Histopathology 2016, 68, 367–377. DOI: 10.1111/his.12765
Differential expression of immunohistochemical markers in primary lung and breast cancers enriched for triple-negative tumours Elena Provenzano,* David J Byrne,1,* Prudence A Russell,2 Gavin M Wright,3 Daniele Generali4 & Stephen B Fox1,5 Department of Histopathology, Addenbrooke’s Hospital, Cambridge, UK, 1Department of Pathology, Peter MacCallum Cancer Centre, The University of Melbourne, Melbourne, Vic., Australia, 2Department of Anatomical Pathology, St Vincent’s Hospital, University of Melbourne, Melbourne, Vic., Australia, 3Department of Surgery, St Vincent’s Hospital, University of Melbourne, Melbourne, Vic., Australia, 4Universita Operativa Multidisciplinare di Patologia Mammaria/ US Terapia Molecolare e Farmacogenomica, dell’Azienda Ospedaliera Istituti Ospitalieri di Cremona, Cremona, Italy, and 5 Department of Pathology, The University of Melbourne, Melbourne, Vic., Australia Date of submission 10 March 2015 Accepted for publication 22 June 2015 Published online Article Accepted 28 June 2015
Provenzano E, Byrne D J, Russell P A, Wright G M, Generali D & Fox S B (2016) Histopathology 68, 367–377. DOI: 10.1111/his.12765
Differential expression of immunohistochemical markers in primary lung and breast cancers enriched for triple-negative tumours Aims: In breast cancer patients presenting with a lung lesion, the distinction between lung and breast origin is clinically important. Lung and breast cancers are both CK7+/CK20 , so additional immunohistochemical markers are needed. Methods and results: We examined the expression of oestrogen receptor (ER), progesterone receptor (PR), thyroid transcription factor-1 (TTF-1), gross cystic disease fluid protein-15 (GCDFP-15), p63 and Wilms’ tumour 1 (WT1) in a series of tissue microarrays comprising 266 non-small-cell lung cancers and 837 primary breast cancers enriched for triple-negative tumours (TNBC). Staining for ER, PR, TTF-1 and GCDFP-15 was present in 63%, 49%, 0% and 25% of breast and 6%, 9%, 59% and 1% of lung cancers,
respectively. Strong staining for p63 was present in 63 (97%) lung squamous cell carcinomas and only eight (9%) TNBC. WT1 nuclear staining was rare; however, cytoplasmic staining was identified in 49 (40%) TNBC and 10 (5%) lung cancers. Cluster analysis segregated TNBC from lung cancers with TTF-1 and/or p63 staining favouring lung origin, and GCDFP-15 or WT1 staining favouring breast origin. Cancers negative for all four markers (17%) were 60% breast and 40% lung origin. Conclusion: An immunohistochemical panel incorporating ER, TTF-1, GCDFP-15, p63 and WT1 can help to distinguish lung cancer from metastatic breast cancer, including TNBC.
Keywords: breast cancer, immunohistochemistry, lung cancer, triple negative breast cancer
Introduction
Address for correspondence: Dr E Provenzano, Box 235, Addenbrooke’s Hospital, Hills Road, Cambridge CB2 0QQ, UK. e-mail: [email protected] *These authors contributed equally to this work. © 2015 John Wiley & Sons Ltd.
Breast and lung cancer are the two leading causes of cancer mortality in women.1 Women with a history of breast cancer have an increased risk of developing a second primary cancer, with the lung being a common site.2–4 Conversely, the lung is also a frequent site for breast cancer metastasis, particularly for
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triple-negative [ER/PR/human epidermal growth factor receptor 2 (HER2)-negative] breast cancer (TNBC).5–7 Hence, when a woman with a history of breast cancer presents with a lung lesion, it is important to distinguish whether this represents metastatic breast cancer or a new lung primary in order to determine appropriate treatment, particularly in the era of targeted therapies now available for both lung and breast cancer. Lung biopsies often provide limited material for histological, immunohistochemical and molecular analyses. The distinction between metastatic and primary lung cancer can be difficult, especially when the tumour is poorly differentiated. Immunohistochemistry can be of help, with commonly used panels including cytokeratin 7 (CK7), cytokeratin 20 (CK20), thyroid transcription factor 1 (TTF-1) and oestrogen receptor (ER). The majority of both lung and breast cancers will be CK7-positive and CK20negative.8 TTF-1, a tissue-specific transcription factor expressed by epithelial cells of lung and thyroid origin, is positive in 68–88% of lung adenocarcinomas, but only 1.5% of squamous cell carcinomas (SQCC) and 32% of sarcomatoid carcinomas.4,9–11 In addition, positivity for TTF-1 has been described in tumours from other primary sites, including the breast.12–14 ER is regarded as a marker for tumours of breast and gynaecological tract origin, although positive staining has been described in tumours from other sites, including the lung.15–17 Other markers of breast origin include gross cystic disease fluid protein-15 (GCDFP-15) and mammaglobin. GCDFP-15 positivity is a feature of apocrine lesions, including the molecular apocrine group of TNBC.18,19 However, GCDFP-15 positivity has been reported in up to 15% of lung cancers.4,10,20 CK5/6 and p63 are expressed by up to 94% of SQCCs of the lung, and are now used for the subclassification of non-small-cell lung cancers.11,21,22 Basal breast cancers are poorly differentiated tumours that are frequently triple-negative and show positive staining for basal cytokeratins, including CK5 and CK14, and can be positive for markers of myoepithelial differentiation such as p63. However, immunohistochemical staining for these markers in basal breast cancers is often focal, and may be absent in small biopsy specimens. We have conducted an immunohistochemical study examining the expression of a selected panel of markers, including TTF-1, ER, PR, GCDFP-15, p63 and Wilms’ tumour 1 (WT1), on tissue microarrays (TMAs), representing a well-characterized series of
primary lung and breast cancers. The aim was to investigate the differential expression of these markers to develop an algorithm to distinguish metastatic breast from primary lung cancers. The series is enriched for TNBC, as identifying metastases from this ER-negative group of cancers is clinically important and diagnostically challenging.
Materials and methods PATIENT MATERIAL
TMAs were constructed using one to four 1-mm cores from 266 non-small-cell lung cancers and 837 primary breast cancers (Table S1). The lung cancers were identified from the archives of St Vincent’s Hospital and the Peter MacCallum Cancer Centre, Melbourne, Australia. All slides from the non-smallcell lung cancers were reviewed centrally by an experienced lung pathologist (P.A.R.), and classified according to the 2015 World Health Organization (WHO) classification of lung tumours.23 The breast cancer TMAs were constructed from 837 non-consecutive primary breast cancers diagnosed between 2007 and 2011 at the Peter MacCallum Cancer Centre, Royal Melbourne Hospital, Focus Pathology, and Dorevitch Pathology in Melbourne, Australia and AO Istituti Ospitalieri di Cremona, Cremona, Italy. Full ethics approval for the use of tumour tissue for TMA construction and ongoing research was obtained in each institution (Peter MacCallum 03/90 and 00/81 covering Focus, Dorevitch Pathology and the Royal Melbourne Hospital; St Vincent’s Hospital, Melbourne A03/12; AO Istituti Ospitalieri di Cremona R-LATN07/11). IMMUNOHISTOCHEMISTRY
Immunohistochemistry for ER, PR, TTF-1, p63, WT1, and GCDFP-15 was performed on 4-lm sections using a Ventana BenchMark Ultra autostainer using Ultra Cell conditioning 1 antigen retrieval solution and the Ultraview diaminobenzidine (DAB) detection system (Ventana Biosystems, Tucson, AZ, USA) (Table 1). Appropriate positive and negative control tissues were included for all antibodies. Nuclear staining was scored for TTF-1, p63, ER and PR, cytoplasmic staining was scored for GCDFP15 and nuclear and cytoplasmic staining were scored separately for WT1. For TTF-1, p63, GCDFP-15 and WT1, the percentage of tumour cells with staining was recorded and average staining intensity was graded as absent (0), weak (1), intermediate (2) or © 2015 John Wiley & Sons Ltd, Histopathology, 68, 367–377.
Immunohistochemistry lung versus breast cancer
Table 1. Antibody and immunohistochemistry protocols used for the centralized staining
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6.00 for Windows (GraphPad Software, La Jolla, CA, USA; www.graphpad.com).
Antigen
Clone
Supplier
Dilution
Amplifier
TTF1
8G7G3/1
Dako
1:100
Results
p63
4A4
Ventana
Pre-dilute
IMMUNOHISTOCHEMISTRY
ER
SP1
Ventana
Pre-dilute
PR
1E2
Ventana
Pre-dilute
WT1
6F-H2
Dako
1:50
GCDFP-15
23A3
Vision Biosystems
1:50
Of 224 lung cancers, 130 (58%) showed high expression of TTF-1, 86% of adenocarcinomas, 50% of large cell carcinomas and only 8% of SQCCs. No breast cancers showed any staining with TTF-1 (Table 2). There were 77 (35%) lung cancers that showed high expression of p63, 97% of SQCCs compared with 7% of adenocarcinomas. There were 21 (23%) TNBC that showed p63 staining, with low expression in 61% of positive cases in contrast to 3% of positive lung cancers (Figure 1). The other breast tissue microarrays were not stained for p63. Of the breast cancers, 323 of 511 (63%) with data available were ER-positive, with 311 showing high expression. The relatively low percentage is due to enrichment for TNBC. Only 12 (5%) lung cancers showed ER positivity, 11 of which were adenocarcinomas, with only three showing high expression. There were 212 (43%) breast cancers with high expression of PR compared to two (1%) lung cancers. GCDFP-15 expression was seen in 87 (25%) breast cancers and two (1%) lung cancers. Low nuclear expression for WT1 was observed in one (0.4%) lung cancer; 18 (3%) breast cancers showed nuclear staining for WT1, with 13 showing high expression, none of which were TNBC (data not shown). A striking pattern of cytoplasmic staining for WT1 was seen in 55 (9%) breast cancers, including 49 (40%) TNBC, while cytoplasmic staining for WT1 was seen in only 10 (5%) lung cancers. TTF-1 was the most specific marker for lung adenocarcinoma, with a mean score of 4.8 of six, p63 for SQCC with a mean score of 5.5 of six and WT1 for TNBC with a mean score of 2 of six, all with P-values < 0.005 (Figure 2). ER staining was associated strongly with breast cancer when it was present, with non-TNBC having a mean score of 5.9 of eight compared with less than 1 in lung adenocarcinoma.
