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A previously developed and centrally validated MammaPrint® (MP) and BluePrint® (BP) targeted RNA next-generation sequencing (NGS) kit was implemented and validated in two large academic European hospitals. Additionally, breast cancer molecular subtypes by MP and BP RNA sequencing were compared with immunohistochemistry (IHC). Patients with early breast cancer diagnosed at University Hospitals Leuven and Curie Institute Paris were prospectively included between September 2017 and January 2018. Formalin-fixed paraffin-embedded tissue sections were analyzed with MP and BP NGS technology at the beta sites and with both NGS and microarray technology at Agendia. Raw NGS data generated on Illumina MiSeq instruments at the beta sites were interpreted and compared with NGS and microarray data at Agendia. MP and BP NGS molecular subtypes were compared to surrogate IHC breast cancer subtypes. Equivalence of MP and BP indices was determined by Pearson's correlation coefficient. Acceptable limits were defined a priori, based on microarray data generated at Agendia between 2012 and 2016. The concordance, the Negative Percent Agreement and the Positive Percent Agreement were calculated based on the contingency tables and had to be equal to or higher than 90%. Out of 124 included samples, 48% were MP Low and 52% High Risk with microarray. Molecular subtypes were BP luminal, HER2 or basal in 82%, 8% and 10% respectively. Concordance between MP microarray at Agendia and MP NGS at the beta sites was 91.1%. Concordance of MP High and Low Risk classification between NGS at the beta sites and NGS at Agendia was 93.9%. Concordance of MP and BP molecular subtyping using NGS at the beta sites and microarray at Agendia was 89.5%. Concordance between MP and BP NGS subtyping, and IHC was 71.8% and 76.6%, for two IHC surrogate models. The MP/BP NGS kit was successfully validated in a decentralized setting. ispartof: Translational Oncology vol:12 issue:12 pages:1557-1565 ispartof: location:United States status: Published online

IntroductionWith an increase in neoadjuvant therapy recommendations for most early-stage breast cancer patients due to the COVID-19 pandemic, it has become increasingly imperative to ensure that molecular diagnostic assays provide reliable results from preoperative core needle biopsies. Therefore, the objective of this study was to determine the concordance of MammaPrint results (70-gene signature) and BluePrint results (80-gene signature) between core needle biopsies (CNB) and surgical resection (SR) specimens using prospectively collected matched tissues from patients enrolled in the FLEX trial (NCT03053193).MethodsWe analyzed 113 matched CNB and SR tumor specimens from women with early-stage breast cancer enrolled in the FLEX trial. Each patient enrolled in the trial receives a MammaPrint recurrence risk classification test with or without BluePrint molecular subtyping. Concordance of MammaPrint is reported using overall percentage agreement, positive predictive value (PPV, High Risk), negative predictive value (NPV, Low Risk), and Cohen’s kappa coefficient. Additionally, correlations between sample types are reported using Pearson correlation coefficient.ResultsWe found good concordance for MammaPrint results between CNB and SR tumor samples (90.3%, κ = 0.803), with a 95.1% NPV and 84.6% PPV. There was also a strong correlation of MammaPrint indices between CNB and SR specimens (r = 0.94). In addition to our primary objective, we determined the concordance of BluePrint subtyping in the matched tumor samples, and found excellent concordance (98.2%) and strong correlation of BluePrint scores within each subtype.ConclusionCNB samples demonstrated overall high concordance with paired SR samples for MammaPrint risk classification, ensuring that physicians are provided with accurate prognostic information for therapy decisions based on testing of core biopsy tissue. Further, BluePrint molecular subtyping also had good concordance between the sample types, outperforming concordance rates based on traditional IHC based classification. Overall, with an increase in neoadjuvant therapy, physicans and patients can be assured that MammaPrint and BluePrint provide reliable results that guide timely and appropriate therapies using preoperative CNB specimens.

The MINDACT trial showed excellent 5-year distant metastasis-free survival of 94·7% (95% CI 92·5-96·2) in patients with breast cancer of high clinical and low genomic risk who did not receive chemotherapy. We present long-term follow-up results together with an exploratory analysis by age., info:eu-repo/semantics/published

Background Many countries restrict patient material exchange to central diagnostic laboratories abroad, limiting access to assays like MammaPrint® (MP) and BluePrint® (BP). Both assays are microarray-based, with MP being prognostic for distant recurrence and BP for molecular subtyping of breast cancer (Luminal-, HER2-, and Basal-type). To increase accessibility, decentralization is required with Next Generation Sequencing (NGS) being the preferred testing platform given that most diagnostic laboratories have the technology in place. The aim of this beta testing study is to validate a previously developed and centrally validated MP and BP NGS kit for RNA samples in two large tertiary academic hospitals in Europe. Patients and Methods Patients with early breast cancer diagnosed at the Multidisciplinary Breast Center at University Hospitals Leuven and Curie Institute Paris were prospectively included between September 2017 and January 2018. Patients with bilateral breast cancer or presenting with more than 3 positive lymph nodes were excluded. Only patients with invasive ductal and invasive lobular carcinoma were included. Twenty tissue sections were cut from formalin-fixed, paraffin-embedded (FFPE) blocks; 10 tissue sections were analyzed at the local site using the MP and BP NGS kit, and 10 tissue sections were analyzed at Agendia using the same kit and procedure, as well as with the golden standard method (gene expression microarrays). Targeted RNA sequencing of the 70 MP and 80 BP signature genes was performed on Illumina MiSeq instruments. The raw NGS data generated at the local test sites was sent through a secure file transfer protocol server to Agendia for interpretation and comparison with microarray and NGS performed in the Agendia laboratories. We aimed for a minimum concordance rate between MP and BP outcome of 90% between each local site and Agendia's centralized site. Results In this study, 116 early breast cancer patients were included (73 from University Hospitals Leuven and 43 from Curie Institute). Out of these patients, 52% were MP Low Risk and 48% MP High Risk according to microarray. The patients had a BP luminal, HER2 or basal subtype in respectively 83%, 9% and 8%. Concordance between MP microarray obtained from Agendia and MP NGS obtained from the local sites was 91.4%. Concordance between MP High and Low Risk classification between NGS Leuven versus NGS Agendia was 92.1% and between NGS Curie versus NGS Agendia 95.3%. For BP subtype outcomes, the results from microarray versus NGS for all patients combined from both local sites gave a 98.3% concordance and NGS Agendia versus NGS from each local site gave a 100% concordance. Conclusion The MP and BP NGS kit was successfully validated in a decentralized setting, showing high concordance between results obtained at three different sites. There was a clear benefit of having well-trained NGS experienced diagnostic technical teams. The MP and BP NGS kit the first FFPE targeted RNA sequencing based multigene signature for breast cancer care, will provide a high and equal standard of MP and BP gene expression testing for breast cancer in a decentralized setting. Citation Format: Slembrouck L, Laurent C, Delahaye LJ, Mittempergher L, Vanden Bempt I, Vander Borght S, Darrigues L, Vliegen L, Sintubin P, Raynal V, Bohec M, Reyes C, Rapinat A, Helsmoortel C, Jongen L, Hoste G, Neven P, Wildiers H, Smeets A, Nevelsteen I, Punie K, Van Nieuwenhuysen E, Han S, Salomon AV, Faron EL, Cynober T, Witteveen AT, Neijenhuis S, Glas AM, Reyal F, Floris G. Decentralized beta testing of MammaPrint and BluePrint NGS kit at University Hospitals Leuven and Curie Institute Paris [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-08-25.

Background: The MINDACT trial showed excellent 5-year distant metastasis-free survival (DMFS) of 94·7% (95%CI 92·5-96·2) in clinical high (C-High)/genomic low (G-Low) risk patients who did not receive chemotherapy. Long-term follow-up is presented. Methods: 6693 patients were enrolled 2007 to 2011. We reassessed DMFS rate at 5 years in C-High/G-Low patients not receiving chemotherapy (n=644) (primary analysis), updated DMFS and overall survival in C-High/G-Low patients receiving chemotherapy or not (n=749 and 748, respectively) (secondary analysis), and examined a potential age effect ( 50y) for women with HR+/HER2- disease. Findings: At a median follow-up of 8·7 years, the updated 5-year DMFS rate for C-High/G-Low patients receiving no chemotherapy is 95·1% (95%CI 93·1-96·6), above the predefined non-inferiority boundary of 92%, proving MINDACT as a positive de-escalation trial.The 5-year and 8-year outcome of C-High/G-Low patients show a respective 0·9% (SE ±1·1) 2·6% (SE ±1·6) difference in DMFS for chemotherapy versus no chemotherapy (HR 0·66; 95%CI 0·48-0·92), without differences in overall survival (95·7% vs 94·3%).The same comparisons confined to HR+/HER2- disease (91% of patients) generate different gain estimates from chemotherapy administration for DMFS according to age: 5% (SE ±2·8%) in women ≤50 years versus 0·2% (SE ±2·3%) in women > 50 years. Interpretation: The clinical utility of a low risk 70-gene signature for foregoing adjuvant chemotherapy in the presence of a high clinical risk (and up to 3 positive nodes) remains proven. In C-high/G-low patients > 50 years chemotherapy did not improve outcome across the whole follow-up trajectory. At longer follow-up in younger women, while underpowered, a clinically relevant benefit of about 5% was observed, possibly due to chemotherapy-induced ovarian function suppression, and should be part of informed, shared decision-making. Trial Registration: ClinicalTrials.gov number, NTC00433589; EudraCT number, 2005-002625-31. Funding Statement: Funded by the European Commission Sixth Framework Programme and other grants. Declaration of Interests: None to declare. Ethics Approval Statement: All patients gave written informed consent. The ethics committees of all participating sites approved the study, which involved collaboration of seven member groups under the Breast International Group umbrella.

