Compare the molecular differences of pleural effusion and metastatic lymph node biopsy of lung adenocarcinoma based on NGS and evaluate the Clinical efficacy

doi:10.21203/rs.3.rs-4913713/v1

Objective

The present study is to investigate the next-generation sequencing (NGS) molecular typing results of liquid-based cytology specimens (LBCSs) of pleural effusion in lung adenocarcinoma evaluate the clinical efficacy of epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI) treatment and compare the consistency of the molecular typing results with those of metastatic lymph node biopsy specimens (MLNBSs).

Methods

A total of 222 cases of lung adenocarcinoma pleural effusion LBCSs and 201 cases of MLNBSs were collected to compare the consistency of NGS molecular typing results. The impacts of different tumor cell contents in LBCSs on mutation detection limitation was evaluated. The clinical efficacy evaluation was performed on 91 patients treated with EGFR-TKI was evaluated and the survival curve analysis was conducted using Kaplan-Meier method.

Results

The mutation rates of cancer-related genes detected by NGS were comparable LBCSs and MLNBSs of lung adenocarcinoma(82.0% vs 79.1%, P = 0.455). However, the mutation rate of EGFR T790M was significantly higher in pleural effusion LBCSs than in MLNBSs(12.2%>3.5%, P = 0.001). After EGFR-TKI treatment, the mean progression-free survival time (PFS) was 11.4 months in 91 patients with molecular typing based on LBCSs.

Conclusions

The results of NGS molecular typing of pleural effusion LBCSs from lung adenocarcinoma patients can yield comparable PFS to that of histological specimens following the clinical application of EGFR-TKI treatment.

lung adenocarcinoma

NGS

therapy efficacy

pleural effusion

liquid-based cytology

In recent years, lung cancer has demonstrated the highest incidence and mortality rates (1). Among all lung cancer cases, non-small cell lung cancer accounts for approximately 85% (2), with adenocarcinoma being the most prevalent histological subtype. Unfortunately, some patients have missed the optimal opportunity for surgical intervention (3), necessitating alternative treatment options. Currently, traditional chemotherapy combined with radiotherapy as the first-line treatment results in a 5-year survival rate of only 26.4% (4). The clinical application of molecular targeted therapies in first-line treatment has provided significant benefits to lung adenocarcinoma patients harboring specific genetic aberrations. The mortality rates of patients with epidermal growth factor receptor (EGFR) mutations and anaplastic lymphoma kinase (ALK) fusions/rearrangements have significantly decreased (5–6). Targeted drugs for EGFR mutations, such as EGFR Tyrosine kinase inhibitors (EGFR-TKIs), have been clinically used, and some patients develop resistance within 1–2 years due to EGFR resistant mutations such as T790M or activitions of other EGFR downstream pathways (7–8). Furthermore, other gene mutations may serve as potential therapeutic targets or important prognostic markers for lung adenocarcinoma (9). Therefore, conducting comprehensive and accurate analyses of the gene mutation spectrum in lung adenocarcinoma holds immense significance in guiding clinical treatment options and prognosis assessment.

Due to absence of surgical specimens in advanced staged patients, biopsy and cytology specimens serves as valuable sources for molecular typing (9). Currently, surgical resections or biopsies are the main source of specimens for genetic testing. However, for patients with pleural effusion in advanced staged lung cancer, sampling pleural effusion is simpler and more convenient. The utilization of LBCSs for NGS gene molecular typing has been reported (10). Nevertheless, there is a lack of large-scale clinical studies investigating the reliability of NGS molecular typing results based on LBCSs after targeted therapy and the consistency of gene mutations among different metastatic lesions. This study aims to evaluate the effectiveness of targeted drug therapy based on NGS molecular typing results obtained from pleural effusion LBCSs in lung adenocarcinoma and investigate the concordance between these results and those from MLNBSs.

