Search

This file includes the legends for supplementary figure 1 and supplementary tables 1 and 2

This file shows the significant regions (highlighted in supplementary figure 1) for the comparison B1mutER+ versus SporER+. Subsequently the weighted average frequency in B1mutER+, in SporER+ and the difference between both groups is shown. The last two columns show respectively the p-value and the false discovery rate (q-bound).

This figure shows pairwise comparisons between the BRCA2-mutated ER+ tumors and the sporadic ER+ tumors (upper panel) and the BRCA1-mutated ER+tumors and the sporadic ER+ tumors (lower

Supplementary Figure S1 and Supplementary Tables S1-S3.

This file shows the significant regions (highlighted in supplementary figure 1) for the comparison B2mutER+ versus SporER+. Subsequently the weighted average frequency in B2mutER+, in SporER+ and the difference between both groups is shown. The last two columns show respectively the p-value and the false discovery rate (q-bound).

Purpose: A preliminary study performed on a small cohort of multifocal prostate cancer (PCa) detected BRCA1 allelic imbalances among circulating tumor cells (CTC). The present analysis was aimed to elucidate the biological and clinical roles of BRCA1 losses in metastatic spread and tumor progression in PCa patients.Experimental Design: To map molecular progression in PCa outgrowth, we used fluorescence in situ hybridization analysis of primary tumors and lymph node sections, and CTCs from peripheral blood.Results: We found that 14% of 133 tested patients carried monoallelic BRCA1 loss in at least one tumor focus. Extended molecular analysis of chr17q revealed that this aberration was often a part of larger cytogenetic rearrangement involving chr17q21 accompanied by allelic imbalance of the tumor suppressor gene PTEN and lack of BRCA1 promoter methylation. The BRCA1 losses correlated with advanced T stage (P < 0.05), invasion to pelvic lymph nodes (P < 0.05), as well as biochemical recurrence (P < 0.01). Their prevalence was twice as high within 62 lymph node metastases (LNM) as in primary tumors (27%, P < 0.01). The analysis of 11 matched primary PCa-LNM pairs confirmed the suspected transmission of genetic abnormalities between these two sites. In four of seven patients with metastatic disease, BRCA1 losses appeared in a minute fraction of cytokeratin- and vimentin-positive CTCs.Conclusions: Small subpopulations of PCa cells bearing BRCA1 losses might be one confounding factor initiating tumor dissemination and might provide an early indicator of shortened disease-free survival. Clin Cancer Res; 16(13); 3340–8. ©2010 AACR.

Background Studies have shown that blood platelets contain tumour-specific mRNA profiles tumour-educated platelets (TEPs). Here, we aim to train a TEP-based breast cancer detection classifier. Methods Platelet mRNA was sequenced from 266 women with stage I–IV breast cancer and 212 female controls from 6 hospitals. A particle swarm optimised support vector machine (PSO-SVM) and an elastic net-based classifier (EN) were trained on 71% of the study population. Classifier performance was evaluated in the remainder (29%) of the population, followed by validation in an independent set (37 cases and 36 controls). Potential confounding was assessed in post hoc analyses. Results Both classifiers reached an area under the curve (AUC) of 0.85 upon internal validation. Reproducibility in the independent validation set was poor with an AUC of 0.55 and 0.54 for the PSO-SVM and EN classifier, respectively. Post hoc analyses indicated that 19% of the variance in gene expression was associated with hospital. Genes related to platelet activity were differentially expressed between hospitals. Conclusions We could not validate two TEP-based breast cancer classifiers in an independent validation cohort. The TEP protocol is sensitive to within-protocol variation and revision might be necessary before TEPs can be reconsidered for breast cancer detection.

PurposeInvasive breast cancer patients are increasingly being treated with neoadjuvant chemotherapy, however, only a fraction of the patients respond to it completely. To prevent over-treating patients with a toxic drug, there is an urgent need for biomarkers capable of predicting treatment response before administering the therapy. In this retrospective study, we developed interpretable, deep-learning based biomarkers to predict the pathological complete response (pCR, i.e. the absence of tumor cells in the surgical resection specimens) to neoadjuvant chemotherapy from digital pathology H&E images of pre-treatment breast biopsies.Experimental DesignOur approach consists of two steps: In the first step, using deep learning, mitoses are detected and the tissue segmented into several morphology compartments including tumor, lymphocytes and stroma. In the second step, computational biomarkers are derived from the segmentation and detection output to encode slide-level relationships between the morphological structures with focus on tumor infiltrating lymphocytes (TILs). We developed and evaluated our method on slides from N=721 patients from three European medical centers with triple-negative and Luminal B breast cancers.ResultsThe investigated biomarkers yield statistically significant prediction performance for pCR with areas under the receiver operating characteristic curve between 0.66 and 0.88 depending on the cancer subtype and center.ConclusionThe proposed computational biomarkers predict pathological complete response, but will require more evaluation and finetuning for clinical application. The results further corroborate the potential role of deep learning to automate TILs quantification, and their predictive value in breast cancer neoadjuvant treatment planning.

Ductal carcinoma in situ (DCIS) is the most common form of preinvasive breast cancer and, despite treatment, a small fraction (5–10%) of DCIS patients develop subsequent invasive disease. A fundamental biologic question is whether the invasive disease arises from tumor cells in the initial DCIS or represents new unrelated disease. To address this question, we performed genomic analyses on the initial DCIS lesion and paired invasive recurrent tumors in 95 patients together with single-cell DNA sequencing in a subset of cases. Our data show that in 75% of cases the invasive recurrence was clonally related to the initial DCIS, suggesting that tumor cells were not eliminated during the initial treatment. Surprisingly, however, 18% were clonally unrelated to the DCIS, representing new independent lineages and 7% of cases were ambiguous. This knowledge is essential for accurate risk evaluation of DCIS, treatment de-escalation strategies and the identification of predictive biomarkers.

Ductal Carcinoma in Situ (DCIS) is a non-invasive non-obligate precursor of invasive breast cancer (IBC). DCIS is usually treated by surgery combined with radiotherapy, which can have a large impact on the life of patients. However, many of these DCIS lesions would never progress into IBC. To reduce the overtreatment of DCIS, but assure proper treatment for high risk DCIS, it is crucial to understand the biology underlying DCIS. To study the biology of DCIS we established Mouse INtraDuctal (MIND) patient-derived xenograft (PDX) models by intraductally injecting patient DCIS material into the mammary ducts of female immunocompromised mice. We engrafted 130 samples, which have been incubated in vivo for a period of 12 months. We obtain a take rate of 88% with 46% of our models showing invasive progression. Histology and molecular subtyping by PAM50 classification are well preserved in the MIND models compared to the primary counterpart, ensuring that our MIND models represent the patient disease well. For 102 primary samples we obtained RNAseq profiles as well as for 64 matched MIND-PDX models. In addition whole exome-/panel sequencing data is generated from the same primary DCIS samples together with 12 matched MIND-PDX WES profiles as well as 60 matched Copy Number Variation (CNV) MIND-PDX profiles. Together these data revealed multiple biomarkers related to invasive progression, including factors such as high grade, solid growth, a high copy number aberrations burden, HER2, PTK6 & MYC amplifications and a high Ki67. On top of this we used whole mount imaging of the injected mammary glands extracted from our MIND-PDX models, showing two distinct growth patterns correlated with invasion. And as this is all done in the context of the PRECISION consortium this allows us to confirm and validate our findings in larger sequencing and imaging efforts of human samples. We have also successfully passaged 42 MIND-PDX models which showed minimal changes in pheno- and genotype over time indicating invasive behavior is an intrinsic phenotype of DCIS with minimal evolution, supporting a multiclonal evolution model. Moreover, this provided a collection of 19 stable sequentially transplantable DCIS MIND models including Luminal A, Luminal B, ER+/HER2+ and ER-/HER2+ models. Ultimately these models can be used to validate the biomarkers found to be related to invasive progression, as an example we proved the direct role of HER2 overexpression in invasive progression by inhibiting the HER2 receptor or by overexpressing HER2. In conclusion all this data together enabled us to create a well-characterized biobank of DCIS models with the unique opportunity to follow the natural progression, sequentially transplant 42 models, find genomic and transcriptomic profiles related to high risk DCIS and manipulate gene expression to validate the role of genes in DCIS progression. Citation Format: Stefan J. Hutten, Roebi de Bruijn, Catrin Lutz, Madelon Badoux, Timo Eijkman, Xue Chao, Marta Ciwinska, Andrea Herencia-Ropero, Petra Kristel, Lennart Mulder, Joyce Sanders, Mathilde Almekinders, Alba Llop-Gueverra, Helen R. Davies, Fariba Behbod, Serena Nik-Zainal, Violeta Serra, Jacco van Rheenen, Esther H. Lips, Lodewyk F.A. Wessels, Jelle Wesseling, Colinda Scheele, Jos Jonkers. A living biobank of patient-derived ductal carcinoma in situ (DCIS) Mouse-INtraDuctal (MIND) xenografts identifies multiple risk factors of invasive progression [abstract]. In: Proceedings of the AACR Special Conference on Rethinking DCIS: An Opportunity for Prevention?; 2022 Sep 8-11; Philadelphia, PA. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_1): Abstract nr PR006.

