pharmDxTM Kit Research Articles

P2.01-047 Intra- and Inter-Observer Reproducibility Study of PD-L1 Biomarker in Non-Small Cell Lung Cancer (NSCLC) - The DREAM STUDY

PD-L1 expression in NSCLC correlates with increased response to pembrolizumab, supporting its role as a predictive biomarker but the reproducibility of pathologists’ scoring of PD-L1 requires further investigation. The primary objective of the DREAM study was to assess the reproducibility of scoring PD-L1 staining by evaluating intra- and inter-observer reproducibility for the assessment of PD-L1 expression in NSCLC. The secondary objective was to assess the impact of training on reproducibility. The study was a blinded, pathologist reproducibility study of scoring PD-L1 expression in NSCLC cases stained with PD-L1 22C3 pharmDx™ kit using the Dako Automated Link 48 Platform. Two pathologists previously trained and certified by Dako scored 789 specimens to form the gold standard. From these specimens 60 were randomly selected to evaluate a 1% cut-point and 60 for a 50% cut-point. Both sample sets were designed to include 50% positive/negative specimens and 20-30 close to each cut-point. Ten pathologists were randomly assigned to two subgroups. Subgroup 1 analyzed all samples on two consecutive days. Subgroup 2 performed the same assessments, except they received a one hour training session prior to the second assessment. The overall percent agreement (OPA) for the analysis of the intra-observer reproducibility was 89.7% (95% CI: 85.7; 92.6) for the 1% cut-point sample set and 91.3% (95% CI: 87.6; 94.0) for the 50% cut-point. The OPAs for inter-observer reproducibility of all ten pathologists were 84.2% (95% CI: 82.8; 85.5) and 81.9% (95% CI: 80.4; 83.3) for the 1% and 50% cut-point sample sets, respectively. There was substantial agreement at both the 1% cut-point (prevalence-adjusted bias-adjusted kappa 0.68 (95% CI: 0.65; 0.71)) and the 50% cut-point (prevalence-adjusted bias-adjusted kappa 0.64 (95% CI: 0.61; 0.67)). Training was found to have no or very little impact on the inter- or intra-observer reproducibility in subgroup 2. The OPAs for the inter-observer reproducibility assessments were 82.0% and 82.3% for the first and second assessments of the 1% cut-point sample set, respectively, and 78.3% and 81.7% for the first and second assessments of the 50% cut-point sample set, respectively. The exploratory analyses showed that the sensitivity and specificity of the pathologists assessments, compared with the gold standard assessment, were 84.3% and 91.3%, respectively, for the 1% cut-point and 56.3% and 94.0%, respectively, for the 50% cut-point. There is high intra-observer reproducibility and substantial inter-observer agreement in pathologists’ assessment of PD-L1 expression in NSCLC at 1% and 50% cut-points.

SC10.03 Anti-EGFR Monoclonal Antibodies in Squamous Cell NSCLC

SC10.03 Anti-EGFR Monoclonal Antibodies in Squamous Cell NSCLC

The impact of co-expression of wild-type EGFR and its ligands determined by immunohistochemistry for response to treatment with cetuximab in patients with metastatic colorectal cancer.

Anti-EGFR mAbs cetuximab and panitumumab are routinely used for the treatment of patients with KRAS-wild type metastatic colorectal cancer (mCRC). However, in some patients their efficacy remains modest and with no clear association between the EGFR protein expression determined by PharmDx™ kit, and response to anti-EGFR therapies. Therefore, we investigated the relative expression and predictive value of wild-type EGFR (wtEGFR), mutated EGFRvIII and EGFR ligand proteins in mCRC patients treated with cetuximab. The expression levels of wtEGFR, EGFRvIII, and EGFR ligand were determined by immunohistochemistry (IHC) in 60 tumour specimens using specific antibodies. Sections were scored according to the percentage of positive tumour cells, intensity and cellular location of staining, and these were associated with response, overall survival (OS) and progression-free survival (PFS). At cut-off value > 5%, wtEGFR, and EGFRvIII were present in 44%, and 41%, betacellulin (BTC) in 72%, followed by epigen (67%), TGFα (58%), amphiregulin (34%), EGF (31%) of the cases, respectively and 96% of the wtEGFR positive cases had co-expression of at least one ligand. We found a significant association between the expression of wtEGFR and poor response to cetuximab. In addition, the co-expression of wtEGFR with one ligand at a cut-off value of > 5% and > 10% was associated with worse response to cetuximab (P = 0.021, and P = 0.005 respectively). We found a 3-fold and 5-fold increased risk of shorter OS with expression of BTC and epigen. Interestingly, the expression of wtEGFR and its co-expression with one or two ligands was associated with shorter PFS but not with OS. The relative expression of wtEGFR and its competing ligands, which is the target for therapeutic interventions with anti-EGFR antibodies, could serve as a more reliable predictive biomarker of response to therapy with anti-EGFR mAbs in mCRC patients and warrants further investigation in large prospective studies.

