The availability of targeted therapies has created a need for precise subtyping of non-small cell lung carcinomas (NSCLCs). In contrast with past years, when such subtyping had no therapeutic relevance, differentiating between adenocarcinoma (AC) and squamous cell carcinoma (SCC) is now important because new therapies have been developed that have different therapeutic or adverse effects depending on the histologic type. For example, the epidermal growth factor receptor inhibitors, gefitinib and erlotinib, are more likely to be effective in ACs than in SCC s.14 2,17 30
Although the majority of NSCLCs can be subtyped by examination of hematoxylin-eosin (H&E)-stained slides alone, difficulty is encountered in poorly differentiated tumors, especially in small biopsy specimens. Although earlier series have examined the role of immunohistochemical markers in the diagnosis of AC and SCC, most researchers have studied all tumors regardless of differentiation, including those in which immunohistochemical stains are not needed for diagnosis.1,3,7,11,13,15,23,28,31,32,34,36,37 lung cancers are unresectable at diagnosis, the only available tissue sample in many cases being a biopsy. In addition, we included only poorly differentiated tumors as these are the cases in which immunohistochemistry is most important.
Our aim, therefore, was to assess the utility of immunohistochemical markers in subtyping poorly differentiated NSCLCs on biopsy specimens. To be certain about the tumor classification, we required a resection specimen as the gold standard for diagnosis. Furthermore, as lung cancers are known to show morphologic heterogeneity in different areas of the same tumor, we also assessed whether immunohistochemical staining on biopsies correlated with staining on the subsequent resections.
MATERIALS AND METHODS
Case Selection
Surgical pathology files of the State University of New York Upstate Medical University were searched for cases of poorly differentiated NSCLC diagnosed on small lung biopsies (endobronchial biopsies, transbronchial biopsies, and computed tomography-guided core biopsies) over a 12-year period (1998 to 2009). Using an electronic database, 366 poorly differentiated NSCLCs were retrieved. In 71 of these cases, the tumor was subsequently resected at our institution. These cases were reviewed and included in the study if (1) review of the biopsy confirmed a diagnosis of NSCLC, (2) the tumor in the biopsy showed no differentiating features that would allow it to be subtyped on the basis of H&E morphology, and (3) glass slides and blocks from both biopsy and resection were available for review. Thirty-two of the 71 cases did not meet these criteria, and were therefore excluded. In 26 of these cases, review of the biopsy showed a tumor that could be subtyped by H&E morphology as AC or SCC (23 cases), sarcomatoid carcinoma (2 cases), or small cell carcinoma (1 case). The other 6 cases were excluded because slides or blocks from the biopsy or resection were not available. The 39 remaining cases were included in this study. Of these, 27 cases were computed tomography-guided core biopsies, 4 cases were endobronchial biopsies, and 8 cases were transbronchial biopsies.
The resected tumors were subtyped on the basis of H&E morphology according to the World Health Organization (WHO) criteria by 2 pulmonary pathologists blinded to the immunohistochemical findings. An average of 4 slides was available from each tumor (range, 1 to 9). Tumors were classified as AC if they showed gland formation or mucin production, SCC if they contained intercellular bridges or showed keratinization, and large cell carcinoma (LCC) if they lacked glandular or squamous differentiation. The presence of mucin production was documented in H&E stains by a light basophilic-to-foamy appearance of the cytoplasm, apical clearing with a goblet cell-like appearance or mucin lakes.
Immunohistochemical Analysis
Each biopsy and a representative block from the subsequent resection was stained with CK7 (OV-TL-12/30, 1:100; Neomarkers, Fremont, CA), TTF-1 (8G7G3/1, 1:50; Neomarkers), napsin A (novel aspartic proteinase of the pepsin family) (TMU-Ad02, 1:100; Immuno-Biological Laboratories, Takasaki, Gunma, Japan), p63 (4A4, 1:200; Neomarkers), CK5/6 (D5/16B4, 1:50; Cell Marque, Rocklin, CA), and high molecular weight keratin 34βE12 (34βE12, 1:100; DAKO, Carpinteria, CA). Immunohistochemistry was performed on formalin-fixed, paraffin-embedded sections using a standard streptavidin-biotin-peroxidase technique with appropriate positive and negative controls. For each stain, the percentage of positive cells was recorded. The presence of more than 10% expression of the marker within tumor cells was considered positive. Cases with less than 10% overall staining and no focal areas of positive staining were considered negative. Cases that were mostly negative but contained small areas of tumor in which nearly all cells stained positive were classified as focally positive.
