Table 1.

Key questions and recommendations for diagnostic immunohistochemistry in lung cancer

Key questions	Short answers
1. What is the best combination of markers to use in daily practice?	When IHC is needed for the subtyping of non-small cell carcinoma (NSCC), TTF1 and p40 are the gold standard, and these two markers are usually sufficient in clinical practice if there are no morphological features of neuroendocrine differentiation. p40 is preferable to p63 to identify squamous cell carcinoma.
2. What extent of TTF1 and p40 positive reactions should we consider to be positive?	Focal positivity for TTF1 is considered a positive reaction indicating pulmonary adenocarcinoma in the proper clinical context, whereas for p40 the cut-off rate should be positivity in more than 50% of tumor nuclei. Focal/weak positivity for p40 is not diagnostic of squamous cell carcinoma.
3. Are there any staining differences in lung adenocarcinoma between among TTF1 clones (SPT24, SP141 and 8G7G3/1)?	The staining performance of TTF1 varies among the clones. Among the most commonly used antibodies, 8G7G3/1 is the most specific antibody to identify lung adenocarcinoma.
4. Should a NSCC that is diffusely positive for CK7 but negative for TTF1 and p40 be regarded as “probably adenocarcinoma”?	CK7 is not specific for adenocarcinoma; the marker can be seen in squamous cell carcinoma. The use of CK7 is discouraged for subtyping of NSCC.
5. When should NE markers be applied to a NSCC?	NE markers should only be applied in support of NE morphology.
6. What is the best antibody panel to differentiate NE tumors from other types of NSCC and which one is the most reliable?	A panel of chromogranin A, synaptophysin and CD56 is the best combination to identify NE tumours. The staining significance of each antibody varies among the sample types, histological subtypes and extent/intensity of positive reactions.
7. When should a proliferation marker be used in diagnosis?	The main established role of Ki-67in lung carcinomas is to help distinguish carcinoids from high grade NE carcinomas (large cell neuroendocrine carcinoma and small cell carcinomas), especially in small or crushed biopsy or cytology samples. The role of Ki-67 in separating typical from atypical carcinoids is not established and needs more investigation
8. Is IHC useful to render a specific diagnosis of uncommon lung cancer subtypes (sarcomatoid carcinoma, salivary gland-type tumors, and NUT carcinoma)?	Currently, IHC, as well as molecular testing, are needed to achieve the definitive diagnoses of uncommon lung cancers and to distinguish from the mimics.
9. What portion of the cytology sample is best for immunostaining: the cell block, the air-dried smears or the ethanol-fixed smears? Can destained smears be used adequately?	All cytology preparations including cell blocks, ethanol-fixed and air-dried slides can principally be used for immunostaining. Formalin fixed cell blocks are most straightforward, while rigorous protocol optimization, validation and quality control are required in immunostaining in cytology.
10. Which IHC panel is recommended to differentiate lung mucinous adenocarcinoma from metastatic mimics?	There is no useful marker to differentiate pulmonary mucinous adenocarcinoma from metastatic mimics. Clinicopathological tumor board is crucial for this clinical context.
11. Are there any IHC or other markers to differentiate between primary lung cancers and metastases; between squamous cell carcinomas of lung primary and metastases from thymic, head and neck, endocervical, and the other cancers; and between adenocarcinomas of primary and metastases from gynecologic, mammary, uroepithelial, nonpulmonary NE, prostate, and liver cancers?	In this clinical context, morphological comparison with prior tumor is crucial. There are no absolute IHC markers to make the differential diagnosis, and pathologists should be aware of IHC pitfalls.