HER2 heterogeneity and treatment response-associated profiles in HER2-positive breast cancer in the NCT02326974 clinical trial.

BACKGROUNDHER2-targeting therapies have great efficacy in HER2-positive breast cancer, but resistance, in part due to HER2 heterogeneity (HET), is a significant clinical challenge. We previously described that in a phase II neoadjuvant trastuzumab emtansine (T-DM1) and pertuzumab (P) clinical trial in early-stage HER2-positive breast cancer, none of the patients with HER2-HET tumors had pathologic complete response (pCR).METHODSTo investigate cellular and molecular differences among tumors according to HER2 heterogeneity and pCR, we performed RNA sequencing and ERBB2 FISH of 285 pretreatment and posttreatment tumors from 129 patients in this T-DM1+P neoadjuvant trial. A subset of cases was also subject to NanoString spatial digital profiling.RESULTSPretreatment tumors from patients with pCR had the highest level of ERBB2 mRNA and ERBB signaling. HER2 heterogeneity was associated with no pCR, basal-like features, and low ERBB2 expression yet high ERBB signaling sustained by activation of downstream pathway components. Residual tumors showed decreased HER2 protein levels and ERBB2 copy number heterogeneity and increased PI3K pathway enrichment and luminal features. HET tumors showed minimal treatment-induced transcriptomic changes compared with non-HET tumors. Immune infiltration correlated with pCR and HER2-HET status.CONCLUSIONResistance mechanisms in HET and non-HET tumors are distinct. HER2-targeting antibodies have limited efficacy in HET tumors. Our results support the stratification of patients based on HET status and the use of agents that target downstream components of the ERBB signaling pathway in patients with HET tumors.TRIAL REGISTRATIONClinicalTrials.gov NCT02326974.FUNDINGThis study was funded by Roche and the National Cancer Institute.

Impact of HER2 Heterogeneity on Treatment Response of Early-Stage HER2-Positive Breast Cancer: Phase II Neoadjuvant Clinical Trial of T-DM1 Combined with Pertuzumab.

Intratumor heterogeneity is postulated to cause therapeutic resistance. To prospectively assess the impact of HER2 (ERBB2) heterogeneity on response to HER2-targeted therapy, we treated 164 patients with centrally confirmed HER2-positive early-stage breast cancer with neoadjuvant trastuzumab emtansine plus pertuzumab. HER2 heterogeneity was assessed on pretreatment biopsies from two locations of each tumor. HER2 heterogeneity, defined as an area with ERBB2 amplification in >5% but <50% of tumor cells, or a HER2-negative area by FISH, was detected in 10% (16/157) of evaluable cases. The pathologic complete response rate was 55% in the nonheterogeneous subgroup and 0% in the heterogeneous group (P < 0.0001, adjusted for hormone receptor status). Single-cell ERBB2 FISH analysis of cellular heterogeneity identified the fraction of ERBB2 nonamplified cells as a driver of therapeutic resistance. These data suggest HER2 heterogeneity is associated with resistance to HER2-targeted therapy and should be considered in efforts to optimize treatment strategies. SIGNIFICANCE: HER2-targeted therapies improve cure rates in HER2-positive breast cancer, suggesting chemotherapy can be avoided in a subset of patients. We show that HER2 heterogeneity, particularly the fraction of ERBB2 nonamplified cancer cells, is a strong predictor of resistance to HER2 therapies and could potentially be used to optimize treatment selection.See related commentary by Okines and Turner, p. 2369.This article is highlighted in the In This Issue feature, p. 2355.

Ki-67 (30-9) scoring and differentiation of Luminal A- and Luminal B-like breast cancer subtypes.

INTRODUCTION: Ki-67 labeling index assessed by immunohistochemical assays has been shown useful in assessing the risk of recurrence for estrogen receptor (ER)-positive HER2-negative breast cancers (BC) and distinguishing Luminal A-like from Luminal B-like tumors. We aimed to assess the performance of the Ventana CONFIRM anti-Ki-67 (30-9) Rabbit Monoclonal Primary Antibody. METHODS: We constructed a case-cohort design based on a random sample (n = 679) of all patients operated on for a first primary, non-metastatic, ER-positive, HER2-negative BC at the European Institute of Oncology (IEO) Milan, Italy during 1998-2002 and all additional patients (n = 303) operated during the same period, who developed an event (metastasis in distant organs or death due to BC as primary event) and were not included in the previous subset. Multivariable Cox proportional hazards regression with inverse subcohort sampling probability weighting was used to evaluate the risk of event according to Ki-67 (30-9) and derived intrinsic molecular subtype, using previously defined cutoff values, i.e., respectively 14% and 20%. RESULTS: Ki-67 was < 14% in 318 patients (32.4%), comprised between 14 and 19% in 245 patients (24.9%) and ≥ 20 in 419 patients (42.7%). At multivariable analysis, the risk of developing distant disease was 1.88 (95% CI 1.20-2.93; P = 0.006) for those with Ki-67 comprised between 14 and 19%, and 3.06 (95% CI 1.93-4.84; P < 0.0001) for those with Ki-67 ≥ 20% compared to those with Ki-67 < 14%. Patients with Luminal B-like BC had an approximate twofold risk of developing distant disease (HR = 1.91; 95% CI 1.35-2.71; P = 0.0003) than patients with Luminal A-like BC defined using Ki-67 (30-9). CONCLUSIONS: Ki-67 evaluation using the 30-9 rabbit monoclonal primary antibody was able to stratify patients with ER-positive HER2-negative BC into prognostically distinct groups. Ki-67 assessment, with strict adherence to the international recommendations, should be included among the clinically useful biological parameters for the best treatment of patients with BC.

