Comparison of risk assessment in 1652 early ER positive, HER2 negative breast cancer in a real-world data set: classical pathological parameters vs. 12-gene molecular assay (EndoPredict).

BACKGROUND: Risk assessment on the molecular level is important in predictive pathology to determine the risk of metastatic disease for ERpos, HER2neg breast cancer. The gene expression test EndoPredict (EP) was trained and validated for prediction of a 10-year risk of distant recurrence to support therapy decisions regarding endocrine therapy alone or in combination with chemotherapy. The EP test provides the 12-gene Molecular Score (MS) and the EPclin-Score (EPclin), which combines the molecular score with tumor size and nodal status. In this project we investigated the correlation of 12-gene MS and EPclin scores with classical pathological markers. METHODS: EndoPredict-based gene expression profiling was performed prospectively in a total of 1652 patients between 2017 and 2020. We investigated tumor grading and Ki67 cut-offs of 20% for binary classification as well as 10% and 30% for three classes (low, intermediate, high), based on national and international guidelines. RESULTS: 410 (24.8%) of 1652 patients were classified as 12-gene MS low risk and 626 (37.9%) as EPclin low risk. We found significant positive associations between 12-gene MS and grading (p < 0.001), EPclin and grading (p = 0.001), 12-gene MS and Ki67 (p < 0.001), and EPclin and Ki67 (p < 0.001). However, clinically relevant differences between EP test results, Ki67 and tumor grading were observed. For example, 118 (26.3%) of 449 patients with Ki67 > 20% were classified as low risk by EPclin. Same differences were seen comparing EP test results and tumor grading. CONCLUSION: In this study we could show that EP risk scores are distributed differentially among Ki67 expression groups, especially in Ki67 low and high tumors with a substantial proportion of patients with EPclin high risk results in Ki67 low tumors and vice versa. This suggests that classical pathological parameters and gene expression parameters are not interchangeable, but should be used in combination for risk assessment.

Evaluation of the 12-Gene Molecular Score and the 21-Gene Recurrence Score as Predictors of Response to Neo-adjuvant Chemotherapy in Estrogen Receptor-Positive, HER2-Negative Breast Cancer.

BACKGROUND: Neo-adjuvant chemotherapy (NaCT) facilitates complete surgical resection in locally advanced breast cancer. Due to its association with improved outcome, complete pathologic response (pCR) to neo-adjuvant treatment has been accepted as a surrogate for long-term outcome in clinical trials of human epidermal growth factor receptor 2 (HER2)-positive, triple-negative, or luminal B breast cancer patients. In contrast, NaCT is effective in only ~ 7-10% of estrogen receptor (ER)-positive, HER2-negative disease. Response biomarkers would enable such patients to be selected for NaCT. METHODS: Two commercially available breast cancer prognostic signatures [12-gene molecular score (MS) and the 21-gene Recurrence Score (RS)] were compared in their ability to predict pCR to NaCT in ER-positive, HER2-negative breast cancer in six public RNA expression microarray data sets. Scores were approximated according to published algorithms and analyzed by logistic regression. RESULTS: Expression data were available for 764 ER-positive, HER2-negative breast cancer samples, including 59 patients with pCR. The two scores were well correlated. Either score was a significant predictor of pCR (12-gene MS p = 9.4 × 10-5; 21-gene RS p = 0.0041). However, in a model containing both scores, the 12-gene MS remained significant (p = 0.0079), while the 21-gene RS did not (p = 0.79). CONCLUSIONS: In this microarray study, two commercial breast cancer prognostic scores were significant predictors of response to NaCT. In direct comparison, the 12-gene MS outperformed the 21-gene RS as a predictive marker for NaCT. Considering pCR as surrogate for improved survival, these results support the ability of both scores to predict chemotherapy sensitivity.

Prediction of chemotherapy benefit by EndoPredict in patients with breast cancer who received adjuvant endocrine therapy plus chemotherapy or endocrine therapy alone.

PURPOSE: EndoPredict (EPclin) is a prognostic test validated to inform decisions on adjuvant chemotherapy to endocrine therapy alone for patients with oestrogen receptor-positive, HER2-negative breast cancer. Here, we determine the performance of EPclin for estimating 10-year distant recurrence-free interval (DRFI) rates for those who received adjuvant endocrine therapy (ET) alone compared to those with chemotherapy plus endocrine therapy (ET + C). METHODS: A total of 3746 women were included in this joint analysis. 2630 patients received 5 years of ET alone (ABCSG-6/8, TransATAC) and 1116 patients received ET + C (GEICAM 2003-02/9906). The primary objective was to evaluate the ability of EPclin to provide an estimate of the 10-year DR rate as a continuous function of EPclin separately for ET alone and ET + C. Cox proportional hazard models were used for these analyses. RESULTS: EPclin was highly prognostic for DR in women who received ET alone (HR 2.79 (2.49-3.13), P < 0.0001) as well as in those who received ET + C (HR 2.27 (1.99-2.59), P < 0.0001). Women who received ET + C had significantly smaller increases in 10-year DR rates with the increasing EPclin score than those receiving ET alone (EPclin = 5; 12% ET + C vs. 20% ET alone). We observed a significant positive interaction between EPclin and treatment groups (P-interaction = 0.022). CONCLUSIONS: In this comparative non-randomised analysis, the rate of increase in DR with EPclin score was significantly reduced in women who received ET + C versus ET alone. Our indirect comparisons suggest that a high EPclin score can predict chemotherapy benefit in women with ER-positive, HER2-negative disease.