Abstract 5014: Predictive value of wild-type EGFR determined by immunohistochemistry for response to treatment with cetuximab in patients with metastatic colorectal cancer

Abstract The aberrant expression of the epidermal growth factor receptor (EGFR) has been reported in patients with colorectal cancer, and EGFR is an important therapeutic target in such patients. To date, of the anti-EGFR monoclonal antibodies (mAbs), only cetuximab and panitumumab have been approved for the treatment of patients with metastatic colorectal cancer (mCRC). While treatment with a combination of antibodies and cytotoxic drugs improves response rate and median time to progression in some colorectal cancer patients, the duration of response is often limited. In addition, no clear association has been found between the expression level of the EGFR protein in the tumours, determined by the FDA-approved EGFR PharmDx kit, or other standard anti-EGFR antibodies, and the response to the EGFR inhibitors. We have previously shown that kits such as the EGFR PharmDx™ kit, used to determine the expression of EGFR, does not discriminate between the wild-type EGFR (wtEGFR) and the type III deletion mutant EGFR (EGFRvIII), and as such could have contributed to the lack of association between the expression of EGFR and response to anti-EGFR antibodies. Therefore, the aim of this study was to determine the predictive value of wtEGFR for response to therapy with cetuximab. The expression levels of wtEGFR, mutated EGFRvIII, and phosphorylated EGFR (pEGFR Tyr1068 & Tyr1173) were determined using immunohistochemistry and specific antibodies in 61 FFPE tumour specimens from patients with mCRC treated with cetuximab. Sections were scored by the percentage of positive tumour cells, location and intensity of staining. Their associations with response and progression free survival were evaluated using Chi-squared and Kaplan-Meier survival curves and log-rank test respectively. Overall 11% and 46% of the cases were found to express membranous and cytoplasmic EGFR, using mAb specific for wtEGFR (DAK-H1-WT). On the other hand using anti EGFR.113 clone antibody, which does not discriminate between the wtEGFR and EGFRvIII, 11.5% and 18% of the cases were found to express the cytoplasmic and membranous EGFR respectively. While all cases were negative for the pEGFR, the expression of EGFRvIII was mainly cytoplasmic, occurring in 41% of the cases examined. Of these, only the cytoplasmic expression of wtEGFR was found to be significantly associated with poor response to treatment (P = 0.002) and progression free survival (95% CI, 0-3 months vs 4.5-17 months, P = 0.001). Taken together, our results suggest that cytoplasmic expression of wtEGFR is associated with a significantly shorter progression free survival, and is a predictive marker for poor response to treatment with the anti-EGFR mAb cetuximab. These findings underlie the need for further investigations, in a larger population of patients, to validate the predictive value of wtEGFR for response to therapy with anti-EGFR antibodies in patients with mCRC. Citation Format: Said Abdullah Khelwatty, Sharadah Essapen, Izhar Bagwan, Margaret Green, Alan Seddon, Helmout Modjtahedi. Predictive value of wild-type EGFR determined by immunohistochemistry for response to treatment with cetuximab in patients with metastatic colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5014.

Changes in human epidermal growth factor receptor 2 status between primary breast/gastric carcinomas and synchronous metastatic lymph nodes: how can we explain them?

Aim: Previous studies demonstrated discordant expression of human epidermal growth-factor receptor 2 (HER2) between primary cancer and their recurrence/metastasis. This study further evaluated HER2 status between primary gastric and breast invasive carcinomas and paired metastatic disease to lymph nodes. Methods: This study collected formalin-fixed paraffin-embedded representative tissue blocks from 62 gastric and 65 breast primary carcinomas as well as synchronous metastatic lymph nodes (male:female = 39:88; age ranged between 44 and 95 years with mean age of 69.32 years) for immunohistochemical staining of HER2 expression (DAKO HercepTest ™ kit). If immunohistochemical HER2 score reached to 2+, HER2 amplification was then assessed using fluorescence in situ hybridization (PharmDx ™ kit DAKO). Results: The discordant HER2 pooled rate, regardless either negative or positive conversion, was 9.67% in primary gastric carcinoma and corresponding nodal metastasis, while the changes in HER2 expression were revealed in 4.61% of mammary and lymph node neoplastic samples. A high-level concordance in HER2 expression between primary carcinoma and synchronous metastatic lymph nodes was confirmed in both types of cancer; the observed event of discordant HER2 status should be ascribed to intra-tumor heterogeneity, mostly appreciable in gastric cancer. Conclusion: In any case, the shift from positive to negative HER2 expression suggests that trastuzumab could be the targeted treatment choice whereas the opposite shift should be evaluated by a simultaneous HER2 determination in both primary and metastatic lymph nodes.