The State University of New York Upstate Medical University Institutional Review Board for Protection of Human Subjects approved the study.
RESULTS
There were 20 ACs, 15 SCCs, and 4 LCCs in the resections. In ACs, the proportion of resected tumor showing gland formation ranged from 1% to 75% (mean, 22%; median, 10%). It is of interest to note that almost half (9 of 20) were moderately differentiated, with gland formation ranging from 30% to 75% of the resected tumor. The remaining 11 tumors were poorly differentiated, showing glands in only 1% to 10% of the resected tumor. Five of these poorly differentiated tumors showed only minimal, focal (1%) gland formation. The proportion of resected tumors showing keratinization or intercellular bridges in SCCs ranged from 1% to 50% (mean, 12%; median, 5%). Only 5 of 15 SCCs were moderately differentiated, showing keratinization or intercellular bridges in 20% to 50% of the tumor. The remaining 10 resected SCCs were poorly differentiated, with keratinization or intercellular bridges discernible in only 1% to 5% of the tumor. LCCs showed no evidence of gland formation, mucin, keratinization, or intercellular bridges on resection.
Immunohistochemistry in Biopsy SpecimensThe results of immunohistochemical staining on biopsy specimens for each histologic subtype of carcinoma are summarized in Table 1 , and the sensitivity, specificity, positive predictive value, and negative predictive value of the immunohistochemical stains for AC and SCC are summarized in Tables 2 and 3 .
TABLE 1: Immunohistochemical Findings in Lung Biopsies of Poorly Differentiated Non-small Cell Carcinomas
TABLE 2: Sensitivity and Specificity of Immunohistochemical Markers for Adenocarcinoma in Lung Biopsies
TABLE 3: Sensitivity and Specificity of Immunohistochemical Markers for Squamous Cell Carcinoma in Lung Biopsies
CK7 was positive in all 19 biopsies from ACs (in the 20th case, no tissue was present on the CK7-stained slide), in 9 of 15 biopsies from SCCs (60%), and in 3 of 4 LCCs (75%). TTF-1 was positive in 16 of 20 ACs (80%) and 2 of 4 LCCs (50%) but none of 15 SCCs. Napsin A was positive in 11 of 19 ACs (58%) in which tissue was available for evaluation (in the 20th case, no tissue was present on the napsin A -stained slide), and was negative in all SCCs and LCCs. All napsin A -positive cases were also positive for TTF-1 (Fig. 1 ). Four TTF-1 -positive cases were negative for napsin A (Fig. 2 ); these were also negative for p63 and CK5/6 . There were no napsin A -positive, TTF-1 -negative ACs. A few isolated napsin A -positive cells (<10% threshold) were present in 1 case of SCC. The 2 TTF-1 -positive LCCs were negative for napsin A , p63 , and CK5/6 . With both TTF-1 and napsin A , alveolar lining cells in adjacent or entrapped lung parenchyma stained strongly, and there was occasional difficulty distinguishing entrapped alveolar lining cells from tumor cells. Interpretation of napsin A staining was also complicated by strong staining of alveolar macrophages (Fig. 3 ).
FIGURE 1: TTF-1 and napsin A positivity in adenocarcinoma . A, Transbronchial biopsy shows a poorly differentiated non-small cell carcinoma . Glands are not evident (H&E, ×200). B, The tumor is positive for TTF-1 (left, ×200) and napsin A (right, ×200). p63 and CK5/6 were negative. C, Resection shows gland formation in 30% of the tumor, confirming the diagnosis of adenocarcinoma (hematoxylin-eosin, ×100).
