Central Immunohistochemistry Research Articles

Abstract P6-08-10: Prospective study of the impact of the Prosigna™ assay on adjuvant clinical decision-making in women with estrogen receptor-positive, HER2-negative, node-negative breast cancer: A GEICAM study

Abstract Background: Prosigna™ (PAM50) is a standardized test that measures the expression levels of 50 classifier genes in formalin-fixed paraffin-embedded (FFPE) breast tissue tumor samples. It provides subtype classification based on the fundamental biology of an individual patient’s tumor (referred to as "intrinsic subtyping"), as well as a prognostic score (referred to as "risk of recurrence [ROR] score") that predicts the probability of distant recurrence over 10 years. This decision impact study examines whether the Prosigna™ test influences both physician and patient adjuvant treatment selection, beyond standard immunohistochemistry (IHC) testing. Methods: The analytic sample was comprised of postmenopausal women with node-negative, estrogen-receptor positive (ER+), HER2 negative (HER2-), early-stage breast cancer with tumors <5 cm (T1-T2). FFPE surgical specimens were analyzed using Prosigna™ in a central laboratory. Patients were classified according to the intrinsic tumor subtype (i.e., Luminal A, Luminal B, HER2-enriched, basal-like) and ROR score (low, intermediate or high risk groups). The primary endpoint was the effect of the Prosigna™ test on oncologists’ treatment recommendations, and the actual treatment received by patients (hormonal therapy [HT], chemotherapy [CT], chemotherapy and hormonal therapy [CHT]). All samples were analyzed in two independent laboratories to measure site-to-site concordance. Prosigna™ subtypes were compared with IHC intrinsic subtypes based on the St. Gallen 2013 criteria (cut-points: PgR≥20% and Ki67≥14%). Results: A total of 200 patients met eligibility criteria and were enrolled in the study between June 2013 and January 2014. According to Prosigna™ results, intrinsic tumor subtypes of these patients were distributed as follows: 129 Luminal A (64.5%), 66 Luminal B (33.0%), 3 HER2-enriched (1.5%) and 2 basal-like (1.0%). Modifications to the adjuvant treatment recommendations by ROR score can be seen in the following table: Change in physician pre- to post- Prosigna recommendation Low ROR (N=101), N (%)Intermediate ROR (N=66), N (%)High ROR (N=33), N (%)TOTAL (N=200), N (%)HT to CHT0 (0.0)8 (12.1)10 (30.3)18 (9.0)CHT to HT11 (10.9)9 (13.6)0 (0.0)20 (10.0)TOTAL11 (10.9)17 (25.7)10 (30.3)38 (19.0) Treatment decisions changed for 19.0% of all patients: 10.0% and 9.0% of patients went from CHT to HT and HT to CHT, respectively. The percentage of patients who received chemotherapy in the low, intermediate and high risk groups was 5%, 36% and 88%, respectively. Both the central and each local laboratory analyzed the samples using IHC. We found 60% concordance between central IHC and Prosigna™ intrinsic subtypes (Kappa=0.2365). Prosigna™ results were consistent across labs (Kappa = 0.89). Conclusions: The Prosigna™ test can be reliably performed in hospital laboratories to provide useful information beyond standard clinical-pathological variables that oncologists can use to optimize adjuvant treatment decisions in clinical practice. Subtype determined using IHC is not an interchangeable proxy for subtype determined by Prosigna™. *Two last authors have contributed equally to the study. Citation Format: Miguel Martín, Milagros González-Rivera, Serafín Morales, Juan de la Haba, Lucía González-Cortijo, Luis Manso, Joan Albanell, Antonio González-Martín, Sónia González, Angels Arcusa, Luis de la Cruz-Merino, Federico Rojo, Maria Vidal, Uxue Goicoechea, Patricia Galván, Rosalía Caballero, Eva Carrasco, Steven Michalopoulos, John Hornberger, Aleix Prat. Prospective study of the impact of the Prosigna™ assay on adjuvant clinical decision-making in women with estrogen receptor-positive, HER2-negative, node-negative breast cancer: A GEICAM study [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-08-10.

Abstract P2-15-02: HER2 discordant results in local vs. central testing in the phase 3 nelipepimut-S trial and implementation of Leica Bond Oracle HER2 Immunohistochemistry (IHC) System for low and intermediate levels (1+, 2+) of HER2 protein expression as a companion diagn