Background: The PARP inhibitor veliparib in combination with carboplatin (VC) was one of the experimental regimens evaluated in the phase 2 neoadjuvant I-SPY 2 standing trial for high risk breast cancer patients. VC graduated in the triple negative (TN) signature. However, not all TN patients achieved pathologic complete response (pCR) and some HR+HER2- patients responded. The I-SPY 2 biomarker component provides a platform for rigorous evaluation of mechanism-of-action-based markers in the context of established biomarkers (HR, HER2, MammaPrint) within the trial. Here, we report results from 5 investigator-submitted biomarker proposals and the MammaPrint High1/High 2 (MP1/2) classification as specific predictors of VC response. Methods: Data from 116 HER2- patients (VC: 72 and concurrent controls: 44) were available. BRCA1/2 germline mutation was assessed by Myriad Genetics. 3 expression signatures relating to DNA damage repair deficiency (PARPi-7, BRCAness and CIN70) and MP1/2 classification were evaluated on Agilent 44K arrays. PARP1 levels were measured using reverse phase protein arrays. We used logistic modeling to assess biomarker performance. A biomarker is considered a specific predictor of VC response if it associates with response in the V/C arm but not the control arm, and if the biomarker x treatment interaction is significant (likelihood ratio test, p Results: BRCA1/2 germline mutation status associates with VC response, but its low prevalence in the control arm precludes further evaluation. Of the biomarkers evaluated, three (PARPi-7, BRCAness, and MP1/2) associate with response in the VC arm but not the control arm, and have biomarker x treatment interactions with p < 0.05 that retains significance upon adjusting for HR status. These signatures are only moderately correlated. When we combined the PARPi-7 and MP1/2 classifications using a simple voting scheme, the 40% of TN patients who are PARPi7-high and MP2 have an estimated pCR rate of 79% in the VC arm. In contrast, TN patients negative for at least one of these signatures (PARPi7-low and/or MP1) only have an estimated pCR rate of 35%. Only 9% of HR+/HER2- patients are PARPi7-high and MP2; but these patients also appear more responsive to VC with estimated pCR rates of 49%, compared to 13% in 'biomarker-negative' HR+HER2- patients. Conclusion: If verified in a larger trial, PARPi7, BRCAness and MP1/2 signatures may help refine predictions of VC response, thereby improving patient care. Evaluation of the combined signature for patients treated with platinum-based chemotherapy is ongoing. Citation Format: Wolf DM, Yau C, Sanil A, Glas A, Petricoin C, Wulfkuhle J, Brown-Swigart L, Hirst G, I-SPY 2 TRIAL Investigators, Buxton M, DeMichele A, Hylton N, Symmans F, Yee D, Paoloni M, Esserman L, Berry D, Rugo H, Olapade O, van 't Veer L. DNA repair deficiency biomarkers and MammaPrint high1/(ultra)high2 risk as predictors of veliparib/carboplatin response: Results from the neoadjuvant I-SPY 2 trial for high risk breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr S2-06.

BACKGROUND: The 70-gene signature test (MammaPrint) has been shown to improve prediction of clinical outcome in women with early-stage breast cancer. We sought to provide prospective evidence of the clinical utility of the addition of the 70-gene signature to standard clinical-pathological criteria in selecting patients for adjuvant chemotherapy. METHODS: In this randomized, phase 3 study, we enrolled 6693 women with early-stage breast cancer and determined their genomic risk (using the 70-gene signature) and their clinical risk (using a modified version of Adjuvant! Online). Women at low clinical and genomic risk did not receive chemotherapy, whereas those at high clinical and genomic risk did receive such therapy. In patients with discordant risk results, either the genomic risk or the clinical risk was used to determine the use of chemotherapy. The primary goal was to assess whether, among patients with high-risk clinical features and a low-risk gene-expression profile who did not receive chemotherapy, the lower boundary of the 95% confidence interval for the rate of 5-year survival without distant metastasis would be 92% (i.e., the noninferiority boundary) or higher. RESULTS: A total of 1550 patients (23.2%) were deemed to be at high clinical risk and low genomic risk. At 5 years, the rate of survival without distant metastasis in this group was 94.7% (95% confidence interval, 92.5 to 96.2) among those not receiving chemotherapy. The absolute difference in this survival rate between these patients and those who received chemotherapy was 1.5 percentage points, with the rate being lower without chemotherapy. Similar rates of survival without distant metastasis were reported in the subgroup of patients who had estrogen-receptor-positive, human epidermal growth factor receptor 2-negative, and either node-negative or node-positive disease. CONCLUSIONS: Among women with early-stage breast cancer who were at high clinical risk and low genomic risk for recurrence, the receipt of no chemotherapy on the basis of the 70-gene signature led to a 5-year rate of survival without distant metastasis that was 1.5 percentage points lower than the rate with chemotherapy. Given these findings, approximately 46% of women with breast cancer who are at high clinical risk might not require chemotherapy. (Funded by the European Commission Sixth Framework Program and others; ClinicalTrials.gov number, NCT00433589; EudraCT number, 2005-002625-31.).

MammaPrint is an FDA-cleared microarray-based test that uses expression levels of the 70 MammaPrint genes to assess distant recurrence risk in early-stage breast cancer. The prospective RASTER study proved that MammaPrint Low Risk patients can safely forgo chemotherapy, which is further subject of the prospective randomized MINDACT trial. While MammaPrint diagnostic results are obtained from mini-arrays, clinical trials may be performed on whole-genome arrays. Here we demonstrate the equivalence and reproducibility of the MammaPrint test. MammaPrint indices were collected for breast cancer samples: (i) on both customized certified array types (n = 1,897 sample pairs), (ii) with matched fresh and FFPE tissues (n = 552 sample pairs), iii) for control samples replicated over a period of 10 years (n = 11,333), and iv) repeated measurements (n = 280). The array type indicated a near perfect Pearson correlation of 0.99 (95 % CI: 0.989–0.991). Paired fresh and FFPE samples showed an excellent Pearson correlation of 0.93 (95 % CI 0.92–0.94), in spite of the variability introduced by intratumoral tissue heterogeneity. Control samples showed high consistency over 10 year's time (overall reproducibility of 97.4 %). Precision and repeatability are overall 98.2 and 98.3 %, respectively. Results confirm that the combination of the near perfect correlation between array types, excellent equivalence between tissue types, and a very high stability, precision, and repeatability demonstrate that results from clinical trials (such as MINDACT and I-SPY 2) are equivalent to current MammaPrint FFPE and fresh diagnostics, and can be used interchangeably. Electronic supplementary material The online version of this article (doi:10.1007/s10549-016-3764-5) contains supplementary material, which is available to authorized users.

Hepatitis C virus infection is causing chronic liver disease, cirrhosis, and hepatocellular carcinoma. By combining direct-acting antivirals (DAAs), high sustained virologic response rates (SVRs) can be achieved. Resistance-associated substitutions (RASs) are commonly observed after DAA failure, and especially nonstructural protein 5A (NS5A) RASs may impact retreatment options.1-3 Data on retreatment of DAA failure patients using first-generation DAAs are limited.4-7 Recently, a second-generation protease- and NS5A-inhibitor plus sofosbuvir (voxilaprevir/velpatasvir/sofosbuvir [VOX/VEL/SOF]) was approved for retreatment after DAA failure.8 However, this and other second-generation regimens are not available in many resource-limited countries or are not reimbursed by regular insurance, and recommendations regarding the selection of retreatment regimens using first-generation DAAs are very important. This study aimed to analyze patients who were re-treated with first-generation DAAs after failure of a DAA combination therapy.

To better understand the expression pattern of programmed death-ligand 1 (PD-L1) expression in different breast cancer types, we characterized PD-L1 expression in tumor and tumor-infiltrating immune cells, in relation to mutation rate, BRCA1-like status and survival. We analyzed 410 primary treatment-naive breast tumors comprising 162 estrogen receptor-positive (ER+) and HER2−, 101 HER2+ and 147 triple-negative (TN) cancers. Pathologists quantified tumor-infiltrating lymphocytes (TILs) and PD-L1 expression in tumor cells and TILs using whole slides and tissue microarray. Mutation rate was assessed by DNA sequencing, BRCA1-like status using multiplex ligation-dependent probe amplification, and immune landscape by multiplex image analyses of CD4, CD68, CD8, FOXP3, cytokeratin, and PD-L1. Half of PD-L1 scores evaluated by tissue microarray were false negatives compared to whole slide evaluations. We observed at least 1% of PD-L1-positive (PD-L1+) cells in 53.1% of ER+HER2−, 73.3% of HER2+, and 84.4% of TN tumors. PD-L1 expression was higher in ductal compared to lobular carcinomas, also within ER+HER2− tumors (p = 0.04). High PD-L1+ TILs score (> 50%) was independently associated with better outcome in TN tumors (HR = 0.27; 95%CI = 0.10–0.69). Within TN tumors, PD-L1 and TIL scores showed a modest but significant positive association with the number of silent mutations, but no association with BRCA1-like status. Multiplex image analyses indicated that PD-L1 is expressed on multiple immune cells (CD68+ macrophages, CD4+, FOXP3+, and CD8+ T cells) in the breast tumor microenvironment, independent of the PD-L1 status of the tumor cells. We found no evidence that levels of PD-L1+ TILs in TN breast cancer are driven by high mutation rate or BRCA1-like status.