221P - Evaluation of Met Staining in Gastric/Gastroesophageal Junction (G/Gej) Tumor Samples As a Biomarker for Rilotumumab (R) Benefit

ABSTRACT Aim: R is an investigational, fully human monoclonal antibody against hepatocyte growth factor, the only known ligand for the MET proto-oncogene. In a phase 2 study in G/GEJ cancer, trends towards improved overall survival (OS) and progression-free survival (PFS) were seen with R + epirubicin, cisplatin, and capecitabine (ECX) vs placebo + ECX, with benefit due to the MET-positive patients (pts). We describe a methodology to select a cutoff for defining MET positivity. Methods: Eligible pts had unresectable locally advanced or metastatic G/GEJ adenocarcinoma, ECOG performance status ≤ 1, and no prior systemic therapy for this disease. Pts were randomized 1:1:1 to R 15 mg/kg, R 7.5 mg/kg, or placebo IV day 1 Q3W plus ECX (50 mg/m2 IV day 1, 60 mg/m2 IV day 1, 625 mg/m2 BID orally days 1–21, respectively). Formalin-fixed paraffin-embedded archival tumor tissues were stained with the Dako MET IHC pharmDx™ kit, which uses the MET4 antibody. Membrane, cytoplasmic, and total (cytoplasmic and membrane) tumor cell staining were evaluated separately. Percent staining at different intensities (0, 1 + , 2 + , 3+) and combinations of intensities were scored directly, whereas overall percent positive, H-score, and predominant staining intensity were derived scores. OS and PFS for subgroups defined by potential cutoff values (5–95%) at 5% increments were analyzed by Cox proportional hazards regression, Kaplan-Meier, and the log rank test, yielding hazard ratios (HRs), median OS and PFS, and p values. Results: 121 pts were randomized; 91 had tumor samples evaluable for MET IHC. Pts whose tumors showed ≥ 25% membranous staining were classified as MET-positive. A strong treatment benefit (R vs placebo) was seen in MET-positive pts (n = 58; OS: HR = 0.46, 95% CI, 0.24–0.87; PFS: HR = 0.46, 95% CI, 0.25–0.85); median OS: 10.6 vs 5.7 mo; median PFS: 6.8 vs 4.4 mo. No benefit or detriment was seen with R in MET-negative pts (n = 33; OS: HR = 1.23; 95% CI, 0.56–2.70; PFS: HR = 1.00; 95% CI, 0.46–2.16). Cutoff values 25–50% showed a similar benefit of R in MET-positive pts. Conclusions: The subgroup defined by a cutoff of 25% MET-positive membranous staining using the Dako MET IHC pharmDx™ kit showed benefit from R + ECX; no negative impact of R was seen in MET-negative pts. This cutoff is being used to select pts in an ongoing phase 3 study of R + ECX in MET-positive G/GEJ cancer (NCT01697072). Disclosure: M.D. Hale, K.S. Oliner, R. Tang, L. Chen, E. Loh and S.D. Patterson have declared:is an employee of and owns stock in Amgen Inc.; S. Webster: Scott Webster is an employee of Dako North America, an Agilent Technologies Company. All other authors have declared no conflicts of interest.

The HER2 CISH pharmDx™ Kit in the assessment of breast cancer patients for anti-HER2 treatment

Testing for amplification of the human EGF receptor 2 (HER2) gene by in situ hybridization is a central principle for the identification of breast cancer patients likely to respond to treatments directed toward HER2. However, its application in clinical routine has been somewhat restricted by the typical use of a visualization system based on fluorescence (FISH), which requires skilled, work-intensive, high-magnification quantitative microscopy. The US FDA has recently approved the HER2 CISH pharmDx™ Kit, which is characterized by employing a chromogenic visualization system that allows quantification of the HER2 gene and centromere 17 reference signals by relatively low-magnification brightfield microscopy. It is indicated as an aid in the assessment of patients for whom Herceptin® (trastuzumab) treatment is being considered.

Abstract P1-07-02: New high-quality HER2 IQFISH pharmDx™ assay with a ½ working day procedure and high concordance to HER2 FISH pharmDx™

Abstract Introduction: HER2 assessment for selection of patients that may benefit from HER2 targeting treatment can be performed by either immunohistochemistry (IHC), fluorescence or chromogen in situ hybridization (FISH or CISH). FISH is a robust and reliable technique for direct visualization and quantitative determination of gene amplifications, deletions and translocations in human cancer cells, but FISH protocols are time-consuming and involve toxic reagents. By introducing a new non-toxic ethylene carbonate based hybridization buffer that perform with very short hybridization times, the total FISH assay time on breast cancer tissue sections can be reduced from the traditional 16–20 hours to 3½–4½ hours. Material and methods: The new Dako HER2 IQFISH pharmDx™ was compared with Dako HER2 FISH pharmDx™ in a comparative study on 120 breast cancer specimens, and reproducibility of the HER2 IQFISH pharmDx™ assay was investigated in a study comprising 3 different sites and a total of 6 different observers. Samples for the comparative study was evaluated by Dako HercepTest™ to include all IHC scoring groups (0, 1+, 2+, 3+). Slides were stained according to manufacturer's instructions using microwave oven for heat pretreatment and RTU pepsin for 3–5 minutes at 37 °C. Hybridization was performed for 2 hours when using HER2 IQFISH pharmDx™ and for 17–20 hours when using HER2 FISH pharmDx™ Kit. All slides were blinded before evaluation. HER2 status was classified as “Non-amplified” when the HER2/CEN17 ratio < 2.0 and “Amplified” when the HER2/CEN17 ratio ≥ 2.0. Results: The new non-toxic hybridization buffer introduces a major safety improvement since formamide is no longer needed. Significantly shorter hybridization times are required to generate the same signal intensity (1–2 hour hybridization versus overnight). HER2 IQFISH pharmDx™ was compared with the traditional HER2 FISH pharmDx™ in a comparative study on 120 breast tissue specimens of human breast carcinoma. The preliminary data on HER2 status for 78 patients obtained by the two assays gave an overall agreement of 98.7% with lower and upper 95% confidence limits at 94.2% and 99.9%. The Kappa value was 0.96 (95% CL: 0.89–1.00). The p-value for McNemars test was 1.00 indicating absence of bias between the two assays. Disagreement between the two assays was observed for one specimen - a heterogeneous tissue with a small amplified area. Data from the reproducibility study that included site-to-site variation, day-to-day variation and inter-observer variation showed that the assay has a high degree of reproducibility. Conclusion: The validation studies of the new HER2 IQFISH pharmDx™ showed a very high concordance to the traditional HER2 FISH pharmDx™ and also that the assay is robust and reproducible. Reduction of the overall assay time from a two-day to a half-day procedure for HER2 FISH, offers more flexible laboratory routines and same day reporting for all working days of the week, which could be used for fast and simultaneous FISH and IHC answers and improved patient care. Taken together, the study demonstrates the potential of a new revolutionary platform that enables optimization and acceleration of FISH analysis to the benefit of cancer patients and laboratory personnel. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-07-02.

