Nicole L. Michmerhuizen

Identification of Targetable ERBB2 Aberrations in Head and Neck Squamous Cell Carcinoma

IMPORTANCE ERBB2 (formerly HER2) is an important drug target in breast cancer, where anti-ERBB2 therapy has been shown to lead to improvements in disease recurrence and overall survival. ERBB2 status in head and neck squamous cell carcinoma (HNSCC) has not been well studied. Identification of ERBB2-positive tumors and characterization of response to ERBB2 therapy could lead to targeted treatment options in HNSCC. OBJECTIVE To identify ERBB2 aberrations in HNSCCs and investigate the potential for ERBB2-targeted therapy in HNSCCs. DESIGN, SETTING, AND PARTICIPANTS A retrospective case series of patients with laryngeal (42 tumor specimens) and oral cavity (94 tumor specimens) SCC enrolled in the University of Michigan Head and Neck Specialized Program of Research Excellence was conducted. Publicly available sequencing data (The Cancer Genome Atlas), as well as data from other studies, were reviewed to identify additional mutations and overexpression in ERBB2 in HNSCC. Established HNSCC cell lines were used for follow-up in vitro analysis. The study was conducted from October 1, 2014, to August 30, 2015. INTERVENTIONS With the use of targeted, amplicon-based sequencing with the Oncomine Cancer Panel, the copy number and mutation status of commonly altered genes in HNSCCs were assessed. Immunohistochemical staining was performed on tissue microarrays of HNSCCs to assess the expression of ERBB2. Western blotting for HNSCC cell line ERBB2 expression and cell survival assays after treatment with ERBB2 inhibitors were performed. MAIN OUTCOMES AND MEASURES The prevalence of ERBB2 genetic aberrations and ERBB2 overexpression in laryngeal and oral cavity SCCs, prevalence of ERBB2 aberrations in HNSCC in The Cancer Genome Atlas, ERBB2 protein expression in HNSCC cell lines, and response of HNSCC cell lines to targeted ERBB2 inhibitors. RESULTS Of the 42 laryngeal SCC samples screened by targeted sequencing, 4 (10%) were positive for ERBB2 amplification. Two of these samples showed ERBB2 overexpression on immunohistochemistry. Two of the 94 oral cavity SCC samples (2%) were positive for ERBB2 on immunohistochemistry. Analysis of 288 patients from publicly available HNSCC sequencing data revealed 9 amplifications (3%) in ERBB2. Protein expression was variable across HNSCC cell lines, and a subset of these cell lines showed responsiveness to anti-ERBB2 therapy. CONCLUSIONS AND RELEVANCE ERBB2 aberrations were identified in a subset of HNSCCs. These tumors may be responsive to targeted therapy against ERBB2. Screening for ERBB2 aberrations and applying targeted therapy in ERBB2-positive patients may be useful in personalized therapy trials, particularly in patients who are refractory to current treatment paradigms.

Correlation of Crtc1/3-Maml2 fusion status, grade and survival in mucoepidermoid carcinoma

OBJECTIVE Mucoepidermoid carcinoma (MEC) is the most common malignant tumor of the salivary glands. Tumor stage and grade have historically been important predictors of survival. An oncogenic CRTC1- or CRTC3-MAML2 gene fusion has been identified in a number of MECs. Historically, these gene fusions have been associated with lower grade tumors and better survival. However, reported gene fusion rates and prognosis varies widely across studies, and have not controlled for tumor grade. We sought to identify gene fusion rates and outcomes in our cohort of MEC patients. MATERIALS AND METHODS An IRB-approved retrospective cohort of patients with MEC was identified at the University of Michigan. Clinical, histologic, and outcome data was collected from medical records. RNA was isolated from formalin fixed paraffin-embedded tumor sections, and qRT-PCR was performed to identify CRTC1/3-MAML2 gene fusions. Sanger sequencing of qRT-PCR products was used to confirm gene fusions. RESULTS Overall, 90 patient MEC tumors were collected (58 low-grade, 25 intermediate-grade, and 7 high-grade). Gene fusions were identified in 59% (53/90) of tumors. On univariate and bivariate analysis, fusion status did not significantly associate with grade or survival. CONCLUSION We have identified a high rate of CRTC1/3-MAML2 gene fusions in a large cohort of MEC. We do not identify any correlation between fusion status with tumor grade or survival. These findings suggest further characterization of MECs is needed before considering the CRTC1/3-MAML2 gene fusion as a prognostic biomarker. Additional genetic drivers may account for survival and grade in MECs.

