AJCP / Original Article
VE1 Antibody Immunoreactivity in Normal Anterior Pituitary and Adrenal Cortex Without Detectable BRAF V600E Mutations Daniel A. Mordes, MD, PhD, Kerry Lynch, Sharon Campbell, Dora Dias-Santagata, PhD, Vania Nose, MD, PhD, David N. Louis, MD, and Mai P. Hoang, MD From the Department of Pathology, Massachusetts General Hospital and Harvard Medical School, Boston, MA.
CME/SAM
Key Words: VE1; BRAF V600E; Immunohistochemistry; Pituitary gland; Adrenal gland Am J Clin Pathol June 2014;141:811-815 DOI: 10.1309/AJCP37TLZLTUAOJL
ABSTRACT Objectives: The VE1 monoclonal antibody was developed to recognize the V600E mutation in BRAF, which is found in various tumors. Methods: We report that the VE1 antibody stains normal anterior pituitary gland and adrenal cortex, which lack detectable BRAF V600E mutations. Results: Staining with the VE1 antibody was seen in the adenohypophysis and correlated well with adrenocorticotropic hormone (ACTH)–positive cells. ACTHpositive cells were typically most concentrated in the central mucoid wedge and pars intermedia, and VE1 staining was strong in these regions. Moreover, VE1 staining was seen in ACTH-expressing pituitary adenomas without detectable BRAF mutations. VE1 staining of the adrenal cortex was also significant, with the strongest staining seen in the inner segment of the zona fasciculata. Parathyroid glands, pancreatic islets, or parafollicular C cells in the thyroid showed no VE1 staining. Conclusions: Overall, VE1 staining of endocrine tissues strongly suggests limitations on the use of this antibody for the detection of BRAF mutations.
© American Society for Clinical Pathology
Upon completion of this activity you will be able to: • describe the distribution of reactivity to VE1 monoclonal antibody in endocrine tissues. • correlate the VE1 monoclonal antibody staining of endocrine organs with BRAF V600E mutation. • outline the role of concordant genetic testing when using VE1 monoclonal antibody as a screening tool for BRAF V600E mutation in tumors. The ASCP is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians. The ASCP designates this journal-based CME activity for a maximum of 1 AMA PRA Category 1 Credit ™ per article. Physicians should claim only the credit commensurate with the extent of their participation in the activity. This activity qualifies as an American Board of Pathology Maintenance of Certification Part II Self-Assessment Module. The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. Questions appear on p 896. Exam is located at www.ascp.org/ajcpcme.
The BRAF gene is found to be mutated in various hematologic as well as solid tumors, including hairy cell leukemia,1 pleomorphic xanthoastrocytoma,2,3 malignant melanoma,4 papillary thyroid carcinomas,5 gastrointestinal carcinomas,6,7 and lung adenocarcinomas.8 The mutantspecific BRAF V600E (clone VE1) antibody was developed to recognize the most common activating mutation in BRAF using immunohistochemistry on formalin-fixed paraffinembedded tissue, and was initially validated in melanoma and papillary thyroid carcinoma.9 This antibody was also shown to detect BRAF V600E mutations in colon carcinoma, lung carcinoma, and brain metastases, including metastatic melanoma.2,10-13 We report that the VE1 antibody stains normal anterior pituitary gland and adrenal cortex without detectable BRAF V600E mutations.
