
==== Front
J Cytol
J Cytol
JCytol
J Cytol
Journal of Cytology
0970-9371
0974-5165
Wolters Kluwer - Medknow India

JCytol-41-171
10.4103/joc.joc_116_23
Original Article
Methodological and TNM Focus-Based Comparison of EGFR Mutation Status in Non-Small-Cell Lung Carcinomas
Akca Yasemin
Erkilic Suna
Department of Pathology, Faculty of Medicine, Gaziantep University, Gaziantep, Turkey
Address for correspondence: Dr. Yasemin Akca, Department of Pathology, Faculty of Medicine, Gaziantep University, Gaziantep, Turkey. E-mail: dryaseminakca@gmail.com
Jul-Sep 2024
18 7 2024
41 3 171175
07 7 2023
23 5 2024
20 6 2024
Copyright: © 2024 Journal of Cytology | Indian Academy of Cytologists
2024
https://creativecommons.org/licenses/by-nc-sa/4.0/ This is an open access journal, and articles are distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as appropriate credit is given and the new creations are licensed under the identical terms.
Background:

Epidermal growth factor receptor (EGFR) mutations in non-small-cell lung carcinomas (NSCLC) are a frequent class of driver mutations, and tyrosine kinase inhibitor (TKI) therapy provides considerable clinical benefits. Using the most effective and also easiest method for EGFR analysis is cost-effective and time-saving. In this study, we aimed to determine which method could be more effective by comparing the incidences of EGFR mutations in cytological and histological samples which were obtained by different methods also, whether there was a difference in the incidences of EGFR mutations between the primary foci, mediastinal lymph nodes, and distant metastatic foci.

Materials and Methods:

We retrospectively reviewed 420 cases of cytological materials, small biopsies, and surgical samples reported as NSCLC underwent EGFR analysis in our department between 2016 and 2022. We collected the data and interpreted the results from two different perspectives.

Results:

We identified 36 EGFR mutations in 362 biopsies (9.94%) and 17 in 58 cytology samples (29.31%). There is a significant difference between the two methods (P = 0.01*). We observed 38 EGFR mutations in 320 primary foci (11.87%), 7 EGFR mutations in 36 mediastinal or subcarinal lymph nodes (19.44%), and 8 EGFR mutations in 64 distant metastatic foci (12.50%). A significant difference was also observed in pleural samples (P = 0.005*).

Conclusion:

We observed more successful results with cell blocks obtained from liquid-based cytological specimens than with formalin-fixed, paraffin-embedded tissues obtained from resection or otherwise in our clinical routine. Our study results highlight the benefits of cytological specimens in molecular treatments and current therapy modalities.

Cell block
EGFR
liquid-based cytology
metastatic tumor
non-small-cell lung carcinoma
ThinPrep
==== Body
pmcINTRODUCTION

Lung cancer is the leading cause of cancer-related deaths worldwide, due to its poor prognosis.[1] EGFR mutations are characteristic of a subset of pulmonary adenocarcinomas and represent an effective therapeutic target for some TKIs. In patients whose tumors harbor somatic activating mutations in EGFR, treatment with the inhibitors gefitinib and erlotinib provided significantly better response rates than tumors without mutations.[23] Some studies have shown that the response to anti-EGFR therapy was also superior to that seen with standard platinum-based chemotherapy.[4] Therefore the detection of the EGFR mutation status is becoming increasingly important.

Although there are several methods of detection, EGFR mutations are generally detected in DNA from samples of tumor tissue obtained during biopsy or surgical resection, in the form of formalin-fixed paraffin-embedded diagnostic blocks, using gene sequencing.[5] The cost of the test is high, especially in developing countries, and the time to start treatment in lung cancer patients should be as soon as possible. Therefore, clinicians usually want to use the most effective method for EGFR analysis.

There are many studies in the literature about EGFR mutation in various ethnic groups and populations. However, we could not find any study that shows methodological and tumor-node-metastasis (TNM)-based results. For this purpose, we first compared the incidences of EGFR mutations in biopsy samples and cytological materials obtained with different methods. Second, we compared the incidences of EGFR mutations in biopsy samples from the primary foci, mediastinal-paravertebral lymph nodes, and distant metastatic foci in patients with NSCLC. We interpreted the results in light of the current literature.

