Next Generation Sequencing of Amplified DNA of Circulating Tumor Cells in Resectable Non-Small Cell Lung Cancer: A Comparative Analysis with Primary Cancer Tissue and Cell-Free DNA

doi:10.21203/rs.3.rs-4278611/v1

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Next Generation Sequencing of Amplified DNA of Circulating Tumor Cells in Resectable Non-Small Cell Lung Cancer: A Comparative Analysis with Primary Cancer Tissue and Cell-Free DNA

https://doi.org/10.21203/rs.3.rs-4278611/v1

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Purpose

Circulating tumor cells (CTCs) are thought to play a major role in the distant metastasis of lung cancer. However, their mutational profile has not been extensively studied. This study analyzed the amplified DNA from CTCs, using next generation sequencing to identify mutations and compare them with those found in primary cancer tissue and cell-free DNA (cfDNA).

Methods

Fifty patients with resectable primary lung cancer were prospectively enrolled from August 2023 to December 2023. Whole blood samples and cancer tissues were collected during routine curative surgery. Targeted panel sequencing was performed on the cancer tissues, cfDNA, and amplified DNA from CTC.

Results

Thirty-seven patients (74.0%) had adenocarcinoma, and 33 (66.0%) were classified as stage 1. Library construction for cancer tissue, cfDNA, and CTCs was successful in 50 (100%), 49 (98%), and 34 (68%) of patients, respectively. The top 10 mutated genes differed significantly across the sample types. CTCs predominantly harbored mutations in tumor suppressor genes, whereas cancer tissues exhibited mutations in both oncogenes and tumor suppressor genes. The most frequently mutated gene in CTCs was MSH6, followed by FANCE, while EGFR and CDH1 were the most common in cancer tissue and cfDNA, respectively. Compared to cancer tissue, the mutational profile of CTCs was more closely associated with the following cancer hallmarks: evasion of anti-growth signaling, replicative immortality, and invasion/metastasis.

Conclusion

Amplified CTC DNA had specific mutations distinct from cancer tissue and cfDNA in resectable primary lung cancer. Some of these mutations may be essential for the transformation of cancer cells into CTCs.

Cell-free DNA

Circulating tumor cell

Next generation sequencing

NSCLC

Lung cancer is a well-known malignant disease worldwide due to its high recurrence rate despite proper management.[1] Liquid biopsy is considered a breakthrough in various aspects, including prognosis, treatment monitoring, and drug resistance, particularly in advanced-stage lung cancer, due to its advantages such as reduced invasiveness and ease of repetition.[2, 3] Furthermore, many physicians anticipate that liquid biopsy will have significant diagnostic, predictive, and staging value for early-stage lung cancer.[4, 5]

Distant recurrence of early-stage lung cancer frequently occurs despite curative surgery.[6, 7] Circulating tumor cells (CTCs) are believed to have a direct relationship with metastasis through the process of the epithelial-mesenchymal transition (EMT).[8] Several studies have suggested that the presence of CTCs, as indicated by immunofluorescence imaging, is associated with a poor prognosis or recurrence in lung cancer patients.[9, 10]

We hypothesize that CTCs possess unique gene mutations that facilitate the transition to EMT from primary cancer tissue. By analyzing the mutational information of CTCs, we may gain insights into the additional characteristics associated with distant recurrence in early-stage lung cancer. However, many studies have been limited to sequencing analysis for Cell-Free DNA (cfDNA) or the immunofluorescence imaging of CTCs.[4, 9–11] Consequently, there is a scarcity of research on the mutational profile of CTCs or on direct comparisons between CTCs, primary cancer tissue, and cfDNA in the context of early-stage lung cancer.

Thus, we aimed to compare the amplified DNA from CTCs with that of cancer tissue and cfDNA by employing next generation sequencing in patients with resectable early-stage lung cancer scheduled for curative surgery.

Ethical Statement

The study protocol received approval from the Institutional Review Board of Seoul National University Bundang Hospital (approval number: B-2211-791-304) and adhered to the principles outlined in the Declaration of Helsinki. Prior to enrollment, each participant provided written informed consent.

