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Keywords:

  • lung neoplasms;
  • nonsmall cell lung carcinoma;
  • prognosis;
  • fine-needle aspiration biopsy;
  • cytology;
  • tumor-infiltrating lymphocytes;
  • multivariate analysis

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

BACKGROUND

The prognostic significance of tumor-infiltrating lymphocytes (TILs) in surgically resected carcinomas was reported. To apply this to inoperable nonsmall cell lung carcinomas (NSCLC) of Stage IIIB–IV, the authors estimated the occurrence of TILs using percutaneous fine-needle aspiration biopsy specimens, and tested the validity of this method.

METHODS

The authors defined the L-N index as [LS/(LS + NS) − LB/(LB + NB)], in which LS and NS denoted lymphocyte and neutrophil counts in the aspiration smear, and LB and NB denoted lymphocyte and neutrophil counts in the peripheral blood specimen. The cutoff value was set at twice the standard deviation of the L-N index of 41 smears contaminated with abundant blood. Retrospectively, the authors compared the survival rate of the group with a high L-N index (lymphocyte-dominant group) (n = 12) with the survival rate of the group with a low L-N index (lymphocyte-nondominant group) (n = 60). Then, they performed a prospective study and compared the survival rates of these 2 groups (n=21 and n = 54). The Cox proportional hazards model was used to determine the effect of the L-N index as a continuous variable and other prognostic factors. The correlation (r) between the L-N index-based grouping (L-N grouping) and the histologic grade of TILs was studied among resected lung tumor specimens (n = 164).

RESULTS

In the retrospective and prospective studies, the survival rate was significantly higher in the lymphocyte-dominant group than in the lymphocyte-nondominant group (P = 0.0019 and P = 0.0001). Using multivariate analysis, the L-N index was an independent prognostic factor. A significant correlation was noted between L-N grouping and histologic grade of TILs (r = 0.476).

CONCLUSIONS

The L-N index of aspiration smears was found to be an independent prognostic factor for patients with advanced-stage NSCLC. L-N grouping was correlated with the histologic assessment of TILs. Cancer 2005. © 2005 American Cancer Society.

The grade of lymphocytic infiltration of the primary tumor is significantly correlated with the survival rate among patients with various types of carcinomas.1–4 Some studies have focused on patients with lung carcinoma and showed the prognostic value of tumor-infiltrating lymphocytes (TILs).5–8 However, because the materials used in these studies were surgically resected lung tumor specimens, their results were applicable only to surgical patients and not to nonsurgical patients with advanced-stage lung carcinoma. TILs may also play a favorable role in the survival of patients with advanced-stage carcinomas. A clinical investigation of the relation between TILs and survival among patients with advanced-stage lung carcinoma is warranted, but it is difficult to measure the degree of infiltration of TILs without resected specimens. We tried to estimate the degree of infiltration of TILs using percutaneous fine-needle aspiration biopsy (FNAB) specimens of nonsmall cell carcinomas of the lung (NSCLC). However, because aspiration specimens are contaminated by blood and the degree of contamination varies, the number of lymphocytes in the smear does not always reflect the grade of infiltration of TILs. Because of this difficulty, instead of trying to estimate the infiltration of TILs quantitatively, we selected TIL-rich tumors qualitatively. We defined the L-N ratio as the ratio of the number of lymphocytes to the number of lymphocytes and neutrophils [lymphocyte count/(lymphocyte count + neutrophil count)]. We assumed that an aspiration smear that has a much higher L-N ratio than the L-N ratio in a blood sample obtained from the same patient was derived from a TIL-rich tumor. We hypothesized that aspiration smears that meet the criterion of [(L-N ratio of the smear) − (L-N ratio of peripheral blood) ≫ 0] were obtained from TIL-rich tumors and that they are associated with good prognosis. To test these hypotheses, we performed retrospective and prospective studies of patients with NSCLC.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Retrospective Study

Between September 1991 and August 1998, 962 patients were diagnosed with NSCLC at the National Hospital Organization Himeji Medical Center (Himeji-city, Japan). Of the 962 patients, percutaneous FNAB cytology had been performed in 252 patients whose diagnosis could not be confirmed by sputum cytology, transbronchial technique, or cytology of pleural effusion. Two hundred seventeen patients were diagnosed with NSCLC by percutaneous FNAB cytology, and 72 of these patients had carcinoma of TNM Stage IIIB or IV.9 This retrospective study included these 72 patients.

