Citations

Abstract Identification of HER2 as an important cellular marker in the biology and treatment of breast cancer has highlighted the importance of reliable testing methodology. Controversy exists regarding type of test, reliability, definition of positivity, and which test may best predict for patient (pt) efficacy to anti-HER2 therapies. Purpose: To determine the concordance between HER2 results by 3 central laboratories, impact of round-robin adjudication of discordant cases, and heterogeneity in HER2 results using specimens from 3 adjuvant trials where HER2 testing was performed by local and central laboratories for enrollment. Methods: We performed a blinded round-robin exchange of randomly sampled, HER2-normal (IHC-& FISH-) and HER2-positive breast tumors among 3 central laboratories (NCCTG, BCIRG and NSABP) for confirmatory HER2 testing. The 3 laboratories performed immunohistochemistry (IHC) for HER2 using the HercepTest kit (Dako, Carpenteria, CA) and fluorescence in situ hybridization (FISH) for HER2 using the PathVysion HER2 DNA probe kit/HER2/CEN17 probe mixture (Abbott Molecular, Des Plaines, IL) on 389 tumor specimens obtained from N9831/BCIRG006 (37 IHC+/FISH+, 33 IHC+/FISH-, 36 IHC-/FISH+, 62 IHC-& FISH-) and BCIRG005 (96 IHC-& FISH-); 123 cases had ≥2 blocks examined from the same pt. HER2 positivity was defined according to FDA-approved guidelines used in the clinical trials (IHC+: 3+ complete membrane staining in >10% cells; FISH+: HER2/CEN17 ratio ≥2.0). The HER2 status (IHC: 3+ vs 0-2+; FISH: amplified vs not) for each block was independently determined at each site; discordant IHC and FISH cases were adjudicated at a face-to-face meeting. Results: Independent reads were concordant across pathologists in IHC status in 351/381 (92%) cases and in FISH status in 343/373 (92%) cases. Upon adjudication, a consensus was reached on 16 and 18 of the discordant IHC and FISH cases, respectively. Among 96 BCIRG005 cases, IHC-and FISH-were confirmed in the primary block in all 96 cases. Among 59 evaluable N9831 HER2-normal cases, IHC-and FISH-were confirmed in the primary block in 57 (97%) cases (but another block tested HER2+ for 4 cases). Among 102 N9831/BCIRG006 HER2+ cases, HER2 positivity was confirmed in the primary block in 73 (72%) cases. Among 118 cases with IHC results for > 1 block, the adjudicated IHC result agreed across blocks in 106 (90%) cases. Among 113 cases with FISH results for >1 block, the adjudicated FISH result agreed in 107 (95%) cases. Among 53 N9831 HER2-normal cases adjudicated as IHC-& FISH-(although they all had a previous local HER2+ test), there was significant improvement in disease-free survival associated with trastuzumab given concurrently with paclitaxel after doxorubicin and cyclophosphamide compared to chemotherapy alone (HR=0.31, 95% CI 0.11-0.91; A 23 pts, 10 events; C 30 pts, 5 events). Conclusions: Excellent agreement (96%) was observed among the pathologists at adjudication, suggesting that standardized methods improve assay proficiency. HER2 heterogeneity across blocks was observed more at the protein than at the gene level. In the small subset of N9831 pts adjudicated as HER2-normal trastuzumab benefit was observed. This work was supported by NCI and Genentech. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr PD10-02.