Patients

We enrolled 50 patients diagnosed with lung cancer postoperatively at Seoul National Bundang Hospital from September to December 2023. The following exclusion criteria were applied: 1) patients with primary tumors from which obtaining a solid specimen was considered difficult, 2) patients diagnosed with another type of malignancy within the past 5 years, 3) patients suspected of having any distant metastasis, and 4) patients who refused to give informed consent.

Specimen Processing

In total, 30 mL of fresh blood was drawn and distributed into two Cell-Free DNA BCT tubes (20 mL each, Streck Inc., Omaha, NE, USA) for cfDNA analysis, and one ACD-A tube (10 mL) for CTC analysis from peripheral arterial blood following anesthesia. After the surgical specimen was removed, a tissue sample measuring 5 mm × 5 mm was excised from the cancerous area. These samples were promptly transported to BeyondDx Inc. (Gwangmyeong, Republic of Korea) for centrifugation and CTC extraction, which occurred within 6 hours of collection. Subsequently, the isolated plasma, amplified DNA from CTCs, and cancer tissue were sent to Geninus (Seoul, Republic of Korea) for sequencing (Fig. 1).

Cancer Tissue Sequencing

The DNA from cancer tissue was extracted using a QIAamp DNA Mini Kit (Qiagen Inc.). The concentration and purity of the DNA were assessed with a NanoDrop One UV-Vis spectrophotometer (Thermo Scientific, Waltham, MA, USA) and a Qubit 4.0 Fluorometer (Life Technologies, Grand Island, NY, USA). The degree of DNA degradation was evaluated using a 4200 TapeStation instrument (Agilent Technologies, Santa Clara, CA, USA). Subsequently, the DNA was used to construct a library with the Complete Library Preparation Kit (Twist Bioscience, San Francisco, CA, USA) and CancerSCAN panel probes (Geninus), which targeted 405 cancer-associated genes (Supplemental Table 1). Sequencing was performed on the Illumina NovaSeq 6000 sequencing platform.

Cell-free DNA Sequencing

Plasma was prepared by performing two centrifugation steps at 4℃, first at 1600 ×g for 15 minutes and subsequently at 16,000 × g for 10 minutes. Mononuclear cells were isolated using Ficoll gradient centrifugation, and genomic DNA was extracted from these cells with the QIAamp DNA Mini Kit (Qiagen Inc., Valencia, CA, USA). Plasma DNA was purified from the plasma samples using the QIAamp Circulating Nucleic Acid Kit (Qiagen Inc.). For library construction, hybrid selection was carried out using the LiquidSCAN panel (Geninus) according to the manufacturer's protocol. The LiquidSCAN panel consists of a pool of DNA baits designed to target 44 cancer-associated genes, including regions with hotspot mutations (Supplemental Table 2). Sequencing was performed on the Illumina NovaSeq 6000 sequencing platform.

CTC Sequencing

From an ACD-A tube stored at room temperature, peripheral blood mononuclear cells (PBMCs) were isolated using the Ficoll-Hypaque density gradient centrifugation method. Candidate CTCs for sequencing were isolated based on a proprietary CTC capture technology, iDXGate TO (BeyondDx, Inc., Korea). iDXGate TO, which is specifically designed for early-stage lung cancer, utilizes EpCAM, EGFR, and HER2 biomarkers for the targeted capture of CTCs. Genomic DNA from the CTCs was extracted and amplified using the REPLI-g Single Cell kit (Cat. No. 150343; Qiagen Inc.) following the manufacturer’s protocol. The amplified DNA was then used to construct a library. The library construction and sequencing process were identical to those used for cancer tissue, employing the CancerSCAN panel.

Definitions

The presence of one or more SNV/indel mutations, as identified by sequencing with the CancerSCAN panel, defined a positive mutation status for cancer tissue and CTCs. Similarly, cfDNA was considered positive for mutations when sequenced with the LiquidSCAN panel. Mutational genes were classified according to their function, with oncogenes including growth factor receptors, transcription factors, and signal transducers, and tumor suppressors comprising genes that function as caretakers and gatekeepers.

