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Supplementary Tables S1-S3 from Association of an Extracellular Matrix Gene Cluster with Breast Cancer Prognosis and Endocrine Therapy Response

Background In breast cancer (BC), recurrent fusion genes of estrogen receptor alpha (ESR1) and AKAP12, ARMT1 and CCDC170 have been reported. In these gene fusions the ligand binding domain of ESR1 has been replaced by the transactivation domain of the fusion partner constitutively activating the receptor. As a result, these gene fusions can drive tumor growth hormone independently as been shown in preclinical models, but the clinical value of these fusions have not been reported. Here, we studied the prognostic and predictive value of different frequently reported ESR1 fusion transcripts in primary BC. Methods We evaluated 732 patients with primary BC (131 ESR1-negative and 601 ESR1-positive cases), including two ER-positive BC patient cohorts: one cohort of 322 patients with advanced disease who received first-line endocrine therapy (ET) (predictive cohort), and a second cohort of 279 patients with lymph node negative disease (LNN) who received no adjuvant systemic treatment (prognostic cohort). Fusion gene transcript levels were measured by reverse transcriptase quantitative PCR. The presence of the different fusion transcripts was associated, in uni- and multivariable Cox regression analysis taking along current clinico-pathological characteristics, to progression free survival (PFS) during first-line endocrine therapy in the predictive cohort, and disease- free survival (DFS) and overall survival (OS) in the prognostic cohort. Results The ESR1-CCDC170 fusion transcript was present in 27.6% of the ESR1-positive BC subjects and in 2.3% of the ESR1-negative cases. In the predictive cohort, none of the fusion transcripts were associated with response to first-line ET. In the prognostic cohort, the median DFS and OS were respectively 37 and 93 months for patients with an ESR1-CCDC170 exon 8 gene fusion transcript and respectively 91 and 212 months for patients without this fusion transcript. In a multivariable analysis, this ESR1-CCDC170 fusion transcript was an independent prognostic factor for DFS (HR) (95% confidence interval (CI): 1.8 (1.2–2.8), P = 0.005) and OS (HR (95% CI: 1.7 (1.1–2.7), P = 0.023). Conclusions Our study shows that in primary BC only ESR1-CCDC170 exon 8 gene fusion transcript carries prognostic value. None of the ESR1 fusion transcripts, which are considered to have constitutive ER activity, was predictive for outcome in BC with advanced disease treated with endocrine treatment.

Purpose: Almost all cervical cancers are caused by human papillomavirus (HPV) and patients with advanced stage are at high risk for relapse. Circulating HPV DNA (HPV ctDNA) may serve as a residual tumor marker at the end of chemoradiation or to predict relapse during the follow-up period. Experimental Design: We analyzed serum samples from 94 HPV16- or HPV18-related CCs from the BioRAIDs prospective cohort. Samples were collected before and after treatment and during an 18-month follow-up period. Using digital droplet PCR (ddPCR), we assessed the relevance of circulating HPV E7 gene as a marker for residual disease compared to HPV integration site and PIK3CA mutations. Finally, the prognostic impact of circulating HPV E7 gene was assessed with its prediction value of relapse. Results: HPV E7 gene was the most sensitive tumor marker, superior to both HPV integration sites and PIK3CA mutations in serum. Circulating HPV DNA (HPV ctDNA) was detected in 63% (59/94) of patients, before treatment. HPV ctDNA detection in serum sample was associated with high FIGO stage (P = 0.02) and para-aortic lymph node involvement (P = 0.01). The level of HPV ctDNA was positively correlated with HPV copy number in the tumor (R = 0.39, P < 0.001). Complete clearance of HPV ctDNA by the end of treatment was significantly associated with a longer PFS (P < 0.0001). Patients with persistent HPV ctDNA in serum relapsed with a median time of 10 months (range, 2–15) from HPV ctDNA detection. Conclusions: HPV ctDNA detection is a useful marker to predict relapse in cervical cancer. See related commentary by Wentzensen and Clarke, p. 5733

