Epidermal Growth Factor Receptor Mutation-specific Antibodies Research Articles

Mini Review: Immunohistochemistry Using EGFR-Mutant Specific Antibodies in Non-Small Cell Lung Carcinoma: Accuracy and Reliability

Epidermal growth factor receptor (EGFR) is a predictive biomarker in many solid cancers including non-small cell lung carcinoma (NSCLC). Patients’ responsiveness to therapy is based on the prior determination of EGFR status. Many techniques were used to detect the potential predictive biomarker and considered as gold standards based on molecular genetic techniques as direct sequencing and polymerase chain reaction (PCR). Immunohistochemistry (IHC) using EGFR mutation-specific antibodies were generated as an alternative simple tool to identify EGFR status and its response to tyrosine kinase inhibitors (TKIs). EGFR gene copy number and wild-type EGFR are other parameters which determine the response to TKIs. The aim of this mini-review is to analyze the studies which investigated the IHC EGFR mutation-specific antibodies, in lung adenocarcinoma (ADC), to determine its accuracy and reliability as a pre-selection tool for candidate patients for TKIs therapy as well as the interplay with other EGFR biomarkers which are EGFR gene copy number and the wild-type EGFR in lung NSCLC. The later determine the response to TKIs and their detection methodology is standardized making them good candidates for comparison with the EGFR-mutation.

Expression of epidermal growth factor receptor mutation specific antibodies in lung adenocarcinoma: evaluation of sensitivity, specificity and relationship to histologic subtypes

To evaluate the expression of epidermal growth factor receptor (EGFR) mutation specific antibodies in invasive lung adenocarcinomas, and their sensitivity, specificity, as well as relationship to histological subtypes. Immunostaining with EGFR mutation-specific antibodies, del E746-A750 in exon 19 and L858R in exon 21, was performed in tissue microarrays of 884 cases of resection specimens to study the relationship between the immunophenotypes and morphologic subtypes. The sensitivity and specificity of the stains were compared with gene mutations detected by amplified refractory mutation system-polymerase chain reaction (ARMS-PCR). Of the 884 cases, the expression of del E746-A750 in exon 19 was 3+ , 2+ , 1+ and 0 in 7 cases (0.79%), 38 cases (4.30%), 129 cases (14.59%) and 710 cases (80.32%), respectively. For L858R in exon 21, 3+ , 2+ , 1+ and 0 staining were seen in 82 cases (9.28%), 93 cases (10.52%), 82 cases (9.28%) and 627 cases (70.93%), respectively. For both antibodies, positive expression (1+ or more) was mainly observed in lepidic, acinar and papillary predominant subtypes, and rarely seen in solid subtype or invasive mucinous adenocarcinoma (P=0.014 and 0.016). If 1+ to 3+ expression was set as positive, the specificity of exon 19/exon 21 reached 98.59%/92.98%, while the sensitivity was relatively lower (62.86%/88.89%). If 2+ to 3+ expression was read as positive, the specificity and sensitivity were 99.30%/97.37% and 25.71%/74.60% for exon 19/exon 21. If only 3+ expression was considered positive, the specificity was 100.0% for both antibodies, with a low sensitivity (8.57% for exon 19 and 34.92% for exon 21). Of the 18 cases with E746-A750 del in exon 19 based on molecular detection, the sensitivity of immunohistochemistry for exon 19 was 88.89% if a positive cutoff value ≥1+ was used; in contrast, of the 8 cases harboring other deletions in exon 19, only two cases were positive as 1+ . Both the EGFR mutation specific antibodies del E746-A750 in exon 19 and L858R in exon 21 demonstrate high specificity and relatively low sensitivity, and are mostly expressed in lepidic, acinar and papillary predominant subtypes, but rarely in solid subtype or invasive mucinous adenocarcinoma. For cases with 3+ expression, a mutational statue for EGFR is likely. For the 2+ positive cases, the accuracy to predict mutation almost reaches 90%, but molecular detection for confirmation is desirable. For the 1+ and negative cases, DNA-based test is essential to avoid false negativity.

Overexpression of mutant EGFR protein indicates a better survival benefit from EGFR-TKI therapy in non-small cell lung cancer.