In all patients, percutaneous FNAB was performed with conventional fluoroscopic guidance using a 22-gauge needle. The depth of the center of the tumor from the skin surface had been measured on computed tomographic scans in advance. In percutaneous FNAB, after the needle was inserted into the tumor, a 20-mL disposable syringe containing approximately 3 mL of normal saline was attached to the needle. The piston was pulled strongly and moved forward and backward with the needle, and then the material was aspirated from a broad area of the tumor. The aspirate was spouted with the normal saline into a test tube. The cells were recovered by centrifugation at 1500 revolutions per minute at room temperature for 5 minutes and were immediately smeared onto 3 glass slides. One slide was air-dried and stained with Giemsa stain. The remaining 2 smears were immediately fixed in 95% alcohol and stained with Papanicolaou (Pap) stain. In the Giemsa-stained smear, the number of lymphocytes and neutrophils was counted and the L-N ratio was calculated by the formula [LS/(LS + NS)], where LS and NS denote the lymphocyte count and neutrophil count on the smear, respectively. Depending on the cellularity of the smear, either cells in the entire smear were counted or cells were counted until the sum of lymphocytes and neutrophils reached 100 cells. Lymphocytes and neutrophils were identified morphologically. We obtained a peripheral blood sample on the day that aspiration was performed. Using the data from the peripheral blood cell count, we calculated LB/(LB + NB), in which LB and NB are the lymphocyte count and neutrophil count of the peripheral blood specimen, respectively. We defined the L-N index as [LS/(LS + NS) − LB/(LB + NB)]. For the control group, from the total of 468 smears that had been obtained by percutaneous FNAB between September 1991 and August 1999, we selected 41 smears that were considered to be contaminated with abundant blood and that fulfilled the following criteria: 1) the smear was negative for malignant cells; 2) the specimen had been aspirated from a tumor that was later confirmed to be carcinoma by reexamination or surgery; 3) the specimen contained many red blood cells without necrotic matter; and 4) histiocytes comprised < 5% of all of the nucleated cells on the smear. We calculated the L-N index of the 41 control smears, and determined the mean and standard deviation (SD). The cutoff value was set as the mean of the L-N index of the 41 control smears plus twice the SD. Patients who had an L-N index of greater than or equal to the cutoff value were placed in the lymphocyte-dominant group, and patients who had an L-N index below the cutoff value were placed in the lymphocyte-nondominant group.

Patients received chemotherapy with or without palliative radiotherapy. Chemotherapy included cisplatin/carboplatin and vindesine. Chemotherapy was repeated every 4 weeks for a maximum of 6 cycles. If progression of the disease was observed, the chemotherapy regimen was stopped and the patient was managed symptomatically including palliative radiotherapy if possible. If the toxicity of the chemotherapy was severe, the treatment was postponed. Patients who refused all these therapies and patients with poor performance status were managed only symptomatically. Survival was defined as the interval between the date of aspiration and the date of death or the date that last follow-up information was obtained for living patients. Survival was compared between the lymphocyte-dominant group and the lymphocyte-nondominant group on September 16, 1999.

Prospective Study

The prospective study included patients who were diagnosed with TNM Stage IIIB/IV NSCLC by aspiration cytology between November 1999 and May 2004, and from whom consent to participate in the study was obtained. The L-N index was calculated as described for the retrospective study. The patients were placed into the lymphocyte-dominant group or lymphocyte-nondominant group using the criteria that had been determined in the retrospective study, and their survival was observed prospectively. The definition of survival was the same as that in the retrospective study. Among the patients in the prospective study, survival was compared between the lymphocyte-dominant group and the lymphocyte-nondominant group on January 11, 2005. Patients were treated with the same regimen as that described in the retrospective study except that docetaxel, vinorelbine, paclitaxcel, gemcitabine, and irinotecan were added to the choice of chemotherapy drugs, and if progression of the disease was observed during the chemotherapy, the regimen was shifted to another regimen as long as a benefit from the chemotherapy was expected.

