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Touch print cytology shows higher sensitivity than pleural lavage cytology for pleural micro-metastasis in lung cancer
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     1 Department of Thoracic and Cardio-vascular Surgery, Seoul Veterans Hospital, Seoul, Korea

    2 Department of Pathology, College of Medicine, Hallym University, Seoul, Korea

    3 Department of Pathology, Seoul Veterans Hospital, Seoul, Korea

    Abstract

    Many authors have used pleural lavage cytology for confirming pleural micro-metastasis in lung cancer, but the results showed relatively low sensitivity having positive range of 4–14%. So, if we have a method with higher sensitivity than lavage cytology, the significance of pleural micro-metastasis will be elucidated clearly. To detect micro-metastasis, we underwent touch print cytology using glass slides and lavage cytology. Just after thoracotomy, touch print was carried with 2 pieces of glass slide from the visceral pleural surface of tumor mass. Then, two lavage cytology were collected at the times of pre-resection and post-resection (after distilled water irrigation) using each 1 liter of saline. The glass slides and lavages were examined for confirming micro-metastasis of cancer cells. From June 1998 to May 2004, 146 tumor masses of lung cancer have been subjected. Among them, 127 sets of cytology slides were reviewed completely in the last one month. Touch print cytology was found to be positive in 81.9% (104/127), while lavage cytology in 4.7% (6/127) of pre-resection and in 0.8% (1/127) of post-resection. To detect pleural micro-metastasis, touch print cytology using a glass slide can be considered as an easy and effective method having higher sensitivity than lavage cytology.

    Key Words: micro-metastasis; touch print cytology; lung cancer; pleural cytology

    1. Introduction

    Pleural micro-metastasis of lung cancer is defined as a metastasis found microscopically at pleural cavity in patients without any clinical or radiological evidence of pleural metastasis. Pleural lavage cytology has been applied to detect pleural micro-metastasis during surgical intervention. Many reports have been published on the prognostic value of pleural lavage cytology in lung cancer patients [1–3], but pleural lavage cytology is not generally accepted yet as a definite staging factor in lung cancer. The positive rate of pleural lavage cytology varied widely, from 4% [4] to 14% [5], due to the different techniques used and different stages of patients enrolled. The impact of pleural micro-metastasis on clinicopathologic factors such as visceral pleural invasion, tumor size, lymph nodal status, cell type and tumor biology (aggressiveness of tumor) remain to be elucidated [6]. So, if we have a method with higher positive rate than pleural lavage cytology, the significance of pleural micro-metastasis will be elucidated clearly.

    We conducted this study to compare touch print cytology with pleural lavage cytology in the same patients, to understand better the significance of pleural micro-metastasis as a prognostic factor.

    2. Patients and methods

    Between June 1998 and May 2004, 146 patients had underwent both touch print cytology test and pleural lavage cytology test during surgical resection of non-small cell lung cancer in the department of Thoracic and Cardio-vascular Surgery at Seoul Veterans Hospital, Seoul, Korea. Patients with macroscopic pleural metastasis and preoperative chemo-radiotherapy were excluded. Among them, 127 sets of cytology slides were collected and reviewed completely in the last one month, for accurate diagnosis and minimizing bias from slide review at different times. Nineteen cases were excluded because of some missing slides among their sets, in spite of pathologic reports. Therefore 127 patients’ data were enrolled. There were 123 men and 4 women aged 43–78 years (mean, 67.2 years). Complete surgical resection consisted of pneumonectomy (n=17), bilobectomy (n=14) and lobectomy (n=96). The subjects included 78 patients with stage I (IA 16, IB 62), 17 with stage II (IIA 1, IIB 16) and 32 with stage III (IIIA 29, IIIB 3) disease. There were 82 squamous cell carcinoma, 34 adenocarcinoma, 8 bronchioloalveolar cell carcinoma and 3 others.

