Abstract
Purpose: In the present study, we examined the relationship between intraoperative pleural lavage cytology findings and presence of epidermal growth factor receptor (EGFR) gene mutations.
Methods: We investigated 160 patients who underwent surgical treatment for primary lung adenocarcinoma at our hospital from January 2011 to December 2013 to determine the presence of EGFR gene mutations and pleural lavage cytology.
Results: In all, 52 subjects (31.5%) were positive EGFR gene mutations, of whom 38 were found to possess the Exon 21 L858R mutation. Intraoperative pleural lavage cytology examinations were performed in 160 subjects and 12 had positive results, of whom 6 were positive for EGFR gene mutations, which was the Exon 21 L858R mutation in all. In a comparison between subjects possessing the Exon 21 L858R mutation and those negative for EGFR gene mutations, lavage cytology-positive (p = 0.02) and vascular infiltration-negative (p = 0.01) were characteristics of the Exon 21 L868R mutation-positive group.
Conclusion: Subjects positive for the EGFR Exon 21 L858R mutation had a higher positive rate of intraoperative pleural lavage cytology than those not possessing EGFR mutations.
Keywords: lung cancer, pleural lavage cytology, EGFR mutation
Introduction
Primary lung adenocarcinoma is the most frequent tumor associated with lung cancer and found in approximately 40% of affected patients.1) Furthermore, epidermal growth factor receptor (EGFR) gene mutations have been confirmed in some lung adenocarcinoma patients and are considered to be an important target of EGFRtyrosine kinase inhibitor (EGFR-TKI) treatment. It has also been found that EGFR gene mutations occur more frequently in Asian populations as compared to Western individuals, while Shigematsu and Gazdar2) confirmed the presence of those mutations in 48% of East Asian lung adenocarcinoma patients.
Intraoperative pleural lavage cytology is often performed for patients without pleural effusion during surgery for lung cancer to determine whether cancerous cells are contained in physiologic saline collected after infusion into the thoracic cavity immediately after a thoracotomy.3) Although not incorporated in the present edition of the TNM (tumor nodes metastasis) classification, several reports have suggested that positive lavage cytology findings are a factor for poor prognosis.3–5) It has also been shown that EGFR gene mutation-positive lung cancer patients frequently have malignant pleural effusion6) that develops during the postoperative follow-up course.7) Thus, an association between EGFR gene mutation positivity and carcinomatous pleuritis has been pointed out. Although patients with positive pleural lavage cytology findings do not always develop carcinomatous pleuritis, pleural lavage cytology positivity and carcinomatous pleuritis might not be identical. Accordingly, it has been speculated that they might be similar pathologic conditions.
There are no known reports regarding the relationship between EGFR gene mutation-positive lung cancer and the rate of positive intraoperative pleural lavage cytology findings. In the present study, we examined the relationship between the presence of EGFR gene mutations and pleural lavage cytology findings in patients with primary lung adenocarcinoma who underwent surgery at our hospital.
Methods
Study design and population
Among patients with a primary lung adenocarcinoma who underwent surgery from January 1, 2011 to December 31, 2013 at Dokkyo Medical University Hospital, we examined 160 to determine the presence of EGFR gene mutations. The Ethical Committee of Dokkyo Medical University Hospital approved this retrospective study (#28146). We investigated specimens obtained by bronchoscopy from patients whose diagnosis of adenocarcinoma was established by preoperative findings as well as those obtained during surgery from patients who did not undergo a confirmative diagnosis procedure. For EGFR gene mutation searching, we used the PNA-LNA PCR Clamp method (LSI Medicine. Tokyo, Japan).8) For intraoperative pleural lavage cytology, 50 mL of physiologic saline was infused into the thoracic cavity immediately after performance of a thoracotomy and then as much as possible was recovered for determination of the presence of carcinomatous cells.
Statistical analysis
A chi-squared test was used for statistical analysis between two groups, with a risk rate of less than 5% defined as a significant difference. SPSS, Ver. 23 (IBM, Chicago, IL, USA) was employed for the analyses.
Results
Patient characteristics are shown in Table 1. The mean age of the 160 subjects was 69.7 years (range: 32–90 years) and mean tumor size was 28.2 mm (7–120 mm). Pathologic stage was re-determined according to the TNM classification, 8th edition, with 57% of the subjects classified as Stage I and 22% as Stage III. For the surgical procedure, a lobectomy was performed in 80.0%, a pneumonectomy in 1.8%, a segmentectomy in 8.8%, and a wedge resection in 9.4%.