Predictors of Survival: Univariate and Multivariate Analyses

The prognostic significance of clinical factors, type of treatment, cytologic features of malignant cells, and the L-N index was studied by univariate and multivariate analyses. The patients in the prospective study were included in the univariate and multivariate analyses. The L-N index was used as a categorical variable (lymphocyte-dominant or lymphocyte-nondominant group) in the univariate analysis and as a continuous variable in the multivariate analysis. The cytologic features of malignant cells were evaluated using the Pap-stained smears of each patient. For each patient, the largest malignant cell on the smears excluding spindle-shaped cells was identified. Then, the widest dimensions of the cytoplasm and nucleus of this cell were measured with an eyepiece micrometer disc (× 40 objective), and the nuclear-to-cytoplasmic ratio (N/C ratio), which was defined as the widest dimension of the nucleus divided by the widest dimension of the cytoplasm of the largest malignant cell, was calculated. For each continuous variable, the patients were divided into two groups according to the median value of that variable. They were also grouped according to the prominence of nucleoli, the presence of mitosis, and cell type (squamous cell carcinoma [SCC] type vs. non-SCC type). The degree of the variation in cell size was classified according to whether the ratio of the dimension of the largest malignant cell to the dimension of the smallest malignant cell did or did not exceed threefold. The amount of necrotic matter was estimated in relation to cellularity. All of the smears were analyzed by two of the authors independently (Y.N and Y.N.). If the results of grouping did not agree, a final decision was made by discussion. The clinical factors that were analyzed were age, gender, Eastern Cooperative Oncology Group (ECOG) performance status, body weight loss, TNM stage, T classification, serum albumin level, lactate dehydrogense level, C-reactive protein level, and type of treatment.

Correlation between the L-N Index-Based Grouping (L-N Grouping) and Histologic Grade of TILs

Between November 1999 and May 2004, 310 patients were diagnosed with NSCLC by aspiration cytology, and 235 of the patients had Stage IA–IIIA disease. Among the 235 patients, 164 patients underwent surgical resection as the first line of treatment. The grade of TILs in these 164 resected tumors was classified into 5 groups according to the method of Black et al.10 The Spearman correlation coefficient between the histologic grade and L-N grouping was calculated.

Statistical Analyses

The Pearson chi-square test (minimum expected count ≥ 5), the Fisher exact test (minimum expected count < 5), or the Mann–Whitney test was used to assess the statistical significance of differences in the clinical characteristics, cytologic variables, and types of treatment between the lymphocyte-dominant and lymphocyte-nondominant groups. Survival curves were estimated according to the Kaplan–Meier method, and the log-rank test was used for univariate analysis of survival. To assess the joint effect of clinical factors, types of treatment, and cytologic variables on survival, the Cox proportional hazards regression model was used in which variables were selected by a stepwise method. Then, the statistically significant parameters were retained, the factor of the L-N index was added to the Cox model, and the prognostic influence of the L-N index was determined after adjustment for these clinical and cytologic factors. We checked the assumptions of the proportional hazards model by plotting the cumulative hazards functions for each categoric variable. The degree of correlation between the L-N grouping and the histologic grade of TILs was calculated by the Spearman's correlation coefficient. The level of statistical significance was set at P < 0.05. The statistical analyses were performed using SPSS for Windows computer software (version 10.0; SPSS Inc., Chicago, IL).