Abstract Background : The distinction between HER2-positive (IHC 3+ or 2+ with FISH ratio >/= 2) and not overexpressing HER2 has been the focus of many diagnostic tests over the past years in association with development of HER2-targeted therapies. The paucity of therapies developed for the low to intermediate HER2 protein expression populations has resulted in limited attention to their diagnostic precision and accuracy. The development of NeuVaxTM (nelipepimut-S; Galena Biopharma, Inc.) in the defined population requires a HER2 IHC 1+/2+ diagnostic that precisely and accurately ensures identification of targeted patients. We describe discordance rates between local and central testing performed to identify tumors with HER2 IHC 1+/2+ expression that supports the development of a method to validate HER2 1+ and 2+ (FISH < 2.2) patients who receive nelipepimut-S adjuvant therapy. Methods : The Prevention of Recurrence in Early Stage, Node-Positive Breast Cancer with Low to Intermediate HER2 Expression with NeuVaxTM Treatment (PRESENT) study, is a multicenter, multinational, prospective, randomized, double-blind, controlled Phase 3 study assessing efficacy and safety of the peptide vaccine nelipepimut-S, in HLA A2 or A3 positive patients with early stage, node positive breast cancer expressing low and intermediate levels (IHC 1+/2+) of HER2 protein. PRESENT 2-step screening includes HLA testing and central lab confirmation of HER2 1+ or 2+ expression using the DAKO HercepTest. Results : As of 2 June 2014, 1454 patients underwent central IHC testing for HER2 and had a quantifiable result of 0, 1+, 2+, or 3+ for both local and central test. Per local testing, 61% (HER2 1+, n=612; HER2 2+, n=275) were eligible and 39% (HER2 0, n=468; HER2 3+, n=99) were ineligible. Of those eligible by local testing, 67.5% (n=599) were confirmed as eligible per central testing for a discordance rate of 32.5% (n=288). Of the 288 discordant samples tested centrally, 73.6% (n=212) and 26.4% (n=76) were reported as HER2 0 and 3+, respectively. 8.7% (76/877) of patients found to be HER2 1+ or 2+ by local testing were determined to be HER2+ (IHC3+) by central testing. Conclusions : Current tests for HER2 expression are defined by their ability to determine 3+ positivity, yet significant discordance still occurs with nearly 9% false negative rate in this trial. Similarly, marked discordance exists between local and central laboratory test results for HER2 by IHC at 1+/2+ levels of expression. The relatively high discordance rate observed may be due, in part, to the lack of a validated IHC assay for low-to-intermediate expression of HER2 (0, 1+, and 2+). In order to improve accuracy of testing and to develop a companion diagnostic for nelipepimut-S, the Leica Bond Oracle HER2 IHC System has been validated to determine samples across the IHC spectrum (0, 1+, 2+ and 3+) and is now incorporated into HER2 screening for the PRESENT trial as a companion diagnostic to increase accuracy, precision, and specificity in discerning HER2 1+ and 2+ patients. Citation Format: Michelle Melisko, Elizabeth A Mittendorf, Sufia Safina, Michael Schenker, Murray A Brunt, Maria Litwiniuk, John Mackey, Katarina Petrakova, Svitiana Alieva, Lacey Chance, Gavin S Choy, Mark Ahn, Adamm Hamm, Sonia Kumar, Hope S Rugo. HER2 discordant results in local vs. central testing in the phase 3 nelipepimut-S trial and implementation of Leica Bond Oracle HER2 Immunohistochemistry (IHC) System for low and intermediate levels (1+, 2+) of HER2 protein expression as a companion diagn [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-15-02.

Abstract P2-01-02: Methods impacting the false negative rate of sentinel lymph node surgery in patients presenting with node positive breast cancer (T0-T4,N1-2) who receive neoadjuvant chemotherapy – Results from a prospective trial – ACOSOG Z1071 (Alliance)

Abstract Background: The American College of Surgeons Oncology Group (ACOSOG) Z1071 trial (Alliance) reported a false negative rate (FNR) of 12.6% with sentinel lymph node (SLN) surgery after neoadjuvant chemotherapy in women presenting with node-positive breast cancer. Proposed methods to decrease the FNR include clip placement in the positive node at initial diagnosis with confirmation of resection of the clipped node at surgery and inclusion of residual metastatic cells identified by immunohistochemistry (IHC) for cytokeratins and disease measuring <0.2mm on H&E in the definition of node positivity after chemotherapy. Herein we evaluate the impact of these methods on FNR. Methods: Z1071 was a prospective multi-institutional trial in which women with clinical T0-4,N1-2,M0 breast cancer underwent both SLN surgery and axillary dissection (ALND) after neoadjuvant chemotherapy. The primary endpoint defined nodal metastasis as disease >0.2mm on H&E. Slides were submitted for central IHC staining when H&E-negative, unless performed locally. For this analysis, we expanded the definition of node positivity to include metastases less than 0.2mm on H&E and metastatic deposits identified by IHC. In 171 cases, a clip was placed in the node at initial biopsy and in these cases the SLNs and ALND were evaluated by x-ray or pathology to document clip location. FNR in the clip cohort is reported using the primary endpoint definition of node positive disease (>0.2mm on H&E). Results: In the 113 cases where the clip was found in the SLN the FNR was 6.4% (1.8-15.5%). In the 29 cases where the clip was found in the ALND specimen, the FNR was 22.2% (6.4-47.6%). IHC was available on 470 of 525 patients with cN1 disease and 2 SLNs resected. Using the definition of H&E metastasis >0.2mm, the FNR in these patients was 11.3% (34/301, 8.0-15.4%) which decreased to 8.7% (27/311, 5.6-11.8%) when including any disease <0.2mm. SLNs from 16 patients had disease <0.2mm in size. Seven patients previously classified as false negative SLN changed to true positive with identification of disease in the SLN. Nine patients changed from node-negative to node-positive with the only disease being <0.2mm disease found in the SLN. Nodal pathologic complete response rate changed from 36.0% to 33.8% with the inclusion of metastases <0.2mm. NResidual disease identified in SLNs or ALNDFNR (%)95% CINode positive definitionSLN metastases >0.2mm by H&E470301 (64.0%)11.38.0-15.4SLN metastasis any size on IHC or H&E470311 (66.2%)8.75.6-11.8Clip locationClip in SLN11462 (54.4%)6.41.8-15.5Clip in ALND2918 (62.1%)22.26.4-47.6Clip location unknown2920 (69.0%)14.33.0-36.3Clip not placed354207 (58.5%)13.59.1-18.9 Conclusion: Placement of a clip at initial diagnosis of node positive disease with identification of the clip during the SLN surgery reduces the FNR. Use of IHC with inclusion of metastases <0.2mm in the definition of residual nodal disease in women with node positive breast cancer after chemotherapy also improves the accuracy of SLN surgery. Use of one or both of these methods should be considered when performing SLN in this setting. Citation Format: Judy C Boughey, Karla V Ballman, William F Symmans, Linda M McCall, Elizabeth A Mittendorf, Gretchen M Ahrendt, Lee G Wilke, Bret Taback, Kelly K Hunt. Methods impacting the false negative rate of sentinel lymph node surgery in patients presenting with node positive breast cancer (T0-T4,N1-2) who receive neoadjuvant chemotherapy – Results from a prospective trial – ACOSOG Z1071 (Alliance) [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-01-02.

Quantitative measurement of HER2 expression in breast cancers: comparison with 'real-world' routine HER2 testing in a multicenter Collaborative Biomarker Study and correlation with overall survival.

