive tumor cells from irradiation.Conclusions: Levels of SMG-1 expression negatively correlated with HPV status in cancer cell lines and tumors. Diminished SMG-1 expression may contribute to the enhanced response to therapy exhibited by HPV-positive HNSCCs. Clin Cancer Res; 18(5); 1257–67. ©2012 AACR.

Purpose: Although the existence of cancer stem cells (CSC) in adenoid cystic carcinoma (ACC) has been proposed, lack of assays for their propagation and uncertainty about molecular markers prevented their characterization. Our objective was to isolate CSC from ACC and provide insight into signaling pathways that support their propagation.Experimental Design: To isolate CSC from ACC and characterize them, we used ROCK inhibitor-supplemented cell culture, immunomagnetic cell sorting, and in vitro/in vivo assays for CSC viability and tumorigenicity.Results: We identified in ACC CD133-positive CSC that expressed NOTCH1 and SOX10, formed spheroids, and initiated tumors in nude mice. CD133+ ACC cells produced activated NOTCH1 (N1ICD) and generated CD133− cells that expressed JAG1 as well as neural differentiation factors NR2F1, NR2F2, and p27Kip1. Knockdowns of NOTCH1, SOX10, and their common effector FABP7 had negative effects on each other, inhibited spheroidogenesis, and induced cell death pointing at their essential roles in CSC maintenance. Downstream effects of FABP7 knockdown included suppression of a broad spectrum of genes involved in proliferation, ribosome biogenesis, and metabolism. Among proliferation-linked NOTCH1/FABP7 targets, we identified SKP2 and its substrate p27Kip1. A γ-secretase inhibitor, DAPT, selectively depleted CD133+ cells, suppressed N1ICD and SKP2, induced p27Kip1, inhibited ACC growth in vivo, and sensitized CD133+ cells to radiation.Conclusions: These results establish in the majority of ACC the presence of a previously uncharacterized population of CD133+ cells with neural stem properties, which are driven by SOX10, NOTCH1, and FABP7. Sensitivity of these cells to Notch inhibition and their dependence on SKP2 offer new opportunities for targeted ACC therapies. Clin Cancer Res; 22(8); 2083–95. ©2016 AACR.

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Suppressive effects of FABP7 depletion on gene expression in Accx11 cells.

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