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Retrospective study

Figure 1 shows a histogram of the L-N index of the 41 control NSCLC specimens that had been obtained by percutaneous FNAB and that were contaminated with abundant blood. The histogram was interpreted as a normal distribution with a mean of 0.00 and an SD of 0.11. We set the cutoff value at 0.22 (mean + 2 × SD). Patients with an L-N index ≥ 0.22 were placed in the lymphocyte-dominant group, and patients with an L-N index < 0.22 were placed in the lymphocyte-nondominant group. The retrospective study included 72 patients who were diagnosed with NSCLC of TNM Stage IIIB or IV by percutaneous FNAB cytology. The characteristics of the 72 patients are summarized in Table 1. Twelve patients had an L-N index ≥ 0.22 and were placed in the lymphocyte-dominant group, whereas 60 patients had an L-N index < 0.22 and were placed in the lymphocyte-nondominant group (Fig. 2). The median survival time of the lymphocyte-dominant group was 32.9 months (95% confidence interval [95% CI], 25.1–40.7 months) (n = 12), whereas that of the lymphocyte-nondominant group was 8.9 months (95% CI, 5.6–12.3 months) (n = 60). The survival rate of the lymphocyte-dominant group was significantly higher than that of the lymphocyte-nondominant group (log-rank test, P = 0.0019) (Fig. 3).

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Figure 1. Histogram of the L-N index of 41 control specimens of nonsmall cell lung carcinoma that were obtained by percutaneous fine-needle aspiration biopsy and that were contaminated by a large amount of blood. The L-N index was defined as [LS/(LS + NS) − LB/(LB + NB)], in which LS and NS denote lymphocyte and neutrophil counts in the aspiration smear, and LB and NB denote lymphocyte and neutrophil counts in the peripheral blood specimen. The L-N indices were interpreted as having a normal distribution with a mean of 0.00 and a standard deviation of 0.11.

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Table 1. Characteristics of the Patients with TNM Stage IIIB or Stage IV Nonsmall Cell Lung Carcinoma in the Retrospective Study and the Types of Treatment They Received
CharacteristicsNo. of patients
  1. ECOG: Eastern Cooperative Oncology Group; BSC: best supportive care.

Age in (yrs) 
 <6938
 ≥6934
Gender 
 Male52
 Female20
ECOG performance status 
 0–161
 2–311
Body weight loss ≥5% in the past 3 mos 
 Yes4
 No68
TNM stage 
 IIIB35
 IV37
Cell type: squamous cell carcinoma type 
 Yes25
 No47
Type of treatment 
 BSC5
 Chemotherapy67
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Figure 2. Histogram of the L-N index of the aspiration smears from 72 patients with nonsmall cell lung carcinoma of TNM Stage IIIB/IV in the retrospective study. The curved line denotes the distribution of the L-N index of the controls. The specimens were divided into the lymphocyte-dominant and lymphocyte-nondominant groups using the cutoff value (0.22), which had been defined as the mean (0.00) plus × 2 the standard deviation of the L-N index of the controls. The L-N index was defined as [LS/(LS + NS) − LB/(LB + NB)], in which LS and NS denote lymphocyte and neutrophil counts in the aspiration smear, and LB and NB denote lymphocyte and neutrophil counts in the peripheral blood specimen..

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thumbnail image

Figure 3. Comparison of the survival of the patients in the lymphocyte-dominant and lymphocyte-nondominant groups in the retrospective study. The survival rate of the patients in the lymphocyte-dominant group was significantly higher than that of the patients in the lymphocyte-nondominant group (log-rank test, P = 0.0019). LD: lymphocyte-dominant group; LnD: lymphocyte-nondominant group.

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Prospective Study

Seventy-five patients who were diagnosed with Stage IIIB/IV NSCLC by aspiration cytology were enrolled in the prospective study. Figure 4 shows the histogram of the L-N index of the lung carcinoma specimens from these 75 patients, which resembled the histogram of the L-N index of specimens from those in the retrospective study. Of the patients in the prospective study, 21 patients had an L-N index ≥ 0.22 and were placed in the lymphocyte-dominant group, whereas 54 patients had an L-N index < 0.22 and were placed in the lymphocyte-nondominant group. The background characteristics of the patients in the lymphocyte-dominant group (n = 21) and those in the lymphocyte-nondominant group (n = 54) did not significantly differ (Table 2). The median survival time of the lymphocyte-dominant group was 35.7 months (95% CI, 11.6–59.8 months) (n = 21), whereas that of the lymphocyte-nondominant group was 8.5 months (95% CI, 4.1–13.0 months) (n = 54). The survival rate of the lymphocyte-dominant group was significantly higher than that of the lymphocyte-nondominant group (log-rank test, P = 0.0001) (Fig. 5).