IntroductionAccurate assessment of HER2 status is critical in determining appropriate therapy for breast cancer patients but the best HER2 testing methodology has yet to be defined. In this study, we compared quantitative HER2 expression by the HERmark™ Breast Cancer Assay (HERmark) with routine HER2 testing by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH), and correlated HER2 results with overall survival (OS) of breast cancer patients in a multicenter Collaborative Biomarker Study (CBS).MethodsTwo hundred and thirty-two formalin-fixed, paraffin-embedded breast cancer tissues and local laboratory HER2 testing results were provided by 11 CBS sites. HERmark assay and central laboratory HER2 IHC retesting were retrospectively performed in a blinded fashion. HER2 results by all testing methods were obtained in 192 cases.ResultsHERmark yielded a continuum of total HER2 expression (H2T) ranging from 0.3 to 403 RF/mm2 (approximately 3 logs). The distribution of H2T levels correlated significantly (P <0.0001) with all routine HER2 testing results. The concordance of positive and negative values (equivocal cases excluded) between HERmark and routine HER2 testing was 84% for local IHC, 96% for central IHC, 85% for local FISH, and 84% for local HER2 status. OS analysis revealed a significant correlation of shorter OS with HER2 positivity by local IHC (HR = 2.6, P = 0.016), central IHC (HR = 3.2, P = 0.015), and HERmark (HR = 5.1, P <0.0001) in this cohort of patients most of whom received no HER2-targeted therapy. The OS curve of discordant low (HER2 positive but H2T low, 10% of all cases) was aligned with concordant negative (HER2 negative and H2T low, HR = 1.9, P = 0.444), but showed a significantly longer OS than concordant positive (HER2 positive and H2T high, HR = 0.31, P = 0.024). Conversely, the OS curve of discordant high (HER2 negative but H2T high, 9% of all cases) was aligned with concordant positive (HR = 0.41, P = 0.105), but showed a significantly shorter OS than concordant negative (HR = 41, P <0.0001).ConclusionsQuantitative HER2 measurement by HERmark is highly sensitive, accurately quantifies HER2 protein expression and correlates well with routine HER2 testing. When HERmark and local HER2 results were discordant, HERmark more accurately predicted overall survival.

A randomized, double-blind, phase II study of first-line FOLFOX plus bevacizumab with onartuzumab versus placebo in patients with metastatic colorectal cancer (mCRC).

663 Background: In mCRC, MET overexpression has been associated with poor prognosis and resistance to anti-VEGF therapy. We initiated a phase II study to evaluate the combination of onartuzumab (O), a ligand-blocking monoclonal antibody directed against the MET receptor, plus bevacizumab and FOLFOX, in first-line mCRC (GO27827; NCT01418222). Methods: This double-blind, randomized, multicenter phase II study randomized patients 1:1 to receive O (10 mg/kg iv) or placebo (P), plus mFOLFOX6 and bevacizumab (5 mg/kg iv). Stratification was by prior adjuvant therapy. All treatments were given on day 1–3 of a 2-week cycle. Oxaliplatin was given for up to 8–12 cycles; all other agents were continued until progression, unacceptable toxicity or death. Primary endpoint was progression-free survival (PFS) in ITT and MET+ subgroup by immunohistochemistry (IHC). MET status was determined by central laboratory IHC evaluation, with scores of 2+ or 3+ considered MET+. Results: From September 2011 to November 2012, 194 patients were enrolled. A recommendation was made to stop O after an interim efficacy and safety analysis in September 2013, due to lack of efficacy. The final analysis (cut-off Feb 2014) found that O did not improve PFS vs. P in the ITT (HR 0.75 [0.52–1.08]; p=0.12) or MET IHC+ populations (n=79; HR 1.03 [0.56–1.89]; p=0.93), although improvement was noted in the MET IHC− population (n=108; HR 0.60 [0.37–0.97]; p=0.03). Neither overall survival (OS) nor response rate (RR) was improved with O vs. P in any of the groups (OS HR 0.96 [0.61–1.50], p=0.85 for ITT; OS HR 1.24 [0.63–2.43], p=0.54 for MET IHC+; OS HR 0.83 [0.44–1.56], p=0.56 for MET IHC−; RR 57.3% vs. 57.7% for ITT, 43.2% vs. 57.1% for MET IHC+, 66.1% vs. 60.8% for MET IHC−). More edema (65.7% vs. 12.9%) and venous thromboembolic events (30.3% vs. 16.1%) were seen with O vs. P, respectively. Grade ≥3 events were similar (86.9% vs. 84.9%) and events leading to discontinuation were increased (48.5% vs. 37.6%) with O vs. P. Conclusions: Adding onartuzumab to FOLFOX/bevacizumab did not prolong PFS in first-line unselected or MET IHC+ mCRC. A trend towards PFS benefit was seen in those with MET IHC− mCRC, contrary to prior reports in other tumor types. Clinical trial information: NCT01418222.

HER2 Status by Standardized Immunohistochemistry and Silver-EnhancedIn SituHybridization in Korean Breast Cancer

PurposeAmplification of the human epidermal growth factor receptor 2 (HER2) gene occurs in 18% to 20% of breast cancers, and it is recognized as a prognostic and predictive marker. We investigated the HER2 status in Korean breast cancer by immunohistochemistry (IHC) and silver-enhanced in situ hybridization (SISH), as the first step toward building a nationwide quality assurance program for HER2 testing.MethodsA total of 1,198 breast carcinoma samples were collected from six institutions and IHC and SISH were performed using tissue microarrays in central laboratories. The results were compared to those of local laboratories.ResultsAvailable data were obtained from 959 samples. Central IHC results were negative, equivocal, and positive for 756 (78.8%; range among institutions, 76.8-81.8%), 37 (3.9%; 1.9-6.2%), and 166 (17.3%; 13.6-20%), respectively. SISH results were negative, equivocal, and positive for 756 (78.8%; 77.4-79.9%), 2 (0.2%; 0-0.7%), and 201 (21%; 20.1-22.2%), respectively. HER2 gene amplification was observed in 4.4%, 19%, and 73.9% of the negative, equivocal and positive groups stratified by local IHC results, respectively. When central SISH was considered to be the gold standard method for measuring HER2 status, the false-negative and false-positive rates of local IHC were 14.4% (29/201) and 7.1% (54/756). The concordance rate between central IHC and SISH was 98.4%.ConclusionCentral IHC and SISH markedly decreased the interlaboratory variability of HER2 status and the results of the two were highly concordant. The quality control program for HER2 testing must be focused on decreasing both the false negativity and positivity of IHC in local laboratories.