MATERIALS AND METHODS

Study Design

We retrospectively evaluated 420 NSCLC cases diagnosed, and the EGFR mutation statuses was tested by polymerase chain reaction (PCR) in our department between 2016 and 2022. Local ethics committee approval was obtained with the number 2023/127.

We designed this study and analyzed the data in 2 stages. First, we methodologically determined the incidences of EGFR mutation and compared the results between different biopsy and cytology methods that were included in the study. Second, we determined the incidences of EGFR mutation in biopsy samples taken from the primary foci, various distant foci, and the mediastinal-subcarinal lymph nodes (TNM-based). The results were compared. To further detail our study, we included the important points in the subheadings of both evaluations. Finally, we indicated the most common mutations in our study group.

Distribution of cases

The study included 362 biopsy specimens obtained by the methods of bronchoscopic biopsy, tru-cut biopsy, wedge resection, lobectomy, pneumonectomy, or metastasectomy, and 58 cytological specimens obtained from pleural fluids, bronchial and bronchioalveolar fluids, endobronchial ultrasound-guided fine needle aspirations, transbronchial fine needle aspirations of lung parenchyma, or mediastinal lymph nodes. The types and numbers of biopsy specimens are shown in detail in Table 1.

Table 1 Numbers of cases obtained by different biopsy methods, numbers of positive cases, and incidences

Specimen types	Numbers of Cases	Numbers of positive EGFR mutations	EGFR incidences	
Total number of cases	420	53	12.61%	
Biopsy	362	36	9.94%	
    • Bronchoscopie and tru-cut biopsy	191	22	11.51%	
    • Metastasectomy or lymph node excision	63	7	11.11%	
    • Resection	108	7	6.48%	
Cytology	58	17	29.31%	
    • Pleural fluid	15	5	33.33%	
    • Bronchial and bronchioalveolar fluid	11	4	36.36%	
    • Endobronchial ultrasound-guided or transbronchial fine needle aspiration	32	8	25%	

From the other perspective, 320 of the cases were from the primary foci, 36 of the cases were from the mediastinal and subcarinal lymph nodes, and 66 of the cases were from the distant metastatic foci. The biopsy sites and numbers are detailed in Table 2.

Table 2 Numbers of TNM-based biopsy foci, numbers of positive cases, and incidences

Focus	Numbers of Cases	Numbers of positive EGFR mutations	EGFR incidences	
• Primary foci	320	38	11.87%	
    Lung	280	28	10%	
    Pleura and pericardium	21	8	38.09%	
    Chest wall and vertebral tissues	19	2	10.52%	
• Mediastinal and subcarinal lymph nodes	36	7	19.44%	
• Distant foci	64	8	12.50%	
    Lymph nodes	17	3	17.64%	
    Liver	6	1	16.66%	
    Brain	17	2	11.76%	
    Bone and soft tissues	20	2	10%	
    Surrenal	2	-	-	
    Other (esophagus and stomach)	2	-	-	

We used the American Joint of Cancer Community (AJCC)[6] TNM staging system and used the radiological and scintigraphic computerized data of the patients for the determination of the primary and distant metastatic foci. Primary foci include lung parenchyma, bronchus, pleura, direct extension to mediastinal structures, chest wall, hearth and pericardium, vertebra, and associated soft tissue. Metastatic foci like liver, brain, distant bones, and vertebral structures, distant lymph nodes (submandibular, neck, supraclavicular, axillary, and paratracheal), and mediastinal and subcarinal lymph nodes are listed as a separate topic.

Tissue preparation and molecular evaluation

We use the ThinPrep® liquid-based cytologic system for all cytological specimens in our laboratory. After preparing the slides from the specimen, we prepare cell blocks from residual material. After the diagnostic and immunohistochemical evaluations, we use cell block sections for DNA analyses. The cell block is prepared by first centrifuging the remaining liquid at 4000 rpm for 4 minutes after slide preparation, then adding 3 ml of 96% ethyl alcohol and 0.5 ml of 10% formaldehyde to the bottom sediment, waiting for 60 minutes, and collecting the remaining sediment on filter paper. It is then placed in a cassette and kept in 96% ethyl alcohol until it is placed in the tissue processing device. Finally, it is processed starting from the alcohol stage in the tissue processing device.