Clinical relevance and concordance of HER2 status in local and central testing-an analysis of 1581 HER2-positive breast carcinomas over 12 years.

Human epidermal growth factor receptor 2 (HER2) is a central predictive biomarker in breast cancer. Inaccurate HER2 results in different laboratories could be as high as 20%. However, this statement is based on data generated more than 13 years ago and may not reflect the standards of modern diagnostic pathology. We compared central and local HER2 testing in a total of 1581 HER2-positive tumors from five clinical trials. We evaluated the clinical relevance for pathological complete response (pCR) and disease-free survival in a subgroup of 677 tumors, which received an anti-HER2 therapy. Over the period of 12 years, the discordance rate for HER2 decreased from 52.4 (GeparTrio) to 8.4% (GeparSepto). Discordance rates were significantly higher in hormone receptor (HR)-positive tumors (26.6%), compared to HR-negative tumors (16.3%, P<0.0001), which could be explained by a different distribution of HER2 mRNA levels in HR-positive and HR-negative tumors. pCR rates were significantly lower in discordant tumors (13.7%) compared to concordant tumors (32.2%, GeparQuattro and GeparQuinto, P<0.001). In survival analysis, tumors with discordant HER2 testing had a reduced overall survival (OS) in the HR-negative group (P=0.019) and a trend for improved OS in the HR-positive group (P=0.125). The performance of local HER2 testing was considerably improved over time and has reached a 92% concordance, which shows that quality initiatives in diagnostic pathology are working. Tumors with discordant HER2 testing had a reduced therapy response and different survival rates.

Prognostic value of circulating Bcl-2/IgH levels in patients with follicular lymphoma receiving first-line immunochemotherapy.

Bcl-2/IgH rearrangements can be quantified in follicular lymphoma (FL) from peripheral blood (PB) by polymerase chain reaction (PCR). The prognostic value of Bcl-2/IgH levels in FL remains controversial. We therefore prospectively studied PB Bcl-2/IgH levels from 173 first-line FL patients who were consecutively enrolled, randomized, and treated within the multicenter phase 3 clinical trial NHL1-2003 comparing bendamustine-rituximab (B-R) with rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone. From April 2005 to August 2008, 783 pre- and posttreatment PB samples were quantified by quantitative PCR. At inclusion, 114 patients (66%) tested positive and 59 (34%) were negative for Bcl-2/IgH. High pretreatment Bcl-2/IgH levels had an adverse effect on progression-free survival (PFS) compared with intermediate or low levels (high vs intermediate: hazard [HR], 4.28; 95% confidence interval [CI], 1.70-10.77; P = .002; high vs low: HR, 3.02; 95% CI, 1.55-5.86; P = .001). No PFS difference between treatment arms was observed in Bcl-2/IgH-positive patients. A positive posttreatment Bcl-2/IgH status was associated with shorter PFS (8.7 months vs not reached; HR, 3.15; 95% CI, 1.51-6.58; P = .002). By multivariate analysis, the pretreatment Bcl-2/IgH level was the strongest predictor for PFS. Our data suggest that pre- and posttreatment Bcl-2/IgH levels from PB have significant prognostic value for PFS in FL patients receiving first-line immunochemotherapy. This trial was registered at www.clinicaltrials.gov as #NCT00991211 and at the German Federal Institute for Drugs and Medical Devices as #BfArM-4021335.

Prognostic ability of EndoPredict compared to research-based versions of the PAM50 risk of recurrence (ROR) scores in node-positive, estrogen receptor-positive, and HER2-negative breast cancer. A GEICAM/9906 sub-study.