Ductal carcinoma in situ (DCIS) is the most common form of preinvasive breast cancer and, despite treatment, a small fraction (5-10%) of DCIS patients develop subsequent invasive disease. A fundamental biologic question is whether the invasive disease arises from tumor cells in the initial DCIS or represents new unrelated disease. To address this question, we performed genomic analyses on the initial DCIS lesion and paired invasive recurrent tumors in 95 patients together with single-cell DNA sequencing in a subset of cases. Our data show that in 75% of cases the invasive recurrence was clonally related to the initial DCIS, suggesting that tumor cells were not eliminated during the initial treatment. Surprisingly, however, 18% were clonally unrelated to the DCIS, representing new independent lineages and 7% of cases were ambiguous. This knowledge is essential for accurate risk evaluation of DCIS, treatment de-escalation strategies and the identification of predictive biomarkers.

Pure ductal carcinoma in situ (DCIS) is being diagnosed more frequently through breast screening programmes and is associated with an increased risk of developing invasive breast cancer. We assessed the clonal relatedness of 143 cases of pure DCIS and their subsequent events using a combination of whole exome, targeted and copy number sequencing, supplemented by single cell analysis. Unexpectedly, 18% of all invasive events after DCIS were clonally unrelated to the primary DCIS. Single cell sequencing of selected pairs confirmed our findings. In contrast, synchronous DCIS and invasive disease (n=44) were almost always (93%) clonally related. This challenges the dogma that almost all invasive events after DCIS represent invasive transformation of the initial DCIS and suggests that DCIS could be an independent risk factor for developing invasive disease as well as a precursor lesion. Our findings support a paradigm shift that confirms a more complex role for DCIS than previously recognized, and that the future management of DCIS should take into account both the precursor and risk factor implications of this diagnosis.

Purpose For optimal management of ductal carcinoma in situ (DCIS), reproducible histopathological assessment is essential to distinguish low-risk from high-risk DCIS. Therefore, we analyzed interrater reliability of histopathological DCIS features and assessed their associations with subsequent ipsilateral invasive breast cancer (iIBC) risk. Methods Using a case-cohort design, reliability was assessed in a population-based, nationwide cohort of 2767 women with screen-detected DCIS diagnosed between 1993 and 2004, treated by breast-conserving surgery with/without radiotherapy (BCS ± RT) using Krippendorff’s alpha (KA) and Gwet’s AC2 (GAC2). Thirty-eight raters scored histopathological DCIS features including grade (2-tiered and 3-tiered), growth pattern, mitotic activity, periductal fibrosis, and lymphocytic infiltrate in 342 women. Using majority opinion-based scores for each feature, their association with subsequent iIBC risk was assessed using Cox regression. Results Interrater reliability of grade using various classifications was fair to moderate, and only substantial for grade 1 versus 2 + 3 when using GAC2 (0.78). Reliability for growth pattern (KA 0.44, GAC2 0.78), calcifications (KA 0.49, GAC2 0.70) and necrosis (KA 0.47, GAC2 0.70) was moderate using KA and substantial using GAC2; for (type of) periductal fibrosis and lymphocytic infiltrate fair to moderate estimates were found and for mitotic activity reliability was substantial using GAC2 (0.70). Only in patients treated with BCS-RT, high mitotic activity was associated with a higher iIBC risk in univariable analysis (Hazard Ratio (HR) 2.53, 95% Confidence Interval (95% CI) 1.05–6.11); grade 3 versus 1 + 2 (HR 2.64, 95% CI 1.35–5.14) and a cribriform/solid versus flat epithelial atypia/clinging/(micro)papillary growth pattern (HR 3.70, 95% CI 1.34–10.23) were independently associated with a higher iIBC risk. Conclusions Using majority opinion-based scores, DCIS grade, growth pattern, and mitotic activity are associated with iIBC risk in patients treated with BCS-RT, but interrater variability is substantial. Semi-quantitative grading, incorporating and separately evaluating nuclear pleomorphism, growth pattern, and mitotic activity, may improve the reliability and prognostic value of these features.

Purpose Gene expression (GE) profiling for breast cancer classification and prognostication has become increasingly used in clinical diagnostics. GE profiling requires a reasonable tumor cell percentage and high-quality RNA. As a consequence, a certain amount of samples drop out. If tumor characteristics are different between samples included and excluded from GE profiling, this can lead to bias. Therefore, we assessed whether patient and tumor characteristics differ between tumors suitable or unsuitable for generating GE profiles in breast cancer. Methods In a consecutive cohort of 738 breast cancer patients who received neoadjuvant chemotherapy at the Netherlands Cancer Institute, GE profiling was performed. We compared tumor characteristics and treatment outcome between patients included and excluded from GE profiling. Results were validated in an independent cohort of 812 patients treated with primary surgery. Results GE analysis could be performed in 53% of the samples. Patients with tumor GE profiles more often had high-grade tumors [odds ratio 2.57 (95%CI 1.77–3.72), p

Purpose: As estrogen receptor–positive (ER+) breast cancer in BRCA1 mutation carriers arises at an older age with less aggressive tumor characteristics than ER-negative (ER−) BRCA1-mutated breast cancer, it has been suggested that these tumors are “sporadic” and not BRCA1 driven. With the introduction of targeted treatments specific for tumors with a nonfunctioning BRCA1 or BRCA2 gene, the question whether the BRCA genes are impaired in the tumor is highly relevant. Therefore, we performed genomic profiling of BRCA1-mutated ER+ tumors. Experimental Design: Genomic profiling, BRCA1 promoter methylation assessment, and loss of heterozygosity analysis were done on 16 BRCA1-mutated ER+ tumors. Results were compared with 57 BRCA1-mutated ER− tumors, 36 BRCA2-mutated ER+-associated tumors, and 182 sporadic ER+ tumors. Results: The genomic profile of BRCA1-mutated ER+ tumors was different from BRCA1-mutated ER− breast tumors, but highly similar to BRCA2-mutated ER+ tumors. In 83% of the BRCA1-mutated ER+ tumors, loss of the wild-type BRCA1 allele was observed. In addition, clinicopathologic variables in BRCA1-mutated ER+ cancer were also more similar to BRCA2-mutated ER+ and sporadic ER+ breast cancer than to BRCA1-mutated ER− cancers. Conclusions: As BRCA1-mutated ER+ tumors show a BRCAness copy number profile and LOH, it is likely that the loss of a functional BRCA1 protein plays a role in tumorigenesis in BRCA1-mutated ER+ tumors. Therefore, we hypothesize that these tumors are sensitive to drugs targeting the BRCA1 gene defect, providing new targeted treatment modalities for advanced BRCA-deficient, ER+ breast cancer. Clin Cancer Res; 23(5); 1236–41. ©2016 AACR.