Although there is no well-defined precise numerical criterion for cellular adequacy in the guidelines, in our laboratory, we accept samples with a minimum of 50 tumor cells and a minimum tumor cell ratio of 25% for testing, so the number of cells has been standardized in this way. We use real-time PCR technology to sequence exons 18, 19, 20, and 21 using the Cobas® EGFR Mutation Test and System in our department.

On the other hand, we use formalin-fixed, paraffin-embedded tissue blocks for biopsy specimens. We elaborate on choosing the most appropriate tissue blocks with a high tumor amount and rate, which does not contain necrosis, haemorrhage, and autolytic findings to optimize the test.

Data analysis and statistical method

The descriptive statistics of the data obtained from the study are presented, with an analysis of frequencies and percentages for the categorical variables. Chi-square analysis was used to test differences between categorical variables. Analyses were performed using SPSS 22.0. P < 0.05 significance level was chosen.

RESULTS

When we analyzed the data, we found 53 EGFR mutations in a total of 420 different patients (12.61%). We found 6 different mutation types in the test, and the most common mutation is Exon 19 deletion (30/53), followed by Exon 21 L858R mutation (15/53). The detailed numbers and percentages of the different types of mutations are shown in Table 3.

Table 3 Mutation types, numbers, and percentages

Mutation types	Numbers	Percentages	
Exon19 Deletion	30	56.60%	
Exon 21 L858R	15	28.30%	
Exon20 lns mutation	3	5.66%	
Exon20 insertion	3	5.66%	
Exon 21 L861Q mutation	1	1.88%	
Exon18 6719X mutation	1	1.88%	
Total	53	100%	

When we analyzed the results from the first perspective, we found 36 positive cases in 362 biopsy specimens (9.94%) and 17 positive cases in 58 cytological specimens (%29.31) and there is a quite obvious difference (P = 0.001*). There was no significant difference between the obtaining methods of cytological (P = 0.716) and biopsy samples (P = 0.355). Even if it did not show statistical significance (P = 0.114), there was a difference between the resection samples (6.48%) and small biopsy samples (11.51%). The detailed findings are presented in Table 1.

When we analyzed the results from the second perspective, we found the highest rate was in mediastinal and subcarinal lymph nodes with a 19.44% incidence. The primary foci and distant foci rates are 11.87% and 12.50%, respectively. The highest rate belonged to the pleural samples with 33.33% in primary foci, and the highest rate belonged to distant lymph nodes with 17.64% in metastatic foci. Although no statistically significant difference was observed in the comparison of primary foci, mediastinal lymph nodes, and distant metastases (P = 0.431), the incidence was higher in mediastinal lymph nodes. The only statistically significant difference was found in pleural samples (P = 0.005*). The findings are presented in detail in Table 2.

DISCUSSION

The prevalence of EGFR mutations in patients with advanced NSCLC differed with geographic region. The highest prevalence for all EGFR mutations was observed in Asian patients (49.1%) compared with other continents (11.9–33.0%). The lowest prevalence is in Europe (11.9%).[7] These results are similar to another systematic review which found that the overall rate of EGFR mutations was lowest in Europe (14.1%) and highest in Asia (38.4%), with a combined North and South America region in the middle (24.4%).[8] The overall incidence is 11.87% in our study. The patients included in the study were from the southeastern region of Turkiye. This rate seems to be correlated with the European incidence. One of the possible reasons for the low incidence may be the possible false negatives in the resection specimens in our study. Although the incidence in small biopsies and especially in cytological materials is quite high, resection specimens reduce this rate.

The most striking result is the difference between the incidences of liquid-based cytological material and the tissue biopsy specimens especially resection specimens. We now know that protecting DNA molecular structure is crucial for molecular analyses such as EFGR. From this point of view, the time of contact between the tumor tissue and the fixative solution is very important. In large specimens such as lobectomy, pneumonectomy, and wedge resection, this time may be prolonged for various reasons, but in liquid-based specimens, the tumor cells come into direct contact with the alcohol.

