Glenn J. Hanna

Response and Acquired Resistance to Everolimus in Anaplastic Thyroid Cancer

Everolimus, an inhibitor of the mammalian target of rapamycin (mTOR), is effective in treating tumors harboring alterations in the mTOR pathway. Mechanisms of resistance to everolimus remain undefined. Resistance developed in a patient with metastatic anaplastic thyroid carcinoma after an extraordinary 18-month response. Whole-exome sequencing of pretreatment and drug-resistant tumors revealed a nonsense mutation in TSC2, a negative regulator of mTOR, suggesting a mechanism for exquisite sensitivity to everolimus. The resistant tumor also harbored a mutation in MTOR that confers resistance to allosteric mTOR inhibition. The mutation remains sensitive to mTOR kinase inhibitors.

Genomic Analysis of Metastatic Cutaneous Squamous Cell Carcinoma

Purpose: A rare 5% of cutaneous squamous cell carcinomas (cSCC) metastasize, lack FDA-approved therapies, and carry a poor prognosis. Our aim was to identify recurrent genomic alterations in this little-studied population of metastatic cSCCs. Experimental Design: We performed targeted sequencing of 504 cancer-associated genes on lymph node metastases in 29 patients with cSCC and identified mutations and somatic copy-number alterations associated with metastatic cSCC. We determined significantly mutated, deleted, and amplified genes and associated genomic alterations with clinical variables. Results: The cSCC genome is heterogeneous with widely varying numbers of genomic alterations and does not appear to be associated with human papillomavirus. We found previously identified recurrently altered genes (TP53, CDKN2A, NOTCH1/2) but also a wide spectrum of oncogenic mutations affecting RAS/RTK/PI3K, squamous differentiation, cell cycle, and chromatin remodeling pathway genes. Specific mutations in known oncogenic drivers and pathways were correlated with inferior patient outcomes. Our results suggest potential therapeutic targets in metastatic cSCC, including PIK3CA, FGFR3, BRAF, and EGFR, similar to those reported in SCCs of the lung and head and neck, suggesting that clinical trials could be developed to accrue patients with SCCs from multiple sites of origin. Conclusions: We have genomically characterized a rare cohort of 29 metastatic cSCCs and identified a diverse array of oncogenic alterations that can guide future studies of this disease. Clin Cancer Res; 21(6); 1447–56. ©2015 AACR.

Ex Vivo Profiling of PD-1 Blockade Using Organotypic Tumor Spheroids

Ex vivo systems that incorporate features of the tumor microenvironment and model the dynamic response to immune checkpoint blockade (ICB) may facilitate efforts in precision immuno-oncology and the development of effective combination therapies. Here, we demonstrate the ability to interrogate ex vivo response to ICB using murine- and patient-derived organotypic tumor spheroids (MDOTS/PDOTS). MDOTS/PDOTS isolated from mouse and human tumors retain autologous lymphoid and myeloid cell populations and respond to ICB in short-term three-dimensional microfluidic culture. Response and resistance to ICB was recapitulated using MDOTS derived from established immunocompetent mouse tumor models. MDOTS profiling demonstrated that TBK1/IKKε inhibition enhanced response to PD-1 blockade, which effectively predicted tumor response in vivo Systematic profiling of secreted cytokines in PDOTS captured key features associated with response and resistance to PD-1 blockade. Thus, MDOTS/PDOTS profiling represents a novel platform to evaluate ICB using established murine models as well as clinically relevant patient specimens.Significance: Resistance to PD-1 blockade remains a challenge for many patients, and biomarkers to guide treatment are lacking. Here, we demonstrate feasibility of ex vivo profiling of PD-1 blockade to interrogate the tumor immune microenvironment, develop therapeutic combinations, and facilitate precision immuno-oncology efforts. Cancer Discov; 8(2); 196-215. ©2017 AACR.See related commentary by Balko and Sosman, p. 143See related article by Deng et al., p. 216This article is highlighted in the In This Issue feature, p. 127.

Induction Chemotherapy for Locoregionally Advanced Head and Neck Cancer: Past, Present, Future?