Abstract In the Breast International Group (BIG) and North Central Cancer Treatment Group (NCCTG) co-led phase III adjuvant breast cancer trial, ALTTO (Adjuvant Lapatinib and/or Trastuzumab Treatment Optimisation), two central laboratories, the European Institute of Oncology (IEO; Milan, Italy) and Mayo Clinic (Rochester, MN), are responsible for confirming the human epidermal growth factor receptor-2 (HER2), estrogen receptor α(ER), and progesterone receptor status of the primary breast tumors for the Rest of World (excluding China, which conducts a separate central review) and North American patients, respectively, prior to patient study entry. As of December 2009, discordance in HER2 and ER testing between local and central laboratories was observed by both central laboratories. For IEO, 14.5% of 8,037 HER2 cases locally positive [by either immunohistochemistry (IHC) or fluorescence in situ hybridization (FISH)] were not able to be confirmed as centrally positive; whereas for Mayo, 5.8% of 412 HER2 cases locally positive were not able to be confirmed as centrally positive (Table 1). Central and local IHC ER discordance was 12.1% and 11.7% for 9,021 IEO screened cases and 419 Mayo screened cases, respectively (Table 2). In particular, IEO observed more false-positive (locally positive/centrally negative) HER2 findings than Mayo and Mayo observed more false-positive ER findings than IEO. Purpose: Motivated by the above findings, we launched a ring study to assess whether the central lab results of a subset of local/central discordant ALTTO cases could be confirmed in the other central lab. Methods: IEO and Mayo exchanged and retested a subset of FFPE breast tumors collected in ALTTO. IEO sent 20 HER2 false-positive, 5 ER false-positive, and 5 ER false-negative (locally negative/centrally positive) ALTTO breast tumors to Mayo. Mayo sent 5 HER2 false-positive, 20 ER false-positive, and 5 ER false-negative ALTTO breast tumors to IEO. IEO and Mayo performed IHC for ER according to their own methodology: DAKO cocktail of ER 1D5 and 2.123 monoclonal antibodies and monoclonal ER 1D5 antibody, respectively. The two laboratories performed IHC for HER2 according to the HercepTest® manufacturers’ instructions (Dako, Carpenteria, CA) and FISH for HER2 using the PathVysion HER2 DNA probe kit and the HER2/centromere 17 probe mixture (Abbott Molecular, Des Plaines, IL). Results: IEO and Mayo confirmed the central HER2-negative result in 100% of 25 cases. Analyses of ER are ongoing and ER results will be presented. Conclusions: In this subset of patients, enrollment ineligibility did not change when HER2 testing was performed by either IEO or Mayo Clinic central laboratories. Table 1. HER2 Local/Central Lab Results Table 2. ER Local/Central Lab Results Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-10-36.

Abstract Previous findings suggested that patients (pts) with copy number alterations of MYC (8q24) and centromere 8 (CEN 8) in the setting of HER2-positive breast cancer may be associated with improved outcome to adjuvant trastuzumab. Our tissue microarray (TMA) data suggested that alternate cutpoints for MYC copy number anomalies [of 802 pts, those with MYC:CEN8 ratio ≥1.3 or <1.3 & polysomy 8 (N=658; 82%) had a hazard ratio (HR) of 0.44 (P<0.001) and those with MYC copy number ≥2.5 (N=669; 84%) had a HR of 0.42 (P<0.001)] were potentially predictive of benefit from adjuvant trastuzumab when administered concurrently with chemotherapy in the NCCTG N9831 HER2+ phase III trial. Purpose: We were interested in determining whether MYC is the target protein responsible for the suggested benefit from trastuzumab. We evaluated the association between MYC protein expression and disease-free survival (DFS) of pts randomized to receive chemotherapy alone (Arm A) or chemotherapy with sequential (Arm B) or concurrent trastuzumab (Arm C) using N9831 TMAs. Patients/Methods: This analysis included 1220 pts from Arms A, B, and C. The percentage of strong (3+) nuclear staining was determined in TMA sections containing 3 cores per block using immunohistochemistry (IHC; clone 9E10, Sigma-Aldrich, St. Louis, MO). A patient's tumor was considered positive (MYC+) if the maximum nuclear 3+ staining percentage across cores was >30% a priori. Median follow-up was 5.8 years. Results: Of 1220 pts with completed IHC analyses, 557 (46%) were MYC+. MYC+ was associated with a higher rate of hormone receptor positivity (58% vs 48%, chi-sq P<0.001) and higher rate of nodal positivity (92% vs 84%, chi-sq P<0.001) but not associated with age, race, tumor histology, tumor grade, or tumor size. In comparing DFS between arms C and A, MYC+ and MYC-pts had HRs of 0.46 (p=0.002; A: 187 pts, 57 events, C: 161 pts, 25 events) and 0.70 (p=0.09; A: 213 pts, 59 events, C: 208 pts, 38 events), respectively (interaction p=0.20). In comparing DFS between arms B and A, MYC+ and MYC-pts had HRs of 0.74 (p=0.11; B: 209 pts, 52 events) and 0.81 (p=0.29; B: 242 pts, 53 events), respectively (interaction p=0.80) (Figure). In addition, pts with nuclear MYC staining of 3+ in 0-9%, 10-30%, 31-60%, and 61-100% of cells had HRs (C vs A) of 0.98 (95% CI 0.53-1.82), 0.54 (95% CI 0.30-0.97), 0.55 (95% CI 0.27-1.12), and 0.37 (95% CI 0.19-0.75). Conclusions: In our analysis of 1220 pts, we observed that MYC protein expression positively correlated with hormone receptor positivity and nodal status. We observed a trend towards greater trastuzumab benefit among MYC+ as compared to MYC-pts. A rising trend in trastuzumab benefit was observed with increasing MYC protein expression. Analyses including pts with whole sections and outcome by MYC protein and gene copy number will be presented. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P2-09-08.