P3.05 A New Non-Toxic Hybridization Buffer Reduces the Fish Assay Time From 16-20 Hours to 3½ Hours Without Compromising the Quality

ABSTRACT Fluorescence in-situ hybridization (FISH) is a powerful technique for direct visualization and quantitative determination of gene amplifications, deletions and translocations in human cancer cells. It is recognized as a reliable and reproducible method, and FISH assays are often referred to as 'Gold Standard' for determining the patient status with predictive tests for HER2 and ALK. But FISH is also known to have characteristics which limit a more widespread use, including the long assay time (staining and scoring), requirement for a fluorescence microscope and complexity involved in handling small volumes and toxic reagents. Dako has addressed the toxicity and long hybridization time by introducing a new hybridization buffer. The new non-toxic hybridization buffer (IQFISH buffer), which allows for significant reduction in hybridization time, brings the total assay time for a FISH assay down from 16-20 hours to just 3½ hours without compromising the quality. The new Dako HER2 IQFISH pharmDx™ (assay time 3½ hours) has been compared with the traditional Dako HER2 FISH pharmDx™ Kit (assay time 16-20 hours) in a comparative study on 78 breast tissue specimens of human breast carcinoma ( Fig 1 ). Cross tabulation of HER2 status obtained by the two assays gave an overall agreement of 98.7% (95% CI: 94.2%-99.9%). Disagreement between the two assays was observed for one specimen that most likely was a heterogeneous tissue with a very small area of amplification. A similar comparative study on 79 gastric adenocarcinoma specimens showed an overall agreement of 98.7% (95% CI: 94.2%-99.9%). The disconcordant sample was a heterogeneous tissue with a very small area of amplification. A third concordance study on 80 breast cancer tissues compared TOP2A IQFISH pharmDx™ with the traditional TOP2A FISH pharmDx™ Kit ( Fig 2 ). A 100% overall agreement (95% CI: 96.9%-100%) was found. Conclusion: We have developed new non-toxic hybridization buffer that by reduction of the hybridization time from 16 to 2 hours allows for a ½ day FISH protocol. The IQFISH buffer has been validated for HER2 on breast and gastric cancer and for TOP2A on breast cancer. Both assays showed a very high agreement with the traditional 2 day FISH procedure. Download : Download full-size image Fig. 1 . Agreement between the new Dako HER2 IQFISH pharmDx™ and the traditional Dako HER2 FISH pharmDx™ Kit. Download : Download full-size image Fig. 2 . Agreement between the new Dako TOP2A IQFISH pharmDx™ and the traditional Dako TOP2A FISH pharmDx™ Kit.

Membranous and cytoplasmic expression of epidermal growth factor receptor in metastatic pancreatic ductal adenocarcinoma

Recent studies indicate the clinical significance of the cellular localization of epidermal growth factor receptor (EGFR) in a variety of cancer types. Internalization of activated EGFR is reported to be closely associated with patient prognosis. This study investigated the clinical significance of the immunohistochemical localization of EGFR in patients with metastatic pancreatic cancers compared to those with surgically resected pancreatic cancers. Using 44 surgically resected primary pancreatic cancers and 40 primary or meta-static tumors from 20 autopsied patients with far advanced pancreatic cancers, the incidence of membranous and cytoplasmic EGFR overexpression was compared between primary tumors and far advanced tumors by immunohistochemistry using the Dako EGFR pharmDx™ kit, a global standard kit for EGFR assay. In the 44 surgically resected cancers, 13 (30%) exhibited membranous overexpression of EGFR, comprising 1 case (2%) with score 3+ and 12 cases (27%) with score 2+ and 10 (23%) exhibited cytoplasmic overexpression of EGFR. In the 40 tumors at a far advanced stage, the percentage of samples exhibiting positivity for membranous and cytoplasmic EGFR overexpression was 48% (19 of 40) comprising 7 (18%) with score 2+ and 12 (30%) with score 3+ and 33% (13 of 40), respectively. The far advanced tumors tended to show membranous and cytoplasmic EGFR overexpression more frequently than the surgically resected tumors, although the difference was not significant. These findings suggest that membranous and cytoplasmic overexpression of EGFR may be indicative of the potential aggressiveness of pancreatic cancers.