Background Neoadjuvant chemotherapy is standard of care for locally advanced breast cancer. Unfortunately not all patients benefit from this treatment. Even after decades of research, we still cannot predict which tumor will or will not respond. This may in part be due to tumor heterogeneity, as the sample taken before treatment not necessarily represents the tumor cell population that causes therapy resistance. Methods To test this hypothesis, we collected pre-treatment biopsies, resection specimens, and matched blood from 21 breast cancer patients treated with doxorubicin and cyclophosphamide in a neoadjuvant setting. Specifically, tumors were selected with a tumor percentage >50% after chemotherapy to enrich for resistant samples and ensure high quality data. RNA and whole exome sequencing were performed to characterize somatic mutations, copy number alterations and gene expression profiles. Histopathological characteristics were determined to obtain a comprehensive profile of all tumor samples. Results The comparisons of somatic variants and copy number alterations revealed a very diverse image: in several cases, high-level amplifications, large genomic gains or losses, and mutations in known oncogenes or tumor suppressors such as MAP3K1 and RUNX1 were either lost or gained during treatment, while in other cases no such changes were detected. We observed a remarkable number of genetic alterations involved in cell cycle progression and DNA damage checkpoints, including amplification of MDM2, CCND1 and CDK4, and copy number loss or mutations in CDKN1B and ATM. Strikingly, both cases of CDKN1B loss were identified in pre-treatment biopsies and no longer detectable in the surgery specimen. In contrast, CCND1, CDK4 and MDM2 amplifications were retained, although CCND1 expression decreased significantly in CCND1 amplified tumors. In addition, eighty percent of tumors showed a decreased cell proliferation after chemotherapy, where the high-proliferative ER+ (Luminal B) tumors were most strongly affected. This trend was also visible in a validation cohort of 94 ER+ samples, but the prognosis of Luminal B tumors that showed a decrease in proliferation was still significantly worse than that of Luminal A tumors that did not show an altered proliferation rate. Conclusion Our results confirm that biologically relevant genomic alterations can differ between pre- and post-treatment samples, which greatly impacts biomarker discovery. In addition, our findings emphasize the chemotherapy insensitivity of CCND1 amplified ER+ breast cancers, and stress the need for better treatment regimens for these patients. In contrast, genomic loss of CDKN1B may be a marker for sensitivity to doxorubicin. Citation Format: Lips EH, Hoogstraat M, Mulder L, Nederlof PM, Sonke GS, Rodenhuis S, Wesseling J, Wessels LFA. Comprehensive characterization of matched pre-treatment biopsies and residual disease of doxorubicin treated 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 PD1-07.

Background As estrogen receptor (ER)-positive breast cancer in BRCA1 mutation carriers arises at an older age with less aggressive tumor characteristics than ER negative BRCA1 mutated breast cancer, it has been suggested that these tumors are 'sporadic' and not BRCA1-driven. With the introduction of targeted treatments specific for tumors with a non-functioning BRCA1 or BRCA2 gene, the question whether the BRCA genes are impaired in the tumor, is highly relevant. Therefore, we performed genomic profiling of BRCA1-mutated ER+ tumors. Methods Genomic profiling, BRCA1 promoter methylation assessment, and loss of heterozygosity (LOH) analysis were done on 16 BRCA1-mutated ER+ tumors. Results were compared with 57 BRCA1-mutated ER- tumors, 36 BRCA2-mutated ER+ associated tumors, and 182 sporadic ER+ tumors. Results The genomic profile of BRCA1-mutated ER+ tumors was different from BRCA1-mutated ER- breast tumors, but highly similar to BRCA2-mutated ER+ tumors. In 83% of the BRCA1-mutated ER+ tumors, loss of the wildtype BRCA1 allele was observed. Clinico-pathological variables in BRCA1-mutated ER+ cancer were more similar to BRCA2-mutated ER+ and sporadic ER+ breast cancer than to BRCA1 mutated ER- cancers. Conclusions As BRCA1-mutated ER+ tumors showed a BRCAness copy number profile and LOH, it is likely that the loss of a functional BRCA1 protein plays a role in tumorigenesis in BRCA1-mutated ER+ tumors. Therefore, we hypothesize that these tumors are sensitive to drugs targeting the BRCA1 gene defect. Citation Format: Lips EH, Debipersad R, Scheerman E, Mulder L, Sonke GS, van der Kolk LE, Wessling J, Hogervorst FBL, Nederlof PM. Estrogen receptor-positive breast cancer in BRCA1 mutation carriers shows a BRCAness profile, suggesting sensitivity to drugs targeting homologous recombination deficiency [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 P4-12-03.

Background. Ductal carcinoma in situ (DCIS) is a potential precursor of invasive breast cancer, because: DCIS often accompanies invasive breast cancer; its risk factors are similar to those of invasive breast cancer; and genetic markers found in DCIS are similar to the ones found in invasive breast cancer. However, clinical behavior of DCIS is still poorly understood, as there is only limited information on its long-term natural history. Altogether, this makes it difficult to understand the relatedness of DCIS and its subsequent ipsilateral invasive breast cancer (iIBC). Here, we set-up a comparison between primary DCIS and matched subsequent iIBC, by making use of pathological and molecular data. Patients and methods. For this study, we used a unique series of 155 DCIS cases which developed a subsequent iIBC during a median follow up period of 12.6 years. We assessed histological characteristics, tumor location, estrogen and progesterone receptor status, p16 expression, and HER2 and p53 overexpression. RNA sequencing and copy number sequencing was done on 78 DCIS lesions and 78 matched invasive breast cancer relapses. We determined if the iIBC lesion and DCIS lesion were related, with respect to tumor location, immunohistochemical (IHC) markers, and genomic features. Results. Based on tumor location and histological grade, >95% of the subsequent invasive breast cancers reflected outgrowth of residual disease. HER2 was the only IHC marker that showed a significant difference in expression between DCIS and matched iIBC: 40% of the HER2 positive DCIS was followed by a HER2 negative invasive recurrence. In addition, RNAseq data was used to classify DCIS and IBC lesions into PAM50 subtypes. 77% of the DCIS IBC pair belonged to the same subtype. The DCIS lesions showed copy number aberrations on typical breast cancer-associated loci. However, when we compared the DCIS with its matched iIBC, we saw in 41% of the cases very distinct copy number profiles, indicating either outgrow of a different tumor subclone or a second primary. Conclusion. This is the first time that a sound comparison could be made between primary DCIS and its subsequent invasive breast cancer with such a large patient group, integrating pathological and molecular data. Our results strongly suggest that many subsequent iIBCs after treatment of pure DCIS could be considered as second primary breast cancer lesions. To provide definite proof for this, in depth DNA sequencing and heterogeneity studies will be presented at SABCS 2018. Citation Format: Visser LL, Hoogstraat M, Elshof LE, van de Vijver K, Groen EJ, Almekinders MM, Bierman C, Nieboer F, de Maaker M, Kristel P, Mulder L, Schaapveld M, Schmidt MK, Lips E, Wesseling J. Approximately 40% of invasive recurrences after treatment of ductal carcinoma in situ is likely to be a second primary tumor [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 PD8-09.

Background: Neoadjuvant chemotherapy is the standard of care for locally advanced triple negative breast cancer (TNBC). A complete remission after chemotherapy is associated with a good prognosis. However, patients with a poor response often relapse and die of metastatic disease. Biomarkers are urgently needed to predict which patients will respond to standard chemotherapy. In the current study we deep-sequenced responding and non-responding tumors to find response prediction markers. In addition, the encountered mutations might provide clues for targeted treatment options. Methods: Next generation sequencing (NGS) was performed for 2,000 genes involved in oncogenesis with an average coverage of 150 reads. DNA from 56 pre-treatment TNBC-biopsies was sequenced, as was non-tumor DNA from each patient for comparison. Biopsies were obtained from patients scheduled to receive neoadjuvant chemotherapy with doxorubicin/cyclophosphamide. The median follow up was 2.5 years. The presence of a BRCA1 germline mutation or BRCA1 promoter methylation was also known. We assessed three variables. First, we compared samples with a pathological complete remission (pCR) with those that did not achieve a pCR, to find mutations predicting chemotherapy response. Second, we performed the same comparison with relapse free survival as outcome measure. Third, we compared tumors with BRCA mutations or BRCA1 promoter methylation with tumors with a functional copy of the BRCA genes. The goal of this analysis was to detect mutations associated with BRCAness. Results: The mutations observed were diverse and few recurrent mutations were detected. Most mutations were in TP53, TTN, and PIK3CA (57%, 22%, 9%). The mutation rates were similar between responders and non-responders (average mutation rate of 13 versus 16 mutations per tumor, p=0.27). The analysis of individual genes did not reveal significant differences between responders and non-responders. NOTCH4 mutations showed an association with relapse free survival, with three out of nine relapsing patients bearing a mutation and no mutations occurring in the relapse-free patients (p=0.008). Interestingly, PIK3CA mutations were only observed in patients with a functional BRCA gene (5/26 (19%) versus 0/30, p=0.017). Tumors with BRCA impairment may develop via different routes than tumors with functioning BRCA genes, and the PI3K pathway may play a role in the latter. This finding has also been observed in an independent dataset (Severson et al, 2014, submitted). Conclusions: Although few recurrent mutations were found, two interesting leads for follow up studies were identified. First, three out of nine tumors with a relapse had a NOTCH4 mutation while NOTCH4 mutations were not occurring in the relapse free group. Second, PIK3CA mutations were associated with BRCA proficient tumors. After validation in larger series, triple negative tumors with PI3K or NOTCH4 mutations can be candidates for agents targeting these oncogenic pathways. In this manner, a more individualized treatment of triple negative breast cancer might become possible in the near future. Citation Format: Esther H Lips, Magali Michaut, Marlous L Hoogstraat, Lennart Mulder, Nicolle Besselink, Marco J Koudijs, Emile E Voest, Rene Bernards, Petra M Nederlof, Jelle Wesseling, Sjoerd Rodenhuis, Lodewyk FA Wessels. Next generation sequencing to find predictors for chemotherapy response in triple negative breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-06-21.