Background: Patients with BRCA1-like tumors correlate with improved response to DNA double-strand break-inducing therapy. A gene expression-based classifier was developed to distinguish between BRCA1-like and non-BRCA1-like tumors. We hypothesized that these tumors may also be more sensitive to PARP inhibitors than standard treatments.Methods: A diagnostic gene expression signature (BRCA1ness) was developed using a centroid model with 128 triple-negative breast cancer samples from the EU FP7 RATHER project. This BRCA1ness signature was then tested in HER2-negative patients (n = 116) from the I-SPY 2 TRIAL who received an oral PARP inhibitor veliparib in combination with carboplatin (V-C), or standard chemotherapy alone. We assessed the association between BRCA1ness and pathologic complete response in the V-C and control arms alone using Fisher's exact test, and the relative performance between arms (biomarker × treatment interaction, likelihood ratio p Results: We developed a gene expression signature to identify BRCA1-like status. In the I-SPY 2 neoadjuvant setting the BRCA1ness signature associated significantly with response to V-C (p = 0.03), but not in the control arm (p = 0.45). We identified a significant interaction between BRCA1ness and V-C (p = 0.023) after correcting for HR.Conclusions: A genomic-based BRCA1-like signature was successfully translated to an expression-based signature (BRC1Aness). In the I-SPY 2 neoadjuvant setting, we determined that the BRCA1ness signature is capable of predicting benefit of V-C added to standard chemotherapy compared to standard chemotherapy alone.Trial registration: I-SPY 2 TRIAL beginning December 31, 2009: Neoadjuvant and Personalized Adaptive Novel Agents to Treat Breast Cancer (I-SPY 2), NCT01042379.

This study compares immunohistochemical (IHC) versus molecular subtyping (BluePrint and MammaPrint) in the population of patients enrolled in MINDACT and outcome based on molecular subtyping (MS) versus surrogate pathological subtyping (PS) as defined by the 2013 St. Gallen guidelines. MS classified patients in the following subtypes: Luminal A, Luminal B, HER-2-, and Basal-type. IHC/FISH for pathological subtyping (ER, PgR, HER-2, and Ki67) was centrally assessed in the European Institute of Oncology (n = 5806). Hazard ratios for distant-metastasis-free survival (DMFS) by subtype were adjusted for chemotherapy and endocrine therapy administration and thus independent of adjuvant treatment allocation. PS Luminal cancers classified as HER-2+ or Basal-type by MS did not have a significantly lower DMFS than the Luminal-type cancers by MS (95.9%): HR = 1.40, 95% CI 0.75–2.60 (p = 0.294). More patients were identified with Luminal A disease by MS (63%) as compared with PS (47%) with comparable 5-year DMFS (≥96.0%). Among the 500 patients with PS TN cancers, MS identified 24 (5%) patients as Luminal-type with 5-year DMFS estimated at 100% versus 71.4% for MS HER-2+ or 90.1% for MS Basal-type. MS was able to re-stratify 54% of patients with a Luminal-B PS subtype to a low-risk Luminal A-type group with comparable outcome. Among TN EBC, 5% were classified as Luminal by MS with Luminal-like outcome. Molecular classification can help to identify a larger group of patients with low risk of recurrence compared with the more contemporarily used classification methodology including high-quality assessed Ki67.

To investigate the correlation of TargetPrint with local and central immunohistochemistry/fluorescence in situ hybridization assessment of estrogen (ER), progesterone (PgR), and human epidermal growth factor receptor 2 (HER2) in the first 800 patients enrolled in the MINDACT trial.Data from local (N = 800) and central (N = 626) assessments of receptor status were collected and compared with TargetPrint results.For ER, the positive agreement (the percentage of central pathology positive assessments that were also TargetPrint/local laboratory positive) for TargetPrint in comparison to centralized assessment was 98% with a negative agreement (the percentage of central pathology negative assessments that were also TargetPrint/local laboratory negative) of 96%. For PgR, the positive agreement was 83% with a negative agreement of 92%. For HER2, the positive agreement was 75% with a negative agreement of 99%. Even though the local assessment showed higher positive agreement for PgR (89%) and higher positive agreement for HER2 (85%), the range of discordant local versus central assessments were as high as 6.7% for ER, 12.9% for PgR, and 4.3% for HER2.TargetPrint and local assessment of ER, PgR, and HER2 show high concordance with central assessment in the first 800 MINDACT patients. However, there are concerns about the higher discordance rates for some local sites. TargetPrint can improve the reliability of hormone receptor and HER2 testing for those centers with a lower rate of concordance with the reference laboratory, with the limitation of a positive agreement of 75% for HER2. TargetPrint consequently has important implications for treatment decisions in clinical practice and is a reliable alternative to local assessment for ER.NCT00433589.

Background MammaPrint® (MP) is a 70-gene based prognostic assay that stratifies early-stage breast cancer patients into low and high-risk of relapse. Recently, further stratification of the 70-gene risk results identified extreme low and high-risk subgroups with specific clinical outcomes (Delahaye et al. 2017) and treatment response characteristics (Wolf et al. 2017). However, the biological profiles of these extreme MP subgroups are not fully investigated. In this study, we aim to gain more insight into their biological significance using differentially expression genes (DEGs) analysis. Methods We selected 400 samples from the whole MP range and defined 4 subgroups (Ultra high [UH], High risk [HR], Low risk [LR], Ultra low [UL]), for which FFPE microarray full-transcriptome data were available at Agendia. DEGs analysis was performed with limma and subsequent pathway analysis with Enrichr and GOrilla. Results Two separate comparative analyses were carried out to unravel biological processes associated with extreme risk subgroups: UL vs. LR and UH vs. HR. We found 101 DEGs (logFC > =0.485 & FDR Conclusions Our preliminary findings give additional insights into the biological processes associated with extreme MP groups, which might open new avenues for therapeutic intervention in breast cancer. Legal entity responsible for the study Agendia Inc. Funding Agendia Inc. Disclosure R. Bhaskaran: Full / Part-time employment: Agendia Inc. C. Griffioen: Full / Part-time employment: Agendia Inc. D. Wehkamp: Full / Part-time employment: Agendia Inc. L. Mittempergher: Full / Part-time employment: Agendia Inc. A.M. Glas: Full / Part-time employment: Agendia Inc.

Background: The majority of breast cancer patients are diagnosed with ERα-positive breast cancer. Most ERα-positive patients are treated with adjuvant endocrine therapy — typically tamoxifen or aromatase inhibitors — to block cellular proliferation. Although these treatments are considered successful, resistance is common. Notably, cross-resistance between the two types of therapies is not always observed suggesting molecular heterogeneity and underlining the need for development of personalized treatments. The Anastrozole, Fulvestrant or Tamoxifen Exposure — Response in molecular profile study (AFTER study, NCT00738777) aims to investigate prospectively whether short-term treatment can induce molecular changes indicative of pre-operative therapy response. Study Design: ERα-positive breast cancer patients are included in this open-label multicenter study. Post-menopausal patients are randomized between tamoxifen, anastrozole and fulvestrant and pre-menopausal and male patients receive tamoxifen. Treatment occurs during the pre-operative window between diagnosis and surgery (4±2 weeks). Clinical characteristics collected are ERα/PR and HER2 status as well as lymph-node status. The primary endpoint is the decrease in tumor cell proliferation, as assessed by Ki67 gene expression and published cell proliferation gene expression signatures. All data are collected from both pre- and post-treatment samples. Additionally, we will compare the changes induced by treatment in gene expression, ERα/DNA binding interactions, DNA copy number, endoxifen and estradiol levels. Results: Among 67 patients currently enrolled, we examined the data from the subset of 28 tamoxifen treated patients. ERα and PR levels did not differ significantly between pre- and post-treatment. All tumors were HER2-negative. Proliferation examined by Ki67 (IHC and gene expression, MKI67) was significantly lower in post-treatment samples (P < 0.01). A significant association was identified with the change in gene expression proliferation signature score and change in MKI67 (rho = 0.7, P < 0.001). We identified two samples, which changed from MammaPrint (MP) low-risk to high-risk among 17 pairs with data. One sample's score was on the cutoff for high-risk definition. Interestingly, the second sample also had an increase in Ki67 gene expression and proliferation gene signature score in the post-treatment sample. Overall, ERα/DNA binding interaction regions overlapped significantly more among post-treatment samples as compared to pre-treatment samples (P Citation Format: Linn SC, Severson TM, Nevedomskaya E, Peeters J, van Rossum A, Kuilman T, Krijgsman O, Goossens I, Glas A, Koornstra R, Peeper D, Wesseling J, Simon I, Wessels L, Zwart W. Neoadjuvant tamoxifen therapy synchronizes ERα binding and gene expression profiles. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-08-06.