Dual color chromogenic in situ hybridization for determination of HER2 status in breast cancer: a large comparative study to current state of the art fluorescence in situ hybridization

BackgroundChromogenic in situ hybridization (CISH) is fast becoming a well established technique for easy and sensitive determination of HER2 gene status in breast cancer. However, for the chromogenic method to achieve status as a safe and reliable technique, the method needs to be validated against already known and validated FISH techniques.MethodsHere it is reported from a comparative study where HER2 gene status obtained by HER2 CISH pharmDx™ Kit was compared to HER2 gene status obtained by the FDA approved HER2 FISH pharmDx™ Kit and the PathVysion HER-2 DNA probe Kit. The study included 365 formalin fixed and paraffin-embedded invasive breast cancer tissue specimens collected consecutively at a US reference laboratory.ResultsThe data obtained revealed an overall HER2 status concordance of approximately 98% for comparisons of HER2 CISH pharmDx™ Kit to both HER2 FISH pharmDx™ Kit and PathVysion HER-2 DNA Probe Kit.ConclusionsThe concordance between results obtained using the recently FDA approved HER2 CISH pharmDx™ Kit with previously FDA approved FISH techniques for HER2 gene status determination indicate that the HER2 CISH pharmDx™ Kit is a reliable chromogenic alternative to fluorescence-based methods.

Epidermal growth factor receptor overexpression in resected pancreatic cancer.

317 Background: The effect of overexpression of epidermal growth factor receptor (EGFR) in pancreatic carcinoma is uncertain. In this study, we examine the association between EGFR overexpression (membrane and cytoplasmic) and histopathologic features and clinical outcomes in patients post resection. Methods: Seventy three patients underwent curative resection for pancreatic cancer from 4/08 to 4/10. Tumor EGFR expression was determined immunohistochemically using EGFR pharmDxTM kit. Cytoplasmic overexpression was considered positive if EGFR expression was seen in the cytoplasm in ≥10% of cells. Cell membrane staining scores were assigned: 0 (no membrane staining), 1+ (incomplete membrane staining in ≥ 10% of cells, 2+ (complete weak/moderate membrane staining in ≥ 10% of cells) and 3+ (complete heavy staining in ≥ 10% of cells). A score of 2+ and 3+ were considered as membrane overexpression. Comparisons between EGFR overexpression and clinicopathologic features were assessed by chi-square test with p-value < 0.05 as statistically significant. Proportional hazard regression was used to estimate progression free survival and overall survival. Results: Out of 73 patients, 43 (59%) and 50 (68%) patients had membrane and cytoplasmic EGFR overexpression respectively. AJCC stages were: stage I: 8, stage II: 63, stage III: 1 and stage IV: 1 patient. There was statistically significant correlation between cell membrane EGFR overexpression and lymph node positivity (p=0.04). Membrane EGFR overexpression was not significantly associated with local invasion, margin positivity, vascular invasion, perineural invasion, histologic grade or stage. Cytoplasmic EFGR overexpression was significantly associated with margin positivity (p=0.01) only. The hazard ratios (95% CI) for PFS and OS in patients with membrane EGFR overexpression was 1.37 (0.72-2.60) and 1.27 (0.62-2.61) respectively. The corresponding values for patients with cytoplasmic overexpression were 1.65 (0.80-3.37) and 1.25 (0.58-2.71) respectively. Conclusions: Membrane EGFR overexpression was associated with lymph node status and cytoplasmic EFGR overexpression with margin positivity. There was no association of EFGR expression and clinical outcomes. No significant financial relationships to disclose.

Study on breast carcinoma Her2/neu and hormonal receptors status assessed by automated images analysis systems: ACIS III (Dako) and ScanScope (Aperio).

