Napsin A is an independent prognostic factor in surgically resected adenocarcinoma of the lung

Lung Cancer 77 (2012) 156–161

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Napsin A is an independent prognostic factor in surgically resected adenocarcinoma of the lung Jin Gu Lee a , Sewha Kim b , Hyo Sup Shim b,∗ a b

Department of Thoracic and Cardiovascular Surgery, Yonsei University College of Medicine, Seoul, Republic of Korea Department of Pathology, Yonsei University College of Medicine, Seoul, Republic of Korea

a r t i c l e

i n f o

Article history: Received 15 December 2011 Received in revised form 6 February 2012 Accepted 17 February 2012 Keywords: Lung Adenocarcinoma Napsin A TTF-1 Prognosis

a b s t r a c t Introduction: Napsin A is regarded as a marker of lung adenocarcinoma. However, no comprehensive analyses of napsin A-positive lung ADCs or the prognostic significance of napsin A expression have been reported to date. Methods: 110 primary lung adenocarcinoma cases were analyzed for clinicopathologic parameters, including overall survival, stage, histology, napsin A and TTF-1 expression, EGFR mutation, and ALK rearrangement. Results: Napsin A-positive adenocarcinomas were significantly more prevalent among tumors characterized as relatively small (p = 0.023), non-solid predominant (p < 0.001), non-mucinous/enteric (p < 0.001), positive for TTF-1 expression (p < 0.001), and positive for EGFR mutation (p = 0.001). Multivariate analysis of overall survival demonstrated that the absence of napsin A was an independent prognostic factor for reduced survival time (p = 0.002). Conclusion: Clinicopathologic characteristics associated with napsin A-positive lung ADC are similar to and overlap with those of TTF-1-positve ADCs. The absence of napsin A is an independent poor prognostic factor in surgically resected adenocarcinoma. Further studies are necessary to determine the role of napsin A in the progression of lung adenocarcinoma. © 2012 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Recent advances in pathology, radiology, and molecular targeted therapy have brought about a new era of lung cancer treatment. A new classification for lung adenocarcinoma (ADC) has been proposed [1], the results of low-dose computed tomographic screening have been reported [2], and targeted therapies based primarily on EGFR mutation status and ALK rearrangement have been implemented [3,4]. The histologic diagnosis of lung cancer is becoming increasingly emphasized, and the newly proposed lung ADC classification scheme holds that histologic classification itself reflects prognosis [1]. The subclassification of non-small cell lung carcinomas (NSCLCs) is essential in small biopsies or cytology specimens because of the differential activities (e.g., pemetrexed) or limited indications (e.g., bevacizumab) of newer agents [1]. Different histologic subtypes are associated with unique molecular alterations, such as EGFR mutation and ALK rearrangement [5–7]. Immunohistochemical staining is an important technique in the accurate classification of lung cancer [8,9]. Recently, napsin A has

been reported as a specific and sensitive immunohistochemical marker for ADC [10–13]. Napsin A is an aspartic protease expressed in the lung and kidney [14,15], that is capable of cleaving the preform of surfactant protein B expressed in type II pneumocytes [16,17]. As the significance of NSCLC subclassification becomes more apparent, napsin A has garnered attention as a sensitive or specific marker that may complement immunohistochemical analyses using thyroid transcription factor-1 (TTF-1) [10–13,18]. However, a comprehensive analysis of napsin A-positive ADC has not been reported to date, and the prognostic significance of napsin A is unknown. In this study, we evaluated the expression of napsin A in surgically resected lung ADC samples and correlated napsin A expression with clinicopathologic parameters such as stage, histology, TTF1 expression, EGFR mutation, and ALK rearrangement. Here, we demonstrate that the absence of napsin A expression in lung ADC samples is an independent poor prognostic indicator of overall survival. 2. Materials and methods

∗ Corresponding author at: Department of Pathology, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752, Republic of Korea. Tel.: +82 2 2228 1760; fax: +82 2 2227 7939. E-mail address: [email protected] (H.S. Shim). 0169-5002/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.lungcan.2012.02.013

