Paul Zolkind

Biomarker and Tumor Responses of Oral Cavity Squamous Cell Carcinoma to Trametinib: A Phase II Neoadjuvant Window of Opportunity Clinical Trial

Purpose: Ras/MEK/ERK pathway activation is common in oral cavity squamous cell carcinoma (OCSCC). We performed a neoadjuvant (preoperative) trial to determine the biomarker and tumor response of OCSCC to MEK inhibition with trametinib. Experimental Design: Patients with stage II–IV OCSCC received trametinib (2 mg/day, minimum 7 days) prior to surgery. Primary tumor specimens were obtained before and after trametinib to evaluate immunohistochemical staining for p-ERK1/2 and CD44, the primary endpoint. Secondary endpoints included changes in clinical tumor measurements and metabolic activity [maximum standardized uptake values (SUVmax) by F-18 fluorodeoxyglucose positron emission tomography/CT), and in tumor downstaging. Drug-related adverse events (AE) and surgical/wound complications were evaluated. Results: Of 20 enrolled patients, 17 (85%) completed the study. Three patients withdrew because of either trametinib-related (n = 2: nausea, duodenal perforation) or unrelated (n = 1: constipation) AEs. The most common AE was rash (9/20 patients, 45%). Seventeen patients underwent surgery. No unexpected surgical/wound complications occurred. Evaluable matched pre- and posttrametinib specimens were available in 15 (88%) of these patients. Reduction in p-ERK1/2 and CD44 expression occurred in 5 (33%) and 2 (13%) patients, respectively. Clinical tumor response by modified World Health Organization criteria was observed in 11 of 17 (65%) evaluable patients (median 46% decrease, range 14%–74%). Partial metabolic response (≥25% reduction in SUVmax) was observed in 6 of 13 (46%) evaluable patients (median 25% decrease, range 6%–52%). Clinical-to-pathologic tumor downstaging occurred in 9 of 17 (53%) evaluable patients. Conclusions: Trametinib resulted in significant reduction in Ras/MEK/ERK pathway activation and in clinical and metabolic tumor responses in patients with OCSCC. Clin Cancer Res; 23(9); 2186–94. ©2016 AACR.

Checkpoint immunotherapy in head and neck cancers

Checkpoint inhibitors have recently gained FDA approval for the treatment of cisplatin-resistant recurrent and metastatic head and neck squamous cell carcinoma (HNSCC) by outperforming standard of care chemotherapy and inducing durable responses in a subset of patients. These monoclonal antibodies unleash the patient’s own immune system to target cancer cells. HNSCC is a good target for these agents as there is ample evidence of active immunosurveillance in the head and neck and a number of immune evasion mechanisms by which HNSCCs form progressive disease including via the PD-1/PD-L1 axis. As HNSCCs typically possess a moderately high mutation burden, they should express numerous mutation-derived antigen targets for immune detection. However, with response rates less than 20% in clinical trials, there is a need for biomarkers to screen patients as well as clinical trials evaluating novel combinations to improve outcomes. The aim of this review is to provide historical and mechanistic context for the use of checkpoint inhibitors in head and neck cancer and provide a perspective on the role of novel checkpoints, biomarkers, and combination therapies that are evolving in the near term for patients with HNSCC.

Cancer immunogenomic approach to neoantigen discovery in a checkpoint blockade responsive murine model of oral cavity squamous cell carcinoma

Head and neck squamous cell carcinomas (HNSCC) are an ideal immunotherapy target due to their high mutation burden and frequent infiltration with lymphocytes. Preclinical models to investigate targeted and combination therapies as well as defining biomarkers to guide treatment represent an important need in the field. Immunogenomics approaches have illuminated the role of mutation-derived tumor neoantigens as potential biomarkers of response to checkpoint blockade as well as representing therapeutic vaccines. Here, we aimed to define a platform for checkpoint and other immunotherapy studies using syngeneic HNSCC cell line models (MOC2 and MOC22), and evaluated the association between mutation burden, predicted neoantigen landscape, infiltrating T cell populations and responsiveness of tumors to anti-PD1 therapy. We defined dramatic hematopoietic cell transcriptomic alterations in the MOC22 anti-PD1 responsive model in both tumor and draining lymph nodes. Using a cancer immunogenomics pipeline and validation with ELISPOT and tetramer analysis, we identified the H-2Kb-restricted ICAM1P315L (mICAM1) as a neoantigen in MOC22. Finally, we demonstrated that mICAM1 vaccination was able to protect against MOC22 tumor development defining mICAM1 as a bona fide neoantigen. Together these data define a pre-clinical HNSCC model system that provides a foundation for future investigations into combination and novel therapeutics.