BackgroundEpidermal growth factor receptor (EGFR) is a novel target for therapy in a subset of non-small cell lung cancer (NSCLC). Tumors with EGFR mutations showed good response to EGFR tyrosine kinase inhibitors (TKIs). We aimed to identify the discriminating capacity of immunohistochemistry (IHC) to detect EGFR L858R and del E746-A750 mutations in NSCLC patients and predict EGFR TKIs response.MethodsWe collected specimens from 200 patients with NSCLC whose EGFR mutation status had been validated by direct DNA sequencing. IHC analyses using EGFR mutation-specific antibodies were employed for all samples. After staining and scoring, the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were calculated.ResultsThe sensitivity, specificity, PPV, and NPV of IHC using EGFR del E746-A750 and L858R mutation antibodies were 95.0%/95.1%, 85.7%/94.1%, 74.0%/91.8%, and 97.6%/96.5%, respectively. When score 2+ and 3+ were considered as positive, the sensitivity, specificity, PPV, and NPV were 53.3%/36.6%, 99.3%/100%, 97.0%/100%, and 83.2%/65.3%, respectively. The median progression-free survival (PFS) after the start of gefitinib treatment was significantly longer in patients with a high score for mutant EGFR expression than in those with a low score (31.0 versus 13.0 months, p <0.05).ConclusionsIHC with EGFR mutation-specific antibodies is a promising screening method for detecting EGFR mutations in NSCLC patients. Otherwise, quantitative analysis of mutant EGFR expression might also predict the efficacy of TKIs treatment for NSCLC patients harboring sensitive EGFR mutation.

Prognostic implications of immunohistochemistry markers for EGFR-TKI therapy in Chinese patients with advanced lung adenocarcinoma harboring EGFR mutations.

BackgroundEpidermal growth factor receptor (EGFR) mutations in non-small-cell lung cancer predict dramatic clinical responses to tyrosine kinase inhibitor (TKI) treatment. The conclusion on EGFR mutation-specific antibodies by immunohistochemistry (IHC) is not consistent. We evaluated the clinical availability of EGFR mutation-specific antibodies, investigating the prediction role of mutant EGFRs and other IHC markers in TKI therapy in patients with advanced lung adenocarcinoma.Materials and methodsWe analyzed 637 primary lung adenocarcinomas from an unselected Chinese population. For IHC, antibodies against EGFR exon 19 E746_A750 deletions, exon 21 L858R mutations, thyroid transcription factor-1 (TTF-1), and Napsin-A were applied. Positivity was defined as staining score >0.ResultsSpecificities of E746_A750 and L858R antibodies were 99.6% and 99.3%, while sensitivities were 86.0% and 82.7%, respectively. Tumors with Napsin-A positivity, TTF-1 positivity, EGFR mutations, and lepidic pattern showed a lower marker of proliferation index (Ki67). Higher expression scores of mutant EGFR protein, TTF-1 positivity, lower Ki67 proliferation index, and lepidic pattern were associated with longer progression-free survival.ConclusionHigh scores of mutant EGFR, Napsin-A positivity, TTF-1 positivity, lower Ki67 index, and lepidic pattern were favorable predictors for TKI therapy in patients with advanced lung adenocarcinoma.

Evaluation of epidermal growth factor receptor mutations based on mutation specific immunohistochemistry in non-small cell lung cancer: A preliminary study.

Background & objectives:Studies have shown that immunohistochemical (IHC) staining using epidermal growth factor receptor (EGFR) mutation specific antibodies, is an easy and cost-effective, screening method compared with molecular techniques. The purpose of present study was to assess the percentage positivity of IHC using EGFR mutation specific antibodies in lung biopsy samples from patients with primary lung adenocarcinoma (ADC).Methods:Two hundred and six biopsies of primary lung ADC were subjected to EGFR mutation specific antibodies against del E746-A750 and L858R. Detection of EGFR mutation done by high resolution melting analysis (HRM) was used as gold standard. A concordance was established between molecular and IHC results. Frequency of IHC positivity was assessed.Results:Of the 206 patients, 129 were male and 77 were female patients, with a mean age of 54.1 yr. Fifty five (26.6%) patients (36 men; 19 women) showed positivity for IHC of del E746-A750 (33) and L858R (22). HRM results were available in 14 patients which showed EGFR mutations in correspondence with del E746-750 or L858R in 64.2 per cent cases. Positive cases on HRM were further confirmed by DNA sequencing and fragment analysis. Three patients showed exon20 variation. Two cases were negative for mutation. The genotype of del E746-750 mutation was more common than L858R. A concordance was established between molecular mutation and IHC in 85.7 per cent cases.Interpretation & conclusions:In this preliminary study from India mutation specific IHC was used for assessment of mutation status of EGFR. Although the number tested was small, a good concordance was observed between molecular EGFR mutation and IHC expression. IHC methodology is a potentially useful tool to guide clinicians for personalized treatment in lung ADC, especially where facilities for molecular analysis are not readily available and for use in small biopsies where material is scant for molecular tests.