2.1. Clinical Statistics

We collected pleural effusion LBCSs and clinical data from lung adenocarcinoma patients from January 2018 to December 2022 at the Cancer Hospital, Chinese Academy of Medical Sciences, totaling 222 cases, including 101 males and 121 females, aged 30 to 85 years with a median age of 63 years. The specimens were obtained from the thoracic, abdominal, and pericardial cavities.There were 201 cases of MLNBSs, including 99 males and 102 females, aged 34–81 years with a median age of 62 years. The lymph node sites include the neck, supraclavicular, inguinal, axillary regions. Thirteen cases had paired specimens of pleural effusion and lymph node puncture from the same patient. All cases were confirmed as lung adenocarcinoma by immunohistochemistry.

2.2. Specimen Collection and Preparation

Pleural effusion specimens: The fluid specimens were centrifuged, the supernatant was discarded, and the cell layer was transferred to a liquid-based preservation solution for preparation of liquid-based thin slices, followed by Papanicolaou staining.

Biopsy specimens: The metastatic lymph node tissues were obtained using a 19G needle, and were made into paraffin blocks, followed by HE staining.

2.3. Genomic DNA extraction

The quantity and tumor cell content in LBCSs and MLNBSs were assessed by two pathologists. DNA was extracted using a QIAamp® DNA mini kit (Qiagen, Germany) following the manufacturer's instructions, and the quantity of DNA was determined using the Qubit 2.0 fluorometer (Thermo Fisher Scientific, Carlsbad, CA, USA).

2.4. Mutation analysis by NGS

The mutation status of the samples was analyzed using the Ion AmpliSeq Colon and Lung Cancer Panel on the Personal Genome Machine (PGM) platform. The panel include 92 pairs of primers for 22 cancer-related genes, including EGFR, KRAS, ALK, etc., and was operated according to the instructions. Variants were annotated by the Torrent Variant Caller and were reconfirmed by the Integrative Genomics Viewer browser. Variants were determined when the coverage was > 1000 and the mutant allele frequency (MAF) was ≥ 5%. The clinicopathological and follow-up data of the patients were retrieved from the electronic medical record system. Among the 91 patients with complete EGFR-TKI treatment data. The efficacy was evaluated using Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1. Disease control rate (DCR) was defined as the percentage of the sum of the numbers of patients with complete remission (CR), partial remission (PR), and stable disease (SD). Progression-free survival (PFS) was defined as the duration from treatment initiation to either disease progression or death due to any cause.

2.5. Statistics analysis

The statistical analysis was performed using SPSS 21.0 software. The chi-square test and Fisher’s exact test were employed to compare the differences in gene molecular typing results between pleural effusion and lymph node, as well as to investigate the impact of tumor cell content on mutation detection rate. The consistency test of paired specimens used Kappa statistics, where Kappa < 0.20 indicated complete inconsistency, Kappa of 0.21–0.40 indicated general consistency, Kappa of 0.41–0.60 indicated moderate consistency, Kappa of 0.61–0.80 indicated substantial consistency, and Kappa of 0.81-1.00 indicated complete consistency. The Kaplan-Meier method was used for survival analysis, and the corresponding survival curve was plotted. The comparison of survival rates between the two groups was conducted using the Log-rank test at a significance level of α = 0.05.

3.1. Analysis of NGS molecular typing results of pleural effusion LBCSs and MLNBSs.

The NGS gene molecular typing results were classified based on the types of genetic aberrations, including mutations of EGFR, KRAS and BRAF, fusions/rearrangements of ALK,ROS-1, and RET, and amplification of HER-2 and MET. The mutation rates of cancer-related genes in 222 pleural effusion samples and 201 metastatic lymph node biopsy samples were 82.0% (182/222) and 79.1% (159/201), respectively. There was no statistical difference between the two types of specimens (P = 0.455). The most common mutation gene in both types of specimens was EGFR mutation(62.6% vs. 53.7% ). Additionally, the detection rate of EGFR T790M mutation in pleural effusion samples was significantly higher than that in lymph node biopsy specimens (12.2 vs. 3.5%, P = 0.001), while there was no significant difference in the mutation rate of other genes except EGFR(24.3% vs. 29.4%, P>0.05).The specific test results are shown in Fig. 1.

