Material and Method: Immunohistochemically, TTF-1, CK 5/6 and p63 were used in 72 cases of squamous cell carcinoma, 19 cases of adenocarcinoma, and 29 cases of non-small cell lung cancer whose final diagnosis was decided with the subsequent resection material. The specificity, sensitivity, and positive and negative predictive value were calculated for each marker.
Results: TTF-1 positivity was seen in none of the 72 squamous cell carcinomas but in all of 19 adenocarcinoma cases. CK5/6 negativity was seen in all cases of adenocarcinoma and in two cases of squamous cell carcinoma. p63 was positive in all squamous cell carcinomas and in 4 adenocarcinomas. Cytokeratin 5/6, p63 positivity and TTF-1 negativity were observed in 17 non-small cell lung cancers whose final diagnosis was squamous cell carcinoma. None of the 12 non-small cell lung cancers whose final diagnosis was adenocarcinoma exhibited positive staining for CK5/6. However, p63 staining was not seen in the biopsy but was focal in the surgical specimen in one case. All the 12 non-small cell lung cancers whose certain diagnosis was adenocarcinoma were positive for TTF-1. TTF-1, CK 5/6 and p63 seem to be useful for differentiating adenocarcinoma from squamous cell carcinoma with 100% specificity, 100% sensitivity and 100% specificity, 97% sensitivity and 87% specificity, and 100% sensitivity, respectively.
Conclusion: We concluded that TTF-1 is a reliable marker for subtyping lung cancer. Different staining patterns can be seen with CK5/6 and p63; however, if they are used together with TTF-1 and interpreted correctly, they can be of help for the final diagnosis even in cases in which the morphology is unclear.
Our study aimed to study the efficiency of Thyroid Transcription Factor-1 (TTF-1), cytokeratin 5/6 (CK5/6) and p63 in distinguishing between AC and SCC and to study the contribution of these markers to the diagnosis in NSCLC.
The cases were included in the study[1] if the biopsy had typical AC morphology[2], if the biopsy had typical SCC morphology[3] when the pathologists agreed on the diagnosis of NSCLC and if the tumor was subsequently resected at our institution[4] if blocks from biopsy and lobectomy specimens were available for immunohistochemical study. Fifty-five of the 175 cases did not meet these criteria and were excluded. The remaining 120 cases were included in this study. Using morphological evaluation, the cases were divided into four groups: AC, SCC, NSCLC-SCC whose final diagnosis was SCC in the subsequent resection material, and NSCLC-AC whose final diagnosis was AC in the subsequent resection material.
Immunohistochemistry
Four-micron-thick sections were obtained for IHC
investigation. Immunohistochemistry was performed by a
standard protocol on a Ventana Discovery XT automated
stainer (Ventana Medical Systems, Tucson, AZ, USA). Only
primary antibodies, cytokeratin 5/6 (Clone D5/16B4, Dako,
1:200 dilution), p63 (Clone 4A4, Dako, 1:700 dilution) and
TTF-1 (Clone SPT24, NovoCastra, 1:50 dilution) were
added manually and incubated in 37°C for 32 minutes.
Pneumocytes were considered as internal controls for TTF-
1 positivity, and bronchial basal cells for p63 and CK5/6
positivity.
Immunohistochemically, cytoplasmic staining was considered positive for cytokeratin 5/6 and nuclear staining was considered positive for TTF-1 and p63. All the areas of biopsy materials and at least 10 high power fields in tumor surgical materials were evaluated and the mean percentage of the positive tumor cells was determined. The staining rate was divided into four categories as follows: 0, <1%; 1+, 1–33%; 2+, 34–66%; 3+, >66%. Intensity of staining was graded as negative (0, no reactivity); weak (1+, less than normal cells); moderate (2+, same as normal cells); and strong (3+, stronger than normal cells)[9,10]. When the staining was heterogeneous, the prominent pattern became the basis. The demographic features (age, gender) were obtained from the hospital archive system. For NSCLC cases, we also assessed whether immunohistochemical staining on biopsies correlated with staining on the subsequent surgical specimens as lung cancers are known to show morphologic heterogeneity in different areas of the same tumor.