Diamond: immunohistochemistry versus sequencing in EGFR analysis of lung adenocarcinomas

AimsIdentification of epidermal growth factor receptor (EGFR) mutations in lung adenocarcinomas is the single most important predictor of clinical response and outcome using EGFR tyrosine kinase inhibitors (TKIs). EGFR E746-A750del...

Detection of epidermal growth factor receptor (EGFR) exon 19 E746-A750 deletion and EGFR exon 21 point mutations in lung adenocarcinoma by immunohistochemistry: a comparative study to EGFR exons 19 and 21 mutations analysis using PCR followed by high-resolution melting and pyrosequencing

Activating mutations in the epidermal growth factor receptor (EGFR) gene in non-small cell lung cancer (NSCLC) with adenocarcinoma histology confers susceptibility to treatment with EGFR tyrosine kinase inhibitors (TKI). Analysis of these activating mutations is typically performed by molecular techniques including polymerase chain reaction (PCR) amplification followed by deoxyribonucleic acid (DNA) melting curve analysis and/or direct sequencing. Recently developed mutation-specific immunohistochemical stains for the L858R point mutation in exon 21 and an E746-A750 deletion in exon 19 of the EGFR gene may offer a fast, cost-effective alternative to molecular testing. In this study, EGFR exons 19 and 21 mutations were analyzed using EGFR E746-A750 del and EGFR L585R point mutation-specific antibodies and the results were compared with the exon 19 deletions and exon 21 L588R point mutation as detected by molecular testing. The findings demonstrate a high concordance rate (100% in 12 cases) between the two methodologies for EGFR exon 21 L585R point mutation but a low concordance rate (54% in 22 cases) for exon 19 deletions when using EGFR E746-A750 mutation-specific antibody compared to molecular testing. This low concordance rate between the two methods is due to the fact that EGFR exon 19 mutations include deletions other than E746-A750 as well as insertion/deletions that will not be covered by exon 19 E746-A750 del mutation-specific antibody. Despite this limitation, the current EGFR mutation-specific antibodies can prove useful as an alternative to molecular tests for exon 21 L585R mutations as well as a subset of EGFR exon 19 deletion mutations, particularly in cases with low percentage of tumor concentration and in decalcified tissue specimens that are not suitable for DNA-based molecular assays.

Identification of EGFR Mutations by Immunohistochemistry with EGFR Mutation-Specific Antibodies in Biopsy and Resection Specimens from Pulmonary Adenocarcinoma.

PurposeMutation-specific antibodies have recently been developed for identification of epidermal growth factor receptor (EGFR) mutations by immunohistochemistry (IHC). This study was designed to investigate whether the type of specimen (biopsy vs. resection) would make a difference in determining mutation status by IHC, and to evaluate whether biopsies are suitable for detection of mutant EGFR protein.Materials and MethodsIHC was performed using mutation-specific antibodies for E746-A750 deletion (DEL) and L858R point mutation (L858R) in biopsies and tissue microarrays of resected tumors from 154 patients with pulmonary adenocarcinoma. Results were then compared with DNA sequencing data.ResultsMolecular-based assays detected EGFR mutations in 62 patients (40.3%), including 14 (9.1%) with DEL, and 31 (20.1%) with L858R. IHC with two mutation-specific antibodies showed a homogeneous staining pattern, and correctly identified EGFR mutation status in 89% (137/154). Overall (biopsy/resection) sensitivity, specificity, positive predictive value, and negative predictive value were 75.6% (78.3%/72.7%), 94.5% (90.9%/96.3%), 85% (78.3%/88.9%), and 90.4% (90.9%/89.7%), respectively.ConclusionOur data showed that IHC using EGFR mutation–specific antibodies is useful for detection of EGFR mutations with high specificity and good sensitivity not only for resection specimens but also for biopsy materials. Therefore, IHC using EGFR mutation–specific antibodies may preclude a second biopsy procedure to obtain additional tissues for identification of EGFR mutations by molecular assays in biopsies from advanced cancer, particularly when tumor cells in the samples are limited.