3.2 Analysis of molecular typing results of LBCSs of pleural effusion with varying tumor cell content .

According to the evaluation results of tumor cell content, the patients were classified into three groups: group 1 (G1) with a tumor cell content ranging from 10–15%, group 2 (G2) with a tumor cell content ranging from 15–20%, and group 3 (G3) with a tumor cell content ≥ 20%. There were ten cases in G1, 16 in G2, and 196 in G3. The results showed that the mutation rate of the EGFR gene was G3 > G2 > G1, but there was no statistical difference between any two groups (P > 0.05). Regarding mutations in genes other than EGFR, each gene type exhibited higher mutation rates in G3 compared to both G2 and G1. Among the 182 pleural effusion LBCSs with detected gene mutations, there were 4 cases in G1, 9 cases in G2, and 169 cases in G3. The gene mutation rate showed G3 > G2 > G1, and the gene mutation detection rate in G3 was significantly higher than that in G2 and G1, with statistical significance (P < 0.001). Furthermore, all samples exhibiting EGFR T790M mutation were exclusively detected in G3. The specific results of gene molecular typing are presented in Fig. 2.

3.3. Consistency Analysis of Gene Mutation Rates in paired specimens

The molecular typing results of 13 pairs of LBCSs from pleural effusion and MLNBSs were analyzed. The results demonstrated complete consistentcy in gene mutations among 10 out of 13 pairs (76.9%), indicating a high level of consistency (Kappa = 0.669, P < 0.001). Among them, 11 cases (11/13, 84.6%) had completely consistent EGFR gene mutation results (Kappa = 0.740, P < 0.001). In the three pairs of specimens with inconsistent gene detection results, all three cases exhibited EGFR and KRAS mutations in pleural effusion LBCSs but not in MLNBSs, suggesting a potentially higher sensitivity of pleural effusion samples. Detailed test outcomes are presented in Table 1.

Table 1

The molecular typing results of 13 paired LBCSs and MLNBSs
No.	Type	Pleural Effusion		Lymph Node biopsy
No.	Type	Source of Sample	Mutation	Source of Sample	Mutation
1	Adenocarcinoma	Pleural effusion	EGFR L858R	Left supraclavicular	EGFR L858R
2	Adenocarcinoma	Pleural effusion	EGFR E709K	Left cervical	-
3	Adenocarcinoma	Pleural effusion	EGFR 19Del	4/7 R	-
4	Adenocarcinoma	Pleural effusion	EGFR L858R and KRAS G12A	Left supraclavicular	EGFR L858R
5	Adenocarcinoma	Pleural effusion	EGFR L858R	Right supraclavicular	EGFR L858R
6	Adenocarcinoma	Pleural effusion	EGFR L858R and intergenic-ALK	Left supraclavicular	EGFR L858R and intergenic-ALK
7	Adenocarcinoma	Pleural effusion	EGFR 19Del	Right axillary	EGFR 19Del
8	Adenocarcinoma	Pleural effusion	EGFR 19Del	Left supraclavicular	EGFR 19Del
9	Adenocarcinoma	Pleural effusion	EGFR 19Del	Left axillary	EGFR 19Del
10	Adenocarcinoma	Pleural effusion	EGFR L858R	Right supraclavicular	EGFR L858R
11	Adenocarcinoma	Pleural effusion	EGFR 19Del	Left inguinal	EGFR 19Del
12	Adenocarcinoma	Pleural effusion	EGFR L858R	Left supraclavicular	EGFR L858R
13	Adenocarcinoma	Pleural effusion	EGFR L858R	Left axillary	EGFR L858R

3.4. Comparison of Gene Mutation Rates in Pleural Effusion Samples before and after EGFR-TKI treatment

Among 222 cytology samples of pleural effusion, the patients were divided into two groups based on whether they received EGFR-TKI treatment: 173 patients underwent NGS test before EGFR-TKI treatment, while 49 patients underwent NGS testing after EGFR-TKI treatment. The results revealed that the incidence rates of EGFR mutation and other point mutation genes in patients who received EGFR-TKI treatment were slightly higher compared to those who did not receive such treatment; however, this difference was not statistically significant (85.7% vs 80.9%, P = 0.441)(Fig. 3).