We use liquid-based cytology and prepare cell blocks for immunohistochemistry and molecular analysis in our laboratory. This is an alcohol-based specimen; however, we use buffered formalin for the fixation in tissue samples. Some studies show that total genomic DNA and RNA yields from alcohol-fixed tissues were significantly higher and of better quality in nucleic acid analysis than formalin-fixed tissues and alcohol penetrated tissues faster than formalin.[910]

In addition, during our literature search, we found some articles on the superiority of liquid-based cytology specimens over paraffin-embedded tissue for molecular testing. In one study, they found that the positive rate of EGFR mutation in liquid-based cytology-tested cases was 47.18%, higher than the 41.37% tested by the formalin-fixed paraffin-embedded sample using the sediment for DNA extraction after centrifugation (P < 0.01).[11]

Liquid-based cytology is the preferred specimen for molecular detection at the Cleveland Clinic in the United States, according to another study. They mentioned that liquid-based cytology specimens are the primary resource used for molecular testing of NSCLC. In most cases, sufficient DNA can be extracted from the residual cell pellet for next-generation sequencing, and ThinPrep slides can be reliably used for fluorescence in situ hybridization testing for ALK gene rearrangements. In occasional cases where the cell pellet material is insufficient for molecular testing, cell blocks and/or surgical pathology specimens are secondary options, and they mentioned that the role of the cytopathologist in specimen handling and triage is essential for successful molecular testing.[12]

In another study, 42 liquid-based cytology samples were used for DNA extraction twice. One sample was obtained directly from the CytoLyt solution, whereas the other DNA sample was obtained after smear preparation and laser capture microdissection of Papanicolaou-stained cells and, applied direct sequencing. They found that liquid-based cytology is the gold standard if using laser capture microdissection.[13] Similarly, a result of another study reveals the overall concordance rate of EGFR gene mutation status, including minor mutations and ALK status according to immunostaining between histological and paired liquid-based specimens, was 100% (105/105) and 100% (48/48), respectively. Thus, genotyping and protein expression studies can be reliably performed using liquid-based samples.[14] Unlike these studies, we prepare the cell blocks and obtain the DNA from here to use the cells in the cytological specimen for immunohistochemical stains in our daily routine.

According to research with 82 patients who underwent EBUS-TBNA and received a confirmed diagnosis of lymph node metastasis of lung adenocarcinoma, they found 87.5% agreement between cell block and liquid-based cytology to perform DNA analysis and EGFR mutation testing,[15] and we found a satisfactory incidence of EGFR in this study, which we made from the DNA we obtained from the cell block.

There is no statistically significant difference in EGFR incidence in biopsies obtained from the primary focus, mediastinal lymph nodes, and distant metastases. We found only one study in the current literature comparing primary or distant focus, but it is not an incidence-based study. They analyzed EGFR exons 18-21 from paired primary and metastatic tumors in 67 lung cancer patients who had not received TKI before tissue sampling and found that the discordant rate reached 27% (18 of 67 cases).[16] Although there is no statistically significant difference between the foci, there are some notable points and one significant difference in pleural and pericardial samples. First, the EGFR incidence of pleura and pericardium is strikingly high (P = 0.005*) in the primary foci. One of the possible reasons for this could be that cytologic specimens represent the majority of cases in both foci. This could be correlated with the method rather than the TNM-based focus; however this hypothesis needs to be proven. Second, the EGFR incidence is highest in the mediastinal and subcarinal lymph nodes. In addition, it is an important detail that the incidence of distant metastatic focus is not significantly higher than the incidence of primary focus, although there is little difference.

If we look at the mutation types, the majority of cases, approximately 85%, are parallel to the world average and consist of exon 19 deletion and exon 21 L858R mutations, which have a higher response rate to TKI treatment.[7]

As a result of this study, we experienced that the liquid-based cytology specimens with cell block give more successful results than formalin-fixed paraffin-embedded tissue obtained by resection or other ways, in EGFR mutation testing. Although the number of cells in the cytologic sample significantly affects the applicability of the test, when the cell number is standardized as in our study, it could be more effective due to the independence of other factors that may negatively affect tissue follow-up.

CONCLUSION

According to modern treatment, targeted therapies are mostly used in patients with locally advanced or metastatic disease when conventional chemotherapeutic agents are insufficient. Being able to perform genetic testing safely from cytological material allows avoiding more invasive biopsy methods and their complications, especially in these patients.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.
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