Synchronous Multiple Pulmonary Ground-Glass Nodules That Arose from Different Lesions Rather than from Multiple Metastases of a Single Lesion

doi:10.21203/rs.3.rs-3140880/v1

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Research Article

Synchronous Multiple Pulmonary Ground-Glass Nodules That Arose from Different Lesions Rather than from Multiple Metastases of a Single Lesion

https://doi.org/10.21203/rs.3.rs-3140880/v1

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Purpose

This study aimed to investigate the expression of lung cancer driver genes in patients with synchronous multiple ground-glass nodules (SMGGNs), to calculate the relationships of these genes with clinical features, and to probe into the origin of multiple primary pulmonary ground-glass nodules (GGNs) from the perspective of molecular biology.

Methods

The clinicopathological data of 50 patients with SMGGNs who were admitted to the Department of Thoracic Surgery of the Fourth Hospital of Hebei Medical University from January 2018 to January 2021 were collected for retrospective analysis. All the samples resected were genotyped at 16 lung cancer driver gene loci by next-generation sequencing technology. The relationships between gene mutations and clinical features were analysed, and suspected pulmonary nodules of the same origin were analysed by the whole-exome sequencing.

Results

Fifty patients were included in the study, 86.0% (43/50) of whom had gene mutations. Some 133 pulmonary nodules were resected, which were pathologically classified into the following types: atypical adenomatous hyperplasia (21 nodules), adenocarcinoma in situ (32), micro-invasive adenocarcinoma (30), and invasive adenocarcinoma (50). Lung cancer driver gene mutations were identified in 73 (54.9%) nodules, 63.0% (46/73) of which were in the epidermal growth factor receptor gene (EGFR). Significant differences were observed in the gene mutation status between nodule types, particularly EGFR mutation, which differed between histological and imaging subtypes. Multiple gene mutations were not discovered in the same tissue. A low-probability mutation (RET fusion) was detected in three nodules of one patient, and further whole-exome sequencing showed that the three nodules had different gene mutations and were not metastatic.

Conclusions

The driver gene mutations in patients with SMGGNs are quite different and mutually exclusive, and nodules can arise from multiple primary lesions. Surgical resection is considered a superior approach to treating SMGGNs.

synchronous multiple pulmonary ground-glass nodules

non-small cell lung cancer

next generation sequencing

driver gene mutation

tumor heterogeneity

Synchronous multiple primary lung cancer (SMPLC) is characterized by multiple primary lung cancer lesions identified in the same period [1, 2]. SMPLC is quite rare, with an incidence rate of 0.5% [3]. With advancements in imaging diagnostic technologies and the large-scale screening of high-risk people using low-dose computed tomography (LDCT), a growing number of pure ground-glass nodules (pGGNs) or mixed ground-glass nodules (mGGNs) have been detected [4], and more and more synchronous multiple ground-glass nodules (SMGGNs) have been discovered. Multiple pulmonary nodules may result from the intrapulmonary spread of a single primary tumour or the synchronous progression of multiple lesions [5, 6]. Tumour heterogeneity has been identified in several types of solid tumours [7–13]. Despite the difficulty in distinguishing SMPLC from metastatic disease, the use of molecular markers has contributed to the differentiation of SMPLC from metastatic tumours [14, 15], since the heterogeneity of lesions may result from the cloning and replication of different tumours [14].

Patients with SMGGNs have distinct clinical features and prognoses [16–19]. Epidermal growth factor receptor gene (EGFR) mutations are highly inconsistent between the tumours of SMPLC patients, and surgical treatment is available for these patients because of their good prognosis [20]. However, there is still no consensus on the best protocol for the treatment of multiple pulmonary nodules. Some studies have found large differences in driver gene mutations in non-small-cell lung cancer (NSCLC) patients with GGNs and favourable prognoses after resection of multiple lesions, supporting the rationality of surgical resection [21, 22]. The identification of the genetic heterogeneity in SMGGNs by molecular-biological means may be conducive to formulating strategies for the diagnosis and treatment of SMGGNs different from those for multiple metastatic lung diseases [23]. Moreover, unnecessary adjuvant means of treating synchronous primary lung cancer with similar histology can be averted when next-generation sequencing (NGS) is routinely used for the detection of multiple lesions [24].