Her-2/neu is overexpressed in 20-30% of breast cancer patients and is associated with a more aggressive disease. Identification of Her-2/c-erbB-2-neu overexpression is based on immunohistochemical [ihc] detection of protein and/or gene amplification in fluorescence in situ hybridization test (FISH). Also Estrogen receptors [ER] and Progesterone receptors [PR] are the prognostic and predictive biomarkers, recently analysed by ihc methods. Subjective, manual scoring of the ihc Her-2/neu expression and expression of the ER/PR reported as the percentage of immunopositive cells are the most common mode of interpretation among pathologists. Automated microscopy and computerised processing have provided increased accuracy in quantification and standardisation. to evaluate the scoring reproducibility of Her-2 /neu ihc expression tested by two automated systems: ACIS (Dako) and ScanScope (Aperio); to estimate the ER/PR expression in ihc staining methods with different anti-ER/anti-PR antibodies (the monoclonal and the ER/PR pharmDx TM Kit) by the ACIS system. Her-2/neu ihc expression was measured in 114 primary invasive breast carcinomas by the manual and the automated scoring (ACIS and Aperio system). 106 slides stained ihc with two types of anti-ER/anti-PR antibodies entered the quantisation. The results of our investigations showed very high reproducibility of Her-2/neu scores in intra- and interobserver analysis by ACIS evaluation. The major concordance was present in strong 3+ ihc cases; very small discordance was shown by cases with low expression of Her-2/neu. The accuracy of scoring by the Aperio was little lower in comparison to ACIS but it might result from the smaller and variable series of samples analysed by Aperio. The concordance in scoring of two automated systems was 86.5% (p<0.0001; gamma=0.887); the discordance was referred only to the lower expression of Her-2/neu. The concordance in manual scoring performed by the single observer and the panel was 84.2% (p<0.0001, gamma = 0.99); the discordance comprised a few cases with strong expression (2+ vs 3+). Very high intra- and interobserver reproducibility of the ER/PR ihc measurements was present in the readers results (referred to the percentage of immunoreactive carcinomatous cell population in the breast carcinomas acc. to the ACIS algorithm). No differences were disclosed in the percentage of ER-immunoreactive and PR-immunoreactive carcinomatous cell populations when used 2 different type of antibodies, in the ACIS automated method.

Abstract 694: Immunohistochemical discrimination of wild type EGFR from EGFRvIII in fixed tumor specimens using anti-EGFR mAbs ICR9 and ICR10

Abstract Abnormal expression of the epidermal growth factor receptor (EGFR) has been reported in a wide range of human epithelial malignancies and in some studies has been associated with poor prognosis and treatment resistance. Currently, three different types of EGFR inhibitors have been approved by the FDA for the treatment of patients with head and neck, metastatic colorectal, pancreatic or breast cancers: anti-EGFR monoclonal antibodies [(mAbs), cetuximab and panitumumab], small molecule EGFR tyrosine kinase inhibitors [(TKIs) gefitinib, erlotinib] or a dual EGFR and HER-2 tyrosine kinase inhibitor (lapatinib) However, there has been no clear association between the expression levels of EGFR in the tumours determined by the FDA approved EGFR PharmDx™ kit (DakoCytomation) or other standard anti-EGFR antibodies and the response to the EGFR inhibitors. One reason could be that the antibody used in the diagnosis of EGFR is not specific to the wild type EGFR and can also bind to the type-III mutant form of EGFR (EGFRvIII). We have extensively profiled our unique panel of high affinity rat anti-EGFR mAbs for use in the diagnosis and therapy of human cancers. The aim of the present investigation was to evaluate the potential of several of these antibodies (ICR9, ICR10, ICR16) for immunohistochemical diagnosis of wild type EGFR and/or EGFRvIII in formalin fixed paraffin embedded tumour specimens. We prepared 10% (v/v) neutral buffered formalin paraffin embedded sections of HN5 and HC2 20d2/c pelleted cells, which overexpress wild type EGFR and EGFRvIII respectively. Tumour sections were incubated with primary anti-EGFR mAbs followed by HRP linked secondary antibody (ABD Serotec Ltd., UK). The sections were then treated with DAB+ substrate-chromogen solution (Dako), counterstained with haematoxylin and mounted. The EGFR PharmDx™ kit, containing positive and negative control cell lines was purchased from Dako (UK) and the immunostaining of sections was conducted according to the manufacturer's protocol. We found that the mouse anti-EGFR mAb in the EGFR pharmDx™ kit stained both wild type and EGFRvIII expressing cells in the formalin fixed paraffin embedded sections. This pattern of EGFR immunostaining was also found with our anti-EGFR mAb ICR16. In contrast, mAbs ICR9 and ICR10, which are directed against two distinct epitopes on the external domain of the EGFR, were specific for wild-type EGFR in formalin-fixed paraffin embedded tumour specimens. We conclude that mAbs ICR9 and ICR10 are ideal tools for investigating the expression patterns of wild type EGFR (membranous, nuclear, and cytoplasmic) in tumour specimens using immunohistochemistry and to determine their prognostic significance as well as predictive value for response to therapy with EGFR antibodies. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 694.

TOP2A, TIMP-1 and responsiveness to adjuvant anthracycline containing chemotherapy in high risk breast cancer patients.