Pathological response of breast cancer to neoadjuvant chemotherapy (NAC) presents great variability, and new prognostic biomarkers are needed. Our aim was to evaluate the association of the epidermal growth factor receptor gene (EGFR) polymorphism R497K (rs2227983) with prognostic features and clinical outcomes of breast cancer, including the pathological response to NAC and the recurrence-free survival (RFS). Tumoral complete response (tCR) was defined by no remaining invasive cancer in the excised breast, whereas pathological complete response (pCR) was defined by no remaining invasive cancer both in the excised breast and lymph nodes. Two independent cohorts were analyzed: one from Brazil (INCA, n = 288) and one from The Netherlands (NKI-AVL, n = 255). In the INCA cohort, the variant (Lys-containing) genotypes were significantly associated with lower proportion of tCR (ORadj = 0.92; 95%CI = 0.85–0.99), whereas in the NKI-AVL cohort they were associated with tumor grade 3 (p = 0.035) and with triple-negative subtype (p = 0.032), but not with clinical outcomes. Such distinct prognostic associations may have arisen due to different neoadjuvant protocols (p < 0.001), or to lower age at diagnosis (p < 0.001) and higher proportion of tumor grade 3 (p = 0.018) at the NKI-AVL cohort. Moreover, NKI-AVL patients achieved better proportion of pCR (21.2% vs 8.3%, p < 0.001) and better RFS (HRadj = 0.48; 95% adjCI = 0.26–0.86) than patients from INCA. In conclusion, large scale studies comprehending different populations are needed to evaluate the impact of genome variants on breast cancer outcomes.

Introduction Detection of early-stage breast cancer from blood is a highly desirable screening application of liquid biopsies. Tumor-educated platelets (TEP) contain distinct spliced RNA repertoires and can function as a blood-based biosource for the detection of cancer (Best et al. Cancer Cell 2015). Here, we investigated the potential of TEP RNA profiles as an early breast cancer diagnostics algorithm. Material and methods We sequenced platelets of women with stage I-III breast cancer (n=134) and asymptomatic non-cancer females (n=121). Samples were divided over a training, evaluation and validation set. We developed a TEP RNA-based classification algorithm, which was tailored towards two clinically relevant diagnostic applications. The first application is to rule out breast cancer in women with an abnormal mammography. The second application is to rule in breast cancer in asymptomatic women carrying an elevated baseline breast cancer risk. We examined if classifications depended on patient age, tumour stage, subtype, BRCA mutation status, and breast density. In addition, we tested if the algorithm was breast cancer specific by testing our algorithm in a pan-cancer cohort (n=192). Results and discussions In the evaluation cohort (n=48 of which n=24 early breast cancer cases) the TEP RNA-based breast cancer classification had an accuracy of 90% with an AUC of 0.96 (95% confidence interval (CI) 0.92–1.00; p Conclusion We show that platelet RNA signatures may enable blood-based screening for early breast cancer, and warrant validation in a confirmatory and screening setting.

Previously, we employed bacterial artificial chromosome (BAC) array comparative genomic hybridization (aCGH) profiles from BRCA1 and -2 mutation carriers and sporadic tumours to construct classifiers that identify tumour samples most likely to harbour BRCA1 and -2 mutations, designated 'BRCA1 and -2-like' tumours, respectively. The classifiers are used in clinical genetics to evaluate unclassified variants, and patients for which no good quality germline DNA is available. Furthermore, we have shown that breast cancer patients with BRCA-like tumour aCGH profiles benefit substantially from platinum-based chemotherapy, potentially due to their inability to repair DNA double strand breaks (DSB), providing a further important clinical application for the classifiers. The BAC array technology has been replaced with oligonucleotide arrays. To continue clinical use of existing classifiers, we mapped oligonucleotide aCGH data to the BAC domain, such that the oligonucleotide profiles can be employed as in the BAC classifier. We demonstrate that segmented profiles derived from oligonucleotide aCGH show high correlation with BAC aCGH profiles. Furthermore, we trained a support vector machine score to objectify aCGH profile quality. Using the mapped oligonucleotide aCGH data, we show equivalence in classification of biologically relevant cases between BAC and oligonucleotide data. Furthermore, the predicted benefit of DSB inducing chemotherapy due to a homologous recombination defect is retained. We conclude that oligonucleotide aCGH data can be mapped to and used in the previously developed and validated BAC aCGH classifiers. Our findings suggest that it is possible to map copy number data from any other technology in a similar way.

Response rates to chemotherapy remain highly variable in breast cancer patients. We set out to identify genes associated with chemotherapy resistance. We analyzed what is currently the largest single-institute set of gene expression profiles derived from breast cancers prior to a single neoadjuvant chemotherapy regimen (dose-dense doxorubicin and cyclophosphamide). We collected, gene expression-profiled, and analyzed 178 HER2-negative breast tumor biopsies ("NKI dataset"). We employed a recently developed approach for detecting imbalanced differential signal (DIDS) to identify markers of resistance to treatment. In contrast to traditional methods, DIDS is able to identify markers that show aberrant expression in only a small subgroup of the non-responder samples. We found a number of markers of resistance to anthracycline-based chemotherapy. We validated our findings in three external datasets, totaling 456 HER2-negative samples. Since these external sets included patients who received differing treatment regimens, the validated markers represent markers of general chemotherapy resistance. There was a highly significant overlap in the markers identified in the NKI dataset and the other three datasets. Five resistance markers, SERPINA6, BEX1, AGTR1, SLC26A3, and LAPTM4B, were identified in three of the four datasets (p value overlap < 1 × 10(-6)). These five genes identified resistant tumors that could not have been identified by merely taking ER status or proliferation into account. The identification of these genes might lead to a better understanding of the mechanisms involved in (clinically) observed chemotherapy resistance and could possibly assist in the recognition of breast cancers in which chemotherapy does not contribute to response or survival

A pathological complete remission (pCR) is rarely achieved by neoadjuvant chemotherapy in estrogen receptor-positive (ER+) HER2-negative (HER2−) tumors. Therefore, its use might be questionable in specific groups of this tumor type. To select which patients benefit and which could be spared neoadjuvant chemotherapy, we tested standard pathology and molecular markers in ER+ HER2− breast tumors. Pretreatment biopsies were available from 211 ER+ HER2− tumors, who had been treated with neoadjuvant chemotherapy (adriamycin/cyclophosphamide). mRNA expression data were available for 132 tumors. We determined progesterone receptor expression (PR), endocrine sensitivity, HER2 expression, histology, proliferation, and molecular subtypes. We correlated these data to chemotherapy response using pCR rates and the previously published neoadjuvant response index (NRI). PR-negative tumors (n = 65, 30.8%) and luminal B type tumors (n = 43, 20.4%) responded significantly better to chemotherapy than other tumors. These associations remained significant in multivariate analysis. However, even in the subgroup of patients with the lowest response rate, comprising tumors that had both a positive-PR expression and the luminal A subtype (n = 58, 44%), the majority of the patients had downstaging because of chemotherapy. For histology (lobular vs. ductal), endocrine sensitivity, and proliferation, no associations with chemotherapy response were observed. Gene expression array analysis resulted in 28 significant genes (FDR