P1-07-01: Comparison of Four HER2 Testing Methods in the Detection of HER2−Positive Breast Cancer: Results from the FinHer Study Cohort.

Abstract Background: Accurate assessment of the HER2 status is essential for identifying patients who may benefit from HER2 targeted therapy. The current methods, immunohistochemistry (IHC) and in situ hybridization (ISH), determine HER2 status semi-quantitatively as positive (+), equivocal (+/−) and negative (−) with predefined cutoff values. Recent studies have suggested that current HER2 cutoffs may not be optimal for all clinical settings of HER2 targeted therapy. In a small subset of adjuvant NCCTG N9831 patients confirmed as HER2−normal by round-robin review of HER2 testing, trastuzumab benefit was observed (Perez et al, SABCS 2010). Quantification of HER2 as continuous variable may enable a more accurate optimization of HER2 cutoffs for various HER2 targeted therapies. In this study, we measured continuous HER2 protein expression by the HERmark™ assay and continuous mRNA expression by quantitative real time polymerase chain reaction (qPCR), and compared these results with central IHC and central chromogenic in situ hybridization (CISH) results of FinHer. Methods: Total HER2 protein expression (H2T) was quantified using the HERmark assay as previously described (Huang et al. Am J Clin Pathol 2010;134:303). HER2 mRNA expression (H2N) was measured by qPCR as previously published (Noske et al. Br Cancer Res Treat 2011;126:109). The results of H2T and H2N as continuous variables and as predefined categories were compared with central CISH results from FinHer (Joensuu et al, N Engl J Med 2006;354), and central IHC retesting. Results: H2T in 899 evaluable samples described a continuum of 0.4 to 721.2 (relative HERmark unit); while H2N in 915 evaluable samples showed a continuum of 31.4 to 42.8 (delta-Ct). Significant correlation between H2T and H2N as continuous variable was found (R2= 0.56, P&amp;lt; .0001). Paired method comparison was performed for samples with valid results in any two of the four testing methods. Overall concordance of H2T and H2N with predefined categories (+, +/−, -) was 81%, and concordance of (+) and (−) subsets was 95% when (+/−) cases (H2T 11%; H2N 6%) were excluded. Overall concordance of central IHC and H2T categories (+, +/−, -) was 75%, and concordance of (+) and (−) subsets was 96% when (+/−) cases (IHC 16%; H2T 11%) were excluded. Overall concordance of IHC and H2N categories (+, +/−, -) was 84%, and concordance of (+) and (−) subsets was 99% when (+/−) cases (IHC 16%; H2N 6%) were excluded. Concordance of central CISH (+, -) with H2T and H2N categories (+, -) was 89% and 91%, respectively, when (+/−) cases were excluded from H2T (13%) and H2N (8%), respectively. Conclusions: All four methods identified HER2−positive breast cancers. The discordance rate between the methods tested was approximately 10 to 20% despite careful delineation of cancerous tissue in the sample and analysis of adjacent tumor sections. No combination of assays could be identified with concordance rate &amp;gt;95% when the equivocal subsets were included in comparisons. Exclusion of the equivocal subsets (about 10% of samples) yielded high concordance rates of approximately 95% or higher. H2T and H2N showed comparable continuous distribution patterns and significant concordance with standard HER2 status by central IHC and CISH. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-07-01.

P1-07-22: A Venezuelan Study of Breast Cancer Estrogen Receptor, Progesterone Receptor and HER2 Receptor Expression by the Standard Method, Immunohistochemistry (IHC), Compared to a New Method, Quantitative Reverse Transcription Polymerase Chain Reaction (RT-PCR).

Abstract Background: Assessment of ER, PR and HER2 is of significant importance in breast cancer diagnosis and treatment. We have performed the first large assessment of central IHC for ER, PR and HER2 by a South American (Caracas, Venezuela) Central Lab to Central Lab RT-PCR by Oncotype DX®. Design: Breast cancer specimens from the Fundación BADAN were evaluated by IHC for ER (clone 1D5), PR (clone 636) using 1% staining for positivity using ASCO/CAP guidelines and HER2 (clone A0485) using ASCO/CAP guidelines of 3+ in ≥30%. Standardized quantitative RT-PCR analysis for ER and PR used Oncotype DX with the pre-defined cutoffs of 6.5 units and 5.5 units for positivity, respectively. For HER2 the standard pre-defined cutoffs were used: positive ≥11.5 units, equivocal &amp;gt;10.7-&amp;lt;11.5 units, and negative ≥10.7 units. For HER2 concordance analysis the equivocal range was excluded from both assays according to ASCO/CAP Guidelines (Wolff et al, 2006). For all quantitative RT-PCR assessments the reference normalized expression measurements ranged from 0 to 15, where each 1-unit increase reflects about a 2-fold increase in RNA. Result: Evaluable data was obtained in 96 pts for ER, 95 pts for PR (1 patient assessment unavailable) and 89 pts for HER2 (7 patient assessments unavailable). Two-by-two tables (below) compare IHC versus RT-PCR for ER, PR and HER2. The overall concordance for ER between IHC and RT-PCR was 94% (Kappa 0.467; 95% CI 0.107, 0.828); for PR between IHC and RT-PCR was 92% (Kappa 0.668; 95% CI 0.457, 0.878); and for HER2 between IHC and RT-PCR was 97% (Kappa = 0.649; 95% CI 0.280, 1.000). Three IHC 2+ cases and 4 equivocal cases by Oncotype DX were excluded from Concordance and Kappa statistics. Conclusion: RT-PCR by Oncotype DX for ER, PR and HER2 status is useful for active monitoring of IHC assays as mandated by ASCO/CAP guidelines. There is a high degree of overall concordance between central IHC performed in South America (Caracas, Venezuela) and central RT-PCR for ER, PR and HER2 status. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-07-22.