Abstract Abstract #36 Background: Anthracyclins are potent poisons for topoisomerase II, an indispensable enzyme for DNA replication, repair and transcription. Apoptosis is often the consequence of anthracycline induced DNA breaks but cancer cells may escape through activation of the cell survival pathway by Tissue Inhibitor of Metalloproteinases-1 (TIMP-1). The aim of the present study was to determine the predictive value of the combination of the two biomarkers (TOP2A and TIMP-1) for benefits observed with CEF compared to CMF in DBCG trial 89D.&amp;#x2028; Material and Methods: The DBCG 89-D trial randomized 980 high-risk Danish breast cancer patients to nine series of CMF or CEF, without endocrine therapy. Overall CEF was superior to CMF in terms of DFS and OS (Ejlertsen et al, EJC 2007). TOP2A copy number changes were determined with the TOP2A FISH pharmDx™ Kit (Dako A/S, Glostrup) as described previously (Knoop et al, JCO 2005). TMAs were constructed and analyzed centrally for TIMP-1 expression using the anti-TIMP-1 monoclonal antibody VT7 (Sørensen et al, J. Hist. Cytochem. 2006). A combined marker (2T) for anthracycline sensitivity was constructed as follows: 2T non-responsive (TOP2A normal and TIMP-1 positive) and 2T responsive (TOP2A abnormal and/or TIMP-1 negative). Relationships between IDFS, OS and the 2T profile were analyzed using multivariate regression analysis.&amp;#x2028; Results: The assessable 623 patients differed significantly (P&amp;lt;0.05) from the non-assessable patients with regard to menopausal status, tumor size, malignancy grade, ER and HER2 status. Among the 347 patients treated with CMF 150 (43%) had tumors with a 2T responsive profile, compared with 119 (43%) of the 276 patients treated with CEF.&amp;#x2028; Among patients with a 2T responsive profile the Cox regression analysis showed that treatment with CEF was superior to CMF in terms of both invasive disease-free survival (IDFS) (adjusted hazard ratio 0.48; 95% CI 0.34 to 0.69; P&amp;lt;0.001) and overall survival (OS) (adjusted hazard ratio 0.54; 95% CI, 0.38 to 0.77; P&amp;lt;0.001). In contrast, among patients with a 2T non-responsive profile, no significant difference between CEF and CMF was demonstrated in terms of IDFS (hazard ratio 1.18; 95% CI 0.87 to 1.60; P=0.29) or OS (hazard ratio 1.07; 95% CI, 0.78 to 1.47; P=0.68). A highly significant 2T profile (responsive or non-responsive) versus treatment (CEF or CMF) interaction was detected in the Cox analysis by the Wald-test for both IDFS (P&amp;lt;0.001) and OS (P=0.004). Discussion: TOP2A aberrations and absence of TIMP-1 immunoreactivity are associated with clinical benefits from anthracycline-containing therapy in patients with early breast cancer. In combination the two biomarkers identify a substantial part of the patients (43%) and the use of the 2T profile seems advantageous compared to using either marker alone. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 36.

High TOP2A mRNA expression occurs in the absence of TOP2A or ERBB2 gene amplification

22057 Background: Topoisomerase II alpha, encoded by TOP2A, is the putative molecular target of anthracycline drug action. Recently, it has been shown that TOP2A gene amplification occurs almost exclusively in conjunction with ERBB2 amplification. Since no benefit of anthracylines was observed in ERRB2 negative patients it was hypothesized that any anthracycline benefit might be restricted to those patients with TOP2A and ERBB2 co-amplification. Since alternative treatment options exist for ERBB2 positive breast cancer, the role of anthracycline-based therapy in the adjuvant treatment of breast cancer has been questioned Methods: In a series of 53 formalin-fixed paraffin-embedded (FFPE) tissue samples from patients with invasive breast carcinomas we analysed TOP2A and ERBB2 gene amplification as well as RNA expression. Amplifications were determined using the HER2 FISH pharmDx™ kit and the TOP2A FISH pharmDxTM kit (both from DakoCytomation, Denmark A/S, Glostrup). In addition, RNA was isolated from corresponding formalin fixed tumors and TOP2A and ERBB2 expression was determined by real time RT-PCR. Results: Neither ERBB2 nor TOP2A amplifications were detected in 40 samples (75.5%). ERBB2 and TOP2A co-amplification was observed in six samples (11.3%). Additional six samples showed ERBB2 but no TOP2A amplification (11.3%). Only one sample (1.9%) exhibited TOP2A amplification without concomitant ERBB2 amplification. ERBB2 m-RNA expression was distinctly higher in ERBB2 amplified compared to non-amplified tumors. In contrast, a considerable fraction of TOP2A non-amplified tumors showed a similar high TOP2A mRNA expression as TOP2A amplified or TOP2A/ERBB2 co-amplified tumors. Conclusions: Our data confirm that TOP2A amplification predominantly occurs as co-amplification with ERBB2. However, TOP2A gene amplification is not a prerequisite for high TOP2A RNA expression. Therefore, it should be considered that anthracylines might still be a valuable treatment option in ERBB2 negative tumors with high TOP2A mRNA expression. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Siemens Medical Solutions Diagnostics

Controversial evaluation of EGFR protein and gene status in predicting response to anti-EGFR monoclonal antibodies in metastatic colorectal cancer: a case report and review of the literature

Clinical studies showed that only 10% of patients with metastatic colorectal cancer (mCRC) respond to treatment with the anti-epidermal growth factor receptor (EGFR) monoclonal antibodies panitumumab or cetuximab, regardless of the line of treatment. The current tool used to select patients, i.e. immunohistochemistry (IHC) evaluation of EGFR expression by EGFR pharmDx™ Kit, is not reliable in predicting response. Retrospective analyses of factors such as increased EGFR gene copy number and KRAS and/or BRAF mutations showed that such molecular changes could affect clinical benefit from anti-EGFR monoclonal antibodies. We report here the case of a 66-year-old man with chemorefractory mCRC, considered not eligible to salvage treatment with the anti-EGFR monoclonal antibody cetuximab and irinotecan because the primary adenocarcinoma of the rectum was found not expressing EGFR protein by IHC. However, FISH analysis of EGFR gene copy number and evaluation of KRAS and/or BRAF specific mutations by gene sequencing showed characteristics associated with favourable clinical outcome to anti-EGFR therapy. Based on the EGFR protein expression by IHC in a liver metastasis, the patient was then treated with cetuximab plus irinotecan, obtaining symptoms improvement and a dramatic objective tumor response in all sites of disease, lasting 4.2 months. We also discuss literature findings about the role of different biological characteristics in predicting clinical benefit from anti-EGFR therapy in patients with mCRC.