There are several prognostic multigene-based tests for managing breast cancer (BC), but limited data comparing them in the same cohort. We compared the prognostic performance of the EndoPredict (EP) test (standardized for pathology laboratory) with the research-based PAM50 non-standardized qRT-PCR assay in node-positive estrogen receptor-positive (ER+) and HER2-negative (HER2-) BC patients receiving adjuvant chemotherapy followed by endocrine therapy (ET) in the GEICAM/9906 trial. EP and PAM50 risk of recurrence (ROR) scores [based on subtype (ROR-S) and on subtype and proliferation (ROR-P)] were compared in 536 ER+/HER2- patients. Scores combined with clinical information were evaluated: ROR-T (ROR-S, tumor size), ROR-PT (ROR-P, tumor size), and EPclin (EP, tumor size, nodal status). Patients were assigned to risk-categories according to prespecified cutoffs. Distant metastasis-free survival (MFS) was analyzed by Kaplan-Meier. ROR-S, ROR-P, and EP scores identified a low-risk group with a relative better outcome (10-year MFS: ROR-S 87 %; ROR-P 89 %; EP 93 %). There was no significant difference between tests. Predictors including clinical information showed superior prognostic performance compared to molecular scores alone (10-year MFS, low-risk group: ROR-T 88 %; ROR-PT 92 %; EPclin 100 %). The EPclin-based risk stratification achieved a significantly improved prediction of MFS compared to ROR-T, but not ROR-PT. All signatures added prognostic information to common clinical parameters. EPclin provided independent prognostic information beyond ROR-T and ROR-PT. ROR and EP can reliably predict risk of distant metastasis in node-positive ER+/HER2- BC patients treated with chemotherapy and ET. Addition of clinical parameters into risk scores improves their prognostic ability.

Clinical validation of the EndoPredict test in node-positive, chemotherapy-treated ER+/HER2- breast cancer patients: results from the GEICAM 9906 trial.

INTRODUCTION: EndoPredict (EP) is an RNA-based multigene test that predicts the likelihood of distant recurrence in patients with estrogen receptor-positive (ER+), human epidermal growth factor receptor 2-negative (HER2-) breast cancer (BC) who are being treated with adjuvant endocrine therapy. Herein we report the prospective-retrospective clinical validation of EP in the node-positive, chemotherapy-treated, ER+/HER2- BC patients in the GEICAM 9906 trial. METHODS: The patients (N = 1,246) were treated either with six cycles of fluorouracil, epirubicin and cyclophosphamide (FEC) or with four cycles of FEC followed by eight weekly courses of paclitaxel (FEC-P), as well as with endocrine therapy if they had hormone receptor-positive disease. The patients were assigned to EP risk categories (low or high) according to prespecified cutoff levels. The primary endpoint in the clinical validation of EP was distant metastasis-free survival (MFS). Metastasis rates were estimated using the Kaplan-Meier method, and multivariate analysis was performed using Cox regression. RESULTS: The molecular EP score and the combined molecular and clinical EPclin score were successfully determined in 555 ER+/HER2- tumors from the 800 available samples in the GEICAM 9906 trial. On the basis of the EP, 25% of patients (n = 141) were classified as low risk. MFS was 93% in the low-risk group and 70% in the high-risk group (absolute risk reduction = 23%, hazard ratio (HR) = 4.8, 95% confidence interval (CI) = 2.5 to 9.5; P < 0.0001). Multivariate analysis showed that, in this ER+/HER2- cohort, EP results are an independent prognostic parameter after adjustment for age, grade, lymph node status, tumor size, treatment arm, ER and progesterone receptor (PR) status and proliferation index (Ki67). Using the predefined EPclin score, 13% of patients (n = 74) were assigned to the low-risk group, who had excellent outcomes and no distant recurrence events (absolute risk reduction vs high-risk group = 28%; P < 0.0001). Furthermore, EP was prognostic in premenopausal patients (HR = 6.7, 95% CI = 2.4 to 18.3; P = 0.0002) and postmenopausal patients (HR = 3.3, 95% CI = 1.3 to 8.5; P = 0.0109). There were no statistically significant differences in MFS between treatment arms (FEC vs FEC-P) in either the high- or low-risk groups. The interaction test results between the chemotherapy arm and the EP score were not significant. CONCLUSIONS: EP is an independent prognostic parameter in node-positive, ER+/HER2- BC patients treated with adjuvant chemotherapy followed by hormone therapy. EP did not predict a greater efficacy of FEC-P compared to FEC alone.

HER2 and ESR1 mRNA expression levels and response to neoadjuvant trastuzumab plus chemotherapy in patients with primary breast cancer.

