- Email: [email protected]
Loss of BRCA1-associated protein 1 (BAP1) expression is rare in non–small cell lung cancer
Loss of BRCA1-associated Protein 1 (BAP1) Expression Is Rare in Non-Small Cell Lung Cancer Daniel Owen MD, Brandon S. Sheffield MD, Diana Ionescu MD, Andrew Churg MD PII: DOI: Reference:
S0046-8177(16)30266-0 doi: 10.1016/j.humpath.2016.10.005 YHUPA 4040
To appear in:
Human Pathology
Received date: Revised date: Accepted date:
29 June 2016 10 October 2016 14 October 2016
Please cite this article as: Owen Daniel, Sheffield Brandon S., Ionescu Diana, Churg Andrew, Loss of BRCA1-associated Protein 1 (BAP1) Expression Is Rare in Non-Small Cell Lung Cancer, Human Pathology (2016), doi: 10.1016/j.humpath.2016.10.005
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1
Revised Oct 9, 2016
T
YHUPA D-16-00437
RI P
Loss of BRCA1-associated Protein 1 (BAP1) Expression Is Rare
SC
in Non-Small Cell Lung Cancer
Daniel Owena, MD; Brandon S. Sheffieldb, MD; Diana Ionescuc, MD; Andrew Churga,
NU
MD a
MA
Department of Pathology, Vancouver General Hospital, Vancouver BC, Canada,
V5Z 1M9 b
ED
Department of Pathology, Abbotsford Regional Hospital and Cancer Center,
Department of Pathology, British Columbia Cancer Agency, Vancouver BC, Canada,
AC
V5Z 4E6
CE
c
PT
Abbotsford BC, Canada V2S 0C2.
Address for correspondence: Dr. Andrew Churg, Dept of Pathology, JPPN1401 Vancouver General Hospital, 910 W 10th Ave, Vancouver, BC V5Z 1M9, Canada. Phone 604 875-4111 x 68185; fax 604 875-4797; email: [email protected]. Other authors’ e-mail addresses: [email protected]; [email protected]; [email protected]
Running Title: Loss of BAP1 is rare in non-small cell lung cancer
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Conflicts of interest: none This research did not receive any specific grant from funding agencies in the public,
AC
CE
PT
ED
MA
NU
SC
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T
commercial, or not-for-profit sectors.
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Abstract BRCA1-associated protein 1 (BAP1) is a tumor suppressor gene involved in regulation
distinction
of
malignant
mesothelioma
from
RI P
T
of the cell cycle, cellular differentiation, repair of DNA damage and apoptosis. In the benign
mesothelial
proliferations,
SC
immunohistochemical loss of BAP1, the protein expressed by the BAP1 gene, has proven highly specific for malignant mesothelioma. However, few studies have
NU
investigated the rate of BAP1 loss in tumors that commonly metastasize to the pleura.
MA
Our objective is to determine the rate of BAP1 loss in non-small cell lung cancer (NSCLC). Immunohistochemistry for BAP1 was performed using tissue microarrays
ED
containing 133 confirmed cases of NSCLC (80 of lung adenocarcinoma and 53 of squamous cell carcinoma). Cases were interpreted as showing BAP1 loss if nuclear
PT
staining was completely absent in all tumor cells and present in stromal and
CE
inflammatory cells that served as internal controls. Cases showing no BAP1 staining in the internal controls were excluded. After exclusion of 32 cases for technical reasons,
AC
only one case of pulmonary adenocarcinoma out 101 cases of NSCLC (69 adenocarcinoma and 32 squamous cell carcinoma, 1.0% of cases) showed BAP1 loss. We conclude that loss of BAP1 expression is a rare event in NSCLC. Therefore, BAP1 is a potentially useful addition to the immunohistochemical markers used to distinguish mesothelioma from pleural metastasis of NSCLC. Keywords: BAP1, mesothelioma, lung adenocarcinoma, non-small cell lung cancer, pleural metastasis, mesothelial hyperplasia
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1. Introduction BRCA1-associated protein 1 (BAP1) is a nuclear ubiquitin hydrolase that suppresses
RI P
T
tumorigenesis through its influence on cellular proliferation, cellular response to DNA damage, regulation of the cell cycle, and apoptosis [1]. A relatively high rate of somatic
SC
and germline mutation in the BAP1 gene has been identified in uveal and cutaneous melanoma, clear cell renal cell carcinoma, intrahepatic cholangiocarcinoma and
NU
malignant mesothelioma [2,3]. Biallelic loss of the BAP1 gene has been shown to
MA
correlate with immunohistochemical loss of BAP1 protein expression (referred to here as BAP1 loss) [4,5]. The exact frequency of BAP1 loss in mesotheliomas is unclear, but
epithelial mesotheliomas [6].