Background: Germline BRCA1/2 mutation status is currently used as predictive biomarker for response to Poly-(ADP-Ribose)-Polymerase (PARP) inhibitors in breast cancer (BC) patients. However, non-germline BRCA1/2 mutated tumors and homologous recombination repair deficient (HRD) tumors with unknown etiology are probably also PARP inhibitor-sensitive. Various HRD biomarkers have been proposed and their clinical validity and utility are under investigation. We recently described the functional REpair CAPacity (RECAP) test as a real-time method to select HRD tumors based on their inability to form RAD51 foci. Here, we investigated whether this functional test defines a similar group of HRD tumors as DNA-based tests for primary and metastatic BCs. Materials and Methods: A cohort (n=71; 52 primary and 19 metastatic BCs) enriched for HRD tumors was selected based on the RECAP test, consisting of 26 RECAP-HRD (37%), 9 RECAP-HR intermediate (HRi; 13%) and 36 RECAP-HR proficient (HRP; 51%) tumors. Whole genome sequencing (WGS) was carried out on matched germline and tumor DNA samples for 38 primary BCs and the 19 metastatic lesions, for which material was available. Three samples were excluded due to a low purity score ( Results: The RECAP test identified all bi-allelic BRCA deficient samples (n=15) in this cohort as HRD/HRi. Within the remaining non-BRCA RECAP-HRD/HRi samples, there were no pathogenic mutations in other relevant genes that could explain the HRD status of these tumors. In the RECAP-HR proficient tumors no bi-allelic BRCA1/2 deficiencies were identified. The RECAP status partially correlated with the DNA-based HRD test outcomes using the HRDetect, CHORD and BRCA1/2-like classifier algorithms (70% concordance for RECAP-CHORD and RECAP-BRCA1/2-like classifier, and 66% for CHORD-BRCA1/2-like classifier). However, RECAP identified additional samples unable to form RAD51 foci. The discordance among the tests were evenly distributed among the primary and metastatic BCs. Conclusions: The difference between RECAP and other HRD tests in BC patients is relatively large (30-34% discordance), implying that RECAP may be identifying deficiencies in other components of DNA repair which could also result in PARP inhibitor sensitivity. Therefore, direct comparison of these HRD tests in clinical trials will be required to evaluate the optimal predictive test for clinical decision making. Citation Format: Titia G. Meijer, Luan Nguyen, Arne van Hoeck, Marjolijn M. Ladan, Nicole S. Verkaik, Kirsten Ruigrok-Ritstier, Carolien H. van Deurzen, Harmen J. van de Werken, Esther H. Lips, Sabine C. Linn, Yasin Memari, Helen Davies, Serena Nik-Zainal, Roland Kanaar, John W. Martens, Edwin Cuppen, Agnes Jager, Dik C. van Gent. RECAP (REpair CAPacity) identifies a subset of breast cancers unable to form RAD51 foci which are undetected by DNA-based BRCAness tests [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 805.

PIK3CA is one of the most frequently mutated genes in invasive breast cancer (IBC). These mutations are generally associated with hyper-activation of the phosphatidylinositol 3-kinase signaling pathway, which involves increased phosphorylation of AKT (p-AKT). This pathway is negatively regulated by the tumor suppressor PTEN. Data are limited regarding the variant allele frequency (VAF) of PIK3CA, PTEN and p-AKT expression during various stages of breast carcinogenesis. Therefore, the aim of this study was to gain insight into PIK3CA VAF and associated PTEN and p-AKT expression during the progression from ductal carcinoma in situ (DCIS) to IBC. We isolated DNA from DCIS tissue, synchronous IBC and metastasis when present. These samples were pre-screened for PIK3CA hotspot mutations using the SNaPshot assay and, if positive, validated and quantified by digital PCR. PTEN and p-AKT expression was evaluated by immunohistochemistry using the Histo-score (H-score). Differences in PIK3CA VAF, PTEN and p-AKT H-scores between DCIS and IBC were analyzed. PIK3CA mutations were detected in 17 out of 73 DCIS samples, 16 out of 73 IBC samples and 3 out of 23 lymph node metastasis. We detected a significantly higher VAF of PIK3CA in the DCIS component compared to the adjacent IBC component (P = 0.007). The expression of PTEN was significantly higher in DCIS compared to the IBC component in cases with a wild-type (WT) PIK3CA status (P = 0.007), while it remained similar in both components when PIK3CA was mutated. There was no difference in p-AKT expression between DCIS and the IBC component. In conclusion, our data suggest that PIK3CA mutations could be essential specifically in early stages of breast carcinogenesis. In addition, these mutations do not co-occur with PTEN expression during DCIS progression to IBC in the majority of patients. These results may contribute to further unraveling the process of breast carcinogenesis, and this could aid in the development of patient-specific treatment.

