Oncogenic activation of BRAF fuels cancer growth by constitutively promoting RAS-independent mitogen-activated protein kinase (MAPK) pathway signalling. Accordingly, RAF inhibitors have brought substantially improved personalized treatment of metastatic melanoma. However, these targeted agents have also revealed an unexpected consequence: stimulated growth of certain cancers. Structurally diverse ATP-competitive RAF inhibitors can either inhibit or paradoxically activate the MAPK pathway, depending whether activation is by BRAF mutation or by an upstream event, such as RAS mutation or receptor tyrosine kinase activation. Here we have identified next-generation RAF inhibitors (dubbed 'paradox breakers') that suppress mutant BRAF cells without activating the MAPK pathway in cells bearing upstream activation. In cells that express the same HRAS mutation prevalent in squamous tumours from patients treated with RAF inhibitors, the first-generation RAF inhibitor vemurafenib stimulated in vitro and in vivo growth and induced expression of MAPK pathway response genes; by contrast the paradox breakers PLX7904 and PLX8394 had no effect. Paradox breakers also overcame several known mechanisms of resistance to first-generation RAF inhibitors. Dissociating MAPK pathway inhibition from paradoxical activation might yield both improved safety and more durable efficacy than first-generation RAF inhibitors, a concept currently undergoing human clinical evaluation with PLX8394.

Human Papillomavirus-16 (HPV-16) associated squamous carcinoma of the oropharynx has a favorable prognosis. Patients with HPV-16 positive cancers have elevated peripheral blood CD8+ T lymphocyte levels that correlate with response to chemotherapy and survival. Tumor infiltrating lymphocyte subpopulations (TIL) were assessed in pretreatment biopsies from a prospective patient cohort to determine if TIL subsets differed by HPV status, clinical factors, patient outcome or correlated with peripheral blood T cell levels. Methods Measured were CD8, CD4, CD68 and Treg (FoxP3) lymphocytes by immunohistochemistry in a tissue microarray created from patients (n=46) with advanced oropharynx cancer. Correlations with peripheral blood levels, HPV status, expression of EGFR, clinical tumor and patient characteristics and outcome were determined. Patients were treated with a single course of neoadjuvant chemotherapy (cisplatin, 5-fluorouracil) followed by either surgery (non-responders) or chemoradiation (cisplatin 100 mg/m2 every 3 weeks × 3; 70 Gy, 2 Gy daily × 7 wks) for responders. Median follow up was 6.6 years. Results HPV-16 positive patients had improved survival (p=0.016). Degree of T cell infiltration did not differ by HPV status but was significantly related to disease specific (DSS) and overall survival (OS). Higher infiltration by CD8, CD4 and FoxP3 subsets was significantly associated with lower T stage and survival. Even after adjusting for HPV status, CD8, FoxP3 and total T cells were significantly associated with DSS (p=0.0236; 0.0040; 0.0197) and OS (p=0.0137; 0.0158; 0.0115, respectively). Less T cell infiltration (p=0.0130) and CD4 cells in particular (p=0.0792) were associated with higher EGFR expression. FoxP3 infiltration correlated significantly and directly with CD4 and CD8 infiltration but not with peripheral blood levels. Conclusions Improved outcomes are associated with increased TILs independent of HPV status and suggest the local immune response to HPV-16 may be related in part to factors such as tumor size, EGFR expression, smoking history, performance status or innate immunity. Assessment of TILs in tissue microarrays is difficult due to small core sample size and variation in tumor representation in tissue cores. Further study of larger numbers of patients and infiltrates in whole tumor sections combined with functional analysis of individual subsets may be necessary to detect differences in local immunity in HPV-16 related cancers.

Diabetic neuropathy is a severe complication of long-standing diabetes and one of the major etiologies of neuropathic pain. Diabetes is associated with an increased formation of reactive oxygen species and the electrophilic dicarbonyl compound methylglyoxal (MG). Here we show that MG stimulates heterologously expressed TRPA1 in CHO cells and natively expressed TRPA1 in MDCK cells and DRG neurons. MG evokes [Ca2+]i-responses in TRPA1 expressing DRG neurons but is without effect in neurons cultured from Trpa1−/− mice. Consistent with a direct, intracellular action, we show that methylglyoxal is significantly more potent as a TRPA1 agonist when applied to the intracellular face of excised membrane patches than to intact cells. Local intraplantar administration of MG evokes a pain response in Trpa1+/+ but not in Trpa1−/− mice. Furthermore, persistently increased MG levels achieved by two weeks pharmacological inhibition of glyoxalase-1 (GLO-1), the rate-limiting enzyme responsible for detoxification of MG, evokes a progressive and marked thermal (cold and heat) and mechanical hypersensitivity in wildtype but not in Trpa1−/− mice. Our results thus demonstrate that TRPA1 is required both for the acute pain response evoked by topical MG and for the long-lasting pronociceptive effects associated with elevated MG in vivo. In contrast to our observations in DRG neurons, MG evokes indistinguishable [Ca2+]i-responses in pancreatic β-cells cultured from Trpa1+/+ and Trpa1−/− mice. In vivo, the TRPA1 antagonist HC030031 impairs glucose clearance in the glucose tolerance test both in Trpa1+/+ and Trpa1−/− mice, indicating a non-TRPA1 mediated effect and suggesting that results obtained with this compound should be interpreted with caution. Our results show that TRPA1 is the principal target for MG in sensory neurons but not in pancreatic β-cells and that activation of TRPA1 by MG produces a painful neuropathy with the behavioral hallmarks of diabetic neuropathy.

Background Despite better prognosis, there is a group of oropharyngeal squamous cell carcinoma (SCC) human papillomavirus (HPV)+ patients who experience treatment failure and succumb to distant metastasis. Methods Seventy-eight previously untreated patients nested in a concurrent chemoradiation protocol were reviewed to correlate patterns of local-regional tumor extent to distant metastasis. Biomarker assessment was: HPV in situ hybridization and epidermal growth factor receptor (EGFR) immunointensity. Results The 3-year disease-specific survival (DSS) for patients presenting with and without matted nodes was 69% and 94%, respectively (p = .003). Matted nodes were a poor prognostic factor independent of T classification, HPV, EGFR, and smoking status. For patients who were HPV+, 7 of 11 died of distant metastasis and 6 of 7 with distant metastasis had matted nodes. Conclusion Matted nodes are a novel marker of poor prognosis in oropharyngeal SCC independent of established prognostic factors. Matted nodes may identify patients at risk for the development of distant metastasis who could benefit from systemic therapy, whereas patients without matted nodes may be candidates for de-escalation of therapy.