2.1. Patients Medical records and archival slides from a collection of surgically resected NSCLCs were analyzed at our institution from 2005

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Fig. 1. Representative sections of napsin A-positive (A and B) and napsin A-negative (C and D) ADCs (A and C: hematoxylin–eosin; B and D: napsin A immunohistochemistry; all 200×).

to 2009. The Institutional Review Board approved this retrospective study. Cases were selected on the basis of archival slide and tissue availability rather than consecutive surgeries. Of 216 NSCLC cases selected, 106 cases displaying non-ADC histologies were excluded. Large cell carcinoma was differentiated from solid-type ADC by negative mucin staining. A total of 110 ADC cases were selected. All of the cases were chemotherapy-naïve. Clinical data, including age, sex, smoking history, tumor size, and tumor stage were obtained from each patient’s medical records. Staging was performed in accordance with the standards of the American Joint Committee on Cancer, 7th Edition. 2.2. Histologic evaluation Samples were classified as lepidic, acinar, papillary, micropapillary, or solid according to their predominant histologic patterns [1]. Tumors with invasive components that were similar to formerly termed mucinous bronchioloalveolar carcinomas were classified as mucinous variants. Primary ADCs that were morphologically similar to colorectal ADCs and that were cytokeratin 20- or CDX2-positive were classified as enteric variants. Of the 110 cases, 10 ADCs were classified as mucinous or enteric variants. 2.3. Immunohistochemical staining and interpretation Briefly, 3-␮m-thick sections were deparaffinized, rehydrated, and treated with 3% H2 O2 to block endogenous peroxidase activity. Antigen retrieval was performed by immersing slides in citrate buffer (pH 6.0) and microwaving on high power for 25 min. Sections were incubated with primary monoclonal antibodies against

napsin A (clone KCG1.1; 1:100; Abcam, Cambridge, UK) and TTF-1 (clone 8G7G3/1; 1:100; Dako, Glostrup, Denmark). All antibodies were visualized using the Dako EnVision kit (Dako) with diaminobenzidine as chromogen. Sections were counterstained with Mayer’s hematoxylin (Fig. 1). TTF-1 positivity was scored upon observation of sole nuclear staining; napsin A-positivity was assessed by granular cytoplasmic staining. Histologic (H) scores were assigned by multiplying the percentage of stained cells (0–100%) by an intensity score (0, absent; 1, weak; 2, moderate; 3, strong). After analyzing the distribution of H scores, we set minimum thresholds for positivity of moderate intensity or 10% of cells stained [13].

2.4. EGFR mutation and ALK rearrangement analysis To determine EGFR mutation status, a representative formalinfixed, paraffin-embedded block containing at least 75% of viable tumor was selected for each sample. Each tumor was dissected from five 4-␮m-thick unstained histologic sections by microscopic examination. DNA was extracted following proteinase K digestion using DNeasy DNA isolation kits (Qiagen, Valencia, CA) according to the manufacturer’s instructions. Direct DNA sequencing of exons 18 through 21 of the EGFR gene was performed. Each case was classified as positive or negative for the EGFR mutation based on comparison to the wild-type sequence. To identify ALK rearrangements, fluorescent in situ hybridization (FISH) was performed on formalin-fixed, paraffin-embedded tumors using a break apart ALK probe (Vysis LSI ALK Dual Color, Break Apart Rearrangement Probe; Abbott Molecular, Abbot Park, IL). ALK rearrangement was scored as positive when >15% of tumor

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Table 1 Patient characteristics according to napsin A expression.a Variable Age, mean (range) <61 y ≥61 y Gender Male Female Smoking status Never smokers Former or current smokers Tumor size, cm, mean (range) ≤3 cm >3 cm Nodal metastasis Yes No Pathologic stage I II III Predominant histologic type Lepidic Acinar Papillary Micropapillary Solid Solid predominant type Positive Negative Mucinous/enteric variant Positive Negative TTF-1 expression Positive Negative EGFR mutation Positive Negative ALK rearrangement Positive Negative a b

All (n = 110)