Quantum dots immunofluorescence histochemical detection of EGFR gene mutations in the non-small cell lung cancers using mutation-specific antibodies.

BackgroundEpidermal growth factor receptor (EGFR) mutation status plays an important role in therapeutic decision making for non-small cell lung cancer (NSCLC) patients. Since EGFR mutation-specific antibodies (E746-A750del and L858R) have been developed, EGFR mutation detection by immunohistochemistry (IHC) is a suitable screening test. On this basis, we want to establish a new screening test, quantum dots immunofluorescence histochemistry (QDs-IHC), to assess EGFR gene mutation in NSCLC tissues, and we compared it to traditional IHC and amplification refractory mutation system (ARMS).Materials and methodsEGFR gene mutations were detected by QDs-IHC, IHC, and ADx-ARMS in 65 cases of NSCLC composed of 55 formalin-fixed, paraffin-embedded specimens and ten pleural effusion cell blocks, including 13 squamous cell carcinomas, two adenosquamous carcinomas, and 50 adenocarcinomas.ResultsPositive rates of EGFR gene mutations detected by QDs-IHC, IHC, and ADx-ARMS were 40.0%, 36.9%, and 46.2%, respectively, in 65 cases of NSCLC patients. The sensitivity of QDs-IHC when detecting EGFR mutations, as compared to ADx-ARMS, was 86.7% (26/30); the specificity for both antibodies was 100.0% (26/26). IHC sensitivity was 80.0% (24/30) and the specificity was 92.31% (24/26). When detecting EGFR mutations, QDs-IHC and ADx-ARMS had perfect consistency (κ =0.882; P<0.01). Excellent agreement was observed between IHC and ADx-ARMS when detecting EGFR mutations (κ =0.826; P<0.01).ConclusionQDs-IHC is a simple and standardized method to detect EGFR mutations with its high sensitivity and specificity, as compared with real-time polymerase chain reaction. In addition, the development of specific antibodies against EGFR mutation proteins might be useful for the diagnosis and treatment of lung cancer.

Immunohistochemical Screening Using Egfr Mutation-Specific Antibodies in Lung Adenocarcinomas: Diamond Project

ABSTRACT Aim: Epidermal growth factor receptor (EGFR) mutations is the single most important predictor of clinical response and outcome using EGFR tyrosine kinase inhibitors (TKI) in lung adenocarcinomas. The most common gene alterations are EGFR deletions involving exon 19 and L858R point mutation in exon 21. We evaluated the accuracy of EGFR mutation-specific antibodies in different cytology and histologic samples. Methods: A series of 111 lung adenocarcinomas diagnosed on cytology (21 cases), biopsy (58) and surgical resections (32) were selected; all cases were previously investigated by Sanger sequencing and/or pyrosequencing (EGFR-KRAS mutation) and by immunohistochemistry(IHC)/FISH (ALK status). IHC using EGFR mutation-specific antibodies were tested using an automated immunostainer. Cases were quoted as positive when at least 2+ staining was present in at least 5% of tumor cells. Results: Conventional molecular analyses showed EGFR mutations in 52 cases (47%), KRAS mutations in 44 cases (40%), ALK positivity in 5 cases (4%) and triple negative setup in 10 cases. There were 19 and 25 tumors harboring L858R and delE746-A750, respectively, while 4 cases had uncommon mutations in exon 19, 2 cases harbor L861Q in exon 21, 2 T790M in exon 20, G719G and G719A in exon 18. At IHC, overall sensitivity was 76.5% and specificity was 98.3%. The sensitivity and specificity of antibodies for exon 19 was 68% and 100%, respectively, for exon 21 was 91% and 99%. Sensitivity and specificity in non-EGFR mutated tumors was absolute (100%). No significant difference in sensitivity and specificity was observed when results were statistically matched with different types of samples. Conclusions: The EGFR mutation-specific antibodies have a high specificity and fair-to-high sensitivity in classic EGFR mutations, while they do not recognized uncommon EGFR mutations, but no cross-reaction with tumors harboring different molecular settings was evidenced. The antibodies work equally well on biopsies and cytology, permitting a useful and rapid screening in cases with poor material. Negative results do not permit to robustly exclude the presence of EGFR mutations, requiring further molecular analysis, possibly microdissecting the same slides tested at IHC. Disclosure: All authors have declared no conflicts of interest.