811
Am J Clin Pathol 2014;141:811-815 DOI: 10.1309/AJCP37TLZLTUAOJL
811 811
Mordes et al / VE1 Immunoreactivity in Pituitary and Adrenal Glands
Materials and Methods Archival, formalin-fixed, paraffin-embedded materials on five samples each from postmortem pituitary glands, adrenocorticotropic hormone (ACTH)–producing pituitary adenomas, adrenal glands, parathyroid glands, pancreas, and thyroid glands were retrieved from the pathology files of Massachusetts General Hospital, Boston, MA. Immunohistochemistry Immunohistochemical studies were performed on 5-mmthick sections of formalin-fixed, paraffin-embedded tissue in a Bond 3 automated immunostainer (Leica Microsystems, Bannockburn, IL), and primary antibodies against BRAF V600E (clone VE1, 1:100, Spring Bioscience, Pleasanton, CA), ACTH (AH26, 1:200, BioGenex, San Ramon, CA), prolactin (A0569, 1:1000, Dako, Carpinteria, CA), growth hormone (1:70, BioGenex), thyroid-stimulating hormone (5404, 1:5000, BioGenex), luteinizing hormone (3LH5B6YH4, 1:9000, BioGenex), follicle-stimulating hormone (83/12/2A82C7, 1:800, BioGenex), and human chorionic gonadotropin a subunit (823, 1:6500, BioGenex). The sections were deparaffinized on the Leica Bonds using Bond dewax solution. Leica Polymer Refine Kit was used for diaminobenzidine staining. Appropriate positive and negative controls were included. Mutational Analysis The SNaPshot genotyping assay for BRAF V600E and BRAF variant mutations developed by our group was performed on these tissues and consists of multiplexed polymerase chain reaction followed by a single-base extension reaction and uses the commercially available SNaPshot platform (Applied Biosystems, Carlsbad, CA).14
Results Immunohistochemistry Staining with the BRAF V600E VE1 antibody was observed in all pituitary gland samples studied. Staining with the VE1 antibody was seen in the anterior lobe of the pituitary gland but not in the posterior lobe ❚Image 1A❚. Notably, strong VE1 staining of a subset of anterior pituitary cells was seen in a background of variably weak staining in most of the other cells ❚Image 1B❚ and ❚Image 1C❚. To determine which hormone-producing cells strongly reacted with VE1 antibody, a standard pituitary hormonal panel (ACTH, prolactin, growth hormone, thyroid-stimulating hormone, luteinizing hormone, follicle-stimulating hormone, and a subunit) was performed on the postmortem pituitary glands. 812 812
Am J Clin Pathol 2014;141:811-815 DOI: 10.1309/AJCP37TLZLTUAOJL
The intensity and distribution of the VE1 staining correlated well with ACTH-positive cells ❚Image 1D❚, ❚Image 1E❚, and ❚Image 1F❚ in the anterior lobe. Moreover, ACTH-positive cells are typically most concentrated in the central mucoid wedge and pars intermedia, and VE1 staining was strong in these regions. So-called “basophil infiltration”—in which anterior pituitary cells, primarily ACTH-producing cells, “invade” the posterior lobe—is a common age-related finding. In this regard, it is significant that the scattered ACTHpositive cells in the posterior lobe were also strongly stained with the VE1 antibody (Image 1). This pattern of crossreactivity of ACTH-producing cells with the VE1 antibody was observed in all pituitary glands studied and in the ACTHproducing pituitary adenomas. Given that VE1 immunoreactivity was present in the endocrine portion of the pituitary gland, additional endocrine organs were tested with this antibody. Parathyroid glands, pancreatic islets, and parafollicular C cells in the thyroid showed no VE1 staining. On the other hand, VE1 staining of the adrenal cortex, but not of the adrenal medulla, was significant ❚Image 2❚. The strongest VE1 staining was seen in the inner segment of the zona fasciculata and to a lesser degree in the zona reticularis, which borders the medulla. Mutational Analysis Mutational analyses for BRAF V600E, V600M, V600K, L597S, L597V, G466V, and G469V mutations were negative in five pituitary gland, ACTH-producing pituitary adenoma, and adrenal gland samples.