ED
a review of the literature suggests that it is probably in the range of 60 to 70% for
PT
Recent studies have clarified the diagnostic significance of BAP1 loss in select tumor
CE
types. For example, in the distinction of malignant mesothelioma from reactive
AC
proliferations of mesothelium, BAP1 loss has been reported as 100% specific for malignant mesothelioma, since no instance of BAP1 loss in a benign proliferation of mesothelium has been identified to date [6]. Loss of BAP1 can similarly be used to distinguish benign mesothelial proliferation from malignant mesothelioma in pleural fluid cytology specimens [7]. However, since other types of tumors can show loss of BAP1, BAP1 loss is specific for malignant mesothelioma only when identified in cells proven to be of mesothelial origin [6,7]. Non-small cell lung cancer (NSCLC) is a common source of pleural metastasis and of malignant pleural fluid cytology. However, few data on the diagnostic and prognostic
ACCEPTED MANUSCRIPT 5
significance BAP1 expression in NSCLC are available. A study by Fan and colleagues reported that high immunohistochemical expression of BAP1 is associated with
T
decreased risk of lymph node metastases and increased median survival in NSCLC [8].
RI P
The authors also found that, among NSCLC, high expression of BAP1 is more common in squamous cell carcinoma than in adenocarcinoma. However, the study reported no
SC
cases of NSCLC showing immunohistochemical loss of BAP1 expression.
NU
Gene sequencing studies of lung adenocarcinoma have identified alterations of the
MA
BAP1 gene in about 1% of cases [9]. In addition, genetic analyses of families with multiple BAP1-associated cancers have identified individuals with germline BAP1
ED
mutations who developed lung cancer. In one Danish family harboring a germline BAP1 mutation, an individual who developed lung cancer was determined, by co-segregation
PT
analysis, to be an obligate carrier of the BAP1 mutation [10]. In another case from a
CE
different family, an individual harboring a germline BAP1 mutation developed uveal melanoma as well as a lung adenocarcinoma in which biallelic inactivation of the BAP1
AC
gene and corresponding immunohistochemical loss of nuclear expression of BAP1 were demonstrated [11].
Two recent studies screening larger numbers of individuals with NSCLC have identified a small percentage of cases which appear to show BAP1 loss. Jaouen and colleagues reported a 48% rate of BAP1 loss in 26 patients with pleural mesothelioma and a 5% rate of loss in 24 patients with lung adenocarcinoma [12]. However, the largest study on BAP1 loss in NSCLC to date identified one case of lung adenocarcinoma with BAP1 loss out of 257 cases of NSCLC (0.4% rate of BAP1 loss) [13].
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If the rate of BAP1 loss in NSCLC is confirmed to be very low, BAP1 immunohistochemistry may become a useful addition to the immunohistochemical
T
markers used to distinguish mesothelioma from pleural metastasis of NSCLC. To that
RI P
end, we report the rate of BAP1 loss in a cohort of 101 cases of NSCLC from our
NU
SC
institutions.
2. Materials and Methods
MA
This study was approved by the ethics review boards of the participating institutions. Two tissue microarrays (TMAs), one comprising 80 cases of surgically resected lung
ED
adenocarcinoma and another comprising 53 cases of surgically resected lung
PT
squamous cell carcinoma, retrospectively identified from the archives of the British Columbia Cancer Agency and Vancouver General Hospital, were used. The
CE
adenocarcinomas were all positive for TTF-1 and negative for p40. Conversely, the
AC
squamous carcinomas were all positive for p40 and negative for TTF-1. Immunohistochemistry for BAP1 was performed in the pathology laboratory at Vancouver General Hospital under the supervision of A.M.C. After pretreatment using a DAKO AutostainerLink 48, deparaffinized 4-µm-thick sections of the TMA were placed in a steamer for 20 minutes in pH 9 Tris-EDTA buffer. Mouse monocloncal anti-human BAP1 clone C4 (Santa Cruz Biotechnology Inc., Dallas, TX) was used at 1:400 dilution. The Ultravision detection system (Thermo Scientific, Waltham, MA) was used for BAP1 detection.