Surveilling the Potential for Precision Medicine-driven PD-1/PD-L1-targeted Therapy in HNSCC

Immunotherapy is becoming an accepted treatment modality for many patients with cancer and is now approved for use in platinum-refractory recurrent or metastatic head and neck squamous cell carcinoma (HNSCC). Despite these successes, a minority of patients with HNSCC receiving immunotherapy respond to treatment, and few undergo a complete response. Thus, there is a critical need to identify mechanisms regulating immune checkpoints in HNSCC such that one can predict who will benefit, and so novel combination strategies can be developed for non-responders. Here, we review the immunotherapy and molecular genetics literature to describe what is known about immune checkpoints in common genetic subsets of HNSCC. We highlight several highly recurrent genetic lesions that may serve as biomarkers or targets for combination immunotherapy in HNSCC.

Genomic sequencing and precision medicine in head and neck cancers

Head and neck squamous cell carcinoma (HNSCC) remains a common and deadly disease. Historically, surgical and chemoradiation treatments have been met with modest success, and understanding of genetic drivers of HNSCC has been limited. With recent next generation sequencing studies focused on HNSCC, we are beginning to understand the genetic landscape of HNSCCs and are starting to identify and advance targeted options for patients. In this review, we describe current knowledge and recent advances in sequencing studies of HNSCC, discuss current limitations and future directions for further genomic analysis, and highlight the translational advances being undertaken to treat this important disease.

Proportion of CD4 and CD8 tumor infiltrating lymphocytes predicts survival in persistent/recurrent laryngeal squamous cell carcinoma

Tumor infiltrating lymphocytes (TILs) have been shown to be an important prognostic factor in patients with previously untreated head and neck cancer. After organ preservation therapy for laryngeal cancer and subsequent persistence/recurrence, the prognostic value of TILs is unknown. Our goal was to determine if TILs have value as a prognostic biomarker in patients with surgically salvageable persistent/recurrent laryngeal squamous cell carcinoma. Levels of TILs were quantified on tissue microarrays from 183 patients undergoing salvage total laryngectomy for persistent/recurrent laryngeal cancer after radiation or chemoradiation between 1997 and 2014. Demographic and clinical data were abstracted. Immunohistology evaluation included CD4, CD8, PDL-1, p16, CD31, Vimentin, EGFR, and p53. Elevated levels of either CD8 or CD4 positive TILs were associated with improved disease specific survival (CD8: HR 0.46, 95% CI 0.24-0.88, CD4: HR 0.43; 95% CI 0.21-0.89) and disease free survival (CD8: HR 0.53, 95% CI 0.29-0.94, CD4: HR 0.52; 95% CI 0.27-0.99). Levels of CD8 (HR 0.74; 95% CI 0.47-1.17) or CD4 (HR 0.66; 95% CI 0.40-1.08) TILs were not significantly associated with overall survival. In bivariate analysis, patients with elevated CD4 and/or CD8 TILs had significantly improved disease specific survival (HR 0.42; 95% CI 0.21-0.83) and disease free survival (HR 0.45; 95% CI 0.24-0.84) compared to patients with low levels of CD4 and CD8. PDL-1, p16, CD31, Vimentin, EGFR, and p53 were not significant prognostic factors. On multivariate analysis, elevated CD8 TILs were associated with improved disease specific survival (HR 0.35; 95% CI 0.14-0.88, p = .02) and disease free survival (HR 0.41; 95% CI 0.17-0.96, p = .04). CD8, and possibly CD4, positive TILs are associated with favorable disease free and disease specific survival for recurrent/persistent laryngeal cancer.