Background Our group has previously employed array Comparative Genomic Hybridization (aCGH) to assess the genomic patterns of BRCA1-mutated breast cancers. It is reasonable to assume that this pattern indicates ‘BRCAness’ and thus serves as a marker for homologous recombination deficiency. This BRCA1-like aCGH profile is also present in about half of all triple negative sporadic breast cancers and has been shown to be predictive for benefit from intensive chemotherapy with DNA crosslinking agents. To study BRCA1-like tumors and conventional dose chemotherapy sensitivity in more detail, we compared clinical factors and survival rates in a uniform cohort of triple negative breast tumors treated with alkylating agents. Patients and methods 103 patients with triple negative tumors received conventional dose adjuvant chemotherapy with doxorubicin/cyclophosphamide. DNA was extracted from tumor samples and BRCA1-like profiles were assessed. Tumors were classified as BRCA1 -like or non-BRCA1-like. Standard clinical and histopathological factors were determined and compared between both groups. Relapse free survival (RFS), disease specific survival (DSS) and overall survival (OS) after diagnosis were compared between BRCA1-like and non-BRCA1-like tumors. Results 66 tumors (65%) had a BRCA1-like profile, while 35 tumors (35%) did not show such a profile. Patients with BRCA1-like tumors tended to be younger and had more often node-negative disease compared to the patients with non-BRCA1-like tumors (p=0.058 and p=0.034, respectively). There was no significant difference in survival between BRCA1-like and non BRCA1-like patients after treatment with alkylating agents: the median RFS was 121 vs. 109 months, median DSS was 129 vs. 114 months and OS was 127 vs. 110 months, for BRCA1-like versus non-BRCA1-like tumors. T-stage was the only variable significantly associated with survival. Conclusion BRCA1-like tumors occurred in younger patients and were more often node negative, which are features shared with tumors in BRCA1-mutation carriers. We did not observe a difference in survival between BRCA1-like and non-BRCA1-like triple negative breast cancers after treatment with conventional dose chemotherapy with alkylating agents. These results confirm our previous findings that BRCA1-like tumors have similar sensitivity to anthracycline-based adjuvant chemotherapy as other triple-negative tumors. It will be important to establish whether BRCA1-like tumors also share the exquisite sensitivity of BRCA-mutated tumors to PARP-inhibitors. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr PD03-08.

Background: Tumors with homologous recombination deficiency (HRD), such as BRCA1-associated breast cancers, are not able to reliably repair DNA double-strand breaks (DSBs) and are therefore highly sensitive to both DSB-inducing chemotherapy and poly (ADP-ribose) polymerase inhibitors. We have studied markers that may indicate the presence of HRD in HER2-negative breast cancers and related them to neoadjuvant chemotherapy response. Patients and methods: Array comparative genomic hybridization (aCGH), BRCA1 promoter methylation, BRCA1 messenger RNA (mRNA) expression and EMSY amplification were assessed in 163 HER2-negative pretreatment biopsies from patients scheduled for neoadjuvant chemotherapy. Results: Features of BRCA1 dysfunction were frequent in triple-negative (TN) tumors: a BRCA1-like aCGH pattern, promoter methylation and reduced mRNA expression were observed in, respectively, 57%, 25% and 36% of the TN tumors. In ER+ tumors, a BRCA2-like aCGH pattern and the amplification of the BRCA2 inhibiting gene EMSY were frequently observed (43% and 13%, respectively) and this BRCA2-like profile was associated with a better response to neoadjuvant chemotherapy. Conclusions: Abnormalities associated with BRCA1 inactivation are present in about half of the TN breast cancers but were not predictive of chemotherapy response. In ER+/HER22 tumors, a BRCA2-like aCGH pattern was predictive of chemotherapy response. These findings should be confirmed in independent series.

Introduction Response to cancer therapeutics, whether in cell lines or patients, is variable and often difficult to predict. While many oncogenic drivers and drug resistance mechanisms have been described, understanding of how the interplay of these factors impacts response is still limited, precluding implementation in clinical practice. Material and methods To better understand the mechanisms behind the variability in drug response in breast cancer, we profiled both cell lines and tumour samples, in the absence of drug treatment, for the presence of mutations, copy number aberrations, mRNA and protein expression as well as protein phosphorylation. Response of the cell lines to several drugs in the RTK/PI3K/MAPK pathways was also determined. The molecular characteristics together with response data were used to construct a knowledge-based, Bayesian computational model that integrates all data types and estimates the relative contribution of the various drug sensitivity mechanisms. Using a set of patient samples, with known response to a combination treatment of trastuzumab, paclitaxel and carboplatin in a neoadjuvant setting (TRAIN trial), we have constructed a preliminary model to explain the observed variability in pathological complete response (pCR). Upon further refinement, the predictive ability of this model will be tested using an independent validation set of patient samples. Results and discussions Our model of regulatory signalling is able to explain most of the variability observed in drug response in cell lines. It also identified cell lines with an unexplained response, and provided an opportunity for us to search for novel explanatory factors. Among others, we found that the 4E-BP1 protein expression level – and not just the extent of its phosphorylation – is a determinant of mTOR inhibitor sensitivity, which we validated experimentally. Extending the modelling approach to focus on a predictive clinical application, specifically response to chemotherapy combined with trastuzumab treatment in a neoadjuvant setting for HER2 +breast cancer, we have constructed a preliminary model able to explain part of the variability in pCR, the refinement and validation of which will be presented. Conclusion Combining molecular characterisation with integrative modelling can be used to systematically test and extend our understanding of the variability in anticancer drug response. Such approaches pave the way for establishing effective personalised treatments.

ER, PR and HER2 status in breast cancer are important markers for the selection of drug therapy. By immunohistochemistry (IHC), three major breast cancer subtypes can be distinguished: Triple negative (TN(IHC)), HER2+(IHC) and Luminal(IHC) (ER+(IHC)/HER2-(IHC)). By using the intrinsic gene set defined by Hu et al. five molecular subtypes (Basal(mRNA), HER2+(mRNA), Luminal A(mRNA), Luminal B(mRNA) and Normal-like(mRNA)) can be defined. We studied the concordance between analogous subtypes and their prediction of response to neoadjuvant chemotherapy. We classified 195 breast tumors by both IHC and mRNA expression analysis of patients who received neoadjuvant treatment at the Netherlands Cancer institute for Stage II-III breast cancer between 2000 and 2007. The pathological complete remission (pCR) rate was used to assess chemotherapy response. The IHC and molecular subtypes showed high concordance with the exception of the HER2+(IHC) group. 60% of the HER2+(IHC) tumors were not classified as HER2+(mRNA). The HER2+(IHC)/Luminal A or B(mRNA) group had a low response rate to a trastuzumab-chemotherapy combination with a pCR rate of 8%, while the HER2+(mRNA) group had a pCR rate of 54%. The Luminal A(mRNA) and Luminal B(mRNA) groups showed similar degrees of response to chemotherapy. Neither the PR status nor the endocrine responsiveness index subdivided the ER+(IHC) tumors accurately into Luminal A(mRNA) and Luminal B(mRNA) groups. Molecular subtyping suggests the existence of a HER2+(IHC)/Luminal(mRNA) group that responds poorly to trastuzumab-based chemotherapy. For Luminal(IHC) and triple negative(IHC) tumors, further subdivision into molecular subgroups does not offer a clear advantage in treatment selection.

Background Although BRCA1-deficient tumors are extremely sensitive to DNA-damaging drugs and poly(ADP-ribose) polymerase (PARP) inhibitors, recurrences do occur and, consequently, resistance to therapy remains a serious clinical problem. To study the underlying mechanisms, we induced therapy resistance in patient-derived xenograft (PDX) models of BRCA1-mutated and BRCA1-methylated triple-negative breast cancer. Methods A cohort of 75 mice carrying BRCA1-deficient breast PDX tumors was treated with cisplatin, melphalan, nimustine, or olaparib, and treatment sensitivity was determined. In tumors that acquired therapy resistance, BRCA1 expression was investigated using quantitative real-time polymerase chain reaction and immunoblotting. Next-generation sequencing, methylation-specific multiplex ligation-dependent probe amplification (MLPA) and Target Locus Amplification (TLA)-based sequencing were used to determine mechanisms of BRCA1 re-expression in therapy-resistant tumors. Results BRCA1 protein was not detected in therapy-sensitive tumors but was found in 31 out of 42 resistant cases. Apart from previously described mechanisms involving BRCA1-intragenic deletions and loss of BRCA1 promoter hypermethylation, a novel resistance mechanism was identified in four out of seven BRCA1-methylated PDX tumors that re-expressed BRCA1 but retained BRCA1 promoter hypermethylation. In these tumors, we found de novo gene fusions that placed BRCA1 under the transcriptional control of a heterologous promoter, resulting in re-expression of BRCA1 and acquisition of therapy resistance. Conclusions In addition to previously described clinically relevant resistance mechanisms in BRCA1-deficient tumors, we describe a novel resistance mechanism in BRCA1-methylated PDX tumors involving de novo rearrangements at the BRCA1 locus, demonstrating that BRCA1-methylated breast cancers may acquire therapy resistance via both epigenetic and genetic mechanisms.