Background PIK3CA is the most frequent somatic mutated oncogene in estrogen receptor ( ER ) positive breast cancer. We previously observed an association between PIK3CA genotype and aromatase inhibitors (AI) treatment outcome. This study now evaluates whether expression of mRNAs and miRs are linked to PIK3CA genotype and are independently related to AI therapy response in order to define potential expressed biomarkers for treatment outcome. Materials and methods The miR and mRNA expression levels were evaluated for their relationship with the PIK3CA genotype in two breast tumor datasets, i.e. 286 luminal cancers from the TCGA consortium and our set of 84 ER positive primary tumors of metastatic breast cancer patients who received first line AI. BRB Array tools class comparison was performed to define miRs and mRNAs whose expression associate with PIK3CA exon 9 and 20 status. Spearman correlations established miR–mRNA pairs and mRNAs with related expression. Next, a third dataset of 25 breast cancer patients receiving neo-adjuvant letrozole was evaluated, to compare expression levels of identified miRs and mRNAs in biopsies before and after treatment. Finally, to identify potential biomarkers miR and mRNA levels were related with overall survival (OS) and progression free survival (PFS) after first-line AI therapy. Results Expression of 3 miRs (miR-449a, miR-205-5p, miR-301a-3p) and 9 mRNAs ( CCNO , FAM81B , LRG1 , NEK10 , PLCL1 , PGR , SERPINA3 , SORBS2 , VTCN1 ) was related to the PIK3CA status in both datasets. All except miR-301a-3p had an increased expression in tumors with PIK3CA mutations. Validation in a publicly available dataset showed that LRG1 , PGR , and SERPINA3 levels were decreased after neo-adjuvant AI-treatment. Six miR–mRNA pairs correlated significantly and stepdown analysis of all 12 factors revealed 3 mRNAs ( PLCL1 , LRG1 , FAM81B ) related to PFS. Further analyses showed LRG1 and PLCL1 expression to be unrelated with luminal subtype and to associate with OS and with PFS, the latter independent from traditional predictive factors. Conclusion We showed in two datasets of ER positive and luminal breast tumors that the expression of 3 miRs and 9 mRNAs associate with the PIK3CA status. Expression of LRG1 is independent of luminal (A or B) subtype, decreased after neo-adjuvant AI-treatment, and is proposed as potential biomarker for AI therapy outcome.