Discussion Mutant-specific antibodies are a powerful tool to gain important diagnostic and prognostic information on specimens, especially when tissue specimens available for genetic studies are limited. The BRAF V600E antibody VE1 has been demonstrated to recognize this common oncogenic mutation in various tumor types. We now report that this antibody also recognizes some normal endocrine tissues. Staining of the anterior, but not posterior, pituitary gland was consistent, with the strongest staining in ACTH-positive cells. This raises the possibilities that the antibody recognizes an epitope that is highly expressed in ACTH-producing cells, and to a lesser degree in other pituitary cells, or also recognizes an additional epitope in ACTH-negative cells. In addition, VE1 immunoreactivity was observed in the adrenal cortex, especially in the zona fasciculata, which contains cortisol-producing, ACTH-responsive cells. This suggests that this antibody may be recognizing an epitope common to cells of the ACTH-cortisol hormonal axis. We observed strong staining of VE1 in the ACTH-producing adenomas associated with Cushing disease. Genotyping for © American Society for Clinical Pathology
AJCP / Original Article
A
D
B
E
C
F
❚Image 1❚ Corresponding cells in anterior pituitary stained with VE1 (BRAF V600E) (A, B, and C) and adrenocorticotropic hormone (D, E, and F) immunostains in three postmortem specimens of pituitary glands. © American Society for Clinical Pathology
813
Am J Clin Pathol 2014;141:811-815 DOI: 10.1309/AJCP37TLZLTUAOJL
813 813
Mordes et al / VE1 Immunoreactivity in Pituitary and Adrenal Glands
❚Image 2❚ VE1 (BRAF V600E) immunostaining of the adrenal cortex while the adrenal medulla is negative.
BRAF mutations, including V600E, were negative in all cases. Our findings are consistent with those of Sperveslage et al,15 who found that the VE1 antibody stained 21% of pituitary adenomas in the absence of the BRAF V600E mutation. In their study, the case with the strongest VE1 staining was an ACTH-expressing adenoma, and they also observed staining in growth hormone–producing and mammosomatotroph adenomas. In addition, BRAF mutations are rare in pituitary adenomas, with only one V600E mutation reported in a nonfunctioning adenoma.2,16 Thus, it is highly unlikely that our observed VE1 immunoreactivity in the pituitary gland reflects an undetectable BRAF mutation. Occasional nonspecific cytoplasmic staining with the VE1 antibody also has been reported in gliomas,17 but we have not recognized the same strong degree of nonspecific VE1 staining in our series of genotyped gliomas.13 Nonspecific nuclear staining without cytoplasmic staining has been reported in the colonic epithelium.18 VE1 immunoreactivity was reported to be absent in the normal ovary, fallopian tube, brain, and thyroid tissue.9,19 In the report originally describing the BRAF V600E VE1 antibody, a Western blot demonstrated that the VE1 antibody detected bands corresponding to mutant BRAF and a suspected splice variant of BRAF in several melanoma cell lines containing the V600E mutation. No bands were detected in the HEK 293T cells, which served as a negative control. However, an additional unspecified band was detected on Western blot in two melanoma cells lines.9 This supports our observation that the VE1 clone may be a recognized epitope other than mutant BRAF, because SNaPshot genotyping for BRAF V600E was negative in all cases. 814 814
Am J Clin Pathol 2014;141:811-815 DOI: 10.1309/AJCP37TLZLTUAOJL
To identify proteins that might contain significant homology with mutant BRAF V600E, protein searches were performed using the Basic Local Alignment Search Tool, which did not reveal any endocrine-related candidate protein. For example, human AlkB homolog 7 shares an identical seven– amino acid region within the 11–amino acid immunogen used to produce the VE1 antibody, but it is a widely expressed nuclear-encoded mitochondrial protein. One possibility is that VE1 antibody may also recognize other mutant forms of BRAF. In our previous study, one metastatic melanoma in the brain with V600K (p.Val600Lys) mutation (confirmed with repeated mutational analyses) was positive for monoclonal VE1.13 This could be because of a protein conformation change that is close enough to be picked up by the antibody. However, genotyping for all BRAF variant mutations (including V600K) was negative in this study for all cases studied. Overall, these findings urge caution when using the VE1 antibody for detecting primary or metastatic tumors in the pituitary and adrenal glands. The use of this antibody as a screening tool for BRAF V600E mutation in tumors in the absence of concordant genetic testing may risk false-positive results. Address reprint requests to Dr Hoang: Dept of Pathology, Massachusetts General Hospital, 55 Fruit St, Warren 820, Boston, MA 02114; [email protected].