Clinical and analytical validation of Ki-67 in 9069 patients from IBCSG VIII + IX, BIG1-98 and GeparTrio trial: systematic modulation of interobserver variance in a comprehensive in silico ring trial.

PURPOSE: Ki-67 has been clinically validated for risk assessment in breast cancer, but the analytical validation and cutpoint-definition remain a challenge. Intraclass correlation coefficients (ICCs) are a statistical parameter for Ki-67 interobserver performance. However, the maximum degree of variance among pathologists allowed for meaningful biomarker results has not been defined. METHODS: Different amounts of variance were added to central pathology Ki-67 data (n = 9069) from three cohorts (IBCSGVIII + IX, BIG1-98, GeparTrio) by simulation of 4500 evaluations for each cohort, which were grouped by ICCs, ranging from excellent (ICC = 0.9) to poor concordance (ICC = 0.1). Endpoints were disease-free survival (DFS) and pathological complete response (pCR, GeparTrio). RESULTS: Ki-67 was a significant continuous prognostic marker for DFS over a wide range of cutpoints between 8% and 30% in all three cohorts. In our modelling approach, Ki-67 was a stable prognostic marker despite increased interpathologist variance. Even for a poor ICC of 0.5, one or more significant Ki-67 cutoffs were detected in 86.8% (GeparTrio), 92.4% (IBCSGVIII + IX) and 100% of analyses (BIG1-98). Similarly, in GeparTrio, even with an extremely low ICC of 0.2, 99.6% of analyses were significant for pCR. CONCLUSIONS: Our study shows that Ki-67 is a continuous marker which is extremely robust to pathologist variation. Even if only 50% of variance is attributable to true Ki-67-based proliferation (ICC = 0.5), this information is sufficient to obtain statistically significant differences in clinical cohorts. This stable performance explains the observation that many Ki-67 studies achieve significant results despite relevant interobserver variance and points to a high clinical validity of this biomarker. For clinical decisions based on analysis of individual patient data, ongoing efforts to further reduce interobserver variability, including ring trials and standardized guidelines as well as image analysis approaches, should be continued.

The Dilemma of HER2 Double-equivocal Breast Carcinomas: Genomic Profiling and Implications for Treatment.

The American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) 2013 guidelines for HER2 assessment have increased the number of HER2 equivocal breast carcinomas following in situ hybridization reflex testing, that is, HER2 "double equivocal" (equivocal protein expression and equivocal gene copy number). Forty-five double-equivocal carcinomas were subjected to Prosigna analysis. Twenty-seven cases were investigated for the expression of genes found to be differentially expressed between estrogen receptor (ER)-positive/HER2-positive (N=22) and ER-positive/HER2-negative (N=22) control cases. Twenty-nine of the 45 cases were also analyzed by targeted sequencing using a panel of 14 genes. We then explored the pathologic complete response rates in an independent series of double-equivocal carcinoma patients treated with trastuzumab-containing chemotherapy. All cases were ER-positive, with a mean Ki67 of 28%. Double-equivocal carcinomas were predominantly luminal B (76%); 9 cases (20%) were luminal A, and 2 cases (4%) HER2-enriched. The majority (73%) showed a high risk of recurrence by Prosigna, even when the carcinomas were small (<2 cm), node-negative/micrometastatic, and/or grade 2. Double-equivocal carcinomas showed TP53 (6/29, 20%), PIK3CA (3/29, 10%), HER2 (1/29, 3%), and MAP2K4 (1/29, 3%) mutations. Compared with grade-matched ER-positive/HER2-negative breast carcinomas from METABRIC, double-equivocal carcinomas harbored more frequently TP53 mutations and less frequently PIK3CA mutations (P<0.05). No significant differences were observed with grade-matched ER-positive/HER2-positive carcinomas. Lower pathologic complete response rates were observed in double-equivocal compared with HER2-positive patients (10% vs. 60%, P=0.009). Double-equivocal carcinomas are preferentially luminal B and show a high risk of recurrence. A subset of these tumors can be labeled as HER2-enriched by transcriptomic analysis. HER2 mutations can be identified in HER2 double-equivocal cases.

