Anti-PD-1 mAb pre-radiotherapy (RT) loading dose and fractionated RT induce better tumor-specific immunity and tumor shrinkage than sequential administration in an HPV+ head and neck cancer model

Radiotherapy (RT) is a standard therapeutic strategy in the treatment in head and neck cancer (HNC), but many patients still experience recurrence and metastasis. Interestingly, radiotherapy (RT) may also induce immunomodulatory effects. Given the recent, exciting responses seen using anti-PD-1 (programmed death-1) checkpoint blockade immunotherapy in recurrent/metastatic disease, including HNC, we evaluated the combination of RT with anti-PD-1 therapy in a pre-clinical mouse model of locally advanced, untreated HPV-positive HNC. We compared utilizing PD-1 blockade before, during or after RT, as well as whether a single large faction (12Gy) or multiple smaller RT doses (2 Gy X 10 fractions) confers optimal antitumor immune responses and tumor shrinkage. We observed that fractionated doses of RT induced the highest PD-L1 (programmed death-ligand 1) expression on HNC cells in vitro and in treated mice. A loading dose of anti-PD-1 mAb therapy prior to RT appeared to be important for best therapeutic outcome, with greatest tumor response and tumor-specific immunity using PD-1 Ab loading dose than sequential administration of anti-PD-1 mAb after fractionated RT (p < 0.0001). Expression and intensity of PD-1 receptor expression on circulating T cells differentially impacted the T cell phenotype and anti-tumor outcome, with loss of PD-1(high) exhausted T cells during the best tumor response (p < 0.05). The combination of fractionated RT + anti-PD-1 Ab optimally upregulated the frequency of HPV E7 tumor antigen-specific T cells (p < 0.05). This study may facilitate strategies required for the combination of RT and immune checkpoint inhibitor in clinical trials, enabling more effective clinical activity and biomarker evaluation

A Polymorphic Variant of AFAP-110 Enhances cSrc Activity12

Enhanced expression and activity of cSrc are associated with ovarian cancer progression. Generally, cSrc does not contain acti- vating mutations; rather, its activity is increased in response to signals that affect a conformational change that releases its auto- inhibition. In this report, we analyzed ovarian cancer tissues for the expression of a cSrc-activating protein, AFAP-110. AFAP-110 activates cSrc through a direct interaction that releases it from its autoinhibited conformation. Immunohistochemical analysis re- vealed a concomitant increase of AFAP-110 and cSrc in ovarian cancer tissues. An analysis of the AFAP-110 coding sequence revealed the presence of a nonsynonymous, single-nucleotide polymorphism that resulted in a change of Ser403 to Cys403. In cells that express enhanced levels of cSrc, AFAP-110403C directed the activation of cSrc and the formation of podosomes indepen- dently of input signals, in contrast to wild-type AFAP-110. We therefore propose that, under conditions of cSrc overexpression, the polymorphic variant of AFAP-110 promotes cSrc activation. Further, these data indicate a mechanism by which an inherited genetic variation could influence ovarian cancer progression and could be used to predict the response to targeted therapy

Strategies for Discovery of Small Molecule Radiation Protectors and Radiation Mitigators

Mitochondrial targeted radiation damage protectors (delivered prior to irradiation) and mitigators (delivered after irradiation, but before the appearance of symptoms associated with radiation syndrome) have been a recent focus in drug discovery for (1) normal tissue radiation protection during fractionated radiotherapy, and (2) radiation terrorism counter measures. Several categories of such molecules have been discovered: nitroxide-linked hybrid molecules, including GS-nitroxide, GS-nitric oxide synthase inhibitors, p53/mdm2/mdm4 inhibitors, and pharmaceutical agents including inhibitors of the phosphoinositide-3-kinase pathway and the anti-seizure medicine, carbamazepine. Evaluation of potential new radiation dose modifying molecules to protect normal tissue includes: clonogenic radiation survival curves, assays for apoptosis and DNA repair, and irradiation-induced depletion of antioxidant stores. Studies of organ specific radioprotection and in total body irradiation-induced hematopoietic syndrome in the mouse model for protection/mitigation facilitate rational means by which to move candidate small molecule drugs along the drug discovery pipeline into clinical development

A genetic variant of the adaptor protein, AFAP -110, efficiently activates c-Src resulting in podosome formation.

