Mutation-Specifi c Antibodies for the Detection of EGFR … · 2017. 5. 16. ·...
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Mutation-Specific Antibodies for the Detection of EGFR Mutations in Non-Small-Cell Lung Cancer Summary Activating mutations within the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) are found in approximately 10-20% of non-small cell lung cancer (NSCLC) patients and are associated with improved response to the EGFR inhibitors Gefitinib and Erlotinib. The most common NSCLC-associated EGFR mu- tations are the exon 19 deletion (E746-A750del) and the exon 21 point mutation (L858R). Together these mutations account for 90% of EGFR mutations. Here, we generate mutation-specific rabbit monoclonal antibodies against each of these EGFR mutations. The mutation-specific antibodies detect the corresponding mutant form of EGFR but not wild type EGFR by Western blotting, immunofluorescence (IF), and immunohistochemistry (IHC). IHC screening of a large panel of paraffin-embedded NSCLC tumor samples demonstrates these antibodies are highly sensitive and spe- cific. This IHC assay provides a rapid, sensitive, specific and cost-effective method to identify lung cancer patients likely to respond to EGFR-targeted therapies. Introduction The most common NSCLC-associated EGFR mutations, the exon 19 deletion (E746_A750del) and the exon 21 substitution (L858R) account for 85-90% of EGFR mutations. The remaining mutations occur at much lower frequencies including those in exon-20 and codon 719. Direct DNA sequencing to detect EGFR mutations in patient tumor tissue is currently the standard method. However, acceptance of this technique has been hindered by the high cost of equipment and reagents, technical difficulties of performing the as- say and length of the procedure. While other DNA-based methods have been devel- oped, these methods are not routine procedures in clinical labs and remain expen- sive and time-consuming. In contrast, immunohistochemistry (IHC) is a well-established method routinely used in solid tumor diagnosis in clinical laboratories. IHC also allows for the analy- sis of small tissue samples or individual cells obtained from a variety of sources including circulating tumor cells. Thus, the development of antibodies that specifi- cally detect mutant EGFR protein by IHC would be a valuable addition to the current protocols utilized in the diagnosis and treatment of lung cancer. Materials and Methods Human NSCLC Tumor Tissues: IRB approval was granted by the Second Xiang- ya Hospital, Central South University (Changsha, Hunan, P.R.China). Human sam- ples of NSCLC paraffin blocks were provided by the Second Xiangya hospital. Conclusions Recent clinical results suggest that the treatment of NSCLC patients with EGFR-targeted therapies based on the status of EGFR mutations can improve patient response. To ad- dress the need to characterize the mutational status of patients, we generated two mutation-specific rabbit monoclonal antibodies that specifically detect the two most com- mon EGFR mutations in NSCLC. We showed that these antibodies are highly sensitive and specific and can be used in conventional IHC assays to identify tumors harboring these EGFR mutations. Importantly, this assay maintains tumor morphology, allows tumor heterogeneity to be studied and eliminates the false negatives seen in DNA sequenc- ing methods due to an inadequate percentage of tumor cells. 