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Figure 4. Histogram of the L-N index of the aspiration smears from the 75 patients with nonsmall cell lung carcinoma of TNM Stage IIIB or IV in the prospective study. The specimens were divided into the lymphocyte-dominant and lymphocyte-nondominant groups based on the cutoff value (0.22). The L-N index was defined as [LS/(LS + NS) − LB/(LB + NB)], in which LS and NS denote lymphocyte and neutrophil counts in the aspiration smear, and LB and NB denote lymphocyte and neutrophil counts in the peripheral blood specimen..

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Table 2. Relation between L-N grouping and Various Clinical and Cytologic Characteristics and Type of Treatment among the Patients in the Prospective Study with TNM Stage IIIB or Stage IV Nonsmall Cell Lung Carcinoma
VariableL-N groupingTotalP value
LnDLD†
  • LnD: Lymphocyte-non-dominant group; † LD: lymphocyte-dominant group; ECOG: Eastern Cooperative Oncology Group; LDH: lactate dehydrogenase; CRP: C-reactive protein; BSC: best supportive care; N/C: nuclear-to-cytoplasmic ratio.

  • a

    Pearson chi-square test.

  • b

    Fisher's exact test.

  • c

    Mann–Whitney test.

Age in (yrs)    
 < 69271138 
 ≥ 692710370.853a
Gender    
 Male411253 
 Female139220.109a
ECOG performance status    
 0–1451863 
 2–393121.000b
Body weight loss ≥ 5% in the past 3 mos    
 Yes7310 
 No4718651.000b
TNM classification    
 IIIB211031 
 IV3311440.491a
T-classification    
 110212 
 217724 
 3617 
 42111320.310c
Serum albumin level < 3.5 mg/dL    
 Yes15419 
 No3917560.435a
Serum LDH level > upper limit of the normal range    
 Yes17522 
 No3716530.512a
CRP ≥ 0.25 mg/dL    
 Yes421658 
 No125171.000b
Type of treatment    
 BSC224 
 Chemotherapy5219710.311b
Size of the largest cell ≥ 21 μm    
 Yes241236 
 No309390.323a
Size of the largest nucleus ≥ 14 μm    
 Yes251338 
 No298370.225a
N/C ratio (widest dimension of the nucleus/widest dimension of the cytoplasm) ≥ 0.71    
 Yes271340 
 No278350.353a
Prominent nucleoli    
 Yes12618 
 No4215570.563a
Variation in cell size: dimension of the largest cell/dimension of the smallest cell ≥ 3    
 Yes516 
 No4920691.000b
Mitosis    
 Present213 
 Absent5220721.000b
Necrosis    
 Present23831 
 Absent3113440.722a
Cell type: squamous cell carcinoma type    
 Yes18523 
 No3616520.422a
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Figure 5. Comparison of the survival of the patients in the lymphocyte-dominant group and lymphocyte-nondominant group in the prospective study. The survival rate of the lymphocyte-dominant group was significantly higher than that of the lymphocyte-nondominant group (log-rank test, P = 0.0001). LD: lymphocyte-dominant group; LnD: lymphocyte-nondominant group.

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Predictors of Survival: Univariate and Multivariate Analyses

Univariate and multivariate analyses of survival were performed for the 75 patients in the prospective study. Univariate analysis of the clinical characteristics, type of treatment, and cytologic features of malignant cells showed that the following factors were found to be predictors of poorer survival: ECOG performance status of 2–3, Stage IV disease, serum albumin level < 3.5 mg/dL, and the presence of mitosis (Table 3). Multivariate analysis using the Cox proportional hazards regression model revealed that Stage IV disease, an ECOG performance 2–3, and a low serum albumin level were significant predictors of poorer survival (Table 4, Model 1). When the L-N index was introduced into this model as a continuous variable, we found that a low L-N index was a significant predictor of poorer survival, and that the addition of the L-N index into the clinical and cytologic model increased the log likelihood significantly (Table 4, Model 2).

