College of American Pathologists
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  Q & A





August 2012

Fredrick L. Kiechle, MD, PhD

Question Q. What are the best immunohistochemical markers to distinguish squamous cell carcinoma from adenocarcinoma in the lung?

A. In the publication describing the new International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society international multidisciplinary classification of lung adenocarcinoma (ADC), the importance of distinguishing ADC from squamous cell carcinoma (SCC) was emphasized.1 While these authors noted that there is no need for immunohistochemical studies if the standard WHO 2004 histologic and cytologic criteria for these latter diagnoses are met, ancillary immunohistochemical studies are not infrequently required, particularly in the context of small biopsies. Furthermore, as it is important to preserve as much tissue as possible for potential molecular studies, it is best to select a limited panel of immunostains. Fortunately, a number of recent publications have highlighted the utility of a small panel of markers to reliably make this distinction. Most of the authors of these recent publications are in agreement that the most robust markers for ADC include TTF-1, napsin A, and cytokeratin 7, and those most robust for the diagnosis of SCC include p63, cytokeratin 5 (or cytokeratin 5/6), and the high-molecular-weight cytokeratins identified by antibody 34βE12.

Two recent papers discuss the application of subsets of these markers to small biopsy specimens. Using a logistic regression model, Terry, et al., determined the optimal marker panel (out of nine potential markers) to separate SCC from ADC by looking at the receiver operating characteristic area under the curve (ROC AUC), noting that the single marker, p63, alone, had an ROC AUC of 0.843, which approached 0.95 when adding TTF-1, CK5/6, and CK7.2 Addition of further markers only marginally improved sensitivity and specificity. Mukhopadhyay and Katzenstein, using a similar set of markers, concluded that antibodies to TTF-1, napsin A, p63, and CK5/6 allowed accurate classification in 77 percent of cases.3 By analyzing whole tissue as well as small specimens, using a similar set of markers (but also including antibody 34βE12, to high-molecular-weight cytokeratins, and incorporating semiquantitative immunostaining data), Rekhtman, et al., demonstrated that the combination of p63 (greater than 50 percent of cells positive) and 34βE12 (greater than 50 percent of cells positive) showed a 96 percent sensitivity and 98 percent specificity for SCC, although comparable results could be obtained using combinations of other antibodies. As a single marker, only diffusely positive TTF-1 was specific for ADC, whereas none of the squamous markers were entirely specific for SCC. In contrast, the combination of TTF-1 and p63 (with addition of CK5/6 in rare indeterminate cases) yielded a panel of immunostains that showed 100 percent accuracy in distinguishing ADC from SCC.4

A survey of more novel markers, including desmocollin-3, glypican 3, S100A2, S100A7, and SOX-2, demonstrated that all were inferior to markers such as CK5/6 and TTF-1 in the distinction of SCC from ADC.5 Recent data suggest, however, that antibodies to p40 (a p63 isoform) might be even more efficacious as a squamous marker than the 4A4 monoclonal antibody to p63, which cross-reacts with both major p63 isoforms.6,7

In summary, there is general agreement that the number of antibodies required to reliably distinguish ADC from SCC ranges from two to four and that they should be selected from a limited group of markers that have been extensively investigated. For ADC, these include TTF-1, napsin A, and cytokeratin 7; for SCC, these include p63, cytokeratin 5 (or cytokeratin 5/6), and the high-molecular-weight cytokeratins identified by antibody 34βE12.


1. Travis WD, Brambilla E, Noguchi M, et al. International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society: international multidisciplinary classification of lung adenocarcinoma. J Thorac Oncol. 2011;6:244–285.

2. Terry J, Leung S, Laskin J, et al. Optimal immunohistochemical markers for distinguishing lung adenocarcinomas from squamous cell carcinomas in small tumor samples. Am J Surg Pathol. 2010;34:1805–1811.

3. Mukhopadhyay S, Katzenstein AL. Subclassification of non-small cell lung carcinomas lacking morphologic differentiation on biopsy specimens: utility of an immunohistochemical panel containing TTF-1, napsin A, p63, and CK5/6. Am J Surg Pathol. 2011;35:15–26.

4. Rekhtman N, Ang DC, Sima CS, et al. Immunohistochemical algorithm for differentiation of lung adenocarcinoma and squamous cell carcinoma based on large series of whole-tissue sections with validation in small specimens. Mod Pathol. 2011;24:1348–1359.

5. Tsuta K, Tanabe Y, Yoshida A, et al. Utility of 10 immunohistochemical markers including novel markers (desmocollin-3, glypican 3, S100A2, S100A7, and Sox-2) for differential diagnosis of squamous cell carcinoma from adenocarcinoma of the lung. J Thorac Oncol. 2011;6:1190–1199.

6. Pelosi G, Fabbri A, Bianchi F, et al. ΔNp63 (p40) and thyroid transcription factor-1 immunoreactivity on small biopsies or cellblocks for typing non-small cell lung cancer: a novel two-hit, sparing-material approach. J Thorac Oncol. 2012;7: 281–290.

7. Bishop JA, Teruya-Feldstein J, Westra WH, et al. p40 (ΔNp63) is superior to p63 for the diagnosis of pulmonary squamous cell carcinoma. Mod Pathol. 2012;25:405–415.

Allen M. Gown, MD
PhenoPath Labs

Member, CAP
Immunohistochemistry Committee

Question Q. What is the standard of practice for confirmation testing with Acetest tablets?

A. Testing urine for ketones using the nitroprusside reaction has been a common practice for many decades.1

During that time, a common practice evolved to “confirm” a positive ketone reaction in a multianalyte dipstick using a tablet test, Acetest being the long-standing brand. At this time, the wisdom of such a practice is questionable for several reasons. First, the tablet uses the same simple chemical reaction as the strip; hence, the test can hardly be regarded as a rigorous confirmation. Second, experience has shown that a positive dipstick ketone test will fail to confirm with the tablet only rarely—about two percent of the time in one published study.2 Third, the clinical implications of this simple, qualitative test are meager. They don’t justify the trouble and expense of a confirmatory test. Serious evaluation of ketosis, as in the setting of diabetic ketoacidosis, warrants rigorous modern tests such as quantitative measurement of beta-hydroxybutyrate, a ketone body that the nitroprusside reaction does not even detect. The Clinical and Laboratory Standards Institute says: “Many of the historical confirmatory chemical urinalysis tests such as sulfosalicylic acid (SSA) test for protein, tablet test for ketones, and the tablet test for bilirubin may not be relevant to current laboratory practice.”3


1. Free HM, Smeby RR, Cook MH, et al. A comparative study of qualitative tests for ketones in urine and serum. Clin Chem. 1958;4:323–330.

2. Abdelaziz HM, Billett HH. Follow-up testing for ketonuria. Is it necessary? Am J Clin Pathol. 1994;101:346–348.

3. Clinical and Laboratory Standards Institute. Urinalysis; approved guideline—3rd edition. CLSI document GP16-A3. Wayne, Pa.: CLSI. 2009.

Jay L. Bock, MD, PhD
Department of Pathology
Stony Brook University Medical Center
Stony Brook, NY

Member, CAP Point-of-Care
Testing Committee

Dr. Kiechle is medical director of clinical pathology, Memorial Healthcare, Hollywood, Fla.