Association of Somatic Driver Alterations With Prognosis in Postmenopausal, Hormone Receptor-Positive, HER2-Negative Early Breast Cancer: A Secondary Analysis of the BIG 1-98 Randomized Clinical Trial.

Importance: A range of somatic driver alterations has been described in estrogen receptor-positive, HER2-negative (ER+/HER2-) early breast cancer (BC); however, the clinical relevance is unknown. Objective: To investigate associations of driver alterations with prognosis and the role of PIK3CA mutations in prediction of benefit associated with endocrine therapy in postmenopausal patients with ER+/HER2- early BC treated with tamoxifen or letrozole. Design, Setting, and Participants: The Breast International Group (BIG) 1-98 trial randomized 8010 postmenopausal patients with hormone receptor-positive, operable, invasive BC to monotherapy with letrozole, tamoxifen, or a sequential strategy for 5 years. Driver alterations were characterized using next-generation sequencing in primary tumors from a subset of 764 patients from 7329 eligible patients with ER+/HER2- BC, with 841 distant recurrences after a median of 8.1 years of follow-up. To correct for the oversampling of distant recurrences, weighted analysis methods were used. This analysis was conducted from April 4, 2016, to November 30, 2016. Main Outcomes and Measures: The prevalence of driver alterations, associations with clinicopathologic factors, distant recurrence-free interval, and treatment interactions were analyzed. Multivariable analyses were performed to adjust for clinicopathologic factors. Results: Of 764 samples, 538 (70.4%), including 140 distant recurrence events, were successfully sequenced. Nineteen driver alterations were observed with 5% or greater frequency, with a mean of 4 alterations (range, 0-15) per tumor. PIK3CA mutations were the most common (49%) and were significantly associated with reduction in the risk for distant recurrence (hazard ratio [HR], 0.57; 95% CI, 0.38-0.85; P = .006). TP53 mutations (HR, 1.92; 95% CI, 1.21-3.04; P = .006), amplifications on 11q13 (HR, 2.14; 95% CI, 1.36-3.37; P = .001) and 8p11 (HR, 3.02; 95% CI, 1.88-4.84; P < .001), and increasing number of driver alterations (HR per additional alteration, 1.18; 95% CI, 1.11-1.25; P < .001) were associated with significantly greater risk. Amplifications on 11q13 and 8p11 remained significant predictors in multivariable analysis, but not PIK3CA and TP53 mutations. Patients with tumors harboring kinase or helical domain PIK3CA mutations derived significantly greater benefit from letrozole over tamoxifen than patients whose tumors did not (P interaction = .002). Conclusions and Relevance: In ER+/HER2- postmenopausal, early-stage BC, amplifications on 11q13 and 8p11 were significantly associated with increased risk for distant recurrence and PIK3CA mutations were predictive of greater magnitude of benefit from letrozole. With these findings, DNA-based classification may aid adjuvant treatment decision making in this setting. Trial Registration: ClinicalTrials.gov Identifier: NCT00004205.

HER2 status predicts for upfront AI benefit: A TRANS-AIOG meta-analysis of 12,129 patients from ATAC, BIG 1-98 and TEAM with centrally determined HER2.

BACKGROUND: A meta-analysis of the effects of HER2 status, specifically within the first 2-3 years of adjuvant endocrine therapy, has the potential to inform patient selection for upfront aromatase inhibitor (AI) therapy or switching strategy tamoxifen followed by AI. The pre-existing standardisation of methodology for HER2 (immunohistochemistry/fluorescence in situ hybridization) facilitates analysis of existing data for this key marker. METHODS: Following a prospectively designed statistical analysis plan, patient data from 3 phase III trials Arimidex, Tamoxifen, Alone or in Combination Trial (ATAC), Breast International Group (BIG) 1-98 and Tamoxifen Exemestane Adjuvant Multicentre Trial (TEAM)] comparing an AI to tamoxifen during the first 2-3 years of adjuvant endocrine treatment were collected and a treatment-by-marker analysis of distant recurrence-free interval-censored at 2-3 years treatment - for HER2 status × AI versus tamoxifen treatment was performed to address the clinical question relating to efficacy of 'upfront' versus 'switch' strategies for AIs. RESULTS: A prospectively planned, patient-level data meta-analysis across 3 trials demonstrated a significant treatment (AI versus tamoxifen) by marker (HER2) interaction in a multivariate analysis; (interaction hazard ratio [HR] = 1.61, 95% CI 1.01-2.57; p < 0.05). Heterogeneity between trials did not reach statistical significance. The HER2 negative (HER2-ve) group gained greater benefit from AI versus tamoxifen (HR = 0.70, 95% CI 0.56-0.87) than the HER2-positive (HER2+ve) group (HR = 1.13, 95% CI 0.75-1.71). However, the small number of HER2+ve cases (n = 1092 across the 3 trials) and distant recurrences (n = 111) may explain heterogeneity between trials. CONCLUSIONS: A patient-level data meta-analysis demonstrated a significant interaction between HER2 status and treatment with AI versus tamoxifen in the first 2-3 years of adjuvant endocrine therapy. Patients with HER2-ve cancers experienced improved outcomes (distant relapse) when treated with upfront AI rather than tamoxifen, whilst patients with HER2+ve cancers fared no better or slightly worse in the first 2-3 years. However, the small number of HER2+ve cancers/events may explain a large degree of heterogeneity in the HER2+ve groups across all 3 trials. Other causes, perhaps related to subtle differences between AIs, cannot be excluded and warrant further exploration.