Objectives: The aim of this study was to independently validate a genomic signature developed both to assess recurrence risk in stage II patients and to assist in treatment decisions. Background: Adjuvant therapy is recommended for high-risk patients with stage II colon cancer, but better tools to assess the patients’ prognosis accurately are still required. Methods: Previously, an 18-gene signature had been developed and validated on an independent cohort, using full genome microarrays. In this study, the gene signature was translated and validated as a robust diagnostic test (ColoPrint), using customized 8-pack arrays. In addition, clinical validation of the diagnostic ColoPrint assay was performed on 135 patients who underwent curative resection (R0) for colon cancer stage II in Munich. Fresh-frozen tissue, microsatellite instability status, clinical parameters, and follow-up data for all patients were available. The diagnostic ColoPrint readout was determined blindly from the clinical data. Results: ColoPrint identified most stage II patients (73.3%) as at low risk. The 5-year distant-metastasis free survival was 94.9% for low-risk patients and 80.6% for high-risk patients. In multivariable analysis, ColoPrint was the only significant parameter to predict the development of distant metastasis with a hazard ratio of 4.28 (95% confidence interval, 1.36–13.50; P = 0.013). Clinical risk parameters from the American Society of Clinical Oncology (ASCO) recommendation did not add power to the ColoPrint classification. Technical validation of ColoPrint confirmed stability and reproducibility of the diagnostic platform. Conclusions: ColoPrint is able to predict the development of distant metastasis of patients with stage II colon cancer and facilitates the identification of patients who may be safely managed without chemotherapy.

Veliparib combined with carboplatin (VC) was an experimental regimen evaluated in the biomarker-rich neoadjuvant I-SPY 2 trial for breast cancer. VC showed improved efficacy in the triple negative signature. However, not all triple negative patients achieved pathologic complete response and some HR+HER2− patients responded. Pre-specified analysis of five DNA repair deficiency biomarkers (BRCA1/2 germline mutation; PARPi-7, BRCA1ness, and CIN70 expression signatures; and PARP1 protein) was performed on 116 HER2− patients (VC: 72 and concurrent controls: 44). We also evaluated the 70-gene ultra-high risk signature (MP1/2), one of the biomarkers used to define subtype in the trial. We used logistic modeling to assess biomarker performance. Successful biomarkers were combined using a simple voting scheme to refine the ‘predicted sensitive’ group and Bayesian modeling used to estimate the pathologic complete response rates. BRCA1/2 germline mutation status associated with VC response, but its low prevalence precluded further evaluation. PARPi-7, BRCA1ness, and MP1/2 specifically associated with response in the VC arm but not the control arm. Neither CIN70 nor PARP1 protein specifically predicted VC response. When we combined the PARPi-7 and MP1/2 classifications, the 42% of triple negative patients who were PARPi7-high and MP2 had an estimated pCR rate of 75% in the VC arm. Only 11% of HR+/HER2− patients were PARPi7-high and MP2; but these patients were also more responsive to VC with estimated pathologic complete response rates of 41%. PARPi-7, BRCA1ness and MP1/2 signatures may help refine predictions of VC response, thereby improving patient care., Biomarkers: Gene expression tests predict response to PARP inhibitor combined with carboplatin Several predictive gene signatures can help identify breast cancer patients likely to respond to veliparib, an investigational PARP inhibitor, combined with the chemotherapy agent carboplatin. A team led by Denise Wolf, Christina Yau, and Laura van ‘t Veer from the University of California, San Francisco, used data from the I-SPY 2 trial to assess the predictive value of six different biomarkers in determining which women with early stage and locally advanced, aggressive breast cancer would have no signs of disease after veliparib—carboplatin treatment. They found three biomarkers with predictive value: a 7-gene expression signature that predicts breast cancer cell line sensitivity to another PARP inhibitor called olaparib; a 77-gene expression signature that detects molecular features shared with BRCA1-mutant tumours; and a 70-gene signature of recurrence risk called MammaPrint.

Purpose Early-stage hormone-receptor positive breast cancer is treated with endocrine therapy and the recommended duration of these treatments has increased over time. While endocrine therapy is considered less of a burden to patients compared to chemotherapy, long-term adherence may be low due to potential adverse side effects as well as compliance fatigue. It is of high clinical utility to identify subgroups of breast cancer patients who may have excellent long-term survival without or with limited duration of endocrine therapy to aid in personalizing endocrine treatment. Methods We describe a new ultralow risk threshold for the 70-gene signature (MammaPrint) that identifies a group of breast cancer patients with excellent 20 year, long-term survival prognosis. Tumors of these patients are referred to as “indolent breast cancer.” We used patient series on which we previously established and assessed the 70-gene signature high–low risk threshold. Results In an independent validation cohort, we show that patients with indolent breast cancer had 100% breast cancer-specific survival at 15 years of follow-up. Conclusions Our data indicate that patients with indolent disease may be candidates for limited treatment with adjuvant endocrine therapy based on their very low risk of distant recurrences or death of breast cancer. Electronic supplementary material The online version of this article (doi:10.1007/s10549-017-4262-0) contains supplementary material, which is available to authorized users.

Background: However hormone receptor–positive, early-stage breast cancer is a disease with a long natural history and improved survival with 5-year adjuvant endocrine treatments. Yet late recurrence remains an important issue in adjuvant therapy. Predictors of late recurrence are not yet well characterized. Method: A total of 252 breast primary tumors were selected at the Netherlands Cancer Institute from retrospective series of ER+, HER2− breast cancer patients with a follow-up of at least 10 years. Gene expression analysis was performed using Agilent 4×44K microarrays. In order to identify genes associated to late survival differences, we used the survdiff function implemented in the R package survival and we set the parameter rho to −1 to give weight to the later part of the survival curve. The survdiff function was applied to each probe individually for DMFS time considering the probe as a covariate dichotomized into 2 groups (above and below the median expression across all samples). The parameter “strata” was used to stratify the 140 patients based on additional clinico-pathological parameters (Grade, Diameter, Lymph node status and MammaPrint), in order to find genes that add prognostic value to those parameters already known. This approach uses the distant-metastasis free survival (DMFS) time as a continuous variable. Results: After univariate analysis, MammaPrint, diameter, lymph node status and grade were significantly associated to late DMFS differences (Chi-square test p-values equal to 0.016, 0.004, In order to independently validate the prognostic power of these two genes, we tested their performance in the validation set of treated patients (n = 112) and in three publicly available datasets. In all datasets, the CH25H gene confirmed to be significantly associated to metastasis-free survival time in all tested series. Conclusions: These results might indicate that CH25H is an independent marker of late metastatic relapses. CH25H catalyzes the formation of 25-hydroxycholesterol from cholesterol, leading to repress cholesterol biosynthetic enzymes. In the last years, it is emerging that lipid metabolism plays an important role in breast cancer development and progression. Taken together, these findings make the CH25H gene a potential target for late metastasis control in breast cancer. These results warrant further prospective investigation and functional characterization of CH25H in this setting. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-09-05.

Background Previous reports have described the use of microarrays to assess the molecular classification of human breast cancers and defined new subgroups based on gene expression that are relevant to patient management through their ability to predict metastatic relapse and survival relapse. However, different mechanisms may be associated with the development of early and late distant metastases. With the hypothesis that tumors may lead to early or distant metastases based on their intrinsic biological initial features, we aimed at defining molecular profiles for several subgroups of patients based on their outcome over time. Material and methods Breast primary tumors were selected from retrospective series of patients with frozen material available. These series include patients of all ages, LN- and LN+; Estrogen or Progesteron-receptor positive, Her2-negative, no adjuvant treatment, with a follow-up of more than 10 years (y) for the control group or distant metastatic relapse as first event (DM) for the study group (n=144). Patients tumors were classified in 4 groups: no relapse at 10 y (M0), DM before 3 y (M0-3, n=30), DM between 3 and 7 y (M3-7), DM after 7 y (M7+). Samples were collected in 2 different institutions (NKI series for identifying the signature and IGR series for validation). Gene expression analysis of breast tumor samples was performed using custom-made Agilent 44K high-density microarrays and hybridized against the Mammaprint® reference pool (MRP). Tumors were also assessed for their Mammaprint® status, wound-healing signature status and their intrinsic subtypes based on the Blueprint® signature. Moreover, we identified the pathway-level activities of the patient groups using PARADIGM. Results and Discussion For the NKI series, A subset of 144 samples was included based on the selection criteria: 57 M0, 31 M0-3, 25 M3-7, 31 M7+. None of the 3 previously mentioned signatures correctly identified M0 vs. M7+ patients. In order to identify a predictive signature of late relapse (after 7y) we considered M0 and M7+ MammaPrint-Low Risk patients and we split them in a training (n=41) and in a test (n=23) sets. A 73-gene signature was able to classify M7+ patients with 75% of sensitivity and 66% of specificity on the test set. DM after 7yr showed significant activation of pathway related to inflammatory response and angiogenesis. Detailed results and validation results on the independent IGR series will be presented at the meeting. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr S1-6.