Differential compensation mechanisms define resistance to PI3K inhibitors in PIK3CA amplified HNSCC

OBJECTIVE Recent sequencing studies of head and neck squamous cell carcinomas (HNSCCs) have identified the phosphatidylinositol 3-kinase (PI3K) pathway as the most frequently mutated, oncogenic pathway in this cancer type. Despite the frequency of activating genomic alterations in PIK3CA (the gene encoding the catalytic subunit of PI3K, targeted inhibitors of PI3K have not shown clinical efficacy as monotherapies. We hypothesized that co-dependent pathways, including the Ras-MEK-ERK pathway, may still be functional in the presence of PI3K inhibitors and might serve as mediators of this resistance. METHODS We assessed the hypothesis using resazurin cell viability and trypan blue exclusion assays. We also used Western blot to characterize Ras-MEK-ERK pathway activity. STUDY DESIGN We evaluated this hypothesis in six PIK3CA-amplified, PI3K inhibitor-resistant HNSCC cell lines following treatment with pan and alpha-isoform selective PI3K inhibitors (BKM120 and HS-173 respectively). We also tested the effect of combination treatment with PI3K inhibitor HS-173 and MEK inhibitor trametinib or EGFR inhibitor gefitinib. RESULTS Our results displayed maintenance of Ras-MEK-ERK pathway activity in 4 of 6 HNSCC cell lines after PI3K inhibitor treatment. We also found that UM-SCC-69 and UM-SCC-108 cells display synergistic responses to dual therapy. CONCLUSION This study suggests that inhibition of the PI3K and Ras-MEK-ERK pathways might be effective in some HNSCC patients; however, it also prompts the study of additional resistance mechanisms to identify synergistic combination therapies for tumors resistant to these di-therapies.

Comprehensive review of genetic factors contributing to head and neck squamous cell carcinoma development in low-risk, nontraditional patients

The past 2 decades have seen an increased incidence of head and neck squamous cell carcinoma (HNSCC) in a nontraditional, low‐risk patient population (ie, ≤45 years of age, no substance use history), owing to a combination of human papillomavirus (HPV) infection and individual genetic variation.

Abstract 4092: Targeting compensatory mechanisms of resistance to phosphatidylinositol 3-kinase inhibitors in head and neck squamous cell carcinoma

Recent sequencing studies of head and neck squamous cell carcinomas (HNSCCs) have identified the phosphatidylinositol 3-kinase (PI3K) pathway as the most frequently mutated, oncogenic pathway in this cancer type. Despite the frequency of activating mutations or amplification in PIK3CA (the gene encoding the catalytic subunit of PI3K), targeted inhibitors of PI3K have not shown clinical efficacy as monotherapies. We tested a panel of more than 20 patient-derived oral cavity squamous cell carcinoma (OCSCC) cell lines, which were profiled by Nimblegen V3 exome sequencing, and observed resistance to PI3K inhibitors despite PIK3CA copy number amplification and/or mutation. Of six inhibitors tested, only alpha-isoform selective and pan-PI3K agents were somewhat effective; these inhibitors, despite on-target and downstream activity, did not cause an appreciable reduction in cell viability when administered at submicromolar concentrations. We hypothesized that other oncogenic pathways might still be functional in the presence of PI3K inhibitors and might serve as mediators of this resistance. For example, our group and others have evaluated co-dependence on the Ras-MEK-ERK pathway in OCSCC, showing that this serves as a compensatory mechanism in some models. In order to more fully characterize other novel mechanisms of resistance, we have also developed a high-throughput screening approach that utilizes a resazurin cell viability assay. This screen serves as an unbiased means of testing ~1400 inhibitors as monotherapies and in combination with alpha-isoform selective PI3K inhibitor HS-173 and pan-PI3K inhibitor BKM120 in several OCSCC models. Further validation of “hits” from this screen, which are drugs that are effective in combination but not as monotherapies, may identify additional resistance mechanisms and lead to combination therapies to improve HNSCC patient prognosis. Citation Format: Nicole L. Michmerhuizen, Elizabeth Leonard, Susan K. Foltin, Aditi Kulkarni, Thomas E. Carey, Carol R. Bradford, Hui Jiang, Chad Brenner. Targeting compensatory mechanisms of resistance to phosphatidylinositol 3-kinase inhibitors in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4092. doi:10.1158/1538-7445.AM2017-4092

Abstract 3712: Mechanistic link between phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) activity and PDL1 expression in head and neck squamous cell carcinoma (HNSCC)