    Just after thoracotomy, the visceral pleural surface of the tumor was carefully imprinted with two pieces of glass slide (TPC, Fig. 1). Then, pleural cavity was washed with 1 l of normal saline, the lavage fluid was collected for cytology (PLC1). When the planned pulmonary resection and radical lymph node dissection had been finished, pleural cavity was washed with 4 l of distilled water to prevent cancer dissemination during manipulation of the lung in pleural cavity. After sucking dry completely, 1 l of new saline was washed repeatedly as another lavage fluid (PLC2), just before closure of thoracotomy. Touch print glass slides and two lavage fluids were sent to the department of pathology with the resected specimen after surgery. Two lavage fluids (PLC1 and PLC2) were centrifuged for 5 min at 1500 rpm to make cellblock. Touch print glass slides and cell blocks were fixed in 10% buffered formalin for paraffin embedding, stained with hematoxylin and eosin for examination of cancer cells (Fig. 2). One hundred and twenty-seven sets of both cytology slides were collected and reviewed completely by a single pathologist in the last one month.

    The results of the cytologic examination of touch print and lavage fluids were divided into two categories: negative and positive. For lavage cytology slide, Papanicolaou classes I to III were regarded as negative, and classes IV to V as positive, because of its low sensitivity. For touch print cytology slides, which have high sensitivity, we divided as negative and positive, and then we divided again positive slides into four subgroups according to the number of tumor cells, less than 10 per slide (a few cells, subgroup 1), 10 to 50 per slide (cell nests, subgroup 2), 50 to 100 (cell clusters, subgroup 3) and 100 or more (cell sheet, subgroup 4). We compared touch print cytology with lavage fluid cytology in their positive rate. Positive and negative groups of touch print cytology were compared with regard to the sex, age at intervention, cell type, differentiation, tumor size, lymph node involvement, visceral pleural invasion, recurrences and survival rates. Among the subgroups of positive touch print cytology, the patients’ survival data were compared. Age groups were divided into 64 years or less and more than 65 years. Histologic cell types were grouped into squamous cell carcinoma and non-squamous cell carcinoma, and differentiation into well, moderate and poor. Tumor sizes were divided into 3 cm or less and larger than 3 cm. For lymph node involvement, we used the classification of Mountain and Dresler[7]. Recurrence was defined as clinical or pathologic confirmation during follow-up periods and divided into loco-regional and distant metastasis. Follow-up data were obtained from patients’ clinic visits, and follow-up loss was defined as no-show after the last 6 months visit to our clinic. Follow-up was completed in 95 patients (loss in 32 patients), and ranged from 0.03 to 70.23 months (mean 54.13 months, median more than 70 months).

    The 2 test was used to evaluate the significance of the relationship between the positivity of touch print cytology and each of the clinicopathologic factors. Survival curves were calculated using Kaplan–Meier method, and the date of tumor resection at my hospital was used as the starting time; statistical comparisons were made using the log-rank test. Univariate analysis was conducted according to each factor, such as sex, age at intervention, histologic type, differentiation, tumor size, nodal status, visceral pleural invasion and touch print cytology. Cox's proportional hazards model was used for the multivariate analysis to evaluate the independent prognostic roles of these factors in overall survival. The level of significance was set at 5% (P<0.05).

    3. Results

    Among the 127 sets of cytology slides, touch print cytology was found to be positive in 81.9% (104/127), while pleural lavage cytology in 4.7% (6/127) of pre-resection and in 0.8% (1/127) of post-resection. All cases of positive pleural lavage cytology are included in positive touch print cytology cases, and post-resection case of positive pleural lavage cytology is included in pre-resection cases of positive pleural lavage cytology (Fig. 3).

    Each of the clinicopathologic factors, such as sex, age at intervention, histology, differentiation, tumor size, nodal status, TNM stage, and visceral pleural invasion shows no statistically significant relationship with positivity of touch print cytology (Table 1). Lymphatic permeation, angio-invasion, perineural invasion, bronchial resection margin involvement and extra-capsular lymph node involvement might have also no significant relationship, not shown in this paper due to inadequate data acquisition. During the follow-up, 18 cases of recurrence were reported. We found only one recurrence (4.3%) of 23 patients of negative touch print cytology, whereas 17 recurrence (16.3%) of 104 patients of positive touch print cytology, but not significant in statistics (P=0.306). In the loco-regional recurrence of same pleural cavity, positive touch print cytology have 11 recurrences (10.6%) of 104 patients, whereas one recurrence (4.3%) of 23 negative touch print cytology patients (Table 2). Overall 5-year survival rate of all patients is 74% and mean survival time is 54 months. Five-year survival rate is 83% and mean survival time is 50 months in negative touch print cytology patients, 73% and 53 months of positive patients (Fig. 4). There is no statistical difference between the two groups yet (P=0.4366). Among the subgroups of positive touch print cytology, there are also no differences in survival yet.