Mouse antibody amp
TTF-1: thyroid transcription factor-1; ER: oestrogen receptor; PR: progestogen receptor; WT1: Wilms’ tumour 1; GCDFP-15: gross cystic disease fluid protein-15.
strong (3) and the staining percentage was divided into four groups, as published previously:24 none = 0, 1– 9% = 1, 10–50% = 2 and > 50% = 3. The intensity and proportion scores were then added together to give a final value of 0–6. A score of 2 was regarded as low expression, and 3–6 was regarded as high expression. Where historical data for ER, PR and HER2 were available results were normalized, including ER and PR weighted H-scores,25 which were converted into a combined Allred score26 (Figure S1). ER and PR staining on TMAs was given a combined Allred score. Based on these data, the breast cancers were divided into non-TNBC (ER/PR and/or HER2-positive), TNBC (ER, PR and HER2-negative) and unknown (ER, PR and/or HER2 data missing). For ER and PR an Allred score of 3 or 4 was regarded as low expression, and 5–8 was regarded as high expression. CLUSTER AND TREE VIEW ANALYSIS
Unsupervised hierarchical cluster analysis was performed to compare lung and TNBC results for TTF-1, p63, GCDFP-15 and WT1 using Cluster and TreeView software, as published previously.27–29 The combined IHC expression scores were converted into microarray format modified from the TMA-Deconvoluter software,30 with a score of 0 = 2 (negative), 2 = 1, 3 = 2, 4 = 3, 5 = 4, 6 = 5, 7 = 6 and 8 = 7. STATISTICAL ANALYSIS
Kruskal–Wallis with Dunn’s multiple comparisons test was performed using GraphPad Prism version © 2015 John Wiley & Sons Ltd, Histopathology, 68, 367–377.
CLUSTER ANALYSIS
A clustering approach was used to try to identify the optimal combination of markers to distinguish TNBC from lung cancers. The resulting clustergram showed seven main nodes (Figure 3). Node 1 is characterized by expression of WT1 (96% TNBC). Node 2 is positive for both WT1 and p63 (82% TNBC). Node 3
4 (50%) 2 (50%)
Carcinoid (n = 8)
Sarcomatoid (n = 6)
8 (100%) 4 (100%)
Carcinoid (n = 8)
Sarcomatoid (n = 6)
202 (94%)
4 (100%)
Adenosquamous (n = 7)
Total (n = 266)
8 (100%)
Large cell (n = 10)
9 (4%)
0
0
0
0
1 (2%)
3 (2%)
0
0
0
0
0
194 (91%)
4 (100%)
7 (88%)
4 (100%)
5 (63%)
61 (92%)
113 (91%)
65 (98%)
3 (3%)
Squamous cell (n = 74)
8 (6%)
113 (91%)
Lung cancers Adenocarcinoma (n = 161)
High
72 (77%)
Neg
Low
0
–
Neg
0
0
72 (77%)
–
137 (63%)
3 (75%)
8 (100%)
0
8 (100%)
2 (3%)
116 (91%)
PR
614 (100%)
Total (n = 837)
0
0
0
130 (58%)
2 (50%)
4 (50%)
0
4 (50%)
5 (8%)
115 (86%)
ER
114 (100%)
Unknown (n = 171)
0
0
3 (1%)
0
0
0
0
2 (2%)
1 (1%)
Marker Score
121 (100%)
379 (100%)
TNBC (n = 130)
Breast cancers Non-TNBC (n = 536)
91 (41%)
5 (100%)
Adenosquamous (n = 7)
Total (n = 266)
4 (50%)
59 (90%)
Squamous cell (n = 74)
Large cell (n = 10)
17 (13%)
Neg
High
Neg
Low
p63
TTF1
Lung cancers Adenocarcinoma (n = 161)
Marker Score
Table 2. Immunohistochemistry results
18 (8%)
0
1 (12%)
0
3 (37%)
5 (8%)
9 (7%)
Low
13 (14%)
–
13 (14%)
–
2 (1%)
0
0
0
0
0
2 (2%)
Low
2 (1%)
0
0
0
0
0
2 (2%)
High
8 (9%)
–
8 (9%)
–
78 (36%)
1 (25%)
0
4 (100%)
0
63 (97%)
10 (7%)
High
222 (99%)
4 (100%)
7 (88%)
4 (100%)
8 (100%)
65 (98%)
134 (100%)
Neg
GCDFP-15
544 (91%)
101 (98%)
72 (60%)
371 (99%)
212 (95%)
3 (75%)
8 (100%)
4 (100%)
7 (88%)
64 (97%)
126 (95%)
Neg
WT1
1 (0.5%)
0
1 (12%)
0
0
0
Low
4 (1%)
0
4 (3%)
0
6 (3%)
0
0
0
0
2 (3%)
4 (3%)
Low
1 (0.5%)
0
0
0
0
1 (2%)
0
High
51 (8%)
2 (2%)
45 (37%)
4 (1%)
4 (2%)
1 (25%)
0
0
1 (12%)
0
2 (2%)
High
370 E Provenzano et al.
© 2015 John Wiley & Sons Ltd, Histopathology, 68, 367–377.
188 (37%) Total (n = 837)
TTF-1: thyroid transcription factor-1; ER: oestrogen receptor; PR: progestogen receptor; WT1: Wilms’ tumour 1; GCDFP-15: gross cystic disease fluid protein-15; TNBC: triple-negative breast cancer.
66 (19%) 21 (6%) 255 (75%) 212 (43%) 30 (6%) 253 (51%) 311 (61%)
– – – – – – Unknown (n = 171)
12 (2%)
15 (23%) 9 (14%) 40 (63%)
17 (15%) 0 94 (85%) 0 4 (3%) 127 (97%) 0 2 (2%) 128 (98%) TNBC (n = 130)
34 (20%) 12 (7%) 121 (73%) 26 (7%) 126 (35%) 311 (82%) 10 (2%) 60 (16%) Breast cancers Non-TNBC (n = 536)
Low Marker Score
Table 2. (Continued)
212 (58%)
Low Neg Neg Neg
High
PR ER
Low
High
GCDFP-15
High
Immunohistochemistry lung versus breast cancer
© 2015 John Wiley & Sons Ltd, Histopathology, 68, 367–377.
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represents a small group that only express GCDFP-15 (88% TNBC). Node 4 is negative for all four markers (60% TNBC, 40% lung). Node 5 expresses p63 (84% lung); two of the TNBC in this group coexpress GCDFP15. Nodes 6 and 7 contain lung cancers only; the former express TTF-1 (n = 92) and the latter coexpress TTF-1 and p63 (n = 11) (Table 3). A suggested algorithm for determination of primary lung versus metastatic breast origin is presented in Figure 4.
Discussion Classification of a lung lesion in a patient with a past history of breast cancer often poses a diagnostic challenge. In patients with a solitary lung nodule, the lesion is almost twice as likely to represent a primary lung cancer rather than a breast metastasis.31,32 The risk of developing lung cancer is increased in smokers, and is compounded by breast radiotherapy.2,3,27,33,34 The lung is also a major metastasis site for breast cancer.35,36 Primary lung or breast metastatic origin can sometimes be distinguished on histological grounds by comparison with the previous cancer, or by the presence of typical morphological features of one of the breast or lung cancer subtypes; however, this is highly subjective and can be inaccurate. Knowledge of the histological type, ER and HER2 status of the breast primary can be of additional value, although a not insignificant proportion of breast metastases show different receptor status to the primary.37,38 Triple-negative/basal breast cancers have a particular tendency to metastasize to lung; these are commonly poorly differentiated, high-grade tumours that may show sarcomatoid or squamous differentiation, making distinction from a lung cancer on morphological grounds impossible.5–7 The most specific markers for determining breast or lung origin in this study were ER and TTF-1, respectively. TTF-1 positivity has been reported in 73–88% of primary lung adenocarcinomas, with positive staining in only 0–3% of breast cancers.4,9–15 Coexpression of ER and TTF-1 has been reported in both lung and breast cancer;13–15 however, none of the primary breast cancers in our series showed TTF-1 positivity. In other lung cancer subtypes, TTF-1 positivity is present in 0–1% of SQCCs, 44–60% of large cell carcinomas, 32% of sarcomatoid carcinomas and 40–53% of adenosquamous carcinomas.11,16 We found similar results for large cell carcinoma (50%); however, 11% of SQCCs and none of the adenosquamous carcinomas were TTF-1-positive. Hence, TTF-1 positivity is helpful in diagnosing primary lung adenocarcinoma,
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A
B
Figure 1. p63 staining in A, triple-negative breast cancer (TNBC) showing weak staining in a small proportion of cells and B, lung squamous cell carcinoma with strong diffuse staining present in tumour cells.
8
Total expression score
7
****
****
6 **** 5 4 3
****
2
**** **
0
nTNBC TNBC ADC SQCC nTNBC TNBC SQCC ADC TNBC SQCC ADC nTNBC TNBC SQCC ADC nTNBC TNBC SQCC ADC
1
ER
TTF1 p63 WT1 Immunohistochemistry stain
GCDFP15
Figure 2. Comparison of expression of oestrogen receptor (ER), thyroid transcription factor-1 (TTF-1), p63 and Wilms’ tumour 1 (WT1) in non-triple-negative breast cancer (nTNBC), TNBC, lung squamous cell carcinomas (SQCC) and lung adenocarcinoma (ADC). Mean expression level and 95% confidence intervals indicated.
but is less useful for other types of non-small cell lung carcinoma, particularly SQCC and sarcomatoid carcinoma. The ER positivity rate among breast cancers in this series was only 63%; however, we deliberately included a large number of TNBC, given the propensity for lung metastasis and associated diagnostic challenges. In unselected, population-based series the ER positivity rate is approximately 83%.39 In the literature, the positivity rate for ER in primary lung cancers ranges from 0 to 67%.15 The large range reflects differences in methodology, with higher rates associated with use of the 6F11 antibody.15,40 We used the SP1 antibody, and found that 6% of all lung cancers and 8% of adenocarcinomas showed positive staining for ER. A recent study using the same antibody found that 15% of lung adenocarcinomas were positive for ER using a 1% cut-off.16
GCDFP-15 is regarded as a relatively specific marker of breast origin and is expressed in 40–73% of primary breast cancers, although positive staining is also seen in tumours of skin adnexal, salivary and prostate glands.4,41 We identified positive staining for GCDFP-15 in 25% of breast cancers, which is lower than reported in the literature. In TNBC, positivity rates for GCDFP-15 in primary tumours are 3–14%, similar to the 13% seen in this study, and in only 8% of metastases to lung.42,43 GCDFP-15 in lung cancer has positivity rates of 0–15%4,10,20 compared with 1% in our series. Overall, GCDFP-15 is of limited use in determining the nature of a lung lesion due to its low sensitivity; however, specificity is high when positive staining is present. Alternative markers of breast origin include mammaglobin and GATA binding protein 3 (GATA3), both of which show higher sensitivity but lower specificity for breast origin.43,44 Mammaglobin positivity has been reported in up to 80% of primary breast cancers, including 21–25% of TNBC, but only 0–1.2% of lung cancers.4,20,42,43 GATA3 positivity is seen in 67–95% of breast cancers; 43–73% of TNBCs are positive, including 20–54% of metaplastic cancers, although staining is often focal and of weaker intensity.45–47 In studies looking specifically at breast metastases to lung, up to 87% were positive, 54% of metastatic TNBC, compared with only 0.3–8% of lung adenocarcinomas and 12% of lung SQCCs.45,48 We did not look at mammaglobin staining in this study, as this has been extensively published previously.20,42,49,50 We have investigated GATA3 expression in breast, and found positive staining in 46% of TNBC using the L50-832 clone (manuscript in preparation). We examined some of this series and, interestingly, only six of the 31 TNBC negative for all four markers were GATA3-positive (data not shown). p63 is a nuclear protein expressed in squamous cells and myoepithelial cells at several sites, including breast. Immunohistochemical panels including p63 are being used increasingly to help to differentiate © 2015 John Wiley & Sons Ltd, Histopathology, 68, 367–377.
188 (37%) Total (n = 837)
TTF-1: thyroid transcription factor-1; ER: oestrogen receptor; PR: progestogen receptor; WT1: Wilms’ tumour 1; GCDFP-15: gross cystic disease fluid protein-15; TNBC: triple-negative breast cancer.
66 (19%) 21 (6%) 255 (75%) 212 (43%) 30 (6%) 253 (51%) 311 (61%)
– – – – – – Unknown (n = 171)
12 (2%)
15 (23%) 9 (14%) 40 (63%)
17 (15%) 0 94 (85%) 0 4 (3%) 127 (97%) 0 2 (2%) 128 (98%) TNBC (n = 130)
34 (20%) 12 (7%) 121 (73%) 26 (7%) 126 (35%) 311 (82%) 10 (2%) 60 (16%) Breast cancers Non-TNBC (n = 536)
Low Marker Score
Table 2. (Continued)
212 (58%)
Low Neg Neg Neg
High
PR ER
Low
High
GCDFP-15
High
Immunohistochemistry lung versus breast cancer
© 2015 John Wiley & Sons Ltd, Histopathology, 68, 367–377.
371
represents a small group that only express GCDFP-15 (88% TNBC). Node 4 is negative for all four markers (60% TNBC, 40% lung). Node 5 expresses p63 (84% lung); two of the TNBC in this group coexpress GCDFP15. Nodes 6 and 7 contain lung cancers only; the former express TTF-1 (n = 92) and the latter coexpress TTF-1 and p63 (n = 11) (Table 3). A suggested algorithm for determination of primary lung versus metastatic breast origin is presented in Figure 4.
Discussion Classification of a lung lesion in a patient with a past history of breast cancer often poses a diagnostic challenge. In patients with a solitary lung nodule, the lesion is almost twice as likely to represent a primary lung cancer rather than a breast metastasis.31,32 The risk of developing lung cancer is increased in smokers, and is compounded by breast radiotherapy.2,3,27,33,34 The lung is also a major metastasis site for breast cancer.35,36 Primary lung or breast metastatic origin can sometimes be distinguished on histological grounds by comparison with the previous cancer, or by the presence of typical morphological features of one of the breast or lung cancer subtypes; however, this is highly subjective and can be inaccurate. Knowledge of the histological type, ER and HER2 status of the breast primary can be of additional value, although a not insignificant proportion of breast metastases show different receptor status to the primary.37,38 Triple-negative/basal breast cancers have a particular tendency to metastasize to lung; these are commonly poorly differentiated, high-grade tumours that may show sarcomatoid or squamous differentiation, making distinction from a lung cancer on morphological grounds impossible.5–7 The most specific markers for determining breast or lung origin in this study were ER and TTF-1, respectively. TTF-1 positivity has been reported in 73–88% of primary lung adenocarcinomas, with positive staining in only 0–3% of breast cancers.4,9–15 Coexpression of ER and TTF-1 has been reported in both lung and breast cancer;13–15 however, none of the primary breast cancers in our series showed TTF-1 positivity. In other lung cancer subtypes, TTF-1 positivity is present in 0–1% of SQCCs, 44–60% of large cell carcinomas, 32% of sarcomatoid carcinomas and 40–53% of adenosquamous carcinomas.11,16 We found similar results for large cell carcinoma (50%); however, 11% of SQCCs and none of the adenosquamous carcinomas were TTF-1-positive. Hence, TTF-1 positivity is helpful in diagnosing primary lung adenocarcinoma,
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A
B
Figure 1. p63 staining in A, triple-negative breast cancer (TNBC) showing weak staining in a small proportion of cells and B, lung squamous cell carcinoma with strong diffuse staining present in tumour cells.
8
Total expression score
7
****
****
6 **** 5 4 3
****
2
**** **
0
nTNBC TNBC ADC SQCC nTNBC TNBC SQCC ADC TNBC SQCC ADC nTNBC TNBC SQCC ADC nTNBC TNBC SQCC ADC
1
ER
TTF1 p63 WT1 Immunohistochemistry stain
GCDFP15
Figure 2. Comparison of expression of oestrogen receptor (ER), thyroid transcription factor-1 (TTF-1), p63 and Wilms’ tumour 1 (WT1) in non-triple-negative breast cancer (nTNBC), TNBC, lung squamous cell carcinomas (SQCC) and lung adenocarcinoma (ADC). Mean expression level and 95% confidence intervals indicated.
but is less useful for other types of non-small cell lung carcinoma, particularly SQCC and sarcomatoid carcinoma. The ER positivity rate among breast cancers in this series was only 63%; however, we deliberately included a large number of TNBC, given the propensity for lung metastasis and associated diagnostic challenges. In unselected, population-based series the ER positivity rate is approximately 83%.39 In the literature, the positivity rate for ER in primary lung cancers ranges from 0 to 67%.15 The large range reflects differences in methodology, with higher rates associated with use of the 6F11 antibody.15,40 We used the SP1 antibody, and found that 6% of all lung cancers and 8% of adenocarcinomas showed positive staining for ER. A recent study using the same antibody found that 15% of lung adenocarcinomas were positive for ER using a 1% cut-off.16
GCDFP-15 is regarded as a relatively specific marker of breast origin and is expressed in 40–73% of primary breast cancers, although positive staining is also seen in tumours of skin adnexal, salivary and prostate glands.4,41 We identified positive staining for GCDFP-15 in 25% of breast cancers, which is lower than reported in the literature. In TNBC, positivity rates for GCDFP-15 in primary tumours are 3–14%, similar to the 13% seen in this study, and in only 8% of metastases to lung.42,43 GCDFP-15 in lung cancer has positivity rates of 0–15%4,10,20 compared with 1% in our series. Overall, GCDFP-15 is of limited use in determining the nature of a lung lesion due to its low sensitivity; however, specificity is high when positive staining is present. Alternative markers of breast origin include mammaglobin and GATA binding protein 3 (GATA3), both of which show higher sensitivity but lower specificity for breast origin.43,44 Mammaglobin positivity has been reported in up to 80% of primary breast cancers, including 21–25% of TNBC, but only 0–1.2% of lung cancers.4,20,42,43 GATA3 positivity is seen in 67–95% of breast cancers; 43–73% of TNBCs are positive, including 20–54% of metaplastic cancers, although staining is often focal and of weaker intensity.45–47 In studies looking specifically at breast metastases to lung, up to 87% were positive, 54% of metastatic TNBC, compared with only 0.3–8% of lung adenocarcinomas and 12% of lung SQCCs.45,48 We did not look at mammaglobin staining in this study, as this has been extensively published previously.20,42,49,50 We have investigated GATA3 expression in breast, and found positive staining in 46% of TNBC using the L50-832 clone (manuscript in preparation). We examined some of this series and, interestingly, only six of the 31 TNBC negative for all four markers were GATA3-positive (data not shown). p63 is a nuclear protein expressed in squamous cells and myoepithelial cells at several sites, including breast. Immunohistochemical panels including p63 are being used increasingly to help to differentiate © 2015 John Wiley & Sons Ltd, Histopathology, 68, 367–377.
Immunohistochemistry lung versus breast cancer
68% of TNBC.59 High WT1 mRNA expression levels have been associated with poor prognosis in breast cancer, and show a correlation with ER-negative, basal-like and HER2 molecular subtypes.62,63 In conclusion, the differential diagnosis of primary lung cancer and metastatic breast cancer can be challenging, particularly in the case of poorly differentiated tumours where ER and TTF1 are often negative. The addition of p63, GCDFP-15 and WT1 may be helpful in this setting, with high-level expression of p63 favouring lung origin, while positive staining for GCDFP-15 or cytoplasmic staining for WT1 favours breast origin. Unfortunately, approximately 17% of cases are negative with all five markers, and these cancers have a roughly equal chance of being either breast or lung in origin. Novel markers are still required to determine the true nature of these lesions, which will hopefully emerge from integrated ‘omics’ studies in the future.
7.
8.
9.
10.
11.
12.
13.
Acknowledgements We gratefully acknowledge Associate Professor Ben Solomon, Head of the Molecular Therapeutics and Biomarkers Laboratory, Peter MacCallum Cancer Centre, for allowing us access to the lung cancer TMAs and patient data used in the project.
14.
15.
16.
Conflicts of interest The authors declare no conflicts of interest.
17.
References
18.
1. Ferlay JSI, Ervik M, Dikshit R et al. GLOBOCAN 2012 v1.0. Cancer incidence and mortality worldwide: IARC Cancerbase no. 11 (internet). Lyon, France: International Agency for Research on Cancer, 2012. 2. Ford MB, Sigurdson AJ, Petrulis ES et al. Effects of smoking and radiotherapy on lung carcinoma in breast carcinoma survivors. Cancer 2003; 98; 1457–1464. 3. Deutsch M, Land SR, Begovic M, Wieand HS, Wolmark N, Fisher B. The incidence of lung carcinoma after surgery for breast carcinoma with and without postoperative radiotherapy. Results of National Surgical Adjuvant Breast and Bowel Project (NSABP) clinical trials B-04 and B-06. Cancer 2003; 98; 1362–1368. 4. Yang M, Nonaka D. A study of immunohistochemical differential expression in pulmonary and mammary carcinomas. Mod. Pathol. 2010; 23; 654–661. 5. Kennecke H, Yerushalmi R, Woods R et al. Metastatic behavior of breast cancer subtypes. J. Clin. Oncol. 2010; 28; 3271– 3277. 6. Sihto H, Lundin J, Lundin M et al. Breast cancer biological subtypes and protein expression predict for the preferential distant © 2015 John Wiley & Sons Ltd, Histopathology, 68, 367–377.
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metastasis sites: a nationwide cohort study. Breast Cancer Res. 2011; 13; R87. Luck AA, Evans AJ, Green AR, Rakha EA, Paish C, Ellis IO. The influence of basal phenotype on the metastatic pattern of breast cancer. Clin. Oncol. 2008; 20; 40–45. Tot T. Cytokeratins 20 and 7 as biomarkers: usefulness in discriminating primary from metastatic adenocarcinoma. Eur. J. Cancer 2002; 38; 758–763. Bishop JA, Sharma R, Illei PB. Napsin A and thyroid transcription factor-1 expression in carcinomas of the lung, breast, pancreas, colon, kidney, thyroid, and malignant mesothelioma. Hum. Pathol. 2010; 41; 20–25. Striebel JM, Dacic S, Yousem SA. Gross cystic disease fluid protein-(GCDFP-15): expression in primary lung adenocarcinoma. Am. J. Surg. Pathol. 2008; 32; 426–432. Warth A, Muley T, Herpel E et al. Large-scale comparative analyses of immunomarkers for diagnostic subtyping of nonsmall-cell lung cancer biopsies. Histopathology 2012; 61; 1017–1025. Klingen TA, Chen Y, Gundersen MD, Aas H, Westre B, Sauer T. Thyroid transcription factor-1 positive primary breast cancer: a case report with review of the literature. Diagn. Pathol. 2010; 5; 37. Sakurai A, Sakai Y, Yatabe Y. Thyroid transcription factor-1 expression in rare cases of mammary ductal carcinoma. Histopathology 2011; 59; 145–148. Ni YB, Tsang JY, Shao MM et al. TTF-1 expression in breast carcinoma: an unusual but real phenomenon. Histopathology 2014; 64; 504–511. Lau SK, Chu PG, Weiss LM. Immunohistochemical expression of estrogen receptor in pulmonary adenocarcinoma. Appl. Immunohistochem. Mol. Morphol. 2006; 14; 83–87. Brunnstrom H, Johansson L, Jirstrom K, Jonsson M, Jonsson P, Planck M. Immunohistochemistry in the differential diagnostics of primary lung cancer: an investigation within the Southern Swedish Lung Cancer Study. Am. J. Clin. Pathol. 2013; 140; 37–46. Tennis M, Singh B, Hjerpe A et al. Pathological confirmation of primary lung cancer following breast cancer. Lung Cancer 2010; 69; 40–45. Lehmann-Che J, Hamy AS, Porcher R et al. Molecular apocrine breast cancers are aggressive estrogen receptor negative tumors overexpressing either HER2 or GCDFP15. Breast Cancer Res. 2013; 15; R37. Mazoujian G, Pinkus GS, Davis S, Haagensen DE Jr. Immunohistochemistry of a gross cystic disease fluid protein (GCDFP15) of the breast. A marker of apocrine epithelium and breast carcinomas with apocrine features. Am. J. Pathol. 1983; 110; 105–112. Takeda Y, Tsuta K, Shibuki Y et al. Analysis of expression patterns of breast cancer-specific markers (mammaglobin and gross cystic disease fluid protein 15) in lung and pleural tumors. Arch. Pathol. Lab. Med. 2008; 132; 239– 243. Rekhtman N, Ang DC, Sima CS, Travis WD, Moreira 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. Whithaus K, Fukuoka J, Prihoda TJ, Jagirdar J. Evaluation of napsin A, cytokeratin 5/6, p63, and thyroid transcription factor 1 in adenocarcinoma versus squamous cell carcinoma of the lung. Arch. Pathol. Lab. Med. 2012; 136; 155–162.
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23. Travis WD, Brambilla E, Burke AP, Marx A, Nicholson AG eds. Pathology and genetics of tumours of the lung, pleura, thymus and heart. 4th ed. Lyon: IARC Press, 2015. 24. Loo PS, Thomas SC, Nicolson MC, Fyfe MN, Kerr KM. Subtyping of undifferentiated non-small cell carcinomas in bronchial biopsy specimens. J. Thorac. Oncol. 2010; 5; 442–447. 25. McCarty KS Jr, Szabo E, Flowers JL et al. Use of a monoclonal anti-estrogen receptor antibody in the immunohistochemical evaluation of human tumors. Cancer Res. 1986; 46; 4244s– 4248s. 26. Harvey JM, Clark GM, Osborne CK, Allred DC. Estrogen receptor status by immunohistochemistry is superior to the ligandbinding assay for predicting response to adjuvant endocrine therapy in breast cancer. J. Clin. Oncol. 1999; 17; 1474–1481. 27. Eisen MB, Spellman PT, Brown PO, Botstein D. Cluster analysis and display of genome-wide expression patterns. Proc. Natl Acad. Sci. USA 1998; 95; 14863–14868. 28. van de Rijn M, Gilks CB. Applications of microarrays to histopathology. Histopathology 2004; 44; 97–108. 29. Makretsov NA, Huntsman DG, Nielsen TO et al. Hierarchical clustering analysis of tissue microarray immunostaining data identifies prognostically significant groups of breast carcinoma. Clin. Cancer Res. 2004; 10; 6143–6151. 30. Liu CL, Prapong W, Natkunam Y et al. Software tools for highthroughput analysis and archiving of immunohistochemistry staining data obtained with tissue microarrays. Am. J. Pathol. 2002; 161; 1557–1565. 31. Quint LE, Park CH, Iannettoni MD. Solitary pulmonary nodules in patients with extrapulmonary neoplasms. Radiology 2000; 217; 257–261. 32. Cahan WG, Shah JP, Castro EB. Benign solitary lung lesions in patients with cancer. Ann. Surg. 1978; 187; 241–244. 33. Andersson M, Jensen MB, Engholm G, Henrik Storm H. Risk of second primary cancer among patients with early operable breast cancer registered or randomised in Danish Breast Cancer Cooperative Group (DBCG) protocols of the 77, 82 and 89 programmes during 1977-2001. Acta Oncol. 2008; 47; 755– 764. 34. Berrington de Gonzalez A, Curtis RE, Gilbert E et al. Second solid cancers after radiotherapy for breast cancer in SEER cancer registries. Br. J. Cancer 2010; 102; 220–226. 35. Cummings MC, Simpson PT, Reid LE et al. Metastatic progression of breast cancer: insights from 50 years of autopsies. J. Pathol. 2014; 232; 23–31. 36. Berman AT, Thukral AD, Hwang WT, Solin LJ, Vapiwala N. Incidence and patterns of distant metastases for patients with early-stage breast cancer after breast conservation treatment. Clin. Breast Cancer 2013; 13; 88–94. 37. Criscitiello C, Andre F, Thompson AM et al. Biopsy confirmation of metastatic sites in breast cancer patients: clinical impact and future perspectives. Breast Cancer Res. 2014; 16; 205. 38. Amir E, Miller N, Geddie W et al. Prospective study evaluating the impact of tissue confirmation of metastatic disease in patients with breast cancer. J. Clin. Oncol. 2012; 30; 587–592. 39. Howlader N, Altekruse SF, Li CI et al. US incidence of breast cancer subtypes defined by joint hormone receptor and HER2 status. J. Natl Cancer Inst. 2014; 106; dju055. 40. Dabbs DJ, Landreneau RJ, Liu Y et al. Detection of estrogen receptor by immunohistochemistry in pulmonary adenocarcinoma. Ann. Thorac. Surg. 2002; 73; 403–405; discussion 406. 41. Lee AH. The histological diagnosis of metastases to the breast from extramammary malignancies. J. Clin. Pathol. 2007; 60; 1333–1341.
42. Huo L, Zhang J, Gilcrease MZ et al. Gross cystic disease fluid protein-15 and mammaglobin A expression determined by immunohistochemistry is of limited utility in triple-negative breast cancer. Histopathology 2013; 62; 267–274. 43. Lewis GH, Subhawong AP, Nassar H et al. Relationship between molecular subtype of invasive breast carcinoma and expression of gross cystic disease fluid protein 15 and mammaglobin. Am. J. Clin. Pathol. 2011; 135; 587–591. 44. Miettinen M, McCue PA, Sarlomo-Rikala M et al. GATA3: a multispecific but potentially useful marker in surgical pathology: a systematic analysis of 2500 epithelial and nonepithelial tumors. Am. J. Surg. Pathol. 2014; 38; 13–22. 45. Cimino-Mathews A, Subhawong AP, Illei PB et al. GATA3 expression in breast carcinoma: utility in triple-negative, sarcomatoid, and metastatic carcinomas. Hum. Pathol. 2013; 44; 1341–1349. 46. Krings G, Nystrom M, Mehdi I, Vohra P, Chen YY. Diagnostic utility and sensitivities of GATA3 antibodies in triple-negative breast cancer. Hum. Pathol. 2014; 45; 2225–2232. 47. Clark BZ, Beriwal S, Dabbs DJ, Bhargava R. Semiquantitative GATA-3 immunoreactivity in breast, bladder, gynecologic tract, and other cytokeratin 7-positive carcinomas. Am. J. Clin. Pathol. 2014; 142; 64–71. 48. Hattori Y, Yoshida A, Yoshida M, Takahashi M, Tsuta K. Evaluation of androgen receptor and GATA binding protein 3 as immunohistochemical markers in the diagnosis of metastatic breast carcinoma to the lung. Pathol. Int. 2015; 65; 286–292. 49. Han JH, Kang Y, Shin HC et al. Mammaglobin expression in lymph nodes is an important marker of metastatic breast carcinoma. Arch. Pathol. Lab. Med. 2003; 127; 1330–1334. 50. O’Brien N, Maguire TM, O’Donovan N et al. Mammaglobin A: a promising marker for breast cancer. Clin. Chem. 2002; 48; 1362–1364. 51. Rakha EA, El-Sayed ME, Green AR, Paish EC, Lee AH, Ellis IO. Breast carcinoma with basal differentiation: a proposal for pathology definition based on basal cytokeratin expression. Histopathology 2007; 50; 434–438. 52. Thike AA, Cheok PY, Jara-Lazaro AR, Tan B, Tan P, Tan PH. Triple-negative breast cancer: clinicopathological characteristics and relationship with basal-like breast cancer. Mod. Pathol. 2010; 23; 123–133. 53. Livasy CA, Karaca G, Nanda R et al. Phenotypic evaluation of the basal-like subtype of invasive breast carcinoma. Mod. Pathol. 2006; 19; 264–271. 54. Hirose M. The role of Wilms’ tumor genes. J. Med. Invest. 1999; 46; 130–140. 55. Scharnhorst V, van der Eb AJ, Jochemsen AG. WT1 proteins: functions in growth and differentiation. Gene 2001; 273; 141– 161. 56. Hwang H, Quenneville L, Yaziji H, Gown AM. Wilms’ tumor gene product: sensitive and contextually specific marker of serous carcinomas of ovarian surface epithelial origin. Appl. Immunohistochem. Mol. Morphol. 2004; 12; 122– 126. 57. Foster MR, Johnson JE, Olson SJ, Allred DC. Immunohistochemical analysis of nuclear versus cytoplasmic staining of WT1 in malignant mesotheliomas and primary pulmonary adenocarcinomas. Arch. Pathol. Lab. Med. 2001; 125; 1316– 1320. 58. Nakatsuka S, Oji Y, Horiuchi T et al. Immunohistochemical detection of WT1 protein in a variety of cancer cells. Mod. Pathol. 2006; 19; 804–814. © 2015 John Wiley & Sons Ltd, Histopathology, 68, 367–377.
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59. Duhig EE, Kalpakos L, Yang IA, Clarke BE. Mesothelial markers in high-grade breast carcinoma. Histopathology 2011; 59; 957–964. 60. Niksic M, Slight J, Sanford JR, Caceres JF, Hastie ND. The Wilms’ tumour protein (WT1) shuttles between nucleus and cytoplasm and is present in functional polysomes. Hum. Mol. Genet. 2004; 13; 463–471. 61. Choi EJ, Yun JA, Jeon EK, Won HS, Ko YH, Kim SY. Prognostic significance of RSPO1, WNT1, p16, WT1, and SDC1 expressions in invasive ductal carcinoma of the breast. World J. Surg. Oncol. 2013; 11; 314. 62. Qi XW, Zhang F, Yang XH et al. High Wilms’ tumor 1 MRNA expression correlates with basal-like and ERBB2 molecular subtypes and poor prognosis of breast cancer. Oncol. Rep. 2012; 28; 1231–1236.
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Supporting Information Additional Supporting Information may be found in the online version of this article: Figure S1. Summary of the scoring systems used. Table S1. Breakdown of the lung and breast cancer samples used.
Differential expression of immunohistochemical markers in primary lung and breast cancers enriched for triple-negative tumours Elena Provenzano,* David J Byrne,1,* Prudence A Russell,2 Gavin M Wright,3 Daniele Generali4 & Stephen B Fox1,5 Department of Histopathology, Addenbrooke’s Hospital, Cambridge, UK, 1Department of Pathology, Peter MacCallum Cancer Centre, The University of Melbourne, Melbourne, Vic., Australia, 2Department of Anatomical Pathology, St Vincent’s Hospital, University of Melbourne, Melbourne, Vic., Australia, 3Department of Surgery, St Vincent’s Hospital, University of Melbourne, Melbourne, Vic., Australia, 4Universita Operativa Multidisciplinare di Patologia Mammaria/ US Terapia Molecolare e Farmacogenomica, dell’Azienda Ospedaliera Istituti Ospitalieri di Cremona, Cremona, Italy, and 5 Department of Pathology, The University of Melbourne, Melbourne, Vic., Australia Date of submission 10 March 2015 Accepted for publication 22 June 2015 Published online Article Accepted 28 June 2015
Provenzano E, Byrne D J, Russell P A, Wright G M, Generali D & Fox S B (2016) Histopathology 68, 367–377. DOI: 10.1111/his.12765
Differential expression of immunohistochemical markers in primary lung and breast cancers enriched for triple-negative tumours Aims: In breast cancer patients presenting with a lung lesion, the distinction between lung and breast origin is clinically important. Lung and breast cancers are both CK7+/CK20 , so additional immunohistochemical markers are needed. Methods and results: We examined the expression of oestrogen receptor (ER), progesterone receptor (PR), thyroid transcription factor-1 (TTF-1), gross cystic disease fluid protein-15 (GCDFP-15), p63 and Wilms’ tumour 1 (WT1) in a series of tissue microarrays comprising 266 non-small-cell lung cancers and 837 primary breast cancers enriched for triple-negative tumours (TNBC). Staining for ER, PR, TTF-1 and GCDFP-15 was present in 63%, 49%, 0% and 25% of breast and 6%, 9%, 59% and 1% of lung cancers,
respectively. Strong staining for p63 was present in 63 (97%) lung squamous cell carcinomas and only eight (9%) TNBC. WT1 nuclear staining was rare; however, cytoplasmic staining was identified in 49 (40%) TNBC and 10 (5%) lung cancers. Cluster analysis segregated TNBC from lung cancers with TTF-1 and/or p63 staining favouring lung origin, and GCDFP-15 or WT1 staining favouring breast origin. Cancers negative for all four markers (17%) were 60% breast and 40% lung origin. Conclusion: An immunohistochemical panel incorporating ER, TTF-1, GCDFP-15, p63 and WT1 can help to distinguish lung cancer from metastatic breast cancer, including TNBC.
Keywords: breast cancer, immunohistochemistry, lung cancer, triple negative breast cancer
Introduction
Address for correspondence: Dr E Provenzano, Box 235, Addenbrooke’s Hospital, Hills Road, Cambridge CB2 0QQ, UK. e-mail: [email protected] *These authors contributed equally to this work. © 2015 John Wiley & Sons Ltd.
Breast and lung cancer are the two leading causes of cancer mortality in women.1 Women with a history of breast cancer have an increased risk of developing a second primary cancer, with the lung being a common site.2–4 Conversely, the lung is also a frequent site for breast cancer metastasis, particularly for
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triple-negative [ER/PR/human epidermal growth factor receptor 2 (HER2)-negative] breast cancer (TNBC).5–7 Hence, when a woman with a history of breast cancer presents with a lung lesion, it is important to distinguish whether this represents metastatic breast cancer or a new lung primary in order to determine appropriate treatment, particularly in the era of targeted therapies now available for both lung and breast cancer. Lung biopsies often provide limited material for histological, immunohistochemical and molecular analyses. The distinction between metastatic and primary lung cancer can be difficult, especially when the tumour is poorly differentiated. Immunohistochemistry can be of help, with commonly used panels including cytokeratin 7 (CK7), cytokeratin 20 (CK20), thyroid transcription factor 1 (TTF-1) and oestrogen receptor (ER). The majority of both lung and breast cancers will be CK7-positive and CK20negative.8 TTF-1, a tissue-specific transcription factor expressed by epithelial cells of lung and thyroid origin, is positive in 68–88% of lung adenocarcinomas, but only 1.5% of squamous cell carcinomas (SQCC) and 32% of sarcomatoid carcinomas.4,9–11 In addition, positivity for TTF-1 has been described in tumours from other primary sites, including the breast.12–14 ER is regarded as a marker for tumours of breast and gynaecological tract origin, although positive staining has been described in tumours from other sites, including the lung.15–17 Other markers of breast origin include gross cystic disease fluid protein-15 (GCDFP-15) and mammaglobin. GCDFP-15 positivity is a feature of apocrine lesions, including the molecular apocrine group of TNBC.18,19 However, GCDFP-15 positivity has been reported in up to 15% of lung cancers.4,10,20 CK5/6 and p63 are expressed by up to 94% of SQCCs of the lung, and are now used for the subclassification of non-small-cell lung cancers.11,21,22 Basal breast cancers are poorly differentiated tumours that are frequently triple-negative and show positive staining for basal cytokeratins, including CK5 and CK14, and can be positive for markers of myoepithelial differentiation such as p63. However, immunohistochemical staining for these markers in basal breast cancers is often focal, and may be absent in small biopsy specimens. We have conducted an immunohistochemical study examining the expression of a selected panel of markers, including TTF-1, ER, PR, GCDFP-15, p63 and Wilms’ tumour 1 (WT1), on tissue microarrays (TMAs), representing a well-characterized series of
primary lung and breast cancers. The aim was to investigate the differential expression of these markers to develop an algorithm to distinguish metastatic breast from primary lung cancers. The series is enriched for TNBC, as identifying metastases from this ER-negative group of cancers is clinically important and diagnostically challenging.
Materials and methods PATIENT MATERIAL
TMAs were constructed using one to four 1-mm cores from 266 non-small-cell lung cancers and 837 primary breast cancers (Table S1). The lung cancers were identified from the archives of St Vincent’s Hospital and the Peter MacCallum Cancer Centre, Melbourne, Australia. All slides from the non-smallcell lung cancers were reviewed centrally by an experienced lung pathologist (P.A.R.), and classified according to the 2015 World Health Organization (WHO) classification of lung tumours.23 The breast cancer TMAs were constructed from 837 non-consecutive primary breast cancers diagnosed between 2007 and 2011 at the Peter MacCallum Cancer Centre, Royal Melbourne Hospital, Focus Pathology, and Dorevitch Pathology in Melbourne, Australia and AO Istituti Ospitalieri di Cremona, Cremona, Italy. Full ethics approval for the use of tumour tissue for TMA construction and ongoing research was obtained in each institution (Peter MacCallum 03/90 and 00/81 covering Focus, Dorevitch Pathology and the Royal Melbourne Hospital; St Vincent’s Hospital, Melbourne A03/12; AO Istituti Ospitalieri di Cremona R-LATN07/11). IMMUNOHISTOCHEMISTRY
Immunohistochemistry for ER, PR, TTF-1, p63, WT1, and GCDFP-15 was performed on 4-lm sections using a Ventana BenchMark Ultra autostainer using Ultra Cell conditioning 1 antigen retrieval solution and the Ultraview diaminobenzidine (DAB) detection system (Ventana Biosystems, Tucson, AZ, USA) (Table 1). Appropriate positive and negative control tissues were included for all antibodies. Nuclear staining was scored for TTF-1, p63, ER and PR, cytoplasmic staining was scored for GCDFP15 and nuclear and cytoplasmic staining were scored separately for WT1. For TTF-1, p63, GCDFP-15 and WT1, the percentage of tumour cells with staining was recorded and average staining intensity was graded as absent (0), weak (1), intermediate (2) or © 2015 John Wiley & Sons Ltd, Histopathology, 68, 367–377.
Immunohistochemistry lung versus breast cancer
Table 1. Antibody and immunohistochemistry protocols used for the centralized staining
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6.00 for Windows (GraphPad Software, La Jolla, CA, USA; www.graphpad.com).
Antigen
Clone
Supplier
Dilution
Amplifier
TTF1
8G7G3/1
Dako
1:100
Results
p63
4A4
Ventana
Pre-dilute
IMMUNOHISTOCHEMISTRY
ER
SP1
Ventana
Pre-dilute
PR
1E2
Ventana
Pre-dilute
WT1
6F-H2
Dako
1:50
GCDFP-15
23A3
Vision Biosystems
1:50
Of 224 lung cancers, 130 (58%) showed high expression of TTF-1, 86% of adenocarcinomas, 50% of large cell carcinomas and only 8% of SQCCs. No breast cancers showed any staining with TTF-1 (Table 2). There were 77 (35%) lung cancers that showed high expression of p63, 97% of SQCCs compared with 7% of adenocarcinomas. There were 21 (23%) TNBC that showed p63 staining, with low expression in 61% of positive cases in contrast to 3% of positive lung cancers (Figure 1). The other breast tissue microarrays were not stained for p63. Of the breast cancers, 323 of 511 (63%) with data available were ER-positive, with 311 showing high expression. The relatively low percentage is due to enrichment for TNBC. Only 12 (5%) lung cancers showed ER positivity, 11 of which were adenocarcinomas, with only three showing high expression. There were 212 (43%) breast cancers with high expression of PR compared to two (1%) lung cancers. GCDFP-15 expression was seen in 87 (25%) breast cancers and two (1%) lung cancers. Low nuclear expression for WT1 was observed in one (0.4%) lung cancer; 18 (3%) breast cancers showed nuclear staining for WT1, with 13 showing high expression, none of which were TNBC (data not shown). A striking pattern of cytoplasmic staining for WT1 was seen in 55 (9%) breast cancers, including 49 (40%) TNBC, while cytoplasmic staining for WT1 was seen in only 10 (5%) lung cancers. TTF-1 was the most specific marker for lung adenocarcinoma, with a mean score of 4.8 of six, p63 for SQCC with a mean score of 5.5 of six and WT1 for TNBC with a mean score of 2 of six, all with P-values < 0.005 (Figure 2). ER staining was associated strongly with breast cancer when it was present, with non-TNBC having a mean score of 5.9 of eight compared with less than 1 in lung adenocarcinoma.
Mouse antibody amp
TTF-1: thyroid transcription factor-1; ER: oestrogen receptor; PR: progestogen receptor; WT1: Wilms’ tumour 1; GCDFP-15: gross cystic disease fluid protein-15.
strong (3) and the staining percentage was divided into four groups, as published previously:24 none = 0, 1– 9% = 1, 10–50% = 2 and > 50% = 3. The intensity and proportion scores were then added together to give a final value of 0–6. A score of 2 was regarded as low expression, and 3–6 was regarded as high expression. Where historical data for ER, PR and HER2 were available results were normalized, including ER and PR weighted H-scores,25 which were converted into a combined Allred score26 (Figure S1). ER and PR staining on TMAs was given a combined Allred score. Based on these data, the breast cancers were divided into non-TNBC (ER/PR and/or HER2-positive), TNBC (ER, PR and HER2-negative) and unknown (ER, PR and/or HER2 data missing). For ER and PR an Allred score of 3 or 4 was regarded as low expression, and 5–8 was regarded as high expression. CLUSTER AND TREE VIEW ANALYSIS
Unsupervised hierarchical cluster analysis was performed to compare lung and TNBC results for TTF-1, p63, GCDFP-15 and WT1 using Cluster and TreeView software, as published previously.27–29 The combined IHC expression scores were converted into microarray format modified from the TMA-Deconvoluter software,30 with a score of 0 = 2 (negative), 2 = 1, 3 = 2, 4 = 3, 5 = 4, 6 = 5, 7 = 6 and 8 = 7. STATISTICAL ANALYSIS
Kruskal–Wallis with Dunn’s multiple comparisons test was performed using GraphPad Prism version © 2015 John Wiley & Sons Ltd, Histopathology, 68, 367–377.
CLUSTER ANALYSIS
A clustering approach was used to try to identify the optimal combination of markers to distinguish TNBC from lung cancers. The resulting clustergram showed seven main nodes (Figure 3). Node 1 is characterized by expression of WT1 (96% TNBC). Node 2 is positive for both WT1 and p63 (82% TNBC). Node 3
4 (50%) 2 (50%)
Carcinoid (n = 8)
Sarcomatoid (n = 6)
8 (100%) 4 (100%)
Carcinoid (n = 8)
Sarcomatoid (n = 6)
202 (94%)
4 (100%)
Adenosquamous (n = 7)
Total (n = 266)
8 (100%)
Large cell (n = 10)
9 (4%)
0
0
0
0
1 (2%)
3 (2%)
0
0
0
0
0
194 (91%)
4 (100%)
7 (88%)
4 (100%)
5 (63%)
61 (92%)
113 (91%)
65 (98%)
3 (3%)
Squamous cell (n = 74)
8 (6%)
113 (91%)
Lung cancers Adenocarcinoma (n = 161)
High
72 (77%)
Neg
Low
0
–
Neg
0
0
72 (77%)
–
137 (63%)
3 (75%)
8 (100%)
0
8 (100%)
2 (3%)
116 (91%)
PR
614 (100%)
Total (n = 837)
0
0
0
130 (58%)
2 (50%)
4 (50%)
0
4 (50%)
5 (8%)
115 (86%)
ER
114 (100%)
Unknown (n = 171)
0
0
3 (1%)
0
0
0
0
2 (2%)
1 (1%)
Marker Score
121 (100%)
379 (100%)
TNBC (n = 130)
Breast cancers Non-TNBC (n = 536)
91 (41%)
5 (100%)
Adenosquamous (n = 7)
Total (n = 266)
4 (50%)
59 (90%)
Squamous cell (n = 74)
Large cell (n = 10)
17 (13%)
Neg
High
Neg
Low
p63
TTF1
Lung cancers Adenocarcinoma (n = 161)
Marker Score
Table 2. Immunohistochemistry results
18 (8%)
0
1 (12%)
0
3 (37%)
5 (8%)
9 (7%)
Low
13 (14%)
–
13 (14%)
–
2 (1%)
0
0
0
0
0
2 (2%)
Low
2 (1%)
0
0
0
0
0
2 (2%)
High
8 (9%)
–
8 (9%)
–
78 (36%)
1 (25%)
0
4 (100%)
0
63 (97%)
10 (7%)
High
222 (99%)
4 (100%)
7 (88%)
4 (100%)
8 (100%)
65 (98%)
134 (100%)
Neg
GCDFP-15
544 (91%)
101 (98%)
72 (60%)
371 (99%)
212 (95%)
3 (75%)
8 (100%)
4 (100%)
7 (88%)
64 (97%)
126 (95%)
Neg
WT1
1 (0.5%)
0
1 (12%)
0
0
0
Low
4 (1%)
0
4 (3%)
0
6 (3%)
0
0
0
0
2 (3%)
4 (3%)
Low
1 (0.5%)
0
0
0
0
1 (2%)
0
High
51 (8%)
2 (2%)
45 (37%)
4 (1%)
4 (2%)
1 (25%)
0
0
1 (12%)
0
2 (2%)
High
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188 (37%) Total (n = 837)
TTF-1: thyroid transcription factor-1; ER: oestrogen receptor; PR: progestogen receptor; WT1: Wilms’ tumour 1; GCDFP-15: gross cystic disease fluid protein-15; TNBC: triple-negative breast cancer.
66 (19%) 21 (6%) 255 (75%) 212 (43%) 30 (6%) 253 (51%) 311 (61%)
– – – – – – Unknown (n = 171)
12 (2%)
15 (23%) 9 (14%) 40 (63%)
17 (15%) 0 94 (85%) 0 4 (3%) 127 (97%) 0 2 (2%) 128 (98%) TNBC (n = 130)
34 (20%) 12 (7%) 121 (73%) 26 (7%) 126 (35%) 311 (82%) 10 (2%) 60 (16%) Breast cancers Non-TNBC (n = 536)
Low Marker Score
Table 2. (Continued)
212 (58%)
Low Neg Neg Neg
High
PR ER
Low
High
GCDFP-15
High
Immunohistochemistry lung versus breast cancer
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represents a small group that only express GCDFP-15 (88% TNBC). Node 4 is negative for all four markers (60% TNBC, 40% lung). Node 5 expresses p63 (84% lung); two of the TNBC in this group coexpress GCDFP15. Nodes 6 and 7 contain lung cancers only; the former express TTF-1 (n = 92) and the latter coexpress TTF-1 and p63 (n = 11) (Table 3). A suggested algorithm for determination of primary lung versus metastatic breast origin is presented in Figure 4.
Discussion Classification of a lung lesion in a patient with a past history of breast cancer often poses a diagnostic challenge. In patients with a solitary lung nodule, the lesion is almost twice as likely to represent a primary lung cancer rather than a breast metastasis.31,32 The risk of developing lung cancer is increased in smokers, and is compounded by breast radiotherapy.2,3,27,33,34 The lung is also a major metastasis site for breast cancer.35,36 Primary lung or breast metastatic origin can sometimes be distinguished on histological grounds by comparison with the previous cancer, or by the presence of typical morphological features of one of the breast or lung cancer subtypes; however, this is highly subjective and can be inaccurate. Knowledge of the histological type, ER and HER2 status of the breast primary can be of additional value, although a not insignificant proportion of breast metastases show different receptor status to the primary.37,38 Triple-negative/basal breast cancers have a particular tendency to metastasize to lung; these are commonly poorly differentiated, high-grade tumours that may show sarcomatoid or squamous differentiation, making distinction from a lung cancer on morphological grounds impossible.5–7 The most specific markers for determining breast or lung origin in this study were ER and TTF-1, respectively. TTF-1 positivity has been reported in 73–88% of primary lung adenocarcinomas, with positive staining in only 0–3% of breast cancers.4,9–15 Coexpression of ER and TTF-1 has been reported in both lung and breast cancer;13–15 however, none of the primary breast cancers in our series showed TTF-1 positivity. In other lung cancer subtypes, TTF-1 positivity is present in 0–1% of SQCCs, 44–60% of large cell carcinomas, 32% of sarcomatoid carcinomas and 40–53% of adenosquamous carcinomas.11,16 We found similar results for large cell carcinoma (50%); however, 11% of SQCCs and none of the adenosquamous carcinomas were TTF-1-positive. Hence, TTF-1 positivity is helpful in diagnosing primary lung adenocarcinoma,
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A
B
Figure 1. p63 staining in A, triple-negative breast cancer (TNBC) showing weak staining in a small proportion of cells and B, lung squamous cell carcinoma with strong diffuse staining present in tumour cells.
8
Total expression score
7
****
****
6 **** 5 4 3
****
2
**** **
0
nTNBC TNBC ADC SQCC nTNBC TNBC SQCC ADC TNBC SQCC ADC nTNBC TNBC SQCC ADC nTNBC TNBC SQCC ADC
1
ER
TTF1 p63 WT1 Immunohistochemistry stain
GCDFP15
Figure 2. Comparison of expression of oestrogen receptor (ER), thyroid transcription factor-1 (TTF-1), p63 and Wilms’ tumour 1 (WT1) in non-triple-negative breast cancer (nTNBC), TNBC, lung squamous cell carcinomas (SQCC) and lung adenocarcinoma (ADC). Mean expression level and 95% confidence intervals indicated.
but is less useful for other types of non-small cell lung carcinoma, particularly SQCC and sarcomatoid carcinoma. The ER positivity rate among breast cancers in this series was only 63%; however, we deliberately included a large number of TNBC, given the propensity for lung metastasis and associated diagnostic challenges. In unselected, population-based series the ER positivity rate is approximately 83%.39 In the literature, the positivity rate for ER in primary lung cancers ranges from 0 to 67%.15 The large range reflects differences in methodology, with higher rates associated with use of the 6F11 antibody.15,40 We used the SP1 antibody, and found that 6% of all lung cancers and 8% of adenocarcinomas showed positive staining for ER. A recent study using the same antibody found that 15% of lung adenocarcinomas were positive for ER using a 1% cut-off.16
GCDFP-15 is regarded as a relatively specific marker of breast origin and is expressed in 40–73% of primary breast cancers, although positive staining is also seen in tumours of skin adnexal, salivary and prostate glands.4,41 We identified positive staining for GCDFP-15 in 25% of breast cancers, which is lower than reported in the literature. In TNBC, positivity rates for GCDFP-15 in primary tumours are 3–14%, similar to the 13% seen in this study, and in only 8% of metastases to lung.42,43 GCDFP-15 in lung cancer has positivity rates of 0–15%4,10,20 compared with 1% in our series. Overall, GCDFP-15 is of limited use in determining the nature of a lung lesion due to its low sensitivity; however, specificity is high when positive staining is present. Alternative markers of breast origin include mammaglobin and GATA binding protein 3 (GATA3), both of which show higher sensitivity but lower specificity for breast origin.43,44 Mammaglobin positivity has been reported in up to 80% of primary breast cancers, including 21–25% of TNBC, but only 0–1.2% of lung cancers.4,20,42,43 GATA3 positivity is seen in 67–95% of breast cancers; 43–73% of TNBCs are positive, including 20–54% of metaplastic cancers, although staining is often focal and of weaker intensity.45–47 In studies looking specifically at breast metastases to lung, up to 87% were positive, 54% of metastatic TNBC, compared with only 0.3–8% of lung adenocarcinomas and 12% of lung SQCCs.45,48 We did not look at mammaglobin staining in this study, as this has been extensively published previously.20,42,49,50 We have investigated GATA3 expression in breast, and found positive staining in 46% of TNBC using the L50-832 clone (manuscript in preparation). We examined some of this series and, interestingly, only six of the 31 TNBC negative for all four markers were GATA3-positive (data not shown). p63 is a nuclear protein expressed in squamous cells and myoepithelial cells at several sites, including breast. Immunohistochemical panels including p63 are being used increasingly to help to differentiate © 2015 John Wiley & Sons Ltd, Histopathology, 68, 367–377.
188 (37%) Total (n = 837)
TTF-1: thyroid transcription factor-1; ER: oestrogen receptor; PR: progestogen receptor; WT1: Wilms’ tumour 1; GCDFP-15: gross cystic disease fluid protein-15; TNBC: triple-negative breast cancer.
66 (19%) 21 (6%) 255 (75%) 212 (43%) 30 (6%) 253 (51%) 311 (61%)
– – – – – – Unknown (n = 171)
12 (2%)
15 (23%) 9 (14%) 40 (63%)
17 (15%) 0 94 (85%) 0 4 (3%) 127 (97%) 0 2 (2%) 128 (98%) TNBC (n = 130)
34 (20%) 12 (7%) 121 (73%) 26 (7%) 126 (35%) 311 (82%) 10 (2%) 60 (16%) Breast cancers Non-TNBC (n = 536)
Low Marker Score
Table 2. (Continued)
212 (58%)
Low Neg Neg Neg
High
PR ER
Low
High
GCDFP-15
High
Immunohistochemistry lung versus breast cancer
© 2015 John Wiley & Sons Ltd, Histopathology, 68, 367–377.
371
represents a small group that only express GCDFP-15 (88% TNBC). Node 4 is negative for all four markers (60% TNBC, 40% lung). Node 5 expresses p63 (84% lung); two of the TNBC in this group coexpress GCDFP15. Nodes 6 and 7 contain lung cancers only; the former express TTF-1 (n = 92) and the latter coexpress TTF-1 and p63 (n = 11) (Table 3). A suggested algorithm for determination of primary lung versus metastatic breast origin is presented in Figure 4.
Discussion Classification of a lung lesion in a patient with a past history of breast cancer often poses a diagnostic challenge. In patients with a solitary lung nodule, the lesion is almost twice as likely to represent a primary lung cancer rather than a breast metastasis.31,32 The risk of developing lung cancer is increased in smokers, and is compounded by breast radiotherapy.2,3,27,33,34 The lung is also a major metastasis site for breast cancer.35,36 Primary lung or breast metastatic origin can sometimes be distinguished on histological grounds by comparison with the previous cancer, or by the presence of typical morphological features of one of the breast or lung cancer subtypes; however, this is highly subjective and can be inaccurate. Knowledge of the histological type, ER and HER2 status of the breast primary can be of additional value, although a not insignificant proportion of breast metastases show different receptor status to the primary.37,38 Triple-negative/basal breast cancers have a particular tendency to metastasize to lung; these are commonly poorly differentiated, high-grade tumours that may show sarcomatoid or squamous differentiation, making distinction from a lung cancer on morphological grounds impossible.5–7 The most specific markers for determining breast or lung origin in this study were ER and TTF-1, respectively. TTF-1 positivity has been reported in 73–88% of primary lung adenocarcinomas, with positive staining in only 0–3% of breast cancers.4,9–15 Coexpression of ER and TTF-1 has been reported in both lung and breast cancer;13–15 however, none of the primary breast cancers in our series showed TTF-1 positivity. In other lung cancer subtypes, TTF-1 positivity is present in 0–1% of SQCCs, 44–60% of large cell carcinomas, 32% of sarcomatoid carcinomas and 40–53% of adenosquamous carcinomas.11,16 We found similar results for large cell carcinoma (50%); however, 11% of SQCCs and none of the adenosquamous carcinomas were TTF-1-positive. Hence, TTF-1 positivity is helpful in diagnosing primary lung adenocarcinoma,
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A
B
Figure 1. p63 staining in A, triple-negative breast cancer (TNBC) showing weak staining in a small proportion of cells and B, lung squamous cell carcinoma with strong diffuse staining present in tumour cells.
8
Total expression score
7
****
****
6 **** 5 4 3
****
2
**** **
0
nTNBC TNBC ADC SQCC nTNBC TNBC SQCC ADC TNBC SQCC ADC nTNBC TNBC SQCC ADC nTNBC TNBC SQCC ADC
1
ER
TTF1 p63 WT1 Immunohistochemistry stain
GCDFP15
Figure 2. Comparison of expression of oestrogen receptor (ER), thyroid transcription factor-1 (TTF-1), p63 and Wilms’ tumour 1 (WT1) in non-triple-negative breast cancer (nTNBC), TNBC, lung squamous cell carcinomas (SQCC) and lung adenocarcinoma (ADC). Mean expression level and 95% confidence intervals indicated.
but is less useful for other types of non-small cell lung carcinoma, particularly SQCC and sarcomatoid carcinoma. The ER positivity rate among breast cancers in this series was only 63%; however, we deliberately included a large number of TNBC, given the propensity for lung metastasis and associated diagnostic challenges. In unselected, population-based series the ER positivity rate is approximately 83%.39 In the literature, the positivity rate for ER in primary lung cancers ranges from 0 to 67%.15 The large range reflects differences in methodology, with higher rates associated with use of the 6F11 antibody.15,40 We used the SP1 antibody, and found that 6% of all lung cancers and 8% of adenocarcinomas showed positive staining for ER. A recent study using the same antibody found that 15% of lung adenocarcinomas were positive for ER using a 1% cut-off.16
GCDFP-15 is regarded as a relatively specific marker of breast origin and is expressed in 40–73% of primary breast cancers, although positive staining is also seen in tumours of skin adnexal, salivary and prostate glands.4,41 We identified positive staining for GCDFP-15 in 25% of breast cancers, which is lower than reported in the literature. In TNBC, positivity rates for GCDFP-15 in primary tumours are 3–14%, similar to the 13% seen in this study, and in only 8% of metastases to lung.42,43 GCDFP-15 in lung cancer has positivity rates of 0–15%4,10,20 compared with 1% in our series. Overall, GCDFP-15 is of limited use in determining the nature of a lung lesion due to its low sensitivity; however, specificity is high when positive staining is present. Alternative markers of breast origin include mammaglobin and GATA binding protein 3 (GATA3), both of which show higher sensitivity but lower specificity for breast origin.43,44 Mammaglobin positivity has been reported in up to 80% of primary breast cancers, including 21–25% of TNBC, but only 0–1.2% of lung cancers.4,20,42,43 GATA3 positivity is seen in 67–95% of breast cancers; 43–73% of TNBCs are positive, including 20–54% of metaplastic cancers, although staining is often focal and of weaker intensity.45–47 In studies looking specifically at breast metastases to lung, up to 87% were positive, 54% of metastatic TNBC, compared with only 0.3–8% of lung adenocarcinomas and 12% of lung SQCCs.45,48 We did not look at mammaglobin staining in this study, as this has been extensively published previously.20,42,49,50 We have investigated GATA3 expression in breast, and found positive staining in 46% of TNBC using the L50-832 clone (manuscript in preparation). We examined some of this series and, interestingly, only six of the 31 TNBC negative for all four markers were GATA3-positive (data not shown). p63 is a nuclear protein expressed in squamous cells and myoepithelial cells at several sites, including breast. Immunohistochemical panels including p63 are being used increasingly to help to differentiate © 2015 John Wiley & Sons Ltd, Histopathology, 68, 367–377.
Immunohistochemistry lung versus breast cancer
68% of TNBC.59 High WT1 mRNA expression levels have been associated with poor prognosis in breast cancer, and show a correlation with ER-negative, basal-like and HER2 molecular subtypes.62,63 In conclusion, the differential diagnosis of primary lung cancer and metastatic breast cancer can be challenging, particularly in the case of poorly differentiated tumours where ER and TTF1 are often negative. The addition of p63, GCDFP-15 and WT1 may be helpful in this setting, with high-level expression of p63 favouring lung origin, while positive staining for GCDFP-15 or cytoplasmic staining for WT1 favours breast origin. Unfortunately, approximately 17% of cases are negative with all five markers, and these cancers have a roughly equal chance of being either breast or lung in origin. Novel markers are still required to determine the true nature of these lesions, which will hopefully emerge from integrated ‘omics’ studies in the future.
7.
8.
9.
10.
11.
12.
13.
Acknowledgements We gratefully acknowledge Associate Professor Ben Solomon, Head of the Molecular Therapeutics and Biomarkers Laboratory, Peter MacCallum Cancer Centre, for allowing us access to the lung cancer TMAs and patient data used in the project.
14.
15.
16.
Conflicts of interest The authors declare no conflicts of interest.
17.
References
18.
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Supporting Information Additional Supporting Information may be found in the online version of this article: Figure S1. Summary of the scoring systems used. Table S1. Breakdown of the lung and breast cancer samples used.