Introduction: HNSCC is an immunosuppressive disease, with multiple functional and quantitative defects contributing to immune evasion and tumor escape. One of the identified areas for therapy development is the Programmed Death-1 (PD-1)/Programmed Death Ligand 1 (PD-L1) pathway, as this pathway has been hypothesized to allow cancer cells to evade the immune system by promoting T cell anergy and apoptosis. Pembrolizumab, a PD-L1 inhibitor, has recently been approved for the treatment of recurrent/metastatic HNSCC. As regulation of PD-L1 expression could play an important role in the effectiveness of therapy, we further explored the regulation of PD-L1 expression in HNSCC cells. Specifically, we targeted our investigation by evaluating the effects on expression of one of the most frequently mutated genes in HNSCC, PIK3CA. We evaluated this with and without interferon-γ, which has previously been shown to affect PD-L1 expression, possibly through activation of the STAT1 pathway. Methods: HPV+ and HPV- HNSCC cell lines were grown in cell culture and treated with selective and non-selective PI3K inhibitors, in combination with interferon-γ. After 72 hours, cells were harvested and flow cytometry was used to measure the expression of PD-L1. Protein expression of various pathway intermediaries was evaluated via western blot to better delineate the mechanism of PD-L1 upregulation. Results: Treatment with selective PI3K inhibitors in combination with interferon-γ in several cell lines significantly increased expression of PD-L1, beyond the increase noted after treatment with interferon-γ alone. Maintenance of STAT1 phosphorylation correlated with upregulation of PD-L1 expression, while total STAT1 expression remained stable. The majority of the cell lines maintaining STAT1 phosphorylation were HPV+, but a few HPV- cell lines also maintained this phosphorylation with a correlating upregulation in PD-L1 expression. Additionally, mutation in PIK3Ca despite HPV- status was noted to maintain phosphorylation of STAT1 with upregulation of PD-L1 expression. Conclusions: Treatment with PI3K inhibitors in combination with interferon-γ significantly upregulated PD-L1 expression in several cell lines, suggesting a possible synergistic effect. Since PD-L1 expression correlated with maintenance of phosphorylation of STAT1, these results suggest a pivotal link between PIK3CA signaling, STAT1 activity and PD-L1 expression in PIK3CA aberrant HNSCC. Citation Format: Rebecca C. Hoesli, Nicole L. Michmerhuizen, Vivek Nair, Chloe Matovina, Elizabeth Leonard, Matthew E. Spector, Carol R. Bradford, Mark E. Prince, Andrew C. Birkeland, J Chad Brenner. Mechanistic link between phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) activity and PDL1 expression in head and neck squamous cell carcinoma (HNSCC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3712. doi:10.1158/1538-7445.AM2017-3712

Abstract 1850: Development of a NOTCH1 loss of function and PIK3CA mutant transgenic model of HNSCC

Head and neck squamous cell carcinoma (HSNCC) is a debilitating disease with a poor 5-year survival rate. The use of in vivo models to study tumorigenesis and to identify potential targeted therapies, especially those that work in combination with immune checkpoint inhibitors, is essential in improving overall patient care. However, the overall number of HNSCC transgenic models with recurrent genetic lesions identified by the HNSCC TCGA project has been limited. For example, TCGA data has shown both Notch1 loss of function mutations and PIK3CA activating mutations as some of the most common in HNSCC, but bi-genic models of these lesions do not yet exist. Here, we have designed a model which utilizes K14-CreERT to drive a bi-genic model with Notch1flox and PIK3CAH1047R mutations in epithelial compartments. Characterization of this line both in the context of spontaneous and carcinogen induced tumor formation is a tool which can be utilized to better understand common mutations in HNSCC. Immunohistochemistry confirms the overexpression of PIK3CA in tamoxifen induced mice. Furthermore, early characterization of this model with chronic treatment of the smoking analogue 4-Nitroquinoline N-oxide (4NQO) in drinking water shows tumor formation on the tongue and surrounding head and neck regions in as little as 12-16 weeks. Hematoxylin and Eosin staining confirmed the squamous tumor formation, while Ki67 staining showed enhanced proliferation of the transformed cells. In the future, this model can be used to study tumor formation over time and in response to targeted inhibitors and common therapies that are being advanced as companion diagnostic strategies for either NOTCH1 deficient or PIK3CA aberrant HNSCC tumors. Further detailed characterization of this model will allow for a deeper understanding of the mechanisms which drive HNSCC and will translate to improved patient care. Citation Format: Samantha N. Devenport, Nicole L. Michmerhuizen, Elizabeth Leonard, Brittany Jewell, Jacob Swanson, Sunny Wong, Thomas E. Carey, J Chad Brenner. Development of a NOTCH1 loss of function and PIK3CA mutant transgenic model of HNSCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1850. doi:10.1158/1538-7445.AM2017-1850