3.5. Clinical Efficacy Evaluation of EGFR-TKI treatment

A total of 113 patients underwent EGFR-TKI treatment following NGS molecular subtypes based on pleural effusion LBCS. Among them, 91 patients with complete imaging and clinical follow-up data were eligible for EGFR-TKI efficacy evaluation. No cases achieved CR, while PR was observed in three cases, SD in twelve cases, and progressive disease (PD) in seventy-six cases. Among the seventy-six patients who experienced disease progression, sixty-seven were found to have EGFR mutations and nine had wild-type EGFR. The results demonstrated that the average PFS for the ninety-one patients treated with EGFR-TKI was 11.4 months (95% CI: 9.9–12.9). The average PFS of patients with EGFR mutation type was 12.3 months, 95% CI: 10.8 ~ 13.9, which was significantly longer than that of patients with wild-type EGFR, with an average PFS of 4.1 months, 95% CI: 2.1 ~ 6.2, and statistically significant (χ²=32.154, P0.001)(Fig. 4).

The advancement of molecular pathological techniques and the identification of additional targeted therapy sites for lung adenocarcinoma necessitate a refined molecular subtypes of non small cell lung cancer(NSCLC) patients as an essential prerequisite for personalized treatment (10). The molecular testing results derived from cytology specimens play a crucial role in formulating clinical treatment plans, particularly for patients with advanced diseases where surgical specimens are unavailable. Some previous studies in East Asian population have shown that the mutation rate of the EGFR gene in LBCSs of NSCLC detected by RT-PCR and ARMS-PCR was 30.1%-50.5%(11–12). In our study, the EGFR mutation rates detected by NGS in pleural effusion LBCSs were found to be 62.6% (139/222), which are similar to previous literature reported by NGS detection results using cytological specimens in East Asian population (55.3–68.0%) (13–14). And surpassing the EGFR gene mutation rates by RT-PCR or AMRS-PCR.This may be due to the fact the selected cases were confirmed by immunohistochemistry as lung adenocarcinoma, excluding cases of adenosquamous carcinoma or squamous cell carcinoma. Additionally, the homogeneous selection of cases in this study, all being stage III to IV cases either with pleural effusion or with metastatic lymph nodes, may contribute to the higher mutation rates observed. Furthermore, the use of NGS methods in this study may have increased the sensitivity of gene mutation detection.

In the analysis of 13 paired pleural effusion LBCSs and MLNBSs, 76.9% showed complete consistency in molecular typing results (Kappa = 0.669, P < 0.001). The consistentcy rate of EGFR reached 84.6% (Kappa = 0.740, P < 0.001), indicating a high consistency with MLNBSs. The inconsistent cases all exhibited EGFR and KRAS mutations in pleural effusion specimens, but not in lymph node biopsy specimens, suggesting that pleural effusion specimens may be more sensitive than metastatic lymph node specimens in detecting EGFR and KRAS genes, although the specific mechanism requires further study. It has also been found in literature and previous studies that the expression rate of PD-L1 in cytology specimens is higher than that in primary tumor histological specimens, and some literature approximately 25.0% in pleural effusion, underscoring the role of pleural effusion as an optimal candidate for molecular typing (15–18). The specific mechanism, however, necessitates further investigation.