Diamond project: Immunohistochemical screening using EGFR mutation-specific antibodies in lung adenocarcinomas.

e19035 Background: Identification in lung adenocarcinomas (LACs) of epidermal growth factor receptor (EGFR) mutations is the single most important predictor of clinical response and outcome using EGFR tyrosine kinase inhibitors (TKI). We evaluated the accuracy of EGFR mutation-specific antibodies in LACs and showing different molecular settings and detected on cytology and histologic samples. Methods: 111 LACs diagnosed on cytology (21 cell blocks), biopsy (58 cases) and surgical resections (32 cases) were selected for the study. All cases were previously investigated by Sanger sequencing and/or pyrosequencing (EGFR exons 18 to 21; KRAS exon 2) and ALK status by Immunohistochemistry (IHC)/FISH. IHC using EGFR mutation-specific antibodies (clone 6B6 for delE746-A750 and clone 43B2 for L858R; Cell Signalling Technologies, Danvers, MA) were tested using an automated immunostainer (Benchmark, Ventana Inc, Tucson, AZ). Cases were quoted as positive when at least 2+ staining was present in at least 5% of tumor ...

Novel EGFR mutation-specific antibodies for lung adenocarcinoma: Highly specific but not sensitive detection of an E746_A750 deletion in exon 19 and an L858R mutation in exon 21 by immunohistochemistry

ObjectivesActivating mutations in the epidermal growth factor receptor (EGFR) kinase domain are correlated with dramatic clinical responses in non-small cell lung cancer patients treated with EGFR-tyrosine kinase inhibitors (TKIs). The two most common EGFR mutations, representing 85–90% of EGFR mutations, are the E746_A750 deletion in exon 19 and the L858R point mutation in exon 21. We conducted this study to evaluate the suitability of mutation-specific antibodies that can detect E746_A750 deletion and L858R mutant EGFR proteins by immunohistochemistry (IHC). Materials and methodsIn a cohort of consecutive patients with surgically resected lung adenocarcinomas (n=240), mutant EGFR protein expression was assessed by IHC using specific antibodies (clone SP111 and SP125) to the 2 major forms of EGFR mutations. Immunoreactivity was scored as 0–3, and the results were compared with the EGFR-mutational status. ResultsWith a cutoff value of IHC 2+ for SP 111 (anti-EGFR E746_A750 del antibody) and SP 125 (anti-EGFR L858R antibody), both antibodies showed high specificity (99.0% and 89.7%, respectively) and sensitivity (70.6% and 80.4%, respectively). While cases with IHC scores of 3 using these 2 antibodies positively correlated with the EGFR-mutational status, cases with IHC scores lower than 3+ showed variable results regarding EGFR-mutational status. ConclusionAlthough each antibody showed relatively high specificity, some EGFR-mutant cases were not detected by the mutation-specific antibodies. Various forms of exon 19 deletions, except E746_A750, were rarely detected by the mutant-specific antibody. Therefore, IHC-negative cases require further molecular analysis to confirm the presence of EGFR mutations.