Napsin A-positive (n = 91)

Napsin A-negative (n = 19)

61.0 (37–80) 52 (47.3) 58 (52.7)

60.7 (40–80) 46 (41.8) 45 (40.9)

62.7 (37–78) 6 (5.5) 13 (11.8)

53 (48.2) 57 (51.8)

41 (37.3) 50 (45.5)

12 (10.9) 7 (6.4)

67 (60.9) 43 (39.1) 3.6 (1.1–15.0) 49 (44.5) 61 (55.5)

59 (53.6) 32 (29.1) 3.3 (1.1–8.5) 45 (40.9) 46 (41.8)

8 (7.3) 11 (10.0) 4.8 (1.5–15.0) 4 (3.6) 15 (13.6)

60 (54.5) 50 (45.5)

50 (45.5) 41 (37.3)

10 (9.1) 9 (8.2)

43 (39.1) 22 (20.0) 45 (40.9)

35 (31.8) 21 (19.1) 35 (31.8)

8 (7.3) 1 (0.9) 10 (9.1)

8 (7.3) 34 (30.9) 32 (29.1) 12 (10.9) 24 (21.8)

7 (6.4) 28 (25.5) 30 (27.3) 12 (10.9) 14 (12.7)

1 (0.9) 6 (5.5) 2 (1.8) 0 (0.0) 10 (9.1)

24 (21.8) 86 (78.2)

14 (12.7) 77 (70.0)

10 (9.1) 9 (8.2)

10 (9.1) 100 (90.9)

2 (1.8) 89 (80.9)

8 (7.3) 11 (10.0)

79 (71.8) 31 (28.2)

77 (70.0) 14 (12.7)

2 (1.8) 17 (15.5)

56 (50.9) 54 (49.1)

53 (48.2) 38 (34.5)

3 (2.7) 16 (14.5)

6 (5.5) 104 (94.5)

5 (4.5) 86 (78.2)

1 (0.9) 18 (16.4)

p valueb 0.132

0.151

0.065

0.023

0.854

0.190

0.004

<0.001

<0.001

<0.001

0.001

0.968

Where number and percentage are given, data represent the number (% of 110 cases). Statistical significance was calculated by the 2 test.

cells displayed split signals or isolated red signals, as previously described [4]. 2.5. Statistical analysis Relationships between clinicopathologic parameters were evaluated using the 2 test. The overall survival rate was evaluated using the Kaplan–Meier method, and statistical differences in survival times were determined using the log-rank test. The Coxproportional hazards model was applied for multivariate survival analysis. Differences were considered significant for p < 0.05. All statistical analyses were conducted using SPSS v.17 (SPSS, Chicago, IL). 3. Results 3.1. Patient characteristics Patient characteristics are summarized in Table 1. Lung ADC samples were derived from 53 men and 57 women aged 37–80 years (mean = 61.0 years). This population included 67 nonsmokers and 43 former or current smokers. Tumor sizes ranged from 1.0 to 15.0 cm (mean = 3.6 cm). Forty-three samples were classified as pathologic stage I, 22 samples were stage II, and 45 samples were stage III.

3.2. Clinicopathologic characteristics associated with TTF-1 expression TTF-1-positive expression was detected in 79 of 110 cases (71.8%). TTF-1-positive ADCs were significantly more prevalent among samples characterized as relatively small (p = 0.013), nonsolid predominant (p = 0.001), non-mucinous/enteric (p < 0.001), and EGFR mutation-positive (p = 0.004). 3.3. Clinicopathologic characteristics associated with napsin A expression Napsin A-positive expression was detected in 91 of 110 cases (82.7%). Positive napsin A expression was correlated with smaller tumor size (p = 0.023), non-solid predominant types (p < 0.001), non-mucinous/enteric variants (p < 0.001), positive TTF-1 expression (p < 0.001), and the presence of EGFR mutation (p = 0.001) (Table 1). 3.4. Clinicopathologic characteristics associated with both TTF-1 and napsin A expression Seventy-seven of 110 cases were positive for both TTF-1 and napsin A; 14 were TTF-1-negative/napsin A-positive, 2 were TTF1-positive/napsin A-negative, and 17 were TTF-1-negative/napsin A-negative. Comparison of the 77 TTF-1-positive/napsin A-positive

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Fig. 2. The overall survival curve between two groups according to napsin A expression (p < 0.001; log-rank test).