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BAP1 loss was identified by complete absence of nuclear staining in tumor cells in the presence of positive nuclear staining in non-tumor cells, such as lung stromal and
T
inflammatory cells which served as internal controls. Non-nuclear staining was not
RI P
scored. Cases showing completely absent or very faint nuclear BAP1 staining (not clearly identifiable at 4X magnification) in non-tumor epithelial, mesenchymal, or
NU
SC
inflammatory cells, were excluded.
MA
3. Results
Out of a total of 133 cases, 32 were excluded from the TMAs because of very faint or
ED
completely absent nuclear BAP1 staining both in tumor cells and the non-tumor cells
PT
acting as internal controls, or because of loss of the matching BAP1 stained core from the stained section. Of the 101 remaining cases with positive internal controls (69
CE
adenocarcinomas and 32 squamous cell carcinomas), only one case of lung
AC
adenocarcinoma (1.4% of lung adenocarcinomas and 1.0% of NSCLCs) showed BAP1 loss (figure 1). The remaining 100 NSCLCs, including all squamous cell carcinomas, showed nuclear expression of BAP1 both in the tumor and in the internal controls. The original slides from the case showing BAP1 loss on the TMA were reviewed and immunohistochemistry was repeated on whole sections from a representative tumor block. The tumor was morphologically consistent with lung adenocarcinoma, mostly solid type (figure 2A) and showed strong diffuse nuclear staining for TTF-1 (figure 2B) as well as absent staining for p40. Complete loss of BAP1 expression in all tumor cell
ACCEPTED MANUSCRIPT 8
nuclei and retained BAP1 expression in all nuclei of non-tumor control cells was also demonstrated (figure 2C).
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The lung adenocarcinoma showing loss of BAP1 was from a female with no smoking history and no personal or family history of any other BAP1-associated malignancy.
SC
Genetic analysis of the tumor at the time of diagnosis also showed an eighteen base pair deletion in exon 19 of the epidermal growth factor receptor (EGFR) gene. The
NU
patient’s tumor responded to gefitinib therapy and has been stable for the last two years
MA
with no evidence of metastasis.
ED
4. Discussion
PT
The BAP1 gene is located on the short arm of chromosome 3 (3p21.1), along with
CE
several other tumor suppressor genes that are frequently altered in lung cancer [2,13,14]. Although mutation or loss of the tumor suppressor genes on chromosome 3p
AC
is thought to play an important role in the pathogenesis of lung cancer, the mechanisms involved and the relative importance of these genes have yet to be fully resolved. Our study and the recent work of others [13] confirm that BAP1 loss is very rare NSCLC. We suspect that the 5% rate of BAP1 loss reported by Jaouen and colleagues [12] reflects a small sample size, and that the true rate of loss is probably no greater than 1%. The one patient with adenocarcinoma showing BAP1 loss in our series did not undergo genetic testing to look for a germline mutation. However, her lack of a strong family history of BAP1-associated malignancy suggests that loss of BAP1 in her tumor is probably the result of a somatic mutational event.
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Recent work has shown that BAP1 loss, while relatively common in peritoneal malignant mesothelioma, is present in 0.3% of pancreatic ductal carcinomas [15] and 0.3% of
T
serous ovarian carcinomas [16]. Therefore, BAP1 loss may be helpful in distinguishing
RI P
peritoneal malignant mesothelioma from pancreatic ductal adenocarcinoma and serous
SC
ovarian carcinoma metastatic to peritoneum.
The distinction of pleural malignant mesothelioma from NSCLC, and especially
NU
adenocarcinoma, metastatic to pleura or peritoneum is usually straightforward but can
MA
occasionally be problematic, particularly since mesotheliomas and benign mesothelial cells can sometimes stain for “carcinoma” markers such as CEA, MOC-31 or BerEP4,
ED
and, conversely, lung adenocarcinomas do not always stain for TTF-1 or Napsin. This problem applies not only to adenocarcinomas, but to poorly differentiated squamous cell
PT
carcinomas as well, because squamous cell carcinomas can express markers such as
CE
calretinin and cytokeratin 5/6, which are usually associated with mesotheliomas [17]. While BAP1 staining of mesotheliomas is limited by sensitivity, our results and those of
AC
Andrici et al [13], suggest that, in the context of a differential diagnosis of mesothelioma vs metastatic NSCLC, BAP1 loss would effectively rule out NSCLC.
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References [1] Carbone M, Yang H, Pass HI, Krausz T, Testa JR, Gaudino G. BAP1 and cancer.
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Nat Rev Cancer. 2013 Mar;13(3):153-9.
[2] Murali R, Weisner T, Scolyer RA. Tumors associated with BAP1 mutations.
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Pathology. 2013 Feb;45(2):116–26.