Fixation effect of SurePath preservative fluids using epidermal growth factor receptor mutation‐specific antibodies for immunocytochemistry

Cytological diagnosis of respiratory disease has become important, not only for histological typing using immunocytochemistry (ICC) but also for molecular DNA analysis of cytological material. The aim of this study was to investigate the fixation effect of SurePath preservative fluids. Human lung cancer PC9 and 11-18 cell lines, and lung adenocarcinoma cells in pleural effusion, were fixed in CytoRich Blue, CytoRich Red, 15% neutral-buffered formalin, and 95% ethanol, respectively. PC9 and 11-18 cell lines were examined by ICC with epidermal growth factor receptor (EGFR) mutation-specific antibodies, the EGFR mutation DNA assay, and fluorescence in situ hybridization. The effect of antigenic storage time was investigated in lung adenocarcinoma cells in pleural effusion by ICC using the lung cancer detection markers. PC9 and 11-18 cell lines in formalin-based fixatives showed strong staining of EGFR mutation-specific antibodies and lung cancer detection markers by ICC as compared with ethanol-based fixatives. DNA preservation with CytoRich Blue and CytoRich Red was superior to that achieved with 95% ethanol and 15% neutral-buffered formalin fixatives, whereas EGFR mutations by DNA assay and EGFR gene amplification by fluorescence in situ hybridization were successfully identified in all fixative samples. Although cytoplasmic antigens maintained high expression levels, expression levels in nuclear antigens fell as storage time increased. These results indicate that CytoRich Red is not only suitable for ICC with EGFR mutation-specific antibodies, but also for DNA analysis of cytological material, and is useful in molecular testing of lung cancer, for which various types of analyses will be needed in future.

Immunohistochemical staining with EGFR mutation-specific antibodies: high specificity as a diagnostic marker for lung adenocarcinoma

We previously demonstrated a high specificity of immunohistochemistry using epidermal growth factor receptor (EGFR) mutation-specific antibodies in lung adenocarcinoma and correlation with EGFR mutation analysis. In this study, we assessed EGFR mutation status by immunohistochemistry in a variety of extrapulmonary malignancies, especially those that frequently show EGFR overexpression. Tissue microarrays containing triplicate cores of breast carcinomas (n=300), colorectal carcinomas (n=65), pancreatic adenocarcinoma (n=145), and uterine carcinosarcoma or malignant mixed müllerian tumors (n=25) were included in the study. Tissue microarray of lung adenocarcinoma with known EGFR mutation status was used as reference. Immunohistochemistry was performed using antibodies specific for the E746-A750del and L858R mutations. In pulmonary adenocarcinoma, a staining intensity of 2+ or 3+ correlates with mutation status and is therefore considered as positive. Out of 300 breast carcinomas, 293 (98%) scored 0, 5 (2%) had 1+ staining, 2 (1%) were 2+ for the L858R antibody. All breast carcinomas scored 0 with the E746-A750 antibody. All the colorectal, pancreatic carcinomas and malignant mixed müllerian tumors were negative (0) for both antibodies. Molecular analysis of the breast carcinomas that scored 2+ for L858R showed no mutation. Our results show that EGFR mutation-specific antibodies could be an additional tool distinguishing primary versus metastatic carcinomas in the lung. False-positivity can be seen in breast carcinoma but is extremely rare (1%).

Ascertaining an Appropriate Diagnostic Algorithm Using EGFR Mutation-Specific Antibodies to Detect EGFR Status in Non-Small-Cell Lung Cancer

BackgroundEpidermal growth factor receptor (EGFR) mutation status is the most valuable indicator in the screening of non-small-cell lung cancer (NSCLC) patients for tyrosine kinase inhibitor (TKI) therapy. Accurate, rapid and economical methods of detecting EGFR mutations have become important. The use of two mutation-specific antibodies targeting the delE746-A750 mutation in exon 19 and L858R mutation in exon 21 makes this task possible, but the lack of consensually acceptable criteria for positive results limits the application of this antibody based mutation detection.MethodsWe collected 399 specimens from NSCLC patients (145 resection specimens, 220 biopsy specimens, and 34 cytology specimens) whose EGFR mutation status had been detected by TaqMan PCR assay. Immunohistochemical (IHC) analyses using EGFR mutation-specific antibodies were employed for all samples. After staining and scoring, the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were calculated in accordance with different levels of positive grades in comparison with the results of PCR-based assay.ResultsIn IHC-based analyses, 144 cases were scored 0, 104 cases were scored 1+, 103 cases were scored 2+, and 48 cases were scored 3+. With the molecular-based results were set as the “gold standard”, the prevalence of mutation was 6.94% (10/144), 23.08% (24/104), 67.96% (70/103) and 100% (48/48), respectively, for samples with scores 0, 1+, 2+ and 3+. When score 3+ was considered positive, the specificity and PPV were 100%; if only score 0 was considered negative, 93.06% NPV was obtained.ConclusionPatients with score 3+ have a perfect PPV (100%), and may accept TKI treatment directly without any molecular-based assays. Patients with score 0 had high NPV (93.06%), which could reach 97.22% when the detection of total EGFR was applied. However, samples with score 1+ or 2+ are unreliable and need further verification of EGFR mutation status by molecular-based assays.