The incidence of EGFR mutations and EGFR T790M mutations in patients who received EGFR-TKI treatment was found to be significantly higher compared to those without prior EGFR-TKI treatment. Moreover, the frequency of other gene point mutations (KRAS, BRAF) apart from the EGFR gene also exhibited a relatively elevated rate of mutation rates compared to those before treatment, possibly due to the development of EGFR TKI resistance mutations in certain patients. Several previous studies have demonstrated that the majority of patients undergoing EGFR-TKI treatment will eventually develop drug resistance, with approximately 50%-60% of these cases being attributed to either the presence of the EGFR T790M mutation or multiple point mutations in EGFR, such as E709G, G719S, S768I, and L861Q [19]. According to the study conducted by Villadolid J et al. NSCLC patients harboring a KRAS gene mutation exhibited an augmented likelihood of drug recurrence and metastasis, with the presence of this mutation being one of the pivotal mechanisms underlying primary drug resistance in EGFR-TKI naive patients (19–20). Furthermore, our investigation also revealed a significantly higher incidence rate of KRAS mutation in the treated group compared to the untreated group (6.15% > 3.50%), suggesting that KRAS mutation may contribute to resistance to TKI drugs.

NSCLC patients who undergo molecular typing using primary and metastatic lesion puncture histological specimens or surgical resection specimens to detect EGFR mutations and receive EGFR-TKI treatment can achieve an average PFS of 8.5 to 17.2 months (21–25). In our study, the median PFS after EGFR-TKI treatment with EGFR mutations was 12.3 months (95% CI: 10.8–13.9). The PFS of patients with stage III and IV lung adenocarcinoma was consistent with the previous findings (PFS: 10.9–11.2 months) (26). Among the two patients who had an EGFR mutation detected in LBCSs but not in MLNBSs, one of them achieved a significantly longer PFS of 14.0 months after EGFR-TKI treatment compared to the average PFS of 12.3 months, indicating an effective treatment response. Overall, molecular typing results obtained from LBCSs of pleural effusion are reliable and contribute to prolonged survival time following targeted therapy. In addition, the average PFS of EGFR-TKI treated patients with wild type EGFR detected in LBCSs was significantly lower compared to that of patients with EGFR mutation (4.1 vs 12.3, χ²=32.154, P < 0.001), further confirming the reliability of molecular typing results based on pleural effusion LBCSs. Cytology samples are simple to obtain, less invasive, particularly beneficial for late-stage patients with pleural effusion who do not require secondary biopsies. For patients, this approach offers convenience and economic benefits while reducing pain associated with specimen collection. Furthermore, the high sensitivity of pleural effusion LBCSs in gene mutation detection facilitates accurate selection of clinical molecular typing.

The assessment of tumor cell content is a crucial component in routine molecular testing. The recommended threshold for tumor cell content in routine molecular detection is typically set at 20% (27–28), which poses a challenge for analyzing samples with tumor cell content below 20%. It has been reported that EGFR mutations can be detected in cytology samples with tumor cell content below 20% (29). Therefore, we included samples with tumor cell content less than 20% and more than 10% in NGS detection, and the differences in gene mutation detection rates among samples with different tumor cell contents after EGFR-TKI treatment and their relevance to clinical targeted therapy were discussed. Although previous studies by Gu and Forest F et al. (30–31) have reported similar frequencies of EGFR mutation in cytological specimens with tumor cell content > 5% and < 20% compared to those with tumor cell content ≥ 20%, our study found a significantly lower EGFR mutation rates in the G1 and G2 groups compared to the G3 group. However, due to the limited number of cases, there was no statistical difference between the G1 and G2 groups (P = 0.160). Additionally, Forest F observed a lower KRAS mutation rate in samples with tumor cell content > 5% and < 20% compared to those with tumor cell content > 20% (11.1% vs 28.2%). In our study, the KRAS mutation rate in samples with tumor cell content < 20% was slightly lower than that in samples with tumor cell content > 20%, but no statistically significant difference was found (3.8% vs 4.1%, P > 0.05). The cytopathologist's experience in assessing tumor cell content is important. According to Li et al. (32), the frequency of EGFR T790M mutation in biopsy samples with tumor cell content ≥ 20% was significantly higher compared to samples with tumor cell content < 20%. However, there is limited research on the differences in gene mutation detection rates in LBC samples with different tumor cell content after EGFR-TKI treatment. In this study, 49 cases of pleural effusion specimens after EGFR-TKI treatment were categorized into G1 group (2 cases, 10%~15%), G2 group (2 cases, 15%~20%), and G3 group (45 cases, ≥ 20%) based on tumor cell content to investigate the difference in gene mutation detection rates. Our outcome revealed that the EGFR mutation rate was notably higher in G3 compared to G1 and G2 (73.3% vs 50% vs 50%), and only G3 exhibited the presence of EGFR T790M mutation while it was absent in both G1 and G2 groups. In our preliminary study, we conducted genetic testing on 82 liquid-based cytological specimens after EGFR-TKI treatment, including pleural effusion, fine needle aspiration (FNA), and brush biopsy (FOB); similarly, EGFR T790M mutation was not detected in three cytological specimens with tumor cell content < 20%.This is a noteworthy issue, and of course, the small number of specimens warrants further research by increasing the sample size. Combining the above results, for untreated NSCLC patients, when the tumor cell content is less than 20% during molecular typing, NGS testing on other kind samples for gene variations can still be attempted to increase the chances of receiving EGFR-TKI treatment. However, for patients who have already undergone EGFR-TKI therapy, it is advisable to select specimens with a tumor cell content exceeding 20% for molecular typing to obtain more dependable results of molecular detection.