INTRODUCTION: Recent data suggest that benefit from trastuzumab and chemotherapy might be related to expression of HER2 and estrogen receptor (ESR1). Therefore, we investigated HER2 and ESR1 mRNA levels in core biopsies of HER2-positive breast carcinomas from patients treated within the neoadjuvant GeparQuattro trial. METHODS: HER2 levels were centrally analyzed by immunohistochemistry (IHC), silver in situ hybridization (SISH) and qRT-PCR in 217 pretherapeutic formalin-fixed, paraffin-embedded (FFPE) core biopsies. All tumors had been HER2-positive by local pathology and had been treated with neoadjuvant trastuzumab/ chemotherapy in GeparQuattro. RESULTS: Only 73% of the tumors (158 of 217) were centrally HER2-positive (cHER2-positive) by IHC/SISH, with cHER2-positive tumors showing a significantly higher pCR rate (46.8% vs. 20.3%, P <0.0005). HER2 status by qRT-PCR showed a concordance of 88.5% with the central IHC/SISH status, with a low pCR rate in those tumors that were HER2-negative by mRNA analysis (21.1% vs. 49.6%, P <0.0005). The level of HER2 mRNA expression was linked to response rate in ESR1-positive tumors, but not in ESR1-negative tumors. HER2 mRNA expression was significantly associated with pCR in the HER2-positive/ESR1-positive tumors (P = 0.004), but not in HER2-positive/ESR1-negative tumors. CONCLUSIONS: Only patients with cHER2-positive tumors - irrespective of the method used - have an increased pCR rate with trastuzumab plus chemotherapy. In patients with cHER2-negative tumors the pCR rate is comparable to the pCR rate in the non-trastuzumab treated HER-negative population. Response to trastuzumab is correlated to HER2 mRNA levels only in ESR1-positive tumors. This study adds further evidence to the different biology of both subsets within the HER2-positive group.

Molecular monitoring of minimal residual disease in the peripheral blood of patients with multiple myeloma.

The prognostic relevance of minimal residual disease (MRD) in patients with multiple myeloma is still an open question. In bone marrow, the level of residual myeloma cells is associated with treatment outcome, but the role of clonotypic cells in the peripheral blood (PB) for the prognosis of patients is not identified yet. In this study, we retrospectively analyzed MRD by quantitative real-time IgH-PCR (IgH-qPCR) in the PB of 42 patients undergoing high-dose therapy followed by autologous PB stem cell transplantation as first-line therapy for multiple myeloma. The MRD level of PB samples was in median 40-fold lower than in bone marrow samples, collected on the same day, with a wide intra- and interindividual variation (range, .4- to 4628-fold). The presence or absence of detectable MRD levels in PB did not correlate with the serological remission status. Still, patients with negative PCR results in PB 3 months after high-dose therapy and PB stem cell transplantation had lower International Staging System stage (P = .01), lower levels of ß2-microglobulin (P = .02), higher hemoglobin levels (P = .01), and a prolonged event-free (median, 15 versus 4 months; P = .004) and overall (median, 52 versus 17 months; P = .03) survival. Importantly, by sequential monitoring of clonotypic cells in PB, in 19 of 29 patients (66%) with progressive disease, an increase of the 2IgH/ß-actin ratio of at least 1 log step could be detected in median 4 months (range, .8 to 13 months) before the relapse was diagnosed on the basis of the European Group for Blood and Marrow Transplantation criteria. These patients with a molecular relapse in PB before a serological relapse had a significantly shorter overall survival than other patients (median, 17 months versus median not reached, P = .02). In conclusion, IgH-qPCR is a sensitive technique for the detection of clonotypic cells in PB, which precede clinical relapse. Future studies are needed to evaluate whether these circulating tumor cells play a role in promoting disease recurrence.

The prognostic and predictive value of mRNA expression of vascular endothelial growth factor family members in breast cancer: a study in primary tumors of high-risk early breast cancer patients participating in a randomized Hellenic Cooperative Oncology Group trial.

INTRODUCTION: The main prognostic variables in early breast cancer are tumor size, histological grade, estrogen receptor/progesterone receptor (ER/PgR) status, number of positive nodes and human epidermal growth factor receptor 2 (HER2) status. The present study evaluated the prognostic and/or predictive value of vascular endothelial growth factor (VEGF) family members in high-risk early breast cancer patients treated with adjuvant chemo-hormonotherapy. METHODS: RNA was isolated from 308 formalin-fixed paraffin-embedded primary tumor samples from breast cancer patients enrolled in the HE10/97 trial, evaluating adjuvant dose-dense sequential chemotherapy with epirubicin followed by cyclophosphamide, methotrexate, fluorouracil (CMF) with or without paclitaxel (E-T-CMF versus E-CMF). A fully automated method based on magnetic beads was applied for RNA extraction, followed by one-step quantitative RT-PCR for mRNA analysis of VEGF-A, -B, -C and vascular endothelial growth factor receptor (VEGFR) 1, 2, 3. RESULTS: With a median follow-up of 8 years, 109 patients (35%) developed a relapse and 80 patients (26%) died. In high VEGF-C and VEGFR1 mRNA expressing tumors, ER/PgR-negative tumors (Fisher's exact test, P = 0.001 and P = 0.021, respectively) and HER2-positive tumors (P <0.001 and P = 0.028, respectively) were more frequent than in low VEGF-C and VEGFR1 expressing tumors, respectively. From the VEGF family members evaluated, high VEGFR1 mRNA expression (above the 75th percentile) emerged as a significant negative prognostic factor for overall survival (OS; hazard ratio (HR) = 1.60, 95% confidence interval (CI): 1.01 to 2.55, Wald's P = 0.047) and disease-free survival (DFS; HR = 1.67, 95% CI: 1.13 to 2.48, P = 0.010), when adjusting for treatment group. High VEGF-C mRNA expression was predictive for benefit from adjuvant treatment with paclitaxel (E-T-CMF arm) for OS (test for interaction, Wald's P = 0.038), while in multivariate analysis the interaction of VEGF-C with taxane treatment was significant for both OS (Wald's P = 0.019) and DFS (P = 0.041) and continuous VEGF-B mRNA expression values for OS (P = 0.019). CONCLUSIONS: The present study reports, for the first time, that VEGF-C mRNA overexpression, as assessed by qRT-PCR, has a strong predictive value in high-risk early breast cancer patients undergoing adjuvant paclitaxel-containing treatment. Further studies are warranted to validate the prognostic and/or predictive value of VEGF-B, VEGF-C and VEGFR1 in patients treated with adjuvant therapies and to reveal which members of the VEGF family could possibly be useful markers in identifying patients who will benefit most from anti-VEGF strategies. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry (ANZCTR) ACTRN12611000506998.