Immunohistochemistry using epidermal growth factor receptor mutation-specific antibodies of delE746-A750 and L858R in lung adenocarcinomas

To evaluate the immunohistochemical detection of epidermal growth factor receptor(EGFR) mutations using two EGFR mutation-specific monoclonal antibodies: delE746-A750 and L858R. A total of 175 paraffin-embedded lung adenocarcinoma tissue samples previously genotyped by directive DNA sequencing were subject to immunostaining using delE746-A750 and L858R antibodies. There was no significant difference of mutation detection between DNA sequence analysis and delE746-A750 and/or L858R immunostaining (33.7% vs 30.9%, P > 0.05). The overall sensitivity, specificity, positive predictive value and negative predictive value of immunostaining using these two EGFR mutation-specific antibodies were 83.1%, 95.7%, 90.7% and 90.9%, respectively. With high sensitivity and good specificity, immunohistochemistry using EGFR mutation-specific monoclonal antibodies is an adequate, easy and cost-effective prescreening method to detect EGFR mutations using paraffin-embedded tissue specimens of lung adenocarcinomas.

Immunostaining with EGFR mutation–specific antibodies: a reliable screening method for lung adenocarcinomas harboring EGFR mutation in biopsy and resection samples

Mutation analysis of epidermal growth factor receptor (EGFR) is essential in determining the therapeutic strategy for lung adenocarcinoma. Immunohistochemical (IHC) staining with EGFR mutation-specific antibodies of del E746-A750 in exon 19 and L858R in exon 21 has been evaluated in resection specimens in a few studies but rarely in biopsy samples. A total of 169 cases (78 biopsies and 91 resected specimens) of lung adenocarcinoma with EGFR mutation status predefined by direct DNA sequencing were histologically examined, and IHC was performed using EGFR mutation-specific antibodies of del E746-A750 and L858R. The cases with positive results by IHC but negative results by direct DNA sequencing were examined by amplified refractory mutation system. Our results showed that the frequency of EGFR mutations for both E746-A750 deletion and L858R mutation was 38.5% (65/169) by DNA sequencing or amplified refractory mutation system and 34.3% (58/169) by IHC in lung adenocarcinomas. Based on molecular test results, the overall sensitivity, specificity, positive predictive value, and negative predictive value of IHC using these 2 antibodies in all (biopsy/resection) cases were 87.7% (80%/94.3%), 99.0% (97.9%/100%), 98.3% (96%/100%), and 92.8% (88.7%/96.6%), respectively. Lung adenocarcinomas with a predominant acinar, papillary, lepidic, or solid growth pattern more often harbor EGFR mutation of del E746-A750 or L858R. In conclusion, the immunostaining with EGFR del E746-A750 and L858R mutation antibodies is a reliable screening method with high specificity and sensitivity for identifying the EGFR mutation in both resected and biopsied lung adenocarcinomas.

EGFR Mutation-specific Antibodies in Pulmonary Adenocarcinoma

Epidermal growth factor receptor (EGFR) gene mutations are usually detected by direct sequencing to identify patients with advanced pulmonary adenocarcinomas as candidates for tyrosine kinase inhibitor therapy. The aim of the study was to evaluate the efficacy of EGFR mutation-specific antibodies in identifying EGFR-mutated adenocarcinomas. Thirty-three consecutive cases of pulmonary adenocarcinomas sequenced for EGFR mutations were retrieved from our files. Immunohistochemistry was performed with the rabbit monoclonal antibodies E746-A750del (6B6) and L858R (43B2). The results obtained using the 2 procedures were statistically compared by Coehn κ and by calculation of sensitivity and specificity. There were 21 women and 12 men, ranging in age from 48 to 78 years. All cases were lung adenocarcinomas, 23 primaries and 10 metastatic. The mutational spectrum was as follows: 12 cases mutated in exon 19 (9 with E746-A750del, 1 with homozygote L747-T751del, 1 with L747-P753del, 1 with E747-S752del), 6 in exon 21 (5 with L858R, 1 with L861Q+L862L), and 15 EGFR wild type. Immunohistochemistry detected 6/9 cases with an E746-A750del mutation (κ=0.744, sensitivity: 66.7%, specificity: 100%) and 5/5 cases with an L858R mutation (κ=1, sensitivity: 100%, specificity: 100%). Four cases showed faint and focal immunostaining and were interpreted as negative. All other cases were negative. Overall, the 2 antibodies had 61.1% sensitivity and 100% specificity for EGFR mutations. EGFR mutation-specific antibodies may represent a first-line screening tool to identify patients as candidates for tyrosine kinase inhibitor therapy.