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[3] Andrici J, Goeppert B, Sioson L, et al. Loss of BAP1 expression occurs frequently in
MA
intrahepatic cholangiocarcinoma. Medicine. 2016 Jan;95(2):e2491. [4] Nasu M, Emi M, Pastorino S, et al. High incidence of somatic BAP1 alterations in
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sporadic malignant mesothelioma. J Thorac Oncol. 2015 Apr;10:565–76.
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[5] Koopmans AE, Verdijk RM, Brouwer RWW, et al. Clinical significance of
2014;27:1321–30.
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immunohistochemistry for detection of BAP1 mutations in uveal melanoma. Mod Pathol.
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[6] Churg A, Sheffield BS, Galateau-Salle F. New Markers for Separating Benign From Malignant Mesothelial Proliferations: Are We There Yet? Arch Pathol Lab Med. 2016 Apr;140(4):318-21.
[7] Hwang HC, Sheffield BS, Rodriguez S, Thompson K, Tse CH, Gown AM, Churg A. Utility of BAP1 immunohistochemistry and p16 (CDKN2A) FISH in the diagnosis of malignant mesothelioma in effusion cytology specimens. Am J Surg Pathol. 2016 Jan;40:120–6. [8] Fan L, Tang L, Yue L, Yang Y, Gao Z, Shen Z. BAP1 is a good prognostic factor in advanced non-small cell lung cancer. Clin Invest Med. 2012 Aug;35(3): E182-9.
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[9] Cancer Genome Atlas Research Network. Comprehensive molecular profiling of lung adenocarcinoma. Nature. 2014 Jul;511(7511):543-50.
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[10] Aoude LG, Wadt K, Bojesen A, et al. A BAP1 mutation in a Danish family predisposes to uveal melanoma and other cancers. PLoS One. 2013 Aug;8(8):e72144.
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[11] Abdel-Rahman MH, Pilarski R, Cebulla CM, et al. Germline BAP1 mutation
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predisposes to uveal melanoma, lung adenocarcinoma, meningioma, and other cancers. J Med Genet. 2011 Dec;48(12):856-9.
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[12] Jaouen A, Thivolet-Bejui F, Chalabreysse L, et al. Apport de l’expression protéique de BRCA1 associated protein 1 (BAP1) dans le diagnostic des mésothéliomes malins
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diffus pleuraux : une analyse cytologique et histologique comparative sur une série de
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50 patients. Ann Pathol. 2016 Apr;36(2):111-9. French.
CE
[13] Andrici J, Parkhill TR, Jung J, et al. Loss of expression of BAP1 is very rare in non-
AC
small cell lung carcinoma. Pathology. 2016 Jun;48(4):336-40. [14] Zabarovsky ER, Lerman MI, Minna JD. Tumor suppressor genes on chromosome 3p involved in the pathogenesis of lung and other cancers. Oncogene. 2002 Oct 7;21(45):6915-35. [15] Tayao M, Andrici J, Farzin M, et al. Loss of BAP1 Expression Is Very Rare in Pancreatic Ductal Adenocarcinoma. PLoS One. 2016 Mar;11(3):e0150338. [16] Andrici J, Jung J, Sheen A, et al. Loss of BAP1 expression is very rare in peritoneal and gynecologic serous adenocarcinomas and can be useful in the differential diagnosis with abdominal mesothelioma. Hum Pathol. 2016 May;51:9-15.
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[17] Kushitani K, Amatya VJ, Okada Y, Katayama Y, Mawas AS, Miyata Y, Okada M, Inai K, Kishimoto T, Takeshima Y. Utility and pitfall of Immunohistochemistry in the
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differential diagnosis between epithelioid mesothelioma and poorly differentiated lung
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squamous cell carcinoma. Histopathology. 2016 Sep 2. doi: 10.1111/his.13073. [Epub
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CE
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ED
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ahead of print] PubMed PMID: 27589012.
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Figure Legends 1. A) H&E and B) corresponding BAP1 stained sections of the TMA showing the one
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adenocarcinoma with loss of BAP1 staining (top image in 1B). Note the positive internal control staining. The bottom core in 1B shows an adenocarcinoma with positive BAP1
SC
staining.
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2. Images of the adenocarcinoma with loss of BAP1 staining taken from the original resection specimen. A) appearance of the tumor on H&E. B) Stain for TTF-1 is strongly
MA
and diffusely positive. C) Stain for BAP1 is uniformly negative with positive internal
AC
CE
PT
ED
control.
