Notably we have characterized the mechanism of action

Notably, we have characterized the mechanism of action of ALK in the context of our current understanding of NLRP3 activation [5]. Non-priming macrophages, including BMDMs, exhibit no or minimal activation of NLRP3 in response to ATP. In contrast, LPS priming of macrophages makes them highly susceptible to ATP, which causes pyroptosis and IL-1β release. We demonstrated that ALK-mediated NF-κB activation is required for LPS-induced NLRP3 transcription during the priming stage. However, the mechanism responsible for LPS-induced ALK upregulation remains unknown. In some case, NLRP3 inflammasome can be activated independent of NF-κB transcription and dependent on NLRP3 phosphorylation [16]. Future work will aim to define the regulatory feedback loop between ALK upregulation, NLRP3 phosphorylation, and NF-κB activation during the priming process. The process of NLRP3 inflammasome assembly is complex and requires many proteins. Recently, NEK7 was identified as being essential for activation of the NLRP3 inflammasome through the protein-protein interaction [[20], [21], [22]]. NEK7-deficient macrophages show decreased pyroptosis and IL-1β production in response to NLRP3-activating stimuli such as ATP and nigericin [[20], [21], [22]]. Our current evidence suggests that ALK is not a direct component of NLRP3-NEK7 complex. Many signals such as K+ efflux, Ca2+ flux, and ROS have been implicated in the regulation of NLRP3-NEK7 formation depending on stimuli and context [[20], [21], [22],29]. We demonstrated that ALK-mediated lipid peroxidation is required for NLRP3-NEK7 formation, CASP1 activation, and IL-1β release. In particular, the lipid peroxidation-derived inflammatory mediator 4-HNE is responsible for NLRP3 activation. Increased serum levels of lipid peroxidation metabolite including 4-HNE and malondialdehyde have been observed in patients with infection and sterile inflammation including sepsis, diabetes, and neurodegenerative diseases [24]. 4-HNE has been shown to be highly immunogenic and proinflammatory [30,31]. Stimulation of lipid peroxidation may increase the intracellular calcium content and K+ efflux because ion ambroxol hydrochloride harbor reactive groups that are expected to be sensitive to ROS [32,33].
Conflicts of interest
Introduction Lung cancer is one of the most common tumor types (the fourth-prevalent in Italy, accounting for 11% of total diagnosed cancers) and, by far, the leading cause of cancer death among both men and women [1]. There are two major types of lung cancer, non-small cell lung cancer (NSCLC, 85% of lung tumors) and small cell lung cancer (SCLC, 15% of lung tumors) [1]. In recent years, there has been a major paradigm shift in the management of NSCLC. NSCLC can be now sub-classified by histology and driver mutation, if this is known or present [2]. In the last decade, relevant efforts have been addressed to detect mutations of the epidermal growth factor receptor (EGFR) and of the abnormal fusion of the anaplastic lymphoma kinase (ALK) [2]. It is estimated that 4% of NSCLCs have a rearrangement in ALK gene [1]. This genetic rearrangement is most often observed in non-smokers (or light smokers) with adenocarcinoma of NSCLC. The ALK gene rearrangement produces an abnormal ALK protein that causes the cells to grow and spread [3]. Detection of ALK mutation is key to treat NSCLC patients with crizotinib, to date the only ALK-targeted therapy approved in Italy, which has recently received an extension of reimbursement for the treatment of newly diagnosed ALK+ NSCLC patients. [4]. Several diagnostic technologies have been introduced to detect ALK rearrangement. For several years, Fluorescence In Situ Hybridization (FISH) has been considered the gold standard for detecting tumors carrying ALK rearrangement. However, the increasing compelling evidence shown by immunohistochemistry (IHC) in recent years is increasing the diagnostic opportunities of ALK detection, thus offering healthcare professionals an efficient tool to drive treatment choices [5]. Indeed, in its recommendations for crizotinib reimbursement, the Italian Drug Agency (AIFA) references both FISH and IHC as recommended tests to detect ALK+ tumors [6]. More specifically, the IHC tests AIFA is currently recommending are: Ventana ALK (D5F3) CDx (Ventana, from now on), recently developed by Roche Diagnostics; ALK1 Dako; ALK 5A4 Abcam. Ventana results are interpreted by using a binary scoring system (positive vs negative) which has been found an efficient predictor of ALK inhibition outcome, tumor response, and survival [7]. Unlike Ventana, ALK1 Dako and ALK 5A4 Abcam provide four possible results: 0 in case of absence of ALK protein expression; 1+ or 2+ in case of uncertain results; 3+ in case of presence of ALK protein expression. For ALK1 Dako and ALK 5A4 Abcam, a confirmation by one type of FISH test is required if 1+ or 2+ result is obtained, and this could require time, delay treatment and increase test costs. The objective of the present analysis is to evaluate the clinical and economic impact of the adoption of Ventana. As a matter of fact, the recent extension of reimbursement conditions for crizotinib and the expected introduction of new ALK target agents (such as alectinib, ceritinib, brigatinib and lorlatinib) [8] will increase adoption of ALK testing. Therefore, technology assessments of diagnostic tools in this area would inform policy makers about cost-effectiveness and economic sustainability of the alternative options.