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Supplemental Figure S1. Levels of serum cytokines in patients over the course of treatment. Serum levels of the cytokines IFN-γ (A), IP-10 (B), TNF-α (C) MIP-1α (D), MIP-1β (E), RANTES (F), GM CSF (G), and IL-8 (H) were measured at baseline and during cycle 8 using a custom V-Plex assay. Assay was performed in triplicate and the average was plotted for each individual patient. *, p=0.0008 for IFN-γ, p=0.0011 for IP-10, p=0.0102 for TNF-α Supplemental Figure S2. Levels of serum cytokines in patients stratified by length of progression-free survival. Serum levels of the cytokines MIP-1α (A), MIP-1β (B), RANTES (C), GM-CSF (D), and IL-8 (E) were measured at baseline and during cycle 8 using a custom V-Plex assay. The assay was performed in triplicate and the average was plotted for each individual patient stratified by progression-free survival greater than or less than 100 days Supplemental Figure S3. Gating strategy for defining monocytic and granulocytic MDSC. Examples of monocytic-dominant (A) and granulocytic-dominant (B) total MDSC. PBMCs procured from pre-therapy blood draws were stained with anti-CD33-APC, anti-HLA-DR-PECy7, anti-CD11b-PE, anti-CD14-V-450, and anti CD15-FITC antibodies; CD33+/HLA-DR- populations were further characterized by CD15, CD14, and CD11b expression. Percentages gleaned from CD15+CD11b+ and CD14+CD11b+ quadrants were back multiplied by total MDSC (CD33+HLA-DR-) to obtain values presented in Table S3.

Supplemental Figure Legends

Purpose:mAbs including cetuximab can induce antibody-dependent cellular cytotoxicity (ADCC) and cytokine production mediated via innate immune cells with the ability to recognize mAb-coated tumors. Preclinical modeling has shown that costimulation of natural killer (NK) cells via the Fc receptor and the IL12 receptor promotes NK-cell–mediated ADCC and production of cytokines.Patients and Methods:This phase I/II trial evaluated the combination of cetuximab with IL12 for the treatment of EGFR-expressing head and neck cancer. Treatment consisted of cetuximab 500 mg/m2 i.v. every 2 weeks with either 0.2 mcg/kg or 0.3 mcg/kg IL12 s.c. on days 2 and 5 of the 2-week cycle, beginning with cycle 2. Correlative studies from blood draws obtained prior to treatment and during therapy included measurement of ADCC, serum cytokine, and chemokine analysis, determination of NK cell FcγRIIIa polymorphisms, and an analysis of myeloid-derived suppressor cell (MDSC) frequency in peripheral blood.Results:The combination of cetuximab and IL12 was well tolerated. No clinical responses were observed, however, 48% of patients exhibited prolonged progression-free survival (PFS; average of 6.5 months). Compared with patients that did not exhibit clinical benefit, patients with PFS >100 days exhibited increased ADCC as therapy continued compared with baseline, greater production of IFNγ, IP-10, and TNFα at the beginning of cycle 8 compared with baseline values and had a predominance of monocytic MDSCs versus granulocytic MDSCs prior to therapy.Conclusions:Further investigation of IL12 as an immunomodulatory agent in combination with cetuximab in head and neck squamous cell carcinoma is warranted.

Supplementary Table 1. Toxicities; Supplementary Table 2. Patient characteristics and response evaluation; Supplementary Table 3. Patient Correlative Study Results

Neuroblastoma RAS viral oncogene homolog is a commonly mutated oncogene in melanoma, and therapeutic targeting of neuroblastoma RAS viral oncogene homolog has proven difficult. We characterized the expression and phenotypic functions of five recently discovered splice isoforms of neuroblastoma RAS viral oncogene homolog in melanoma. Canonical neuroblastoma RAS viral oncogene homolog (isoform-1) was expressed to the highest degree and its expression was significantly increased in melanoma metastases compared to primary lesions. Isoform-5 expression in metastases showed a significant, positive correlation with survival and tumours over-expressing isoform-5 had significantly decreased growth in a xenograft model. In contrast, over-expression of any isoform resulted in enhanced proliferation, and invasiveness was increased with over-expression of isoform-1 or isoform-2. Downstream signalling analysis indicated that the isoforms signalled differentially through the mitogen-activated protein kinase and PI3K pathways and A375 cells over-expressing isoform-2 or isoform-5 showed resistance to vemurafenib treatment in vitro. The neuroblastoma RAS viral oncogene homolog isoforms appear to play varying roles in melanoma phenotype and could potentially serve as biomarkers for therapeutic response and disease prognosis.