Immunohistochemical Performance of Estrogen and Progesterone Receptor Antibodies on the Dako Omnis Staining Platform: Evaluation in Multicenter Studies.

The analysis of estrogen receptor (ER) and progesterone receptor (PR) expression levels by immunohistochemistry is an important part of the initial evaluation of breast cancer and critically important in treatment planning. Anti-ERα (clone EP1) and anti-PR (clone PgR 1294) antibodies are in development for the Dako Omnis automated staining platform. These antibodies are not yet commercially available and are in performance evaluation, including the 4 international, multicenter studies reported here. For each antibody, a reproducibility study and a method comparison study was done in a randomized manner in order to test the antibodies under conditions closest to real-world user conditions. The reproducibility studies included 5 staining runs on the Dako Omnis with 20 formalin-fixed and paraffin-embedded human breast carcinoma specimens in 3 independent laboratories, and the method comparison studies included several hundred specimens stained on the Dako Omnis and on the Autostainer Link 48 platforms. Stained slides were evaluated for nuclear ER or PR expression according to American Society of Clinical Oncology/College of American Pathologists guidelines (≥1% cut-off for positive) by pathologists who were blinded from the staining method and specimen ID. For both anti-ERα (clone EP1) and anti-PR (clone PgR 1294) on the Dako Omnis, high reproducibility agreement rates were obtained on the interrun, interlaboratory, and interobserver endpoints. High concordance rates were observed between the specimens stained on the Dako Omnis platform and the Autostainer Link 48 platform. Staining quality was excellent for both anti-ERα (clone EP1) and anti-PR (clone PgR 1294) on the Dako Omnis. These results suggest that these antibodies are reliable and reproducible tools for immunohistochemistry analysis of ER and PR expression levels in formalin-fixed and paraffin-embedded breast carcinoma tissues on the Dako Omnis platform.

Assessment of HER2 amplification status in breast cancer using a new automated HER2 IQFISH pharmDx™ (Dako Omnis) assay.

In breast cancer the human epidermal growth factor receptor 2 (HER2) is an important target for a number of different HER2 inhibitors. Different slide-based assays are available for assessment of treatment eligibility, which include fluorescence in situ hybridization (FISH) or other in situ hybridization (ISH) methods for assessment of the HER2 gene status. Here we report a summary of the validation data on HER2 IQFISH pharmDx™ (Dako Omnis), a newly developed assay for the automated staining platform Dako Omnis. The assay uses a non-toxic buffer that significantly reduces the hybridization time, which results in a total turnaround time of 3½ to 4h from deparaffinization to counting of the gene and centromere signals. The data reported in the current summary covers method comparison, assessment of staining quality, observer-to-observer reproducibility as well as reproducibility within and between laboratories. Based on data from the different studies it was concluded that HER2 IQFISH pharmDx (Dako Omnis) is a reliable and robust assay with a high precision that is at least comparable to the manual HER2 IQFISH pharmDx™ assay and the PathVysion(®)HER-2 DNA Probe Kit.

Sensitive and affordable diagnostic assay for the quantitative detection of anaplastic lymphoma kinase (ALK) alterations in patients with non-small cell lung cancer.

Accurate detection of altered anaplastic lymphoma kinase (ALK) expression is critical for the selection of lung cancer patients eligible for ALK-targeted therapies. To overcome intrinsic limitations and discrepancies of currently available companion diagnostics for ALK, we developed a simple, affordable and objective PCR-based predictive model for the quantitative measurement of any ALK fusion as well as wild-type ALK upregulation. This method, optimized for low-quantity/-quality RNA from FFPE samples, combines cDNA pre-amplification with ad hoc generated calibration curves. All the models we derived yielded concordant predictions when applied to a cohort of 51 lung tumors, and correctly identified all 17 ALK FISH-positive and 33 of the 34 ALK FISH-negative samples. The one discrepant case was confirmed as positive by IHC, thus raising the accuracy of our test to 100%. Importantly, our method was accurate when using low amounts of input RNA (10 ng), also in FFPE samples with limited tumor cellularity (5-10%) and in FFPE cytology specimens. Thus, our test is an easily implementable diagnostic tool for the rapid, efficacious and cost-effective screening of ALK status in patients with lung cancer.