References 1. Tiacci E, Trifonov V, Schiavoni G, et al. BRAF mutations in hairy-cell leukemia. N Engl J Med. 2011;364:2305-2315. 2. Schindler G, Capper D, Meyer J, et al. Analysis of BRAF V600E mutation in 1,320 nervous system tumors reveals high mutation frequencies in pleomorphic xanthoastrocytomas, ganglioglioma and extra-cerebellar pilocytic astrocytoma. Acta Neuropathol. 2011;121:397-405. 3. Dias-Santagata D, Lam Q, Vernovsky K, et al. BRAF V600E mutations are common in pleomorphic xanthoastrocytoma: diagnostic and therapeutic implications. PLoS One. 2011;6:e17948. 4. Long GV, Menzies AM, Nagrial AM, et al. Prognostic and clinicopathologic associations of oncogenic BRAF in metastatic melanoma. J Clin Oncol. 2011;29:1239-1246. 5. Tufano RP, Teixeira GV, Bishop J, et al. BRAF mutation in papillary thyroid cancer and its value in tailoring initial treatment: a systematic review and meta-analysis. Medicine. 2012;91:274-286. 6. Tannapfel A, Sommerer F, Benicke M, et al. Mutations of the BRAF gene in cholangiocarcinoma but not in hepatocellular carcinoma. Gut. 2003;52:706-712. 7. Samowitz WS, Sweeney C, Herrick J, et al. Poor survival associated with the BRAF V600E mutation in microsatellitestable colon cancers. Cancer Res. 2005;65:6063-6069. 8. Marchetti A, Felicioni L, Malatesta S, et al. Clinical features and outcome of patients with non–small-cell lung cancer harboring BRAF mutations. J Clin Oncol. 2011;29:35743579. © American Society for Clinical Pathology
AJCP / Original Article
9. Capper D, Preusser M, Habel A, et al. Assessment of BRAF V600E mutation status by immunohistochemistry with a mutation-specific monoclonal antibody. Acta Neuropathol. 2011;122:11-19. 10. Sinicrope FA, Smyrk TC, Tougeron D, et al. Mutationspecific antibody detects mutant BRAF(V600E) protein expression in human colon carcinomas. Cancer. 2013;119:2765-2770. 11. Capper D, Berghoff AS, Magerle M, et al. Immunohistochemical testing of BRAF V600E status in 1,120 tumor tissue samples of patients with brain metastases. Acta Neuropathol. 2012;123:223-233. 12. Long GV, Wilmott JS, Capper D, et al. Immunohistochemistry is highly sensitive and specific for the detection of V600E BRAF mutation in melanoma. Am J Surg Pathol. 2013;37:61-65. 13. Routhier CA, Mochel MC, Lynch K, et al. Comparison of two monoclonal antibodies for immunohistochemical detection of BRAF V600E mutation in malignant melanoma, pulmonary carcinoma, gastrointestinal carcinoma, thyroid carcinoma, and gliomas. Hum Pathol. 2013;44:2563-2570. 14. Dias-Santagata D, Akhavanfard S, David SS, et al. Rapid targeted mutational analysis of human tumors: a clinical platform to guide personalized cancer medicine. EMBO Mol Med. 2010;2:146-158.
© American Society for Clinical Pathology
15. Sperveslage J, Gierke M, Capper D, et al. VE1 immunohistochemistry in pituitary adenomas is not associated with BRAFV600E mutation. Acta Neuropathol. 2013;125:911-912. 16. De Martino I, Fedele M, Palmieri D, et al. B-RAF mutations are a rare event in pituitary adenomas. J Endocrinol Invest. 2007;30:RC1-RC3. 17. Ida CM, Vrana JA, Rodriguez FJ, et al. Immunohistochemistry is highly sensitive and specific for detection of BRAF V600E mutation in pleomorphic xanthoastrocytoma. Acta Neuropathol Commun. 2013;1:20. 18. Mesteri I, Bayer G, Meyer J, et al. Improved molecular classification of serrated lesions of the colon by immunohistochemical detection of BRAF V600E. Mod Pathol. 2013;27:135-144. 19. Bösmüller H, Fischer A, Pham DL, et al. Detection of the BRAF V600E mutation in serous ovarian tumors: a comparative analysis of immunohistochemistry with a mutation-specific monoclonal antibody and allele-specific PCR. Hum Pathol. 2013;44:329-335.