Table 1. Patient characteristics (n = 160).
| Gender | |
| Male | 89 |
| Female | 71 |
| T factor | |
| Tis | 13 |
| T1a | 4 |
| T1b | 41 |
| T1c | 36 |
| T2a | 32 |
| T2b | 10 |
| T3 | 16 |
| T4 | 8 |
| N factor | |
| N0 | 111 |
| N1 | 17 |
| N2 | 30 |
| N3 | 2 |
| Pleural invasion | |
| Pl0 | 114 |
| Pl1 | 30 |
| Pl2 | 6 |
| Pl3 | 10 |
| Pathologic stage | |
| I | 91 |
| II | 28 |
| III | 35 |
| IV | 6 |
| Surgical procedure | |
| Pneumonectomy | 3 |
| Lobectomy | 128 |
| Segmentectomy | 14 |
| Wedge resection | 15 |
EGFR mutation
EGFR gene mutations were confirmed present in 52 (31.5%) subjects, whereas 113 (65.5%) were negative. Of the EGFR mutations noted, the Exon 21 L858R mutation was most frequently seen in 38 (73%) subjects, followed by Exon 19 deletion in 11 (21.1%), and the Exon 18 G719A mutation in 2 (3.8%) and Exon 21 L861Q mutation in 1 (1.9%).
Pleural lavage cytology
Intraoperative pleural lavage cytology examinations were performed in 160 cases. In all, 12 (7.5%) of 160 were pleural lavage cytology-positive (Classes IV and V) and 148 (92.5%) were negative (Classes I, III, and III). The mean age of the 12 cytology-positive subjects was 69.75 years, and this group consisted of eight males and four females. Mean tumor size was 25.2 mm (range: 12–50 mm). For cytological stage, 11 subjects were classified as Class V and 1 as Class IV. Pleural invasion was p10 in 3, p11 in 5, and p12 in 4, whereas lymphatic invasion was positive in 10 subjects and vascular invasion positive in 9. EGFR gene mutations were confirmed in 6 subjects, all of whom possessed the Exon 21 L858R mutation.
Pleural lavage cytology positivity and pathologic findings in L858R group
Since all pleural lavage cytology-positive subjects showed the Exon 21 L858R mutation, subjects negative for EGFR gene mutations were defined as the control group (Table 2). Our results showed that pleural lavage cytology-positive subjects were significantly predominant among those positive for the Exon 21 L858R mutation (p = 0.02). We also examined the relationship between various pathologic factors and EGFR gene mutations. No relationship was seen between Exon 21 L858R mutation positivity and pathologic pleural or lymphatic invasion. However, among the Exon 21 L858 mutation-positive subjects, those negative for vascular invasion were predominant (p = 0.01).
Table 2. Comparison of pathologic factors between EGFR mutation-positive (L858R) and EGFR mutation-negative groups.
| EGFR mutation | Statistics | ||
|---|---|---|---|
| L858R | Negative | ||
| PLC | p = 0.02 | ||
| Positive | 6 | 6 | |
| Negative | 29 | 106 | |
| Pleural invasion | p = 0.95 | ||
| Positive | 11 | 30 | |
| Negative | 27 | 72 | |
| Lymphatic invasion | p = 0.25 | ||
| Positive | 11 | 40 | |
| Negative | 26 | 59 | |
| Vascular invasion | p = 0.01 | ||
| Positive | 9 | 47 | |
| Negative | 28 | 52 | |
EGFR: epidermal growth factor receptor; PLC: pleural lavage cytology
Discussion
The present study revealed that the ratio of subjects with positive pleural lavage cytology findings was high among those showing EGFR gene mutation positivity, all of whom possessed the Exon 21 L858R mutation. To the best of our knowledge, this is the first study to investigate and report the relationship between EGFR gene mutations and pleural lavage cytology findings. Moreover, there are few reports regarding the relationship between the Exon 21 L858R mutation and pathologic factors. Although we observed no relationship of that mutation with pleural or lymphatic invasion, few of our subjects with the Exon 21 L858R mutation showed vascular invasion.
It is thought that some lung adenocarcinoma cases are positive for EGFR gene mutations, which is important when considering treatment targeting EGFR-TKI. There are also ethnic differences in regard to EGFR gene mutations. Among lung adenocarcinoma patients, it has been reported that the positive rate was 48% (270 of 563) in Asian patients as compared to only 12% (63 of 519) in non-Asian, mostly Caucasian, patients.2) The positive rate of our Japanese subjects was 31.5%, slightly lower than that rate noted for Asians. Furthermore, various types of EGFR gene mutations have been reported although the Exon 21 L858R mutation and Exon 19 deletion are considered to be predominant. Shigematsu and Gazdar2) noted that the Exon 21 L858R mutation was found in 41% and the Exon 19 deletion in 44% in their study. In our subjects, 73% of the EGFR mutations were the Exon 21 L858 mutation, a positive rate greater than in their report.