High-resolution computed tomography features of lung adenocarcinomas can be used to differentiate multiple lung adenocarcinomas from intrapulmonary metastases. In synchronous multiple primary lung adenocarcinomas, GGNs (pGGNs or mGGNs) are present in at least one lesion, while solid nodules (SNs) are more frequently seen in lung adenocarcinomas with lung metastasis. Intrapulmonary metastases of lung cancer are mainly SNs with pleural attachment, and the concomitant lesions in the lung are also SNs without lobules and bubble-like transparency [25]. However, heterogeneity between pulmonary nodules determined by imaging features alone is not convincing. Intrapulmonary metastases can exist in multiple GGNs, and the number of GGNs is independent of metastasis [26]. Tumour-related protein expression is thought to be effective in differentiating multiple primary lung cancer lesions from intrapulmonary metastases and in determining the appropriate biological stage of lung cancer [27]. Further gene analysis and clinical trials will help clarify the biological characteristics of pre-invasive adenocarcinoma and develop minimally invasive strategies for treating GGNs [28].

In 133 nodules from 50 patients with SMGGNs in this study, 16 lung cancer driver genes were sequenced by NGS technology to analyse the mutation statuses of EGFR, KRAS, ERBB2, HER2, BRAF, ALK, ROS1, MET, RET, PIK3CA, KIT, ESR1, PDGFRA, DDR2, HRAS, and NRAS so as to infer the origins of these patients’ multiple lesions. In addition, suspected pulmonary nodules of the same origin with concurrent low-probability mutations were subjected to whole-exome sequencing to further clarify the heterogeneity between tumours.

Patients and tissue samples. Patients with SMGGNs who underwent surgical treatment in the Department of Thoracic Surgery of the Fourth Hospital of Hebei Medical University from January 2018 to January 2021 were selected as research subjects. Gene sequencing analysis of formalin-fixation and paraffin-embedding (FFPE) samples surgically resected was performed using NGS technology to analyse the mutation statuses of EGFR, KRAS, ERBB2, HER2, BRAF, ALK, ROS1, MET, RET, PIK3CA, KIT, ESR1, PDGFRA, DDR2, HRAS, and NRAS. On this basis, whole-genome sequencing was conducted on suspected tumour samples of the same origin infer the origin of multiple lesions through molecular biology methods.

Inclusion criteria were as follows: (1) patients with at least 2 SMGGNs resected, (2) those who did not receive neoadjuvant chemotherapy or any other anti-tumour treatment before surgery, (3) those with complete medical records and imaging data, and (4) those who signed the informed consent.

This study was approved by the Ethics Committee of the Fourth Hospital of Hebei Medical University (Ethics Approval No.: 2021ky103).

Molecular-biological analysis. All patients underwent postoperative tumour–node–metastasis (TNM) staging per the TNM Staging of Lung Cancer (8th Edition) released by the International Union against Cancer (UICC) [29]. All samples were examined and histologically classified by two pathologists according to the Histological Classification of Lung Tumors published by the World Health Organization (WHO) in 2015 [30]. Genomic analysis was carried out in the Laboratory of Pathology Department of the Fourth Hospital of Hebei Medical University.

Detection of lung cancer-related genes. At least 30 ng of DNA was extracted from each FFPE tumour sample using a DNA extraction kit (QIAamp DNA FFPE Tissue Kit; Qiagen, Hilden, Germany) following the manufacturer's instructions. Next, the DNA concentration was determined by Qubit dsDNA assay. DNA was cleaved by a Covaris M220, followed by end repair, phosphorylation, and linker ligation. Fragments of 200–400 bp in length were selected by magnetic beads (Agencourt AMPure XP Kit, Beckman Coulter, California, USA), then hybridized with a capture probe. Later, magnetic beads were applied for hybrid selection and amplification by polymerase chain reaction (PCR). The quality and size of DNA fragments were measured using a biological analyser with high sensitivity, and the samples were sequenced. Paired-end reads were completed using the MiSeq DX sequencer (Illumina, Inc., California, USA). The genetic maps of captured tissue samples were subjected to targeted deep sequencing using the lung cancer 16-gene panel, involving a 76-kb human genome region (kit: Burning Rock Dx, Lung cure 16-gene panel). The carcinogenic driver mutations of EGFR, KRAS, ERBB2, HER2, BRAF, ALK, ROS1, MET, RET, PIK3CA, KIT, ESR1, PDGFRA, DDR2, HRAS, and NRAS were detected by the 16-gene panel. The sequencing depth of the FFPE samples was 1000×.

Whole-exome sequencing. For patients with suspected SMGGNs of the same origin, all nodules were further analysed by whole-exome sequencing.

1. DNA sample detection

DNA concentrations were accurately quantified by Qubit 3.0, and DNA samples with a concentration ≥ 20 ng/µL and a total amount > 0.4 µg were used for the library construction.

