Research & Education

Diagnostic Immunohistochemistry in Lung Cancer

Yatabe Y, Dacic S, Borczuk AC and other members of the IASLC Pathology Committee published Best Practices Recommendations for Diagnostic Immunohistochemistry in Lung Cancer in the Journal of Thoracic Oncology, December 2018. Key questions and answers from this article are summarized below: 

 

1.   What is the best combination of markers to use in daily practice?

Short answer: 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 (NE). p40 is preferable to p63 to identify squamous cell carcinoma (Fig 1).  ALK receptor tyrosine kinase (ALK)-positive adenocarcinoma of the lung

Figure 1. ALK receptor tyrosine kinase (ALK)-positive adenocarcinoma of the lung (A). A vast majority of ALK positive lung cancers are also positive for thyroid transcription factor 1, as in this case (B). Another characteristic of an ALK-positive tumor is discordant expression between p63 (C) and p40 (D), which can be a pitfall when p63 is used alone as a marker of squamous cell carcinoma. 

 

2.   What extent of TTF1 and p40 positive reactions should we consider to be positive?

Short answer: Focal positivity for TTF1 is considered a positive reaction indicating pulmonary adenocarcinoma in the proper clinical context (Fig 2A, B), 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 (Fig 2C, D).

  Thyroid transcription factor

Figure 2. (A-B) A case with focal positivity of thyroid transcription factor 1. Histologically, the tumor cells do not show clear morphologic differentiation (A). The displayed thyroid transcription factor 1 staining (B) should be evaluated as positive; thus, the tumor is diagnosed as non-small cell carcinoma, favor adenocarcinoma. (C-D) Another case with unclear morphologic differentiation (C). AS the definition of a positive reaction with p40 is defined as 50% or more positive staining of the tumor cells, the widely scattered but sparse positive reaction with p40 (D) should not be considered as a definite diagnosis of squamous cell carcinoma (D). 

 

3.  Are there any staining differences in lung adenocarcinoma between TTF1 clones (SPT24, SP141 and 8G7G3/1)?

Short answer:  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 (Table 3, 4 and Fig 3).

 

Table 3. TTF1 expression in lung adenocarcinoma and squamous cell carcinoma.

TTF1 expression in lung adenocarcinoma and squamous cell carcinoma

 

Table 4. Results of TTF1 expression in tumors from primary sites including female genital tract, breast, colon and stomach in some of the published studies in the literature (reproduced from Ordonez NG. Value of thyroid transcription factor -1 immunostaining in tumor diagnosis: a review and update. Appl Immunohistochem Mol Morphol. 2012 Oct;20(5):4429-44).

                                                                               8G7G3/1                                       SPT24                                    

                                                                               n=               positive (%)                 n=               positive (%)      
                                                                                                                                                                                 

 Ovarian carcinoma                                             615              22 (3.6%)                   161             16 (9.9%)

 Endometrial adenocarcinoma                          215              17 (7.9%)                   68               19 (27.9%)

 Uterine cervical squamous carcinoma           92                 3 (3.3%)                    39                6 (15.4%)

 Breast adenocarcinoma                                    297               4 (1.5%)                    580              13 (2.4%)

 Colon adenocarcinoma                                     594               11 (1.8%)                  258              15 (5.8%)

 Gastric adenocarcinoma                                   170                3 (1.8%)                   110              1 (0.9%)

 

 Thyroid transcription factor 1

Figure 3. Thyroid transcription factor 1 expression according to antibody clones in primary squamous cell carcinoma of the lung (hematoxylin and eosin staining) (A). Positive reactions with clone SPT24 staining (B) is contrasted with weak or negative with clone 8G7G3/1 (C).  

 

4.   Should a NSCC that is diffusely positive for CK7 but negative for TTF1 and p40 be regarded as “probably adenocarcinoma”?

Short answer:  CK7 is not specific for adenocarcinoma; the marker can be seen in squamous cell carcinoma (Fig 4). The use of CK7 is discouraged for subtyping of NSCC.

  Cytokeratin expression of squamous cell carcinoma in a bronchial biopsy specimen

Figure 4. Cytokeratin expression of squamous cell carcinoma in a bronchial biopsy specimen (A). Nuclear staining with p40 (BC28 Ab) supports the diagnosis (B). The results of staining with both cytokeratin 7 (OV-TL 12/30 antibody) (C) and cytokeratin 5/6 (D5/16B4 antibody) (D) are positive.  

 

5.   When should NE markers be applied to a NSCC?

Short answer:  NE markers should only be applied in support of NE morphology. Positive NE markers may be encountered in approximately 10-30% of NSCC without overt NE morphology by light microscopy. Such tumors may be termed “non-small cell carcinoma with NE differentiation”, however it is recommended that resected tumors be classified primarily as squamous cell carcinoma, adenocarcinoma or large cell carcinoma, as applicable, with a comment regarding the positive NE markers. Therefore, the case shown in Figure 6 should be diagnosed as adenocarcinoma with a positive NE marker.

