<p>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.</p>
<div>AbstractPurpose:<p>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.</p>Patients and Methods:<p>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/m<sup>2</sup> 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.</p>Results:<p>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.</p>Conclusions:<p>Further investigation of IL12 as an immunomodulatory agent in combination with cetuximab in head and neck squamous cell carcinoma is warranted.</p></div>
Abstract 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.
<p>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.</p>
Stroke results in varying levels of motor and sensory disability that have been linked to the neurodegeneration and neuroinflammation that occur in the infarct and peri-infarct regions within the brain. Specifically, previous research has identified a key role of the corticospinal tract in motor dysfunction and motor recovery post-stroke. Of note, neuroimaging studies have utilized magnetic resonance imaging (MRI) of the brain to describe the timeline of neurodegeneration of the corticospinal tract in tandem with motor function following a stroke. However, research has suggested that alternate motor pathways may also underlie disease progression and the degree of functional recovery post-stroke. Here, we assert that expanding neuroimaging techniques beyond the brain could expand our knowledge of alternate motor pathway structure post-stroke. In the present work, we will highlight findings that suggest that alternate motor pathways contribute to post-stroke motor dysfunction and recovery, such as the reticulospinal and rubrospinal tract. Then we review imaging and electrophysiological techniques that evaluate alternate motor pathways in populations of stroke and other neurodegenerative disorders. We will then outline and describe spinal cord neuroimaging techniques being used in other neurodegenerative disorders that may provide insight into alternate motor pathways post-stroke.
Abstract Myeloid derived suppressor cells (MDSC) interfere with anti tumor immune responses. MDSC have also been shown to antagonize the effectiveness of immune based therapies including immune checkpoint blockade. As a result, MDSC have received attention as potential targets for immune based combination therapies. There has been limited success in the identification of clinically active agents with the ability to inhibit the function or generation of MDSC. Ibrutinib is an orally available irreversible inhibitor of Bruton's tyrosine kinase (BTK) that is FDA approved for the treatment of B cell malignancies. In addition to B cells, cells of the myeloid lineage including monocytes and macrophages express BTK, and treatment with ibrutinib has been shown to alter their function and differentiation. As a result, it was hypothesized that ibrutinib would interfere with the function or generation of MDSC in the setting of cancer. MDSC isolated from the spleens of multiple murine tumor models (EMT6, 4T1, and C26) as well as MDSC from patients with metastatic melanoma expressed BTK. Treatment with ibrutinib at doses ranging from 0.1-5 μM inhibited the phosphorylation of BTK in both murine and human MDSC. Ibrutinib treatment of murine and human MDSC resulted in a significant reduction in nitric oxide (NO) production (p< 0.05), but had only modest effects on MDSC levels of IDO and arginase. Ibrutinib was also able to inhibit murine MDSC migration in response to EMT6 cell line conditioned media and the chemokine CXCL12 (p< 0.05). In addition, ibrutinib inhibited human MDSC migration in response to GM CSF (p< 0.05). Ibrutinib reduced the expression of the myeloid adhesion molecules CD11a (p< 0.05) and CD49D (p< 0.01) by MDSC, which could explain the reduction in migration. Importantly, ibrutinib significantly reduced the ability of MDSC to suppress CD8+ T cell proliferation compared to DMSO (21.98% vs. 12.49% proliferation, p< 0.05). Daily treatment with ibrutinib effectively inhibited the in vitro generation of human MDSC from monocytes by promoting HLA DR expression (p< 0.05). Using the EMT6 mammary carcinoma model in vivo, ibrutinib treatment resulted in a significant reduction of MDSC in both the spleen and tumor (p< 0.05). Ibrutinib also reduced the frequency of splenic MDSC in wild type B16F10 tumor bearing mice, but not in BTK mutant XID mice. In addition, both murine and human MDSC did not express significant levels of alternative ibrutinib targets including ITK, Bmx, and Blk. These results suggest that inhibition of BTK is the primary driver behind the observed effects of ibrutinib on MDSC function and generation. Finally, the combination of ibrutinib and anti PDL1 therapy was significantly more effective than either agent alone (p< 0.01 and p< 0.05) producing complete tumor regression in 50% of EMT6 tumor being mice. The results support further investigation of ibrutinib in combination with immune based therapies for solid tumors. Citation Format: Andrew R. Stiff, Prashant Trikha, Robert Wesolowski, Kari Kendra, Sarvani Uppati, David Abood, Elizabeth McMichael, Megan Duggan, Amanda Campbell, Natarajan Muthusamy, Susheela Tridandapani, Michael Caliguiri, John C. Byrd, William E. Carson. Ibrutinib, a BTK inhibitor, impairs the generation and function of myeloid derived suppressor cells. [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 553.
<div>AbstractPurpose:<p>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.</p>Patients and Methods:<p>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/m<sup>2</sup> 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.</p>Results:<p>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.</p>Conclusions:<p>Further investigation of IL12 as an immunomodulatory agent in combination with cetuximab in head and neck squamous cell carcinoma is warranted.</p></div>
