Morphologic and molecular study of lung cancers associated with idiopathic pulmonary fibrosis and other pulmonary fibroses

Idiopathic pulmonary fibrosis (IPF) is a chronic parenchymal lung disease of severe prognosis, with a median survival of about 3 years from diagnosis [1]. An increased incidence of lung cancer has been described in IPF patients, with a significantly adverse impact on survival [2‐6]. IPF and lung cancer are both strongly associated with tobacco-smoking. Incidence of lung cancer is also increased in non-idiopathic pulmonary fibrosis suggesting a role for inflammation and fibrosis in the development of lung tumors [7]. Common pathogenic pathways and epigenetic alterations have been described in both IPF and cancer but specific molecular analysis of lung fibrosis-associated tumors has not been published so far [8].

Lung cancer in IPF patients is a therapeutic challenge as both surgery and radiotherapy are limited by lung dysfunction and are at high risk of respiratory exacerbation. Moreover chemotherapy can also be deleterious [5, 9]. However, over the past decade a better knowledge of lung cancer biology led to major changes in the management of lung cancer patients. Targeted therapies based on biomarkers have shown clinical success. Genetic alterations differ according to histologic subtypes. In adenocarcinoma (ADC), the most common cancer type, molecular characterization is now an established procedure before any therapeutic decision [10]. In squamous cell carcinoma (SCC), some targets have been identified but need to be validated [11]. Molecular alterations in oncogenes may confer constitutive activation and oncogenic addiction as for EGFR, the first target identified in lung ADC. More recently mutated BRAF and MET were also demonstrated to be addictive oncogenes. Finally, gene fusions, for instance ALK and ROS1 are other molecular mechanisms leading to oncogene activation and are validated targets [12]. In parallel identification of the tumor immune-evasion mechanisms is the basis for innovative therapies, particularly targeting the PD-1/PD-L1 pathway. Although in need of standardization, PD-L1 expression as detected by immunohistochemistry may be a predictive biomarker of anti PD-1/PD-L1 drug’s efficacy [13].

The aim of this study was to describe a retrospective cohort of lung cancers developed on IPF and other pulmonary fibroses, and to search for molecular alterations that could either represent therapeutic targets or specific oncogenic pathways in these interstitial lung diseases (ILD).

The aim of this study was to describe a cohort of lung cancers developed on IPF and other pulmonary fibroses, and to characterize their molecular alterations. SCC was the most frequent histologic subtype in our IPF group, as mostly reported in previous studies encompassing a large period of time [3, 21]. This squamous histology could suggest specific oncogenic events in the IPF micro-environment where peripheral honeycomb-associated squamous metaplasia and dysplasia has been reported [22]. In contrast, ADC was the most frequent subtype in the heterogeneous non-IPF group, like in the general population. Acinar subtype was the most frequent ADC subtype in our cohort (43%), and invasive mucinous subtype was rare (7%), as reported in a 89 idiopathic interstitial pneumonia-associated ADC cases recent Japanese series (35.95% and 11.24% respectively), described by Kojima [23]. In another recent Japanese series on 44 UIP-associated ADC reported by Masai, invasive mucinous subtype was predominant (29.5% of ADC) [6].

We detected TP53 mutations in 8 SCC (80% of SCC) and 6 ADC (50% of ADC), with the same frequency as reported in the literature [11]. We also detected TP53 mutations in all other cancer subtypes. Allelic ratios suggest a loss of the second TP53 allele, as usually in cancers [24]. Detected mutations occurring in the DNA binding domain (from codon 125 to 300), especially the hotspot codons in CpG sites, are similar to those already described, according to the COSMIC public database [16]. More than one third are G > T transversions, in accordance with the high proportion of smokers [25]. Thus a specific carcinogenesis process differing from tobacco smoke DNA signature and linked to chronic lung inflammation could not be inferred from this molecular analysis.

Four MET mutations were detected in our cohort: p.Arg359Gln and p.Arg988Cys in SCC (20%), p.Thr1011Ala in one ADC (8%) and c.2942-36G > A in one ADS. In the literature, MET mutations are reported in 2% to 7% of lung ADC and in 1% of lung SCC [12]. Codon 359 is located within the SEMA domain, involved in binding with the MET-specific ligand HGF. Codons 988 and 1011 are located in the exon 14, and c.2942-36G > A in the intronic region before the exon 14, required for negative regulation of MET. Mutations involving exon 14 splicing site have been described in lung ADC, they mostly result in exon 14 skipping and ultimately in MET protein stabilization [12, 26]. Case reports have demonstrated responses to MET-inhibitors in ADC patients with METex14 alterations [26]. METex14 mutations were, so far, not reported in lung SCC. These three exonic mutations have been described as rare polymorphisms. However their functional impact remains unclear as discordant results are obtained with pathogenicity prediction softwares. For instance p.Arg988Cys, although described as a germline polymorphism (rs34589476), has been reported in numerous lung cancers, and its pathogenic role remains elusive, in vitro data supporting functional consequences [27, 28]. Interestingly, in our cohort, three MET mutations occurred in IPF and 1 in CTD-ILD with an UIP pattern on CT-scan. Whether these variants represent true oncogenic drivers or significant polymorphisms in the fibrotic process, this could suggest a specific pathway in IPF/UIP lung with activation of the HGF/MET axis [29]. The search for MET mutations in non-tumoral IPF lung would be mandatory to test these hypotheses. Of note, we looked for mutations in flanking introns of exon 14 in only three cases. Thus we cannot exclude the possibility of more MET mutations. Whether such alterations could be targetable would deserve specific clinical trials.

A p.Trp259Arg DDR2 mutation was observed in an ADS. In the literature, DDR2 mutations are found in 4% of lung SCC and in 1% of lung ADC, without hotspot mutations. Clinical response to dasatinib was reported in rare case-reports of patients with lung SCC [30].

