Arun Khattri

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.

A phase II study of lapatinib in recurrent/metastatic squamous cell carcinoma of the head and neck.

Purpose: This study sought to determine the efficacy and safety profile of lapatinib in patients with recurrent/metastatic squamous cell carcinoma of the head and neck (SCCHN). Experimental Design: This phase II multiinstitutional study enrolled patients with recurrent/metastatic SCCHN into two cohorts: those without (arm A) and those with (arm B) before exposure to an epidermal growth factor receptor (EGFR) inhibitor. All subjects were treated with lapatinib 1,500 mg daily. Primary endpoints were response rate (arm A) and progression-free survival (PFS; arm B). The biologic effects of lapatinib on tumor growth and survival pathways were assessed in paired tumor biopsies obtained before and after therapy. Results: Forty-five patients were enrolled, 27 in arm A and 18 in arm B. Diarrhea was the most frequent toxicity occurring in 49% of patients. Seven patients experienced related grade 3 toxicity (3 fatigue, 2 hyponatremia, 1 vomiting, and 1 diarrhea). In an intent-to-treat analysis, no complete or partial responses were observed, and stable disease was the best response observed in 41% of arm A (median duration, 50 days, range, 34–159) and 17% of arm B subjects (median, 163 days, range, 135–195). Median PFS was 52 days in both arms. Median OS was 288 (95% CI, 62–374) and 155 (95% CI, 75–242) days for arms A and B, respectively. Correlative analyses revealed an absence of EGFR inhibition in tumor tissue. Conclusion: Lapatinib as a single agent in recurrent/metastatic SCCHN, although well tolerated, appears to be inactive in either EGFR inhibitor naive or refractory subjects. Clin Cancer Res; 18(8); 2336–43. ©2012 AACR.

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.

Growth factor expression mediates resistance to EGFR inhibitors in head and neck squamous cell carcinomas.

OBJECTIVES Epidermal growth factor receptor (EGFR)-targeted therapy is frequently used in the treatment of advanced head and neck squamous cell carcinoma (HNSCC). However, constitutive or acquired resistance is common and underlying resistance mechanisms remain poorly understood. We investigated the expression levels of growth factors (GF) in tumor-associated stroma and tumor from HNSCC patients and determined the influence of GFs on EGFR inhibitor efficacy in vitro. MATERIALS AND METHODS The Chicago HNC Genomic Cohort (CHGC) was queried for GF and receptor tyrosine kinase (RTK) expression. Viability assays were used to evaluate the effect of EGFR inhibition (gefitinib), GF treatment, or both in HNSCC cell lines. Caspase-based assays were used to measure apoptotic activity. Expression of RTKs was determined and correlated with GF treatment effects. RESULTS Amphiregulin (AREG), transforming growth factor (TGFβ1), insulin like growth factor (IGF1), fibroblast growth factors (FGF1/FGF2) and the corresponding RTKs were highly expressed in 30-50% of HNSCC, and expression was usually concurrent. While EGFR inhibition was markedly efficacious in HNC cell lines (HN5/HN13/H400/SCC61), co-treatment with most GFs increased viability up to 100%. Only TGFβ1 treatment was additive to EGFR inhibition. GFs also reduced apoptotic effects of EGFR inhibition. RTK expression showed strong positive correlation with respective GF treatment effect for IGF1-IGF1R, less strong for HGF-MET/AREG-EGFR and a moderate negative correlation for TGFβ1-TGFBR1/2. CONCLUSION High expression of GFs/RTKs occurs in HNSCC. Co-expression is common. GF expression contributes to EGFR inhibition resistance in our model system, and may be a common mechanism of constitutive or acquired resistance to EGFR inhibition in HNSCC.