The level of minimal residual disease in the bone marrow of patients with multiple myeloma before high-dose therapy and autologous blood stem cell transplantation is an independent predictive parameter.

The prognostic relevance of minimal residual disease (MRD) in patients with multiple myeloma is still an open question. We measured MRD levels in bone marrow (BM) samples of 53 patients treated with high-dose therapy (HDT) and autologous peripheral blood stem cell transplantation using real-time quantitative (RQ)-IgH-PCR with allel-specific oligonucleotide probes. We identified a prognostically relevant threshold level of 0.2% 2IgH/ß-actin ratio in the BM before HDT. Twenty-six patients with MRD levels below this value were termed as the "low-MRD group," whereas 27 patients with levels above this threshold were allocated to the "high-MRD group." Median event-free-survival (EFS) in the low-MRD group was significantly (P = .001) longer than in the high-MRD group with 35 versus 20 months, respectively. Overall survival (OS) within the low-MRD group was also significantly longer with 70 versus 45 months (P = .04). Using multivariate analysis, we found that the pretransplantation MRD level was an independent prognostic factor for EFS (P = .003) and OS (P = .05). Further, EFS of patients in the high-MRD could be improved (P = .005) when they achieved a low MRD level after HDT. In conclusion, measuring MRD is of prognostic relevance in patients with MM, and low MRD levels should be a goal of treatment.

HER2 and TOP2A in high-risk early breast cancer patients treated with adjuvant epirubicin-based dose-dense sequential chemotherapy.

BACKGROUND: HER2 and TOP2A parameters (gene status, mRNA and protein expression) have individually been associated with the outcome of patients treated with anthracyclines. The aim of this study was to comprehensively evaluate the prognostic/predictive significance of the above parameters in early, high-risk breast cancer patients treated with epirubicin-based, dose-dense sequential adjuvant chemotherapy. METHODS: In a series of 352 breast carcinoma tissues from patients that had been post-operatively treated with epirubicin-CMF with or without paclitaxel, we assessed HER2 and TOP2A gene status (chromogenic in situ hybridization), mRNA expression (quantitative reverse transcription PCR), as well as HER2 and TopoIIa protein expression (immunohistochemistry). RESULTS: HER2 and TOP2A amplification did not share the same effects on their downstream molecules, with consistent patterns observed in HER2 mRNA and protein expression according to HER2 amplification (all parameters strongly inter-related, p values < 0.001), but inconsistent patterns in the case of TOP2A. TOP2A gene amplification (7% of all cases) was not related to TOP2A mRNA and TopoIIa protein expression, while TOP2A mRNA and TopoIIa protein were strongly related to each other (p < 0.001). Hence, TOP2A amplified tumors did not correspond to tumors with high TOP2A mRNA or TopoIIa protein expression, while the latter were characterized by high Ki67 scores (p = 0.003 and p < 0.001, respectively). Multivariate analysis adjusted for nodal involvement, hormone receptor status, Ki67 score and HER2/TOP2A parameters revealed HER2/TOP2A co-amplification (21.2% of HER2 amplified tumors) as an independent favorable prognostic factor for DFS (HR = 0.13, 95% CI: 0.02-0.96, p = 0.046); in contrast, increased HER2/TOP2A mRNA co-expression was identified as an independent adverse prognostic factor for both DFS (HR = 2.41, 95% CI: 1.31-4.42, p = 0.005) and OS (HR = 2.83, 95% CI: 1.42-5.63, p = 0.003), while high TOP2A mRNA expression was an independent adverse prognostic factor for OS (HR = 2.06, 95% CI: 1.23-3.46, p = 0.006). None of the parameters tested was associated with response to paclitaxel. CONCLUSIONS: This study confirms the favorable prognostic value of HER2/TOP2A co-amplification and the adverse prognostic value of high TOP2A mRNA expression extending it to the adjuvant treatment setting in early high-risk breast cancer. The strong adverse prognostic impact of high HER2/TOP2A mRNA co-expression needs further validation in studies designed to evaluate markers predictive for anthracyclines. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry ACTRN12611000506998.