2. Library construction

Genomic DNA was randomly broken up into fragments of 180–280 bp in length using the Covaris crusher. Library construction and capture experiments were conducted using the Agilent SureSelect Human All Exon V5/V6 kit. Following end repair, phosphorylation, and addition of A tails, the two ends of the fragments were ligated with a linker to construct the DNA library. The library with a specific index was hybridized with up to 543,872 biotin-labelled probes in the liquid phase, and 334,378 exons of 20,965 genes were captured using streptavidin magnetic beads. After linear amplification by PCR, the quality of the library was tested, and the qualified DNAs were sequenced.

3. Quality control of the library

After library construction, DNAs were preliminarily quantified using Qubit 3.0. Then, the insert size of the library was detected using Agilent 2100. After the insert size complied with expectations, the effective concentration (3 nmol/L) of DNA in the library was accurately quantified by quantitative PCR to ensure the library quality.

4. On-board sequencing

After the library’s quality was confirmed, Illumina HiSeq PE150 sequencing was carried out in light of the effective concentration and data output requirements of the library.

Statistical analysis. GraphPad Prism 9.4.0 software was adopted for statistical analysis and visualization. Enumeration data are expressed as [n (%)] and were compared between groups by the χ² test. Measurement data conforming to a normal distribution are expressed as mean ± standard deviation (‾x ± s) and were compared between groups by Student’s t-test. Non-normally distributed data are described as M (P25, P75), and the rank sum test was run to compare them between groups. The significance level was p = 0.05 (two-sided).

Patient and sample data. A total of 133 nodules were resected from the 50 enrolled patients. They were 48–73 years old (median: 58 years). The clinical data of the samples were collected (Table 1).

Table 1

Patient and sample information table
	Category		Quantity
Total number of patients (n = 50)	Sex	Male	16
	Sex	Female	34
	Age	< 60	27
	Age	≥ 60	23
	Operation mode	Unilateral surgery	41
	Operation mode	Bilateral surgery	9
	Nodule origin	Same lobe	16
	Nodule origin	Different lobes	34
Total number of nodules (n = 133)	Nodule position	Left lung	54
	Nodule position	Right lung	79
	Histological type	AAH	21
		AIS	32
		MIA	30
		IAC	50
	Image credit type	pGGN	75
	Image credit type	mGGN	58

Mutation statuses of driver genes. Lung cancer driver gene mutations were presented in 73 out of the 133 (54.9%) pulmonary nodules, among which EGFR mutations made up 63.0% (46/73), including the E18G719X mutation in two nodules, exon 19 deletion mutation in 16 nodules, exon 20 insertion mutation in three nodules (all of which were the YVMA insertion mutation), and exon 21 L858R mutation in 24 nodules. Other mutations included ALK mutation in three nodules, ESR1 mutation in one nodule, ROS1 mutation in three nodules, NRAS mutation in one nodule, BRAF mutation in two nodules, MET mutation in five nodules, KRAS mutation in seven nodules, RET mutation in three nodules, and PIK3CA mutation in two nodules. No ERBB2, HER2, KIT, PDGFRA, DDR2, or HRAS mutations were detected. No lung nodules with multiple gene mutation types were detected, signifying that the gene mutation types were mutually exclusive (Fig. 1a、Fig. 1b). The gene mutation rate in right pulmonary nodules was 57.0% (45/79), slightly higher than that in left pulmonary nodules [51.9% (28/54)] (p > 0.05) (Fig. 1c). Compared to patients with pulmonary nodules in the same lobe (n = 16), those with pulmonary nodules in different lobes (n = 34) displayed a slightly higher gene mutation probability (75.0% vs. 91.2%), but again there was no significant difference (p > 0.05) (Fig. 1d).

Mutation status in patients. Taken all together, gene mutations were identified in 86.0% (43/50) patients (Fig. 2a). Female patients had a slightly higher probability of gene mutation than male patients (91.7% vs. 71.4%) (Fig. 2b), and the gene mutation rate was higher in younger patients than older patients (92.6% vs. 78.3%), but these differences were not statistically significant (p > 0.05) (Fig. 2c).

Histological classification and driver gene mutation analysis. A total of 21 nodules of atypical adenomatous hyperplasia (AAH), 32 nodules of adenocarcinoma in situ (AIS), 30 nodules of micro-invasive adenocarcinoma (MIA), and 50 nodules of invasive adenocarcinoma (IAC) were resected surgically (Fig. 3a). The probability of driver gene mutations in them was 33.3% (7/21), 56.3% (18/32), 70.0% (21/30), and 54% (27/50), respectively, showing no significant differences between groups (p > 0.05, Fig. 3b). The probability of EGFR gene mutation in these entities was 14.2% (3/21), 25.0% (8/32), 40.0% (12/30), and 46.0% (23/50), respectively, which were significantly different from each other (p < 0.05, Fig. 3c).