Analysis of HER2 status in gastroesophageal tumor specimens using a new automated HER2 IQFISH pharmDx™ (Dako Omnis) assay.

The human epidermal growth factor receptor 2 (HER2) is an important target for treatment of gastroesophageal cancer. Different slide-based assays are available for assessment of HER2 status. Overexpression of the HER2 protein is assessed by immunohistochemistry (IHC) whereas amplification of the HER2 gene is assessed by fluorescence in situ hybridization (FISH) or other in situ hybridization (ISH) methods. Here we report a summary of the validation data on HER2 IQFISH pharmDx™ (Dako Omnis), a newly developed assay for the automated staining platform Dako Omnis. This assay uses a non-toxic buffer that significantly reduces the hybridization time, which results in a total turnaround time of less than 4 hours from deparaffinization to counting of the gene and centromere signals. The data reported in the current summary cover method comparison, assessment of staining quality, observer-to-observer reproducibility as well as reproducibility within and between laboratories. Based on data from the different studies it was concluded that HER2 IQFISH pharmDx (Dako Omnis) is a reliable and robust assay, with high precision and at least comparable to the manual HER2 IQFISH pharmDx™ assay. The HER2 IQFISH pharmDx (Dako Omnis) assay is currently not commercially available outside the Europe Union.

Discordant assessment of tumor biomarkers by histopathological and molecular assays in the EORTC randomized controlled 10041/BIG 03-04 MINDACT trial breast cancer : Intratumoral heterogeneity and DCIS or normal tissue components are unlikely to be the cause of discordance.

Accurate identification of breast cancer patients most likely to benefit from adjuvant systemic therapies is crucial. Better understanding of differences between methods can lead to an improved ER, PgR, and HER-2 assessment. The purpose of this preplanned translational research is to investigate the correlation of central IHC/FISH assessments with microarray mRNA readouts of ER, PgR, and HER-2 status in the MINDACT trial and to determine if any discordance could be attributed to intratumoral heterogeneity or the DCIS and normal tissue components in the specimens. MINDACT is an international, prospective, randomized, phase III trial investigating the clinical utility of MammaPrint in selecting patients with early breast cancer for adjuvant chemotherapy (n = 6694 patients). Gene-expression data were obtained by TargetPrint; IHC and/or FISH were assessed centrally (n = 5788; 86 %). Macroscopic and microscopic evaluation of centrally submitted FFPE blocks identified 1427 cases for which the very same sample was submitted for gene-expression analysis. TargetPrint ER had a positive agreement of 98 %, and a negative agreement of 95 % with central pathology. Corresponding figures for PgR were 85 and 94 % and for HER-2 72 and 99 %. Agreement of mRNA versus central protein was not different when the same or a different portion of the tumor tissue was analyzed or when DCIS and/or normal tissue was included in the sample subjected to mRNA assays. This is the first large analysis to assess the discordance rate between protein and mRNA analysis of breast cancer markers, and to look into intratumoral heterogeneity, DCIS, or normal tissue components as a potential cause of discordance. The observed difference between mRNA and protein assessment for PgR and HER-2 needs further research; the present analysis does not support intratumoral heterogeneity or the DCIS and normal tissue components being likely causes of the discordance.

The Genomic Grade Assay Compared With Ki67 to Determine Risk of Distant Breast Cancer Recurrence.