Table 3. Comparison of the Survival of Patients in the Prospective Study with TNM Stage IIIB or Stage IV Nonsmall Cell Lung Carcinoma with Various Demographic, Clinical, and Cytologic Characteristics and Type of Treatment
VariableNo. of patientsMST in mos (95% CI)P valuea
  • MST: median survival time; 95% CI: 95% confidence interval; ECOG: Eastern Cooperative Oncology Group; LDH: lactate dehydrogenase; CRP: C-reactive protein; BSC: best supportive care; N/C: nuclear-to-cytoplasmic ratio.

  • a

    Log-rank test.

Age in (yrs)   
 < 693815.3 (6.2–24.4) 
 ≥ 69378.6 (4.6–12.7)0.2364
Gender   
 Male5310.0 (7.2–12.7) 
 Female2214.5 (5.6–23.4)0.3280
ECOG performance status   
 0–16314.5 (9.6–19.4) 
 2–3123.7 (2.0–5.5)0.0000
Body weight loss ≥ 5% in the past 3 mos   
 Yes104.1 (2.1–6.1) 
 No6511.1 (6.2–16.0)0.3059
TNM stage   
 IIIB3120.1 (15.3–24.8) 
 IV446.0 (3.0–9.0)0.0013
T-classification   
 11214.0 (7.0–21.0) 
 22410.0 (4.4–15.6) 
 378.5 (1.0–16.1) 
 43211.4 (2.8–20.0)0.8607
Serum albumin level < 3.5 mg/dL   
 Yes195.8 (3.1–8.4) 
 No5614.5 (9.4–20.0)0.0024
Serum LDH level > the upper limit of the normal range   
 Yes228.4 (2.6–14.3) 
 No5311.4 (6.7–16.1)0.9491
CRP ≥0.25 mg/dL   
 Yes588.7 (6.4–11.1) 
 No1717.3 (11.7–22.8)0.2317
Treatment   
 BSC44.6 (0.0–14.3) 
 Chemotherapy7110.8 (5.5–16.1)0.6153
Size of the largest cell ≥ 21 μm   
 Yes3610.8 (2.8–18.8) 
 No3910.3 (5.6–15.0)0.6046
Size of the largest nucleus ≥ 14 μm   
 Yes388.5 (1.7–15.3) 
 No3711.4 (6.5–16.2)0.6762
N/C ratio (widest dimension of the nucleus/widest dimension of the cytoplasm) ≥ 0.71   
 Yes409.5 (6.6–12.4) 
 No3514.0 (7.2–20.9)0.5751
Prominent nucleoli   
 Yes1814.5 (0.0–34.4) 
 No5710.3 (7.5–13.0)0.4242
Variation in cell size: dimension of the largest cell/dimension of the smallest cell ≥ 3   
 Yes68.5 (2.5–14.6) 
 No6911.1 (5.9–16.3)0.7630
Mitosis   
 Present35.3 (0.0–10.7) 
 Absent7211.1 (6.6–15.6)0.0075
Necrosis   
 Present318.6 (4.7–12.6) 
 Absent4414.0 (9.4–18.6)0.2928
Cell type: squamous cell carcinoma type   
 Yes238.7 (5.0–12.5) 
 No5211.1 (6.4–15.8)0.7360
Table 4. Multivariate Analysis among the Patients in the Prospective Study with Nonsmall Cell Lung Carcinomaa
VariableModel 1: Clinical and cytologic grouping factors and types of treatmentModel 2: Model 1 with L-N index
CoefficientSEHR (95% CI)P valueCoefficientSEHR† (95% CI)P value
  • SE: standard error of coefficient; HR: hazard ratio; 95% CI: 95% confidence interval; ECOG: Eastern Oncology Oncology Group.

  • The L-N index: L-N index was treated as a negative, continuous variable in the Cox proportional hazards model.

  • a

    The likelihood ratio of Model 1 is 26.69 (degrees of freedom: 3), and that of Model 2 is 39.94 (degrees of freedom: 4).

  • b

    There is significant improvement in the likelihood ratio in Model 2 (P = 0.000).

