Citations

Abstract Purpose: This study investigated the association between tumor MYC protein expression and disease-free survival (DFS) of patients randomized to receive chemotherapy alone (Arm A) or chemotherapy with sequential (Arm B) or concurrent trastuzumab (Arm C) in the N9831 (Alliance) adjuvant HER2+ trastuzumab breast cancer trial. Experimental Design: This analysis included 1,736 patients randomized to Arms A, B, and C on N9831. Nuclear MYC protein expression was determined in tissue microarray sections containing three biopsies per patient or whole tissue sections using standard immunohistochemistry (clone 9E10). A tumor was considered positive for MYC protein overexpression (MYC+) if the nuclear 3+ staining percentage was more than 30%. Results: Five hundred and seventy-four (33%) tumors were MYC+. MYC+ was associated with hormone receptor positivity (χ2, P = 0.006), tumors 2 cm or more (χ2, P = 0.02), and a higher rate of nodal positivity (χ2, P < 0.001). HRs for DFS (median follow-up: 6.1 years) for Arm C versus A were 0.52 (P = 0.006) and 0.65 (P = 0.006) for patients with MYC+ and MYC− tumors, respectively (Pinteraction = 0.40). For Arm B versus A, HRs for patients with MYC+ and MYC− tumors were 0.79 (P = 0.21) and 0.74 (P = 0.04), respectively (Pinteraction = 0.71). For Arm C versus B, HRs for patients with MYC+ and MYC− tumors were 0.56 (P = 0.02) and 0.89 (P = 0.49), respectively (Pinteraction = 0.17). Conclusions: Our data do not support an impact of tumor MYC protein expression on differential benefit from adjuvant trastuzumab. Clin Cancer Res; 19(20); 5798–807. ©2013 AACR.

Purpose It has been suggested that PTEN, a negative regulator of PI3K/AKT signaling, is involved in tumor sensitivity to trastuzumab. We investigated the association between tumor PTEN protein expression and disease-free survival (DFS) of patients randomly assigned to receive chemotherapy alone (arm A) or chemotherapy with sequential (arm B) or concurrent trastuzumab (arm C) in the phase III early-stage human epidermal growth factor receptor 2 (HER2) –positive trial—North Central Cancer Treatment Group (NCCTG) N9831. Patients and Methods The intensity and percentage of invasive cells with cytoplasmic PTEN staining were determined in tissue microarray sections containing three cores per block (n = 1,286) or in whole tissue sections (WS; n = 516) by using standard immunohistochemistry (138G6 monoclonal antibody). Tumors were considered positive for PTEN (PTEN-positive) if any core or WS had any invasive cells with ≥ 1+ staining. Median follow-up was 6.0 years. Results Of 1,802 patients included in this analysis (of 3,505 patients registered to N9831), 1,342 (74%) had PTEN-positive tumors. PTEN positivity was associated with hormone receptor negativity (χ2 P < .001) and nodal positivity (χ2 P = .04). PTEN did not have an impact on DFS within the various arms. Comparing DFS of arm C to arm A, patients with PTEN-positive and PTEN-negative tumors had hazard ratios (HRs) of 0.65 (P = .003) and 0.47 (P = .005), respectively (interaction P = .16). For arm B versus arm A, patients with PTEN-positive and PTEN-negative tumors had HRs of 0.70 (P = .009) and 0.85 (P = .44), respectively (interaction P = .47). Conclusion In contrast to selected preclinical and limited clinical studies suggesting a decrease in trastuzumab sensitivity in patients with PTEN-negative tumors, our data show benefit of adjuvant trastuzumab for patients with HER2-positive breast cancer, independent of tumor PTEN status.