Background Classification of breast cancers into molecular subtypes may be important for the proper selection of therapy for patients as tumors with seemingly similar biology can have strikingly different clinical outcomes. We analyzed response to neo-adjuvant chemotherapy as a function of molecular sub-types and show patient survival from the multi-center neo adjuvant trial, I-SPY I (CALGB 150007/150012; ACRIN 6657). Previous analyses had shown that breast cancer subtypes as identified by immunohistochemistry or molecular analyses, have distinct clinical outcome (Esserman, ASCO 2009). Here, we analyze how the 70-gene signature (MammaPrint) together with an 80-gene molecular subtyping profile (BluePrint=Basal-type, Luminal-type, ERBB2-type; de Snoo, ASCO, 2010 and manuscript under review) can sub-classify patients. Materials: This study was carried out on genomics data of core needle biopsies from 149 out of 221 patients enrolled in the I-SPY I trial, a multi-center trial designed to identify predictive markers of pathological complete response (pCR) and disease-free survival (DFS) in women with locally advanced breast cancer (3 cm or greater). All women received neoadjuvant Doxorubicin & Cytoxan followed by paclitaxel chemotherapy. The 70-gene and the subtyping signatures, Basal, Luminal and ERBB2-type were determined on 44K Agilent arrays available through the I-SPY 1 data portal (Esserman, ASCO, 2009 and manuscript under review). Results: The 70-gene signature classified 9% of patients (13/149) as Low Risk, of whom one patient was ERBB2-type, and the other 12 were Luminal-type. None of these patients experienced a pCR. The remaining 136 were classified as 70-gene High Risk (91%), of whom 47% were classified as Luminal-type with a pCR rate of 13%, 41% were Basal-type with a pCR rate of 34%, and 12% were ERBB2-type with a pCR rate of 56%. Patients with BluePrint Basal-type tumors had a 5-year DFS of 61%; ERBB2-type had a 5-year DFS of 78%; 70-gene High Risk/Luminal-type had a 5-year DFS of 73% and 70-gene Low Risk/Luminal-type showed 5-year DFS of 100%. The molecular subtype classification shows significant association to clinically assessed receptor status. However, clinically assessed HER2+ patients were distributed across all molecular subtypes, where ER+HER+ are predominantly classified as Luminal-type. Conclusion: This study was performed with the I-SPY I dataset, which provides a platform to compare, contrast & combine marker signatures to tailor therapy. We show how combining BluePrint, with MammaPrint risk-classification can detect specific groups of patients who are at high risk of recurrence and who would have a higher likelihood to benefit from chemotherapy. Furthermore, MammaPrint Low Risk patients do not benefit from neo-adjuvant therapy, though have excellent survival rates, suggesting that the Low Risk Luminal-type patients could be managed conservatively. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-06-20.

Background Previously, the micro-array readout of ER, PR and HER2 by TargetPrint was shown to be strongly correlated with high quality immunohistochemistry (IHC)/FISH assessment, especially for ER and HER2. Concordance rates were 93% (k=0.79) for ER; 83% (k=0.65) for PR and 96% for HER2 (k=0.88) in 636 patients (Roepman et al., Clin Cancer Res, 2009). This study analysis was undertaken to further determine the correlation of microarray readout with IHC/FISH assessment both locally and centrally determined in the 1st 800 pts enrolled in the MINDACT trial. This work is essential to determine the quality of biological data in the two risk assessment methods used in MINDACT based upon which adjuvant chemotherapy decision is made, in order to exclude bias. Methods: ER/PR/HER2 IHC assessment was performed on the 1st 800 primary breast cancers (BC) of pts enrolled in the MINDACT study. The assessment was performed locally at each center (n=800) and by central review at the laboratory of the European Institute of Oncology (n=626). A tumor was classified positive for ER and PR when 1% of tumor cells showed positive staining. HER2 IHC status was scored as 0, 1+, 2+ or 3+; a score of 3+ was considered positive. In 2+ cases FISH was performed to assess final HER2 status. Gene expression data for ER, PR and HER2 were obtained by TargetPrint stratified as receptor positive or negative using previously determined and validated thresholds for ER, PR and HER2 mRNA levels (n=800). Results: Comparison of local assessment (IHC & FISH for HER2) with central review indicated highly similar results for receptor readout with a concordance of 98% (k=0.90) for ER; and 96% for HER2 (k=0.80) and slightly lower for PR (90% (k=0.72)). Comparison of central assessment (IHC & FISH for HER2) with micro array readout by TargetPrint indicated highly similar results for receptor readout with a concordance of 97% (k=0.88) for ER and 95% for HER2 (k=0.76). For PR the concordance was lower but still quite acceptable (85% (k=0.62)). Conclusion: Local and centrally assessed ER, PR and HER2 status in the first 800 MINDACT patient samples indicate a high level of quality for pathology in the local participating hospitals. These results exclude any bias induced by a lower quality of “traditional” pathology results as compared to the centrally assessed MammaPrint, both used for risk assessment and adjuvant chemotherapy decision in the MINDACT trial. The microarray-based assessment of ER, PR and HER2 gives results comparable to IHC & FISH and provides an objective and quantitative assessment of tumor receptor status. These results indicate that TargetPrint can serve as a second pathology assessment for locally assessed parameters, especially since TargetPrint is part of a multi-profile platform for breast cancer treatment management. This work was funded by the Breast Cancer Research Foundation and the EU Framework Program VI. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-07-06.

We sought to compare the molecular signature of node negative cancers from two cohorts 15 years apart, to determine if there is molecular evidence of increase in low and ultralow risk cancers over time. We studied the impact of age, time period of diagnosis, and mammographic screening on biology of tumors where The Netherlands Cancer Institute 70-gene prognosis signature was generated as part of 2 validation series, one retrospective (1984-1992), Cohort 1, and one prospective (2004-2006), Cohort 2. A total of 866 patients were analyzed. Regardless of time period of diagnosis, the proportion of T1, grade 1, hormone receptor positive (HR) tumors, and good prognosis by 70-gene signature significantly increases as age increases (P < 0.01). In women aged 49-60, the time period of diagnosis significantly affects the proportion of cancers that were NKI 70-gene low risk: 40.6% (67/165) compared with 58% (119/205) for Cohorts 1 and 2, respectively. This is in contrast to the absence of a significant change for women under age 40, where 25% (17/68) and 30% (17/56) were low risk in Cohorts 1 and 2, respectively. In women aged 49-60, using an ultralow risk threshold of the 70-gene signature, 10% of tumors in Cohort 1 were ultralow risk compared with 30% for women with screen-detected cancers in Cohort 2. Older age and method of detection (screening) are associated with a higher likelihood of a biologically low risk tumor. In women over age 50, biologically low risk tumors are frequent and tools that classify risk may minimize overtreatment.

Background MammaPrint® (MP) and BluePrint® (BP) are microarray-based tests with MP being prognostic for distant recurrence and BP enabling stratification into breast cancer molecular subtypes (Luminal, HER2, Basal-type). Recently, a CE marked MP and BP targeted RNA Next Generation Sequencing (NGS)-based kit was developed at Agendia and validated at University Hospitals Leuven and Curie Institute Paris. Here we compare breast cancer molecular subtype stratification defined by immunohistochemistry (IHC) and by MP and BP NGS- and microarray- based tests. Patients and Methods In this study, 124 primary operable invasive breast cancer patients were included at University Hospitals Leuven and at Curie Institute (n=80 Leuven; n=44 Curie) with the following histological subtypes: ductal-NOS (n=100), lobular (n=16), mucinous (n=3), tubular (n=2), others (n=3). Patients with bilateral breast cancer or with >3 positive lymph nodes were excluded. Surrogate breast cancer subtypes based on IHC were defined as follows: luminal if ≥10% estrogen receptor (ER) expression; triple negative if Results Concordance between IHC and MP/BP NGS subtyping was 75.0% (93/124), while concordance between MP/BP on NGS and microarray was 89.5% (111/124). MP/BP NGS subtyping identified more low risk Luminal A tumors compared to IHC (54.0%, (67/124) vs 44.3% (55/124)). Notably, concordance was excellent for triple-negative and, to less extent for HER2 driven tumors (Luminal B-like-HER2 positive and HER2+). IHC vs. MP/BP NGS molecular subtyping (n=124) MP/BP NGSIHCLuminal ALuminal BHER2-positiveBasalTotalLuminal A-like4690055Luminal B-like, HER2-negative16210037Luminal B-like, HER2-positive565016HER2-positive00303Triple negative0101213Total6737812124Microarray vs MP/BP NGS molecular subtyping (n=124) MP/BP NGSMicroarrayLuminal ALuminal BHER2 positiveBasalTotalLuminal A6040064Luminal B7310038HER2-positive028010Basal0001212Total6737812124 Conclusion This study shows a discordance of 25.0% between IHC and BP/MP NGS subtyping. This is in line with previous findings where IHC was compared to molecular subtyping based on microarray (Viale 2017, Whitworth 2014) underlining the complementarity of genomic testing in early stage breast cancer. Moreover, we observed a high concordance between NGS and microarray molecular subtyping, which suggests a successful translation of the MP/BP microarray test to a MP/BP NGS test. Citation Format: Darrigues L, Slembrouck L, Mittempergher L, Delahaye LJ, Vanden Bempt I, Vander Borght S, Vliegen L, Sintubin P, Raynal V, Bohec M, Reyes C, Rapinat A, Helsmoortel C, Jongen L, Hoste G, Neven P, Wildiers H, Smeets A, Nevelsteen I, Punie K, Van Nieuwenhuysen E, Han S, Laurent C, Vincent-Salomon A, Laas-Faron E, Witteveen AT, Neijenhuis S, Glas AM, Floris G, Reyal F. Comparison of breast cancer molecular subtyping by Immunohistochemistry and by BluePrint® next generation RNA sequencing-based test at University Hospitals Leuven and Curie Institute Paris [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-02-07.

