Prognostic Value of Intraoperative Pleural Lavage Cytology as an Independent Prognostic Factor in Non-Small Cell Lung Cancer: A Retrospective Study

Clinical records of in were retrospectively reviewed and analysed for the relevance of intraoperative PLC and clinical outcomes.


Abstract Background
The aim of this study is to demonstrate that intraoperative PLC has a role in predicting clinical outcomes in NSCLC patients.

Methods
Intraoperative PLC was performed in NSCLC patients who had no pleural effusion before the operation. PLC was performed three times for each patient. PLC1 was performed after the thoracotomy; PLC2 was performed immediately after complete operation; and PLC3 was performed after complete operation and washed the pleural cavity with 5,000 ml of normal saline solution. Clinical records of 178 patients in Ramathibodi Hospital from 2012 to 2016 were retrospectively reviewed and analysed for the relevance of intraoperative PLC and clinical outcomes.
Results 178 patients were included in this study; 67 patients were male (37.6%). Metastatic tumour from primary lung cancer occurred in 56 patients (31.4%). Positive intraoperative PLC was signi cantly associated with higher metastatic rate (p < 0.05). Survival rate in the positive intraoperative PLC group was signi cantly worse than that in the negative PLC group (p < 0.05).

Conclusions
This study shows positive intraoperative PLC was statistically signi cant for increasing metastatic rate and decreasing survival rate in NSCLC patients. Intraoperative PLC could provide important information for the prediction of disease progression and treatment planning.

Background
Lung cancer is the third most common cancer in Thailand [1][2][3]. Amongst them, NSCLC is the most common type, contributing 80-85 %. The current diagnosis and staging system for NSCLC is based on the TNM system, which is de ned by using tumour size, lymph node metastasis, and distant metastasis. The TNM staging system is e ciently used for determining disease prognosis and treatment options. The overall ve-year survival rate for NSCLC is 24%. However, the survival rate depends on several factors, especially the stages of disease. For patients with stage 1A disease, the overall ve-year survival rate is more than 90%, whereas it is less than 10% for stage 4 patients. A different stage leads to a different treatment. In the early stage NSCLC, upfront surgery is the treatment of choice while chemoradiation is preferred in metastatic patients. In NSCLC, pleural effusion, especially malignant pleural effusion, demonstrates an advanced stage of NSCLC.
Pleural effusion with hemorrhagic or serosanguinous appearance is more likely to be malignant. In biochemical analysis, malignant pleural effusion is usually exudate. The simplest method to diagnose malignant pleural effusion is the presence of malignant cells from pleural uid cytology. Another method is to demonstrate tumour cells in the pleural tissues which can be obtained from pleural biopsy. According to the TNM staging system for NSCLC, patients with malignant effusion are categorised as M1a or stage 4 disease [4]. However, pleural effusion is only present in 7-23% of lung cancer patients and malignant pleural effusion is found in only 20% of patients with an advanced stage of disease [5,6]. Several studies show that cancer cells can be detected in the thoracic cavity from pleural lavage, even in the absence of pleural effusion [7,8].
IPL is the procedure done by using physiologic saline solution irrigate in the thoracic cavity over the lung surface. In our study, we performed 3 IPLs for each patient and sent specimens for cytologic analysis. Interestingly, IPL cytology has been reported to be associated with clinical outcomes in many studies [9][10][11][12]. Nevertheless, IPL cytology is not included in current methods for diagnosis and prognosis prediction [13]. We demonstrated here to determine whether intraoperative PLC can reveal micro metastasis and assess the prognostic signi cance in clinical outcomes.

Patients
This retrospective study was approved by the Ethics Committee of the Institute (MURA2017/695; ID 10-60-19) and compliance with Helsinki declaration. The need of informed consent was waived by ethical committee of Faculty of Medicine Ramathibodi Hospital, Mahidol University, Thailand. No identifying information was recorded by research team. We included all patients with NSCLC who underwent lung surgery and intraoperative pleural lavage in Ramathibodi Hospital from January 2012 to December 2016. Patients who had preoperative pleural effusion and patients whose data had been lost were excluded from this study. The nal study sample consisted of 178 patients.

Procedure
We performed intraoperative PLC three times for each patient. The 1 st PLC (PLC 1) was performed after the thoracotomy. The 2 nd PLC (PLC 2) was performed after complete the operation and the 3 rd PLC (PLC 3) was performed after complete the operation and carefully washed the pleural cavity with 5,000 ml of normal saline solution. 500 ml of pleural lavage uid for each specimen was collected and sent for cytologic examination. The cytologic report of malignant cells or atypical cells was considered to be positive. The cytologic result came back within 2 weeks after the operations. PLC technique and surgical procedure were standardised in all patients and a single-surgeon surgeries were performed throughout the study. Pathological staging was performed by using the International Staging System for lung cancer [13].

Statistical analysis
Continuous data in this study were reported as mean with SD or median with IQR. The difference between mean or media was conducted using the independent t-test or the Mann-Whitney U-test. Categorical variables were presented and analysed by chi-squared test or Fisher's exact test. The survival and recurrent rate were analysed by Kaplan-Meier method. The differences between the survival curves were assessed by using the log-rank test.