A subset of genes was selected as cancer hallmark genes using the methodology described by Liang et al.[12] Cancer hallmark genes were classified into 11 genes or pathways including deregulated metabolism, evasion of anti-growth signaling, angiogenesis, immune system evasion, resistance to cell death, replicative immortality, sustained proliferative signaling, tissue invasion and metastasis, tumor-promoting inflammation, tumor microenvironment, and genome instability.

Statistical Analysis

The primary objective of this study was to compare the mutational differences between cfDNA and CTCs relative to primary tumor tissue based on the sequencing profiles of resectable lung cancer. The secondary objective is to compare the classification and roles of mutations among the three sample types. Categorical variables were described as numbers and percentages, and continuous variables were presented as medians with interquartile ranges (IQR). Statistical analyses were conducted using IBM SPSS Statistics for Windows, Version 25 (IBM Corp., Armonk, NY, USA).

Baseline and Pathologic Characteristics

The mean age of the 50 patients was 68.7 ± 8.3 years, with 30 (60%) being male. Additional baseline and preoperative characteristics are presented in Supplemental Table 3. Thirty-seven patients (74%) were diagnosed with adenocarcinoma. The pathologic stages, based on the AJCC 8th edition, were distributed as follows: 33 patients (66%) were diagnosed with stage I, 8 patients (16%) with stage II, and 9 patients (18%) with stage III or higher (Table 1).

Table 1. Pathologic status

	Total (n=50)
Histology
Adenocarcinoma	37 (74.0%)
Others
Squamous cell carcinoma	8 (16.0%)
Adenosquamous cell carcinoma	1 (2.0%)
Large cell neuroendocrine carcinoma	1 (2.0%)
Carcinoid tumor	1 (2.0%)
Sarcomatoid	1 (2.0%)
Small cell lung cancer	1 (2.0%)
Pathologic stage
1A1	1 (2.0%)
1A2	10 (20.0%)
1A3	13 (26.0%)
1B	9 (18.0%)
2A	2 (4.0%)
2B	6 (12.0%)
3A	5 (10.0%)
3B	3 (6.0%)
4	1 (2.0%)
Lymph node metastasis	10 (20.0%)

Panel Sequencing Data

Library construction for sequencing was successfully completed in 50 (100%) of cancer tissue samples, 49 (98%) of cfDNA samples, and 34 (68%) of CTC samples. No alterations were detected in the cancer tissue of 1 patient (2%) and in the cfDNA of 4 patients (8%).

Figure 2 displays the top 10 mutated genes in respective cancer tissues, cfDNA, and CTCs. The most frequently mutated genes in cancer tissue were EGFR (N=20, 40%), followed by TP53 (N=17, 34%). In cfDNA, the most prevalent mutations were found in CDH1 (N=23, 47.0%), with FBXW7 mutations following closely (N=21, 43.0%). In CTCs, MSH6 mutations were universal (N=34, 100%), and FANCE mutations were the second most common (N=31, 91.2%). The mutation profiles of the top 10 genes in CTCs were significantly different from those in cancer tissue and cfDNA, exhibiting a higher percentage of mutations compared to the other sample types.

Figure 3 shows the confirmed mutation gene profiles for cancer tissue and CTCs, reflecting the roles of each gene. The cancer tissue exhibited a range of oncogenes across three samples, whereas CTCs predominantly harbored tumor suppressor genes rather than oncogenes. cfDNA contained a mix of both oncogenes and tumor suppressor genes. Among the patients with positive CTC mutations (N=34), 32 (94.1%) shared common mutations.

Cancer hallmark pathways

The total number of genes identified in cancer tissue, CTCs, and cfDNA was 194, 279, and 34, respectively. Of the identified genes, 52 from CTCs were classified as cancer hallmarks, which was the highest number among the three sample types (vs. 34 genes in cancer tissue and 20 genes in cfDNA). Cancer tissue and CTCs shared 144 common genes, which is a higher number than any other intersection of sample types. This may suggest that CTCs originate from cancer tissue and reflect the genetic profile of the original tumor (Fig. 4a).

Figures 4b and 4c illustrate the hallmark pathways present in each cancer tissue and in CTCs. The cancer tissue's hallmark pathways were characterized by sustained proliferation, resistance to cell death, and altered metabolism. In contrast, CTCs exhibited additional hallmark pathways, including evasion of anti-growth signaling, replicative immortality, and invasion/metastasis (Fig. 4d).