815
Am J Clin Pathol 2014;141:811-815 DOI: 10.1309/AJCP37TLZLTUAOJL
815 815
VE1 Antibody Immunoreactivity in Normal Anterior Pituitary and Adrenal Cortex Without Detectable BRAF V600E Mutations Daniel A. Mordes, MD, PhD, Kerry Lynch, Sharon Campbell, Dora Dias-Santagata, PhD, Vania Nose, MD, PhD, David N. Louis, MD, and Mai P. Hoang, MD From the Department of Pathology, Massachusetts General Hospital and Harvard Medical School, Boston, MA.
CME/SAM
Key Words: VE1; BRAF V600E; Immunohistochemistry; Pituitary gland; Adrenal gland Am J Clin Pathol June 2014;141:811-815 DOI: 10.1309/AJCP37TLZLTUAOJL
ABSTRACT Objectives: The VE1 monoclonal antibody was developed to recognize the V600E mutation in BRAF, which is found in various tumors. Methods: We report that the VE1 antibody stains normal anterior pituitary gland and adrenal cortex, which lack detectable BRAF V600E mutations. Results: Staining with the VE1 antibody was seen in the adenohypophysis and correlated well with adrenocorticotropic hormone (ACTH)–positive cells. ACTHpositive cells were typically most concentrated in the central mucoid wedge and pars intermedia, and VE1 staining was strong in these regions. Moreover, VE1 staining was seen in ACTH-expressing pituitary adenomas without detectable BRAF mutations. VE1 staining of the adrenal cortex was also significant, with the strongest staining seen in the inner segment of the zona fasciculata. Parathyroid glands, pancreatic islets, or parafollicular C cells in the thyroid showed no VE1 staining. Conclusions: Overall, VE1 staining of endocrine tissues strongly suggests limitations on the use of this antibody for the detection of BRAF mutations.
© American Society for Clinical Pathology
Upon completion of this activity you will be able to: • describe the distribution of reactivity to VE1 monoclonal antibody in endocrine tissues. • correlate the VE1 monoclonal antibody staining of endocrine organs with BRAF V600E mutation. • outline the role of concordant genetic testing when using VE1 monoclonal antibody as a screening tool for BRAF V600E mutation in tumors. The ASCP is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians. The ASCP designates this journal-based CME activity for a maximum of 1 AMA PRA Category 1 Credit ™ per article. Physicians should claim only the credit commensurate with the extent of their participation in the activity. This activity qualifies as an American Board of Pathology Maintenance of Certification Part II Self-Assessment Module. The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. Questions appear on p 896. Exam is located at www.ascp.org/ajcpcme.
The BRAF gene is found to be mutated in various hematologic as well as solid tumors, including hairy cell leukemia,1 pleomorphic xanthoastrocytoma,2,3 malignant melanoma,4 papillary thyroid carcinomas,5 gastrointestinal carcinomas,6,7 and lung adenocarcinomas.8 The mutantspecific BRAF V600E (clone VE1) antibody was developed to recognize the most common activating mutation in BRAF using immunohistochemistry on formalin-fixed paraffinembedded tissue, and was initially validated in melanoma and papillary thyroid carcinoma.9 This antibody was also shown to detect BRAF V600E mutations in colon carcinoma, lung carcinoma, and brain metastases, including metastatic melanoma.2,10-13 We report that the VE1 antibody stains normal anterior pituitary gland and adrenal cortex without detectable BRAF V600E mutations.
811
Am J Clin Pathol 2014;141:811-815 DOI: 10.1309/AJCP37TLZLTUAOJL
811 811
Mordes et al / VE1 Immunoreactivity in Pituitary and Adrenal Glands
Materials and Methods Archival, formalin-fixed, paraffin-embedded materials on five samples each from postmortem pituitary glands, adrenocorticotropic hormone (ACTH)–producing pituitary adenomas, adrenal glands, parathyroid glands, pancreas, and thyroid glands were retrieved from the pathology files of Massachusetts General Hospital, Boston, MA. Immunohistochemistry Immunohistochemical studies were performed on 5-mmthick sections of formalin-fixed, paraffin-embedded tissue in a Bond 3 automated immunostainer (Leica Microsystems, Bannockburn, IL), and primary antibodies against BRAF V600E (clone VE1, 1:100, Spring Bioscience, Pleasanton, CA), ACTH (AH26, 1:200, BioGenex, San Ramon, CA), prolactin (A0569, 1:1000, Dako, Carpinteria, CA), growth hormone (1:70, BioGenex), thyroid-stimulating hormone (5404, 1:5000, BioGenex), luteinizing hormone (3LH5B6YH4, 1:9000, BioGenex), follicle-stimulating hormone (83/12/2A82C7, 1:800, BioGenex), and human chorionic gonadotropin a subunit (823, 1:6500, BioGenex). The sections were deparaffinized on the Leica Bonds using Bond dewax solution. Leica Polymer Refine Kit was used for diaminobenzidine staining. Appropriate positive and negative controls were included. Mutational Analysis The SNaPshot genotyping assay for BRAF V600E and BRAF variant mutations developed by our group was performed on these tissues and consists of multiplexed polymerase chain reaction followed by a single-base extension reaction and uses the commercially available SNaPshot platform (Applied Biosystems, Carlsbad, CA).14
Results Immunohistochemistry Staining with the BRAF V600E VE1 antibody was observed in all pituitary gland samples studied. Staining with the VE1 antibody was seen in the anterior lobe of the pituitary gland but not in the posterior lobe ❚Image 1A❚. Notably, strong VE1 staining of a subset of anterior pituitary cells was seen in a background of variably weak staining in most of the other cells ❚Image 1B❚ and ❚Image 1C❚. To determine which hormone-producing cells strongly reacted with VE1 antibody, a standard pituitary hormonal panel (ACTH, prolactin, growth hormone, thyroid-stimulating hormone, luteinizing hormone, follicle-stimulating hormone, and a subunit) was performed on the postmortem pituitary glands. 812 812
Am J Clin Pathol 2014;141:811-815 DOI: 10.1309/AJCP37TLZLTUAOJL
The intensity and distribution of the VE1 staining correlated well with ACTH-positive cells ❚Image 1D❚, ❚Image 1E❚, and ❚Image 1F❚ in the anterior lobe. Moreover, ACTH-positive cells are typically most concentrated in the central mucoid wedge and pars intermedia, and VE1 staining was strong in these regions. So-called “basophil infiltration”—in which anterior pituitary cells, primarily ACTH-producing cells, “invade” the posterior lobe—is a common age-related finding. In this regard, it is significant that the scattered ACTHpositive cells in the posterior lobe were also strongly stained with the VE1 antibody (Image 1). This pattern of crossreactivity of ACTH-producing cells with the VE1 antibody was observed in all pituitary glands studied and in the ACTHproducing pituitary adenomas. Given that VE1 immunoreactivity was present in the endocrine portion of the pituitary gland, additional endocrine organs were tested with this antibody. Parathyroid glands, pancreatic islets, and parafollicular C cells in the thyroid showed no VE1 staining. On the other hand, VE1 staining of the adrenal cortex, but not of the adrenal medulla, was significant ❚Image 2❚. The strongest VE1 staining was seen in the inner segment of the zona fasciculata and to a lesser degree in the zona reticularis, which borders the medulla. Mutational Analysis Mutational analyses for BRAF V600E, V600M, V600K, L597S, L597V, G466V, and G469V mutations were negative in five pituitary gland, ACTH-producing pituitary adenoma, and adrenal gland samples.
