Discussion

TTF-1 is a homeodomain-containing nuclear factor that plays a crucial role in the development of the thyroid and lung, and in the normal development of the ventral brain and pituitary gland.1 Within the lung, TTF-1 stimulates the synthesis of lung-specific surfactant proteins,11–14 as well as Clara cell secretory protein.1 In mice, genetic ablation of the NKX2 gene on chromosome 14q13—which encodes TTF-1 protein1 ,15—results in agenesis of the thyroid gland and lung hypoplasia, as well as forebrain defects.1 ,16 TTF-1 protein is a polypeptide of 371 amino acids,1 and can be detected by immunohistochemistry in fetal lung epithelial cell nuclei by about 11 weeks of gestation.17 IHC demonstration of TTF-1 protein is also useful for the identification of some primary lung tumours, especially non-mucinous adenocarcinomas, for which it is considered to be a reliable marker, with labelling of 60%–100% of such cases.1–3 ,18

The 8G7G3/1 MAb was the first TTF-1 MAb to become available commercially,15 and is the most widely used.1 ,4 As shown in tables 1–9 in Ordóñez’ 2012 review of multiple published studies on TTF-1 MAbs (as well as polyclonal antibodies),1 those studies that used the 8G7G3/1 MAb greatly outnumbered those that used the SPT24 MAb: for example, his table 2 lists 2614 cases of adenocarcinoma of the lung across 37 references, in comparison with 579 cases in seven reports for SPT24, and the same table lists 17 studies for 428 cases of adenocarcinoma subtypes, whereas the table does not list any studies for the same adenocarcinoma subtypes using SPT24; His table 1 (thyroid tumours) lists 16 references where the 8G7G3/1 MAb was used; no studies are listed for SPT24. In its 2014 Quality Assurance Program for immunohistochemistry, the Royal College of Pathologists of Australasia19 gave the SP141 TTF-1 MAb a higher score than the 8G7G3/1 MAb for TTF-1 expression because of the intensity and clarity of its labelling, but it was used by only 8% of the laboratories tested, and aberrant staining of plasma cells was noted; the 8G7G3/1 clone was used by 56% of laboratories, but labelling was more commonly scored as borderline or unsatisfactory (the SPT24 MAb was ranked as intermediate between the SP141 and 8G7G3/1 MAbs, and was used by 31% of the laboratories). An assessment performed by Nordic Quality Control found similar results with the SP141 clone, which was considered more sensitive than the 8G7G3/1 MAb.20 Matoso et al4 also depicted stronger labelling of lung adenocarcinomas with the SPT24 MAb with a higher detection rate for ‘… all histologic subtypes of non-small cell pulmonary primary tumors with a clear advantage over 8G7G3/1 in squamous cell carcinomas’.

It has become clear that labelling with TTF-1 MAbs is not restricted to adenocarcinomas of the lung and thyroid follicular epithelial neoplasms (and medullary carcinoma of thyroid), but it also occurs with small cell neuroendocrine (NE) carcinomas and non-small cell NE bronchopulmonary tumours and also—although with lesser frequency—extrapulmonary NE carcinomas, as well as some sarcomatoid carcinomas of the lung. Across four series of sarcomatoid carcinoma cases for which the 8G7G3/1 MAb was used, Ordóñez1 listed 19/63 cases with positive labelling (∼30%); in one of the four studies cited by Ordóñez,1 43% of the pleomorphic carcinomas showed positive labelling, and 55% of the spindle-cell carcinomas were labelled positively.21 In contrast, Lucas et al22 found that all 10 cases of sarcomatoid carcinoma were negative with the 8G7G3/1 MAb, whereas Takeshima et al23 reported positive labelling of 3/20 cases (15%) using the same MAb. With the SPT24 MAb, Terra et al9 recorded positive TTF-1 labelling in a higher proportion of pleomorphic/sarcomatoid carcinomas of the lung or a carcinoma comprising spindle and giant cells: they assessed only the sarcomatoid component and detected any degree of TTF-1 positivity in 18/27 pleomorphic carcinomas (67%), 1/5 pure spindle cell carcinomas and 3/3 carcinosarcomas (this study also recorded labelling for/with 0–4 mesothelioma markers—calretinin, WT1, D2-40 and CK5/6—in some cases).

Labelling of variable proportions of other extrapulmonary neoplasms, including colonic adenocarcinomas, is also recorded—reviewed in detail by Ordóñez1 who recorded labelling of 11/594 cases of colonic adenocarcinoma (1.8%) across multiple studies where the 8G7G3/1 MAb was used versus 15/258 cases in three studies where the SPT24 was used (5.8%). Studies have also shown that the SPT24 MAb labels a wider spectrum of both pulmonary and extrapulmonary neoplasms—and with greater frequency—than the 8G7G3/1 MAb,4 ,5 ,20 and includes, as but a few examples, thymomas, gliomas including glioblastomas, and carcinomas of the female reproductive tract.