IMPORTANCE: The Genomic Grade Index (GGI) was previously developed, evaluated on frozen tissue, and shown to be prognostic in early breast cancer. To test the GGI in formalin-fixed, paraffin-embedded breast cancer tumors, a quantitative reverse transcriptase polymerase chain reaction assay was developed and named the Genomic Grade (GG). The GG assay has the potential to increase the clinical application of the GGI, but robust demonstration of the clinical validity of the GG assay is required. OBJECTIVE: To evaluate the prognostic ability of the GG assay to detect breast cancer recurrence compared with centrally reviewed immunohistochemical testing of Ki67 antigen proliferation. DESIGN, SETTING, AND PARTICIPANTS: This is an internationally collaborative substudy of a large phase 3 4-arm adjuvant trial. Patients had endocrine receptor-positive, node-positive, or node-negative nonmetastatic primary breast cancer. Patients included in this study had available formalin-fixed, paraffin-embedded samples of their primary tumors and were randomized to either a 5-year tamoxifen monotherapy arm or a 5-year letrozole monotherapy arm. Associations between either GG assay results or log2-transformed Ki67 data and survival end points were evaluated using Cox regression models stratified for chemotherapy use; the 2 vs 4 arm randomization option; and endocrine therapy assignment with and without adjustment for clinicopathological parameters, including centrally reviewed histological grade, hormone receptors, and ERBB2 (formerly HER2 or HER2/neu). The likelihood ratio statistic was used to assess the added prognostic value. INTERVENTIONS: Central evaluation and comparison, blinded for clinical information, of the GG assay, breast cancer histological grade, and Ki67. MAIN OUTCOMES AND MEASURES: Distant recurrence-free interval (DRFI). RESULTS: Genomic Grade assay data were obtained in 883 breast cancer samples (62%). At a median follow-up of 8.1 years, 84 (10%) had distant recurrences. Increasing GG or Ki67 were both significantly associated with lower DRFI and added independent prognostic information to the clinicopathological prognostic factors. In patients with early node-negative breast cancer who were endocrine-only treated, 38% were GG1 with a 10-year DRFI of 99% (95% CI, 97%-100%), and 18% were histological grade 1 with a 10-year DRFI of 100% (95% CI, 100%-100%). For GG equivocal patients, the 10-year DRFI was 94% (95% CI, 90%-98%), and for GG3 patients, the 10-year DRFI was 87% (95% CI, 80%-94%). CONCLUSIONS AND RELEVANCE: Either the GG assay or centrally reviewed Ki67 significantly improves clinicopathological models to determine distant recurrence of breast cancer. Compared with the histological grade, the GG assay can identify a higher proportion of endocrine-only treated patients with very low risk of distant recurrence at 10 years. TRIAL REGISTRATION: clinicaltrials.gov Identifiers: NCT00004205 and NCT00004205.

ESR1 and ESR2 polymorphisms in the BIG 1-98 trial comparing adjuvant letrozole versus tamoxifen or their sequence for early breast cancer.

Estrogen receptor 1 (ESR1) and ESR2 gene polymorphisms have been associated with endocrine-mediated physiological mechanisms, and inconsistently with breast cancer risk and outcomes, bone mineral density changes, and hot flushes/night sweats. DNA was isolated and genotyped for six ESR1 and two ESR2 single-nucleotide polymorphisms (SNPs) from tumor specimens from 3691 postmenopausal women with hormone receptor-positive breast cancer enrolled in the BIG 1-98 trial to receive tamoxifen and/or letrozole for 5 years. Associations with recurrence and adverse events (AEs) were assessed using Cox proportional hazards models. 3401 samples were successfully genotyped for five SNPs. ESR1 rs9340799(XbaI) (T>C) variants CC or TC were associated with reduced breast cancer risk (HR = 0.82,95% CI = 0.67-1.0), and ESR1 rs2077647 (T>C) variants CC or TC was associated with reduced distant recurrence risk (HR = 0.69, 95% CI = 0.53-0.90), both regardless of the treatments. No differential treatment effects (letrozole vs. tamoxifen) were observed for the association of outcome with any of the SNPs. Letrozole-treated patients with rs2077647 (T>C) variants CC and TC had a reduced risk of bone AE (HR = 0.75, 95% CI = 0.58-0.98, P interaction = 0.08), whereas patients with rs4986938 (G>A) genotype variants AA and AG had an increased risk of bone AE (HR = 1.37, 95% CI = 1.01-1.84, P interaction = 0.07). We observed that (1) rare ESR1 homozygous polymorphisms were associated with lower recurrence, and (2) ESR1 and ESR2 SNPs were associated with bone AEs in letrozole-treated patients. Genes that are involved in estrogen signaling and synthesis have the potential to affect both breast cancer recurrence and side effects, suggesting that individual treatment strategies can incorporate not only oncogenic drivers but also SNPs related to estrogen activity.

Predictive value and clinical utility of centrally assessed ER, PgR, and Ki-67 to select adjuvant endocrine therapy for premenopausal women with hormone receptor-positive, HER2-negative early breast cancer: TEXT and SOFT trials.

The SOFT and TEXT randomized phase III trials investigated adjuvant endocrine therapies for premenopausal women with hormone receptor-positive (HR+) early breast cancer. We investigated the prognostic and predictive value of centrally assessed levels of estrogen receptor (ER), progesterone receptor (PgR), and Ki-67 expression in women with HER2-negative disease. Of 5707 women enrolled, 4115 with HER2-negative (HR+/HER2-) disease had ER, PgR, and Ki-67 centrally assessed by immunohistochemistry. Breast cancer-free interval (BCFI) was defined from randomization to first invasive local, regional, or distant recurrence or contralateral breast cancer. The prognostic and predictive values of ER, PgR and Ki-67 expression levels were assessed using Cox modeling and STEPP methodology. In this HR+/HER2- population, the median ER, PgR, and Ki-67 expressions were 95, 90, and 18 % immunostained cells. As most patients had strongly ER-positive tumors, the predictive value of ER levels could not be investigated. Lower PgR and higher Ki-67 expression were associated with reduced BCFI. There was no consistent evidence of heterogeneity of the relative treatment effects according to PgR or Ki-67 expression levels, though there was a greater 5-year absolute benefit of exemestane + ovarian function suppression (OFS) versus tamoxifen with or without OFS at lower levels of PgR and higher levels of Ki-67. Women with poor prognostic features of low PgR and/or high Ki-67 have greater absolute benefit from exemestane + OFS versus tamoxifen + OFS or tamoxifen alone, but individually PgR and Ki-67 are of limited predictive value for selecting adjuvant endocrine therapy for premenopausal women with HR+/HER2- early breast cancer.

