Michaela K. Keck

Integrative and Comparative Genomic Analysis of HPV-Positive and HPV-Negative Head and Neck Squamous Cell Carcinomas

Purpose: The genetic differences between human papilloma virus (HPV)–positive and –negative head and neck squamous cell carcinomas (HNSCC) remain largely unknown. To identify differential biology and novel therapeutic targets for both entities, we determined mutations and copy-number aberrations in a large cohort of locoregionally advanced HNSCC. Experimental Design: We performed massively parallel sequencing of 617 cancer-associated genes in 120 matched tumor/normal samples (42.5% HPV-positive). Mutations and copy-number aberrations were determined and results validated with a secondary method. Results: The overall mutational burden in HPV-negative and HPV-positive HNSCC was similar with an average of 15.2 versus 14.4 somatic exonic mutations in the targeted cancer-associated genes. HPV-negative tumors showed a mutational spectrum concordant with published lung squamous cell carcinoma analyses with enrichment for mutations in TP53, CDKN2A, MLL2, CUL3, NSD1, PIK3CA, and NOTCH genes. HPV-positive tumors showed unique mutations in DDX3X, FGFR2/3 and aberrations in PIK3CA, KRAS, MLL2/3, and NOTCH1 were enriched in HPV-positive tumors. Currently targetable genomic alterations were identified in FGFR1, DDR2, EGFR, FGFR2/3, EPHA2, and PIK3CA. EGFR, CCND1, and FGFR1 amplifications occurred in HPV-negative tumors, whereas 17.6% of HPV-positive tumors harbored mutations in fibroblast growth factor receptor genes (FGFR2/3), including six recurrent FGFR3 S249C mutations. HPV-positive tumors showed a 5.8% incidence of KRAS mutations, and DNA-repair gene aberrations, including 7.8% BRCA1/2 mutations, were identified. Conclusions: The mutational makeup of HPV-positive and HPV-negative HNSCC differs significantly, including targetable genes. HNSCC harbors multiple therapeutically important genetic aberrations, including frequent aberrations in the FGFR and PI3K pathway genes. Clin Cancer Res; 21(3); 632–41. ©2014 AACR. See related commentary by Krigsfeld and Chung, p. 495

Integrative Analysis of Head and Neck Cancer Identifies Two Biologically Distinct HPV and Three Non-HPV Subtypes

Purpose: Current classification of head and neck squamous cell carcinomas (HNSCC) based on anatomic site and stage fails to capture biologic heterogeneity or adequately inform treatment. Experimental Design: Here, we use gene expression-based consensus clustering, copy number profiling, and human papillomavirus (HPV) status on a clinically homogenous cohort of 134 locoregionally advanced HNSCCs with 44% HPV+ tumors together with additional cohorts, which in total comprise 938 tumors, to identify HNSCC subtypes and discover several subtype-specific, translationally relevant characteristics. Results: We identified five subtypes of HNSCC, including two biologically distinct HPV subtypes. One HPV+ and one HPV− subtype show a prominent immune and mesenchymal phenotype. Prominent tumor infiltration with CD8+ lymphocytes characterizes this inflamed/mesenchymal subtype, independent of HPV status. Compared with other subtypes, the two HPV subtypes show low expression and no copy number events for EGFR/HER ligands. In contrast, the basal subtype is uniquely characterized by a prominent EGFR/HER signaling phenotype, negative HPV-status, as well as strong hypoxic differentiation not seen in other subtypes. Conclusion: Our five-subtype classification provides a comprehensive overview of HPV+ as well as HPV− HNSCC biology with significant translational implications for biomarker development and personalized care for patients with HNSCC. Clin Cancer Res; 21(4); 870–81. ©2014 AACR.