Novel Epidermal Growth Factor Receptor Mutation-Specific Antibodies for Non-small Cell Lung Cancer: Immunohistochemistry as a Possible Screening Method for Epidermal Growth Factor Receptor Mutations

Epidermal growth factor receptor (EGFR) mutations in non-small cell lung cancer (NSCLC) predict better outcome to EGFR tyrosine kinase inhibitors. The most common mutations are exon 19 deletions (most frequently E746-A750) and L858R point mutation in exon 21. Here, we evaluated the accuracy of novel EGFR mutation-specific antibodies in a Japanese cohort with NSCLC and compared with direct DNA sequencing and clinical outcome. Immunohistochemistry (IHC) using antibodies specific for the E746-A750 and L858R mutations in EGFR was performed on tissue microarrays of tumors from 70 gefitinib treated NSCLC patients. Extracted DNA was sequenced for mutational analysis of EGFR exons 18 to 21. DNA sequencing showed EGFR mutations in 41 patients (58.6%) and exon 19 deletions in 18 patients (25.7%), 11 of 18 (61%) had a deletion in the range of E746-A750 and 12 (17.1%) had exon 21 mutations (L858R). IHC showed, for the E746-A750 and L858R mutations, sensitivity (81.8 and 75%), specificity (100 and 96.6%), positive predictive value (100 and 81.8%), and negative predictive value (96.7 and 94.9%). Analysis for objective response rates and survival were not correlated to IHC staining, although the combined staining showed nonsignificant trends toward better overall survival for patients with EGFR mutations. The mutation-specific IHC antibodies have high sensitivity and specificity for predefined EFGR mutations and may be suitable for screening for these predefined mutations. However, negative IHC results require further mutation analyses before excluding EGFR-targeted therapy.

Molecular Diagnosis of Activating EGFR Mutations in Non–Small Cell Lung Cancer Using Mutation-Specific Antibodies for Immunohistochemical Analysis

Therapeutic responses of non-small cell lung carcinoma (NSCLC) to epidermal growth factor receptor (EGFR)-targeted drugs, such as gefitinib and erlotinib, are closely associated with activating EGFR mutations. The most common mutations are delE746-A750 in exon 19 and L858R in exon 21, accounting for approximately 90% of all EGFR mutations. Recently, EGFR mutation-specific antibodies were developed and did well in immunohistochemical analysis, giving a sensitivity of approximately 90%. We have investigated whether this method detects activating EGFR mutations with sensitivity comparable with direct DNA sequencing, which is used to detect these mutations in NSCLC. We used antibodies specific for the E746-A750 deletion mutation in exon 19 and the L858R point mutation in exon 21 in Western blot analysis and immunohistochemistry to determine the presence of these mutations in NSCLC cell lines. We also examined these EGFR mutations in NSCLC tumor samples from 60 patients by immunohistochemically and direct DNA sequencing. We were able to identify EGFR mutations in NSCLC tumor samples immunohistochemically with a sensitivity of 79% using the anti-delE746-A750 antibody and 83% using the anti-L858R antibody. Additional DNA sequencing markedly improved the sensitivity obtained by immunohistochemistry. This simple and rapid assay for detecting EGFR mutations, even in the small bronchial biopsies obtained in stage IV NSCLC patients, will be useful for diagnosing responsiveness to EGFR-targeted drugs in patients with NSCLC. Combining this with DNA sequencing is recommended for the development of improved personalized EGFR-targeted therapeutics.