 A lesion of typical ground glass attenuation on computered tomography image

Figure 6. A lesion of typical ground glass attenuation on computed tomography image (A) Was surgically removed and histology shows adenocarcinoma (B). This tumor has diffuse expression of synaptophysin (C). Despite the diffuse expression, the tumor should be diagnosed as adenocarcinoma because it does not have any morphologic neuroendocrine features. 

 

6.   What is the best antibody panel to differentiate NE tumors from other types of NSCC and which one is the most reliable?

Short answer:  A panel of chromogranin A, synaptophysin and CD56 is the best combination to identify NE tumors. The staining significance of each antibody varies among the sample types, histological subtypes and extent/intensity of positive reactions (Fig 7 and 8).

  Typical immunoprofile of carcinoid tumor.

Figure 7. Typical immunoprofile of carcinoid tumor. Note the homogenous distribution of tumor cells, strong staining for three neuroendocrine markers and low MIB1 immunoreactions.

 

Typical immunoprofile of small cell lung carcinoma.

Figure 8. Typical immunoprofile of small cell lung carcinoma. Note the irregular distribution of tumor cells, strong staining for two neuroendocrine markers and dot-like positivity with chromogranin A. Also, MIB1 labeling is high. 

 

7.   When should a proliferation marker be used in diagnosis?

Short answer:  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/cytology samples (Fig 9). The role of Ki-67 in separating typical from atypical carcinoids is not established and needs more investigation.

  Transbronchial biopsy sepcimens often have crushed tumor cells as shown

Figure 9. Transbronchial biopsy specimens often have crushed tumor cells as shown (A and C). In this situation, cytologic specimens, if available, can be more useful for diagnosis, and Ki-67 staining (B and D) can help with the differential diagnosis between carcinoid tumor (A and B) and small cell carcinoma (C and D). 

 

8.   Is IHC useful to render a specific diagnosis of uncommon lung cancer subtypes (sarcomatoid carcinoma,  salivary gland-type tumors, and NUT carcinoma)?

Short answer:  Currently, IHC and molecular testing are needed to achieve the definitive diagnoses of uncommon lung cancers such as sarcomatoid carcinoma, salivary gland-type tumors, and NUT carcinoma and to distinguish from the mimics.

 Specific Sarcomas That Mimic Sarcomatoid Carcinoma of the Lung in Thoracic Regions

 

9.   What portion of the cytology sample is best for immunostaining: the cell block, the air-dried smears or the ethanol-fixed smears? Can de-stained smears be used adequately?

Short answer:  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.

  Immunostaining of cytologic specimens.

Figure 11. Immunostaining of cytologic specimens. (A) Thyroid transcription factor 1–positive adenocarcinoma in cell block specimen (brown) (by 3,3’-diaminobenzidine, Ventana Benchmark XT immunostainer, Ventana Medical Systems, Tucson, AZ). (BE) Immunostaining on Papanicolaou-stained, ethanol-fixed, non–cell block specimens (Leica Bond automated immunostainer, Leica Biosystems, Nussloch, Germany). Thyroid transcription factor 1–positive adenocarcinoma (red) (detection by 3-amino-9 ethylcarbazole) (B). p40-positive nonkeratinizing squamous cell carcinoma (C). p40-positive benign hyperplastic basal cells underlying ciliated respiratory cells (bronchial brush cytologic examination) (D). CD56-positive small cell carcinoma (E) with corresponding Papanicolaou-stained specimen (F).

 

10.   Which immunohistochemistry panel is recommended to differentiate lung mucinous adenocarcinoma from metastatic mimics?

Short answer: There is no useful marker to differentiate pulmonary mucinous adenocarcinoma from metastatic mimics (Fig 12 and 13, Table 8). Clinicopathological tumor board is crucial for this clinical context.

Table 8.  Immunoprofiles of pulmonary mucinous adenocarcinomas and their mimics* 

-: <10%, -/+: 10-40%, +/-: 40-70%, +: 70-90%, ++: >90% of examined tumors exhibited positive expression

 

 

TTF1

Napsin-A6)

CK7

CK20

CDX2

Pulmonary adenocarcinomas

         
 

Invasive mucinous adenocarcinoma1)

-/+

-/+

++

+/-

+/-

 

Colloid adenocarcinoma

+/-

+/-

+

+/-

+

 

Signet ring cell carcinoma2)

+

+/-

++

-

-

 

Solid adenocarcinoma with mucin

+

+/-

++

-

-

 

Mucinous adenocarcinoma of the lung, NOS3)