Neoantigens in immunotherapy and personalized vaccines: Implications for head and neck squamous cell carcinoma

The recent success of immunotherapies has demonstrated the potency of tumor-specific immune cells in mediating tumor rejection and generating durable tumor immunity. Our understanding of the scientific basis of these responses results from the confluence of a better comprehension of the cancer immunoediting process and the revolution in next generation sequencing of cancer genomes. Recent evidence suggests that T cell specificity for cancer cell expressed mutant proteins - termed neoantigens - is an important component of immune mediated tumor rejection. Improved neoantigen prediction algorithms have made it possible to predict and monitor immune responses to checkpoint inhibitors and adoptively transferred autologous lymphocytes and have enabled the development of tumor-specific therapeutic vaccines. Herein, we review the current research on cancer neoantigens in immunotherapies and its implications for the future of head and neck cancer management.

Rationale for neoadjuvant immunotherapy in head and neck squamous cell carcinoma

The clinical benefit of immunotherapy in recurrent, metastatic head and neck squamous cell carcinoma has fueled interest in revisiting neoadjuvant approaches to complement definitive treatment. Neoadjuvant strategies incorporating immune checkpoint inhibitors and other novel immune-based therapies in head and neck cancer are reviewed here, with particular attention paid to the rationale for these approaches from both a clinical and biologic discovery standpoint. The potential benefits of neoadjuvant immunotherapy include reduction of extent of surgery and the intensity of adjuvant therapy by tumor downstaging, reduction of the risk of distant metastatic spread by early introduction of systemic therapy, conversion of unresectable to resectable disease, and early evaluation of biomarkers of tumor response. We await early trial results utilizing these approaches to confirm both safety and initial efficacy in head and neck cancer.

Oral Cavity Squamous Cell Carcinoma Xenografts Retain Complex Genotypes and Intertumor Molecular Heterogeneity

SUMMARY Herein, we report an oral cavity squamous cell carcinoma (OCSCC) patient-derived xenograft (PDX) platform, with genomic annotation useful for co-clinical trial and mechanistic studies. Genomic analysis included whole-exome sequencing (WES) and transcriptome sequencing (RNA-seq) on 16 tumors and matched PDXs and additional whole-genome sequencing (WGS) on 9 of these pairs as a representative subset of a larger OCSCC PDX repository (n = 63). In 12 models with high purity, more than 90% of variants detected in the tumor were retained in the matched PDX. The genomic landscape across these PDXs reflected OCSCC molecular heterogeneity, including previously described basal, mesenchymal, and classical molecular subtypes. To demonstrate the integration of PDXs into a clinical trial framework, we show that pharmacological intervention in PDXs parallels clinical response and extends patient data. Together, these data describe a repository of OCSCC-specific PDXs and illustrate conservation of primary tumor genotypes, intratumoral heterogeneity, and co-clinical trial application.

Abstract PR01: Genomic and functional correlates from a phase II clinical trial of trametinib in surgically resectable oral cavity squamous cell carcinoma