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14
Figure 1
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15
Figure 2
ACCEPTED MANUSCRIPT 16 Highlights
BAP1 loss is common in epithelial mesotheliomas
We report here that only 1% of nonsmall cell lung cancers (NSCLC) show BAP1 loss
Loss of BAP1 staining supports a diagnosis of mesothelioma vs metastatic NSCLC
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T
S0046-8177(16)30266-0 doi: 10.1016/j.humpath.2016.10.005 YHUPA 4040
To appear in:
Human Pathology
Received date: Revised date: Accepted date:
29 June 2016 10 October 2016 14 October 2016
Please cite this article as: Owen Daniel, Sheffield Brandon S., Ionescu Diana, Churg Andrew, Loss of BRCA1-associated Protein 1 (BAP1) Expression Is Rare in Non-Small Cell Lung Cancer, Human Pathology (2016), doi: 10.1016/j.humpath.2016.10.005
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1
Revised Oct 9, 2016
T
YHUPA D-16-00437
RI P
Loss of BRCA1-associated Protein 1 (BAP1) Expression Is Rare
SC
in Non-Small Cell Lung Cancer
Daniel Owena, MD; Brandon S. Sheffieldb, MD; Diana Ionescuc, MD; Andrew Churga,
NU
MD a
MA
Department of Pathology, Vancouver General Hospital, Vancouver BC, Canada,
V5Z 1M9 b
ED
Department of Pathology, Abbotsford Regional Hospital and Cancer Center,
Department of Pathology, British Columbia Cancer Agency, Vancouver BC, Canada,
AC
V5Z 4E6
CE
c
PT
Abbotsford BC, Canada V2S 0C2.
Address for correspondence: Dr. Andrew Churg, Dept of Pathology, JPPN1401 Vancouver General Hospital, 910 W 10th Ave, Vancouver, BC V5Z 1M9, Canada. Phone 604 875-4111 x 68185; fax 604 875-4797; email: [email protected]. Other authors’ e-mail addresses: [email protected]; [email protected]; [email protected]
Running Title: Loss of BAP1 is rare in non-small cell lung cancer
ACCEPTED MANUSCRIPT 2
Conflicts of interest: none This research did not receive any specific grant from funding agencies in the public,
AC
CE
PT
ED
MA
NU
SC
RI P
T
commercial, or not-for-profit sectors.
ACCEPTED MANUSCRIPT 3
Abstract BRCA1-associated protein 1 (BAP1) is a tumor suppressor gene involved in regulation
distinction
of
malignant
mesothelioma
from
RI P
T
of the cell cycle, cellular differentiation, repair of DNA damage and apoptosis. In the benign
mesothelial
proliferations,
SC
immunohistochemical loss of BAP1, the protein expressed by the BAP1 gene, has proven highly specific for malignant mesothelioma. However, few studies have
NU
investigated the rate of BAP1 loss in tumors that commonly metastasize to the pleura.
MA
Our objective is to determine the rate of BAP1 loss in non-small cell lung cancer (NSCLC). Immunohistochemistry for BAP1 was performed using tissue microarrays
ED
containing 133 confirmed cases of NSCLC (80 of lung adenocarcinoma and 53 of squamous cell carcinoma). Cases were interpreted as showing BAP1 loss if nuclear
PT
staining was completely absent in all tumor cells and present in stromal and
CE
inflammatory cells that served as internal controls. Cases showing no BAP1 staining in the internal controls were excluded. After exclusion of 32 cases for technical reasons,
AC
only one case of pulmonary adenocarcinoma out 101 cases of NSCLC (69 adenocarcinoma and 32 squamous cell carcinoma, 1.0% of cases) showed BAP1 loss. We conclude that loss of BAP1 expression is a rare event in NSCLC. Therefore, BAP1 is a potentially useful addition to the immunohistochemical markers used to distinguish mesothelioma from pleural metastasis of NSCLC. Keywords: BAP1, mesothelioma, lung adenocarcinoma, non-small cell lung cancer, pleural metastasis, mesothelial hyperplasia
ACCEPTED MANUSCRIPT 4
1. Introduction BRCA1-associated protein 1 (BAP1) is a nuclear ubiquitin hydrolase that suppresses
RI P
T
tumorigenesis through its influence on cellular proliferation, cellular response to DNA damage, regulation of the cell cycle, and apoptosis [1]. A relatively high rate of somatic
SC
and germline mutation in the BAP1 gene has been identified in uveal and cutaneous melanoma, clear cell renal cell carcinoma, intrahepatic cholangiocarcinoma and
NU
malignant mesothelioma [2,3]. Biallelic loss of the BAP1 gene has been shown to
MA
correlate with immunohistochemical loss of BAP1 protein expression (referred to here as BAP1 loss) [4,5]. The exact frequency of BAP1 loss in mesotheliomas is unclear, but
epithelial mesotheliomas [6].
ED
a review of the literature suggests that it is probably in the range of 60 to 70% for
PT
Recent studies have clarified the diagnostic significance of BAP1 loss in select tumor
CE
types. For example, in the distinction of malignant mesothelioma from reactive
AC
proliferations of mesothelium, BAP1 loss has been reported as 100% specific for malignant mesothelioma, since no instance of BAP1 loss in a benign proliferation of mesothelium has been identified to date [6]. Loss of BAP1 can similarly be used to distinguish benign mesothelial proliferation from malignant mesothelioma in pleural fluid cytology specimens [7]. However, since other types of tumors can show loss of BAP1, BAP1 loss is specific for malignant mesothelioma only when identified in cells proven to be of mesothelial origin [6,7]. Non-small cell lung cancer (NSCLC) is a common source of pleural metastasis and of malignant pleural fluid cytology. However, few data on the diagnostic and prognostic
ACCEPTED MANUSCRIPT 5
significance BAP1 expression in NSCLC are available. A study by Fan and colleagues reported that high immunohistochemical expression of BAP1 is associated with
T
decreased risk of lymph node metastases and increased median survival in NSCLC [8].
RI P
The authors also found that, among NSCLC, high expression of BAP1 is more common in squamous cell carcinoma than in adenocarcinoma. However, the study reported no
SC
cases of NSCLC showing immunohistochemical loss of BAP1 expression.
NU
Gene sequencing studies of lung adenocarcinoma have identified alterations of the
MA
BAP1 gene in about 1% of cases [9]. In addition, genetic analyses of families with multiple BAP1-associated cancers have identified individuals with germline BAP1
ED
mutations who developed lung cancer. In one Danish family harboring a germline BAP1 mutation, an individual who developed lung cancer was determined, by co-segregation
PT
analysis, to be an obligate carrier of the BAP1 mutation [10]. In another case from a
CE
different family, an individual harboring a germline BAP1 mutation developed uveal melanoma as well as a lung adenocarcinoma in which biallelic inactivation of the BAP1
AC
gene and corresponding immunohistochemical loss of nuclear expression of BAP1 were demonstrated [11].
Two recent studies screening larger numbers of individuals with NSCLC have identified a small percentage of cases which appear to show BAP1 loss. Jaouen and colleagues reported a 48% rate of BAP1 loss in 26 patients with pleural mesothelioma and a 5% rate of loss in 24 patients with lung adenocarcinoma [12]. However, the largest study on BAP1 loss in NSCLC to date identified one case of lung adenocarcinoma with BAP1 loss out of 257 cases of NSCLC (0.4% rate of BAP1 loss) [13].
ACCEPTED MANUSCRIPT 6
If the rate of BAP1 loss in NSCLC is confirmed to be very low, BAP1 immunohistochemistry may become a useful addition to the immunohistochemical
T
markers used to distinguish mesothelioma from pleural metastasis of NSCLC. To that
RI P
end, we report the rate of BAP1 loss in a cohort of 101 cases of NSCLC from our
NU
SC
institutions.
2. Materials and Methods
MA
This study was approved by the ethics review boards of the participating institutions. Two tissue microarrays (TMAs), one comprising 80 cases of surgically resected lung
ED
adenocarcinoma and another comprising 53 cases of surgically resected lung
PT
squamous cell carcinoma, retrospectively identified from the archives of the British Columbia Cancer Agency and Vancouver General Hospital, were used. The
CE
adenocarcinomas were all positive for TTF-1 and negative for p40. Conversely, the
AC
squamous carcinomas were all positive for p40 and negative for TTF-1. Immunohistochemistry for BAP1 was performed in the pathology laboratory at Vancouver General Hospital under the supervision of A.M.C. After pretreatment using a DAKO AutostainerLink 48, deparaffinized 4-µm-thick sections of the TMA were placed in a steamer for 20 minutes in pH 9 Tris-EDTA buffer. Mouse monocloncal anti-human BAP1 clone C4 (Santa Cruz Biotechnology Inc., Dallas, TX) was used at 1:400 dilution. The Ultravision detection system (Thermo Scientific, Waltham, MA) was used for BAP1 detection.
ACCEPTED MANUSCRIPT 7
BAP1 loss was identified by complete absence of nuclear staining in tumor cells in the presence of positive nuclear staining in non-tumor cells, such as lung stromal and
T
inflammatory cells which served as internal controls. Non-nuclear staining was not
RI P
scored. Cases showing completely absent or very faint nuclear BAP1 staining (not clearly identifiable at 4X magnification) in non-tumor epithelial, mesenchymal, or
NU
SC
inflammatory cells, were excluded.
MA
3. Results
Out of a total of 133 cases, 32 were excluded from the TMAs because of very faint or
ED
completely absent nuclear BAP1 staining both in tumor cells and the non-tumor cells
PT
acting as internal controls, or because of loss of the matching BAP1 stained core from the stained section. Of the 101 remaining cases with positive internal controls (69
CE
adenocarcinomas and 32 squamous cell carcinomas), only one case of lung
AC
adenocarcinoma (1.4% of lung adenocarcinomas and 1.0% of NSCLCs) showed BAP1 loss (figure 1). The remaining 100 NSCLCs, including all squamous cell carcinomas, showed nuclear expression of BAP1 both in the tumor and in the internal controls. The original slides from the case showing BAP1 loss on the TMA were reviewed and immunohistochemistry was repeated on whole sections from a representative tumor block. The tumor was morphologically consistent with lung adenocarcinoma, mostly solid type (figure 2A) and showed strong diffuse nuclear staining for TTF-1 (figure 2B) as well as absent staining for p40. Complete loss of BAP1 expression in all tumor cell
ACCEPTED MANUSCRIPT 8
nuclei and retained BAP1 expression in all nuclei of non-tumor control cells was also demonstrated (figure 2C).
RI P
T
The lung adenocarcinoma showing loss of BAP1 was from a female with no smoking history and no personal or family history of any other BAP1-associated malignancy.
SC
Genetic analysis of the tumor at the time of diagnosis also showed an eighteen base pair deletion in exon 19 of the epidermal growth factor receptor (EGFR) gene. The
NU
patient’s tumor responded to gefitinib therapy and has been stable for the last two years
MA
with no evidence of metastasis.
ED
4. Discussion
PT
The BAP1 gene is located on the short arm of chromosome 3 (3p21.1), along with
CE
several other tumor suppressor genes that are frequently altered in lung cancer [2,13,14]. Although mutation or loss of the tumor suppressor genes on chromosome 3p
AC
is thought to play an important role in the pathogenesis of lung cancer, the mechanisms involved and the relative importance of these genes have yet to be fully resolved. Our study and the recent work of others [13] confirm that BAP1 loss is very rare NSCLC. We suspect that the 5% rate of BAP1 loss reported by Jaouen and colleagues [12] reflects a small sample size, and that the true rate of loss is probably no greater than 1%. The one patient with adenocarcinoma showing BAP1 loss in our series did not undergo genetic testing to look for a germline mutation. However, her lack of a strong family history of BAP1-associated malignancy suggests that loss of BAP1 in her tumor is probably the result of a somatic mutational event.
ACCEPTED MANUSCRIPT 9
Recent work has shown that BAP1 loss, while relatively common in peritoneal malignant mesothelioma, is present in 0.3% of pancreatic ductal carcinomas [15] and 0.3% of
T
serous ovarian carcinomas [16]. Therefore, BAP1 loss may be helpful in distinguishing
RI P
peritoneal malignant mesothelioma from pancreatic ductal adenocarcinoma and serous
SC
ovarian carcinoma metastatic to peritoneum.
The distinction of pleural malignant mesothelioma from NSCLC, and especially
NU
adenocarcinoma, metastatic to pleura or peritoneum is usually straightforward but can
MA
occasionally be problematic, particularly since mesotheliomas and benign mesothelial cells can sometimes stain for “carcinoma” markers such as CEA, MOC-31 or BerEP4,
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and, conversely, lung adenocarcinomas do not always stain for TTF-1 or Napsin. This problem applies not only to adenocarcinomas, but to poorly differentiated squamous cell
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carcinomas as well, because squamous cell carcinomas can express markers such as
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calretinin and cytokeratin 5/6, which are usually associated with mesotheliomas [17]. While BAP1 staining of mesotheliomas is limited by sensitivity, our results and those of
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Andrici et al [13], suggest that, in the context of a differential diagnosis of mesothelioma vs metastatic NSCLC, BAP1 loss would effectively rule out NSCLC.
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Pathology. 2013 Feb;45(2):116–26.