FIGURE 2: TTF-1 -positive, napsin A -negative adenocarcinoma . A, Core biopsy shows a poorly differentiated non-small cell carcinoma [hematoxylin-eosin (H&E), ×200]. B, The neoplastic cells are positive for TTF-1 (left, ×200) but negative for napsin A (right, ×200). p63 and CK5/6 were negative. C, The resected tumor is a poorly differentiated adenocarcinoma with only focal (1%) gland formation. A poorly formed gland is seen in the top half of the picture (arrows) (H&E, ×200).
FIGURE 3: Pitfalls in interpreting napsin A staining. The napsin A -positive cells within this poorly differentiated squamous cell carcinoma in a transbronchial biopsy represent alveolar lining cells (arrows) and must not be misinterpreted as napsin A -positive tumor cells. Scattered macrophages are also positive for napsin A (arrowheads). The tumor is napsin A negative (×200). It was also negative for TTF-1 , and positive for p63 and CK5/6 . The resected tumor showed focal morphologic evidence of squamous differentiation.
34βE12 was positive in all 15 SCCs (100%) and also in 12 of 20 ACs (60%). All LCCs were 34βE12 negative. p63 staining was seen in all 15 SCCs (100%) and also seen in 2 of 20 ACs (10%). All 4 LCCs were p63 negative. In SCCs, p63 was diffusely positive (>90% of tumor cells) in all but 2 cases. In 1 of these cases, p63 was positive in 25% of the tumor and CK5/6 was also positive. In the other case, 20% of tumor cells stained for p63 and CK5/6 were negative. Both cases of p63 -positive AC were positive for TTF-1 and napsin A . In 1 case, 30% of tumor cells stained with p63 and 90% of tumor cells stained with TTF-1 (Fig. 4 ), whereas in the other case, 25% of tumor cells stained with p63 and 90% of tumor cells stained with TTF-1 . CK5/6 was positive in 11 of 15 SCCs (73%), all of which were also p63 positive (Fig. 5 ). All 4 cases that were positive for p63 but negative for CK5/6 lacked TTF-1 and napsin A expression (Fig. 6 ). p63 was diffusely positive (>90%) in 3 cases, but showed only scattered positivity (approximately 20% of tumor cells) in the fourth case. CK5/6 was negative in all ACs and LCCs.
FIGURE 4: p63 positivity in a poorly differentiated adenocarcinoma . A, Top left: Poorly differentiated non-small cell carcinoma in a transbronchial biopsy [hematoxylin-eosin (H&E), ×200]. Top right: The tumor is diffusely (90%) positive for TTF-1 (×200). Bottom left: Napsin A is also positive (×200). Bottom right: The tumor is focally positive for p63 (30% of tumor cells) (×200). B, The corresponding resection showed glands in 40% of the tumor (H&E, ×100). p63 staining was seen in <5% of the resected tumor.
FIGURE 5: p63 and CK5/6 positivity in squamous cell carcinoma . A, Core needle biopsy shows a poorly differentiated non-small cell carcinoma [hematoxylin-eosin (H&E), ×200]. The neoplastic cells are diffusely positive for p63 (B, ×200) and CK5/6 (C, ×200). TTF-1 and napsin A were negative. D, The resected tumor is recognizable on H&E as a squamous cell carcinoma by the presence of intercellular bridges (arrows) in 30% of the tumor (H&E, ×200). Focal keratinization was also present elsewhere in the tumor.
FIGURE 6: p63 -positive, CK5/6 -negative squamous cell carcinoma . A, Core needle biopsy shows a poorly differentiated non-small cell carcinoma intermixed with neutrophils [hematoxylin-eosin (H&E), ×200]. The tumor is strongly positive for p63 (the negative staining portion is the acute inflammatory infiltrate) (B, ×200) but negative for CK5/6 (C, ×200). TTF-1 and napsin A were negative. D, The focal presence of intercellular bridges (arrows) in the resection specimen (1%) confirms that the tumor is a squamous cell carcinoma (H&E, ×200).