Background: Neoadjuvant trametinib treatment of patients with locally advanced oral cavity squamous cell carcinoma (OSCC) demonstrates metabolic- and biomarker-based responses (Uppaluri et al., Clin. Can. Res, 2016). Patient response, defined as both metabolic response and decreased ERK phosphorylation in trametinib-treated tumors in comparison to baseline biopsies, was seen in 4/20 patients. We hypothesized that genomic and transcriptomic analyses of the pre- and post-treatment samples would provide insight into biomarkers of response for stratification of patients in future clinical trials. Methods: Tumor biopsies were collected at baseline and post-treatment at surgical resection. To generate patient-derived xenografts (PDX), biopsies from both baseline and post-treatment were implanted into immunodeficient NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. Whole exome and RNA sequencing (WES, RNAseq) were performed on baseline specimens and RNA-Seq was also performed on matched post-treatment tumor samples. Successful PDX models were also interrogated with whole exome and RNA-Seq. For PDX therapy, tumor bearing NSG mice were treated with trametinib (GSK1120212) via daily oral gavage (3 mg/kg/dose) or vehicle control and monitored for tumor growth. Results:WES—The mutational landscape of our cohort largely reflected the constellation of mutations present in the TCGA OSCC samples. Mutations in TP53 occurred in 15/20 (75%) of patients, CDKN2A in 8/20 (40%), NOTCH1 in 6/20 (30%), FAT1 in 5/20 (25%) and CASP8 in 5/20 (25%) of patients. Although gain of function mutations in RAS are associated with activation of the RAS/RAF/MEK/ERK pathway, no patients in our trial had RAS, RAF or ERK mutations. RNA-Seq—We identified crosstalk between the MEK/RAF/ERK pathway and the Hippo pathway. Responder patients had significantly higher YAP1 expression at baseline than the non-responders (p=0.0141). Furthermore, treatment with trametinib induced substantial reduction in Hippo pathway associated genes. Matched RNA-Seq analysis of two of three responder patients showed decreased expression of YAP1 and its downstream targets FOXM1 and BIRC5, known mediators of cell survival and metastasis. PDX—We successfully established PDXs in 22 of 39 tumor samples. Comparison of the WES in a subset of early passage PDXs to their matched primary tumors (n=9) demonstrated conservation with the primary tumor mutational content. To evaluate if treatment of the PDX model would reflect in vivo responsiveness of the primary tumor we treated the baseline biopsy PDX of a responder with daily trametinib or vehicle control (n=8 each). Consistent with findings in the patient, all PDXs treated with trametinib had significant decrease in tumor size after 14 days of daily treatment whereas the control treated tumors grew progressively (average tumor volume 52 mm3 and 714 mm3, respectively. p=0.013). As the 14-day short window phase did not allow full tumor responses to mature in patients, we modeled continuous treatment beyond 14 days in the PDX to evaluate for clinical response. Whereas 8/8 vehicle treated PDXs displayed progressive growth, 7/8 trametinib-treated PDXs were dramatically attenuated until eventual escape and outgrowth between days 40-60. Conclusion: In this genomic and functional analysis of samples from a trametinib window trial in OSCC, we identified YAP1 overexpression to be associated with clinical and biomarker response. Functional inhibition of this pathway with trametinib was identified in 2/3 patients with reductions in YAP1, FOXM1 and BIRC5 message. Finally, we demonstrated successful prospective establishment of PDX models that prove useful for confirming patient responses and for pathway specific molecular dissection. This abstract is also being presented as Poster 65. Citation Format: Paul A. Zolkind, Katie M. Campbell, Tianxiang Lin, Zach Skidmore, Ashley Winkler, Erica Barnell, Tusar Giri, Douglas R. Adkins, Malachi Griffith, Gavin P. Dunn, Obi L. Griffith, Ravindra Uppaluri. Genomic and functional correlates from a phase II clinical trial of trametinib in surgically resectable oral cavity squamous cell carcinoma [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 PR01.

Abstract CT070: Biomarker and clinical response of oral cavity squamous cell carcinoma to the MEK 1/2 inhibitor trametinib: A phase II neoadjuvant window of opportunity clinical trial