No mutation of EGFR was observed in our cohort, although reported in 10–15% of lung ADC [12]. This result, in addition to the absence of ALK and ROS1 rearrangement, is consistent with the predominance of male smokers in our cohort. Three recent Japanese studies also described a significantly lower EGFR mutation frequency in ILD/IPF patients [5, 6, 23].

Mutations involving the MAP kinase pathway are frequent in ADC [12]. We described a p.Gly469Ala BRAF mutation in a SCC (10% of SCC), a p.Lys601Glu and a p.Gly469Ala BRAF mutation in 2 ADC (17% of ADK). In the literature, BRAF mutations are reported in about 4% of lung SCC and in 10% of lung ADC [11, 12]. BRAF mutations p.Lys601Glu and p.Gly469Ala have already been described in lung ADC. Non-V600E mutations are usual, representing about half of BRAF mutations [31]. Conversely, p.Gly469Ala has never been described in lung SCC. Both are activating BRAF mutations. BRAF and MEK inhibitors can target p.V600E BRAF mutations [31, 32]. Response rates for lung cancer patients with non-V600 mutations are unknown. Only one ADC was KRAS mutated (representing 8% of adenocarcinomas) whereas KRAS mutations are reported in more than 30% of lung ADC [12], especially in smokers. While the absence of EGFR mutation could be explained by the high smoking rate in our population, the low incidence of KRAS mutations could suggest the implication of other oncogenic drivers possibly related to the chronic lung injury during the fibrotic process. Interestingly the recent series described by Masai et al. included frequent invasive mucinous ADC (29,5%), associated with numerous KRAS mutations (30,2%) [6]. This could suggest carcinogenesis differences linked to ethnicity or be the reflect of our limited number of patients. However these results were not confirmed by Kojima et al. who reported a low rate of invasive mucinous subtype (11,24%) and no difference of KRAS mutation rate between non-UIP-ADC and UIP-ADC [23].

One SMAD4 mutation was found in one SCC-IPF tumors. SMAD4 is a tumor-suppressive gene that can cause cell cycle arrest and apoptosis of epithelial cells, and is inactivated by mutation in over half of pancreatic cancers [33]. It acts as a central mediator in the transforming growth factor-β (TGF-β) signalling pathway. SMAD4 mutations are uncommon in lung cancer, according to COSMIC database. However this signalling pathway, targeted by TGF-beta, could be of particular relevance in a lung fibrosis context. pSer343* predicted as pathogenic is located in the MH2 region which is implicated in the oligomerization of the protein which is essential for TGFbeta signalling [34].

A p.Ser37Cys CTNNB1 mutation was detected in an ADC (8%). The codon 37 is a known hotspot mutation, implied in the constitutive activation of the Wnt signalling pathway, and the p.Ser37Cys mutation has been reported in lung ADC [35]. Mutated beta-catenin (CTNNB1) accumulation is followed by translocation to the nucleus and action in a transcriptional complex involving other transcriptional regulators like YAP1 to modulate apoptosis, proliferation or epithelial-mesenchymal transition [36].

A p.His470Tyr FBXW7 mutation was detected in a SCC (10%). FBXW7 mutations are uncommon in lung cancer, according to COSMIC. FBXW7 is implicated in proteasome degradation of specific substrates and control tumorigenesis, acting on cell cycle, differentiation and apoptosis [37]. It is also involved in epithelial-to-mesenchymal transition by controlling mTOR pathway [38]. A p.Arg465His FBXW7 mutation was reported in a lung ADC; the patient benefited from the mTOR inhibitor temsirolimus [39].

Besides molecular targeted therapies, immunotherapy using checkpoint inhibitors is a new efficient therapy against lung cancer. PD-L1 is an immune-checkpoint protein, interacting with its ligand PD-1 expressed by T-cells, used by the tumoral cell to escape the antitumor immune response. Several drugs target the PD-1/PD-L1 interaction. An association between therapeutic response and PD-L1 expression on tumor cells has been described, although it is not a binary predictive marker and the PD-L1 assays need further standardization and validation [13]. PD-L1 expression was assessed in 16 surgical cases in the current work. All ADC but one had less than 5% of stained tumor cells, which, in addition to the pulmonary adverse effects of these molecules, may not plead for a first-line use of immunotherapy in these patients. This has to be investigated in larger series. As far as SCC are concerned PD-L1 expression seems to be less correlated to efficacy, at least in second-line of treatment [40].

We report here for the first time, to our best knowledge, an extensive pathological and molecular analysis of lung fibrosis-associated lung cancers. We found potentially actionable alterations in MET, FGFR3, ERBB4, DDR2, EGFR, BRAF, PI3KCA genes in various histologic subtypes. While most detected mutations are likely tobacco-associated TP53 mutations, others may suggest alternative oncogenesis mechanisms: notably we found MET, FGFR3, SMAD4 and CTNNB1 mutations, all genes that could potentially be involved in the lung fibrosis process, either participating to epithelial-mesenchymal transition or the regulation or TGFβ pathway. Conversely, the low prevalence of KRAS mutations, contrasting with the high percentage of smokers, also supports a role for endogenous carcinogenic mechanisms linked to lung fibrosis. Although limited by the size of the cohort, our series shows the feasibility of such systematic molecular characterization, for both therapeutic and pathophysiological purposes. The high mortality of fibrotic lung diseases implies that cancer remains a rare complication since possibly occurring late in the course of fibrosis. Two recently approved drugs, pirfenidone and nintedanib, have been shown to slow IPF progression [41], and are expected to extend survival. If confirmed this may lead to an increase of challenging cancer cases and encourage to perform a large molecular characterization to every lung fibrosis-associated cancer.