P1-06-11: Comparison of Community and Central Her2 Assessment on Outcome of Neoadjuvant Chemotherapy in the I-SPY Trial.

Abstract Background: Her-2/neu overexpression, by immunohistochemistry (IHC) or fluorescence in-situ hybridization (FISH), is highly correlated with response to trastuzumab and these are currently the gold-standard, FDA-cleared testing methods for assigning treatment to Her-2-directed therapies. However, substantial variability has been documented between community and central laboratory IHC and FISH testing. Biologically, Her-2 overexpression may reflect increased gene copy number, gene expression and/or protein production, and these can be measured by other platforms, including comparative genomic hybridization (CGH), expression arrays and quantitative protein assays, respectively. We sought to determine the degree to which community IHC/FISH results differed from centrally-assessed IHC, FISH, and other assessment platforms within the I-SPY Trial and whether response to neoadjuvant chemotherapy (NAC) differed by platform. Methods: The I-SPY Trial enrolled 237 women 2002–06 with invasive breast tumors at least 3 cm in clinical/radiographic size who subsequently underwent anthracycline/taxane NAC, serial core biopsies and imaging. Pathologic complete response (pCR) was determined at time of surgery and 3-year follow up has been reached. Trastuzumab was given to Her2+ patients at physician discretion, based upon community IHC/FISH results, and became more widespread after 2005. Central I-SPY laboratories determined Her2 copy number by MIP array, gene expression by Affymetrix and Agilent arrays, and Her2 protein by reverse-phase protein array (RPMA). Unsupervised clustering algorithms were used to evaluate expression patterns. Composite variables were constructed for DNA, RNA and protein positivity as well as for community and central IHC/FISH. Platforms were compared and Kaplan-Meier curves were constructed to compare outcomes by platform. Results: 222 women were evaluable, though not all patients had results for all platforms. Community composite IHC/FISH was positive in 64/214 (30%) but only 41 of these (64%) were confirmed by central IHC/FISH and 4 additional cases were centrally positive despite negative community testing. Concordance was high among centrally-assessed Her2 platforms, but was lower between community IHC/protein and central RNA (90%), DNA (91%) and protein (91%). Among patients receiving trastuzumab (n=36), the pCR rate was ∼50% regardless of Her2-assessment platform; in contrast, those not receiving trastuzumab had pCR rates below 30%. Among the 64 patients deemed Her2+ by community IHC/FISH, 30 (48%) had pCR and 15 (25%) have had distant relapse. Five distant relapses have occurred despite pCR; all received trastuzumab, all were Her2 positive by multiple central platforms and 3/5 were ER-positive. Sites of distant relapse included brain, bone and viscera; only 1 of 5 had isolated brain relapse. Conclusions: Community IHC/FISH testing for Her2 expression in the I-SPY Trial overcalled Her2 positivity compared to central testing while central results were highly concordant among DNA, RNA and protein platforms. Despite the high rate of community “false positives”, relapse after pCR occurred only in central Her2 “true positives,” exclusively among those receiving trastuzumab, and was rarely isolated to CNS sanctuary sites. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-06-11.

Local versus central laboratory discrepancies in the determination of triple-negative breast cancer (TNBC) status in a large phase III (CIBOMA/2004-01/GEICAM/2003−11) trial assessing adjuvant capecitabine (C) maintenance therapy after standard chemotherapy (CT) in early breast cancer (EBC) patients (pts).

1022 Background: Appropriate determination of immunohistochemistry (IHC) to define EBC subtypes is crucial for an adequate treatment recommendation. A number of studies have raised questions about the reproducibility of IHC results when comparing local and central laboratories. In this report we explore the differences between local and central determinations of TNBC status in a multinational randomized phase III trial that focuses on adjuvant C maintenance after conventional induction CT in early TNBC: CIBOMA/2004−01/GEICAM/2003−11. Methods: A total of 1.441 specimens of pts locally defined as TNBC were registered and sent to Spain for central laboratory confirmation of negative results for both Estrogen (ER) and Progesterone Receptors (PR) by IHC (<1%) using Clone 1D5 (ER) and Clone PGR 636 (PR), and HER−2 by HercepTest and FISH (HercepTest 2+). CK 5/6 and EGFR status were also assessed. 854 (59%) samples came from Spain and 587 (41%) from 7 Latin American (LA) countries. Results: In 18 (1%) cases it was not possible to centrally assess TNBC status due to lack of tumour tissue in the specimen. In 130 cases (9%) tumours were found not to be TNBC by central determination. 74 (13% of LA samples) discordant cases came from LA countries while 56 (7% of Spanish samples) came from Spain (p<0.0001). Most of the discrepant results involved ER or PR status (71%) while HER-2 discrepancies represented 22% of cases. Conclusions: The considerable emphasis modern management strategies places in an individualized treatment assignment needs to take into consideration the significant discrepancies that are seen between local and central laboratory IHC determinations. In this large trial up to 9% of pts were erroneously diagnosed as TNBC. The translation of these results into routine clinical practice indicates that a significant portion of pts may not receive appropriate therapy due to faulty IHC testing. We need to improve the methodological standards for ICH testing.