Abstract Background Taxane, a class of common anticancer agent, is widely used to treat many cancers. Among the taxanes, paclitaxel and docetaxel are the most commonly used agents. The mechanism of action for taxane drugs is through stabilizing microtubules by enhancing tubulin assembly and against depolymerization, thereby disrupting the dynamics of the microtubule. Large inter-individual variation has been observed in response to taxane treatment. Therefore, in the present study, we conducted a genome-wide association (GWA) study to identify genetic polymorphisms that might contribute to variation in response to taxanes drugs using the “Human Variation Panel” lymphoblastoid cell line (LCL) model system that consists of 287 ethnically defined cell lines. Method Genome-wide SNP and basal gene expression data were obtained for 287 LCLs using Illumina HumanHap 550K, 510S BeadChips, Affymetix SNP Array 6.0 and Affymetrix U133 plus 2.0 GeneChip. Cytotoxicity assay was also performed in the same cell lines for both paclitaxel and docetaxel. Candidate genes were selected based on an integrated analysis of genome-wide SNPs, expression array data and IC50 values (a cytotoxicity phenotype indicating the drug concentration required for 50% inhibition of maximal cell growth) for paclitaxel and docetaxel. SiRNA screening was performed to functionally validate those top candidates, followed by MTS assay in a breast cancer cell line, MDA-MB231. Results Of the 54,000 probe sets considered, 390 probe sets, representing 272 unique annotated genes, were found to be associated with docetaxel cytotoxicity (IC50 value) with p value <10−3, while 176 probe sets, representing 124 unique genes, were associated with paclitaxel IC50. Of these probe sets, 50 probe sets were associated with IC50 for both drugs. Analysis of 1.3 million SNPs identified 11 loci, defined as a region containing at least 1 SNP of p value <10−5 and 1 SNP of p value <10−4 within 50kb, associated with docetaxel IC50, while 7 loci were associated with paclitaxel IC50. One locus was found to be associated with both drugs. Previous studies suggested that gene expression is regulated in a cell-type specific manner. Therefore, based on our analysis and biological implication, we selected 16 top candidate genes that were expressed in our LCLs to perform functional validation studies using siRNA, followed by MTS assay in a breast cancer cell line, MDA-MB231. Functional studies showed that knockdown of SLC44A1 and HSPA5 significantly desensitized the cancer cell to both taxanes treatment. Conclusion GWAS using LCL model system could provide potential pharmacogenomic candidates for future clinical translational studies. Functional validation in tumor cell lines may help determine the genomic mechanism contributing to the inter-individual variation of taxane response. 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 2599.

Abstract The advancement in genotyping technology and large SNP arrays has led to an increase in the number of genome-wide association studies (GWAS) for various complex traits. These GWAS have resulted in the implication of over 1500 SNPs for association with disease traits. However, the SNPs identified from these GWAS are not necessarily the functional variants. Therefore, the next phase in GWAS will involve the validation, augmentation and refining of these putative regions or loci. One possible “next phase” approach would be to catalog all variants found in the study subjects within the loci of interest using sequencing technology, followed by the association analysis. However, sequencing a locus in a large number of subjects is still relatively expensive. By sequencing only a portion of the samples for the follow-up of signals from GWAS, followed by imputation in the remaining samples, one can significantly reduced the cost to localize the punitive variant involved in the etiology of complex phenotypes. A potentially attractive alternative option would be to impute based only on the 1000 Genomes Project; however, this has the drawbacks of using a reference population that does not necessarily match with disease status and LD pattern. Therefore, we conducted a study to investigate the ability to carry-out genotype imputation using a reference panel consisting of sequencing data for a fraction of the study participants. The various approaches were implemented using data from the candidate gene sequencing study conducted at the Mayo Clinic and the 1000 Genomes Project. Our results show that imputation of untyped markers based on sequencing a portion of the study participants is a reasonable, cost-saving, approach for disease mapping and the refinement of putative regions detected with GWAS. We recommend a few guidelines in designing sequencing studies to take advantage of the recent advances in genotype imputation: (1) Select the largest and most diverse reference panel for sequencing, with respect to both haplotypes and phenotypes. One can also use the 1000 Genomes Project in addition to the sequencing of a portion of the study participants; (2) Genotype as many “anchor” markers as possible, in that, the number of markers genotyped on all subjects impacts accuracy. Therefore, additional genotyping of a few common SNP markers not already genotyped on all subjects using a cost effective platform may be needed if the GWAS SNP array does not provide adequate coverage in the locus to be sequenced. 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 4738.