Imaging classification and driver gene mutation analysis. A total of 75 pGGNs and 58 mGGNs were resected surgically (Fig. 4a). They had respective probabilities of driver gene mutations of 44.0% (33/75) and 69.0% (40/58) (p < 0.05, Fig. 4b). The probability of EGFR mutation in the pGGNs and mGGNs was 21.3% (16/75) and 51.7% (30/58), respectively (p < 0.001, Fig. 4c).

Whole-exome sequencing analysis. Three lesions and normal lung tissues in one patient (P40) with a rare mutation (RET fusion) were subjected to whole-exome sequencing. Somatic mutations, six substitution profiles, and 96 substitution profiles were compared between multiple GGNs. The results revealed nine common somatic mutations among the three lesions in the patient (Fig. 5a), and the mutation spectra illustrated that the proportions of single-nucleotide variants (SNVs) in the three nodules were different (Fig. 5b). Significantly discordant mutational spectra were observed between different GGNs in the patient at the level of substitution composition (t-test, p < 0.05), and the profiles of 96 substitutions for each GGN were not similar (Pearson correlation coefficient < 0.8) (Fig. 5c). The mutation features of the three samples were clustered with 78 known mutation features in the COSMIC website, and the three mutation features were not similar to any of the known 78 features (cosine similarity < 0.9), the cosine similarity between the three mutation features being less than 0.9 (Fig. 5d).

In this study, the mutation statuses of lung cancer driver genes in 133 nodules from 50 patients with SMGGNs were analysed, and the regular pattern of driver gene mutations in multiple lesions of patients with SMGGNs was explored, in order to characterize the heterogeneity of GGNs from the perspective of molecular biology. Tumour heterogeneity refers to distinct molecular-biological or genetic alterations of tumour cells after multiple divisions and proliferation during the growth of the tumour in the same patient, leading to differences in various aspects such as tumour growth rate, invasive ability, drug sensitivity, and prognosis [31–33]. Overall, tumour heterogeneity has become a major challenge to individualized cancer treatment.

This study found that there was at least one nodule with a lung cancer driver gene mutation in 86.0% of the 50 patients with SMGGNs. Women, young patients, and those with nodules in different lobes had higher gene mutation probabilities. The probability of lung cancer driver gene mutations in the 133 nodules was 54.9%, and it was higher in patients with mGGNs, right lung nodules, or invasive nodules. It can be seen from the overall mutation profile that multiple GGNs in patients with SMGGNs seem to arise from different primary tumours [31, 33]. Thus, there is a need to develop diagnosis and treatment strategies different from those for patients with multiple metastatic lung diseases. As radiology develops, an increasing number of patients with SMGGNs are detected [34, 35], but a consensus has not been reached on the treatment of these patients. Chang et al. [17] reviewed the natural history of multiple pure ground-glass opacity lung nodules and proposed that subjects without a history of malignancy should be followed up for a time. During the long-term follow-up, approximately 90% of the nodules showed no progression. However, Asamura et al. [36] advised that mGGNs, even if multiple, should be resected surgically. We discovered that the gene mutation probability in nodules in the mGGN group differed from that in the pGGN group, and the mGGN group had a higher EGFR mutation probability than the pGGN group. GGNs with different pathological subtypes and CT features can have different genetic mutation features [37], in line with the findings here. It is therefore reasonable to speculate that multiple GGNs in patients with SMGGNs are independent of each other and thus can be viewed as primary tumours, and that lung cancer driver gene mutations are relevant to the imaging progression of pulmonary nodules. In addition, surgical resection is an appropriate therapeutic regimen and should be the first choice if available.

We analysed the gene mutation statuses in nodules of different pathological types. Mutations of lung cancer driver genes have been detected in precancerous lesions, and the EGFR mutation probability shows a significant increase with the progression of pathological stage, confirming the viewpoint that EGFR mutations occur in the early stage of lung adenocarcinoma progression [38, 39]. Chung et al. [5] detected EGFR mutations in 24 NSCLC patients with multiple ground-glass opacities, and the discordance rate between them in a given patient reached up to 70.8%, in agreement with the findings described in this study. This means that the EGFR gene is essential in the occurrence and progression of lung cancer [40, 41]. Furthermore, the EGFR, KRAS, ALK, or HER2 mutation is positive after 3/4 of GGNs are resected, and quadruple-negative tumours are associated with the absence of GGN growth, while EGFR mutation-positive tumours are associated with GGN growth [42]. Here, the total gene mutation probability in nodules showed no gradual increase with the progression of pathological stage, unlike the EGFR mutation probability. This difference may be attributed to the small sample size and the relatively low incidence of gene mutations except for EGFR, showing no significant difference. Selection bias may exist in this retrospective study due to the small sample size. Hence, multicentre prospective trials are needed to validate this conjecture.