Abstract P4-08-05: Delineation of HER2 Gene Status in Korean Breast Cancers by Standardized Immunohistochemistry (IHC) and Silver In Situ Hybridization (SISH): Comparison of Local IHC Result with Central IHC and SISH Results

Abstract Background: Amplification of the human epidermal growth factor receptor 2 (HER2) gene and concomitant protein overexpression are present in 20-25% of breast cancers. HER2 overexpression and/or gene amplification is associated with a worse clinical outcome and an important predictive marker for sensitivity to anthracycline-based chemotherapy and HER2- targeted therapy. In Korea, the HER2 status has usually been assessed by immunohistochemistry (IHC) in the laboratories. To verify the preanalytic, analytic, and postanalytic factors of IHC in multi-institutions, we assessed the interlaboratory variability of HER2 IHC by comparing the local IHC with the central IHC and SISH results, as a first step toward building a nationwide quality assurance program. Methods: The Breast Pathology Study Group of Korean Society of Pathologists collected 1,198 breast carcinoma samples from 7 university-based hospitals and constructed 56 tissue microarray (TMA) blocks with triplicate of 1 mm cores. Local IHC results for HER2 were obtained from 1,130 patients. We performed IHC (PATHWAY anti-HER2/neu (4B5) rabbit monoclonal antibody, Ventana Medical Systems) and SISH (INFORM HER2 DNA Probe Kit, Ventana Medical Systems) for HER2 on the TMA sections using the automated Benchmark platform (Ventana Medical Systems). We interpreted the results according to the American Society of Clinical Oncology/College of American Pathologists guidelines. Results from local IHC were compared with central IHC and SISH. The concordant rates between central IHC and SISH results were also calculated. Results: The total percentage of cases in each category of local IHC score was: 0/1+, 70.4%; 2+, 8.8% (range among hospitals, 0-36%); and 3+, 20.8% (10.8-28.8%). The percentage of central IHC results (1,110 patients) in each category was: 0/1+, 79.7%; 2+, 3.6% (1.1-8.3%); and 3+, 16.7% (13.1-18.8%). SISH results were obtained from 1,033 patients: negative, 79.5%; equivocal, 0.2% (0-1.6%); and positive, 20.3% (19.2-22.6%). SISH amplifications in each local IHC category were observed as follows: 0/1+, 4.3% (0.6-10%); 2+, 18.8% (5.6-66.7%); and 3+, 71.2% (50-97.3%). When considering the central SISH as gold standard, 3% of cases (30/995) were false negative and 6.3% (63/995) were false positive in local IHC. In central lab, the concordance rates between IHC 3+ and SISH-positive, and IHC 0/1+ and SISH-negative were 95.5% and 98%, respectively. Conclusions: The results of central IHC and SISH markedly decreased the interlaboratory variability of HER2 status. The measurement of HER2 gene amplification by SISH was less affected by the preanalytic factors than the measurement of overexpression of HER2 protein by IHC. Because the rate of false positivity is higher than that of false negativity in the local IHC, the quality assurance program needs to be focused on decreasing not only the false negativity, but also the false positivity of HER2 IHC in local laboratories Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-08-05.

Quantitative HER2 protein levels predict outcome in fluorescence in situ hybridization‐positive patients with metastatic breast cancer treated with trastuzumab

Only a portion of breast cancer patients currently selected for trastuzumab therapy respond. Using a novel assay (HERmark) to quantify total human epidermal growth factor receptor 2 (HER2) expression, the authors examined outcomes in 102 trastuzumab-treated metastatic breast cancer patients previously assessed as immunohistochemistry (IHC) 3+ by local but not central IHC, or fluorescence in situ hybridization (FISH) positive, and then retested by central FISH. Of 102 MBC patients previously scored as IHC 3+ or 2+/FISH-positive and treated with trastuzumab-containing regimens, 98 had both central FISH and HER2 total expression values. Sixty-six of 76 central FISH-positive patients (87%) had high HER2 total expression levels (concordant positive), and 19 of 22 central FISH-negative patients (86%) were HER2 total expression low (concordant negative). Fourteen percent (3 of 22) of central FISH-negative patients were HER2 total expression high (discordant HER2 total expression high), and 13% (10 of 76) of central FISH-positive patients were HER2 total expression low (discordant HER2 total expression low). The concordant positive group had a significantly longer time to progression (TTP, median = 11.3 months) compared with the concordant negative group (median TTP, 4.5 months; hazard ratio [HR] = 0.42, P < .001), and also compared with the discordant HER2 total expression low group (median TTP, 3.7 months; HR = 0.43, P = .01). The discordant HER2 total expression low group behaved similarly compared with concordant negatives (HR = 1, P = .99). In analyses restricted to central FISH-positive patients only (n = 77), Cox proportional hazards multivariate regression identified HER2 total expression as an independent predictor of TTP (HR = 0.29, P = .0015) and overall survival (HR = 0.19, P < .001). A subset of patients with HER2 gene amplification by FISH express low levels of HER2 protein and have reduced response to trastuzumab-containing therapy, similar to FISH-negative patients. This cohort represents a training dataset, and the observed relationships and derived cutoffs require validation in an independent cohort of trastuzumab-treated metastatic breast cancer patients.

