Clinicopathological and molecular features of lung cancers associated with cystic airspaces: an analysis of 34 cases

Introduction

Lung cancer remains the leading cause of cancer-related mortality worldwide (1). Most lung cancer lesions appear as solid masses or nodules, while some manifest as sub-solid with pure ground-glass or part-solid features (2). Lung cancer associated with cystic airspaces (LCCA) is rare (3). To date, there is not a definitive description or histopathological diagnosis of what this cystic component is. In cases of lung cancer with cystic airspaces observed on computed tomography (CT) scans, the imagery typically shows one or more cyst-like features, accompanied by areas of ground-glass opacity or consolidation that are either adjacent to, or interspersed among, the cystic areas. Although the Fleischner Society’s Glossary of Terms for Thoracic Imaging provides a definition for “cyst”, it does not currently offer a definition for this particular manifestation of lung cancer (4).

Unfortunately, while current medical guidelines provide recommendations for handling solid or ground-glass nodules identified in routine scans or lung cancer screening programs (5), they lack specific directives for the management of cystic lung lesions. LCCA often goes undetected on CT scans, resulting in a potential delay in diagnosis, particularly in the early stages of the disease (3).

Next-generation sequencing (NGS) technology allows high-throughput and high-sensitivity multigene sequencing. NGS is becoming more important in the study of tumor genetics and plays a key role in directing targeted therapy after surgery and in tracking the recurrence of early-stage lung cancer. By using NGS technology with a comprehensive panel that covers multiple signaling pathways, it is possible to examine the variations in oncogenes associated with different biological behaviors within the same histological type (6).

In this study, we collected 34 cases of LCCA patients and retrospectively investigated their clinicopathological characteristics. We also studied the molecular features of 34 LCCA cases using NGS, especially for their differences associated with various imaging features. By comparing with typical lung adenocarcinoma (LUAD), we revealed the molecular differences between the two. We present this article in accordance with the STROBE reporting checklist (available at https://jtd.amegroups.com/article/view/10.21037/jtd-24-1310/rc).

Methods

Ethical statement

The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The study was approved by the Ethics Committee for The Second Hospital of Dalian Medical University (No. SHDMUEC-2023-074) and individual consent for this retrospective analysis was waived.

Patient selection

With approval from the Ethics Committee of the Second Hospital of Dalian Medical University, Dalian, China, 34 consecutive patients diagnosed with LCCA between March 2017 and March 2023 were included in this study. The LCCA were classified by morphology based on preoperative CT scans, using a previously described modified classification system (7,8). Type I (thin-walled) described a cystic airspace with an average wall thickness <2 mm, while type II (thick-walled) applied to those circumferential thickening cystic airspaces with an average wall thickness ≥2 mm. Type III (a cystic airspace with a mural nodule) identified a cystic airspace containing a mural nodule (either endophytic or exophytic). Type IV (mixed) referred to solid or nonsolid tissue intermixed within clusters of multiple cystic airspaces. Patient information includes sex, age, histology, morphology, tumor location and recurrence.

NGS methods

Genomic DNA was extracted from formalin-fixed paraffin-embedded (FFPE) samples followed by sequencing library preparation according to published protocols (9). Hybridization capture-based targeted NGS, which was used to selectively target 437 cancer-specific genes, was performed on the Illumina Hiseq platform (Illumina, San Diego, CA, USA) with 2×150 bp pair-end reads. All the libraries reached the mean coverage depth >20×, and the percentage of mapped read >99.9%. In the bioinformatics analysis, quality control of FASTQ files was performed using Trimmomatic48 (10). The sequencing data were mapped to the hg19 human genome reference using the Burrows-Wheeler Aligner (BWA-mem, v0.7.12) (11). Detection of single nucleotide variants (SNVs) and insertions/deletions (indels) was carried out employing SCALPEL (http://scalpel.sourceforge.net) and the Genome Analysis Toolkit (GATK). The estimation of tumor purity was conducted using ABSOLUTE. Additionally, purity-adjusted gene-level and segment-level copy number variations (CNVs) were quantified using the CNV kit (12) and ADTEx tools (http://adtex.sourceforge.net).

The public LUAD dataset

The clinical records, somatic mutations, CNVs and histological images of 305 LUAD patients in East Asians were downloaded from OncoSG (https://src.gisapps.org/OncoSG/) under dataset ‘Lung Adenocarcinoma (GIS, 2019)’ (13). To compare the cystic LUAD cohort with the typical LUAD cohort, we have filtered out 437 genes from the typical LUAD dataset.

Statistical analysis

Fisher’s exact test was used to evaluate differences in the mutation rates. The Kaplan-Meier methods and log-rank test were applied to compare survival between groups. Survival analyses was conducted using the Cox’s proportional regression hazard ratio (HR) method. All P values were bilateral, with a difference of P<0.05 being statistically significant. All statistical analyses were conducted using SPSS 21.0 or R (R 3.6.3).