Stage IV0.640.301.90 (1.06–3.41)0.0330.900.322.46 (1.32–4.60)0.005
ECOG performance status 2–31.290.393.65 (1.71–7.75)0.0010.930.392.52 (1.18–5.41)0.017
Serum albumin level <3.5 mg/dL0.800.312.22 (1.20–4.09)0.0110.830.322.29 (1.22–4.30)0.010
−L-N indexb    3.250.9725.77 (3.84–173.17)0.001

Correlation between L-N Grouping and Histologic Grade of TILs among Resected Specimens

We determined the grade of TILs on histologic analysis of surgically resected NSCLC specimens (Stage I, 110 specimens; Stage II, 24 specimens; Stage IIIA, 30 specimens) (Table 5). There was a significant correlation between the histologic grade of TILs and L-N grouping (Spearman's correlation coefficient, 0.476, P = 0.000) (Table 5).

Table 5. Correlation between L-N Grouping and Histologic Grade of TILs among Resected Nonsmall Cell Carcinomas of the Lung of Stage IA to IIIAa
Grade of TILsbL-N groupingTotal
LnDLD
  • TILs: tumor-infiltrating lymphocytes; LnD: lymphocyte-nondominant group; LD: lymphocyte-dominant group.

  • a

    Spearman's correlation coefficient = 0.476 (P = 0.000).

  • b

    Black's histologic grading of tumor-infiltrating lymphocytes.

019322
149958
2293261
351419
4044
Total10262164

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

To our knowledge, few cytologic studies regarding TILs have been reported to date. Using aspiration smears of mammary carcinomas, Wallgren and Zajicek11 tried to investigate the prognostic value of the grade of lymphocyte occurrence on the smear, but they did not find a significant relation between the lymphocytic factor and 10-year survival. They evaluated the lymphocytic population on the smear by determining the ratio of the number of lymphocytes to the number of carcinoma cells on the smear with allowance for the lymphocytes in admixed blood. In their study, 18 of the 494 aspirates were detected as smears containing more than occasional lymphocytes. The grade of TILs must reflect the ratio of the number of lymphocytes to the number of tumor cells or tumor volume.5 It is possible to determine this ratio in histologic studies. However, in aspiration cytologic preparations, several factors, such as contamination with blood and the degree of tightness of the cell-to-cell connection between tumor cells, interfere with precise evaluation of this ratio. Our L-N index does not proportionately reflect this ratio, but it can be used to select TIL-rich tumors. To determine the cutoff value of the L-N index, we needed aspiration smears from tumors with no infiltration of lymphocytes or neutrophils as controls. However, it was very difficult to obtain such smears. Theoretically, if an aspiration specimen is contaminated by a large amount of blood, the L-N index of the sample would be close to 0 regardless of the degree of infiltration of lymphocytes or neutrophils into the tumor. We used severely blood-contaminated specimens as a substitute for smears from infiltration-free tumors. In fact, the histogram of the L-N index of these control specimens had a normal distribution with a mean of 0. The SD was calculated, and aspirates with an L-N index of greater than or equal to twice the SD were believed to have been derived from TIL-rich tumors, and were placed in the lymphocyte-dominant group. Theoretically, patients with tumors with infiltration of predominantly neutrophils, with an L-N index of less than minus twice the SD (i.e., the neutrophil-dominant group), may exist. However, there was only 1 patient in our retrospective study who met this criterion (L-N index: −0.23, survived for 9.1 months) and this patient was placed in the lymphocyte-nondominant group. In the prospective study, no patient was placed in the neutrophil-dominant group.

It must be noted that our method may misjudge TIL-rich tumors as lymphocyte-nondominant cases if the aspiration specimen is contaminated with a large amount of blood. In our study of the correlation between L-N grouping and histologic grade of TILs, 5 of the 19 specimens with Grade 3 TIL were classified into the lymphocyte-nondominant group (Table 5), and it was ascertained that these 5 aspiration specimens had been contaminated by a large amount of blood.