Discussion Mutant-specific antibodies are a powerful tool to gain important diagnostic and prognostic information on specimens, especially when tissue specimens available for genetic studies are limited. The BRAF V600E antibody VE1 has been demonstrated to recognize this common oncogenic mutation in various tumor types. We now report that this antibody also recognizes some normal endocrine tissues. Staining of the anterior, but not posterior, pituitary gland was consistent, with the strongest staining in ACTH-positive cells. This raises the possibilities that the antibody recognizes an epitope that is highly expressed in ACTH-producing cells, and to a lesser degree in other pituitary cells, or also recognizes an additional epitope in ACTH-negative cells. In addition, VE1 immunoreactivity was observed in the adrenal cortex, especially in the zona fasciculata, which contains cortisol-producing, ACTH-responsive cells. This suggests that this antibody may be recognizing an epitope common to cells of the ACTH-cortisol hormonal axis. We observed strong staining of VE1 in the ACTH-producing adenomas associated with Cushing disease. Genotyping for © American Society for Clinical Pathology
AJCP / Original Article
A
D
B
E
C
F
❚Image 1❚ Corresponding cells in anterior pituitary stained with VE1 (BRAF V600E) (A, B, and C) and adrenocorticotropic hormone (D, E, and F) immunostains in three postmortem specimens of pituitary glands. © American Society for Clinical Pathology
813
Am J Clin Pathol 2014;141:811-815 DOI: 10.1309/AJCP37TLZLTUAOJL
813 813
Mordes et al / VE1 Immunoreactivity in Pituitary and Adrenal Glands
❚Image 2❚ VE1 (BRAF V600E) immunostaining of the adrenal cortex while the adrenal medulla is negative.
BRAF mutations, including V600E, were negative in all cases. Our findings are consistent with those of Sperveslage et al,15 who found that the VE1 antibody stained 21% of pituitary adenomas in the absence of the BRAF V600E mutation. In their study, the case with the strongest VE1 staining was an ACTH-expressing adenoma, and they also observed staining in growth hormone–producing and mammosomatotroph adenomas. In addition, BRAF mutations are rare in pituitary adenomas, with only one V600E mutation reported in a nonfunctioning adenoma.2,16 Thus, it is highly unlikely that our observed VE1 immunoreactivity in the pituitary gland reflects an undetectable BRAF mutation. Occasional nonspecific cytoplasmic staining with the VE1 antibody also has been reported in gliomas,17 but we have not recognized the same strong degree of nonspecific VE1 staining in our series of genotyped gliomas.13 Nonspecific nuclear staining without cytoplasmic staining has been reported in the colonic epithelium.18 VE1 immunoreactivity was reported to be absent in the normal ovary, fallopian tube, brain, and thyroid tissue.9,19 In the report originally describing the BRAF V600E VE1 antibody, a Western blot demonstrated that the VE1 antibody detected bands corresponding to mutant BRAF and a suspected splice variant of BRAF in several melanoma cell lines containing the V600E mutation. No bands were detected in the HEK 293T cells, which served as a negative control. However, an additional unspecified band was detected on Western blot in two melanoma cells lines.9 This supports our observation that the VE1 clone may be a recognized epitope other than mutant BRAF, because SNaPshot genotyping for BRAF V600E was negative in all cases. 814 814
Am J Clin Pathol 2014;141:811-815 DOI: 10.1309/AJCP37TLZLTUAOJL
To identify proteins that might contain significant homology with mutant BRAF V600E, protein searches were performed using the Basic Local Alignment Search Tool, which did not reveal any endocrine-related candidate protein. For example, human AlkB homolog 7 shares an identical seven– amino acid region within the 11–amino acid immunogen used to produce the VE1 antibody, but it is a widely expressed nuclear-encoded mitochondrial protein. One possibility is that VE1 antibody may also recognize other mutant forms of BRAF. In our previous study, one metastatic melanoma in the brain with V600K (p.Val600Lys) mutation (confirmed with repeated mutational analyses) was positive for monoclonal VE1.13 This could be because of a protein conformation change that is close enough to be picked up by the antibody. However, genotyping for all BRAF variant mutations (including V600K) was negative in this study for all cases studied. Overall, these findings urge caution when using the VE1 antibody for detecting primary or metastatic tumors in the pituitary and adrenal glands. The use of this antibody as a screening tool for BRAF V600E mutation in tumors in the absence of concordant genetic testing may risk false-positive results. Address reprint requests to Dr Hoang: Dept of Pathology, Massachusetts General Hospital, 55 Fruit St, Warren 820, Boston, MA 02114; [email protected].