Introduction: PIK3CA is the most frequent (30%) mutated oncogene in breast cancer and may lead to an activation of the PI3K/AKT/mTOR-pathway. Cell lines resistant to tamoxifen have an activated PI3K-pathway. Phase III clinical trials show substantial benefit when mTOR-inhibitors are added to aromatase inhibitor treatment. On the other hand, patients with PIK3CA exon 20 mutation expression signature show better treatment outcome after adjuvant tamoxifen therapy. To address this controversy, we evaluated the PIK3CA mutation status in 1423 primary breast cancer specimens for its relationship with prognosis and treatment outcome after first-line endocrine treatment. Methods: Hotspot mutations in exon 9 and 20 of PIK3CA were detected by multiplex snapshot analyses. Mutation status in ER-positive tumors was related to metastasis free survival (MFS) in 292 untreated lymph node negative (LNN) patients and time to progression (TTP) in patients with metastatic disease treated with first-line tamoxifen (N = 482) or aromatase inhibitors (AIs; N=103). Whole genome mRNA and miRs expression profiling was performed in the latter patient subset to develop a PIK3CA gene signature in 64 specimens. This was validated in 28 independent ER-positive specimens. Results: We could evaluate 1371 specimens and detected 437 hotspot mutations for PIK3CA (32%). Mutations in exon 20 were detected in 256 patients (59%), of which 40 cases with a H1047L (16%) and 216 with a H1047R (84%) mutation. Mutations in PIK3CA exon 9 were discovered in 174 patients (41%), with E542K and E545K mutations in 59 (34%) and 105 (60%) cases, respectively, as the most prevalent ones. Finally, 6 patients had PIK3CA double mutations for both exon 9 and 20 and one patient had a E542K and E545K mutation. Evaluation of the untreated LNN patients for prognosis showed no relationship between MFS and PIK3CA mutations (HR = 1.07 [95% CI: 0.73–1.56]; p = 0.73), neither for exon 9 nor exon 20 compared to wild-type. In the multicenter cohort of 482 patients with advanced disease treated with first-line tamoxifen no link with treatment outcome and PIK3CA mutation status was observed (HR = 1.13 [95% CI: 0.92–1.38]; p = 0.24). However, patients with advanced disease treated with first-line AIs (N = 64) showed a significant longer TTP for patients with a PIK3CA mutation compared to wild-types (HR = 0.46 [95% CI: 0.25–0.87]; p = 0.017). Expression profiles of mRNAs and miRs were integrated and resulted in signatures for PIK3CA status discovered by pathway (17 genes) and expression analyses (10 genes and 9 miRs). Validation and comparison with published signatures in the 28 independent tumors showed that our 10-genes signature had the highest PIK3CA prediction accuracy (75%), with 60% sensitivity and 78% specificity. Moreover, this signature better associates with TTP (HR = 0.38 [95% CI: 0.20–0.72]; P = 0.003). Conclusion: Mutations in PIK3CA are not prognostic value in untreated ER-positive LNN patients, not predictive in ER-positive patients with advanced disease treated with first-line tamoxifen therapy, however, are predictive for favourable outcome after first-line AIs. Moreover, we propose an expression signature of 10 genes as a putative biomarker to predict the PI3K status and response to AIs. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P3-06-01.

For patients with metastatic breast cancer, we previously described that increased EZH2 expression levels were associated with an adverse outcome to tamoxifen therapy. Main objective of the present study is to investigate miR-26a and miR-101 levels, which both target EZH2, for their association with molecular pathways and with efficacy of tamoxifen as first-line monotherapy for metastatic breast cancer. Expression levels were measured using quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) in primary breast cancer specimens of 235 estrogen receptor-α (ER)-positive patients. Pathway analysis was performed on microarray data available for 65 of these tumors. Logistic regression and Cox uni- and multivariate analysis were performed to relate expression levels with clinical benefit and time to progression (TTP). Increasing levels of miR-26a were significantly (P