Decentral gene expression analysis: analytical validation of the Endopredict genomic multianalyte breast cancer prognosis test.

BACKGROUND: EndoPredict (EP) is a clinically validated multianalyte gene expression test to predict distant metastasis in ER-positive, HER2-negative breast cancer treated with endocrine therapy alone. The test is based on the combined analysis of 12 genes in formalin-fixed, paraffin-embedded (FFPE) tissue by reverse transcription-quantitative real-time PCR (RT-qPCR). Recently, it was shown that EP is feasible for reliable decentralized assessment of gene expression. The aim of this study was the analytical validation of the performance characteristics of the assay and its verification in a molecular-pathological routine laboratory. METHODS: Gene expression values to calculate the EP score were assayed by one-step RT-qPCR using RNA from FFPE tumor tissue. Limit of blank, limit of detection, linear range, and PCR efficiency were assessed for each of the 12 PCR assays using serial samples dilutions. Different breast cancer samples were used to evaluate RNA input range, precision and inter-laboratory variability. RESULTS: PCR assays were linear up to Cq values between 35.1 and 37.2. Amplification efficiencies ranged from 75% to 101%. The RNA input range without considerable change of the EP score was between 0.16 and 18.5 ng/µl. Analysis of precision (variation of day, day time, instrument, operator, reagent lots) resulted in a total noise (standard deviation) of 0.16 EP score units on a scale from 0 to 15. The major part of the total noise (SD 0.14) was caused by the replicate-to-replicate noise of the PCR assays (repeatability) and was not associated with different operating conditions (reproducibility). Performance characteristics established in the manufacturer's laboratory were verified in a routine molecular pathology laboratory. Comparison of 10 tumor samples analyzed in two different laboratories showed a Pearson coefficient of 0.995 and a mean deviation of 0.15 score units. CONCLUSIONS: The EP test showed reproducible performance characteristics with good precision and negligible laboratory-to-laboratory variation. This study provides further evidence that the EP test is suitable for decentralized testing in specialized molecular pathological laboratories instead of a reference laboratory. This is a unique feature and a technical advance in comparison with existing RNA-based prognostic multigene expression tests.

Clinical Validation of EndoPredict in Pre-Menopausal Women with ER-Positive, HER2-Negative Primary Breast Cancer.

PURPOSE: The EndoPredict prognostic assay is validated to predict distant recurrence and response to chemotherapy primarily in post-menopausal women with estrogen receptor-positive (ER+), HER2- breast cancer. This study evaluated the performance of EndoPredict in pre-menopausal women. EXPERIMENTAL DESIGN: Tumor samples from 385 pre-menopausal women with ER+, HER2- primary breast cancer (pT1-3, pN0-1) who did not receive chemotherapy in addition to endocrine therapy were tested with EndoPredict to produce a 12-gene EP molecular score and an integrated EPclin score that includes pathologic tumor size and nodal status. Associations of molecular and EPclin scores with 10-year distant recurrence-free survival (DRFS) were evaluated by Cox proportional hazards models and Kaplan-Meier analysis. RESULTS: After a median follow-up of 9.7 years, both the EP molecular score and the molecular-clinicopathologic EPclin score were associated with increased risk of distant recurrence [HR, 1.33; 95% confidence interval (CI), 1.18-1.50; P = 7.2 × 10-6; HR, 3.58; 95% CI, 2.26-5.66; P = 9.8 × 10-8, respectively]. Both scores remained significant after adjusting for clinical factors in multivariate analysis. Patients with low-risk EPclin scores (64.7%) had significantly improved DRFS compared with high-risk patients (HR, 4.61; 95% CI, 1.40-15.17; P = 4.2 × 10-3). At 10 years, patients with low-risk and high-risk EPclin scores had a DRFS of 97% (95% CI, 93%-99%) and 76% (95% CI, 67%-82%), respectively. CONCLUSIONS: The EPclin score is strongly associated with DRFS in pre-menopausal women who received adjuvant endocrine therapy alone. On the basis of these data, pre-menopausal women with EPclin low-risk breast cancer may be treated with endocrine therapy only and safely forgo adjuvant chemotherapy.

Development and validation of a cell cycle progression signature for decentralized testing of men with prostate cancer.