Introduction In triple negative breast cancers (TNBC) the initial response to chemotherapy is often favorable, but relapse and chemotherapy resistance frequently occur in advanced disease. Hence there is an urgent need for targeted treatments in this breast cancer subtype. In the current study we deep sequenced DNA of tumors prior to chemotherapy to search for predictors of response or resistance. Methods Next generation sequencing (NGS) was performed for 1,977 genes involved in tumorigenesis. DNA from 56 pre-treatment TNBC-biopsies was sequenced, as well as matched normal DNA. Following their tumor biopsy, patients started neoadjuvant chemotherapy with doxorubicin and cyclophosphamide. We studied associations between genetic alterations and three clinical variables: chemotherapy response, relapse-free survival and BRCA proficiency. Results The mutations observed were diverse and few recurrent mutations were detected. Most mutations were in TP53, TTN, and PIK3CA (55 %, 14 %, and 9 %, respectively). The mutation rates were similar between responders and non-responders (average mutation rate 9 vs 8 mutations). No recurrent mutations were associated with chemotherapy response or relapse. Interestingly, PIK3CA mutations were exclusively observed in patients proficient for BRCA1. Samples with a relapse had a higher copy number alteration rate, and amplifications of TTK and TP53BP2 were associated with a poor chemotherapy response. Conclusions In this homogenous cohort of TNBCs few recurrent mutations were found. However, PIK3CA mutations were associated with BRCA proficiency, which can have clinical consequences in the near future. Electronic supplementary material The online version of this article (doi:10.1186/s13058-015-0642-8) contains supplementary material, which is available to authorized users.

Background Cancer classification, prognostication, and prediction of treatment sensitivity increasingly rely on DNA and RNA-based tests. This approach requires sufficiently high tumor cell percentages to yield enough DNA or RNA for reliable test results. As a consequence, samples of insufficient quality may drop out, e.g. due to poor cellularity or high numbers of tumor infiltrating lymphocytes. We hypothesized that requiring a high tumor cell percentage and high quality RNA causes systemic bias when interpreting genomic test results in breast cancer, as specific breast cancer subgroups may be over- or underrepresented. Patients and methods For this analysis, we used pre-treatment frozen samples from patients included in neoadjuvant chemotherapy trials at the Netherlands Cancer Institute between 2004 and 2012. Histological features and tumor cell percentage were reviewed and assessed by a consultant breast pathologist. Gene expression profiling was done if the tumor cell percentage exceeded 50% and RNA quantity (>1 µg) and quality (RIN value≥ 6.5) were sufficient. We compared patient and tumor characteristics between patients in whom gene expression profiling could be performed and those in whom it could not be performed. In addition, we performed a systematic review on gene expression profiling for breast cancer, to assess the percentage of sample dropout in published studies. Results Frozen biopsies were available from 658 patients (79% of the total study population) and gene expression profiling could be performed in 60% of the cases, a percentage comparable to what is reported in the literature. Reasons for drop out were a low cellularity, poor quality and quantity of the RNA, or a too small biopsy for processing. These patients had more grade 3 tumors (43% versus 34%, p=0.04) and were more node positive (76% versus 69%, p=0.03). Analysis of neoadjuvant chemotherapy response and survival in these patients is ongoing and will be presented at the AACR Annual Meeting 2017. Conclusion Breast cancers for which gene expression data were successfully obtained were associated with a higher grade and with lymph node metastasis. Such tumors represent a more aggressive phenotype and have a relatively poor prognosis, compared to the patients for whom gene expression data could not be measured. As gene expression arrays are now broadly used in a clinical context, it is important to acknowledge this systematic bias, and to be cautious in applying gene expression based tests on different patient populations than a test was developed on. Citation Format: Esther H. Lips, Antien L. Mooyaart, Maartje van Seijen, Lennart Mulder, Marlous Hoogstraat, Petra M. Nederlof, Lodewyk F. Wessels, Sjoerd Rodenhuis, Gabe S. Sonke, Jelle Wesseling. Systematic bias in genomic breast cancer classification due to selecting cases with high tumor percentage and good RNA quality [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1751. doi:10.1158/1538-7445.AM2017-1751

Background: BRCAness is defined as shared tumour characteristics between sporadic and BRCA-mutated cancers. However, how to exactly measure BRCAness and its frequency in breast cancer is not known. Assays to establish BRCAness would be extremely valuable for the clinical management of these tumours. We assessed BRCAness characteristics frequencies in a large cohort of triple-negative breast cancers (TNBCs). Methods: As a measure of BRCAness, we determined a specific BRCA1-like pattern by array Comparative Genomic Hybridisation (aCGH), and BRCA1 promoter methylation in 377 TNBCs, obtained from 3 different patient cohorts. Clinicopathological data were available for all tumours, BRCA1-germline mutation status and chemotherapy response data were available for a subset. Results: Of the tumours, 66–69% had a BRCA1-like aCGH profile and 27–37% showed BRCA1 promoter methylation. BRCA1-germline mutations and BRCA1 promoter methylation were mutually exclusive events (P=1 × 10−5). BRCAness was associated with younger age and grade 3 tumours. Chemotherapy response was significantly higher in BRCA1-mutated tumours, but not in tumours with BRCAness (63% (12 out of 19) vs 35% (18 out of 52) pathological complete remission rate, respectively). Conclusion: The majority of the TNBCs show BRCAness, and those tumours share clinicopathological characteristics with BRCA1-mutated tumours. A better characterisation of TNBC and the presence of BRCAness could have consequences for both hereditary breast cancer screening and the treatment of these tumours.

Background: Most breast cancers arising in BRCA1-mutation carriers are triple negative (TN). Therefore, it has been suggested that TN status is intrinsic to BRCA1-mutated tumors. However, 10 to 20% of BRCA1-associated breast cancers are estrogen receptor positive (ER+). As these tumors arise more often in older patients, and have less aggressive tumor characteristics, it has been suggested that these tumors are ‘sporadic’ and not related to BRCA1 deficiency. With the introduction of targeted treatments specific for tumors with a non-functioning BRCA1 or BRCA2 gene (i.e. PARP-inhibitors), the question whether the BRCA genes are impaired in the tumor, is highly relevant. Therefore, we performed a genomic analysis of a series of BRCA1-associated ER+ tumors, and compared them with BRCA1-associated TN tumors, as well as BRCA2-associated and sporadic breast cancers. Material and Methods: Genomic profiling of 18 BRCA1-associated ER+ tumors was performed using Nimblegen 135K arrays. We previously developed a BRCA1-like and BRCA2-like genomic profile, containing specific copy number alterations for respectively BRCA1-associated and BRCA2-associated breast cancer. These genomic profiles were applied on the current series. In addition, the BRCA1 promoter methylation status was determined, and LOH analysis was performed to assess if the wildtype or mutant allele was lost in the tumor. Results were compared with BRCA1 associated ER- tumors, BRCA2 associated tumors, and sporadic ER+ and TN tumors. Results: Only 2 out of 18 ER+ BRCA1 associated tumors showed a BRCA1-like genomic profile, while 90% of all ER- BRCA1-associated tumors show this genomic profile. Interestingly, 11 out of 18 ER+ BRCA1-associated tumors showed a BRCA2-like genomic profile. BRCA1 promoter methylation was absent in ER+ BRCA1-associated tumors, similar to TN BRCA1-associated tumors. LOH analysis was possible in 12 tumors, loss of the wildtype BRCA1 allele was shown in 10 out of 12 ER+ BRCA1-mutated tumors. One sample showed loss of the mutant allele, while another sample did not show loss at the BRCA1 locus. The genomic profiles of ER+ BRCA1-associated tumors were more similar to BRCA2-associated breast cancers and sporadic ER+ breast tumors than to BRCA1-associated TN tumors. Conclusion: The majority of BRCA1-associated ER+ tumors did not show a BRCA1-like genomic profile, even though LOH analysis indicated that loss of the wildtype BRCA1 allele was a frequent event. Therefore, it is likely that the complete loss of the BRCA1 gene plays a crucial role in tumorgenesis in this group of breast tumors. Remarkably, a BRCA2-like genomic profile was observed in the majority of ER+ BRCA1 associated tumors in this series. A clinical consequence of our findings is that ER+ BRCA1-associated breast tumors are probably highly sensitive to PARP inhibitors, similar to TN BRCA1-associated breast cancers. However, as ER+ BRCA1-mutated tumors clearly have different tumor characteristics compared to TN BRCA1-mutated tumors, they should be considered as a special group, and response to therapies exploiting the BRCA1 gene defect should be specifically monitored in this subgroup. Citation Format: Esther H. Lips, Rashmie Debipersad, Esther Scheerman, Lennart Mulder, Lizet E. van der Kolk, Jelle Wesseling, Frans B.L. Hogervorst, Petra M. Nederlof. The genomic profile of BRCA1-associated estrogen-receptor positive breast cancer does not resemble BRCA1-associated triple negative cancers, but is more similar to BRCA2-associated breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A27.