+/-

-/+

++

-/+

-/+

Non-pulmonary adenocarcinomas

         
 

GI tract all

-

-

+/-

+

+

 

    Lower GI tract4)

-

-

-/+

++

++

 

    Upper GI tract5)

-

-

+

+/-

+/-

 

    Pancreas

-

-

++

+/-

+/-

 

Breast, mucinous

-

-

++

-

-

 

Ovary, mucinous

-

-

++

+/-

+/-

 *Summary of the references 123-142, 148

1) including mixed mucinous sand non-mucinous adenocarcinoma
2) adenocarcinoma with signet ring cell features
3) NOS: not otherwise specified 
4) colorectum and appendix
5) esophagus, stomach and ampulla
6) immunohistochemistry with monoclonal Napsin-A antibodies

 

An example of invasive mucinous adenocarcinoma of the lung demonstrating lepidic and acinar patterns

Figure 12. An example of invasive mucinous adenocarcinoma of the lung demonstrating lepidic and acinar patterns (A), diffuse expression of cytokeratin 7 (B), focal expression of cytokeratin 20 (C) scattered foci with weak expression of thyroid transcription factor 1 (D) and/or napsin A (E), and weak to moderate expression of caudal type homeobox 2 (F), Of note, the entrapped type II pneumocytes are reactive to cytokeratin 7 (B), thyroid transcription factor 1 (D), and napsin A (E).

 A pancreatic ductal adenocarcinoma metastatic to the lung exhibiting a lepidic pattern at low-power magnification 

Figure 13. A pancreatic ductal adenocarcinoma metastatic to the lung exhibiting a lepidic pattern at low-power magnification (A) and high-power magnification (B), focal cytokeratin 7 expression (C), thyroid transcription factor 1 negativity (D), diffuse weak expression of caudal type homeobox 2 (E), and loss of SMAD family member 4 (F). Of note, strong nuclear expression of thyroid transcription factor 1 in the entrapped pneumocytes may give the impression of a false-positive result. Also, loss of expression of SMAD family member 4 has been reported as a useful marker for the diagnosis of pancreatic adenocarcinoma, but a significant proportion of invasive mucinous adenocarcinomas of the lung harbor this alteration.

 

11.   Are there any IHC or other markers to differentiate primary lung cancers from metastases; squamous cell carcinomas of lung primary vs. metastases from thymic, head & neck, endocervix and the others; and adenocarcinomas of primary vs. metastases from gynecologic, mammary, uroepithelial, non-pulmonary neuroendocrine, prostate and liver cancers?

Short answer: 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 (Fig 15 and 16).

 Useful antibodies and pitfalls for some of the more common differential diagnosis of metasitc lung cancer

Figure 15. Useful antibodies and pitfalls for some of the more common differential diagnosis of metasitc lung cancer. Paired box 8 (monoclonal) positivity in metastatic serous adenocarcinoma from the uterine corpus (A). Focal estrogen receptor positivity in primary lung adenocarcinoma (B). GATA binding protein 3 positivity in metastatic adenocarcinoma from the breast (C). Expression of secretoglobin family 2A in salivary-type 2 in salivary-type adenocarcinoma (mucoepidermoid carcinoma) of the lung (D).

 

Expression of thyroid transcription factor 1 (TTF1) in nonpulmonary carcinoma.

Figure 16. Expression of thyroid transcription factor 1 (TTF1) in nonpulmonary carcinoma. (A-C) Metastasis of ovarian endometrioid carcinoma to the lung (A) express paired box 8 (B) and TTF1 (clone SP24) (C). The diagnosis was confirmed with identical KRAS mutation (G12A) between lung and ovarian cancer. (D-F) Lymph node metastasis of mammary invasive ductal carcinoma (D) displays dual expression of estrogen receptor (E) and TTF1 (clone SPT24) (F).

 

Appendix

Quality control of pre-analytical and analytical phases is recognized as crucial for appropriate assessment for IHC. A brief check could be possible by means of confirmation of positive reactions in the tissue internal controls if available. This is quite efficient procedure and important, particularly in the case of negative reaction of tumor cells. Representative figures as shown in Figure S1. 

Positive reactions as internal control in the normal lung tissue.

Figure S1. Positive reactions as internal control in the normal lung tissue. Whenever evaluate immunostained slides, pathologists should check the internal positive reactions, particularly in the case of negative reaction of tumor cells. (A) TTF-1 expression of type II pneumocytes. (B) Positive reaction of p40 at basal cells of the bronchial epithelium. (C) Synaptophysin expression in normal bronchus. Note positive reactions of fine neural fibers in the smooth muscle in addition to Kulchitsky (neuroendocrine) cells. (D) CD56 expression in neural fibers around the vessel.