In our histologic study of resected carcinoma specimens, we frequently observed that the distribution of TILs was not always uniform in the specimens, and it was reported that the type of distribution (peritumoral or intratumoral infiltration) affected the prognostic importance of TILs.5, 7 However, it is difficult to obtain a sample from a particular part of the tumor by the aspiration method. We also noticed that the presence of specimens with severe necrosis often disturbed the correlation between L-N grouping and the histologic grade of TILs. Nevertheless, the current study revealed a good correlation between L-N grouping and the grade of TILs among the resected tumors. This suggests that the L-N index is a meaningful index for picking TIL-rich tumors using aspiration materials. The reproducibility of the L-N index has not been verified, but its close correlation with the histologic grade of TILs supports that it may be reproducible if vast contamination by blood due to accidental puncture of blood vessels by the aspiration needle is avoided.

To verify the joint effect of the L-N index and other prognostic factors by multivariate analysis, we studied several clinical and cytologic characteristics. We selected clinical variables based on the consensus report of prognostic factors in inoperable NSCLC.12 To our knowledge, there has been no consensus report published to date regarding significant predictors of survival among cytologic variables. In morphometric studies of various neoplasms, it was reported that the nuclear dimension, mitotic frequency, and N/C ratio are prognostic factors.13–17 However, these results are controversial.5, 18–21 The absence of mitosis was found to be a significant prognostic factor for survival in our univariate analysis, although it was not a significant prognostic factor in the multivariate analysis. It must be noted that our results on the assessment of cytologic factors have limited value because the number of malignant cells on each smear varied widely from a few cells to thousands of cells. The influence of this lack of uniformity may not be neglected, especially with regard to the presence of mitosis. According to Lee et al.,5 the mean number of mitotic cells is approximately 1/1000 of the number of all malignant cells in lung carcinomas, and the chance of finding mitotic features in the aspiration smear greatly depends on the cellularity of malignant cells on the smear. In the current prospective study, mitosis was detected in only 3 of the 75 specimens. Hence, our finding regarding the prognostic significance of the absence of mitosis is not definite.

Multivariate analysis revealed that the L-N index had independent prognostic value for survival, with a higher L-N index associated with better prognosis. The association between a high L-N index and better prognosis may be explained by the immunologic reaction of the TILs against the malignant tumor. In addition, FNAB specimens that are contaminated with abundant blood have a low L-N index and likely originated from tumors with high angiogenesis. It was reported that high angiogenesis, as evaluated by the microvessel count in resected tumors, was associated with poorer survival among patients with NSCLC.22, 23 However, a low L-N index in an aspiration smear does not always indicate that the smear originated from a tumor with high angiogenesis, because the low L-N index may simply reflect a low TIL count.

Eerola et al.8 reported that a high TIL count was associated with small tumor size and low tumor stage among resected small cell lung carcinomas. Our study using percutaneous FNAB specimens of advanced-stage NSCLC revealed no significant relations between L-N grouping and neither the TNM stage of disease nor T classification (Table 2). Watanabe et al.24 reported that a large number of TILs was present, particularly in papillary adenocarcinomas, among various histologic types of resected lung carcinomas. However, the results of the current study did not demonstrate a relation between the cell type of malignant cells (SCC vs. non-SCC) and L-N grouping.

The potential risks of percutaneous FNAB include chest wall implantation of malignant cells and spreading of carcinoma cells in the pleural cavity.25–27 However, Kjellberg et al.28 reported that there was no significant correlation between the result of preoperative transthoracic needle biopsy and positivity for malignant pleural lavage cytology. Sawabata et al.29 reported that percutaneous FNAB was not associated with disease recurrence or prognosis among patients with operable lung carcinoma. These findings suggest the safety of this technique.

In the current study, we found that when using percutaneous FNAB specimens, the L-N index was an independent prognostic factor among patients with advanced stage NSCLC. L-N grouping was also found to be well correlated with the histologic grade of TILs among resected tumors. To the best of our knowledge, there has been no report of a valid method of assessing TILs using aspiration materials. Our method theoretically provides reproducible results, can be applied to any stage of carcinomas, and could be used in various types of clinical studies on lung carcinomas.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

The authors thank all members of the Department of Respiratory Medicine, the Department of Respiratory Surgery, and the Department of Pathology Laboratory of Himeji Medical Center for their collaboration.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES