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

Background Primitive lung cancers developed on lung fibroses are both diagnostic and therapeutic challenges. Their incidence may increase with new more efficient lung fibrosis treatments. Our aim was to describe a cohort of lung cancers associated with idiopathic pulmonary fibrosis (IPF) and other lung fibrotic disorders (non-IPF), and to characterize their molecular alterations using immunohistochemistry and next-generation sequencing (NGS). Methods Thirty-one cancer samples were collected from 2001 to 2016 in two French reference centers for pulmonary fibrosis - 18 for IPF group and 13 for non-IPF group. NGS was performed using an ampliseq panel to analyze hotspots and targeted regions in 22 cancer-associated genes. ALK, ROS1 and PD-L1 expressions were assessed by immunohistochemistry. Results Squamous cell carcinoma was the most frequent histologic subtype in the IPF group (44%), adenocarcinoma was the most frequent subtype in the non-IPF group (62%). Forty-one mutations in 13 genes and one EGFR amplification were identified in 25 samples. Two samples had no mutation in the selected panel. Mutations were identified in TP53 (n = 20), MET (n = 4), BRAF (n = 3), FGFR3, PIK3CA, PTEN, STK11 (n = 2), SMAD4, CTNNB1, DDR2, ERBB4, FBXW7 and KRAS (n = 1) genes. No ALK and ROS1 expressions were identified. PD-L1 was expressed in 10 cases (62%) with only one (6%) case >50%. Conclusions This extensive characterization of lung fibrosis-associated cancers evidenced molecular alterations which could represent either potential therapeutic targets either clues to the pathophysiology of these particular tumors. These findings support the relevance of large molecular characterization of every lung fibrosis-associated cancer.


Background
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][3][4][5][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).

Patients and tumors
Cases of lung fibrosis-associated lung cancer diagnosed between 2001 and 2016 were identified from clinical and pathological databases of Bichat-Claude Bernard and Georges Pompidou University hospitals (Paris, France), which are both "Competence Centers for rare pulmonary disorders". Formalin-fixed and paraffin-embedded (FFPE) samples were retrieved from Pathology department archives. Two pathologists (AC, AG) reviewed all samples to confirm diagnoses of lung fibrosis and cancer. Cancers were classified according to the 2015 WHO Classification of Lung Tumors [14]. IPF and Idiopathic Interstitial Pneumonias were diagnosed according to American Thoracic Society-European Respiratory Society consensus criteria [1,15]. The relationship between tumor and UIP lesions was assessed on 2 slides/tumor on surgical cases of the IPF group. This study was reviewed and approved by the CEERB Paris Nord ethics committee, under the number 16-007.

Next-generation sequencing
The percentage of tumor cells was assessed by two pathologists (AC, AG), in a macrodissection area if required. DNA extraction from FFPE tissues was performed using Maxwell® 16 (Promega, Fitchburg, Wisconsin). DNA was quantified by Qubit® 2.0 Fluorometer (Qubit® dsDNA BR Assay kit-Life Technologies-Thermo Fisher Scientific, Saint Aubin, France). Sequencing libraries were prepared from tumor FFPE DNA using Ion AmpliSeq™ Colon and Lung Cancer Research Panel V2 (Life Technologies-Thermo Fisher Scientific). This panel targets over 500 hotspot mutations in 22 colon and lung cancer-associated genes: AKT BRAF CTNNB1 EGFR ERBB2 ERBB4 FBXW7 FGFR1 FGFR2 FGFR3 KRAS MET NOTCH1 NRAS PIK3CA PTEN SMAD4 STK11 TP53 ALK DDR2 MAP2K1. The multiplex barcoded libraries were generated with Ion AmpliSeq Library kit from 3-μL of DNA corresponding to 10-30ng. Using NGS data, we developed an algorithm that was used to test the presence of gene amplifications in this series. Amplifications were subsequently validated by qPCR.

Statistical analysis
Continuous variables are described by their mean and SD, and compared by use of Student's t-test. Categorical variables are described by percentages and compared by Fisher's exact test. Statistical analysis used Prism 5 (GraphPad Software, La Jolla, CA). P < 0.05 was considered statistically significant.

Patients
Thirty-one tumor samples were collected from 30 patients (Table 1). Eighteen were collected from patients diagnosed with IPF and 13 from patients suffering from other lung fibrotic disorders: connective tissue disease-associated interstitial lung disease (CTD-ILD) n = 6, idiopathic non-specific interstitial pneumonia n = 2, pneumoconiosis n = 4, drug-induced lung fibrosis n = 1.
Immunohistochemistry PD-L1 expression was assessed in all surgical resections and in the autopsy specimen, corresponding to 16 cases (6 SCC, 7 ADC and 3 ADS). Among them, 6 had less than 1% of stained tumor cells, 3 had 1% to <5%, 6 had 5% to <50% and one ADC had more than 50% of stained tumor cells. Overall, 10 tumors (62%) should be considered as expressing tumor cell membrane PD-L1 antigen in more than 1% of cells (Table 2 and Fig. 1d), and one (6%) with a high level of expression.
ALK and ROS1 expression was assessed in all ADC from surgical resections and autopsy specimen (n = 10). For two other patients, ALK expression was assessed during the patient management (P9 and P12). In all tested cases, ALK and ROS1 were negative.

Discussion
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 honeycombassociated 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].
Among the genes assessed in the NGS panel, we detected 43 mutations in 13 genes and an EGFR gene amplification in 25 samples.
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  [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 CTscan. 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-tomesenchymal 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 Tcells, 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].

Conclusion
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.