Detection of BRAF V600E in cancers: Focus on BRAF V600E (VE1) Mouse Monoclonal Primary Antibody...

Detection of BRAF V600E in cancers Focus on BRAF V600E (VE1) Mouse Monoclonal Primary Antibody Scientific Overview

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Introduction

BRAF V600E (VE1) Mouse Monoclonal Primary Antibody

Colorectal cancer

Thyroid cancer

Hairy cell leukemia

Conclusion

4

Introduction

BRAF V600E (VE1) Mouse Monoclobal Primary Antibody

Colorectal cancer

Thyroid cancer

Hairy cell leukemia

Conclusion

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v-RAF murine sarcoma viral oncogene homolog B1 (BRAF)

• Part of the RAS/MAPK signaling pathway

• Drives cell proliferation, survival, differentiation1

BRAF is a serine threonine kinase

1.  Wan et al. Cell, Vol. 116, 855–867, March 19, 2004. Roring M, et al. The EMBO Journal (2012) 31, 2629–2647. Davies H, et al. NATURE |VOL 417 | 27 JUNE 2002

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>30 different BRAF mutations have been described, however, “…the T1799A transversion mutation in BRAF accounts for more than 80% of all known BRAF mutations. The protein product resulting from this mutation is a glutamic acid for valine substitution at codon 600 (V600E)….”2

BRAF V600E is the most common mutant

2. Pakneshan S, et al. Pathology (June 2013) 45(4), pp. 346–356.

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BRAF V600E mutations have been detected in –  Colorectal cancer –  Papillary thyroid carcinoma –  Melanoma –  Hairy cell leukemia –  Primary central nervous system cancers –  Lung cancers

The BRAF V600E mutant is found in many cancers

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Distribution of BRAF mutations by cancer

2 Pakneshan S,, et al. Clinicopathological relevance of BRAF mutations in human cancer. Pathology. 2013 Jun; 45(5): 346-356. 3. Capper D, Preusser M, et al. Assessment of BRAF V600E mutation status by immunohistochemistry with a mutation-specific monoclonal antibody. Acta Neuropathol. 2011 Jul; 4. Sharma S. BRAF Mutation Testing in Colorectal Cancer. Arch Pathol Lab Med. 2010; 134: 1225-1228. 5. Bullock M, O’Neill C, Chou A, et al. Utilization of a MAB for BRAF V600E detection in papillary thyroid carcinoma. Endocrine-Related Cancer. 2012; 19: 779-784.

Cancer Percent (%) with BRAF mutations

Percent (%) that are BRAF V600E mutations

Colorectal 5-152 >904

Papillary thyroid 40-703 >985

Hairy cell leukemia ~1003 ~1003

Melanoma* ~40-603 70-903

*Content should be used only for scientific discussions only. The BRAF IHC should not be promoted in Melanoma

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In addition, BRAF V600E accounts for >90% of activating mutations6

“The protein product resulting from this mutation is a glutamic acid for valine substitution at codon 600 (V600E), leading to hyperactivation of the MAPK pathway. The result of this deregulated downstream signaling is unregulated cell proliferation and survival, factors that contribute to oncogenesis.”4

BRAF V600E mutant is constitutively active

4. Sharma S, et al. Arch Pathol Lab Med. 2010;134:1225–1228

6. Pakneshan S, et al. Pathology (June 2013) 45(4), pp. 346–356.

Colorectal cancer positive for BRAF V600E Detected by VE1 with OptiView DAB IHC detection (20x)

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Introduction


Colorectal cancer

Thyroid cancer

Hairy cell leukemia

Conclusion

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•  Mouse monoclonal antibody3,7

•  Specific for BRAF V600E mutant protein3,7

•  The staining pattern for anti-BRAF V600E (VE1) antibody is cytoplasmic staining of tumor cells.

•  Evaluate for the presence of this mutant with tissue context3,7

•  Easily integrated into the anatomic pathology laboratory

BRAF V600E (VE1) mutation specific antibody

3. Capper D et al. Acta Neuropathol (2011) 122:11–19.

7.Capper D, et al. Oncotarget 2012; 3: 907-908. Capper D, et al. Int J Cancer. 2013 Mar 30. doi: 10.1002/ijc.28183.