Abstract C16: Prospective real-time analysis of p-cadherin expression to select patients into a phase I oncology trial

Abstract Effectively personalizing medicine depends upon selecting patients (pts) amenable to targeted therapies. We report the development of a custom immunohistochemistry (IHC) assay to identify P-cadherin positive expression in the archival biopsy of pts with solid malignancies. Validated reagents including a mAB to P-cadherin were incorporated into a custom IHC kit standardized across three Phase I sites (Seoul National University, Royal Melbourne Hospital, and Fox Chase Cancer Center). Pts with a P-cadherin positive archival biopsy are being enrolled into an ongoing trial. Clinical results will be reported separately. P-cadherin positivity is defined by a scoring index (SI) [product of the Tumor Area Positive for P-cad x Staining Intensity]. Based on the literature, a SI ≥ 4 is associated with poor clinical outcome and was therefore required for pt entry into the trial. The IHC protocol was standardized across sites using positive and negative controls run in a masked fashion at each site and cross-examined and repeated by a central lab that had final decision-making authority (ApoCell, Inc., Houston, TX). Each site ran independent IHC assays and analyses by board-certified pathologists, and provided stained and unstained archival biopsy slides for independent, masked analysis by ApoCell. The turn-around time for central IHC analysis was ≤ 48 h. Forty-four of 67 pts (65.7%) were found to be P-cadherin positive and eligible for entry into the trial. The distribution of enrolled patients by site is as follows: Australia (SI ≥ 4, n = 20), Korea (SI ≥ 4, n = 15), and USA (SI ≥ 4, n = 9). Median and mean SI values for the P-cadherin SI were 6 and 5.3 ± 2.9 (SD), respectively, with a range of 0 – 9. Clinical sites produced a SI equal to ApoCell in 56 of 67 pts (85.6% concordance rate). Among solid malignancies tested, a majority of gastric, colon and rectal cancers have demonstrated P-cadherin SI values ≥ 4. Successful execution of this IHC study demonstrates the feasibility of prospectively selecting pts based on IHC expression of P-cadherin using a custom standardized scoring index analysis across multiple clinical sites. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C16.

Local versus central HER2 immunohistochemistry correlates with kinetic RT-PCR but only central immunohistochemistry and RT-PCR predict pathological complete response: results from the neoadjuvant multicenter GeparTrio trial.

Abstract Abstract #1070 Background: There are several methods for determination of HER2 overexpression. Immunohistochemistry (IHC) has been set up as the gold standard in routine diagnostics to identify patients eligible for anti-HER2 therapy. Nonetheless, there is still controversy about the predictive value of HER2 overexpression and pathological response to neoadjuvant anthracycline/taxane-based chemotherapy.&amp;#x2028; Methods: In this study, we investigated the HER2 expression levels in formalin-fixed, paraffin-embedded pre-treatment punch biopsies by kinetic one-step RT-PCR (kRT-PCR); local IHC performed at the pathological institutes of the participating centers; and centrally performed IHC in 150 patients from the neoadjuvant GeparTrio study (NCT00544765), where patients were treated with docetaxel, doxorubicin and cyclophosphamide (TAC) but received no trastuzumab therapy regardless of the individual HER2 status. HER2 was considered positive for IHC if expression was 3+ and for mRNA expression if normalized level was &amp;gt;/= 18.0 arbitrary units (as defined by bimodal expression for this cohort). We evaluated the associations by Spearman's rho correlation between the different methods as well as the best method that might predict a pathological complete response to this neoadjuvant chemotherapy.&amp;#x2028; Results: A randomly selected subset out of 150 of 2,090 patients was included in this analysis. This is a representative collective of TAC-treated patients from the GeparTrio trial with a pCR rate (ypT0; ypTis/ypN0) of 23%. The different methods revealed a HER2 positivity of 31% for the local analyses, 16% for the central HER2 testing and 20% positivity for kRT-PCR analyses for this study collective, compared to 28% for the entire trial population. Local determination of HER2 showed a correlation with central laboratory (RS=.418, P&amp;lt;.001, N=106) and kRT-PCR results (RS=.428, P&amp;lt;.001, N=120), and centrally assessed HER2 expression correlated with the results of kRT-PCR (RS=.923, P&amp;lt;.001, N=115). Binary logistic regression with the locally determined HER2 expression revealed no significant results in prediction of a pathological complete response (P=.802, N=121), whereas the results from central IHC (P=.038, OR 2.84, N=119) as well as the results from kRT-PCR (P=.047, OR 2.47, N=134) were significant.&amp;#x2028; Conclusions: The value of HER2 expression and response to neoadjuvant chemotherapy is still controversial. Our results on this subset of patients show that kRT-PCR of HER2 mRNA expression and centrally performed IHC but not local HER2 assessment predicts pCR for neoadjuvant TAC chemotherapy. Theses results need further confirmation on a larger group of patients. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1070.

Quantitative measurements of HER2 expression and HER2 homodimer using a novel proximity based assay: comparison with HER2 status by immunohistochemistry and chromogenic in situ hybridization in the FinHer study.

Abstract Abstract #2071 Background: The accuracy and reliability of current methods, immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH), to assess HER2 status has recently been a subject of debate. The best method to assess HER2 status remains controversial. We developed a novel assay (HERmark, Monogram Biosciences) that provides precise quantification of HER2 expression (H2T) and HER2 homodimer (H2D) in formalin-fixed paraffin-embedded (FFPE) tissues. We compared H2T and H2D to local IHC, central chromogenic in situ hybridization (CISH) and central IHC retesting of breast cancers from the FinHer study.&amp;#x2028; Methods: H2T and H2D were detected through light-dependent release of fluorescent tags (VeraTag reporters) conjugated to a HER2 antibody, requiring proximity to a second HER2 “scissors” antibody. The VeraTag signal was quantified by capillary electrophoresis and normalized to tumor area. Assay comparisons correlated H2T and H2D with HER2 testing by local IHC and central CISH from FinHer (Joensuu et al, N Engl J Med 2006;354), as well as central HER2 status reassessment by combination of externally performed central IHC retesting (PhenoPath labs, Seattle, WA) and central CISH (FinHer) according to ASCO/CAP guideline for HER2 testing in breast cancer (Wolff et al, Arch Pathol Lab Med 2007;131).&amp;#x2028; Results: H2T and H2D in 899 evaluable FinHer samples described a continuum over a wide dynamic range (∼ 3 logs), in contrast with conventional IHC categories (0-3+). The correlation between H2T and IHC categories was significant (P &amp;lt; .0001). Overlap of H2T among the IHC categories was observed. H2D showed a similar correlation with IHC and a general correlation with H2T (P &amp;lt; .0001). A H2T cutoff value, based on its ability to distinguish high and low responders in a cohort of metastatic breast cancer patients treated with trastuzumab-based regimens (log10 H2T= 1.14, Leitzel et al, 2008 ASCO, abstract 1002), was used to define HERmark negative (-) and positive (+), which were then compared with IHC and CISH results. The concordances between HERmark (-) and local IHC (-), central CISH (-), and central HER2 reassessment (-) were 89%, 84%, and 91%, respectively. The concordances between HERmark (+) and local IHC (+), central CISH (+), and central HER2 reassessment (+) were 71%, 89%, and 92%, respectively. The HERmark test showed greater overall concordance with central HER2 reassessment (91%) than with local IHC (84%) and central CISH (87%)&amp;#x2028; Conclusions: HERmark reliably measures H2T and H2D in FFPE tissues. H2T showed excellent concordance with central HER2 status according to ASCO/CAP guideline for HER2 testing. The precise quantification of H2T and H2D may provide novel, quantifiable, predictive tests for targeted HER2 therapy. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2071.