HPV-related methylation signature predicts survival in oropharyngeal squamous cell carcinomas

High-risk types of human papilloma virus (HPV) are increasingly associated with oropharyngeal squamous cell carcinoma (OPSCC). Strikingly, patients with HPV-positive OPSCC are highly curable with ionizing radiation and have better survival compared with HPV-negative patients, but the underlying molecular mechanisms remain poorly understood. We applied an array-based approach to monitor global changes in CpG island hypermethylation between HPV-negative and HPV-positive OPSCCs and identified a specific pattern of differentially methylated regions that critically depends on the presence of viral transcripts. HPV-related alterations were confirmed for the majority of candidate gene promoters by mass spectrometric, quantitative methylation analysis. There was a significant inverse correlation between promoter hypermethylation of ALDH1A2, OSR2, GATA4, GRIA4, and IRX4 and transcript levels. Interestingly, Kaplan-Meier analysis revealed that a combined promoter methylation pattern of low methylation levels in ALDH1A2 and OSR2 promoters and high methylation levels in GATA4, GRIA4, and IRX4 promoters was significantly correlated with improved survival in 3 independent patient cohorts. ALDH1A2 protein levels, determined by immunohistochemistry on tissue microarrays, confirmed the association with clinical outcome. In summary, our study highlights specific alterations in global gene promoter methylation in HPV-driven OPSCCs and identifies a signature that predicts the clinical outcome in OPSCCs.

Rare occurrence of EGFRvIII deletion in head and neck squamous cell carcinoma

BACKGROUND The epidermal growth factor receptor (EGFR) is a transmembrane tyrosine kinase receptor and is overexpressed in up to 90% of head and neck squamous cell carcinoma (HNSCC) cases. The EGFR truncation mutation, EGFR variant III (EGFRvIII), harbors an in-frame deletion of exons 2-7 (801 bp) that leads to the constitutive activation of downstream signaling. EGFRvIII has been reported in ∼40% of glioblastomas (GBM), but its presence in HNSCC remains controversial. METHODS EGFRvIII deletion in 638 HNSCC samples was analyzed using: (i) quantitative Real-Time polymerase chain reaction (qRT-PCR) on 108 HNSCC samples with direct detection of the EGFRvIII breakpoint, (ii) RNA-Seq analysis on 7 HNSCC tumor tissues and 425 The Cancer Genome Atlas (TCGA) HNSCC samples, and (iii) immunohistochemistry (IHC) for EGFRvIII using an established antibody (L8A4) on a tissue microarray of 105 HNSCC samples. RESULTS qRT-PCR did not show the presence of EGFRvIII in any of the samples analyzed. Furthermore, we could not detect any EGFRvIII transcripts in the RNA-Seq data of the seven HNSCC samples. However, 2 samples out of 425 TCGA HNSCC samples had EGFRvIII specific reads. EGFRvIII IHC results were assessed as negative for all samples. CONCLUSION Our results firmly establish that EGFRvIII is very rare in HNSCC as only 2 out of 638 (0.31%) samples we analyzed overall, or 2 out of 540 (0.37%) using mRNA based approaches, were positive for EGFRvIII. EGFRvIII is extremely rare in HNSCC and the clinical significance remains unclear. We propose not to include EGFRvIII testing in regular diagnostic tests for HNSCC.

Oral cavity tumors in younger patients show a poor prognosis and do not contain viral RNA

BACKGROUND Oral cavity and in particular oral tongue cancers occur with a rising incidence in younger patients often lacking the typical risk factors of tobacco use, alcohol use, and human papilloma virus (HPV) infection. Their prognosis when treated with chemoradiation has not been well studied and responsible risk factors remain elusive. A viral etiology (other than HPV) has been hypothesized. METHODS First we analyzed outcomes from 748 head and neck cancer patients with locoregionally advanced stage tumors treated with curative-intent chemoradiation by anatomic site. Second, we analyzed seven oral tongue (OT) tumors from young, non-smokers/non-drinkers for the presence of viral mRNA using short-read massively-parallel sequencing (RNA-Seq) in combination with a newly-developed digital subtraction method followed by viral screening and discovery algorithms. For positive controls we used an HPV16-positive HNC cell line, a cervical cancer, and an EBV-LMP2A transgene lymphoma. RESULTS Younger patients with oral cavity tumors had worse outcomes compared to non-oral cavity patients. Surprisingly none of the seven oral tongue cancers showed significant presence of viral transcripts. In positive controls the expected viral material was identified. CONCLUSION Oral cavity tumors in younger patients have a poor prognosis and do not appear to be caused by a transcriptionally active oncovirus.