Abstract Background Radiation therapy is used to treat half of the all cancer patients. Response to radiation therapy varies widely, in terms of both response and toxicity related events. Previous studies suggested almost 80% of the variation in normal tissue response to radiation treatment was likely to be due to genetic factors. Therefore, we performed a genome-wide association study (GWAS) to identify biomarkers that might help predict radiation response using a the “Human Variation Panel” lymphoblastoid cell lines (LCLs) that consists 287 ethnically defined cell lines. Method Basal gene expression level, 1.3 million genome-wide SNP data were obtained for 287 human LCLs using Affymetrix U133 plus 2.0 GeneChip, Illumina HumanHap 550K, 510S BeadChips and Affymetix SNP Array 6.0. Radiation cytotoxicity assay was also performed to obtain the area under radiation dose response curve (AUC), a summary measure for cytotoxicity for the same cell lines. Functional validation of candidate genes, selected based on the integrated analysis of SNP, expression and AUC data, was performed in multiple cancer cell lines using specific siRNAs followed by MTS and colony formation assays. Results 27 loci, containing at least 2 SNPs of p values <10−4 within 50kb were associated with radiation AUC. 270 expression probe sets were associated with radiation AUC (p< 10−3). The integrated analysis of 27 loci, 54,000 basal expression probe sets and radiation AUC identified 50 unique SNPs associated with AUC were also associated with gene expression of 39 unique annotated genes. In addition, these 39 genes were associated with radiation AUC with p values <10−3. Based on the GWAS and integrated analysis of SNP, expression array and radiation AUC data, 22 genes were selected for functional validation studies using siRNA knock down followed by MTS and colony formation assays in multiple tumor cell lines. C13orf34 (Bora), MAD2L1, PLK4, TPD52 significantly altered the radiation sensitivity in at least 2 cancer cell lines for both MTS and colony formation assays (p<0.05). Knock down of DEPDC1B, a gene containing 2 SNPs at the 3′-end that was significantly related with Bora gene expression level (p<10−4) significantly desensitized cancer cells to radiation treatment. Both microarray data and QRT-PCR analysis indicated that expression levels for those two genes were significantly correlated. Conclusion Our results using the lymphoblastoid cell lines might help identify novel biomarkers that contribute to variation in response to radiation therapy and help understand the mechanisms underlying the association. 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 3602.

Purpose Findings from the human epidermal growth factor receptor 2 (HER2) –positive National Surgical Adjuvant Breast and Bowel Project (NSABP) B31 trial suggested that MYC/HER2 coamplification (> 5.0 copies/nucleus) was associated with additional benefit from adjuvant trastuzumab in patients with early-stage breast cancer. To further explore this relationship, we investigated associations between MYC amplification and disease-free survival (DFS) in a similar adjuvant trastuzumab HER2-positive breast cancer trial—North Central Cancer Treatment Group (NCCTG) N9831. Patients and Methods This analysis included 799 patients randomly assigned to receive chemotherapy alone or with concurrent trastuzumab on N9831. Fluorescence in situ hybridization (FISH) was performed by using a dual-probe mixture for MYC and centromere 8 (MYC:CEP8) on tissue microarrays. MYC amplification was prespecified as MYC:CEP8 ratio > 2.2 or average MYC copies/nucleus > 5.0. Exploratory variables included polysomy 8. Results In comparing DFS (median follow-up, 4.0 years) between treatments, patients with MYC:CEP8 ratio ≤ 2.2 (n = 618; 77%) and > 2.2 (n = 181; 23%) had hazard ratios (HRs) of 0.46 (P < .001) and 0.67 (P = .33), respectively (interaction P = .38). Patients with MYC copies/nucleus ≤ 5.0 (n = 534; 67%) and > 5.0 (n = 265; 33%) had HRs of 0.52 (P = .002) and 0.48 (P = .02), respectively (interaction P = .94). Patients with MYC:CEP8 ratio < 1.3 with normal chromosome 8 copy number (n = 141; 18%) and ≥ 1.3 or < 1.3 with polysomy 8 (n = 658; 82%) had HRs of 0.66 (P = .28) and 0.44 (P < .001), respectively (interaction P = .23). Patients with MYC copies/nucleus < 2.5 (n = 130; 16%) and ≥ 2.5 (n = 669; 84%) had HRs of 1.07 (P = .87) and 0.42 (P < .001), respectively (interaction P = .05). Conclusion We did not confirm the B31 association between MYC amplification and additional trastuzumab benefit. Exploratory analyses revealed potential associations between alternative MYC/chromosome 8 copy number alterations and differential benefit of adjuvant trastuzumab.

