Megan Ludwig

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.

The Tip of the Iceberg: Clinical Implications of Genomic Sequencing Projects in Head and Neck Cancer.

Recent genomic sequencing studies have provided valuable insight into genetic aberrations in head and neck squamous cell carcinoma. Despite these great advances, certain hurdles exist in translating genomic findings to clinical care. Further correlation of genetic findings to clinical outcomes, additional analyses of subgroups of head and neck cancers and follow-up investigation into genetic heterogeneity are needed. While the development of targeted therapy trials is of key importance, numerous challenges exist in establishing and optimizing such programs. This review discusses potential upcoming steps for further genetic evaluation of head and neck cancers and implementation of genetic findings into precision medicine trials.

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.

Changing the paradigm：the potential for targeted therapy in laryngeal squamous cell carcinoma

Laryngeal squamous cell carcinoma (LSCC) remains a highly morbid and fatal disease. Historically, it has been a model example for organ preservation and treatment stratification paradigms. Unfortunately, survival for LSCC has stagnated over the past few decades. As the era of next-generation sequencing and personalized treatment for cancer approaches, LSCC may be an ideal disease for consideration of further treatment stratification and personalization. Here, we will discuss the important history of LSCC as a model system for organ preservation, unique and potentially targetable genetic signatures of LSCC, and methods for bringing stratified, personalized treatment strategies to the 21st century.

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.

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 B27: Identifying therapeutic combinations with EGFR-targeted therapy through the generation of genome-wide CRISPR-Cas9 knockout libraries in oral cancer cell lines

Oral squamous cell carcinoma (OSCC) remains a disease with poor survival. With recent characterization of the mutational landscape of OSCCs, there is great potential for personalized targeted therapies. However, to date utilization of targeted therapies in OSCC have had limited success with anti-EGFR therapy. Combinations of targeted therapies, which may have greater efficacy by inhibiting compensatory pathways, have not been well studied. To identify synergistic combinations with EGFR-targeted therapy, we introduced Genome-scale CRISPR-Cas9 Knock-Out (GeCKO) libraries into OSCC cell lines. CRISPR-Cas9 generates individual genetic knockouts through targeted gene editing, and by using a pool of CRISPRs targeting over 18,000 genes we can perform genome-scale screening for synergistic targetable pathways. Upon selection of the OSCC GeCKO pool, we identified that knockouts of genes in the FGFR pathway caused sensitivity to the EGFR inhibitor gefitinib. We further investigated the possible synergistic effect of dual EGFR and FGFR inhibition with resazurin viability assays in 14 of our OSCC models. We found 6/14 (43%) of the models were responsive to the combination of EGFR and FGFR inhibition, indicating that the FGFR pathway could be an alternate mechanism of resistance to EGFR-targeted therapy. For further investigation of this combination, we again used CRISPR-Cas9 to generate an EGFR knockout (KO) OSCC cell line. This EGFR KO cell line retains sensitivity to FGFR inhibitors, and we expect that this cell line will be a useful tool in further evaluating the compensatory mechanism of the FGFR pathway. OSCC remains a common and frequently lethal cancer with great potential for the development of personalized targeted therapies. Here, we describe the use of genome-wide CRISPR-Cas9 library to discover the synergistic combination of EGFR and FGFR inhibition. Further investigation suggests the FGFR pathway as a common compensatory mechanism to EGFR inhibition. We hope to use this approach to identify additional compensatory mechanisms of resistance to targeted therapies with the eventual goal of translating these findings to clinic. Citation Format: Megan Ludwig, Andrew Birkeland, Sai Nimmagadda, Sue Foltin, Hui Jiang, Thomas Carey, John Chad Brenner. Identifying therapeutic combinations with EGFR-targeted therapy through the generation of genome-wide CRISPR-Cas9 knockout libraries in oral cancer cell lines [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr B27.

Abstract 54: Genome-wide CRISPR screen identifies potential therapeutic combination of EGFR and FGFR inhibitors in oral cancer

Oral squamous cell carcinoma (OSCC) is a disease with poor survival with few novel treatment options in development. While EGFR is commonly over-expressed, targeting EGFR has had limited success possibly because of compensatory pathways rescuing cell survival. Inhibiting compensatory pathways in combination with EGFR signaling may result in greater efficacy. To identify compensatory pathways, we used a Genome-wide CRISPR-Cas9 Knock-Out (GeCKO) library to identify genetic knockouts that sensitized OSCC cell lines to EGFR inhibition. This CRISPR library established a pool of knockouts targeting over 18,000 genes, and upon selection of the GeCKO pool we identified gene knockouts in the FGFR pathway increased sensitivity to the EGFR inhibitor gefitinib. We further profiled this combination with viability assays and found that 6/14 (43%) cell lines responded to the combination of EGFR and FGFR inhibition, suggesting that the FGFR pathway could be a common compensatory mechanism to loss of EGFR signaling. In complement, we again used CRISPR-Cas9 to generate a syngeneic EGFR knockout (KO) OSCC cell line. The parent OSCC cell line is responsive to the EGFR and FGFR dual inhibition, and the EGFR KO derivative retains sensitivity to FGFR-targeted monotherapies. We expect that this EGFR KO cell line will be a useful tool in further evaluating the compensatory mechanism of the FGFR pathway. In total, we describe the use of genome-wide CRISPR-Cas9 library to discover the synergistic combination of EGFR and FGFR inhibition, with follow-up indicating that the FGFR pathway could be a common compensatory mechanism to EGFR inhibition. Targeting compensatory mechanisms in combination with EGFR-targeted therapy could be clinically beneficial, including the combination of EGFR and FGFR inhibition. Citation Format: Megan Ludwig, Andrew Birkeland, Sai Nimmagadda, Sue Foltin, Aditi Kulkarni, Hui Jiang, Thomas Carey, J. Chad Brenner. Genome-wide CRISPR screen identifies potential therapeutic combination of EGFR and FGFR inhibitors in oral cancer [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr 54.