Activating mutations in BRAF are found in 50% of melanomas and although treatment with BRAF inhibitors (BRAFi) is effective, resistance often develops. We now show that recently discovered NRAS isoform 2 is up-regulated in the setting of BRAF inhibitor resistance in melanoma, in both cell lines and patient tumor tissues. When isoform 2 was overexpressed in BRAF mutant melanoma cell lines, melanoma cell proliferation and in vivo tumor growth were significantly increased in the presence of BRAFi treatment. shRNA-mediated knockdown of isoform 2 in BRAFi resistant cells restored sensitivity to BRAFi compared with controls. Signaling analysis indicated decreased mitogen-activated protein kinase (MAPK) pathway signaling and increased phosphoinositol-3-kinase (PI3K) pathway signaling in isoform 2 overexpressing cells compared with isoform 1 overexpressing cells. Immunoprecipitation of isoform 2 validated a binding affinity of this isoform to both PI3K and BRAF/RAF1. The addition of an AKT inhibitor to BRAFi treatment resulted in a partial restoration of BRAFi sensitivity in cells expressing high levels of isoform 2. NRAS isoform 2 may contribute to resistance to BRAFi by facilitating PI3K pathway activation.

Purpose: mAbs including cetuximab can induce antibody-dependent cellular cytotoxicity (ADCC) and cytokine production mediated via innate immune cells with the ability to recognize mAb-coated tumors. Preclinical modeling has shown that costimulation of natural killer (NK) cells via the Fc receptor and the IL12 receptor promotes NK-cell–mediated ADCC and production of cytokines. Patients and Methods: This phase I/II trial evaluated the combination of cetuximab with IL12 for the treatment of EGFR-expressing head and neck cancer. Treatment consisted of cetuximab 500 mg/m2 i.v. every 2 weeks with either 0.2 mcg/kg or 0.3 mcg/kg IL12 s.c. on days 2 and 5 of the 2-week cycle, beginning with cycle 2. Correlative studies from blood draws obtained prior to treatment and during therapy included measurement of ADCC, serum cytokine, and chemokine analysis, determination of NK cell FcγRIIIa polymorphisms, and an analysis of myeloid-derived suppressor cell (MDSC) frequency in peripheral blood. Results: The combination of cetuximab and IL12 was well tolerated. No clinical responses were observed, however, 48% of patients exhibited prolonged progression-free survival (PFS; average of 6.5 months). Compared with patients that did not exhibit clinical benefit, patients with PFS >100 days exhibited increased ADCC as therapy continued compared with baseline, greater production of IFNγ, IP-10, and TNFα at the beginning of cycle 8 compared with baseline values and had a predominance of monocytic MDSCs versus granulocytic MDSCs prior to therapy. Conclusions: Further investigation of IL12 as an immunomodulatory agent in combination with cetuximab in head and neck squamous cell carcinoma is warranted.

Activating mutations in BRAF are found in 50% of melanomas and are associated with poor outcomes due to constitutive activation of the mitogen activated protein kinase (MAPK) pathway. Treatment with BRAF inhibitors (BRAFi), such as vemurafenib, is effective but resistance can develop. A major mechanism of resistance involves feedback loop-mediated increased expression of the NRAS gene. Recently, we discovered the NRAS transcript is differentially spliced to give 5 novel isoforms (isos), which differ in their expression, cellular localization and downstream effects. We hypothesized that differential expression of one or more of the isos may contribute to the development of BRAFi resistance. We assessed iso-specific NRAS expression in 14 MM cell lines and melanocytes, and noted a curious expression profile of iso2: while undetectable in melanocytes and BRAFwt MM cells, its abundance was increased in BRAFmut cells, and it was the only NRAS iso highly overexpressed in 3 BRAFi resistant cell lines (p This study indicates for the first time that NRAS iso2 may play a role in BRAFi resistance by facilitating alternative survival signaling through the PI3K pathway in the presence of MAPK pathway inhibition. Targeting NRAS iso2 may be a beneficial treatment strategy in the prevention and management of BRAFi resistance in melanoma. Citation Format: Megan Duggan, Andrew Stiff, Gonzalo Olaverria Salavaggione, Maryam Bainazar, Nicholas Latchana, Joseph Markowitz, Albert de la Chapelle, Ann-Kathrin Eisfeld, William Carson. Identification of NRAS isoform 2 overexpression as a novel mechanism facilitating BRAF inhibitor resistance in malignant melanoma (MM). [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 301.