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•  High concordance with Sanger sequencing >95%3,7

•  Can detect mutant protein when low mutant levels limits molecular approaches (minimum ~25% of mutant allele) 3,7

•  Does not require microdissection3,7


3. Capper D et al. Acta Neuropathol (2011) 122:11–19.

7. Capper D, et al. Oncotarget 2012; 3: 907-908. Capper D, et al. Int J Cancer. 2013 Mar 30. doi: 10.1002/ijc.28183.

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VE1 IHC compared to Sanger sequencing in MSI-H colorectal cancers demonstrated

Sensitivity 100% Specificity 98.8% 8


8. Capper D, et al. Int J Cancer. 2013 Mar 30. doi: 10.1002/ijc.28183

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Colorectal cancer positive for BRAF V600E. Detected by VE1 with OptiView DAB IHC detection (20x)

Ventana Medical Systems, Inc.

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Thyroid cancer positive for BRAF V600E. Detected by VE1 with OptiView DAB IHC detection (20x)

Ventana Medical Systems, Inc.

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BRAF V600E (VE1) Mouse Monoclonal Primary Antibody Package insert highlights Intended use statement Anti-BRAF V600E (VE1) Mouse Monoclonal Primary Antibody may be used to aid in the identification of the mutant protein, BRAF V600E. The antibody is intended for qualitative staining in sections of formalin-fixed, paraffin-embedded tissue. This product should be interpreted by a qualified pathologist in conjunction with histological examination, relevant clinical information and proper controls. This antibody is intended for in vitro diagnostic (IVD) use.

Summary and explanation The BRAF gene located on chromosome 7q34 encodes a cytoplasmic serine-threonine kinase that acts downstream of the mitogen-activated protein kinase (MAPK) signaling pathway. Oncogenic mutations in BRAF gene, all within the kinase domain, constitutively activate MAPK signaling pathway resulting in increased cell proliferation and apoptosis resistance. The most common of all activating BRAF mutations (T1799A point mutation) results in a substitution of valine (V) to glutamic acid (E) at the position 600 of the amino acid sequence. BRAF V600E mutations were detected in approximately 8% of all solid tumors, including 43% of melanomas, 39% of papillary thyroid carcinomas, 12% of serous ovarian carcinomas, 12% of colorectal adenocarcinomas, 2% of lung cancers and other cancers. Furthermore, the BRAF V600E mutation has been recently described as a molecular marker of hairy cell leukemia.

The anti-BRAF V600 (VE1) antibody is a mouse monoclonal antibody (clone VE1) produced against synthetic peptide representing the BRAF mutated amino acid sequence from amino acid 596 to 606 (GLATEKSRWSG). This mutation-specific antibody exhibits a cytoplasmic staining pattern. This antibody differentiates V600E mutation in the BRAF protein from the wild type BRAF protein and the other BRAF mutated proteins.

Confidential and proprietary to Ventana Medical Systems, Inc. For internal use only. Do not copy. Do not distribute.

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BRAF V600E (VE1) Mouse Monoclonal Primary Antibody Package insert highlights – Specific limitations Specific Limitations “The user must validate individual laboratory optimized results obtained with this reagent. This assay was optimized with OptiView DAB IHC Detection Kit and it is not recommended to be used with ultraView Universal DAB Detection Kit.

The specimen should be fixed within 2 hours after collection for at least 12 hours with 10% neutral buffered formalin. It is not recommended to fix tissues with 95% alcohol, Prefer fixative, Z-5 fixative or alcohol formalin acetic acid (AFA).

Anti-BRAF antibody (VE1) was found to occasionally exhibit cytoplasmic background staining in smooth muscle and nuclear staining in normal colon epithelial cells, enterocytes, Leydig cells of testis, adrenal gland, pituitary gland and some tumor cells; however, such cases should not be considered as positive for the BRAF V600E mutation”

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BRAF V600E (VE1) Mouse Monoclonal Primary Antibody Package insert highlights… cont.