The treatment of patients with locoregionally advanced squamous cell cancer of the head and neck is still evolving. Induction chemotherapy (IC) is widely used in this patient population and it is unclear how to best incorporate IC into multimodality treatment. Recently, the results of two randomized clinical trials were presented (the PARADIGM and Docetaxel Based Chemotherapy Plus or Minus Induction Chemotherapy to Decrease Events in Head and Neck Cancer trials), which showed no demonstrable benefit of IC followed by concurrent chemoradiation over concurrent chemoradiotherapy alone. However, a lower rate of distant metastatic disease was noted, suggesting that patients who are at high risk for metastatic disease may benefit from IC. This review summarizes how IC has evolved over the years, provides an update of recent developments, and discusses how IC may develop in the future.

Defining an inflamed tumor immunophenotype in recurrent, metastatic squamous cell carcinoma of the head and neck

OBJECTIVES Immune checkpoint inhibitors have demonstrated clinical benefit in recurrent, metastatic (R/M) squamous cell carcinoma of the head and neck (SSCHN), but lacking are biomarkers that predict response. We sought to define an inflamed tumor immunophenotype in this R/M SCCHN population and correlate immune metrics with clinical parameters and survival. METHODS Tumor samples were prospectively acquired from 34 patients to perform multiparametric flow cytometry and multidimensional clustering analysis integrated with next-generation sequencing data, clinical parameters and outcomes. RESULTS We identified an inflamed subgroup of tumors with prominent CD8+ T cell infiltrates and high PD-1/TIM3 co-expression independent of clinical variables, with improved survival compared with a non-inflamed subgroup (median overall survival 84.0 vs. 13.0months, p=0.004). The non-inflamed subgroup demonstrated low CD8+ T cells, low PD-1/TIM3 co-expression, and higher Tregs. Overall non-synonymous mutational burden did not correlate with response to PD-1 blockade in a subset of patients. CONCLUSION R/M SCCHN patients with an inflamed tumor immunophenotype demonstrate improved survival. Further prospective studies are needed to validate these findings and explore the use of immunophenotype to guide patient selection for immunotherapeutic approaches.

Salivary and serum HPV antibody levels before and after definitive treatment in patients with oropharyngeal squamous cell carcinoma

BACKGROUND HPV-associated oropharyngeal squamous cell carcinoma (OPSCC) is increasing in incidence. Laboratory assays that identify virally-mediated disease and predict HPV clearance in OPSCC may have important prognostic implications. OBJECTIVE Here we investigated serum, salivary HPV16 early (E) antibodies before and after definitive chemoradiotherapy (CRT) in patients with locoregionally advanced OPSCC. METHODS We prospectively measured salivary, serum samples at the beginning of and 6-7 weeks following the completion of CRT in 44 patients. IgG antibodies targeting N- and C-terminal fragments of E2 (NE2, CE2), E6 and E7 were measured by programmable enzyme-linked immunosorbent assay. RESULTS We observed serum antibodies directed against HPV-associated E proteins in patients with HPV-associated OPSCC. E7 directed antibodies were detected in saliva in the majority of patients, and were associated with HPV status (p= 0.03). When analyzed longitudinally, median salivary E7 antibody levels decreased significantly post-treatment (p= 0.007). CONCLUSIONS Our results demonstrate the feasibility of measuring HPV16 E antibodies in saliva and serum. E7 antibodies, in particular, are more detectable in saliva as compared with other E protein antibodies. Measuring E7 salivary antibody levels at various time points may have utility in understanding HPV clearance and should be explored for their ability to predict the risk of recurrence.

Genomic Heterogeneity and Exceptional Response to Dual Pathway Inhibition in Anaplastic Thyroid Cancer

Purpose: Cancers may resist single-agent targeted therapies when the flux of cellular growth signals is shifted from one pathway to another. Blockade of multiple pathways may be necessary for effective inhibition of tumor growth. We document a case in which a patient with anaplastic thyroid carcinoma (ATC) failed to respond to either mTOR/PI3K or combined RAF/MEK inhibition but experienced a dramatic response when both drug regimens were combined. Experimental Design: Multi-region whole-exome sequencing of five diagnostic and four autopsy tumor biopsies was performed. Meta-analysis of DNA and RNA sequencing studies of ATC was performed. Results: Sequencing revealed truncal BRAF and PIK3CA mutations, which are known to activate the MAPK and PI3K/AKT pathways, respectively. Meta-analysis demonstrated 10.3% cooccurrence of MAPK and PI3K pathway alterations in ATC. These tumors display a separate transcriptional profile from other ATCs, consistent with a novel subgroup of ATC. Conclusions: BRAF and PIK3CA mutations define a distinct subset of ATC. Blockade of the MAPK and PI3K pathways appears necessary for tumor response in this subset of ATC. This identification of synergistic activity between targeted agents may inform clinical trial design in ATC. Clin Cancer Res; 23(9); 2367–73. ©2016 AACR.