    By univariate analysis, sex, age at intervention, histology, differentiation, tumor size, N stage, visceral pleural invasion, pathologic stage and touch print cytology were evaluated to find predictors of a poor prognosis. As a result, age at intervention (P=0.0011), tumor size (P=0.0027), N stage (P<0.0001) and pathologic stage (P=0.0001) were found statistically significant predictors of a poor prognosis. Touch print cytology was not a prognostic predictor yet, in spite of its high positive rate in our data set.

    The factors above were evaluated by multivariate Cox model analysis. As a result, N stage (relative risk=1.41, P=0.0001) was identified only as independent predictors of a poor prognosis in our data set.

    4. Comments

    To understand better pleural micro-metastasis as a possible prognostic factor in lung cancer patients, we conducted this study and found that touch print cytology shows higher positive rate than pleural lavage cytology. Surprisingly, the positive rate of my touch print cytology test is very high (81.9%), in spite of the relatively low positive rate of lavage cytology test (4.7%) from same patients. Some authors showed as high as 71%–83% of positive results in visceral pleural penetration subgroup (P2) by similar method [8] and by lavage cytology test [9]. However, even in no visceral pleural invasion (Table 1), touch print cytology was positive at same high (76.9%) in my data. Some author [8] reported that positive cytology appeared even in early stage and no visceral pleural invasion, and it maybe due to the limitation of pathologic examination. But, it might not be the problem of pathologic examination. The source of malignant cells in the pleural cavity is still unclear. There are two possible mechanisms to explain positive touch print cytology. One is tumor cell exfoliation from the tumor mass, and the other is lymphatic permeation through sub-pleural lymphatic micro-connection with pleural space [5]. Exfoliation might be associated with visceral pleural invasion [1,5]. However, when the tumor mass is far from the visceral pleural surface, the positive cytology may originate from the lymphatic micro-connection of the tumor. There is need to elucidate their mechanism later.

    Pleural micro-metastasis influences the postoperative long-term survival of a patient of non-small cell lung cancer, therefore it is very important to thoroughly examine during surgery. Many authors have reported the micro-metastasis of cancer cells in pleural cavity by pleural lavage cytology, and the positive rate of pre-resection lavage cytology is varied from 4% to 14%, but their relevance for survival are still uncertain [1–3,9]. These variable differences of results are due to the different lavage methods, relatively low sensitivity of lavage cytology test itself, differences of subjected patients’ profile in staging, histologic cell types, etc.

    We described touch print cytology as a feasible method with high sensitivity to detect pleural micro-metastasis. Therefore, if the patient is suspected as occult pleural dissemination of tumor cells, touch print cytology could be a good and easy method to examine.

    Lavage cytology showed very low result as 4.7% in PLC1 (pleural lavage cytology before resection) and 0.8% in PLC2 (pleural lavage cytology after resection and distilled water irrigation). It might be the dilution effect from large amount as 1 l of saline used in my study. I checked touch print cytology at first just after thoracotomy, and then made lavage cytology before resection (PLC1). If I reverse touch print cytology and lavage cytology before resection, the rate of positive touch print cytology is lower than my result, because of washing (dilution) effect on pleural surface, but the positive lavage cytology may not change. There is need to confirm whether or not. Lavage cytology after resection (PLC2) had only one positive result (0.8%) among 127 cases, it might be very useful to eradicate the possible tumor cells exfoliated in pleural cavity during resection, with washing of 4 l of distilled water. Survival data are 74% in overall 5-year survival rate and 54 months in mean survival time. Survival difference was not elucidated in touch print cytology yet.