Introduction: Preclinical investigations demonstrated that IL-12 significantly enhances the lytic actions and cytokine production of NK cells against cetuximab-coated HER1-positive SCCHN cell lines via synergistic activation of the MAP kinase pathway regardless of tumor cell HPV infection status. This phase I/II trial evaluated the combination of IL-12 with cetuximab to enhance NK cell effector mechanisms in patients with unresectable primary or recurrent squamous cell carcinoma of the head and neck. Patients/Methods: The phase I dose escalation portion of the trial included 6 patients that received 500 mg/m2 cetuximab i.v. on day 1 of a 2 week cycle with either 0.2 mcg/kg or 0.3 mcg/kg IL-12 given s.c. on days 2 and 5 of the 2 week cycle, beginning with cycle 2. The phase II portion of the trial included 17 patients that received a combination of 500 mg/m2 cetuximab i.v. plus 0.3 mcg/kg IL-12 s.c. (MTD) following the phase I schema. Correlative immune studies using patient blood and plasma obtained prior to and during therapy included measurement of antibody-dependent cytotoxicity (ADCC), serum cytokine analysis, determination of NK cell FcγRIIIa polymorphisms, and myeloid derived suppressor cell (MDSC) frequency. Results: 23 patients with unresectable primary or recurrent SCCHN were accrued (22 male, 1 female, avg. age 60.2 yrs). The combination therapy was well tolerated. Grade 4 AST and ALT increase was the only adverse event reported in the phase I portion which accrued 6 patients. The MTD was determined to be 0.3 mcg/kg s.c. 15 patients achieved stable disease for an average of 35 weeks (range 6-87). 60% of patients showed heightened ADCC from a baseline reading by the end of cycle 4, averaging an increase of 8.6% lysis. but there was not correlation with overall survival. Patients with progression-free survival (PFS) greater than 100 days showed an increased secretion of IFNγ, IP-10, MIP-1α, and TNF-α in sera from baseline to end of cycle 5, increasing by 81.7%, 53.3%, and 29.6% respectively. 6 patients carried the VV high affinity NK cell FcγRIIIa polymorphism, and PBMCs from these patients were able to better lyse cetuximab-coated SCCHN tumor cells compared to 10 patients with the FF low affinity receptor (25.7% lysis compared to 5.4% lysis at the 25:1 E:T ratio). MDSCs comprised an average of 5.3% of circulating cells. An analysis of MDSC incidence revealed that 15 of 23 majority of patients had a greater percentage of the monocytic subset of MDSCs versus the granulocytic MDSC subset, and these patients achieved an average of 28 weeks of progression free survival (PFS), compared to 13 week PFS in patients with a higher granulocytic MDSC population. Conclusions: These findings suggest that the addition of IL-12 to cetuximab may lead to enhanced efficacy through the induction of anti-tumor immunity. Citation Format: Elizabeth McMichael, Amanda Campbell, Megan Duggan, Tiffany Noel, Melanie Davis, Kallan Opheim, Kala Levine, Lakhvir Atwal, Gonzalo Olaverria Salavaggione, Akansha Gansu, Sarvani Uppati, Bonnie Paul, Thomas Olencki, Theodoros Teknos, Panayiotis Savvides, Stephen Liu, William Carson. A phase I/II trial of cetuximab in combination with interleukin-12 administered to patients with unresectable primary or recurrent squamous cell carcinoma of the head and neck. [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 CT143.