Staining interpretation “The staining pattern for anti-BRAF V600E (VE1) antibody is cytoplasmic staining of tumor cells. Positive cases must show cytoplasmic staining in tumor cells when the anti-BRAF V600E (VE1)…. Nuclear staining in tumor cells is sometimes observed”

Nuclear staining

Nuclear staining

Cytoplasmic staining

Negative colorectal case for BRAF V600E with Nuclear staining

Positive colorectal cancer for BRAF V600E with (cytoplasmic staining)

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Introduction

BRAF V600E (VE1) Mouse Monoclobal Primary Antibody

Colorectal cancer

Thyroid cancer

Hairy cell leukemia

Conclusion

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Third most common cancer globally11

Fourth leading cause of cancer death11

12,13

Colorectal cancer: key facts

11. Haggar F, et al. CLINICS IN COLON AND RECTAL SURGERY/VOLUME 22, NUMBER 4 2009.

12. GLOBOCAN 2008. Cancer Fact Sheet. Colorectal. American Cancer Society. http://www.cancer.org/cancer/colonandrectumcancer/detailedguide/colorectal-cancer-key-statistics.

13. American Cancer Society http://www.cancer.org/cancer/colonandrectumcancer/detailedguide/colorectal-cancer-survival-rates

Geography Age group Incidence number

Incidence rate (per 100,000)

Population

Worldwide All 1,233,711 17.3 7,095,217,980

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15. Aaltonen et al. Clin Cancer Res. 2007: 356-361.

Sporadic vs. Inherited

- Vast majority attributed to sporadic versus inherited15

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8. Capper et al. 2013. Int J cancer

Microsatellite status

-  Most are Microsatellite stable (MSS)

-  A subset demonstrate microsatellite instability-high (MSI-H) 8

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Retrospective analysis at the Cleveland Clinic, N = 475 CRC

BRAF V600E detected by PCR

BRAF mutations in 56 (12%)

BRAF V600E comprised 55 (98%) of detected mutations

BRAF V600E vs. wildtype, p value <0.001

BRAF V600E in 76% of MSI cases

BRAF wildtype in 16% of MSI cases17

BRAF V600E in colorectal cancer, Kalady et al.: Associated with MSI

17. Kalady MF., et al.Dis Colon Rectum 2012; 55: 128–133.

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Successive CRC patients N=262 evaluated by MMR IHC

MMR deficient n =39, 14.9% (36 MLH1, 3 MSH2)

MLH1 deficient n =36 (92%)

BRAF V600E by direct sequencing 32 (89%) 10

BRAF V600E in colorectal cancer, Jensen et al.: Associated with MLH1 deficiency

10. Jensen LH, et al. 2007 The Association of Coloproctology of Great Britain and Ireland. Colorectal Disease, 10, 490–497.

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BRAF V600E (VE1) in colorectal cancer: Desai et al. Sensitivity, Specificity, NPV, PPV19

19. Desai J, et al. ESMO 2012 Poster. Absract No.XXX

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BRAF V600E (VE1) in colorectal cancer: Desai et al. Sensitivity, Specificity, NPV, PPV19


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BRAF V600E (VE1) in colorectal cancer: Desai et al. Sensitivity, Specificity, NPV, PPV


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VE1 demonstrated a concordance of 99% with sequencing (N=91) 8

Detection of BRAF V600E in CRC: VE1 IHC versus Sanger Sequencing

8. Capper 2013. Int J cancer .

METHOD VE1 IHC

Sanger sequencing Positive Negative Total

Positive 11 0 12

Negative 1 79 79

Total 12 80 91

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Detection of BRAF V600E: Comparison of IHC and PCR in CRC resections

9. Sinicrope et al. Cancer Month 00, 2013.

METHOD VE1 IHC

PCR Positive Negative Total

Positive 49 1 50

Negative 0 25 25

Total 49 26 75

VE1 IHC demonstrated a concordance of 99% with PCR (N=75) 9

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Introduction


Colorectal cancer

Papillary thyroid cancer

Hairy cell leukemia

Conclusion

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Worldwide incidence (2008)

