Major pathologic response on biopsy (MPRbx) in patients with advanced melanoma treated with anti-PD-1: evidence for an early, on-therapy biomarker of response.

BACKGROUND: With increasing anti-PD-1 therapy use in patients with melanoma and other tumor types, there is interest in developing early on-treatment biomarkers that correlate with long-term patient outcome. An understanding of the pathologic features of immune-mediated tumor regression is key in this endeavor. MATERIALS AND METHODS: Histologic features of immune-related pathologic response (irPR) following anti-PD-1 therapy were identified on hematoxylin and eosin (H&E)-stained slides in a discovery cohort of pre- and on-treatment specimens from n = 16 patients with advanced melanoma. These features were used to generate an irPR score [from 0 = no irPR features to 3 = major pathologic response on biopsy (MPRbx, ≤10% residual viable tumor)]. This scoring system was then tested for an association with objective response by RECIST1.1 and overall survival in a prospectively collected validation cohort of pre- and on-treatment biopsies (n = 51 on-treatment at 4-week timepoint) from melanoma patients enrolled on the nivolumab monotherapy arm of CA209-038 (NCT01621490). RESULTS: Specimens from responders in the discovery cohort had features of immune-activation (moderate-high TIL densities, plasma cells) and wound-healing/tissue repair (neovascularization, proliferative fibrosis) compared to nonresponders, (P ≤ 0.021, for each feature). In the validation cohort, increasing irPR score associated with objective response (P = 0.009) and MPRbx associated with increased overall survival (n = 51; HR 0.13; 95%CI, 0.054-0.31, P = 0.015). Neither tumoral necrosis nor pretreatment histologic features were associated with response. Eight of 16 (50%) of patients with stable disease showed irPR features, two of which were MPRbx, indicating a disconnect between pathologic and radiographic features at the 4-week on-therapy timepoint for some patients. CONCLUSIONS: Features of immune-mediated tumor regression on routine H&E-stained biopsy slides from patients with advanced melanoma correlate with objective response to anti-PD-1 and overall survival. An on-therapy biopsy may be particularly clinically useful for informing treatment decisions in patients with radiographic stable disease. This approach is inexpensive, straightforward, and widely available.

Muscarinic signaling in ciliated tracheal epithelial cells: dual effects on Ca2+ and ciliary beating.

To examine cholinergic signal transduction pathways that modulate ciliary beat frequency (CBF), cultured ovine tracheal epithelial cells were imaged using a combination of phase-contrast (CBF) and fluorescence (Ca2+) microscopy techniques. In single cells, acetylcholine (ACh) transiently increased CBF and intracellular Ca2+ concentration ([Ca2+]i), mainly by Ca2+ release from internal stores, with a small delayed contribution from Ca2+ influx. Nicotinic agonists did not alter CBF or [Ca2+]i, whereas atropine blocked the ACh-stimulated transients, consistent with the involvement of muscarinic receptors. 4-Diphenylacetoxy-N-methylpiperidine methiodide was approximately 100 times more potent than pirenzepine in inhibiting the ACh-induced [Ca2+]i peaks, suggesting that the receptor is a pharmacologically defined (M3) subtype. Interestingly, after depletion of intracellular Ca2+ stores by thapsigargin, ACh caused a rapid transient decrease in both CBF and [Ca2+]i, again with an antagonist profile of M3 receptors. We conclude that activation of M3 muscarinic receptors initiates specific signaling pathways that act simultaneously to increase and decrease [Ca2+]i and CBF.