About 10-20% of all breast carcinomas show a basal-like phenotype, while ∼ 90% of breast tumors from BRCA1-mutation carriers are of this subtype. There is growing evidence that BRCA1-mutated tumors are not just a specific subset of the basal-like tumors, but that (the majority of) basal-like tumors show a dysfunctional BRCA1 pathway. This has major treatment implications, because emerging regimens specifically targeting DNA repair mechanisms would then be most effective against these tumors. To further understand the involvement of BRCA1 deficiency in sporadic basal-like tumors, we investigated 41 basal-like tumors for BRCA1 mRNA expression by quantitative real-time polymerase chain reaction, BRCA1 promoter methylation, their genomic profile by array-CGH, and gene expression levels by whole genome expression arrays. Array-CGH results were compared to those of 34 proven BRCA1-mutated tumors. Basal-like tumors were subdivided into two equal groups: deficient and proficient in BRCA1 gene expression. The chromosomal makeup of BRCA1 deficient sporadic basal-like tumors was similar to that of BRCA1-mutated tumors. BRCA1 proficient sporadic basal-like tumors were more similar to nonbasal-like tumors. Only half of the basal-like breast tumors are actually deficient in BRCA1 expression. Gain of chromosome arm 3q is a marker for BRCA1 deficiency in hereditary and sporadic breast tumors.

Purpose: A preliminary study performed on a small cohort of multifocal prostate cancer (PCa) detected BRCA1 allelic imbalances among circulating tumor cells (CTC). The present analysis was aimed to elucidate the biological and clinical roles of BRCA1 losses in metastatic spread and tumor progression in PCa patients. Experimental Design: To map molecular progression in PCa outgrowth, we used fluorescence in situ hybridization analysis of primary tumors and lymph node sections, and CTCs from peripheral blood. Results: We found that 14% of 133 tested patients carried monoallelic BRCA1 loss in at least one tumor focus. Extended molecular analysis of chr17q revealed that this aberration was often a part of larger cytogenetic rearrangement involving chr17q21 accompanied by allelic imbalance of the tumor suppressor gene PTEN and lack of BRCA1 promoter methylation. The BRCA1 losses correlated with advanced T stage (P < 0.05), invasion to pelvic lymph nodes (P < 0.05), as well as biochemical recurrence (P < 0.01). Their prevalence was twice as high within 62 lymph node metastases (LNM) as in primary tumors (27%, P < 0.01). The analysis of 11 matched primary PCa-LNM pairs confirmed the suspected transmission of genetic abnormalities between these two sites. In four of seven patients with metastatic disease, BRCA1 losses appeared in a minute fraction of cytokeratin- and vimentin-positive CTCs. Conclusions: Small subpopulations of PCa cells bearing BRCA1 losses might be one confounding factor initiating tumor dissemination and might provide an early indicator of shortened disease-free survival. Clin Cancer Res; 16(13); 3340–8. ©2010 AACR.

1010 Background: In TNBC initial response to chemotherapy is often favorable, but relapse and chemotherapy resistance frequently occur in advanced disease. Hence there is an urgent need for targeted treatments in this breast cancer subtype. Methods: To identify biomarkers of chemotherapy resistance and putative directed treatment targets, we performed next generation sequencing (NGS) of 2,000 genes implicated in oncogenesis. DNA from 31 pre-treatment biopsies and matched normal blood was sequenced. Biopsies were derived from patients scheduled to receive neoadjuvant chemotherapy with doxorubicin/cyclophosphamide. For the analysis, the tumors were divided in responders and non-responders, depending on whether or not a pathological complete remission (pCR) was achieved. Two definitions of pCR were employed: either the complete absence of infiltrating tumor cells in the breast (n=18) or a pCR of both the breast and lymph nodes (n=15). Tumors with partial or no responses were grouped in the non-responder category. Results: As a positive control, we verified that all patients with a known germline BRCA1 mutation (n=8) could be detected by the NGS analysis. In further analyses, we focused only on somatic mutations. Overall, the mutation rate was slightly higher in the non-responders (per tumor, average=12, range=[3-25]) than in the responders (average=8, range=[3-19]) (p=0.17). The analysis of individual genes did not reveal significant differences between responders and non-responders. However, pathway analysis showed that mutations in phosphatidylinositol signaling (e.g., PIK3CA, CALML5) were significantly more frequent in the non-responders, with mutations present in 10/13 non-responders and 2/18 responders (adjusted p=0.013). The chemokine and integrin signaling pathways also revealed a significantly higher mutation rate in the non-responders. Conclusions: Mutations in genes of the phosphatidylinositol pathway occur frequently in TNBCs that do not achieve a pCR on a neoadjuvant chemotherapy regimen consisting of doxorubicin and cyclophosphamide. After validation, alternative chemotherapy regimens and targeted agents should be investigated for these tumors.

Introduction Previous work from our group and other groups has shown that triple negative breast cancers (TNBC) frequently show BRCAness, the phenotype that some sporadic tumors share with BRCA-mutated cancers. We propose the use of a BRCA1-like genomic profile and of BRCA1 promoter methylation as indicators of BRCAness. In a randomized trial, tumors with this BRCA1-like profile responded more favorably to intensified alkylating chemotherapy. We now assessed the frequency of BRCAness in a large cohort of TNBCs. In addition, we determined the association with clinicopathological variables and treatment response. Methods A total of 388 TNBCs were included in this study. These were obtained from three different sources: a neoadjuvant chemotherapy trial (n=152), a series of patients from breast cancer families but without germline mutations (n=145), and a series of patients treated with adjuvant chemotherapy (n=91). Two assays for BRCAness were employed: array Comparative Genomic Hybridization (aCGH) to asses a BRCA1-like profile and methylation specific Multiplex Ligation-dependent Probe Amplification (MLPA) to determine BRCA1 promoter methylation. BRCA1 germline mutation status was known for half of the patients. Clinicopathological characteristics were available for most patients, and chemotherapy response data was available for the neoadjuvant series. Results In the three series, 66% to 69% of the tumors had an aCGH BRCA1-like profile and 27% to 37% showed BRCA1 promoter methylation. 87% of the tumors with promoter methylation showed a BRCA1-like aCGH profile. A germline mutation in BRCA1 and somatic BRCA1 promoter methylation did not occut together (n=155; p = 10-6). Patients with aCGH BRCA1-like or BRCA1 methylated tumors were younger than patients with non-BRCA1-like or unmethylated tumors (p=0.02 and p Conclusion The majority of the TNBCs in this study show signs of BRCAness, and these tumors share clinicopathological characteristics with BRCA1 mutated tumors. Somatic BRCA1 promoter methylation and BRCA1 germline mutations are mutually exclusive events. A better characterization of TNBC and the presence of BRCAness will not only have consequences for treatment, but also for hereditary breast cancer screening. Citation Format: Esther H. Lips, Lennart Mulder, Lizet E. van der Kolk, Anne M.M. Oonk, Alex L.T. Imholz, Frans B.L. Hogervorst, Jelle Wesseling, Sjoerd Rodenhuis, Petra M. Nederlof. Triple negative breast cancer: BRCAness and concordance of clinical features and treatment response with BRCA1 mutation carriers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1192. doi:10.1158/1538-7445.AM2013-1192

10513 Background: Our group has previously employed array Comparative Genomic Hybridization (aCGH) to assess the genomic patterns of BRCA1-mutated breast cancers. We have shown that the so-called BRCA1-likeaCGH profile is also present in about half of all triple negative sporadic breast cancers and is predictive for benefit from bifunctional alkylating agents. As aCGH is a rather complex method, we translated the BRCA1aCGH profile to a Multiplex Ligation-dependent Probe Amplification (MLPA) assay, to identify both BRCA1-mutated breast cancers and sporadic cases with a BRCA1-likeaCGH profile. Methods: The most important genomic regions of the original aCGH based classifier (3q22-27, 5q12-14, 6p23-22, 12p13, 12q21-23, 13q31-34) were mapped to a set of 34 MLPA probes. The training set consisted of 39 BRCA1-likeaCGH breast cancers and 45 non-BRCA1-likeaCGH breast cancer patients, which had previously been analyzed by aCGH. Part of the sporadic tumors showing a BRCA1-likeaCGH profile had a low BRCA1 gene expre...