Intraoperative pleural lavage cytology examinations are commonly performed and several reports have noted the association of those findings with prognosis3–5) although that is not reflected in the present edition of the TNM classification. Accordingly, additional studies are required. The 5-year survival rate for pleural lavage cytologypositive patients was reported to be 31–44.5%, whereas that for cytology-negative patients was 72.0–72.8%, indicating a worse prognosis in association with positive cytology findings.3–5) There is no consensus regarding the optimal techniques, such as the amounts of physiologic saline infused and recovered, and time until its recovery. Our protocol is to infuse 50 mL of normal saline into the thoracic cavity immediately after performance of a thoracotomy, then recover as much as possible for a cytology examination.3) Kameyama et al.5) reported that the positive rate of intraoperative pleural lavage cytology was 5.2%, whereas others have generally noted positive rates of 10% or lower.3,4) The positive rate in the present subjects was 8.5%, which was consistent with those other results.
The association between EGFR gene mutations and carcinomatous pleuritis is largely unknown. Wu et al.6) examined 136 carcinomatous pleuritic patients with and 91 without pleural effusion who underwent surgery for lung cancer over the same period, and found that the EGFR mutation-positive rate was higher in the carcinomatous pleuritis group (68.4% vs. 50.5%) (Table 3). They also reported that the positive rate was significantly higher for the Exon 21 L858R mutation, while no difference was found regarding the Exon 19 deletion, and speculated that the Exon 21 L858R mutation more than other EGFR gene mutations is related to carcinomatous pleuritis. The same group also examined 448 patients with carcinomatous pleuritis, and compared between those whose carcinomatous pleuritis was found at the first consultation and those who developed the condition during the course of treatment. They reported that the group with pleuritis discovered at the first consultation had a higher rate of EGFR mutation positivity, especially for the Exon21 L858R mutation.7) However, opinions vary regarding the relationship between carcinomatous pleuritis and pleural lavage cytology findings. Higashiyama et al.11) reported that carcinomatous pleuritis at the site of recurrence was more frequent in their lavage cytology-positive group (23.5% vs 2.4%). It is possible that pleural lavage cytology positivity and carcinomatous pleuritis follow the same course of development.
Table 3.
Relationship between malignant pleural effusion and EGFR gene mutation
| EGFR mutation status | Malignant pleural effusion | Statistics | ||
|---|---|---|---|---|
| Positive | Negative | |||
| L858R positive | 50 | 19 | p = 0.01 | Wu et al.6) |
| Mutation negative | 86 | 72 | ||
| L858R positive | 119 | 246 | p = 0.01 | Wu et al.7) |
| Mutation negative | 15 | 68 | ||
| Mutation positive | 9 | 25 | p = 0.02 | Smits et al.9) |
| Mutation negative | 17 | 210 | ||
| Mutation positive | 16 | 19 | p = 0.03 | Zou et al.10) |
| Mutation negative | 26 | 75 | ||
EGFR: epidermal growth factor receptor
As for the mechanism of the increase in number of cases with the Exon 21 L858R mutation in pleural effusion, hyper-expression of CXCR4 has been reported in mutation-positive patients,12) while it was also confirmed that a high expression of CXCL12 was present in effusion samples of patients with non-small-cell lung cancer.13) In another study, a high level of CXCR4 expression was noted in these patients, which reacted with CXCL12 and activated PI3K to accelerate migration, engraftment, and proliferation of cancer cells.14) Thus, it is considered that Exon 21 L858R mutation-positive cases are likely to become pleural lavage cytology-positive due to activation of the CXFR4-CXCL12 pathway.
Few studies have investigated the relationship between the Exon 21 L858R mutation and pathologic factors, with no association confirmed between the Exon 21 L858R mutation and pleural invasion in the present findings. Positive findings of pleural invasion are likely in intraoperative pleural lavage of cytology-positive cases although not all are positive.3) In the present study, the ratio of Exon 21 L858R mutation-positive subjects was high among those with positive pleural effusion cytology results. Accordingly, it is considered likely that pleural lavage cytology becomes positive even in cases with negative results for pleural invasion due to cancer cell migration accelerated by activation of the CXCR4-CXCL12 pathway, as noted above. Regarding lymphatic invasion, we found no difference between cases positive for the Exon 21 L858R mutation and those negative for any EGFR gene mutations. Furthermore, vascular invasion was significantly reduced in the Exon 21 L858R mutation-positive cases. Findings to explain the results of the present study are scarce, thus further investigations are necessary.
Important limitations of the present study include its retrospective nature, use of results from a single institution, and the few subjects examined. These limitations may affect the result of high rate (73%) of EGFR L858R mutation in the present study. In future investigations, increased accumulation of cases from multiple study centers will be important.
Conclusion
EGFR Exon 21 L858R mutation-positive subjects demonstrated a higher rate of positivity in intraoperative pleural lavage cytology findings as compared to those negative for EGFR mutations. Subjects revealed to be positive for the EGFR Exon 21 L858R mutation showed no differences in regard to pleural and lymphatic invasion, while fewer had vascular invasion as compared to subjects negative for EGFR mutations.
Disclosure Statement
The authors declare that there are no conflicts of interest to disclose in regard to this study.
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