1. HCC827 (dEGFR) 2. H1975 (L858R) 3. H3255 (L858R) 4. H1650 (dEGFR) 5. Kyse70 (wtEGFR) 6. Kyse450 (wtEGFR) Western blot analysis of whole cell lysates from cancer cell lines with control EGFR antibody, L858R specific antibody and dEGFR specific antibody. L858R (Clone SP) dEGFR (Clone 6B6) Total EGFR (Clone 86) 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 Immunofluorescence (IF) staining of xenografts. H1975 L858R/-amp EGFR H3255 L858R/+amp H1650 del/-amp HCC827 del/+amp Kyse70 wt/-amp Kyse450 wt/+amp L858R dEGFR H1975 L858R/-amp H3255 L858R/+amp H1650 del/-amp HCC827 del/+amp EGFR L858R dEGFR Kyse70 wt/-amp Kyse450 wt/+amp Immunohistochemistry (IHC) staining of xenografts. Jian Yu 1 , Susan Kane 1 , Daiqiang Li 3 , Herbert Haack 1 , Bradley Smith 1 , Ting-Lei Gu 1 , Massimo Loda 2 , Xinmin Zhou 4* , Michael J. Comb 1* 1. Cell Signaling Technology, Inc., Danvers, MA USA / 2. Department of Medical Oncology and Center for Molecular Oncologic Pathology, Dana-Farber Cancer Institute, Brigham & Women’s Hospital, Boston, MA USA / 3. Department of Pathology / 4. Department of Cardiothoracic Surgery, The Second Xiangya Hospital, Central South University, Changsha, Hunan, P.R.China Exon 19 Deletion L858R MS MS failed Direct DNA Sequencing: Incorrect / Total 5 / 9 3 / 9 2 / 18 IHC: Incorrect / Total 2 / 9 0 / 9 0 / 18 Comparison of MS Sequencing, Direct Sequencing and IHC. Comparison of IHC results and Direct DNA sequencing analysis. A. L858R Mutation IHC DNA Sequencing L858R wt failed L858R (+) 24 2 2 L858R (-) 2 193 25 B. Exon 19 Deletion IHC DNA Sequencing dEGFR wt failed dEGFR (+) 23 0 1 dEGFR (-) 3 196 23 Sensitivity: 92% Specificity: 99% 3% 2% 9% 40% 46% Exon 20 Variants Codon 719 Variants Others L858R Substitution Exon 19 Deletions Immunohistochemical staining of two NSCLC tumor samples. IHC results of NSCLC tumor samples. Pathology Number L858R (+) dEGFR (+) AC 217 28 23 SCC 112 0 1 LCC 11 0 0 Total 340 28 24 Pan-keratin wtEGFR L858R dEGFR 1. 2.
Transcript of Mutation-Specifi c Antibodies for the Detection of EGFR … · 2017. 5. 16. ·...
Mutation-Specifi c Antibodies for the Detection of EGFR Mutations in Non-Small-Cell Lung Cancer
SummaryActivating mutations within the tyrosine kinase domain of the epidermal growth
factor receptor (EGFR) are found in approximately 10-20% of non-small cell lung
cancer (NSCLC) patients and are associated with improved response to the EGFR
inhibitors Gefi tinib and Erlotinib. The most common NSCLC-associated EGFR mu-
tations are the exon 19 deletion (E746-A750del) and the exon 21 point mutation
(L858R). Together these mutations account for 90% of EGFR mutations. Here, we
generate mutation-specifi c rabbit monoclonal antibodies against each of these EGFR
mutations. The mutation-specifi c antibodies detect the corresponding mutant form
of EGFR but not wild type EGFR by Western blotting, immunofl uorescence (IF), and
immunohistochemistry (IHC). IHC screening of a large panel of paraffi n-embedded
NSCLC tumor samples demonstrates these antibodies are highly sensitive and spe-
cifi c. This IHC assay provides a rapid, sensitive, specifi c and cost-effective method
to identify lung cancer patients likely to respond to EGFR-targeted therapies.
IntroductionThe most common NSCLC-associated EGFR mutations, the exon 19 deletion
(E746_A750del) and the exon 21 substitution (L858R) account for 85-90% of
EGFR mutations. The remaining mutations occur at much lower frequencies
including those in exon-20 and codon 719.
Direct DNA sequencing to detect EGFR mutations in patient tumor tissue is currently
the standard method. However, acceptance of this technique has been hindered by
the high cost of equipment and reagents, technical diffi culties of performing the as-
say and length of the procedure. While other DNA-based methods have been devel-
oped, these methods are not routine procedures in clinical labs and remain expen-
sive and time-consuming.