We investigated the gene profiles of amplified DNA from CTCs, which were markedly distinct from those of primary cancer tissue and cfDNA in resectable non-small cell lung cancer (NSCLC). The classifications and roles of mutated genes, based on cancer hallmarks, also varied across the samples.

All but one of the cancer tissue samples exhibited multiple mutations, many of which are well-known to be associated with lung cancer.[13] Mutations were identified in both oncogenes (EGFR, NOTCH, ROS1, and KRAS) and tumor suppressor genes (TP53, FAT1, LRP1B, and PKHD1). The most common mutation was in the EGFR gene, which is now amenable to targeted therapy.[14] The second most common mutation was found in TP53, a gene that is well-recognized in various types of cancer and is associated with the cell cycle.[15] Additional minor gene mutations were detected through panel sequencing, but these were considered to reflect cancer heterogeneity secondary to the primary mutations.

In the analysis of cfDNA, mutations were detected in both oncogenes (NOTCH, ERBB2, and KRAS) and tumor suppressor genes (CDH1, FBXW7, PTEN, ARID1A, SMARCA4, NF1, and CDKN2A) through panel sequencing. The two most frequently mutated genes were tumor suppressor genes, specifically CDH1 and FBXW7. The FBXW7 gene has been linked to treatment outcomes in NSCLC.[16] Mutations in the CDH1 gene are related to cell-cell adhesion, which, unlike mutations in EGFR or TP53, are not directly associated with lung cancer.[17]

Previous studies identified several mutations in cfDNA, including those in EGFR, TP53, KRAS, and CDKN2A which were similar to the mutations found in the cancer tissue of patients with resectable lung cancer.[11, 18] The similarity in their study between the mutations in cancer tissue and cfDNA was observed in cases of relatively advanced lung cancer compared to our cohort. Their studies included over 50% of patients at stage 3 or higher, whereas 66% of our cases were diagnosed at stage 1. We hypothesized that the early stage of the disease did not allow a sufficient number of dominant cancer cells to release cfDNA into the bloodstream.

CTC mutation was detected in only 34 patients. We identified numerous tumor suppressor genes, including MSH6, FANCE, CDK12, HNF1A, and AXIN2. Most of these genes are not directly associated with lung cancer.[13] Research on panel sequencing of CTCs in patients with primary lung cancer is limited. Barbirous et al. conducted a study on single CTC–targeted next generation sequencing across 65 genes in NSCLC.[19] They discovered seven mutations with oncogenic potential, including NF1, PTCH1, TP53, SMARCB1, SMAD4, KRAS, and ERBB2. NF1 and PTCH1 were also present in our cases (71% and 74%, respectively), but the other mutations were not as prevalent. We believe this discrepancy may be due to the number of cases studied and the differences in panel sequencing. Their study included only seven patients with more than two CTC counts, using a 65-gene panel, and all were in advanced stages. In contrast, our study included 50 patients, who were in relatively early stages and were analyzed using a 405-gene panel.

In our study, we detected common genes between cancer tissue and CTCs in the majority of patients (94.1%). Notably, the top 10 mutations in CTCs were present at a high frequency compared to those in tissue samples. This suggests that certain mutations, such as MSH6 and FANCE, may be critical for the differentiation of cancer cells into CTCs in lung cancer. However, this does not necessarily imply that these mutations are essential for actual metastasis, as CTCs undergoing EMT may require additional mutations to complete the reverse process of mesenchymal-epithelial transition. To better understand this process, further studies comparing CTCs with actual metastatic tissue are needed.