    My study presents a feasibility that touch print cytology using glass slides can be considered as an effective method with surprisingly high positive rate to examine pleural micro-metastasis. We are waiting the survival results and collect more cases to elucidate the survival impact and relations between other clinicopathologic factors.

    It is suggested that if we use the new method, touch print cytology, the significance of pleural micro-metastasis can be elucidated clearly.

    Appendix. Conference discussion

    Dr. W. Walker (Edinburgh, UK): With 82% of patients being positive for this test, is there a possibility that it may be so sensitive, so ubiquitous that it doesn't provide us with additional information that we can use Do you think that it's too sensitive

    Dr. Kim: Yes, I think so. I found positive touch print cytology in suprisingly higher rate than pleural lavage cytology from same patients. However, survival difference was not elucidated in touch print cytology positivity yet. So, I think that there is a limit in number of expoliated tumor cells having clinical significance. The optimum number is 105 microorganism in infection and 107 cancer cells in mouse lung cancer inoculation. Below the some limit, there might be no clinical effect on host, I think.

    Dr. Walker: Most of your patients are positive, 82%

    Dr. Kim: Yes.

    Dr. Walker: So I wonder if it would be helpful to have a distinction between very positive and a little positive so that you have more groups.

    Dr. Kim: Yes. In fact, I divided my positive groups into 4 subgroups according to the number of tumor cells, less than 10 per slide, 10 to 50, 50 to 100 and 100 or more. In my preliminary result, between the subgroups, there are no significant survival differences yet, and even between 1,2 and 3,4 subgroups, there is no significant difference.

    Dr. H-B. Ris (Lausanne, Switzerland): Many years ago we have tested this technique in order to predict the outcome of resectable non-small cell lung cancer, and I'm very glad to hear that, in fact, taken your results, there is no relevant impact on the outcome whether or not this test is positive after complete resection, as far as I understood. However, could you identify subgroups of patients within your cohort where the outcome was significantly linked to the positivity of the result, in order to suggest adjuvant treatment in these patients In other words, can you identify patients where the positivity of the result had an impact on survival

    Dr. Kim: In my opinion, survival impact might be originated not from whether the tumor cell detected or not by my test, but from the remained amount of tumor cells in remained tissue, if they have same aggressiveness. If there is some correlation between the amount of tumor cells detected by my method and the unknown remained amount of tumor cells in remained tissue, we should consider adjuvant treatment and it should effects on survival benefit. So, I hope to know the unknowns by my later study. Anyway, I think, after resection, we must try to clean up the pleural cavity from the possible exfoliated tumor cells thoroughly to prevent loco-regional pleural recurrence.

    Dr. Ris: For instance, in stage I or II or III of the disease, was there a difference with respect to survival, long-term survival, with or without a positive result from the touch technique

    Dr. Kim: Unfortunately, there is no significant survival difference yet in overall and in each stages according to the touch print cytology positivity. However, from my preliminary data, in stage III, there is a tendency toward better prognosis in negative TPC, so, I wait it.

    Dr. T. Dosios (Athens, Greece): By definition, stage T1, the tumor T1, has no infiltration of the pleura, but the touch print positivity of your patients, they are supposed to have infiltration of the pleura. So how do you characterize your patients Do you still characterize them as IA or IB after these touch print positive results

    Dr. Kim: There are two possible mechanisms of positive touch print cytology or pleural lavage cytology. One is the tumor cell exfoliation from the tumor mass and the other is lymphatic permeation from the sub-pleural lymphatic micro-channels. So, if the pleural mass is far from the visceral pleural surface, maybe it originated from the lymphatic micro-connection. So, I think that it is need to distinguish the near-pleural mass and the mass in far from the pleura, because of different possible mechanism. However, it is fact that the tumor even in negative pleural invasion has also positive touch print cytology result, and I think it is not only the limitation of pathologic exam.

    Footnotes

    Presented at the joint 18th Annual Meeting of the European Association for Cardio-thoracic Surgery and the 12th Annual Meeting of the European Society of Thoracic Surgeons, Leipzig, Germany, September 12-15, 2004.

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