Background: Genome-wide association studies has identified single nucleotide polymorphisms (SNPs) in several loci being associated with breast cancer risk: fibroblast growth factor receptor 2 (FGFR2), trinucleotide repeat containing 9 (TNRC9 or TOX3), mitogen-activated protein kinase kinase kinase 1 (MAP3K1), lymphocyte-specific protein 1 (LSP1) and the imprinted maternally expressed H19, and loci in gene deserts at 8q24 and 2q35. However, the mechanism by which these loci confer breast cancer risk in patients is still largely unknown. Here, we addressed this question by associating these SNPs with the mRNA expression of the genes located at or nearest to the SNP in the same linkage disequilibrium region. In addition, we associated the SNPs with clinical, pathological and patient characteristics and prognosis. Material and Methods : SNPs tagging breast cancer loci were genotyped in genomic tumor DNA samples of 2,480 breast cancer patients. All samples were collected between 1978-2004. The mean age was 55.6 years and median follow-up up was 106 months. The SNPs were correlated with patients and tumor characteristics. Of the 1,262 patients with lymph-node negative disease and who did not receive any adjuvant systemic therapy, SNP status was associated with distant metastasis-free survival (MFS) using Cox regression analysis. Finally, in a subset of 1,400 of the 2,480 patients, the mRNA expression of FGFR2, TNRC9, MAP3K1, LSP1 and H19 genes was determined by quantitative RT-PCR and correlated with SNP genotypes. Results: The SNP rs2981582 in FGFR2 was significantly associated with ER and PgR status of the tumors (both p=0.001). Besides weak associations with tumor grade (FGFR2, p=0.01), ER (MAP3K1, p=0.03; LSP1, p=0.03), and PgR (LSP1, p=0.05), no other association with any clinical or pathological variable for any of the SNPs was observed. Of the SNPs analyzed, only rs2107425 near H19 was significantly linked in uni-and multivariable analysis with MFS (Hazard ratio [HR]=1.44, 95% Confidence interval [CI]: 1.04-1.98; p=0.026; HR=1.53; 95% CI: 1.09-2.14; p=0.013, respectively) with the more aggressive minor allele displaying a recessive trade. Interestingly, the minor allele of SNP rs3803662 was significantly associated with lower mRNA expression of the 8 kb downstream TNRC9 gene (p=0.0019). However, none of the other risk alleles, including the one in FGFR2, had an association with mRNA expression of the nearest located gene. Conclusions: In agreement with previous studies, a clear correlation of the SNPs in FGFR2 with ER and PR status of tumors was observed. The lower level of TNRC9 mRNA in tumors having the minor allele genotype suggests that TNRC9 may act as a tumor suppressor gene and its expression might have a protective effect. A significant association of the SNP near H19 with poor outcome without apparent effect on H19 mRNA expression suggests that this prognostic SNP in a well-known imprinted region is not linked to prognosis through altering H19 gene expression. This study indicates that most of the studied SNPs that confer breast cancer risk are not linked to prognosis nor do they effect, in primary tumors mRNA expression of the nearest gene. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-07-01.

The purpose of this study is to investigate EZH2 in a large series of breast cancer patients for its prognostic and predictive value, and to evaluate its functional role in treatment response in vitro. EZH2 levels were measured using quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) in primary breast cancer specimens and related to clinicopathologic factors and disease outcome. EZH2 expression was downregulated with siRNAs in MCF7, to assess expression alterations of putative EZH2 downstream genes and to determine cell numbers after treatment with the anti-estrogen ICI 164384. In 688 lymph node-negative patients who did not receive adjuvant systemic therapy, EZH2 was not significantly correlated with metastasis-free survival (MFS). In 278 patients with advanced disease treated with first-line tamoxifen monotherapy, the tertile with highest EZH2 levels was associated with the lowest clinical benefit (OR = 0.48; P = 0.02) and with a shorter progression-free survival (PFS) in both univariate (HR = 1.80; P < 0.001) and multivariate analysis, including traditional factors (HR = 1.61; P = 0.004). In vitro, EZH2 silencing in MCF7 caused a 38% decrease in cell numbers (P < 0.001) whereas ICI 164384 treatment resulted in a 25% decrease (P < 0.001) compared to controls. Combining EZH2 silencing with ICI treatment reduced cell numbers with 67% (P < 0.001) compared to control conditions. EZH2 downregulation was associated with an almost two-fold upregulation of the estrogen receptor alpha (ER) (P = 0.001). In conclusion, EZH2 has no prognostic value in breast cancer. High levels of EZH2 are associated with poor outcome to tamoxifen therapy in advanced breast cancer. Downregulated EZH2 leads to upregulation of the ER and better response to anti-estrogens.