Molecular Testing for Targeted Therapy in Advanced Non-Small Cell Lung Cancer: Suitability of Endobronchial Ultrasound Transbronchial Needle Aspiration.

OBJECTIVES: Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is a minimally invasive procedure that has revolutionized the diagnosis and staging of lung cancer. The goal of the present study was to investigate the yield and applicability of molecular testing in the specimens obtained by EBUS-TBNA from patients with advanced non-small cell lung cancer (NSCLC), comparing the results with a series of patients who underwent diagnostic surgical procedures in the same institution. METHODS: The study followed 306 consecutive patients with clinically diagnosed primary lung cancer who had the EBUS-TBNA procedure. EGFR and KRAS mutations were evaluated on cytologic specimens by Sanger sequencing and Cobas real-time polymerase chain reaction, whereas ALK rearrangement was tested by fluorescence in situ hybridization. The results were compared with those obtained from a series of 1,000 NSCLC surgical samples routinely analyzed. RESULTS: Molecular testing was possible in 96.9% of the samples obtained by EBUS-TBNA. EGFR (exons 18-21) mutations were found in 16.9%, KRAS mutation (exons 2-3) in 31.6%, and ALK rearrangement in 3.9% of the cases. In the surgical series, the mutations' distribution were 14.8%, 29.0%, and 3.4%, respectively. There were no statistical differences between the two series. CONCLUSIONS: Our study demonstrates that EBUS-TBNA can be effectively used not just for diagnosis but also for complete mutational testing.

CYP19A1 polymorphisms and clinical outcomes in postmenopausal women with hormone receptor-positive breast cancer in the BIG 1-98 trial.

To determine whether CYP19A1 polymorphisms are associated with abnormal activity of aromatase and with musculoskeletal and bone side effects of aromatase inhibitors. DNA was isolated from tumor specimens of 4861 postmenopausal women with hormone receptor-positive breast cancer enrolled in the BIG 1-98 trial to receive tamoxifen and/or letrozole for 5 years. Tumors were genotyped for six CYP19A1 polymorphisms using PCR-based methods. Associations with breast cancer-free interval (BCFI), distant recurrence-free interval (DRFI), musculoskeletal and bone adverse events (AEs) were assessed using Cox proportional hazards models. All statistical tests were two-sided. No association between the CYP19A1 genotypes and BCFI or DRFI was observed overall. A reduced risk of a breast cancer event for tamoxifen-treated patients with rs700518 variants was observed (BCFI CC/TC vs. TT: HR 0.53, 95 % CI 0.34-0.82, interaction P = 0.08), but not observed for letrozole-treated patients. There was an increased risk of musculoskeletal AEs for patients with rs700518 variants CC/TC versus TT (HR 1.22, 95 % CI 1.03-1.45, P = 0.02), regardless of treatment. Tamoxifen-treated patients with rs4646 variants had a reduced risk of bone AEs (AA/CA vs. CC: HR 0.76, 95 % CI 0.59-0.98), whereas an increase of minor allele (C) of rs10046 was associated with an increased risk of bone AEs (HR 1.28, 95 % CI 1.07-1.52). rs936308 variants were associated with a reduced risk of bone AEs in letrozole-treated patients (GG/GC vs. CC: HR 0.73, 95 % CI 0.54-0.99), different from in tamoxifen-treated patients (GG/GC vs. CC: HR 1.32, 95 % CI 0.92-1.90, interaction P = 0.01). CYP19A1 rs700518 variants showed associations with BCFI, DRFI, in tamoxifen treated patients and musculoskeletal AEs regardless of treatment. SNPs rs4646, rs10046, and rs936308 were associated with bone AEs.

Biology of breast cancer during pregnancy using genomic profiling.