Background MammaPrint was developed as a diagnostic tool to predict risk of breast cancer metastasis using the expression of 70 genes. To better understand the tumor biology assessed by MammaPrint, we interpreted the biological functions of the 70-genes and showed how the genes reflect the six hallmarks of cancer as defined by Hanahan and Weinberg. Results We used a bottom-up system biology approach to elucidate how the cellular processes reflected by the 70-genes work together to regulate tumor activities and progression. The biological functions of the genes were analyzed using literature research and several bioinformatics tools. Protein-protein interaction network analyses indicated that the 70-genes form highly interconnected networks and that their expression levels are regulated by key tumorigenesis related genes such as TP53, RB1, MYC, JUN and CDKN2A. The biological functions of the genes could be associated with the essential steps necessary for tumor progression and metastasis, and cover the six well-defined hallmarks of cancer, reflecting the acquired malignant characteristics of a cancer cell along with tumor progression and metastasis-related biological activities. Conclusion Genes in the MammaPrint gene signature comprehensively measure the six hallmarks of cancer-related biology. This finding establishes a link between a molecular signature and the underlying molecular mechanisms of tumor cell progression and metastasis.

Background: Previous studies suggest that within the triple positive HR+HER2+ subtype, patients classified as BluePrint (BP) Luminal subtype are more responsive to pertuzumab and trastuzumab (P/H) as opposed to trastuzumab (H) alone. In the I-SPY2 TRIAL, HER2-targeted treatment arms include H, P/H, neratinib (N), and T-DM1/Pertuzumab (T-DM1/P); and patients were classified by BP molecular subtyping in addition to conventional receptors. We evaluated BP subtype as a predictor of response in HR+HER2+ patients and assessed pathway differences between BP molecular subtypes. Methods: 125 HR+HER2+ patients (N: 42; P/H: 29, T-DM1/P: 35; H: 19) with pre-treatment Agilent microarrays and BP subtype assignments were considered. We assess association between BP subtypes and pCR using Fisher's exact test. To identify genes associated with BP Luminal vs. BP HER2 subtype, we (1) apply a Wilcoxon rank sum test and (2) fit a logistic model, with the Benjamini-Hochberg (BH) multiple testing correction (BH p Results: Of the 125 HR+HER2+ patients, 71 were BP HER2-type and 50 were BP Luminal-type. The distribution of pCR rates in BP Luminal/ HER2 subtypes are as follows: Distribution of pCR rates in BP Luminal/ HER2 subtypes by treatment armN (n = 40)P/H (n = 29)T-DM1/P (n = 34)H (n = 18)All arms (n = 121)BP Luminal (n = 50)0 / 9 (0%)1 / 12 (8.3%)2 / 16 (12.5%)1 / 13 (7.7%)4 / 50 (8%)BP HER2 (n = 71)12 / 31 (38.7%)13 / 17 (76.5%)15 / 18 (83.3%)1 / 5 (20%)41 / 71 (57.7%)Odds Ratio6.1; p = 0.12730.2; p = 0.00048429.6; p = 8.60E-052.8; p = 0.49015.3; p = 1.04E-08 In a whole transcriptome analysis, 1725 genes were differentially expressed. By DAVID enrichment analysis, the most significantly enriched pathways were related to immune function, with the BP HER2 subgroup showing higher expression. Conclusion: Our analysis suggests that HR+HER2+ BP Luminal subtype is associated with lower response rates to HER2-targeted agents, including P/H, and may need an alternative strategy. BP HER2 subtype appears associated with higher expression of immune-related genes, relative to BP Luminal; and suggests that immune signaling may contribute to HER2-targeted therapy sensitivity. Citation Format: Pei Rong Evelyn Lee, Zelos Zhu, Denise Wolf, Christina Yau, William Audeh, Annuska Glas, Lamorna Brown-Swigart, Gillian Hirst, Angela DeMichele, I-SPY 2 TRIAL Investigators, Laura Esserman, Laura van 't Veer. BluePrint Luminal subtype predicts non-response to HER2-targeted therapies in HR+/HER2+ I-SPY 2 breast cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2612.

Background The majority of breast cancer patients are postmenopausal women who are increasingly being offered adjuvant chemotherapy. Since the beneficial effect of chemotherapy in postmenopausal patients predominantly occurs in the first 5 years after diagnosis, a prognostic marker for early events can be of use for adjuvant treatment decision making. The aim of this study was to evaluate the prognostic value of the 70-gene prognosis signature for early events in postmenopausal patients. Methods Frozen tumor samples from 148 patients aged 55–70 years were selected (T1–2, N0) and classified by the 70-gene prognosis signature (MammaPrint™) into good or poor prognosis. Eighteen percent received hormonal therapy. Results Breast cancer-specific survival (BCSS) at 5 years was 99% for the good-prognosis signature versus 80% for the poor-prognosis signature group (P = 0.036). The 70-gene prognosis signature was a significant and independent predictor of BCCS during the first 5 years of follow-up with an adjusted hazard ratio of 14.4 (95% confidence interval 1.7–122.2; P = 0.01) at 5 years. Conclusion The 70-gene prognosis signature can accurately select postmenopausal patients at low risk of breast cancer-related death within 5 years of diagnosis and can be of clinical use in selecting postmenopausal women for adjuvant chemotherapy.

Patients with carcinoma of unknown primary (CUP) present with metastatic disease for which the primary site cannot be found, despite extensive standard investigation. Here, we describe the development and implementation of the first clinically available microarray-based test for this cancer type (CUPPrint), based on 633 individual tumors representing 30 carcinoma and 17 noncarcinoma classes. Tissue of origin prediction for either fresh frozen or paraffin-embedded tumor samples is achieved with the use of a custom 8-pack 1.9k microarray and robust classification algorithm. An expression profile of 495 genes was used to predict tumor origin by applying a k-nearest neighbor algorithm. Internal cross-validation and analysis of an independent, previously published, 229-sample dataset revealed that clinically informative predictions were made for up to 94% of samples analyzed. Analysis of 13 previously published CUP specimens yielded predicted tumor origins that supported the clinical suspicion in 12 cases (92%). Microarray profiling presents a promising tool to assist in the identification of the primary tumor and might direct a more tailored treatment for CUP patients.

Purpose: The level of estrogen receptor (ER), progesterone receptor (PR), and HER2 aids in the determination of prognosis and treatment of breast cancer. Immunohistochemistry is currently the predominant method for assessment, but differences in methods and interpretation can substantially affect the accuracy, resulting in misclassification. Here, we investigated the association of microarray-based mRNA expression levels compared with immunohistochemistry. Experimental Design: Microarray mRNA quantification of ER, PR, and HER2 was done by the developed TargetPrint test and compared with immunohistochemical assessment for breast tumors from 636 patients. Immunohistochemistry was done in a central laboratory and in an independent reference laboratory according to American Society of Clinical Oncology/College of American Pathologists guidelines for 100 cases. For HER2 immunohistochemistry 2+ cases, additional chromogenic in situ hybridization (CISH) was used to determine the final status. Results: ER concordance between microarray and central immunohistochemistry was 93 [95 confidence interval (95 CI), 91-95]. Only 4 of immunohistochemistry-positive samples were classified negative using microarray, whereas 18 of immunohistochemistry-negative samples showed a positive microarray ER status. Concordance for PR was 83 (95 CI, 80-86) and 96 of all samples showed an identical classification of HER2 status by microarray and immunohistochemistry/CISH (95 CI, 94-98). Nine percent of immunohistochemistry HER2-positive samples showed a negative microarray classification. Detailed review of 11 cases with discordant classifications by American Society of Clinical Oncology/College of American Pathologists and central immunohistochemistry indicated that microarray assessment was likely to add additional information in 5 cases. Conclusion: Microarray-based readout of ER, PR, and HER2 shows a high concordance with immunohistochemistry/CISH and provides an additional, objective, and quantitative assessment of tumor receptor status in breast cancer. (Clin Cancer Res 2009;15(22):700311)

The tumour antigen PReferentially expressed Antigen of MElanoma (PRAME) is expressed in a variety of malignancies, including breast cancer. We have analysed PRAME gene expression in relation to clinical outcome for 295 primary breast cancer patients. Kaplan–Meier survival curves show a correlation of PRAME expression levels with increased rates of distant metastases and decreased overall patient survival. This correlation existed both for the entire patient group (n=295) and for the subgroup of patients (n=185) who did not receive adjuvant chemotherapy. Multivariable analysis indicated that PRAME is an independent marker of shortened metastasis-free interval in patients who did not receive adjuvant chemotherapy. PRAME expression was associated with tumour grade and negative oestrogen receptor status. We conclude that PRAME expression is a prognostic marker for clinical outcome of breast cancer, independent of traditional clinicopathological markers.