Abstract Background: Unstained recuts from formalin-fixed paraffin-embedded tissues are commonly collected for cooperative group studies. There is concern among pathologists that improper storage conditions can lead to antigen degradation. In an effort to quantify this effect, we compared the expression of HER1 and HER2 on two sets of identical cohort tissue microarrays (TMAs) from the N9831 HER2+ adjuvant phase III trial (NCT00005970; www.clinicaltrials.gov); one freshly cut set (cut April 18, 2011) and a second set stored at 4 degrees for over two years (cut between Nov, 2007 and Jan, 2008). Methods: The two sets of TMA slides containing 1580 tumor samples from the N9831 cohort were treated identically using the AQUA method of quantitative immunofluorescence. HER1 was tested with D38B1 (rabbit monoclonal, Cell Signaling Technology, Inc.) and HER2 with CB11 (mouse monoclonal, Biocare, Inc.) on tumors from 695 patients (712 specimens) in the fresh TMAs and 779 patients (800 specimens) in the old TMAs in up to three-fold redundancy per specimen. Results: Frequency distributions of the expression of HER2 revealed bimodality in the fresh TMAs compared to an attenuated distribution of the old cases. The average score of the entire cohort was significantly lower in old TMAs compared to fresh cuts (paired t-test, p&lt;0.0001). Linear regression of the average HER2 scores from new TMAs versus the average scores from old TMAs showed a slope term of 0.52, which is statistically significantly different from the hypothetical value of 1 (p&lt;0.0001). Regressions between any two fresh slides showed slopes close to 1.0. Similar results were seen for HER1, but fewer positive cases made the changes less dramatic. Conclusions: The storage condition of tissue slides is a critical pre-analytical variable that can dramatically lower the score of HER1 and HER2, artificially. Thus, studies done on inadequately stored slides, either whole sections or TMAs, must be interpreted with caution. Tissue collection and analysis of biomarkers for cooperative group studies should not include unstained recuts, but rather, entire blocks or large cores from tissue blocks. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr PD05-04.

Abstract Background: Prediction of benefit from trastuzumab in patients (pts) with HER2+ breast cancer remains an important goal. We sought to investigate the predictive value of quantitative measurement of HER2, HER3, HER4, EGFR, ER and PTEN protein expression on the benefit of trastuzumab in the phase III HER2+ adjuvant N9831 study for pts randomized to chemotherapy alone (Arm A) or chemotherapy with sequential (Arm B) or concurrent trastuzumab (Arm C). Methods: For each marker, we evaluated quantitative expression, relationship with demographic data, and association with disease-free survival (DFS) of pts. Freshly cut tissue microarray slides with up to three-fold redundancy per specimen from the N9831 cohort were treated identically using the AQUA (Camp, et al; Nat Med 2002, JCO 2008) method of quantitative immunofluorescence for each marker. HER2 was tested with CB11 (mouse monoclonal, Biocare, Inc.) and preliminary results were available for 698 of nearly 1400 pt specimens to be tested. The minimum value per pt was used in statistical analysis. Specimens were classified with high versus low expression based on a median value cutpoint for each marker. Median follow-up was 7.0 yrs. Results: Quantitative HER2 was compared with centrally performed HER2 testing by IHC and FISH. Median quantitative HER2 via AQUA was 10,017 units for the HER2 IHC 3+ group (n=607) versus 1058, 831, and 970 for the HER2 IHC 2+ (n=68), 1+ (n=11), and 0 (n=11) groups, respectively. The Spearman correlation between quantitative HER2 and FISH HER2/CEP17 ratio was 0.32 (p&lt;0.001). High quantitative HER2 was associated with lower percentage of hormone receptor positivity (48% vs 59%, chi-sq p=0.003) but not associated with age, race, nodal positivity, tumor histology, grade, or size. High HER2 did not impact DFS in any arm of the study (See Table). Data for additional HER2 testing, HER3, HER4, EGFR, ER and PTEN are in process and will be ready by September, 2011. Conclusions: Similar to results based on standard HER2 testing by IHC and FISH in N9831, quantitative HER2 did not impact benefit from adjuvant trastuzumab. Results for additional markers will be presented. Our complete quantitative results for a second epitope on HER2, HER3, HER4, ER and EGFR will be the first report of these markers in a large patient cohort in the adjuvant setting. Disease Free Survival Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr PD05-03.

We measured the financial consequences of new CRC treatment regimens. New regimens have increased cost directly through price and indirectly through nonstandard and second-line regimen use.

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.

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.