In contrast, immunohistochemistry (IHC) is a well-established method routinely
used in solid tumor diagnosis in clinical laboratories. IHC also allows for the analy-
sis of small tissue samples or individual cells obtained from a variety of sources
including circulating tumor cells. Thus, the development of antibodies that specifi -
cally detect mutant EGFR protein by IHC would be a valuable addition to the current
protocols utilized in the diagnosis and treatment of lung cancer.
Materials and MethodsHuman NSCLC Tumor Tissues: IRB approval was granted by the Second Xiang-
ya Hospital, Central South University (Changsha, Hunan, P.R.China). Human sam-
ples of NSCLC paraffi n blocks were provided by the Second Xiangya hospital.
Conclusions Recent clinical results suggest that the treatment of NSCLC patients with EGFR-targeted therapies based on the status of EGFR mutations can improve patient response. To ad-
dress the need to characterize the mutational status of patients, we generated two mutation-specifi c rabbit monoclonal antibodies that specifi cally detect the two most com-
mon EGFR mutations in NSCLC. We showed that these antibodies are highly sensitive and specifi c and can be used in conventional IHC assays to identify tumors harboring
these EGFR mutations. Importantly, this assay maintains tumor morphology, allows tumor heterogeneity to be studied and eliminates the false negatives seen in DNA sequenc-
ing methods due to an inadequate percentage of tumor cells.
1. HCC827 (dEGFR)2. H1975 (L858R)
3. H3255 (L858R)4. H1650 (dEGFR)
5. Kyse70 (wtEGFR)6. Kyse450 (wtEGFR)
Western blot analysis of whole cell lysates from cancer cell lines with control EGFR antibody, L858R specifi c antibody and dEGFR specifi c antibody.
L858R (Clone SP) dEGFR (Clone 6B6)Total EGFR (Clone 86)
1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6
Immunofl uorescence (IF) staining of xenografts.
H1975 L858R/-amp
EGFR
H3255 L858R/+amp H1650 del/-amp HCC827 del/+amp Kyse70 wt/-amp Kyse450 wt/+amp
L858R
dEGFR
H1975 L858R/-amp H3255 L858R/+amp H1650 del/-amp HCC827 del/+amp
EGFR
L858R
dEGFR
Kyse70 wt/-amp Kyse450 wt/+amp
Immunohistochemistry (IHC) staining of xenografts.
Jian Yu1, Susan Kane1, Daiqiang Li3, Herbert Haack1, Bradley Smith1, Ting-Lei Gu1, Massimo Loda2, Xinmin Zhou4*, Michael J. Comb1*
1. Cell Signaling Technology, Inc., Danvers, MA USA / 2. Department of Medical Oncology and Center for Molecular Oncologic Pathology, Dana-Farber Cancer Institute, Brigham & Women’s Hospital, Boston, MA USA / 3. Department of Pathology / 4. Department of Cardiothoracic Surgery, The Second Xiangya Hospital, Central South University, Changsha, Hunan, P.R.China
Exon 19 Deletion L858R
MS MS failedDirect DNA Sequencing: Incorrect / Total 5 / 9 3 / 9 2 / 18
IHC: Incorrect / Total 2 / 9 0 / 9 0 / 18
Comparison of MS Sequencing, Direct Sequencing and IHC.
Comparison of IHC results and Direct DNA sequencing analysis.
A. L858R Mutation
IHC DNA SequencingL858R wt failed
L858R (+) 24 2 2
L858R (-) 2 193 25
B. Exon 19 Deletion
IHC DNA SequencingdEGFR wt failed
dEGFR (+) 23 0 1
dEGFR (-) 3 196 23
Sensitivity: 92% Specifi city: 99%
3%
2%9%
40%
46%
Exon 20 Variants Codon 719 Variants OthersL858R Substitution Exon 19 Deletions
Immunohistochemical staining of two NSCLC tumor samples.
IHC results of NSCLC tumor samples.
Pathology Number L858R (+) dEGFR (+)
AC 217 28 23
SCC 112 0 1
LCC 11 0 0
Total 340 28 24
Pan-keratin wtEGFR L858R dEGFR
CL761
CL764
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