Algorithm for Interpreting Immunohistochemistry in Biopsy Specimens
On the basis of these results, an algorithm was devised as outlined in Table 4 for subtyping poorly differentiated NSCLC on biopsy specimens. On account of the low specificity of CK7 and 34βE12 for AC and SCC, respectively, these markers were excluded from the antibody panel. Positivity for either TTF-1 or napsin A was considered diagnostic of AC irrespective of p63 or CK5/6 staining. Positivity for the combination of p63 and CK5/6 indicated SCC. Tumors positive for p63 but negative for CK5/6 were diagnosed as SCC only if they were diffusely and strongly positive for p63 and negative for TTF-1 and napsin A . Using these criteria, it was possible to accurately subtype 30 of 39 (77%) poorly differentiated NSCLCs on biopsy specimens, including 16 ACs and 14 SCCs. Nine tumors could not be further classified on biopsies by this immunohistochemical panel. On resection, 4 of these were ACs, 1 was a SCC, and 4 were LCCs. Of the 4 ACs, 2 were poorly differentiated on resection (5% glands each), whereas 2 were moderately differentiated (50% and 75% glands). The AC with 75% glands on resection was positive for TTF-1 and napsin A in the resection specimen, even though the earlier biopsy was negative for both markers (see discrepancies, below). The single case of SCC that could not be classified on biopsy was negative for TTF-1 , napsin A , and CK5/6 and positive for p63 in only 20% of cells. The tumor was poorly differentiated on resection, showing intercellular bridges in only 5%. As occasional TTF-1 and/or napsin-A-positive ACs can show weak or focal staining for p63 , and as not all ACs show positive TTF-1 or napsin A staining, the possibility of AC with weak or focal p63 staining but negative TTF-1 or napsin A could not be excluded. Weak or focal staining for p63 alone, therefore, was not considered diagnostic of SCC.
TABLE 4: Proposed Algorithm for Subclassification of Poorly Differentiated Non-small Cell Lung Carcinomas Using Immunohistochemical Staining in Lung Biopsies
Comparison of Immunohistochemical Staining in Biopsy and Resection Specimens
In 28 of 39 (72%) cases, the immunohistochemical staining pattern for all 6 antibodies was identical in biopsies and their corresponding resections. A discrepancy in immunohistochemical staining between the biopsy and the corresponding resection was encountered in 11 cases, and included only a single antibody in 10 cases and 2 antibodies in 1 case (Table 5 ). Overall, a total of 468 slides were stained with immunohistochemical markers (39 cases, 6 stains on each biopsy, 6 on corresponding resection). No tissue remained on 2 immunostained slides, yielding a total of 466 slides, 464 of which formed 232 pairs (biopsy and corresponding resection for each immunohistochemical marker). Of these 232 pairs, 220 (95%) showed concordant staining between the biopsy and its corresponding resection and 12 (5%) showed discrepant staining.
TABLE 5: Discrepancies in Immunohistochemical Staining Between Biopsies and Resections
In 8 discrepant pairs, the tumor was negative on biopsy but was positive on resection. Sampling error accounted for 7 of these cases. The cause of 1 biopsy-negative, resection-positive case was uncertain, but technical error was considered because the biopsy lacked a positive internal control, making it impossible to be certain that antibody was adequately applied to the tissue.
In 4 discrepant pairs, the biopsy was positive whereas the corresponding resection was negative. Sampling error accounted for 1 of these cases, in which the biopsy was focally positive for 34βE12. The resected AC showed focal 34βE12 staining similar to the biopsy, but staining did not reach the 10% threshold for positivity. In the remaining 3 biopsy-positive, resection-negative cases, the cause of discrepancy was unclear, but may have been related to fixation issues in the resection. Only 2 discrepancies (both from the same case) impacted the final immunohistochemical diagnosis rendered on biopsy (Table 5 , second row). In this case, an AC was missed because both TTF-1 and napsin A were negative on biopsy.
In both cases of TTF-1 -positive LCC, TTF-1 staining identical to the earlier biopsy was obtained on the corresponding resection. In addition, 1 of these cases showed diffuse napsin A staining on resection that was not present on the biopsy (Table 5 , row 3).