Breast cancer during pregnancy is rare and is associated with relatively poor prognosis. No information is available on its biological features at the genomic level. Using a dataset of 54 pregnant and 113 non-pregnant breast cancer patients, we evaluated the pattern of hot spot somatic mutations and did transcriptomic profiling using Sequenom and Affymetrix respectively. We performed gene set enrichment analysis to evaluate the pathways associated with diagnosis during pregnancy. We also evaluated the expression of selected cancer-related genes in pregnant and non-pregnant patients and correlated the results with changes occurring in the normal breast using a pregnant murine model. We finally investigated aberrations associated with disease-free survival (DFS). No significant differences in mutations were observed. Of the total number of patients, 18.6% of pregnant and 23% of non-pregnant patients had a PIK3CA mutation. Around 30% of tumors were basal, with no differences in the distribution of breast cancer molecular subtypes between pregnant and non-pregnant patients. Two pathways were enriched in tumors diagnosed during pregnancy: the G protein-coupled receptor pathway and the serotonin receptor pathway (FDR <0.0001). Tumors diagnosed during pregnancy had higher expression of PD1 (PDCD1; P=0.015), PDL1 (CD274; P=0.014), and gene sets related to SRC (P=0.004), IGF1 (P=0.032), and ß-catenin (P=0.019). Their expression increased almost linearly throughout gestation when evaluated on the normal breast using a pregnant mouse model underscoring the potential effect of the breast microenvironment on tumor phenotype. No genes were associated with DFS in a multivariate model, which could be due to low statistical power. Diagnosis during pregnancy impacts the breast cancer transcriptome including potential cancer targets.

Central pathology laboratory review of HER2 and ER in early breast cancer: an ALTTO trial [BIG 2-06/NCCTG N063D (Alliance)] ring study.

Choice of therapy for breast cancer relies on human epidermal growth factor receptor-2 (HER2) and estrogen receptor α (ER) status. Before randomization in the phase III Adjuvant Lapatinib and/or Trastuzumab Treatment Optimisation (ALTTO) trial for HER2-positive disease, HER2 and ER were centrally reviewed by Mayo Clinic (Rochester, MN, and Scottsdale, AZ) for North America and by the European Institute of Oncology (IEO; Milan, Italy) for the rest of world (except China). Discordance rates (local vs. central review) differed between Mayo and IEO. Among locally HER2-positive cases, 5.8 % (Mayo) and 14.5 % (IEO) were centrally HER2 negative. Among locally ER-positive cases, 16.2 % (Mayo) and 4.2 % (IEO) were centrally ER-negative. Among locally ER-negative cases, 3.4 % (Mayo) and 21.4 % (IEO) were centrally ER-positive. We, therefore, performed a ring study to identify features contributing to these differing discordance rates. Mayo and IEO exchanged slides for 25 HER2 and 35 ER locally/centrally discordant cases. Both laboratories performed IHC and FISH for HER2 using the HercepTest(®) and PathVysion HER2 DNA probe kit/HER2/centromere 17 probe mixture. IHC for ER was tested centrally using the monoclonal ER 1D5 antibody (Mayo) or the DAKO cocktail of ER 1D5 and 2.123 antibodies (IEO). Mayo and IEO confirmed the central HER2-negative result in 100 % of 25 cases. Mayo and IEO confirmed the central ER result in 29 (85 %) of 34 evaluable cases. The five Mayo-negative/IEO-positive cases were ER-positive when retested at Mayo using the DAKO ER cocktail. In this ring study, ALTTO ineligibility did not change when HER2 testing was performed by either IEO or Mayo central laboratories. However, a dual antibody ER assay had fewer false-negative test results than an assay with a single antibody, and there was more discordance between the two ER reagents than has been previously reported. Using even slightly different assay methods yielded different results, even between experienced central laboratories.

The clinical relevance of micropapillary carcinoma of the breast: a case-control study.

AIMS: To ascertain the prognostic relevance of micropapillary carcinoma, a specific type of breast tumour. METHODS AND RESULTS: We interrogated the clinical records of a series of 49 pure micropapillary carcinoma patients and 13 487 invasive ductal carcinoma patients, diagnosed and treated consecutively in our institution over a 9-year time-frame. Compared with invasive ductal carcinoma, patients with micropapillary carcinoma more frequently had moderately differentiated tumours (P = 0.02) with extensive peritumoral vascular invasion (P < 0.0001), associated with a significantly higher rate of axillary lymph node involvement (P < 0.0001). Survival data obtained by comparing 49 micropapillary carcinoma patients with a set of 98 invasive ductal carcinoma patients matched for age, tumour size and grade, peritumoral vascular invasion, immunohistochemically defined molecular subtype, number of positive lymph nodes and year of surgery showed that the micropapillary histotype did not add any independent information to the risk of locoregional (P = 0.48) or distant (P = 0.79) relapse, or overall survival (P = 0.60). CONCLUSIONS: Our data reinforce the notion that micropapillary carcinoma usually arises as a locally advanced disease, and provide evidence that micropapillary histology does not add any additional information on clinical outcome independent of clinicopathological characteristics such as lymph node status and immunohistochemically defined molecular subtype.