Abstract 2746: An evaluation of poly (ADP-ribose) polymerase inhibitor efficacy in head and neck cancer

Background: Synthetic lethality induced by poly (ADP-ribose) polymerase (PARP) inhibitors can occur in tumors without BRCA mutations. This “BRCAness” phenomenon has been observed in ovarian and triple negative breast cancers, but its role in other malignancies is not known. This study aims to evaluate the potency of PARP inhibitors in head and neck cancer, where BRCA mutations are rare. Methods: First, we compared three PARP inhibitors (veliparib, olaparib and rucaparib). We subsequently established dose response curves for rucaparib for ten head and neck cancer cell lines and compared with two BRCA deficient breast cancer cell lines. Furthermore, we used immunofluorescent staining for γH2AX and RAD51 to study the capability for the DNA repair mechanism homologous recombination. Results: We identified rucaparib as the most potent of the three PARP inhibitors tested and found a subset of tumors that show high rucaparib sensitivity (IC50 values: 7.0µM, 10.3µM and 11.7µM) comparable to a BRCA deficient breast cancer cell line (IC50 value: 8.9µM). Foci formation of the homologous recombination marker RAD51 did not serve as a reliable post-treatment biomarker. Conclusion: In conclusion, we demonstrate that PARP inhibitors are effective in a subset of head and neck cancer cell lines, suggesting that these compounds could play a role in the treatment of a subset of head and neck tumors that exhibit the “BRCAness” phenotype. Further studies regarding the underlying mechanism of this phenotype are warranted. Citation Format: Jana Heitmann, Paul Geeleher, Michaela Keck, Zhixiang Zuo, Arun Khattri, Susanne Tepper, Michael Beckett, Ralph R. Weichselbaum, Sebastian Fetscher, Everett E. Vokes, Tanguy Seiwert. An evaluation of poly (ADP-ribose) polymerase inhibitor efficacy in head and neck cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2746. doi:10.1158/1538-7445.AM2014-2746

Abstract 5472: Role of Harvey Ras (HRAS) mutations in head and neck squamous cell carcinoma (HNSCC)

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background: Harvey Ras (HRAS) was recently reported being mutated in head and neck squamous cell carcinomas (HNSCC) and likely plays an important role as an oncogene. The precise role of HRAS mutations for signaling and carcinogenesis of HNSCC remains to be determined. Methods: We completed mutational screening (Sanger Sequencing) for tissues and cell lines focused on the known hotspot mutations G12X and Q61X. Furthermore we performed viability testing for various cell lines and visualized the signaling-effects by itself, in presence of PI3K-, EGFR inhibitors and likewise in combination, by immunoblotting. After suppression of HRAS using siRNA, we determined the cell-viability. Results: In our study we sequenced 100 HNSCC tumor tissues and HNSCC cell lines and identified several canonical HRAS mutations. One cell line contained a G12D HRAS mutation and was further examined. Additional two cell lines with atypical HRAS variants were identified and compared to the classic hotspot mutated cell line. The viability for the mentioned cell lines were indicative of resistance to EGFR inhibition to different degrees. The protein activation levels in important signaling pathways (PI3K/MAPK) confirmed our viability data. HRAS signaling was primarily via PI3K/AKT. Silencing HRAS showed significantly decreased viability. Conclusions: Previous studies have shown that EGFR-targeting agents remain insufficient as single targeted therapy. HRAS appears to contribute to the EGFR-resistance of HNSCC. The canonical mutation G12D appears to signal primarily via PI3K and PI3K inhibitors may be effective. The G12D cell line model indicates a central role of mutated HRAS for signaling and viability consistent with role as a driver mutation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5472. doi:1538-7445.AM2012-5472

Abstract LB-398: Detection of copy number alterations in 124 head and neck squamous cell carcinomas using the Nanostring nCounter assay