Restricting the analysis of discrepancies to the 4 stains used in the algorithm outlined above, the immunohistochemical staining pattern for these 4 antibodies was identical in biopsies and their corresponding resections in 33 of 39 (85%) cases. A discrepancy was found in 6 cases, including a single antibody in 5 cases and 2 antibodies in 1 (Table 5 ) case. Of the 312 slides stained with these 4 markers (39 cases, 4 stains on each biopsy, 4 stains on corresponding resection), no tissue remained on 1 immunostained slide, yielding a total of 311 slides, 310 of which formed 155 pairs (biopsy and corresponding resection for each marker). Of these 155 pairs, 148 (95%) showed concordant staining between the biopsy and its corresponding resection and 7 (5%) showed discrepant staining. It is important to note that the discrepant staining results were inconsequential in all but 1 case.
DISCUSSION
Most NSCLCs can be easily subtyped as AC or SCC on biopsies without using additional special stains or immunohistochemistry . In fact, the current WHO classification of lung carcinomas is based primarily on H&E morphology. Difficulty in classification, however, is not infrequently encountered in small biopsy specimens, either due to poor sampling or due to the presence of only a small amount of tumor that may not show features of differentiation. Our study shows that the use of a panel of immunohistochemical stains, including TTF-1 , napsin A , p63 , and CK5/6 in such cases allows correct subclassification in more than three fourths of cases. These findings are important because, in contrast to previous years, clinicians currently demand more precise classification of NSCLCs for treatment with targeted therapies. Although earlier reports have noted the utility of many of these stains, most studies have examined resection specimens,5,13,15,16,23,31,32 1,3,11,34,37 36 lung cancers are unresectable at diagnosis, and a biopsy is the only tissue available for diagnosis in a large percentage of cases. Furthermore, most earlier studies included tumors with all degrees of differentiation, most of which do not need immunohistochemistry for classification.1,3,7,11,13,15,23,28,31,32,34,36,37 20 TTF-1 , p63 , CK5/6 , and Alcian blue-periodic acid Schiff, but did not provide specific details regarding staining and criteria for subtyping. We did not perform mucin stains in our cases because we feel that significant mucin production is adequately visualized on H&E-stained slides. Our study, similar to study of Loo et al,20
In our hands, TTF-1 and napsin A were specific for AC and CK5/6 was specific for SCC. p63 , although highly sensitive for SCC, was not entirely specific, because it was also expressed occasionally in ACs. The high specificity of TTF-1 for ACs has been noted by most earlier studies,11,16,18,23,24,37 TTF-1 positive.7,15,22,34 TTF-1 -positive entrapped lung epithelium within SCCs may have been misinterpreted as TTF-1 positivity. Some studies, furthermore, have considered <10% or weak staining as positive.15,22
Napsin A is an antibody that has recently emerged as a marker of pulmonary ACs. Although napsin A occasionally stains nonpulmonary ACs, it is highly useful for separating primary lung ACs from SCCs.3,13,32,37 napsin A was less sensitive than TTF-1 (58% vs. 80%) for AC, but equally specific. Other studies have noted a similar lower sensitivity of napsin A (33% to 69%), but equally high specificity.3,13,32,37 napsin A in our immunohistochemical panel because positive staining may be helpful when TTF-1 staining is equivocal, especially because it is a cytoplasmic rather than nuclear stain. In addition, rare cases of TTF-1 -negative but napsin A -positive pulmonary AC have been reported.3,37
The reported sensitivity of CK5/6 for pulmonary SCC ranges from 75% to 100%.11,15,18,19,25,29 CK5/6 for SCC in our study (100%) is similar to earlier studies,6,15,25 CK5/6 positivity has been reported in a small percentage (2% to 8%) of primary pulmonary ACs.8,10,11,26 CK5/6 positivity has been reported in lung ACs in 2 studies, but these studies considered focal (<10%) and weak staining as positive.18,29
The high sensitivity of p63 for pulmonary SCC in this study (100%) is in agreement with the literature, in which the reported sensitivity ranges from 78% to 100%.1,5,18,28,29,36 CK5/6 , p63 was not entirely specific for SCC, as it was positive in 2 TTF-1 /napsin A -positive ACs. However, staining for p63 in these cases was focal (involving only 25% to 30% of cells), whereas staining for TTF-1 and napsin A was diffuse (involving 90% to 100% of cells). p63 expression in pulmonary ACs has been reported earlier,1,28,29,36 TTF-1 and napsin A can be positive in p63 -positive ACs. Recognition of this fact is important because expression of TTF-1 and napsin A allows poorly differentiated NSCLCs to be diagnosed as ACs despite p63 expression. Another difficult diagnostic situation arises in cases that are positive for p63 but negative for CK5/6 , TTF-1 , and napsin A . Of 4 such cases in our study, diffuse p63 positivity (>90%) was encountered in 3 cases, in which it was considered diagnostic of SCC as none of our ACs showed diffuse p63 positivity. The 1 case in which p63 expression was seen in only 20% of tumor cells was considered unclassifiable because such focal staining could potentially be encountered in a TTF-1 /napsin A -negative, p63 -positive AC.