212,033 cases

3.1/100,000 ASR

Gender disparity

Female predominance ~73% cases

Males ~27% cases

Papillary thyroid carcinoma

Most common thyroid cancer20,21

Thyroid cancer: key facts

20. Nikiforov 2013 USCAP Presentation.

21. Nikiforov YE, et al. J Clin Endocrinol Metab 94: 2092–2098, 2009. Incidence and Prevalence Database. http://www.tdrdata.com/default.aspx

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Thyroid nodules are common….cancer is rare

Papillary thyroid cancer: key facts

21. Nikiforov YE, et al. J Clin Endocrinol Metab 94: 2092–2098, 2009

“…among surgically removed thyroid nodules only 8% to 56% are found to be malignant.”21

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About 10%-40% of thyroid nodule FNAs are indeterminate

On diagnostic lobectomy for indeterminate FNAs – 75% are benign – 25% cancer

•  These patients will need additional treatment: often total thyroidectomy and radioiodine therapy20,21




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For those with an indeterminate FNA20,21




75% over-treated

25% under-treated

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VE1 IHC versus direct DNA sequencing, N =144

Demonstrated 100% concordance22

BRAF V600E (VE1) in papillary thyroid cancer

22. Koperek O, et al. Am J Surg Pathol 2012;36:844–850)

BRAF V600E Status

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Compared VE1 IHC and Sanger sequencing

N=101 well characterized papillary thyroid carcinomas

96 had molecular data, 10 discordant samples5

BRAF V600E (VE1) in papillary thyroid cancer: Bullock et al.

5. Bullock M. Endocrine-Related Cancer (2012) 19 779–784

BRAF V600E status

IHC n (%)

Sanger Sequencing n (%)

Positive 68 (71) 59 (61)

Negative 28 (29) 37 (39)

Total 96 (100) 96 (100)

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Discordant samples were reevaluated by VE1 IHC, Sanger sequencing and massive parallel sequencing on macrodissected samples.

VE1 IHC initially identified 7 more cases (17%) compared to Sanger sequencing5

BRAF V600E (VE1) in papillary thyroid cancer

5. Bullock M. Endocrine-Related Cancer (2012) 19 779–784

BRAF V600E status

IHC n (%)

Sequencing n (%)

Positive 10 (100) 7 (70)

Negative 0 (0) 3 (30)

Total 10(100) 10(100)

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Introduction

BRAF V600E (VE1) Mouse Monoclonal Antibody

Colorectal cancer

Thyroid cancer

Hairy cell leukemia

Conclusion

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A mature B-cell neoplasm

Unlike most hematological cancers, no recurrent characteristic chromosomal abnormalities have been identified23

Hairy cell leukemia: key facts

23. Andrulis M, et al. J Surg Pathol 2012;36:1796–1800)

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Detected by Sanger sequencing

Hairy cell leukemia BRAF V600E positive in 100% (48/48)

Other peripheral B-cell lymphomas (including HCL variant, SMZL)

BRAF V600E positive in 0% (0/195)24

BRAF V600E in hairy cell leukemia, Tiacci et al.

24. Tiacci e, et al. N Engl J Med 2011;364:2305-15.

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Allele specific PCR in 240 cases including 62 HCL

Hairy cell leukemia 100% (62/62) BRAF V600E positive

Other B cell chronic lymphoproliferative disorders 1 % (2/178) BRAF V600E positive25

BRAF V600E in hairy cell leukemia, Arcaini et al.

25. Arcainina L et al. BLOOD, 5 JANUARY 2012 VOLUME 119, NUMBER 1.

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Introduction

BRAF V600E (VE1) Mouse Monoclonal Antibody

Colorectal cancer

Thyroid cancer

Hairy cell leukemia

Conclusion

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Detection of BRAF V600E in cancers: Focus on BRAF V600E (VE1) Mouse Monoclonal Primary Antibody...

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