Abstract Background: N9831 is the only randomized phase III trial comparing safety and efficacy of the addition of trastuzumab (H) to doxorubicin and cyclophosphamide then paclitaxel (Arm A: AC→T) either following (Arm B: AC→T→H) or starting concurrently with paclitaxel (Arm C: AC→T+H→H) for women with resected Stage I-III invasive HER2+ breast cancer. The 3 yr cumulative incidence of NYHA class III or IV congestive heart failure or sudden cardiac death was previously reported: 3.3% in Arm C, 2.8% in Arm B (Perez EA, et al. JCO 2008). The comparison of AC→T to AC→T+H→H was reported in a joint analysis of N9831 and NSABP B-31 in 2005 and updated in 2007, demonstrating a 52% reduction in risk of a disease event (Romond E et al., NEJM 2005; Perez EA, et al. ASCO 2007).Materials and Methods: Primary endpoint is disease-free survival (DFS). At the second planned interim analysis of Arm A vs. Arm B, the O’Brien-Fleming boundary (OFB) was crossed. NCCTG Independent Data Safety Monitoring Committee approved the release of these data as well as the data pertaining to Arm B vs. Arm C due to slow pace of events [expected 647 events in 4 yr follow-up period (f/u) vs. actual 334 events in 4.5 yr f/u]. Shortly thereafter, there were sufficient events to perform the first planned interim analysis of B vs. C. We present the results of each of these pairwise comparisons taking into account the potential for crossover to Arm C after the release of the joint analysis findings in 2005.Results: From 5/2000 to 4/2005, 2448 eligible women were enrolled for the Arm A (n=1087) vs. Arm B (n=1097) comparison. Median f/u is 5.5 yrs. with 386 events. The addition of trastuzumab sequentially to AC→T significantly improved DFS, univariately [HR(Arm B/Arm A)=0.70, 95% CI: 57-86%, logrank p=0.0005] and after adjusting for age, tumor size, number of positive nodes, and ER [PPH: HRadj=0.67 (95% CI: 0.55-0.82)]. 5 yr DFS was increased from 72% with AC→T to 80% with AC→T →H.From 5/2000 to 4/2005, 1903 eligible women were enrolled for the Arm B (n=954) vs. Arm C (n=949) comparison. Median f/u is 5.3 yrs. with 312 events. The log-rank p-value testing whether DFS differs with respect to starting time of trastuzumab was 0.019. [Not crossing pre-specified OFB for statistical significance]. After adjusting for tumor size, number of positive nodes, and ER, HRadj(Arm C/Arm B)=0.75 (95% CI: 0.60-0.94)]. 5 yr DFS was increased from 80% with AC→T →H to 84% for AC→T+H →H.Conclusions: DFS is significantly improved with the addition of 52 weeks of H (sequentially or concurrently) to AC→ T. There is a statistically significant 33% reduction in the risk of an event with the sequential addition of H following AC→T. There is a strong trend for a 25% reduction in the risk of an event with starting H concurrently with T relative to sequentially after T. Therefore, based on a positive risk/benefit ratio, we recommend that trastuzumab be incorporated in a concurrent fashion with T chemotherapy.Acknowledgements: NIH CA25224, Breast Cancer Research Foundation, Genentech. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 80. NOTE: This abstract was accepted for presentation at the Symposium after the Abstract Book went to press.