Fig. 3. The overall survival curve among groups by TTF-1 and napsin A expression (survival difference tested by log-rank test).

cases with the 14 TTF-1-negative/napsin A-positive cases suggested that no significant differences in clinicopathologic variables existed between the two groups.

4. Discussion

3.5. Univariate analysis of overall survival Univariate analysis of overall survival identified a significant relationship between poor prognosis and solid predominant type (p = 0.006), mucinous/enteric variants (p = 0.009), napsin Anegativity (p < 0.001) (Fig. 2), EGFR mutation-negative (p = 0.022), nodal metastasis (p = 0.001), and advanced pathologic stage (p < 0.001) (Table 2). Although TTF-1-positivity tended to be associated with better prognosis, the association was not statistically significant (p = 0.096). 3.6. Overall survival of three groups by TTF-1 and napsin A expression To compare overall survival times, we divided the samples into three groups, as follows: group 1 consisted of samples that were positive for both TTF-1 and napsin A, group 2 was positive for only one marker, and group 3 was negative for both markers. Significant survival differences were detected between groups 1 and 3 (p = 0.002) and between groups 2 and 3 (p = 0.050). No significant difference in survival time was detected between groups 1 and 2 (p = 0.185). These results were unchanged when two TTF-1positive/napsin A-negative samples were excluded from group 2 (Fig. 3). 3.7. Multivariate analysis of overall survival The multivariate analysis of overall survival demonstrated that the absence of napsin A (p = 0.002) and pathologic stage III classification (p < 0.001) was independent prognostic factors of shortened survival time (Table 3). Table 2 Univariate analysis of overall survival. Poor prognostic factor

p value

Solid predominant type Mucinous/enteric variant Napsin A-negative EGFR mutation-negative Nodal metastasis Pathologic stage

0.006 0.009 <0.001 0.022 0.001 <0.001

Statistical significance was calculated by the log-rank test.

Lung ADCs are well known for their clinical, radiologic, pathologic, and molecular heterogeneity, which have brought about changes to the classification of lung ADCs during the past several decades. This was reflected recently in the International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society’s updated classification scheme for lung ADCs [1]. Classifications based on immunohistochemical staining and driver mutations recently have been assigned greater importance because of their clinical relevance. Among immunohistochemical markers, TTF-1 (NKX2-1, and TITF1) is considered the gold standard for primary lung ADC [8]. TTF-1 is a lineage-specific transcription factor that regulates lung development and tissue-specific expression of surfactant apoproteins [19]. A major fraction of lung ADCs is TTF-1 positive, supporting their origin from the terminal respiratory unit [20]. TTF-1 also has been identified as an amplified lineage-survival oncogene in lung ADCs [21–24]. In our study, TTF-1 positivity was detected in 79 of 110 cases (71.8%). TTF-1-positive ADCs were significantly associated with smaller tumor sizes, non-solid predominant types, non-mucinous/enteric variants, and the presence of EGFR mutations. These histopathologic and molecular findings are in agreement with previous studies [20,25]. We also investigated the prognostic significance of TTF-1 expression in lung ADCs. TTF-1 expression is known as a prognostic factor of more favorable outcomes [26–28]. The tumor suppressor role of TTF-1 recently was described [29,30]. TTF-1 down-regulation is associated with tumor progression and acquisition of metastatic ability, in association with de-repression of HMGA2 in a mutant K-ras/p53 conditional knockout model of lung ADC [29]. Myosin binding protein H is a transcriptional target of TTF-1 associated with reduced tumor invasion and metastasis [30]. Our univariate survival analysis suggested that TTF-1 expression trended with better prognosis, but the association was not statistically significant (p = 0.096). Rather, napsin A was a more potent prognostic factor in our cohort (p < 0.001).