References 1. Tiacci E, Trifonov V, Schiavoni G, et al. BRAF mutations in hairy-cell leukemia. N Engl J Med. 2011;364:2305-2315. 2. Schindler G, Capper D, Meyer J, et al. Analysis of BRAF V600E mutation in 1,320 nervous system tumors reveals high mutation frequencies in pleomorphic xanthoastrocytomas, ganglioglioma and extra-cerebellar pilocytic astrocytoma. Acta Neuropathol. 2011;121:397-405. 3. Dias-Santagata D, Lam Q, Vernovsky K, et al. BRAF V600E mutations are common in pleomorphic xanthoastrocytoma: diagnostic and therapeutic implications. PLoS One. 2011;6:e17948. 4. Long GV, Menzies AM, Nagrial AM, et al. Prognostic and clinicopathologic associations of oncogenic BRAF in metastatic melanoma. J Clin Oncol. 2011;29:1239-1246. 5. Tufano RP, Teixeira GV, Bishop J, et al. BRAF mutation in papillary thyroid cancer and its value in tailoring initial treatment: a systematic review and meta-analysis. Medicine. 2012;91:274-286. 6. Tannapfel A, Sommerer F, Benicke M, et al. Mutations of the BRAF gene in cholangiocarcinoma but not in hepatocellular carcinoma. Gut. 2003;52:706-712. 7. Samowitz WS, Sweeney C, Herrick J, et al. Poor survival associated with the BRAF V600E mutation in microsatellitestable colon cancers. Cancer Res. 2005;65:6063-6069. 8. Marchetti A, Felicioni L, Malatesta S, et al. Clinical features and outcome of patients with non–small-cell lung cancer harboring BRAF mutations. J Clin Oncol. 2011;29:35743579. © American Society for Clinical Pathology
AJCP / Original Article
9. Capper D, Preusser M, Habel A, et al. Assessment of BRAF V600E mutation status by immunohistochemistry with a mutation-specific monoclonal antibody. Acta Neuropathol. 2011;122:11-19. 10. Sinicrope FA, Smyrk TC, Tougeron D, et al. Mutationspecific antibody detects mutant BRAF(V600E) protein expression in human colon carcinomas. Cancer. 2013;119:2765-2770. 11. Capper D, Berghoff AS, Magerle M, et al. Immunohistochemical testing of BRAF V600E status in 1,120 tumor tissue samples of patients with brain metastases. Acta Neuropathol. 2012;123:223-233. 12. Long GV, Wilmott JS, Capper D, et al. Immunohistochemistry is highly sensitive and specific for the detection of V600E BRAF mutation in melanoma. Am J Surg Pathol. 2013;37:61-65. 13. Routhier CA, Mochel MC, Lynch K, et al. Comparison of two monoclonal antibodies for immunohistochemical detection of BRAF V600E mutation in malignant melanoma, pulmonary carcinoma, gastrointestinal carcinoma, thyroid carcinoma, and gliomas. Hum Pathol. 2013;44:2563-2570. 14. Dias-Santagata D, Akhavanfard S, David SS, et al. Rapid targeted mutational analysis of human tumors: a clinical platform to guide personalized cancer medicine. EMBO Mol Med. 2010;2:146-158.
© American Society for Clinical Pathology
15. Sperveslage J, Gierke M, Capper D, et al. VE1 immunohistochemistry in pituitary adenomas is not associated with BRAFV600E mutation. Acta Neuropathol. 2013;125:911-912. 16. De Martino I, Fedele M, Palmieri D, et al. B-RAF mutations are a rare event in pituitary adenomas. J Endocrinol Invest. 2007;30:RC1-RC3. 17. Ida CM, Vrana JA, Rodriguez FJ, et al. Immunohistochemistry is highly sensitive and specific for detection of BRAF V600E mutation in pleomorphic xanthoastrocytoma. Acta Neuropathol Commun. 2013;1:20. 18. Mesteri I, Bayer G, Meyer J, et al. Improved molecular classification of serrated lesions of the colon by immunohistochemical detection of BRAF V600E. Mod Pathol. 2013;27:135-144. 19. Bösmüller H, Fischer A, Pham DL, et al. Detection of the BRAF V600E mutation in serous ovarian tumors: a comparative analysis of immunohistochemistry with a mutation-specific monoclonal antibody and allele-specific PCR. Hum Pathol. 2013;44:329-335.
815
Am J Clin Pathol 2014;141:811-815 DOI: 10.1309/AJCP37TLZLTUAOJL
815 815