Human papillomavirus and induction chemotherapy versus concurrent chemoradiotherapy in locally advanced oropharyngeal cancer: The Dana Farber Experience.

Human papillomavirus (HPV) infection indicates favorable prognosis in oropharyngeal squamous cell carcinoma (SCC). The purpose of this study was for us to assess the impact of HPV in patients treated with sequential therapy versus concomitant chemoradiotherapy (CRT).

Frameshift events predict anti–PD-1/L1 response in head and neck cancer

Programmed cell death protein 1 (PD-1) inhibitors have efficacy in treating squamous cell carcinoma of the head and neck (SCCHN), but objective response rates are low. PD-1 ligand (PD-L1) expression alone is not considered a robust predictor of response and additional biomarkers are needed. This 3-year observational cohort followed 126 SCCHN patients treated with anti-PD-1/L1 therapy. Prior to treatment, 81 (64%) had targeted massively parallel tumor sequencing. Of these, 42 (52%) underwent fluorescence-activated cell sorting and PD-L1 immunohistochemistry for tumor immunoprofiling. Six (5%) complete responses (CRs) and 11 (9%) partial responses (PRs) were observed. Those treated with prior chemotherapy (98, 78%) versus only surgery and/or radiation had longer overall survival (OS) (10 vs. 3 months, P = 0.02). Smokers had a higher total mutational burden (TMB) (P = 0.01). Virus-positive patients had a lower TMB (P < 0.01) and improved OS (P = 0.02). Among virus-negative responders, NOTCH1 and SMARCA4 were more frequently mutated and frameshift events in tumor suppressor genes occurred more frequently (P = 0.03). Higher TMB and CD8+ T cell infiltrates predicted anti-PD-1/L1 benefit (P < 0.01, P < 0.01, respectively) among virus-negative tumors. TIM-3/LAG-3 coexpression with PD-1 was higher on T cells among nonresponders (P = 0.03 and 0.02, respectively). Somatic frameshift events in tumor suppressor genes and higher TMB among virus-negative SCCHN tumors predict anti-PD-1/L1 response.

CIC-NUTM1 fusion: A case which expands the spectrum of NUT-rearranged epithelioid malignancies

NUT carcinoma (NC) shows very aggressive clinical behavior, occurs predominantly in the thorax and head and neck region of children and adults, and is defined by the presence of NUT (aka NUTM1) rearrangement, mostly BRD4‐NUTM1 fusion resulting from t(15;19)(q13; p13.1). So‐called “NUT variants” harbor alternate fusions between NUTM1 and BRD3, NSD3, ZNF532, or unknown partners. Rare cases of pediatric tumors with CIC‐NUTM1 fusion were recently reported in somatic soft tissue, brain, and kidney. However, such cases have not been identified in adult patients and the presence of a fusion between CIC, characteristic of CIC‐rearranged sarcoma, and NUTM1—a defining feature of NC—poses a diagnostic challenge. We herein report a case of malignant epithelioid neoplasm with myoepithelial features harboring CIC‐NUTM1 fusion arising in soft tissue of the head in a 60‐year‐old man. Immunohistochemistry revealed strong expression of NUT, but only weak ETV4 staining and negativity for keratins, EMA, p40, CD99, and WT1. SMARCB1 expression was retained. Fluorescence in situ hybridization and targeted next‐generation sequencing identified a CIC‐NUTM1 fusion resulting from t(15;19)(q14;q13.2). In light of morphologic features that overlap with those of NC from typical anatomical sites we have seen previously, the tumor was best classified as falling within the NC spectrum rather than CIC‐associated sarcoma. This case highlights the emerging diagnostic challenges generated by newly detected gene fusions of unknown clinical and biologic significance. Careful integration of cytogenetic, molecular, and immunohistochemical findings with morphologic appearances in the diagnostic workup of undifferentiated neoplasms is essential.