Data Analysis
The statistical analyses were performed with SPSS software,
version 20.0. The specificity, sensitivity, positive predictive
value and negative predictive value were calculated for each
marker. In addition, the relationship between the staining on
biopsy and resection materials was analyzed using Pearson
Chi-Square test, Chi-Square test and Fisher’s Exact Test. p
values of ≤0.05 were considered statistically significant.
Cytokeratin 5/6: Of the 72 SCC cases, 56 had 3+ staining, 4 had 2+ and 10 had 1+. CK5/6 negativity was encountered in two SCC cases that had TTF-1(-)/p63(+) (Figure 1A-C). In the cases that stained positive, intensity of staining was strong in 54 cases, moderate in 13 cases and weak in 3 cases. However, no staining was seen in any of the 19 AC cases.
When focal staining was considered positive, the specificity, sensitivity, positive predictive value and negative predictive value were 1.000 (95% Confidence Interval: 0.1000-1.0000), 0.978 (95% Confidence Interval: 0. 9467-1.0000), 0.939 (95% Confidence Interval: 0.8580-1.0000) and 1.000 (95% Confidence Interval: 0.1000-1.0000), respectively.
Positive staining was observed in all of the 17 NSCLCSCC cases (Figure 2A-B). While the staining rate did not change in the biopsy and the surgical specimen in 15 of the 17 cases, it increased in the surgical specimen in the remaining 2 cases. No statistically significant difference was found between the staining rates of the biopsy and surgical specimens (p=0.500). None of the 12 NSCLC-AC cases exhibited positive staining either in biopsy or surgical specimens so no statistical calculations were performed.
When the staining intensity in the NSCLC-SCC cases and that in surgical specimens were compared, it did not change in 15 cases but it increased in the surgical specimens of 2 cases. In the NSCLC-SCC cases, the intensity of staining displayed no statistically significant difference between biopsy and surgical specimens (p=0.500).
p63: We observed 3+ staining in 71 of the 72 SCC cases and 2+ staining in one. There was no staining in 15 of the 19 AC cases. However, p63 positivity was observed in 4 AC cases that had TTF-1(+)/ CK5/6 (-) (Figure 3A-D). Two cases had 1+ staining. One of the remaining two had 2+ staining and the other had 3+ staining. Intensity of p63 staining was weak in 3 of the 4 AC cases and it was moderate in one. When the focal staining was considered positive, the specificity, sensitivity, positive predictive value and negative predictive value were 0.871 (95% Confidence Interval: 0.7530-1.0000), 1.0000 (95% Confidence Interval: 1.0000-1.0000), 1.000 (95% Confidence Interval: 1.0000-1.0000) and 0.957 (95% Confidence Interval: 0,9158- 1.0000), respectively.
In all of the 17 NSCLC-SCC cases (Figure 2C), the staining rate and intensity were the same both in the NSCLC areas and surgical specimens so no statistical assessment was conducted. Eleven of the 12 NSCLC-AC had no staining both in biopsy and surgical specimens. In the other case, staining was not seen in the biopsy but 1+ staining was seen in the surgical specimen. That finding was not found statistically significant (p=1.000).
TTF-1: TTF-1 staining was not seen in any of the 72 SCC cases while it was seen in all AC cases. In 16 of the AC cases, the staining was 3+ but 2+ in one of them and 1+ in two of them. Staining intensity was strong in 17 cases whereas it was weak in one case. When the focal staining was considered positive, the specificity, sensitivity, positive predictive value and negative predictive value were 1.000 (95% Confidence Interval: 1.0000-1.0000), 1.000 (95% Confidence Interval: 1.0000-1.0000), 1.000 (95% Confidence Interval: 1.0000- 1.0000) and 1.000 (95% Confidence Interval: 1.0000- 1.0000), respectively.