Two of the markers we evaluated—CK7 for AC and 34βE12 for SCC—were highly sensitive but too nonspecific to be diagnostically useful. Similar findings have been reported by other investigators, suggesting that CK7 and 34βE12 should not be used to differentiate between AC and SCC.16,21,24,35
Two biopsies in our study contained carcinomas that were positive for TTF-1 and negative for napsin A , p63 , and CK5/6 , but in which the resected tumor fulfilled the WHO morphologic criteria for LCC (Fig. 7 ). In addition, 1 of these cases, was also positive for napsin A at resection. Similar staining for TTF-1 and napsin A in LCC has been reported by other investigators.3,22,27 4,9,12,33 TTF-1 and napsin A is highly specific for AC, we believe that such tumors should be classified as AC. If this criterion was to be used in our study, the proportion of biopsies accurately subtyped by our immunohistochemical panel would increase from 77% (30 of 39) to 82% (32 of 39). Although we did not observe any p63 or CK5/6 staining in LCC, it is entirely possible that some cases may show immunohistochemical evidence of squamous differentiation, and thus should be classified as SCC. It seems that the reliance of the WHO classification on H&E morphology alone in this era of immunohistochemistry and molecular techniques may no longer be appropriate for these poorly differentiated tumors. Whether these (and other) poorly differentiated carcinomas will respond appropriately to targeted therapy, however, is another question that can only be answered by additional prospective studies.
FIGURE 7: TTF-1 -positivity in large cell carcinoma . A, Poorly differentiated non-small cell carcinoma on transbronchial biopsy [left, hematoxylin-eosin (H&E), ×200]. The tumor is diffusely positive (95%) for TTF-1 (right, ×200). Napsin A , p63 , and CK5/6 were negative. B, Resection shows no morphologic evidence of gland formation, mucin production, keratinization, or intercellular bridges (H&E, ×200). TTF-1 was positive in 50% of the resected tumor.
The morphologic heterogeneity of NSCLCs is well known, raising the question of whether immunohistochemical staining on small biopsies is representative of the resected tumor. We addressed this issue by performing the same set of 6 immunohistochemical stains on both biopsies and corresponding resections. Our data show that discordant staining is uncommon, occurring in only 5% of all biopsy-resection pairs. Not surprisingly, the main reason for discordant staining is sampling error, mainly in cases in which biopsies are negative and resections are positive. Biopsies with minimal amounts of tumor may be especially susceptible to such sampling errors. Technical difficulties may also play a part in some small biopsies that lack an adequate internal control, as it is not possible to be certain in such cases whether antibody was appropriately applied to the tissue. Cases in which the tumor was positive in the biopsy but negative in the resection are more difficult to explain, but may be related to fixation issues in the resection. The diagnostic impact of discrepancies in immunohistochemical staining between the biopsy and resection was minimal in this study, leading to an erroneous diagnosis in only 1 case, an AC in which both TTF-1 and napsin A were negative in the biopsy but positive in the resection.
In conclusion, poorly differentiated NSCLCs can be accurately subtyped on small lung biopsies in more than three fourths of cases using a panel composed of TTF-1 , napsin A , p63 , and CK5/6 . TTF-1 -positive tumors that show focal p63 staining should be classified as ACs. Consideration should be given to the addition of immunohistochemistry to the WHO criteria for classifying poorly differentiated lung carcinomas.
ACKNOWLEDGMENT
The authors thank Ms Deborah Rexine in the Department of Medical Photography for her meticulous attention to detail in preparing photomicrographs for this study.
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