The actin filament associated protein of 110 kDA (AFAP-110) is a well-characterized protein composed of amino-terminal binding motifs that function upstream of a carboxy-terminal actin binding domain. In vitro studies demonstrate that the amino-terminal pleckstrin homology 1 (PH1) domain interacts with activated PKCα, relaying signals that activate Src family kinases via SH3 and SH2 binding. Serving as an adaptor protein, AFAP-110 relays signals that result in the assembly of motility structures. These findings could be of physiological as well pathological significance as AFAP-110 colocalizes with SFKs in the developing brain as well as in the glial-derived tumor, glioblastoma multiforme. More recently, AFAP-110 and c-Src expression were analyzed in ovarian cancer. While c-Src exhibits a diffuse expression pattern, AFAP-110 localized focally. Interestingly, colocalization between AFAP-110 and c-Src always occurs in desmoplastic regions of the tumor. In order to determine if a genetic variant of AFAP-110 exists, a PCR analysis of AFAP cDNA derived from ovarian cancer cell lines, ovarian tumor sections, as well as normal myometrium revealed a nucleotide change of G1426C resulting in an amino-acid change of serine (S) to cysteine (C) at position 403 of AFAP-110 (AFAP-110403S/C). This SNP was found to exist in approximately 25% of the samples. Molecular modeling predicts that the tertiary structure of AFAP-110403S/C could be contorted in a manner that facilitates altered protein binding. This is supported by GST-absorption studies that suggest that AFAP-110403S/C may differ from AFAP WT in its ability to self-associate. Altered self-association could present AFAP in a conformation that facilitates c-Src activation. In a model system that mimics the level of c-Src over-expression in ovarian cancer, transient transfection of AFAP-110403S/C results in the activation of c-Src and the formation of podosomes. This could be of clinical significance as the generation of invasive motility structures could facilitate tumor progression. In addition, the efficient activation of c-Src by AFAP-110403S/C could contribute to rapid development of paclitaxel resistance. Therefore, the genetic variant of AFAP-110 could be used in a gene assay to predict those most likely to exhibit rapid tumor progression as well as those most likely to develop chemotherapy-resistance

Salvage stereotactic body radiotherapy for locally recurrent non-small cell lung cancer after sublobar resection and I125 vicryl mesh brachytherapy

Purpose: Locally-recurrent non-small cell lung cancer (LR-NSCLC) remains challenging treat, particularly in patients having received prior radiotherapy. Heterogeneous populations and varied treatment intent in existing literature result in significant limitations in evaluating efficacy of lung re-irradiation. In order to better establish the impact of re-irradiation in patients with LR-NSCLC following high-dose radiotherapy, we report outcomes for patients treated with prior sublobar resection and brachytherapy that subsequently underwent stereotactic body radiotherapy (SBRT).Methods: A retrospective review of patients initially treated with sublobar resection and I125 vicryl mesh brachytherapy, who later developed LR-NSCLC along the suture line, was performed. Patients received salvage SBRT with curative intent. Dose and fractionation was based on tumor location and size, with a median prescription dose of 48 Gy in 4 fractions (range 20-60 Gy in 1-4 fractions).Results: Thirteen consecutive patients were identified with median follow-up of 2.1 years (range 0.7-5.6 years). Two in-field local failures occurred at 7.5 and 11.1 months, resulting in 2-year local control of 83.9% (95% CI 63.5-100.0%). Two-year disease-free survival and overall survival estimates were 38.5% (95% CI 0.0-65.0%) and 65.8% (95% CI, 38.2-93.4%). Four patients (31%) remained disease-free at last follow-up. All but one patient who experienced disease recurrence developed isolated or synchronous distant metastases. Only one patient (7.7%) developed grade ≥3 toxicity, consisting of grade 3 esophageal stricture following a centrally located recurrence previously treated with radiofrequency ablation.Conclusion: Despite high local radiation doses delivered to lung parenchyma previously with I125 brachytherapy, re-irradiation with SBRT for LR-NSCLC results in excellent local control with limited morbidity, allowing for potential disease cure in a subset of patients