Problems and Solutions in the Evaluation of Hormone Receptors in Breast Cancer

Problems and Solutions in the Evaluation of Hormone Receptors in Breast Cancer

HER2 testing in a population-based study of patients with metastatic breast cancer treated with trastuzumab.

Abstract Context.—The development of trastuzumab has led to new therapeutic strategies for patients with breast cancer. Trastuzumab has been shown to only be effective in those patients whose cancers are HER2 positive by either immunohistochemistry (IHC) or fluorescence in situ hybridization (FISH). Objective.—To determine the accuracy of HER2 testing in 2 provinces in Canada. Design.—Patients with metastatic breast cancer diagnosed between 1999 and 2002 who were HER2 positive in the local laboratories were included in this study. All cases were retested centrally, by IHC (using 4 methods) and FISH. In addition, 205 locally reported HER2-negative metastatic breast cancer cases were retested centrally. Results.—Concordance between the 505 local IHC-positive cases with central IHC testing varied between 79.3% and 89.6% depending on the IHC method and scoring method used. HER2 gene amplification by FISH was present in 86.1% of local IHC-positive cases. Concordance between the 205 local IHC-negative tumors wi...

HER2 Testing in a Population-Based Study of Patients With Metastatic Breast Cancer Treated With Trastuzumab

The development of trastuzumab has led to new therapeutic strategies for patients with breast cancer. Trastuzumab has been shown to only be effective in those patients whose cancers are HER2 positive by either immunohistochemistry (IHC) or fluorescence in situ hybridization (FISH). To determine the accuracy of HER2 testing in 2 provinces in Canada. Patients with metastatic breast cancer diagnosed between 1999 and 2002 who were HER2 positive in the local laboratories were included in this study. All cases were retested centrally, by IHC (using 4 methods) and FISH. In addition, 205 locally reported HER2-negative metastatic breast cancer cases were retested centrally. Concordance between the 505 local IHC-positive cases with central IHC testing varied between 79.3% and 89.6% depending on the IHC method and scoring method used. HER2 gene amplification by FISH was present in 86.1% of local IHC-positive cases. Concordance between the 205 local IHC-negative tumors with central testing ranged between 94.8% and 100% for IHC and was 98.5% for FISH. This study demonstrated that locally reported HER2-negative results were highly accurate, but less accuracy was demonstrated with local HER2-positive results. These results emphasize the need for participation in a quality assurance program.

Concordance of local and central laboratory hormone and HER2 receptor status in ECOG 2197

21022 Background: Central and local laboratory concordance for hormone and HER2 receptor measurement is of national interest. This study compares ER/PR/HER2 by local laboratories using immunohistochemistry (IHC) and central laboratories (IHC &amp; quantitative RT-PCR). Methods: Of 2952 patients in E2197, a case-cohort sample of 776 patients who either did (N=179) or did not recur was studied. Central IHC for ER/PR/HER2 was performed using single 0.6 mm microarrays; Allred score (AS) was used for ER/PR (AS&gt;2 = positive). Positive HER2 was 3+ staining in &gt;10% cells for Central IHC and 2+ or 3+ for Local IHC. RT-PCR analysis by Oncotype DX™ for ER/PR/HER2 was performed using pre-defined cutoffs of 6.5, 5.5 and 11.5 units, respectively. Hormone receptor (HR) pos was defined as ER &amp;/or PR pos. Results: Results from Local IHC (ER/PR in 776 &amp; HER2 in 517 pts) were compared with Central IHC (760 pts) and RT-PCR results (776 pts). The discordance between HR positivity by Local IHC and RT-PCR was very low. However, 12% of HR neg pts by Local IHC (38/321) &amp; Central IHC (39/326) were HR pos by RT-PCR. The relationship between ER and recurrence as a function of AS was examined. Patients with AS of 3–4 were found to be closer to the AS=2 group than to the AS&gt;4 group Patients with AS of 3–4 were found to be closer to the AS ÿ 2 group than to the AS &gt; 4 group (Est.HR for ER 0.97 for AS 3–4 vs. 0–2 and 0.46 for AS 5–8 vs. 0–2, and for PR were 0.84 for AS 3–4 vs. 0–2 and 0.41 for AS 5–8 vs. 0–2). Conclusions: There is a high degree of overall concordance among Local IHC, Central IHC, and Central RT-PCR for ER and PR. The degree of concordance is even greater for HR compared to ER or PR alone. Although the concordance with local labs for HER2 testing was poor, the concordance between Central IHC and RT-PCR was very high. The relatively high incidence (12%) of IHC HR neg pts who are HR pos by RT- PCR is notable. [Table: see text] [Table: see text]

Central HER2 IHC and FISH analysis in a trastuzumab (Herceptin) phase II monotherapy study: assessment of test sensitivity and impact of chromosome 17 polysomy

Aims:To investigate the correlation between centrally assessed human epidermal growth factor receptor 2 (HER2) immunohistochemistry (IHC) and fluorescence in situ hybridisation (FISH) results and response to treatment of patients with...