Aim: The 46-gene Prolaris® cell cycle progression test provides information on the risk of prostate cancer progression. Here we developed and validated a 16-gene kit-based version. Methods: RNA was extracted from prostate cancer biopsy tissue. Amplification efficiency, minimum tumor content, repeatability, reproducibility and equivalence with the 46-gene test were evaluated. Results: Amplification efficiencies for all genes were within the acceptable range (90-110%), and samples with ≥50% tumor content were appropriate for the 16-gene test. Results were repeatable (standard deviation: 0.085) and reproducible (standard deviation: 0.115). Instrument, operator and kit lot had minimal impact on results. Cell cycle progression scores from the 46- and 16-gene tests were highly correlated (r = 0.969; bias = 0.217). Conclusion: The 16-gene test performs consistently and similarly to the 46-gene test.

Prostate cancer does not always require aggressive treatment, and some men with low risk of disease progression may chose active surveillance. One way to measure the risk of disease progression is the Prolaris^® cell cycle progression test, which is performed at a commercial testing facility and measures the expression of 46 genes. However, certain European countries would prefer to run this test at a centralized testing facility. To this end we developed a streamlined kit measuring 16 genes to be used in these testing facilities, and showed that the cell cycle progression scores derived from the kit test are robust and equivalent to those obtained with the larger 46-gene test.

Prognostic utility of ß-tubulin isotype III and correlations with other molecular and clinicopathological variables in patients with early breast cancer: a translational Hellenic Cooperative Oncology Group (HeCOG) study.

We evaluated the prognostic and predictive utility of ß-tubulin isotype III (TUBB3) tumour gene transcription in early breast cancer patients enrolled in a randomised study. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was applied for assessment of TUBB3, ER, PgR, HER2 and MAPT messenger RNA and immunohistochemistry (IHC) for protein expression in 314 patients enrolled in trial HE10/97, evaluating epirubicin-alkylator adjuvant chemotherapy with or without paclitaxel. High TUBB3 mRNA status was associated with advanced T stage, high histological grade, low mRNA and protein levels of ER, PgR and MAPT, and high levels of HER2 (p < 0.001). At a median follow-up of 98 months, multivariate analysis showed high TUBB3 mRNA status to have prognostic significance for DFS (HR = 1.83, 95% CI 1.25-2.68, p = 0.002) and OS (HR = 1.71, 95% CI 1.03-2.83, p = 0.038), along with the number of involved axillary nodes, PgR mRNA status and tumour grade. TUBB3 mRNA levels did not predict benefit from inclusion of paclitaxel in adjuvant chemotherapy (test for interaction p = 0.96 for OS, p = 0.46 for DFS). Transcriptional activity of ß-tubulin isotype III in early breast cancer is an adverse prognostic factor, though not a predictive one for taxane efficacy.

Molecular Drivers of Oncotype DX, Prosigna, EndoPredict, and the Breast Cancer Index: A TransATAC Study.

PURPOSE: The Oncotype DX Recurrence Score (RS), Prosigna Prediction Analysis of Microarray 50 (PAM50) Risk of Recurrence (ROR), EndoPredict (EP), and Breast Cancer Index (BCI) are used clinically for estimating risk of distant recurrence for patients receiving endocrine therapy. Discordances in estimates occur between them. We aimed to identify the molecular features that drive the tests and lead to these differences. PATIENTS AND METHODS: Analyses for RS, ROR, EP, and BCI were conducted by the manufacturers in the TransATAC sample collection that consisted of the tamoxifen or anastrozole arms of the ATAC trial. Estrogen receptor-positive/human epidermal growth factor receptor 2 (HER2)-negative cases without chemotherapy treatment were included in which all four tests were available (n = 785). Clinicopathologic features included in some tests were excluded from the comparisons. Estrogen, proliferation, invasion, and HER2 module scores from RS were used to characterize the respective molecular features. Spearman correlation and analysis of variance tests were applied. RESULTS: There were moderate to strong correlations among the four molecular scores (ρ = 0.63-0.74) except for RS versus ROR (ρ = 0.32) and RS versus BCI (ρ = 0.35). RS had strong negative correlation with its estrogen module (ρ = -0.79) and moderate positive correlation with its proliferation module (ρ = 0.36). RS's proliferation module explained 72.5% of ROR's variance, while the estrogen module explained only 0.6%. Most of EP's and BCI's variation was accounted for by the proliferation module (50.0% and 54.3%, respectively) and much less by the estrogen module (20.2% and 2.7%, respectively). CONCLUSION: In contrast to common understanding, RSs are determined more strongly by estrogen-related features and only weakly by proliferation markers. However, the EP, BCI, and particularly ROR scores are determined largely by proliferative features. These relationships help to explain the differences in the prognostic performance of the tests.

Clozapine mobilizes CD34+ hematopoietic stem and progenitor cells and increases plasma concentration of interleukin 6 in patients with schizophrenia.