In conclusion, the results of NGS molecular typing of pleural effusion specimens indicate that EGFR-TKI treatment can lead to a significantly prolonged progression-free survival. LBCSs serve as an important source for molecular testing in NSCLC patients, especially when obtaining tissue samples is challenging, and they demonstrate good reliability. At the same time, they show clinical efficacy consistent with tissue-based molecular typing results. For patients undergoing EGFR-TKI therapy, it is recommended to utilize specimens containing tumor cells ≥ 20% for molecular typing to obtain more reliable molecular detection results.

Author Contributions

SL and YW conducted the writing and statistical analysis, while JCW, ZHS, and XYX were responsible for data collation. LLZ, YS, CW, and XXC performed specimen collection and production. HQG and HZ contributed to slide interpretation, and evaluation.WHL provided research guidance for genetic testing experimental operations. ZHZ offered research guidance, paper revision assistance, as well as funding support.

Funding Statement

This study was supported by the Beijing Hope Run Special Fund of Cancer Foundation of China (grant numbers: LC2020L04 and LC2022B12).

Data Availability

The datasets during the current study are not publicly available due to it containing information that could compromise the privacy of research participants but are available from the corresponding author on reasonable request.

Ethics approval and consent to participate

This retrospective study was approved by the ethics committee of the Cancer Hospital Chinese Academy of Medical Sciences (No. 21/036-2707). The need for informed consent was waived by the ethics committee of Cancer Hospital Chinese Academy of Medical Sciences. We confirm that all methods were performed in accordance with the relevant guidelines.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Author details

Cytopathology Section, Department of Pathology, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College,

Beijing 100021, China

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No competing interests reported.

Compare the molecular differences of pleural effusion and metastatic lymph node biopsy of lung adenocarcinoma based on NGS and evaluate the Clinical efficacy

Status:

Version 1

Abstract

Objective

Methods

Results

Conclusions

Figures

1. Background

2. Material and Method

2.1. Clinical Statistics

2.2. Specimen Collection and Preparation

2.3. Genomic DNA extraction

2.4. Mutation analysis by NGS

2.5. Statistics analysis

3. Results

3.1. Analysis of NGS molecular typing results of pleural effusion LBCSs and MLNBSs.

3.3. Consistency Analysis of Gene Mutation Rates in paired specimens

3.4. Comparison of Gene Mutation Rates in Pleural Effusion Samples before and after EGFR-TKI treatment

3.5. Clinical Efficacy Evaluation of EGFR-TKI treatment

4. Discussion

5. Conclusion

Declarations

References

Additional Declarations

Status:

Version 1