Implications of EGFR PharmDx™ Kit for cetuximab eligibility

EGF receptor (EGFR) represents an attractive target for anticancer therapies in a variety of malignant neoplasms, including colorectal, non-small-cell lung, head and neck carcinomas and gliomas. Monoclonal antibodies, such as cetuximab, are directed against the extracellular EGFR domain, whereas small molecules are targeting the intracellular tyrosine kinase domain. Particularly for application of drugs against extracellular EGFR parts, knowledge about EGFR levels within the cell membrane is of high import, because only EGFR-depending tumors respond to these therapeutic approaches. Immunohistochemical investigation of tissue slides of the primary tumor are performed to screen for EGFR occurrence in tumor cells. Since 2004, the combination of ‘EGFR PharmDx™ kit’, a diagnostic test for EGFR, and subsequent application of cetuximab in EGFR-positive colon carcinomas has been approved by the US FDA. It represents the second approved combination of diagnostic tools and dependent application of monoclonal antibody therapies after the successful HercepTest™/Herceptin™ for breast carcinomas. This proceeding represents an important step toward a personalized cancer therapy with major advantages for patients, mainly reduction of toxic side effects and dramatically increased efficiency.

No statistical impact of intra-tumoral overexpression of EGFR, VEGF and p53 on the overall survival in head and neck squamous cell cancer (HNSCC): Preliminary results of the GORTEC 99–02 randomized trial

6010 Background: Predictive and prognostic factors are warranted to improve the management of HNSCC patients. We designed a prospective study to analyze the p53 status, vascular endothelial growth factor (VEGF) and epidermal growth factor receptor (EGFR) tumoral overexpression of the patients included in the GORTEC 99–02 prospective phase III trial which compared conventional radiotherapy (RT) plus 5FU and carboplatin (FUP) vs RT with concomitant boost and FUP vs very accelerated RT Methods: This propective study has been designed to include 820 patients. We present here the results in a sub group of 219 patients for whom the immunohistochemical overexpression of EGFR, P53 and VEGF was analyzed. Association beetween these markers and TNM staging, age, sex,disease progression and survival was evaluated. Tumor overexpression was assessed by immunohistochemistry for EGFR (Dako EGFR pharmDx™ kit), VEGF (Santa Cruz Biotech), P53 (Dako). EGFR status was defined as positive or negative.If positive, we evaluated the percentage of cells presenting the same intensity of staining. Staining pattern was qualified as “diffuse” if more than 95% of cancer nests were stained with the same intensity, and as “mosaic” if the staining was heterogeneous Statistical analysis were performed using Spearman correlation coefficient, Kruskal-Wallis test for quantitative variables, chi2 and Fisher exact test for qualitative variables and Logrank test and cox model for survival analysis. Multivariate analysis of survival took into account T and N status, tumor localisation and the type of treatment. Results: Positive expression for P53, VEGF and EGFR were respectively found in 56.4%, 61.1% and 94%.Diffuse EGFR status was associated with higher T status (p=0.0141). VEGF(+) status was associated with increased EGFR staining (p=0.0017). Patients with p53+ status were younger than p53(-) (p=0.0362). P53,VEGF and EGFR expressions were not significatively related to survival Conclusions: These preliminary results don’t found any relationship between intra-tumoral markors and survival in HNSCC.Work supported by a french PHRC from the national Institute of Health No significant financial relationships to disclose.

Protocol modifications influence the result of EGF receptor immunodetection by EGFR pharmDx™ in paraffin-embedded cancer tissues

EGF receptor (EGFR) became a useful target for several recently introduced therapies of various cancer types including colorectal, lung, head and neck cancers and glioblastoma. The successful clinical application of these novel molecularly targeted therapies requires the expression of their target, EGFR, determined by nucleic acid based or immunohistochemical techniques. However, until now, immunohistochemistry has not become a reliable diagnostic approach for this purpose. The golden standard for the determination of EGFR protein expression in paraffin-embedded cancer tissues is the EGFR pharmDxTM kit. Here we show that the recommended protocol may not be optimal for EGFR immunodetection. Microwave antigen retrieval and extended primary antibody incubation time converted four out of eight EGFR-negative tumors into EGFR-positive in a study of 50 lung adenocarcinoma cases. Accordingly, we recommend retesting cases negative for EGFR with EGFR pharmDxTM using protocol modifications optimizing antigen retrieval and the incubation periods.