Table 3 Multivariate analysis of overall survival. Variables

Hazard ratio

Stage III Napsin A-negative Solid predominant type

3.718 3.007 1.976

95% confidence interval 1.956–7.068 1.476–6.125 0.997–3.919

p value <0.001 0.002 0.051

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Some discrepancies exist regarding the prognostic significance of TTF-1 [26]. A meta-analysis of 10 studies reported that only 4 demonstrated an association between TTF-1 positivity and better survival. Five of these studies showed no significant impact on survival, and one identified TTF-1 as a prognostic factor of reduced survival [26]. These heterogeneous results may have resulted from different thresholds for scoring TTF-1 positivity, the use of the monoclonal antibody at different concentrations, dissimilar staining protocols, and/or different patient cohorts [26]. Recent reports indicate that TTF-1-positive ADCs displaying TTF-1 gene amplification were associated with poor prognosis [31,32]. We divided patients into three groups according to their expression of TTF-1 and napsin A. TTF-1-negative/napsin A-positive cases and TTF-1positive/napsin A-positive cases exhibited similar survival rates (Fig. 3). That is, TTF-1-negative cases were not consistently related with poor survival. This suggests that the prognostic significance of TTF-1 expression can be influenced by other factors. Napsin A, first identified in the lung in 1998 [14], shows promise as a diagnostic marker for lung ADCs, particularly given the mounting importance of NSCLC subclassification [10–13,18]. Bishop et al. and Turner et al. reported that napsin A is a more sensitive marker than TTF-1 for pulmonary ADCs (83% vs. 73%, and 87% vs. 64%, respectively) [10,33]. We found that, compared with TTF-1, napsin A also displays a more intense and diffuse pattern of staining (mean of H scores in positive cases: 189 vs. 152), in agreement with recent studies [33,34]. Napsin A examination can be particularly useful in cases for which the TTF-1 signal is weak, focal, or difficult to interpret. We detected TTF-1 positivity in 79 of 110 cases (71.8%) and napsin A positivity in 91 of 110 (82.7%) cases, which is in accordance with the frequencies reported in previous studies. Ninety-three samples were positive for either TTF-1 or napsin A, and the combined prevalence of these markers was 84.5%. The napsin Apositive group was significantly correlated with smaller tumor size (p = 0.023), non-solid predominant types (p < 0.001), nonmucinous/enteric variants (p < 0.001), positive TTF-1 expression (p < 0.001), and the presence of EGFR mutations (p = 0.001). Five of six ALK rearrangement-positive ADC samples also were napsin Apositive. These features overlap with those of previously described TTF-1-positive ADCs [20,25,35]. Our results support that napsin A expression reflects the so-called “terminal respiratory unit” origin of lung ADC [35]. The tumor suppressor role of TTF-1 has been elucidated [29,30]. Prior to this, clinicopathologic studies regarding the prognostic significance of TTF-1 expression were conducted. We identified napsin A as a more potent prognostic factor than TTF-1, and our multivariate analysis revealed that napsin A was an independent prognostic factor. Thus, the functional role of napsin A in lung ADC progression should be examined in subsequent studies. Besides napsin A, the aspartic proteinases include several physiologically important enzymes, such as pepsin, renin, and cathepsin D. As cathepsin D expression was implicated in breast cancer progression [36], napsin A may be implicated in lung cancer progression. Unlike cathepsin D, napsin A expression suppressed tumor growth in the tumorigenic HEK293 kidney cell line [37]. This suggests that napsin A might have a tumor suppressive function. We observed that poorly differentiated lung ADCs expressed napsin A less frequently than more differentiated tumors [10,38]. This supports an inverse association between napsin A and tumor progression. Further studies are needed to determine the role of napsin A in lung ADC progression. In conclusion, the clinicopathologic characteristics of napsin A-positive lung ADCs were similar to and overlapped with those of TTF-1-positve ADCs. This study demonstrated that the absence of napsin A expression is an independent poor prognostic factor regardless of TNM stage in lung ADC.

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