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., SCOPUS: ar.j, info:eu-repo/semantics/published

Diffuse large B-cell lymphomas (DLBCLs) constitute a heterogeneous group of lymphomas in which germinal centre B-cell-like and activated B-cell-like subtypes can be discerned based on pathology, clinical presentation, and gene expression patterns. Testicular DLBCLs form an immune-privileged site-related subgroup of DLBCLs with an unfavourable prognosis. In the present study, cDNA microarray analysis, immunohistochemistry for CD10, Bc16 and NUM1, and somatic hypermutation analysis of the immunoglobulin heavy chain gene rearrangements were used to determine the subtype of primary testicular DLBCL. Immunohistochemistry revealed 14/22 testicular DLBCLs with an activated B-cell-like immunophenotype and 8/22 with an ambiguous immunophenotype co-expressing CD10 and high levels of MUM1. cDNA microarray analysis of these 22 and four additional cases showed a uniform activated B-cell-like gene expression pattern for both immunophenotypes. Somatic hypermutation analysis of immunoglobulin heavy chain genes showed a very high mutation load in seven cases tested, but intraclonal heterogeneity was found at low level in only one of these cases. It is concluded that primary testicular DLBCLs have uniform activated B-cell-like subtype characteristics despite a number of cases showing an ambiguous immunophenotype. Copyright (c) 2006 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Purpose and Experimental Design: Loss of human leukocyte antigen (HLA) expression on tumor cells is frequent in diffuse large B-cell lymphoma (DLBCL) arising in immune-privileged sites, such as the testis and central nervous system, and is associated with small homozygous deletions of HLA-DQ/HLA-DR and larger hemizygous deletions of the MHC region. To better understand the significance of down-regulation of HLA class II expression in relation to the homozygous and hemizygous deletions, we analyzed global gene expression patterns in a series of 26 testicular DLBCL after characterization of these deletions. Results: Low levels of HLA-DR mRNA in whole testicular DLBCL samples were associated with a strong down-regulation of numerous immune-related genes specific for T cells, macrophages, antigen presentation and processing, lymphocyte activation, chemokines and chemokine receptors, and the complement system. The number of CD3+ tumor-infiltrating T cells was also significantly lower in low expressors of HLA-DR mRNA. Interestingly, hemizygous and homozygous deletions in the MHC region did not have any additional effect on global gene expression. Conclusion: In conclusion, we found that loss of HLA class II mRNA expression in testicular DLBCL is associated with a significant change in global gene expression patterns. This effect is independent of the mechanism causing the down-regulation of HLA class II genes in the lymphoma cells.

The axillary lymph node status is the most powerful prognostic factor for breast cancer patients to date. The molecular mechanisms that control lymph node metastasis, however, remain poorly understood. To define patterns of genes or gene regulatory pathways that drive breast cancer lymph node metastasis, we compared the gene expression profiles of 15 primary breast carcinomas and their matching lymph node metastases using microarrays. In general, primary breast carcinomas and lymph node metastases do not differ at the transcriptional level by a common subset of genes. No classifier or single gene discriminating the group of primary tumours from those of the lymph node metastases could be identified. Also, in a series of 295 breast tumours, no classifier predicting lymph node metastasis could be developed. However, subtle differences in the expression of genes involved in extracellular-matrix organisation and growth factor signalling are detected in individual pairs of matching primary and metastatic tumours. Surprisingly, however, different sets of these genes are either up- or downregulated in lymph node metastases. Our data suggest that breast carcinomas do not use a shared gene set to accomplish lymph node metastasis.

Contains fulltext : 47779.pdf (Publisher’s version ) (Closed access) Follicular lymphoma (FL) is a disease characterized by a long clinical course marked by frequent relapses that vary in clinical aggressiveness over time. Therefore, the main dilemma at each relapse is the choice for the most effective treatment for optimal disease control and failure-free survival while at the same time avoiding overtreatment and harmful side effects. The selection for more aggressive treatment is currently based on histologic grading and clinical criteria; however, in up to 30% of all cases these methods prove to be insufficient. Using supervised classification on a training set of paired samples from patients who experienced either an indolent or aggressive disease course, a gene expression profile of 81 genes was established that could, with an accuracy of 100%, distinguish low-grade from high-grade disease. This profile accurately classified 93% of the FL samples in an independent validation set. Most important, in a third series of FL cases where histologic grading was ambiguous, precluding meaningful morphologic guidance, the 81-gene profile shows a classification accuracy of 94%. The FL stratification profile is a more reliable marker of clinical behavior than the currently used histologic grading and clinical criteria and may provide an important alternative to guide the choice of therapy in patients with FL both at presentation and at relapse.

Background A more accurate means of prognostication in breast cancer will improve the selection of patients for adjuvant systemic therapy. Methods Using microarray analysis to evaluate our previously established 70-gene prognosis profile, we classified a series of 295 consecutive patients with primary breast carcinomas as having a gene-expression signature associated with either a poor prognosis or a good prognosis. All patients had stage I or II breast cancer and were younger than 53 years old; 151 had lymph-node–negative disease, and 144 had lymph-node–positive disease. We evaluated the predictive power of the prognosis profile using univariable and multivariable statistical analyses. Results Among the 295 patients, 180 had a poor-prognosis signature and 115 had a good-prognosis signature, and the mean (±SE) overall 10-year survival rates were 54.6±4.4 percent and 94.5±2.6 percent, respectively. At 10 years, the probability of remaining free of distant metastases was 50.6±4.5 percent in the group with a poor-prognosis signature and 85.2±4.3 percent in the group with a good-prognosis signature. The estimated hazard ratio for distant metastases in the group with a poor-prognosis signature, as compared with the group with the good-prognosis signature, was 5.1 (95 percent confidence interval, 2.9 to 9.0; P

Clinical course, outcome, radiological features, severity, and histopathology are heterogenous in multiple sclerosis (MS). Since MS is considered to be a polygenic disease, the genetic background may at least partly be responsible for this variability. Some MS cases are histopathologically characterized by a dramatic oligodendrocyte loss that is in part caused by apoptosis. A dysregulated apoptotic elimination of self-reactive T cells may also contribute to disease susceptibility. To analyze genetic differences in the apoptosis regulating factors bcl-2, bax, bcl-x and p53 we investigated polymorphisms of these genes in 105 patients with a relapsing remitting disease course and 99 controls by PCR-SSCP and direct sequencing. We identified so far unpublished sequence alterations in the promotor region of the bxl-x gene, in exon 7 of the p53 gene, and in exon 1 of the bax gene. No differences were observed between MS patients and controls. Additional known polymorphisms were found in intron 3 of the bax gene and in exon 6 of the p53 gene. No significant differences in the frequency of gene sequence variations were found between MS patients and controls. The apoptosis genes studied here therefore appear less likely to be important effector genes in MS.

Colorectal cancer patients with the BRAF(p.V600E) mutation have poor prognosis in metastatic setting. Personalized treatment options and companion diagnostics are needed to better treat these patients. Previously, we developed a 58-gene signature to characterize the distinct gene expression pattern of BRAF-mutation-like subtype (accuracy 91.1%). Further experiments repurposed drug Vinorelbine as specifically lethal to this BRAF-mutation-like subtype. The aim of this study is to translate this 58-gene signature from a research setting to a robust companion diagnostic that can use formalin-fixed, paraffin-embedded (FFPE) samples to select patients with the BRAF-mutation-like subtype. BRAF mutation and gene expression data of 302 FFPE samples were measured (mutants = 57, wild-type = 245). The performance of the 58-gene signature in FFPE samples showed a high sensitivity of 89.5%. In the identified BRAF-mutation-like subtype group, 50% of tumours were known BRAF mutants, and 50% were BRAF wild-type. The stability of the 58-gene signature in FFPE samples was evaluated by two control samples over 40 independent experiments. The standard deviations (SD) were within the predefined criteria (control 1: SD = 0.091, SD/Range = 3.0%; control 2: SD = 0.169, SD/Range = 5.5%). The fresh frozen version and translated FFPE version of this 58-gene signature were compared using 170 paired fresh frozen and FFPE samples and the result showed high consistency (agreement = 99.3%). In conclusion, we translated this 58-gene signature to a robust companion diagnostic that can use FFPE samples.

The 70 gene-signature (MammaPrint(®)) is a prognostic profile of distant recurrence and survival of primary breast cancer (BC). BC patients with 4-9 positive nodes (LN 4-9) are considered clinically at high-risk. Herein we examined MammaPrint(®) added prognostic value in this group.MammaPrint(®) profiles were generated from frozen tumours of patients operated from primary BC. Samples were classified as genomic Low Risk (GLR) or genomic High Risk (GHR).Among the 173 samples, 70 (40%) were classified as GLR and 103 (60%) as GHR. Tumours in the GHR group were significantly more often ductal carcinomas (93%), grade 3 (60%), oestrogen and progesterone-negative, Her2 positive (25%). In the GLR category, the 5-year overall survival was 97% vs. 76% for in the GHR group (p0.01); Distant Metastasis Free Survival (DMFS) at 5 years was 87% for GLR patients and 63% for GHR patients (p0.01). In the Luminal A subgroup, the genomic profile was the only independent risk factor for DM and BC specific death.In the Luminal A subgroup, MammaPrint(®) is an independent prognostic marker in BC patients with LN 4-9 and may be integrated in a selection strategy of patients candidate for more aggressive therapeutic approaches.