In our study, all of the cases of normal lung showed positive labelling of alveolar epithelium with both the 8G7G3/1 and SP141 MAbs, and all of the SCCs were negative with the 8G7G3/1 MAb; however, six of 13 cases of squamous lesions (12 SCCs and a single case of bronchial squamous dysplasia) showed positive labelling of nuclei with the SP141 MAb (∼46%), and 4/12 sarcomatoid carcinomas of the lung also labelled with this MAb, in comparison with 2/12 cases with 8G7G3/1. The SP141 MAb also labelled basal cells in normal-appearing bronchial mucosa. Based on earlier studies using the 8G7G3/1 MAb, primary lung SCC has been regarded as usually TTF-1 negative,24 ,25 but in his survey of multiple studies, Ordóñez1 commented that across 23 reported series, TTF-1 positivity was recorded for 3%–38% of SCCs, whereas all cases in another 24 published series were negative (table 2 in Ordóñez’s1 review records positive TTF-1 labelling of 42/1057 pulmonary SCCs (4%) with the 8G7G3/1 MAb, in comparison with 23/142 cases with the SPT24 MAb (∼16%) and 18.5% with a polyclonal antibody). It was considered likely that the positive labelling in at least some cases was explicable by the presence of residual terminal airway epithelium entrapped by the carcinomas and misinterpreted as neoplastic cells.1 However, the product information package insert sheet for the SP141 MAb refers to 12/51 SCCs of the lung with positive labelling for TTF-1 (23.5%) in comparison with 3/38 cases for the package insert for the 8G7G3/1 MAb produced by the same manufacturer (∼8%).26 (Other potential factors that can potentially explain the differences reported across different publications include methodological issues such as antibody dilutions, antigen retrieval, detection systems and so forth.4)

Current approaches to the differential diagnosis of poorly differentiated non-small cell carcinoma (for the distinction between SCC vs adenocarcinoma) in limited bronchopulmonary biopsy samples (or cytology specimens) include restricted use of antibodies for this assessment, to optimise the amount of tissue remaining for molecular studies (eg, for epidermal growth factor receptor (EGFR) receptor mutation or naplastic lymphoma kinase (ALK) mutation status).2 Travis et al2 have commented that p63 is often positive in most nuclei of SCCs of the lung, but there may be patchy and/or weak staining in 20%–30% of adenocarcinomas (and that p40 is a superior marker for SCC2), but they also state that for a case ‘… positive for an adenocarcinoma marker such as TTF-1, the tumor should be classified as NSCLC, favor adenocarcinoma, despite any expression of squamous markers’; the same authors2 also comment that TTF-1 and p40 are ‘… generally mutually exclusive’. Our experience with the SP141 MAb suggests that this is probably not so for the SP141 TTF-1 MAb, with the potential for misclassification of some SCCs as adenocarcinoma. Accordingly and pending further investigation, we suggest that p63 may best be replaced by p40 and that for this specific situation, the more extensively investigated 8G7G3/1 MAb may be preferable to the SP141 (or the SPT24) antibody, or the use of both of these MAbs for a particular case.

Based on findings with the 8G7G3/1 clone MAb that only a small proportion of mesotheliomas show positive TTF-1 labelling, this antibody has been extensively used for distinction of mesothelioma from primary tumours intrinsic to the lung,1 ,3 and Hammar et al10 commented in 2008 that strong nuclear labelling for TTF-1 in a pleural epithelioid tumour represents strong evidence against a diagnosis of mesothelioma and favours an alternative diagnosis of adenocarcinoma of the lung. The results of our study further support this statement, but for epithelioid tumours only: all of the 66 epithelioid mesotheliomas were negative with each of the 8G7G3/1 and SP141 MAbs, and all 19 sarcomatoid mesotheliomas were negative with 8G7G3/1, but 8/19 sarcomatoid mesotheliomas (∼42%) were positive with the SP141 MAb. Therefore, our study suggests that there is a significant potential for misclassification of a sarcomatoid mesothelioma as a sarcomatoid carcinoma of the lung when the SP141 MAb is used. Potential solutions to this problem would include continued use of the 8G7G3/1 MAb or the use of 8G7G3/1 in addition to SP141 for serosal sarcomatoid lesions, and to take into account the results for generic and other site-‘specific’ carcinoma-related markers.10 From our study, we conclude that neither negative nor positive labelling of a pleural sarcomatoid tumour with the SP141 MAb in isolation discriminates reliably between sarcomatoid carcinoma of the lung and pleural sarcomatoid mesothelioma, although positive nuclear labelling with the 8G7G3/1 MAb represents evidence against a sarcomatoid mesothelioma and strongly favours the alternative diagnosis of sarcomatoid carcinoma.

We consider that the positive labelling of SCCs and sarcomatoid carcinoma of the lung and pleural sarcomatoid mesotheliomas with the SP141 MAb—and the more widespread labelling of various neoplasms with SPT24 as reported in the literature—may reflect augmented sensitivity of those MAbs, with some resultant loss of specificity (table 1). Gene Expression Omnibus DataSets have shown mRNA expression for TTF-1 at low levels in some malignant pleural mesotheliomas,27 and Scorer et al5 found that, for six cases of colonic adenocarcinoma positive with the SPT24 MAb, reverse transcriptase PCR (RT-PCR) demonstrated fragments of TTF-1 RNA transcripts indicative of TTF-1 gene expression and ‘… three smaller RNA fragments when sequenced provided evidence of a novel splice variant previously not described in the literature’.

Therefore, positive IHC labelling with the SP141 MAb—like the SPT24 MAb—may simply represent high sensitivity for very low levels of TTF-1 protein in a variety of tumours other than primary lung/thyroid adenocarcinomas and pulmonary (and extrapulmonary) small cell and non-small cell NE carcinomas, but it does carry implications for diagnosis and treatment. The differences between these two MAbs also highlight the question of the differential sensitivity/specificity for other antibodies used for diagnosis of other neoplasms, when multiple antibodies for each marker are available from different manufacturers. As stated by Bisceglia et al28 in relation to labelling of breast carcinoma with the SPT24 TTF-1 MAb: ‘The Chosen Clone Matters’.