In all of the 12 NSCLC-AC cases, the rate and intensity of staining in the NSCLC areas were the same as those in the surgical specimens. In all of the 17 NSCLC-SCC cases (Figure 2D), staining was seen in neither NSCLC areas nor surgical specimens. Since TTF-1 staining in NSCLC- SCC and NSCLC-AC was the same, no statistical analyses were performed.
In 29 NSCLC cases whose diagnosis was confirmed by the surgical specimen, no difference was found in the rate and intensity of staining in both biopsy and surgical specimens for all three immunohistochemical markers (p>0.050).
Statistically, no difference was found between the sensitivity of immunohistochemical markers (p=0.506) but the specificity of TTF-1 and CK5/6 was higher than that of p63 (p=0.001). The specificity, sensitivity, positive predictive value and negative predictive value for each immunohistochemical marker are shown in Table III.
The reported sensitivity of CK5/6 for pulmonary SCC ranges from 73% to 100% and the sensitivity of CK5/6 is reported to go down as there is a decrease in the tumor differentiation[7,13,21]. CK5/6 positivity was observed in 43 of 48 SCC cases in the study by Kim et al.[16], but in all 32 SCC cases in the study by Nicholson et al.[22]. One of the reasons for the wide range of CK5/6 sensitivity can be the fact that some studies regard focal or weak staining as positive but others do not. In our study, no CK5/6 staining was detected in only two cases while 75% of SCC cases were diffuse and strong positive. All the NSCLC-SCC cases were CK5/6 positive but six of them were focal. In addition, the staining was diffuse in two surgical specimens but it was focal in their former biopsies. According to our findings, CK5/6 was a highly specific but not very sensitive marker in distinguishing between SCC and AC when focal and weak staining were considered to be positive. Additionally, CK5/6 positivity can be considered as a finding supporting SCC even if it is focal in the cases where histological differentiation is not clear.
The utility of p63 for the identification of lung SCCs is well-known, and its sensitivity ranges from 73% to 100%[5,7,13]. However, p63 specificity is not high because p63 can be stained focal and/or weak in some ACs[12,13,16]. In our study, p63 positivity was observed in 4 AC cases which had TTF-1(+)/ CK5/6 (-). Staining was focal in three cases but diffuse in one. Nevertheless, intensity of staining was weak in the case that had diffuse staining like the other two cases that had focal staining. This finding is in agreement with the literature. All of the NSCLC-SCC in our study were positive for p63 and their staining rate and intensity were the same both in NSCLC areas and surgical specimens. None of the NSCLC-AC cases had staining except one, in which staining was not seen in the biopsy while focal (1–33%) staining was seen in the surgical specimen. In a study contucted by Warth et al.[2], p63 positivity was seen in 16.3% of the histomorphologically unclear ACs and staining intensity was lower than in SCC. Our study suggests that an AC diagnosis can be made despite p63 positivity in some cases, as in the literature. If the p63 positivity is focal and weak or moderate, TTF-1 positivity and CK5/6 negativity are enough for AC diagnosis even if AC is not clear histomorphologically. In our study, SCC had CK5/6 negativity and AC had p63 positivity in a few cases. However, TTF-1 was positive in all the ACs and negative in all the SCCs. Although the specificity, sensitivity, PPV and NPV of TTF-1 were 100%, a statistically significant difference was found in only the specificity value due to the limited number of cases. Our results need to be supported by studies including large series.
Subtyping of NSCLCs is very important due to current treatment modalities. Immunohistochemistry can be used in subtyping of samples with limited tissue and cases in which the morphology is unclear. TTF-1, CK 5/6 and p63 are the markers that can be used for this purpose. Different staining patterns can be seen with CK5/6 and p63; however they can be of help for the final diagnosis even in the cases in which morphology is unclear, if they are used together with TTF-1 and interpreted correctly.
Acknowledgments
We gratefully acknowledge Hatice Uluer in the Department
of Biostatistics and Medical Communication of Ege
University for performing the statistical analyses.
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