Liang et al. introduced the concept of "cancer hallmarks" to identify mutations specifically associated with carcinogenesis based on gene function.[12] We can infer that the amplified CTC DNA originated from cancerous tissue, as both the cancer tissue and CTCs shared common genes (Fig. 4.A). CTCs exhibited a greater number of cancer hallmarks than the cancer tissue did. This suggests that CTCs possess more mechanisms conducive to carcinogenesis, potentially enabling them to initiate other cancer types, such as distant metastases. Although the overall composition of cancer hallmarks was similar between cancer tissue and CTCs, the latter had a higher representation of genes in categories such as evasion of anti-growth signaling, replicative immortality, and invasion/metastasis. This suggests that cancer tissue initially arises from errors in proliferation or the cell cycle, and that CTCs are more prone to metastasis through invasion and replication. In our study, mutational changes in CTCs were detected in 68% of patients, even those with early-stage lung cancer. Therefore, it is imperative to regularly monitor these patients for signs of distant recurrence.

We also explored the reasons behind the differing mutation types among cancer tissue, cfDNA, and CTCs. First, the tumor burden in our study may have been low, as most patients were at stage 1 or 2 of cancer. This suggests that there may not have been a dominant mutation in the bloodstream directly linked to the cancer tissue for each patient. Consequently, only a very small number of minor mutations were sensitively detected, which could occur more readily in CTCs due to the amplification process. Second, the panels used for cfDNA analysis differed. Cancer tissue and CTCs were analyzed using a 405-gene panel, in contrast to the 44-gene panel used for cfDNA. Genes such as MSH6, FANCE, FAT1, and LRP1B, which were highly ranked in cancer tissue and CTC analyses, were not included in the cfDNA's 44-gene panel. Since most cfDNA panels for sequencing are smaller than those used for cancer cell sequencing,[20] we believe that there were likely many false-negative results in the cfDNA studies compared to the cancer tissue or CTC analysis.

Several methods exist for capturing CTCs, including targeting EpCAM for inclusion or using a size-based exclusion technique. However, EpCAM-positive CTCs have been reported to be less frequently detected in NSCLC than in epithelial tumors.[21] The size-based exclusion technique has the problem of resulting in implausibly high genomic diversity in colorectal cancer.[22] Therefore, a need has emerged for a new methodology to capture CTCs specifically. We utilized a self-developed CTC capture technology that targets CTCs with EpCAM, EGFR, and HER2 biomarkers. EGFR and HER2 are well-studied markers associated with lung cancer according to previous studies,[23, 24] and EpCAM is also well-known as a characteristic of CTCs. We hypothesized that this method could more specifically detect early-stage lung cancer compared to techniques that rely solely on EpCAM inclusion or size-based exclusion.

Limitations

This study has several limitations. First, the number of patients enrolled was relatively small, which may not fully represent the characteristics of primary lung cancer. Second, the stages and histologic profiles of the cancers were too diverse to evaluate specific types of primary lung cancer, and the patients were not evenly distributed across stages or histologies. Third, the volume of blood sampled for CTC analysis was limited. For cfDNA, we postulated that the concentration might be low because the cases enrolled were in the early stages of cancer. Our approach for CTC involved an amplification process; therefore, we anticipated that the small DNA volume would not be problematic for CTC mutation analysis. We plan to increase the blood sample volume in future studies.

The mutational profiles of amplified CTC DNA differed significantly from those of primary tumor tissue and cfDNA in resectable primary lung cancer. The mutations in CTCs involved specific genes at a high frequency and were associated with cancer metastasis. These findings suggest a deeper understanding of early-stage lung cancer is emerging; however, further research is required.

Funding:

This work was supported by the New Faculty Startup Fund from Seoul National University.

Author Contribution

W.K. was responsible for data curation, formal analysis, investigation, methodology, software, visualization, and writing - original draft. S.C. was responsible for funding acquisition, conceptualization, project administration, resources, supervision, validation, and writing - review & editing. J.L. provided methodology and resources. J.L., S.J. and H.S. was responsible for formal analysis, methodology and visualization. W.J., J.H.J., K.K. and S.J. provided resources. All authors reviewed the manuscript.

Data availability statements

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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No competing interests reported.

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Next Generation Sequencing of Amplified DNA of Circulating Tumor Cells in Resectable Non-Small Cell Lung Cancer: A Comparative Analysis with Primary Cancer Tissue and Cell-Free DNA

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Abstract

Purpose

Methods

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Conclusion

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Introduction

Methods

Results

Discussion

Conclusion

Declarations

Funding:

Author Contribution

Data availability statements

References

Additional Declarations

Supplementary Files

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