Introduction: A pathological complete remission (pCR) is rarely achieved by neoadjuvant chemotherapy (NAC) in ER+HER2- tumors. Therefore, its use might be questionable in specific groups of this tumor type. To select which patients benefit and which could be withheld from NAC, we tested standard pathology and molecular markers in ER+HER2- breast tumors. Methods: Pre-treatment biopsies were available from 211 ER+HER2- tumors, which had been treated with NAC. mRNA expression and aCGH data were available for 132 tumors. We determined progesterone receptor (PR)expression, endocrine responsiveness, histology, proliferation, molecular subtypes and BRCAness aCGH profiles. We correlated these data with chemotherapy response using pCR rates and the neoadjuvant response index (NRI). Results: PR-negative tumors (n = 65, 30.8%) and Luminal B type tumors (n=43, 20.4%) responded significantly better to NAC than other tumors, for both pCR breast and the NRI as outcome measure (table 1). The associations remained significant in multivariate analysis. However, even in the subgroup of patients with the lowest response rate (tumors with both a positive PR and of the Luminal A type (n=58, 44 %)), the majority showed downstaging (median NRI=0.25)(table 1). For endocrine responsiveness and the BRCAness aCGH profile only an association with pCR breast was observed. Histology (lobular versus ductal) and proliferation did not show an association with chemotherapy response (table 1). Discussion: PR expression and molecular subtype are associated with a better response to NAC. However, both markers had only weak predictive power and it was not possible to identify a subgroup with no or only minimal chemotherapy benefit. Therefore, the decision to refrain patients from NAC in ER+HER2- breast tumors should not be based on predictive markers, but exclusively on estimates of prognosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3134. doi:10.1158/1538-7445.AM2011-3134

Motivation: Breast cancer is a heterogeneous disease and although gene expression analysis or immuno histo chemistry can be used to subclassify tumors into distinct subgroups, even within these subgroups there exist significant differences at the molecular level. When it comes to chemotherapy resistance, it appears highly unlikely that all resistant tumors share the same mechanism of action. Classical approaches have been unsuccessful in finding reliable biomarkers for therapy resistance and this may be due to the fact that these approaches are not sensitive to changes in small subgroups of samples. Methods: Traditional analyses approaches, like a t-test or using the SAM approach, compare the two groups of samples for each gene, and identify genes with a significant difference in expression between the two groups. If only a small subset of the resistant tumors would show aberrant expression indicative of a resistance mechanism, then this would not be picked up by such approaches. Using a novel algorithm we try to circumvent this problem and specifically aim to find relatively small subgroups of tumors within the resistant group that show differential expression compared to the sensitive group. Results: Using a positive control set, where we mixed in a small set of HER2-positive tumors in a larger HER2-negative set and by generating an artificial dataset we show that our algorithm is able to pick up all positive controls. These controls are not picked up by either a t-test or the SAM algorithm. Next, we applied the algorithm on a gene expression set of 195 patients that were neoadjuvantly treated and identified a number of genes that show an expression pattern that suggests a role in chemotherapy resistance in ER+ tumors. We validate our findings on a separate set of 90 patients. Discussion: We have developed a novel algorithm that allows the identification of genes that show aberrant expression, relative to the reference group, in a small subset of the samples. Using this approach we identified a number of genes that are linked to chemotherapy resistance. The algorithm can be used in any type of analysis that involves the detection of small subsets of samples within one of the labeled classes, where the small subset shows behavior different from the average behavior of the remaining samples. Our approach can therefore be applied to a wide range of problems. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 22. doi:10.1158/1538-7445.AM2011-22

Background: Tumors with homologous recombination (HR) deficiency are highly sensitive to DNA double strand break (DSB) inducing agents, such as alkylating agents and poly (ADP-ribose) polymerase (PARP)-inhibitors. BRCA1 or BRCA2- mutated tumors, which are HR deficient, have characteristic DNA gains and losses that can be assessed by an array Comparative Genomic Hybridization (aCGH) classifier, one for BRCA1 mutations and one for BRCA2 mutations. We have studied these aCGH profiles together with several other HR deficiency indicators in sporadic breast cancers and we have correlated their presence to neoadjuvant chemotherapy response. Material and Methods: A total of 163 HER2-negative pre-treatment biopsies were examined, procured from sporadic breast cancer patients scheduled to receive neoadjuvant therapy with doxorubicin and cyclophosphamide. Triple negative (TN) and estrogen receptor positive (ER+/HER2-) tumors were analyzed separately. aCGH was performed to assess BRCA1-like and BRCA2-like profiles. In addition, BRCA1 promoter methylation, BRCA1 mRNA expression and amplification of the BRCA2-inhibiting gene EMSY were analyzed. Response to neoadjuvant treatment was assessed by measuring pathological complete remission (pCR) and near pCR at the time of surgery. Results: Inactivation of BRCA1 was frequent in TN tumors: 57% of these tumors showed a BRCA1-like profile at aCGH. BRCA 1 promoter methylation and reduced BRCA1 mRNA expression were observed in 25% and 36% of the TN tumors, respectively. The BRCA1-like aCGH profile was not clearly associated with a better neoadjuvant treatment response (58% vs. 48%, p=0.47). In ER+ tumors, a BRCA2-like aCGH profile and the amplification of the BRCA2 inhibiting gene EMSY were frequently observed (43% and 13% respectively). A BRCA2-like aCGH profile was associated with a significantly higher response rate (35% vs. 14%, p=0.014). EMSY amplification and a BRCA2-like aCGH profile occurred together in only one case, suggesting mutual exclusivity. EMSY was not associated with treatment response, questioning the role of EMSY in HR deficiency. Conclusion: Alterations associated with BRCA1 inactivation are present in about half of the TN breast cancers, but were not predictive of chemotherapy response. In ER+/HER2- tumors, the BRCA2-like aCGH profile predicts sensitivity to DSB-inducing chemotherapy, and possibly as well to new targeted agents, such as the PARP inhibitors. After validation in independent series the aCGH classifiers may lead to a diagnostic test that could assist in neoadjuvant treatment selection. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr PD07-07.

22102 Background: Homologous Recombination (HR) is the only mechanism that can reliably repair double-strand DNA breaks. Alternative DNA repair mechanisms, such as non-homologous end joining exist, but these induce DNA mutations and chromosomal aberrations. Hence, tumors that have deregulating mutations in the key players of HR are extremely sensitive to DNA damaging agents, such as bifunctional alkylators and are targeted specifically by Poly(ADP-ribose) Polymerase (PARP) inhibiting drugs. Defects in homologous recombination arise through dysfunction of BRCA1 or BRCA2 or by abnormalities in the Fanconi Anemia pathway. In addition, the recently discovered oncogene EMSY is amplified in a subgroup of breast tumors and blocks HR by inactivating the Brca2 protein. HR defects have been reported to be present in up to 30% of sporadic breast cancers. This study was designed to develop a clinically useful test to identify patients with tumors deficient in homologous recombination. Methods: We have prospectively t...

Intrinsic subtypes are widely accepted for the classification of breast cancer. Lacking gene expression data, surrogate classifications based on immunohistochemistry (IHC) have been proposed. A recent St. Gallen consensus meeting recommends to use this “surrogate intrinsic subtypes” for predicting adjuvant chemotherapy resistance, implying that “Surrogate Luminal A” breast cancers should only receive endocrine therapy. In this study we assessed both gene expression based intrinsic subtypes as well as surrogate intrinsic subtypes regarding their power to predict neoadjuvant chemotherapy benefit. Single institution data of 560 breast cancer patients were reviewed. Gene expression data was available for 247 patients. Subtypes were determined on the basis of IHC, Ki67, histological grade, endocrine responsiveness, and gene expression, and were correlated with chemotherapy response and recurrence-free survival. In ER+/HER2− tumors, a high histological grade was the best predictor for chemotherapy benefit, both in terms of pCR (p = 0.004) and recurrence-free survival (p = 0.002). The gene expression based and surrogate intrinsic subtype based on Ki67 had no predictive or prognostic value in ER+/HER2− tumors. Histological grade, ER, PR, and HER2 were the best predictive factors for chemotherapy response in breast cancer. We propose to continue the conventional use of these markers. Electronic supplementary material The online version of this article (doi:10.1007/s10549-013-2620-0) contains supplementary material, which is available to authorized users.