ABSTRACTBackground:The relations between hepatic steatosis and histological features of hepatocyte injury in children with nonalcoholic fatty liver disease have yet to be examined. The aims of the present study were to establish associations between steatosis amount, type, and distribution in a well‐characterized group of children with biopsy‐proven nonalcoholic fatty liver disease (NAFLD).Patients and Methods:One hundred eight children with NAFLD seen in 5 centers were studied. Clinical and laboratory data were collected. Hematoxylin‐eosin and Masson trichrome stains were evaluated by 2 expert liver pathologists. Steatosis grade (0–3), type (macrovesicular, microvesicular, or mixed), and zone (1, 3, azonal, or panacinar) were determined. The NAFLD activity score and fibrosis stage were determined.Results:Median patient age was 12 years and median body mass index was 31 kg/m2. Fibrosis was present in 87%. The median NAFLD activity score was 4. Mild, moderate, and severe steatosis were present in 42%, 34%, and 24% of biopsies, respectively. Macrovesicular steatosis was present in 81% and mixed steatosis was present in 19%. Panacinar distribution of steatosis was most frequent (40%), followed by azonal (27%). Steatosis grade positively correlated with portal inflammation (P = 0.018). Azonal distribution positively correlated with presence of hepatocyte ballooning (P = 0.03). Biopsies with mixed steatosis were approximately 20 times more likely to have megamitochondria than those with macrovesicular steatosis alone (95% confidence interval 2.3–204.9). There was no relation between steatosis amount, type, or distribution to fibrosis stage.Conclusions:Specific histological patterns of steatosis in children are associated with histological markers of steatohepatitis. Ballooning and portal inflammation correlated well with features of steatosis.

Abstract Background: Gene expression profiling is an important tool in prediction, prognosis, and cancer modeling. The DASL® assay (cDNA –mediated Annealing, extension, Selection, and Ligation; Illumina; San Diego, CA) is a gene expression profiling system designed for degraded RNAs such as those derived from formalin-fixed paraffin-embedded (FFPE) tumor samples. FFPE tumor samples are invaluable resources for cancer research, being the most widely available materials for which patient outcomes are known. Purpose: The objective of this study was to evaluate the performance of Illumina's Whole Genome DASL platform (24,526; 24K) at distinguishing HER2-positive (HER2+) from HER2-negative (HER2-) FFPE breast tumors. Methods: We measured the relative gene expression of biological and technical replicates using nine HER2+ and 11 HER2- primary breast tumors from patients that were all node-positive and estrogen receptor-positive. The HER2 status of the tumors was determined using the Dako Hercept Test following the 2007 ASCO/CAP guidelines for HER2 positivity. The non-background corrected probe-expression data were exported from Illumina BeadStudio software and normalized using the software R and the function fastlo. Log2 transformed expression values were used to obtain the relative gene expression per probe. Spearman correlations were calculated between both types of replicates. Unequal-variance t-tests were conducted to test for differences in expression levels between HER2+ and HER2- tumors. An overall ERBB2 gene expression level was calculated by taking the average of the three ERBB2 probes for each tumor across all replicates. To simulate the possible outcome if only one replicate of each tumor was measured for gene expression, differential expression was tested for all possible combinations of single replicate probe expression values. Results: Spearman correlations for technical and biological replicates within tumor samples ranged from 0.9804 – 0.9946, and 0.9822 – 0.9945, respectively. The overall ERBB2 gene expression was significantly lower in HER2- tumors than in HER2+ tumors (p = 0.0000018). The top five most differentially expressed genes (p &lt; 0.0005) included ERBB2, GRB7, C17ORF37, PERLD1, and ASF1B. For each of the three ERBB2 probes, there were 65,536 possible ways to combine the individual probe expression values. The p-values for testing differential expression in each of these 65,536 combinations of HER2+ tumors versus HER2- tumors ranged from 0.00029 – 0.0091 for ILMN_1717902, 0.0000078 – 0.00026 for ILMN_1728761, and 0.000000002 – 0.0000032 for ILMN-2352131. Conclusions: Illumina's Whole Genome Platform provided highly reproducible gene expression values among biological and technical replicates of breast tumors. This platform also demonstrated significant differential ERBB2 gene expression between HER2+ and HER- breast tumors, which correctly classified the breast tumors as HER2+ and HER2-. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6127.