Results

Samples and clinical characteristics

The clinicopathological characteristics of all 34 patients with LCCA were analyzed (Table 1). Our series included 17 males and 17 females with a mean age of 61.80±8.88 years. Among them, 33 patients were diagnosed with adenocarcinoma (ADC) and only one patient was diagnosed with squamous cell carcinoma (SCC). With imaging analysis of these 34 patients, 17 cases were type I (50.0%), one case was type II (2.9%), one case was type III (2.9%), and 15 cases were type IV (44.1%). The location of the tumor was in the right upper lobe in 7 patients (20.6%), right middle lobe in 3 patients (8.8%), right lower lobe in 8 patients (23.5%), left upper lobe in 4 patients (11.8%) and left lower lobe in 12 patients (35.3%) respectively. Five patients (14.7%) experienced a recurrence and died during the observation period. Twenty-nine patients (85.3%) were alive and there was no evidence of recurrence.

Molecular features of LCCA

The LCCA patients’ DNA samples were subjected to genetic profiling using NGS by targeting 437 cancer-related genes. A total of 189 mutations was identified in 34 patients, averaging around six mutations per individual. The most common types of mutations were missense and nonsense mutations, comprising 64% (112/189) and 10.6% (20/189) of the mutations, respectively. The genes with the highest frequency of mutations were EGFR, TP53 and KRAS. EGFR mutations were most common, found in 61.8% (21/34) of cases. The most common EGFR mutations were L858R, exon 19 deletions, and L861R. TP53 mutations were found in 20.6% (7/34) of patients. KRAS mutations were less common, found in 14.7% (5/34) of patients. These were of four different types, including G12D, G12C, G12A and G13C (Figure 1A).

Figure 1 Distributions of gene alterations in LCCA and typical LUAD cohorts. (A) Alteration distribution, type, and frequency of genes in LCCA. (B) Alteration distribution, type, and frequency of genes in typical LUAD. LCCA, lung cancer associated with cystic airspaces; LUAD, lung adenocarcinoma.

Association between imaging features and molecular features in LCCA

We conducted a comparison to determine the frequency of differentially mutated genes across different imaging features in LCCA (Figure S1 and Table S1). Among the four LCCA patterns, minimal differences were observed between imaging and molecular features, potentially due to the small size of the LCCA cohort.

Comparison of genetic alterations between cystic LUAD and cystic LUAD

To further explore the differences in the molecular features between cystic LUAD and typical LUAD, the public available typical LUAD cohort of 305 patients (13) was used for the comparison with our cohort. We observed that EGFR (47.4%), TP53 (36.1%) and TTN (22.5%) were the top three mutated genes in typical LUAD (Figure 1B). Overall, the top mutated genes are slightly different between cystic LUAD and typical LUAD. In cystic LUAD cohort, EGFR alterations were the predominant alterations (61.8%), followed by TP53 alterations (20.6%) and KRAS alterations (14.7%). By comparing the gene mutation rates between the two cohorts, we identified 4 genes with statistically significant differences in their mutation frequencies (Figure 2 and Table S2). MET (P=0.01), GNAS (P=0.04), PMS2 (P=0.050) and PTCH1 (P=0.050) had significant higher mutation rates in cystic LUAD.

Figure 2 The frequency comparison of differentially mutated genes between LCCA and typical LUAD. *, P<0.05. LUAD, lung adenocarcinoma; LCCA, lung cancer associated with cystic airspaces.

Correlation between genomic alterations and patient survival

The univariate analysis focused on gene mutations that were present at a frequency exceeding 2% in the entire cohort. We found that the mutations of NF1 (8.8%) and RUNX1T1 (5.9%) were significantly associated with relapse-free survival (RFS) (P<0.001 and P=0.046, Figure S2A,S2B), and only the mutations of NF1 (8.8%) were significantly associated with overall survival (OS) (P=0.002, Figure S2C) (Table S3).

Discussion

LCCA represents a heterogeneous group of primary lung cancer that appears on CT as a cystic airspace with thickened (nodular) wall or cystic airspaces mixed with ground glass or consolidation. This uncommon presentation of lung cancer has been recognized as a reason for overlooked or postponed diagnoses. Being aware of this rare radiological presentation of lung cancer is essential for the early detection of the disease, which is important for prognosis and survival.

We conducted a retrospective study on the clinicopathological characteristics and molecular features of 34 patients with imaging features of LCCA to identify any correlation between them. In our study, patients diagnosed with LCCA had an average age of 62 years. At histologic examination, the majority of cases (97.1%) were ADC, which is consistent with previous studies (2,14). Tumors can develop in any lobe of the lung, with a higher probability of occurring in peripheral than central locations. In contrast with previous study (15), type I (50.0%) was the most common type followed by type IV (44.1%). The proportion of types II and III (2.9% respectively) in our study was consistently low, each at 2.9%. This difference in prevalence of cancer types may be due to the limited size of this study.