The blood of 8 European patients with schizophrenia without manifest comorbidity was studied whether the classical atypical antipsychotic drug clozapine altered the amount of circulating CD34(+) hematopoietic cells. As assessed by flow cytometry, the number of CD34(+) cells increased by 433% (from 1.49 ± 1.07 × 10(6)/L, mean ± SD pretreatment, to a peak of 6.45 ± 3.47 × 10(6)/L) following first-time therapy with clozapine for 12 weeks. The increase of CD34(+) cell, neutrophil, and leukocyte counts was statistically significant (P = 0.012). A transversal investigation of 23 long-term patients and 58 controls showed elevated neutrophil counts in the clozapine-monotreated group, whereas CD34(+) cell numbers were unaltered. A transversal investigation of 12 clozapine-monotreated long-term patients and 10 controls revealed a 1.3-fold elevation of plasma interleukin 6 levels in patients on clozapine (P = 0.017). In conclusion, clozapine treatment results in an initial mobilization of CD34(+) stem and progenitor cells into the peripheral blood and in a slight long-term elevation of interleukin 6.

Prognostic Value of EndoPredict in Women with Hormone Receptor-Positive, HER2-Negative Invasive Lobular Breast Cancer.

PURPOSE: Invasive lobular carcinoma (ILC) accounts for approximately 5%-15% of all invasive breast cancer cases. Most of the correlations between multigene assays and patient outcome were derived from studies based on patients with invasive ductal carcinoma (IDC) or without distinction between the subtypes. Here, we investigate the prognostic value of EndoPredict (EPclin) in a large cohort of ILCs pooled from three phase III randomized trials (ABCSG-6, ABCSG-8, TransATAC). EXPERIMENTAL DESIGN: The primary objective of this analysis was to determine the prognostic value of EPclin for distant recurrence (DR) in years 0-10 in postmenopausal women with ILC. The primary outcome was DR. RESULTS: 470 women (17.9%) presented with ILC, 1,944 (73.9%) with IDC, and 216 (8.2%) with other histologic types. EPclin was highly prognostic in women with ILC [HR = 3.32 (2.54-4.34)] and provided more prognostic value than the Clinical Treatment Score [CTS; HR = 2.17 (1.73-2.72)]. 63.4% of women were categorized into the low EPclin risk group and they had a 10-year DR of 4.8% (2.7-8.4) compared with 36.6% of women in the high-risk group with a 10-year DR risk of 26.6% (20.0-35.0). EPclin also provided highly prognostic information in women with node-negative disease [HR = 2.56 (1.63-4.02)] and node-positive disease [HR = 3.70 (2.49-5.50)]. CONCLUSIONS: EPclin provided highly significant prognostic value and significant risk stratification for women with ILC. Ten-year DR risk in the EPclin low-risk groups were similar between ILC and IDC. Our results show that EPclin is informative in women with ILC and suggest that it is equally valid in both histologic subtypes.

The EndoPredict score predicts response to neoadjuvant chemotherapy and neoendocrine therapy in hormone receptor-positive, human epidermal growth factor receptor 2-negative breast cancer patients from the ABCSG-34 trial.

BACKGROUND: Neoadjuvant chemotherapy (NaCT) and neoadjuvant endocrine therapy (NET) can reduce pre-operative tumour burden in patients with estrogen receptor (ER)-positive, human epidermal growth factor receptor 2 (HER2)-negative early-stage breast cancer. This prospective translational study assessed the ability of a 12-gene molecular score (MS; EndoPredict®) to predict response to NaCT or NET within the ABCSG-34 trial. PATIENTS AND METHODS: Hormone receptor (HR)-positive, HER2-negative samples from patients in the ABCSG-34 randomized phase II trial were selected and EndoPredict testing was performed to generate a 12-gene MS. ABCSG-34 patients were assigned to receive either NaCT or NET based on menopausal status, HR expression, grade and Ki67. Response was measured by residual cancer burden (RCB). RESULTS: Patients selected for NaCT generally had high-risk disease by 12-gene MS (125/134), while slightly more patients treated with NET had low-risk disease (44/83). Low-risk NaCT-treated and high-risk NET-treated tumours responded poorly (NPV 100% [95% CI 66.4%-100%] and NPV 92.3% [95% CI 79.1%-98.4%], respectively]. The 12-gene MS significantly predicted treatment response for NaCT (AUC 0.736 [95% CI 0.63-0.84]) and NET (AUC 0.726 [95% CI 0.60-0.85]). CONCLUSIONS: The 12-gene MS predicted RCB after treatment with neoadjuvant therapies for patients with HR-positive, HER2-negative early-stage breast cancer. Tumours with low MS were unlikely to benefit from NaCT, whereas a high MS predicted resistance to NET. This additional biologic information can aid personalized treatment selection in daily practice and builds a strong rationale to use EndoPredict in biomarker-driven studies in the neoadjuvant setting.