Genetic profiling of advanced radioactive iodine-resistant differentiated thyroid cancer and correlation with axitinib efficacy

Biomarkers predicting which patients with advanced radioiodine-resistant differentiated thyroid cancer (DTC) may benefit from multi-kinase inhibitors are unavailable. We aimed to describe molecular markers in DTC that correlate with clinical outcome to axitinib. Pretreatment thyroid cancer blocks from 18 patients treated with axitinib were collected and genomic DNA was isolated. The OncoCarta™ Mutation Panel was used to test for 238 oncogenic mutations. Copy number of VEGFR1-3 and PIK3CA was determined using qPCR on enriched tumor samples. Genomic DNA was analyzed for all coding regions of VEGFR1-3 with custom primers. Protein expressions of VEGFR1-3, c-Met, and PIK3CA were evaluated with immunohistochemistry. Clinical response to axitinib, including best response (BR) and progression free survival (PFS), was ascertained from corresponding patients. Fishers exact test and logistic regression models were used to correlate BR with molecular findings. Cox proportional hazards regression was used to correlate PFS with molecular defects. A total of 22 pathology samples (10 primary, 12 metastatic) were identified. In patients with 2 samples (n = 4), genetic results were concordant and only included once for analysis. Tumors from 4 patients (22%) harbored BRAF V600E mutations, 2 (11%) had KRAS mutations (G12A, G13D) and 2 (11%) had HRAS mutations (Q61R, Q61K). One metastatic sample with mutated KRAS also harbored a PIK3CA (H1047R) mutation. qPCR showed increased copy numbers of PIK3CA in 6 (33%) tumors, VEGFR1 in 0 (0%) tumors, VEGFR2 in 4 (22%) tumors, and VEGFR3 in 6 (33%) tumors. VEGFR sequencing was significant for a possibly damaging non-synonymous SNP in VEGFR2 (G539R) in 2 samples (11%), a possibly damaging SNP in VEGFR3 (E350V) in 1 sample (6%), and a potentially novel mutation in VEGFR2 (T439I) in 2 samples (11%). Immunohistochemistry (VEGFR1, -2, -3; c-MET; PIK3CA) revealed positive staining in the majority of samples. No significant relationship was seen between BR or PFS and the presence of molecular alterations. Molecular evaluation of DTC specimens did not predict clinical response to axitinib but our data were limited by sample size. We did identify molecular changes in VEGFR that should be further explored. While DTC is genetically heterogeneous, primary and metastatic lesions showed identical oncogenic alterations in four cases.

Characterization of the T-Cell Receptor Repertoire and Immune Microenvironment in Patients with Locoregionally Advanced Squamous Cell Carcinoma of the Head and Neck

Purpose: Squamous cell carcinoma of the head and neck (SCCHN) is a lethal cancer with a suboptimal 5-year overall survival of approximately 50% with surgery and/or definitive chemoradiotherapy. Novel treatments are thus urgently awaited. Immunotherapy with checkpoint blockade has emerged as a promising option for patients with recurrent/metastatic SCCHN; however, it has not been investigated in the curative-intent setting yet. The purpose of this study was to investigate the T-cell receptor repertoire and the tumor microenvironment in tumor tissues of SCCHN patients with locoregionally advanced disease. Experimental Design: We performed T-cell receptor sequencing of tumor tissues from 44 patients with locoregionally advanced SCCHN prior to treatment with definitive chemoradiotherapy and correlated the T-cell clonality and the mRNA expression levels of immune-related genes with clinicopathologic parameters. Results: Clonal expansion of T cells was significantly higher in human papilloma virus (HPV)–negative compared with HPV-positive tumors, signifying more robust antigen presentation in HPV-negative tumors. The latter was supported by the higher percentage of HPV-negative tumors expressing HLA-A protein compared with HPV-positive tumors (P = 0.049). Higher GRZB levels correlated significantly with longer recurrence-free survival (log-rank, P = 0.003) independent of tumor size, nodal stage, and HPV status. Conclusions: Our findings support clonal expansion of T cells in SCCHN patients with locoregionally advanced disease and imply differences in the antigen presentation capacity between HPV-negative and HPV-positive tumors. Elevated GRZB mRNA levels may also serve as a favorable and independent predictor of outcome in SCCHN patients treated with chemoradiotherapy. These data provide rationale for the introduction of immunotherapeutic approaches in the curative-intent setting. Clin Cancer Res; 23(16); 4897–907. ©2017 AACR.

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