Several theories have been proposed to explain the origin of LCCA. Most of the cystic airspaces are reported to result from a check-valve mechanism, which may explain the gradual enlargement of cystic airspace size (8). Another proposed mechanism for the formation of cysts is known as cystification. This process occurs when tumors degrade due to vascular insufficiency with subsequent absorption of degenerated material (14). The majority of patients with LCCA were diagnosed with stage I disease (94.1%), which is consistent with previous study (2). This could be explained by detection bias or reporting bias. Detection bias might arise from the fact that LCCA was identified during a screening program for some patients. Reporting bias could occur as the tumor progresses and takes on a more mass-like appearance, possibly being misinterpreted as a cavity. Meanwhile, other lesions might change by losing their cystic features and turning entirely solid.

The use of NGS to analyze genomic profiles has become a powerful way to characterize the molecular features underlying disease pathology (16). Kobayashi et al. reported that the escalation in tumor malignancy requires additional activation of specific genes, mainly EGFR, KRAS, and TP53 (17). This aligns with the genes that were found to have the highest rate of alteration in our group, with EGFR alterations being the predominant alterations, followed by TP53 alterations and KRAS alterations. The high frequency of EGFR mutations may be due to the Asian cohort (18). However, we also discovered that the frequency of EGFR mutations in Asian LCCA (61.8%) was higher than in the Asian population with typical LUAD (47.4%). EGFR mutations have been demonstrated to play a role in the early stages of tumor development and are often found in early stage LUAD (19). In LCCA cohort, KRAS alterations were among the top three genetic variations, and KRAS mutations are also associated with decreased OS (20). In addition, TTN is among the top three mutated genes in typical LUAD. It was reported that LUAD patients with TTN mutations showed high levels of immune antigens and better prognosis (21,22). By comparing the NGS results of cystic LUAD and typical LUAD, four differentially mutated gene were identified. These genes, MET, GNAS, PMS2 and PTCH1, exhibited higher mutation rates in cystic LUAD. Even though some of the differentially mutated genes have been implicated in tumorigenesis or development (23,24), their mutations in cystic LUAD have not been systematically studied. Therefore, our NGS mutational analysis has played a role in investigating the molecular mechanisms underlying LCCA. The mutational analysis between the four LCCA patterns revealed that there were not much differences between imaging features and molecular features.

We also found the possible association between patient survival and mutations in two genes, NF1 and RUNX1T1. The NF1 gene functions as a tumor suppressor by inhibiting the Ras signaling pathways. Furthermore, the presence of NF1 mutation in NSCLC is associated with worse survival outcomes (25). In our study, NF1-mutated patients presented with worse RFS and OS than patients with the wild-type NF1gene. This finding aligns with the biological role of NF1. RUNX1T1 was believed to act as a tumor suppressor, with changes in its expression observed in various solid tumors. Notably, a decrease in RUNX1T1 expression in triple-negative breast tumors has been documented, along with its impact on the prognosis (26). In our cohort, patients with RUNX1T1 mutations had a worse RFS compared to patients with the wild-type RUNX1T1. Owing to the rarity of LCCA and loss to follow up, further studies with more patient data are necessary to confirm this result.

Several limitations to this study should be considered. First is that the study size was small due to the rarity of LCCA patients. A larger population and more genomic features should be studied in order to further understand the clinical characteristics and molecular features of LCCA. Secondly, this study was conducted retrospectively and was limited by the challenge of incomplete information, such as details regarding smoking history. This hindered our investigation into the link between smoking and LCCA. Furthermore, serial CT follow-up was insufficient, as both the number of our patients and the duration of observation cannot be compared to those of the other studies.

Conclusions

In summary, our retrospective investigation has provided insights into the clinicopathological and molecular characteristics of LCCA, enhancing our understanding of its features in medical research and practice. Using NGS approaches, we not only revealed the molecular characteristics of LCCA, but also analyzed the molecular features across different imaging presentations of LCCA, as well as comparing the molecular differences between LCCA and traditional LUAD. Overall, this study sheds light on the understanding of LCCA, which can further help classify this disease and stratify patients for more targeted treatment options.

Acknowledgments

Funding: This work was supported by the Special Fund for Clinical Research of Wu Jieping Medical Foundation under Grant 320.6750.2022-18-11.

Footnote

Reporting Checklist: The authors have completed the STROBE reporting checklist. Available at https://jtd.amegroups.com/article/view/10.21037/jtd-24-1310/rc

Data Sharing Statement: Available at https://jtd.amegroups.com/article/view/10.21037/jtd-24-1310/dss

Peer Review File: Available at https://jtd.amegroups.com/article/view/10.21037/jtd-24-1310/prf

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jtd.amegroups.com/article/view/10.21037/jtd-24-1310/coif). All authors report that this work was supported by the Special Fund for Clinical Research of Wu Jieping Medical Foundation under Grant 320.6750.2022-18-11. The authors have no other conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The study was approved by the Ethics Committee for The Second Hospital of Dalian Medical University (No. SHDMUEC-2023-074) and individual consent for this retrospective analysis was waived.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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