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[3] Andrici J, Goeppert B, Sioson L, et al. Loss of BAP1 expression occurs frequently in
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intrahepatic cholangiocarcinoma. Medicine. 2016 Jan;95(2):e2491. [4] Nasu M, Emi M, Pastorino S, et al. High incidence of somatic BAP1 alterations in
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sporadic malignant mesothelioma. J Thorac Oncol. 2015 Apr;10:565–76.
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[5] Koopmans AE, Verdijk RM, Brouwer RWW, et al. Clinical significance of
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immunohistochemistry for detection of BAP1 mutations in uveal melanoma. Mod Pathol.
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[6] Churg A, Sheffield BS, Galateau-Salle F. New Markers for Separating Benign From Malignant Mesothelial Proliferations: Are We There Yet? Arch Pathol Lab Med. 2016 Apr;140(4):318-21.
[7] Hwang HC, Sheffield BS, Rodriguez S, Thompson K, Tse CH, Gown AM, Churg A. Utility of BAP1 immunohistochemistry and p16 (CDKN2A) FISH in the diagnosis of malignant mesothelioma in effusion cytology specimens. Am J Surg Pathol. 2016 Jan;40:120–6. [8] Fan L, Tang L, Yue L, Yang Y, Gao Z, Shen Z. BAP1 is a good prognostic factor in advanced non-small cell lung cancer. Clin Invest Med. 2012 Aug;35(3): E182-9.
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[9] Cancer Genome Atlas Research Network. Comprehensive molecular profiling of lung adenocarcinoma. Nature. 2014 Jul;511(7511):543-50.
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[10] Aoude LG, Wadt K, Bojesen A, et al. A BAP1 mutation in a Danish family predisposes to uveal melanoma and other cancers. PLoS One. 2013 Aug;8(8):e72144.
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[11] Abdel-Rahman MH, Pilarski R, Cebulla CM, et al. Germline BAP1 mutation
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predisposes to uveal melanoma, lung adenocarcinoma, meningioma, and other cancers. J Med Genet. 2011 Dec;48(12):856-9.
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[12] Jaouen A, Thivolet-Bejui F, Chalabreysse L, et al. Apport de l’expression protéique de BRCA1 associated protein 1 (BAP1) dans le diagnostic des mésothéliomes malins
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diffus pleuraux : une analyse cytologique et histologique comparative sur une série de
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50 patients. Ann Pathol. 2016 Apr;36(2):111-9. French.
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[13] Andrici J, Parkhill TR, Jung J, et al. Loss of expression of BAP1 is very rare in non-
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small cell lung carcinoma. Pathology. 2016 Jun;48(4):336-40. [14] Zabarovsky ER, Lerman MI, Minna JD. Tumor suppressor genes on chromosome 3p involved in the pathogenesis of lung and other cancers. Oncogene. 2002 Oct 7;21(45):6915-35. [15] Tayao M, Andrici J, Farzin M, et al. Loss of BAP1 Expression Is Very Rare in Pancreatic Ductal Adenocarcinoma. PLoS One. 2016 Mar;11(3):e0150338. [16] Andrici J, Jung J, Sheen A, et al. Loss of BAP1 expression is very rare in peritoneal and gynecologic serous adenocarcinomas and can be useful in the differential diagnosis with abdominal mesothelioma. Hum Pathol. 2016 May;51:9-15.
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[17] Kushitani K, Amatya VJ, Okada Y, Katayama Y, Mawas AS, Miyata Y, Okada M, Inai K, Kishimoto T, Takeshima Y. Utility and pitfall of Immunohistochemistry in the
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differential diagnosis between epithelioid mesothelioma and poorly differentiated lung
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squamous cell carcinoma. Histopathology. 2016 Sep 2. doi: 10.1111/his.13073. [Epub
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ahead of print] PubMed PMID: 27589012.
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Figure Legends 1. A) H&E and B) corresponding BAP1 stained sections of the TMA showing the one
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adenocarcinoma with loss of BAP1 staining (top image in 1B). Note the positive internal control staining. The bottom core in 1B shows an adenocarcinoma with positive BAP1
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staining.
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2. Images of the adenocarcinoma with loss of BAP1 staining taken from the original resection specimen. A) appearance of the tumor on H&E. B) Stain for TTF-1 is strongly
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and diffusely positive. C) Stain for BAP1 is uniformly negative with positive internal
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control.
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Figure 1
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Figure 2
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BAP1 loss is common in epithelial mesotheliomas
We report here that only 1% of nonsmall cell lung cancers (NSCLC) show BAP1 loss
Loss of BAP1 staining supports a diagnosis of mesothelioma vs metastatic NSCLC
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