In this study, a special patient (No. 40) was found to have RET fusion mutation in three nodules. In 2011, a RET (a receptor tyrosine kinase coding gene) fusion mutation was discovered in lung adenocarcinoma tissues for the first time, and its mutation rate in lung adenocarcinoma patients is only 1–2% [43]. The protein encoded by RET gene stimulates intracellular phosphorylation through binding to its ligand, thus activating downstream signals and participating in regulating cell growth and differentiation [44, 45]. In this study, this low-probability mutation was only identified in three nodules of one patient. We speculate that these three nodules may have the same origin. To test this possibility, we subjected these three nodules to whole-exome sequencing, and surprisingly, the mutation spectra and mutation features did not support that the three nodules were early-stage intrapulmonary metastases. This implies that even the presence of a rare driver gene mutation in multiple GGNs cannot prove that these GGNs are early-stage intrapulmonary metastases. Somatic mutations, six-gene substitution profiles, 96-gene substitution profiles, or even copy number variation analysis and clonal evolution analysis are needed to comprehensively evaluate whether multiple pulmonary nodules are early-stage intrapulmonary metastases. Moreover, metastatic tumours may differ from primary tumours at the genetic level due to the gradual accumulation of genetic changes in some cases [33]. In contrast, comprehensive molecular assessment can illuminate the clonal relationship between tumours. In clinical practice, a larger panel as a supplement to histology helps distinguish the cloning relationship of NSCLC [46], and 50 genomes may be a promising auxiliary tool of the AJCC standard [47]. Hence, large-scale, frequent genomic sequencing is needed in the future.

In this study, driver gene mutations in patients with SMGGNs were highly different between tumours. These differences indicated that the separate nodules in patients with SMGGNs were primary lesions independent of each other, rather than metastases, and the prognosis after surgical resection was good. More molecular-biological studies are still needed to verify the heterogeneity and mutual exclusivity between the GGNs.

SMPLC Synchronous multiple primary lung cancer

LDCT Low-dose computed tomography

pGGNs Pure ground-glass nodules

mGGNs Mixed ground-glass nodules

SMGGNs Synchronous multiple ground-glass nodules

EGFR Epidermal growth factor receptor gene

NSCLC Non-small-cell lung cancer

NGS Next-generation sequencing

SNs Solid nodules

FFPE Formalin-fixation and paraffin-embedding

TNM Tumour–node–metastasis

PCR Polymerase chain reaction

AAH Atypical adenomatous hyperplasia

AIS Adenocarcinoma in situ

MIA Micro-invasive adenocarcinoma

IAC Invasive adenocarcinoma

SNVs Single-nucleotide variants

Statement

This study is approved by the ethics committee of the Fourth Hospital of Hebei Medical University (2021ky103), all methods were carried out in accordance with relevant guidelines and regulations in the declaration, and all patients signed informed consent forms.

Consent for publication

Not applicable

Availability of data and materials

The whole exon data of this study has been uploaded to BioProject database (PRJNA1000732, PRJNA1000739).

Competing interests

The authors declare that they have no competing interests.

Funding

Health Commission of Hebei Province (20201076).

Authors' contributions

YQ and WY analyzed and interpreted the patient data regarding the hematological disease and the transplant. SN and QY performed the histological examination of the kidney, and was a major contributor in writing the manuscript. All authors read and approved the final manuscript.

Acknowledgments

Not applicabl

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Table 1: Patient and sample information table

	Category		Quantity
Total number of patients (n=50)	Sex	Male	16
	Sex	Female	34
	Age	<60	27
	Age	≥60	23
	Operation mode	Unilateral surgery	41
	Operation mode	Bilateral surgery	9
	Nodule origin	Same lobe	16
	Nodule origin	Different lobes	34
Total number of nodules (n=133)	Nodule position	Left lung	54
	Nodule position	Right lung	79
	Histological type	AAH	21
		AIS	32
		MIA	30
		IAC	50
	Image credit type	pGGN	75
	Image credit type	mGGN	58

No competing interests reported.

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Synchronous Multiple Pulmonary Ground-Glass Nodules That Arose from Different Lesions Rather than from Multiple Metastases of a Single Lesion

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