Targeted sequencing reveals clonal genetic changes in the progression of early lung neoplasms and paired circulating DNA

Evgeny Izumchenko, Xiaofei Chang, Mariana Brait, Elana Fertig, Luciane T Kagohara, Atul Bedi, Luigi Marchionni, Nishant Agrawal, Rajani Ravi, Sian Jones, Mohammad O Hoque, William H Westra, David Sidransky, Evgeny Izumchenko, Xiaofei Chang, Mariana Brait, Elana Fertig, Luciane T Kagohara, Atul Bedi, Luigi Marchionni, Nishant Agrawal, Rajani Ravi, Sian Jones, Mohammad O Hoque, William H Westra, David Sidransky

Abstract

Lungs resected for adenocarcinomas often harbour minute discrete foci of cytologically atypical pneumocyte proliferations designated as atypical adenomatous hyperplasia (AAH). Evidence suggests that AAH represents an initial step in the progression to adenocarcinoma in situ (AIS), minimally invasive adenocarcinoma (MIA) and fully invasive adenocarcinoma. Despite efforts to identify predictive markers of malignant transformation, alterations driving this progression are poorly understood. Here we perform targeted next-generation sequencing on multifocal AAHs and different zones of histologic progression within AISs and MIAs. Multiregion sequencing demonstrated different genetic drivers within the same tumour and reveal that clonal expansion is an early event of tumorigenesis. We find that KRAS, TP53 and EGFR mutations are indicators of malignant transition. Utilizing droplet digital PCR, we find alterations associated with early neoplasms in paired circulating DNA. This study provides insight into the heterogeneity of clonal events in the progression of early lung neoplasia and demonstrates that these events can be detected even before neoplasms have invaded and acquired malignant potential.

Figures

Figure 1. Histological characteristics of samples.
Figure 1. Histological characteristics of samples.
(a) DNA was extracted from multiple AAH lesions collected from six patients on resection of primary lung adenocarcinoma. Samples from AIS and MIA tumours extracted from five patients each were collected from different zones of histologic progression within the same lesion. (b) AAH is the earliest form of glandular neoplasia of the lung, which characterized by the proliferation of slightly atypical epithelial cells lining the slightly thickened by intact septae. Size is a critical feature in distinguishing AAH from AIS, a scale bar (1.0 mm) was included to underscore the small size of the AAH (magnification × 4). AIS is characterized by atypical cells with enlarged hyperchromatic nuclei lining intact alveolated lung parenchyma (zone 1). With progression towards the centre of the tumour, the septae become increasingly thickened and the cytologic atypia becomes more pronounced (zone 2 and zone 3) (magnification × 40 for normal lung and all AIS panels). MIA exhibits increasing septal thickening and epithelial atypia with progression from the periphery of the lesion (zone 1) towards its centre (zone 2 and zone 3). In addition, there is a small focus (<0.5 cm) of invasion at the core of the lesion (zone 4) characterized by irregular acinar glands in a desmoplastic stroma (magnification × 10 for all MIA panels).
Figure 2. Mutational landscape varies between the…
Figure 2. Mutational landscape varies between the three stages of tumour progression.
(a) Cytoscape bioinformatics toolset was used to create the network of protein–protein interactions of the mutated genes in each respective progression stage. Red nodes indicate the mutated genes, with node size dependent on number of mutations found in AAH, AIS and MIA patients. (b) Venn diagram was created to compare genes mutated in different stages of lung adenocarcinoma evolution (in red—mutations in the same position). (c) The chart demonstrates the percentage of fractional abundance of all mutations found in AAH, AIS and MIA. Pink frame in MIA cohort indicates genes that harbour mutations with fractional abundance higher than 20%. (d) Spectrum of base-pair substitutions in AAH lesions and AIS or MIA tumours (in red, base-pair substitutions that present in all three groups). Silent mutations were not included in the analysis.
Figure 3. Intratumour heterogeneity and branched evolution…
Figure 3. Intratumour heterogeneity and branched evolution in AIS and MIA tumours.
Dendrograms relating relative development of mutations from inferred common ancestors (dashed, white rounded boxes) and tumour zones (solid boxes) for each (a) AIS and (b) MIA tumour. A complete list of mutations in each patient is provided in the white ovals. Mutations in common ancestors are shown in black, while subsequent alterations are depicted in red for mutations and blue for copy number variations. Zones with no mutation (zones 1 and 2 for AIS patient #2 and zone 2 for AIS patient #4) are shown as parallel branches, but may also be ancestors of subsequent mutations in other zones. Synonymous and non-coding mutations were not considered in the analyses.
Figure 4. Detection of lesion-associated DNA in…
Figure 4. Detection of lesion-associated DNA in bodily fluids.
(a) DNA with known NRAS mutation (extracted from the primary tumour of patient 2AAH) was serially diluted with 35 ng of the matched control DNA isolated from the lymph node of the same patient and every mixed mutant:wild-type sample was assessed using PrimePCR ddPCR assay. The blue markers indicate the concentration of mutant DNA (copies per μl) and the green markers indicate the concentration of wild-type DNA (copies per μl) in each sample. All error bars generated by QuantaSoft software represent the 95% confidence interval. Fractional abundance of the mutant DNA in a wild-type DNA background is shown at the bottom of the plot. (b) Table shows the list of mutations detected either in primary lesions, blood or sputum samples from two patients, analysed using NGS or PrimePCR ddPCR assay. (c) Mutant circDNA has been detected by PrimePCR ddPCR assay in blood and (d) sputum samples isolated from patients with known ATM, NRAS and IGF1R mutations. Fractional abundance of these mutations in primary tumours was 35%, 20% and 2% respectively. (e) BRAF mutation that was identified in pre-malignant AAH lesion was detected in DNA extracted from matched plasma and sputum species by ddPCR.

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