No abstract

Recapitulating human disease pathophysiology using genetic animal models is a powerful approach to enable mechanistic understanding of genotype-phenotype relationships for drug development. Na V 1.7 is a sodium channel expressed in the peripheral nervous system with strong human genetic validation as a pain target. Efforts to identify novel analgesics that are nonaddictive resulted in industry exploration of a class of sulfonamide compounds that bind to the fourth voltage-sensor domain of Na V 1.7. Due to sequence differences in this region, sulfonamide blockers generally are potent on human but not rat Na V 1.7 channels. To test sulfonamide-based chemical matter in rat models of pain, we generated a humanized Na V 1.7 rat expressing a chimeric Na V 1.7 protein containing the sulfonamide-binding site of the human gene sequence as a replacement for the equivalent rat sequence. Unexpectedly, upon transcription, the human insert was spliced out, resulting in a premature stop codon. Using a validated antibody, Na V 1.7 protein was confirmed to be lost in the brainstem, dorsal root ganglia, sciatic nerve, and gastrointestinal tissue but not in nasal turbinates or olfactory bulb in rats homozygous for the knock-in allele (HOM-KI). HOM-KI rats exhibited normal intraepidermal nerve fiber density with reduced tetrodotoxin-sensitive current density and action potential firing in small diameter dorsal root ganglia neurons. HOM-KI rats did not exhibit nociceptive pain responses in hot plate or capsaicin-induced flinching assays and did not exhibit neuropathic pain responses following spinal nerve ligation. Consistent with expression of chimeric Na V 1.7 in olfactory tissue, HOM-KI rats retained olfactory function. This new genetic model highlights the necessity of Na V 1.7 for pain behavior in rats and indicates that sufficient inhibition of Na V 1.7 in humans may reduce pain in neuropathic conditions. Due to preserved olfactory function, this rat model represents an alternative to global Na V 1.7 knockout mice that require time-intensive hand feeding during early postnatal development.

BACKGROUND: Programmed death ligand 1 (PD-L1) and its receptor, PD1, are involved in limiting immune response. In the context of cancer, tumor cells expressing PD-L1 can suppress the immune response of PD1-expressing tumor infiltrating lymphocytes (TILs). Disruption of this pathway with antibodies to either the ligand or the receptor has shown promise in the treatment of non small cell lung cancer, melanoma and renal cell cancer. Here we investigate the pathway in breast cancer. METHODS: PD-L1 protein expression was assessed on two Yale TMA breast cancer cohorts with two-fold redundancy by quantitative immunofluorescence (QIF) using AQUA technology. Cohort 1 (YTMA201) consists of 400 patients with has extensive follow-up and adjuvant treatment information. Cohort 2 (YTMA128) consists of 245 patients with limited follow-up and no treatment information. The PD-L1 antibody (Lieping Chen, clone 5H1) has been previously validated for specificity and reproducibility using transfected cell models. TILs were assessed by a pathologist for each cohort using a score from 0-3 based on the amount of TILs within the spot. AQUA scores for PD-L1 were used as a continuous variable and also cut at the median for outcome analysis. RESULTS: PD-L1 protein is positively correlated with TILs (p<0.0001 on cohort 1 and p = 0.0072 on cohort 2) and inversely correlated with estrogen receptor status (p<0.0001 on cohort 1 and p = 0.0188 on cohort 2) on both breast cancer cohorts examined (total n = 594). On cohort 1, PD-L1 protein expression is a marker of good prognosis as a continuous variable (p = 0.0271) as well as when cut at the median (p = 0.0151), particularly in the estrogen receptor positive subset of patients. CONCLUSIONS: PD-L1 expression in breast cancer is positively associated with TILs and inversely associated with estrogen receptor status on two independent breast cancer cohorts. PD-L1 protein expression shows prognostic value in breast cancer patients, particularly the ER positive subset of patients. Further assessment of PD-1 axis marker expression may be valuable as the associated therapeutics are being tested in breast cancer patients. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-10-02.