Purpose: Ras/MEK/ERK pathway activation is common in oral cavity squamous cell carcinoma (OCSCC). To determine biomarker and clinical tumor response of MEK inhibition in patients with OCSCC, we performed a neoadjuvant window of opportunity trial in which the MEK inhibitor trametinib was administered before surgery (NCT01553851). Patients and Methods: Patients with untreated Stage II-IV OCSCC were scheduled to receive trametinib 2 mg/day orally for 7-14 days prior to surgery (last dose 24 hours before surgery). Tumor specimens from the primary site obtained before and after trametinib underwent immunohistochemistry staining for p-ERK1/2 (a marker of Ras/MEK/ERK activation) and CD44 (a protein upregulated by ERK activation), which represented the primary endpoint. Secondary endpoints included comparison of changes in pre- and post-trametinib tumor measurement by clinical examination and in metabolic activity (SUVmax) by FDG-PET/CT (partial response: >25% reduction). Adverse events (AE) and surgical/wound complications were evaluated. Results: Of the 20 enrolled patients, 17 (85%) completed the study as planned. Three patients withdrew from the study due to AE, two (nausea; duodenal perforation) related to trametinib and one (constipation) related to narcotics. The most common drug-related AE was mild rash (9/20 patients, 45%). Nineteen patients (95%) underwent surgery and neck dissection with no unexpected surgical/wound complications. Fifteen patients (75%) were evaluable for the primary biomarker endpoint. 5 (25%) patients either had insufficient pre- or post-treatment biopsies or did not complete the trial. Reduction in p-ERK1/2 expression occurred in 7/15 evaluable patients (47%), whereas a reduction in CD44 occurred in 3/15 (20%). Reduction in tumor size (median 40%, range -74 to +17%) assessed by clinical examination was observed in 12/17 (71%) evaluable patients, and partial metabolic tumor response assessed by FDG-PET/CT was observed in 5/13 (38%) patients. Conclusions: Trametinib was safe to administer as a neoadjuvant treatment in patients with OCSCC and several patients displayed significant reduction in Ras/MEK/ERK pathway activation, and in clinical and metabolic tumor responses. Further exploration of trametinib response in OCSCC patients is warranted. Citation Format: Ravindra Uppaluri, Ashley Winkler, Tianxiang Lin, Jonathan Law, Bruce Haughey, Brian Nussenbaum, Randal Paniello, Jason Rich, Jason Diaz, Loren Michel, Tanya Wildes, Gavin Dunn, Dorina Kallogjeri, Paul Zolkind, Farrokh Dehdashti, Barry Siegel, Rebecca Chernock, James S. Lewis, Jay Piccirillo, Douglas Adkins. Biomarker and clinical response of oral cavity squamous cell carcinoma to the MEK 1/2 inhibitor trametinib: A phase II neoadjuvant window of opportunity clinical trial. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr CT070.

Abstract 5488: Novel death pathway in a p53 null human leukemia cell line treated with PNC-27

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Background: PNC-27 is a p53-derived peptide from the human double minute binding domain (HDM-2). This peptide has been shown to induce rapid tumor cell necrosis of selected cancer cells, while sparing their normal cellular counterparts (PNAS 2010; 107: 1918-1923). PNC-27 binds to HDM-2 in the cancer cell membrane, causing pore-formation and permeabilization of the membrane. We now propose an additional target for PNC-27 involving mitochondrial membrane-bound B-cell lymphoma-2 (Bcl-2), which contains a BH3 protein-protein interaction domain. BH3 serves as a potent mediator of cell death in pro-apoptotic proteins (such as Bax and Bad), as well as anti-apoptotic proteins, Bcl-2 and Bcl-XL. These Bcl-2 proteins have been proposed to control function and stability of the outer mitochondrial membrane. Methods: The anti-cancer activity and mechanism of PNC-27 (p53 aa12-26-penetratin) was studied against p53 null K562, a human leukemia cancer cell line. Necrosis was determined by measuring lactate dehydrogenase (LDH), while apoptosis was determined by measuring early markers of apoptosis (caspases 3, 7). Co-immunoprecipitation (IP) was performed between PNC-27 and subcellular components to identify potential targets for PNC-27 in K562 cells. HDM-2, Bcl-2, Cytochrome C, and ATP synthesis in PNC-27-treated cells were studied. Results: We found that PNC-27, but not control peptide PNC-29, is cytotoxic to K562 cells, inducing nearly 90% inhibition of proliferation with LDH release. PNC-27 peptide induced Cytochrome C release as well as downregulation of ATP synthesis. Furthermore, endoge