Classification of breast cancer into molecular subtypes maybe important for the proper selection of therapy, as tumors with seemingly similar histopathological features can have strikingly different clinical outcomes. Herein, we report the development of a molecular subtyping profile (BluePrint), that enables rationalization in patient selection for either chemotherapy or endocrine therapy prescription. An 80-Gene Molecular Subtyping Profile (BluePrint) was developed using 200 breast cancer patient specimens and confirmed on four independent validation cohorts (n = 784). Additionally, the profile was tested as a predictor of chemotherapy response in 133 breast cancer patients, treated with T/FAC neoadjuvant chemotherapy. BluePrint classification of a patient cohort that was treated with neoadjuvant chemotherapy (n = 133) shows improved distribution of pathological Complete Response (pCR), among molecular subgroups compared with local pathology: 56% of the patients had a pCR in the Basal-type subgroup, 3% in the MammaPrint Low-risk, Luminal-type subgroup, 11% in the MammaPrint High-risk, Luminal-type subgroup, and 50% in the HER2-type subgroup. The group of genes identifying Luminal-type breast cancer is highly enriched for genes having an Estrogen Receptor binding site proximal to the promoter-region, suggesting that these genes are direct targets of the Estrogen Receptor. Implementation of this profile may improve the clinical management of breast cancer patients, by enabling the selection of patients who are most likely to benefit from either chemotherapy or from endocrine therapy.

Purpose This study aims to develop a robust gene expression classifier that can predict disease relapse in patients with early-stage colorectal cancer (CRC). Patients and Methods Fresh frozen tumor tissue from 188 patients with stage I to IV CRC undergoing surgery was analyzed using Agilent 44K oligonucleotide arrays. Median follow-up time was 65.1 months, and the majority of patients (83.6%) did not receive adjuvant chemotherapy. A nearest mean classifier was developed using a cross-validation procedure to score all genes for their association with 5-year distant metastasis–free survival. Results An optimal set of 18 genes was identified and used to construct a prognostic classifier (ColoPrint). The signature was validated on an independent set of 206 samples from patients with stage I, II, and III CRC. The signature classified 60% of patients as low risk and 40% as high risk. Five-year relapse-free survival rates were 87.6% (95% CI, 81.5% to 93.7%) and 67.2% (95% CI, 55.4% to 79.0%) for low- and high-risk patients, respectively, with a hazard ratio (HR) of 2.5 (95% CI, 1.33 to 4.73; P = .005). In multivariate analysis, the signature remained one of the most significant prognostic factors, with an HR of 2.69 (95% CI, 1.41 to 5.14; P = .003). In patients with stage II CRC, the signature had an HR of 3.34 (P = .017) and was superior to American Society of Clinical Oncology criteria in assessing the risk of cancer recurrence without prescreening for microsatellite instability (MSI). Conclusion ColoPrint significantly improves the prognostic accuracy of pathologic factors and MSI in patients with stage II and III CRC and facilitates the identification of patients with stage II disease who may be safely managed without chemotherapy.

The 70-gene expression profile MammaPrint is a powerful prognostic indicator for disease outcome in breast cancer patients with improved prediction of recurrence risk compared to currently used guidelines. The microarray-based test TargetPrint further provides reliable, quantitative assessment of mRNA expression levels of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2). This study was performed as a validation of MammaPrint and TargetPrint in an unselected German breast cancer population and was designed to determine the degree of concordance with currently applied clinical parameters. One hundred and forty cases of breast cancer stage I and II were classified as being low or high risk for distant metastasis using MammaPrint. Results were compared to current clinical risk classifications and adjuvant treatment management. Immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH)/chromogenic in situ hybridization (CISH) assessments of ER, PR and HER2 were further compared with gene expression read-outs using TargetPrint. Thirty-two percent of patients (19/59) with a poor prognosis-signature identified via MammaPrint did not receive adjuvant systemic treatment apart from endocrine therapy and were potentially undertreated; whereas 42% (35/77) of patients with a good prognosis-signature received chemotherapy and were potentially overtreated. Comparison of microarray receptor results with IHC and FISH/CISH were concordant in 97% for ER; 86% for PR; and 94% for HER2. In this German study population, MammaPrint would have resulted in altered treatment advice for adjuvant systemic therapy in 40% of patients. Furthermore, TargetPrint presented high concordance for ER, PR and Her2 with IHC and FISH/CISH analysis.

Objective van't Veer and colleagues developed a 70-gene prognosis profile known as MammaPrint to identify breast cancer patients who were at low risk of developing metastases. We evaluated the prognostic value of the 70-gene MammaPrint profile in Japanese women with node-negative breast cancer. Methods Frozen tumour samples from 102 eligible node-negative breast cancer patients aged 70 or younger were characterized with the MammaPrint array. The patients were treated with breast-conserving therapy or mastectomy with axillary lymph node dissection between December 1998 and August 2001. About 73 percent received adjuvant hormonal therapy and 28 percent received adjuvant chemotherapy. The gene expression profiles obtained by MammaPrint classified the patients as high- or low-genomic risk. The median follow-up was 7.1 years. Results Among the 102 patients, 20 (20%) were classified as low-genomic risk and 82 (80%) were classified as high-genomic risk. The probability of distant metastasis-free survival at five years was 100% for the low-risk group and 94% for the high-risk group. Conclusions The 70-gene MammaPrint prognosis profile accurately identified Japanese breast cancer patients at low risk of developing recurrences. In fact, 100% of the individuals in the low-risk category remained metastasis-free for the duration of the observation period.

The 70-gene signature (MammaPrint™) is a prognostic tool used to guide adjuvant treatment decisions. The aim of this study was to assess its value to predict chemosensitivity in the neoadjuvant setting. We obtained the 70-gene profile of stage II–III patients prior to neoadjuvant chemotherapy and classified the prognosis-signatures. Pathological complete remission (pCR) was used to measure chemosensitivity. Among 167 patients, 144 (86%) were having a poor and 23 (14%) a good prognosis-signature. None of the good prognosis-signature patients achieved a pCR (0/23), whereas 29/144 patients (20%) in the poor prognosis-signature group did (P = 0.015). All triple-negative tumors (n = 38) had a poor prognosis-signature. Within the non triple-negative subgroup, the response of the primary tumor remained associated with the classification of the prognosis-signature (P = 0.023). A pCR is unlikely to be achieved in tumors that have a good prognosis-signature. Tumors with a poor prognosis-signature are more sensitive to chemotherapy.

Background Microarray diagnostics of tumour samples is based on measurement of prognostic and/or predictive gene expression profiles. Typically, diagnostic profiles have been developed using bulk tumour samples with a sufficient amount of tumour cells (usually >50%). Consequentially, a diagnostic results depends on the minimal percentage of tumour cells within a sample. Currently, tumour cell percentage is assessed by conventional histopathological review. However, even for experienced pathologists, such scoring remains subjective and time consuming and can lead to ambiguous results. Methods In this study we investigated whether we could use transcriptional activity of a specific set of genes instead of histopathological review to identify samples with sufficient tumour cell content. Genome-wide gene expression measurements were used to develop a transcriptional gene profile that could accurately assess a sample's tumour cell percentage. Results Supervised analysis across 165 breast tumour samples resulted in the identification of a set of 13 genes which expression correlated with presence of tumour cells. The developed gene profile showed a high performance (AUC 0.92) for identification of samples that are suitable for microarray diagnostics. Validation on 238 additional breast tumour samples indicated a robust performance for correct classification with an overall accuracy of 91 percent and a kappa score of 0.63 (95%CI 0.47–0.73). Conclusion The developed 13-gene profile provides an objective tool for assessment whether a breast cancer sample contains sufficient tumour cells for microarray diagnostics. It will improve the efficiency and throughput for diagnostic gene expression profiling as it no longer requires histopathological analysis for initial tumour percentage scoring. Such profile will also be very use useful for assessment of tumour cell percentage in biopsies where conventional histopathology is difficult, such as fine needle aspirates.

Gene expression assays that are used in daily clinical practice for treating early breast cancer patients have been introduced in the clinic only recently. This review discusses the development of these arrays, summarizes the validation of those that are commercially available and indicates how the information provided by these assays can help in the care of patients. The review also provides an extensive overview of commercially available assays focusing on MammaPrint, the first and only assay for breast cancer management that has been cleared by the FDA.

Purpose The 70-gene prognosis signature (van't Veer et al., Nature 415(6871):530-536, 2002) may improve the selection of lymph node-negative breast cancer patients for adjuvant systemic therapy. Optimal validation of prognostic classifiers is of great importance and we therefore wished to evaluate the prognostic value of the 70-gene prognosis signature in a series of relatively recently diagnosed lymph node negative breast cancer patients. Methods We evaluated the 70-gene prognosis signature in an independent representative series of patients with invasive breast cancer (N = 123