Patient characteristics
From 178 patients in total, 67 patients were male (37.6%). Intraoperative PLC was positive in 20 cases (11.23%). Patient baseline characteristics were not different between the recurrent/metastatic group and non-recurrent group. However, visceral pleural invasion, vascular invasion, lymphatic invasion and post-operative staging were higher in recurrent/metastatic group (Table 1). Similar results were reported when we analysed patient baseline characteristics in death and non-death groups ( Table 2).
Pleural lavage cytology is associated with mortality Patients with positive intraoperative PLC had a signi cantly higher recurrent/metastatic rate (80%) compared to those in the negative PLC group (25%) (p < 0.001; Fig.1). The prognosis of patients with positive intraoperative PLC was worse than that of patients with negative PLC, with 60% and 12% of mortality rate respectively (p < 0.001; Fig.2). The median follow-up time was 53 months. (IQR: 33-66 months) We also separately analysed the recurrent/metastatic rate and survival based on the results of PLC. The patient with either PLC 1, 2 or 3 positive was signi cantly associated with the increasing recurrent rate and the decreasing survival rate, compared to those in the negative PLC group (p < 0.01). Patients with PLC 1 positive had the worst prognosis (Fig.3). The association between PLC status and patient baseline characteristics and clinicopathological characteristics were shown in Table 3. Positive intraoperative pleural lavage cytology was signi cantly associated with visceral pleural invasion (p=0.018), lymphatic invasion (p=0.043), and pathological stage (p=0.003).
In our sub-group analysis, we analysed the association between intraoperative pleural lavage cytologic results and clinical outcomes for each pathological stage. We found that in pathological stage 1A and 1B, patients with positive PLC had signi cantly higher recurrent/metastatic rate compared to those with negative PLC (p< 0.05; Table 4). In addition, patients in pathological stage 1B with positive PLC had both higher recurrent rate and worse survival rate compared to patients with negative PLC (p< 0.05; Table 5) However, the recurrent/metastatic rate and survival rate were not signi cantly different between two patient groups in other pathological stages.

Discussion
Back in 1958, the rst report about cytological examination for cancer cells in pleural cavities from pleural lavage at the time of thoracotomy was published [14]. Later, Kondo et al [15] and Okumura et al [16] analysed the clinical signi cance of intraoperative pleural lavage cytology and reported its prognostic value. Pleural lavage cytology has been proved to be associated with the degree of pleural extension, microscopic pleural dissemination, pathologic stage, and the presence of lymphatic or vascular invasion in these studies. In 2014, Hokka et al [17] published a report showing a prognostic value of pleural lavage cytology in NSCLC patients with stage 1 disease.
In our study, we compiled a ve-year collection of data in Ramathibodi Hospital with the aim to provide the information about the clinical signi cance of intraoperative PLC status in NSCLC patients. The yield of positive intraoperative PLC was 11.2%. This percentage is comparable with other previous studies [16,18]. For primary outcomes, we compared the recurrent/metastatic and survival rate between positive and negative PLC groups.
The results suggested that a positive PLC status is signi cantly associated with poor clinical outcomes: the increasing recurrent rate and the decreasing survival rate. Apart fromthe recurrent/metastatic andsurvival rate, PLC status is signi cantly associated with visceral pleural invasion, vascular invasion and lymphatic invasion.
Patients with positive PLC also seemed to have a higher pathological staging. Moreover, from our sub-group analysis, NSCLC patients with pathological stage 1A and 1B, which current recommendation suggests surveillance follow up without any adjuvant therapy [19], were found to have a statistically signi cant association between positive PLC status and their recurrent and survival rate. Since intraoperative pleural lavage is a noninvasive procedure and requires only simple equipment and a cytological examination, it can be easily added on to the usual operations. Besides, we recommend intraoperative pleural lavage cytologic status to be considered as one of the prognostic factors for further treatment planning and surveillance strategy, especially in NSCLC patients with pathological stage 1A and 1B.

Conclusion
The PLC status was positive in 11.23% of NSCLC patients who underwent lungresection surgery. The positive nding signi cantly associated with higher rates of visceral pleural invasion, vascular invasion, lymphatic invasion, and a higher pathological staging. More importantly, a positive PLC status was signi cantly associated with higher recurrent/metastatic rate and worse survival rate, particularly for patients with early-stage diseases. Availability of data and materials The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.   Figure 1 Kaplan-Meier plot of recurrent/metastatic rate in 178 patients with NSCLC and its association with the PLC result.
The P value was determined using the log-rank test.

Figure 2
Kaplan-Meier plot of overall survival in 178 patients with NSCLC and its association with the PLC result. The P value was determined using the log-rank test.

Figure 3
Kaplan-Meier plot of recurrent/metastatic rate in 178 patients with NSCLC and its association with the PLC result divided by type of specimen. The P value was determined using the log-rank test.