Abstract 3198: Using a genome-wide CRISPR-Cas9 knockout library to identify therapeutic combinations in oral cancer

Oral squamous cell carcinoma (OSCC) has remained a disease with poor survival for decades with few novel treatment options. We anticipate great potential for personalized targeted therapies, facilitated by recent characterizations of the mutational landscape of OSCCs. Combinations of targeted therapies may have greater efficacy through inhibiting compensatory pathways. For example, targeting EGFR has had limited success. To identify synergistic combinations with EGFR-targeted therapy, we introduced Genome-scale CRISPR-Cas9 Knock-Out (GeCKO) libraries into OSCC cell lines. CRISPR-Cas9 generates individual genetic knockouts through targeted gene editing. We used a pool of CRISPRs targeting over 18,000 genes to perform genome-scale screening for drivers of sensitivity to EGFR-targeted therapy. Upon selection of the OSCC GeCKO pool, we identified gene knockouts in the FGFR pathway that increased sensitivity to the EGFR inhibitor gefitinib. Using resazurin viability assays we tested combinations of EGFR and FGFR inhibitors in 14 OSCC cell lines. Six/14 (43%) of the cell lines were responsive to the combination, indicating that the FGFR pathway is an alternate mechanism of resistance to EGFR-targeted therapy in some tumors. In complement, we again used CRISPR-Cas9 to generate a syngeneic EGFR knockout (KO) OSCC cell line. The parent OSCC cell line is responsive to the EGFR and FGFR dual inhibition, and the EGFR KO derivative retains sensitivity to FGFR-targeted monotherapies. We expect that this EGFR KO cell line will be a useful tool in further evaluating the compensatory mechanism of the FGFR pathway. OSCC remains a common and frequently lethal cancer with great potential for the development of personalized targeted therapies. Here, we describe the use of genome-wide CRISPR-Cas9 library to discover the synergistic combination of EGFR and FGFR inhibition. Further investigation suggests the FGFR pathway is a common compensatory mechanism to EGFR inhibition. We hope to use this approach to identify additional compensatory mechanisms of resistance to targeted therapies with the eventual goal of translating these findings to clinic. Citation Format: Megan Ludwig, Andrew Birkeland, Sai Nimmagadda, Sue Foltin, Aditi Kulkarni, Hui Jiang, Thomas Carey, Chad Brenner. Using a genome-wide CRISPR-Cas9 knockout library to identify therapeutic combinations in oral cancer [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 3198. doi:10.1158/1538-7445.AM2017-3198

Abstract 1667: Identifying intrinsic regulators of PD-L1 expression in cancer cells: A genome-scale CRISPR knock-out approach

Head and Neck Squamous Cell Carcinoma (HNSCC) is the sixth most common cancer in the United States and affects 600,000 people each year worldwide. With a five-year survival rate of only 50% and a recent rise in HPV-associated HNSCCs, improved treatment protocols are urgently needed. Evidence of immunosuppression is often reported in HNSCC, making immunotherapy an attractive strategy for the management of this disease. The immune checkpoint inhibitor Pembrolizumab was recently approved for the treatment of metastatic and recurrent HNSCC, but only 18% of initial participants in a trial of Pembrolizumab responded, and it remains difficult to predict patients likely to experience benefit. To increase the number of patients that respond to immune checkpoint inhibition, we sought to identify targetable genetic factors modulating PD-L1, a molecule that serves to dampen the anti-tumor immune response. We show that several HNSCC models exhibit mild (2-4 fold) induction of PD-L1 expression following treatment with interferon-γ (adaptive expression), and recent reports suggest that common oncogenic pathways, including the EGFR and PI3K pathways, may provide a novel strategy to regulate PD-L1 expression. To this end, we developed a high throughput screen to identify targetable pathways that may be used to regulate PD-L1 expression in patients with specific genetic lesions. We have employed a Genome-scale CRISPR Knock-Out (GeCKO) screening technique in HNSCC cell lines and selected for genetic knockouts exhibiting altered PD-L1 expression. Stable knockout pools with representation of approximately 300 gRNAs per target gene (>20,000 target genes in the library) were expanded and serially sorted to create stable sub-populations with enhanced PD-L1 expression. These sub-populations were sequenced to identify gRNAs whose knockout causes a change in PD-L1 expression (e.g. genes that repress or enhance PD-L1 expression). We expect that these large-scale screens, when performed over multiple HNSCC cell lines with diverse genetic lesions, will identify patterns of targetable regulators that may ultimately be manipulated in combination with PD-1/PD-L1 inhibitors. Citation Format: Jacqueline E. Mann, Megan L. Ludwig, Rebecca C. Hoesli, Aditi Kulkarni, Simmy Patel, Judy Kafelghazal, Alexey Nesvizhskii, J Chad Brenner. Identifying intrinsic regulators of PD-L1 expression in cancer cells: A genome-scale CRISPR knock-out approach [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 1667. doi:10.1158/1538-7445.AM2017-1667