Background: Head and Neck Squamous Cell Carcinoma (HNSCC) is the 6th most common cancer worldwide. Little is known about changes in copy number (CN) in many cancer associated genes in this tumor type, which may contribute to carcinogenesis and could become useful treatment targets in the future. Many analyses of CN are complicated by the need to use formalin-fixed paraffin-embedded (FFPE) tissues posing technical challenges. We determined copy number alterations (CNA) using a novel, medium-throughput technology (NanoString nCounter) in order to determine common cancer associated CNAs and assess its performance in FFPE tissues. Results were compared with more established technologies such as SNP array, aCGH, and qPCR. Methods: We investigated CNA in 124 tumor specimens and 22 cell lines for 100 literature curated cancer genes using the NanoString nCounter. Most samples were OCT frozen tissues, with a small subset having both OCT frozen, and FFPE tissues. Slides were assessed for tumor content by a HNC pathologist and samples with at least 60% tumor content selected. DNA was extracted using standard column-based methods (Qiagen). We performed CN analysis in 124 frozen (+4 matching FFPE) HNSCC specimens and cell lines (Nanostring nCounter assay) focusing on a selection of cancer associated genes. Furthermore we used aCGH and SNP-CHIP to analyse 20 and 4 cell lines respectively two of which were covered by all three methods. FGFR1 was assessed by qPCR. For FFPE samples a special Nanostring probeset was used with 3-5 probes per gene to provide redundancy with degraded DNA samples. HPV status of samples was assessed by a nested PCR for E6. Results Copy number changes detected by Nanostring and aCGH correlated well. The Nanostring nCounter assay appeared more accurate in calling deletions, which were detected in MST1R, PBRM1, PTPRD for instance. We found amplifications in multiple samples and genes, e.g. CCND1, EGFR, MDM4, MYC, VEGFA, PAX9, ITGB4, SSND1, CTTN, FADD, FGF19, ORAOV1, PPFIA1, some of which were frequently (n>50 samples) or highly amplified (>30 copies). Some of these amplifications such as ORAOV1 and PPFIA1 seemed higher/more frequent in HPV(-) compared to HPV(+) samples. Samples with FGFR1 amplification were validated using qPCR and correlated very closely. FFPE sample processing was uncomplicated using the FFPE probeset. While some probes failed, using degraded FFPE derived DNA, the redundancy of probes allowed accurate calling of CNA that closely correlated with frozen sample results. Conclusions Copy number alterations are frequent in HNSCC and involve many cancer associated genes, including potentially targetable genes such as EGFR, MDM4, and PIK3CA. Most of the CN changes are recurrent. Amplifications and deletions to some extent differed depending on HPV status. The role and implications of these CN aberrations in a clinical setting need to be further elucidated and validated. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-398. doi:1538-7445.AM2012-LB-398

Abstract 2832: Fibroblast growth factors in head and neck cancer: Genetic alterations and therapeutic targeting with ponatinib

Background: FGF signaling plays an important role in cancer. Little is known about its involvement in head and neck squamous cell carcinoma (HNSCC). FGFR1 focal amplification and high expression (Freier, Schwaenen et al.), expression of FG-BP (Li et al.) and FGF2 autocrine signalling loops (Marshall et al.) have been described in connection with HNSSC. We therefore examined copy number alterations for several FGFR and FGF-related Genes in 159 patient tissues and cell lines as well as expression levels in 116 patient tissues and 40 cell lines. Based upon our findings we tried a combined FGFR-inhibiton with ponatinib in 5 head and neck cancer-cell lines alone and in combination with gefitinib. Methods: Copy Number Data analysis for 144 patient tissues and cell lines (Nanostring) as well as for 20 cell lines (aCGH) of which 5 were covered by both techniques and showed comparable results. Expression data analysis for 116 tissues and 40 cell lines (Agilent). Viability for 5 cell lines treated with gefitinib and ponatinib alone and in combination. Immunoblotting was performed to deterimed PI3K-AKT and MAPK signaling. Results: Frequent copy number increase could be detected for FGF19 in tissues and cell lines. FGFR1 copy number increase could be seen in only 1 tissue sample, but appeared to be deleted in several samples. High relative expression could be detected for FGFBP1 in tissues and cell lines. Viability testing showed high efficacy in 5/5 cell lines tested for ponatinib but was not solely mediated by AKT or MAPK signaling. Combined treatment with ponatinib and gefitinib was more effective than treatment with either agent alone and synergy was present. Conclusions: FGF signaling is important in Head and Neck Cancer. FGF19 amplification is frequent. We were unable to replicate FGFR1 amplification with only one tissue showing FGFR1 copy number increase. High expression levels could be shown for FGFBP1, providing an alternative hypothesis for explaining the efficacy of FGF2 inhibition as previously shown (Marshall et. al.). Ponatinib is effective as a single agent on HNSSC cell line models and shows synergistic effect in combination with gefitinib. It is promising to evaluate FGF pathway inhibition (e.g. with ponatinib) in its ability to overcome EGFR-inhibitor resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2832. doi:1538-7445.AM2012-2832