Purpose Two genes, TSC22 domain family, member 1 (TSC22D1) and prosaposin (PSAP) were identified in an in vitro functional screen for genes having a causative role in tamoxifen resistance. These genes were also present in our previously established 81-gene signature for resistance to first-line tamoxifen therapy. The aim of this study was to investigate the predictive value of these genes for tamoxifen therapy failure in patients with recurrent breast cancer. Experimental Design The mRNA levels of TSC22D1 and PSAP were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) in 223 estrogen receptor-positive primary breast tumors of patients with recurrent disease treated with first-line tamoxifen therapy. The main objective of this study was the length of progression-free survival (PFS). Results High mRNA levels of TSC22D1 and PSAP were significantly associated with shorter PFS and both were independent of the traditional predictive factors (HR = 1.30, 95% CI = 1.04-1.64 P = 0.023; and HR = 1.40, 95% CI = 1.03-1.88, P = 0.029, respectively). In multivariate analysis, patients with high mRNA levels of both genes associated significantly with no clinical benefit (OR = 0.19, 95% CI = 0.06-0.62, P = 0.006) and had the shortest PFS (HR = 2.05, 95% CI = 1.29-3.25, P = 0.002). Conclusion These results confirm our previous in vitro and tumor-related findings and are indicative for the failure of tamoxifen treatment in breast-cancer patients. Both TSC22D1 and PSAP are associated with clinical outcome and may have a functional role in therapy resistance.

Purpose: We previously discovered an extracellular matrix (ECM) gene cluster associated with resistance to first-line tamoxifen therapy of patients with metastatic breast cancer. In this study, we determined whether the six individual ECM genes [collagen 1A1 (COL1A1), fibronectin 1 (FN1), lysyl oxidase (LOX), secreted protein acidic cysteine-rich (SPARC), tissue inhibitor of metalloproteinase 3 (TIMP3), and tenascin C (TNC)] were associated with treatment response, prognosis, or both. Experimental Design: In 1,286 primary breast tumors, mRNA expression (quantitative real-time PCR) was related to clinicopathologic factors and disease outcome in univariate and multivariate analysis including traditional factors. Results: TIMP3, FN1, LOX, and SPARC expression levels (continuous variables) were significantly associated with distant metastasis-free survival (MFS) in 680 lymph node–negative untreated patients (P < 0.03). Using a calculated linear prognostic score, these patients were evenly divided into five prognostic groups with a significant difference in 10-year MFS of ∼40% between the two extreme prognostic groups. Furthermore, high TNC expression as continuous variable was associated with (a) shorter MFS in 139 estrogen receptor–positive and lymph node–positive patients who received adjuvant tamoxifen therapy (hazard ratio, 1.53; P = 0.001), and (b) no clinical benefit (odds ratio, 0.81; P = 0.035) and shorter progression-free survival (hazard ratio, 1.19; P = 0.002) in 240 patients in whom recurrence was treated with tamoxifen as first-line monotherapy. These results were also significant in multivariate analyses. Conclusion: FN1, LOX, SPARC, and TIMP3 expression levels are associated with the prognosis of patients with breast cancers, whereas TNC is associated with resistance to tamoxifen therapy. Further validation and functional studies are necessary to determine the use of these ECM genes in decisions regarding treatment and whether they can serve as targets for therapy.

Background: Patients with Estrogen Receptor alpha-positive (ER+) Inflammatory Breast Cancer (IBC) are less responsive to endocrine therapy compared with ER+ non-IBC (nIBC) patients. The study of ER+ IBC samples might reveal biomarkers for endocrine resistant breast cancer. Materials & methods: Gene expression profiles of ER+ samples from 201 patients were explored for genes that discriminated between IBC and nIBC. Classifier genes were applied onto clinically annotated expression data from 947 patients with ER+ breast cancer and validated with RT-qPCR for 231 patients treated with first-line tamoxifen. Relationships with metastasis-free survival (MFS) and progression-free survival (PFS) following adjuvant and first-line endocrine treatment, respectively, were investigated using Cox regression analysis. Results: A metagene of six genes including the genes encoding for 4-aminobutyrate aminotransferase (ABAT) and Stanniocalcin-2 (STC2) were identified to distinguish 22 ER+ IBC from 43 ER+ nIBC patients and remained discriminatory in an independent series of 136 patients. The metagene and two genes were not prognostic in 517 (neo)adjuvant untreated lymph node-negative ER+ nIBC breast cancer patients. Only ABAT was related to outcome in 250 patients treated with adjuvant tamoxifen. Three independent series of in total 411 patients with advanced disease showed increased metagene scores and decreased expression of ABAT and STC2 to be correlated with poor first-line endocrine therapy outcome. The biomarkers remained predictive for first-line tamoxifen treatment outcome in multivariate analysis including traditional factors or published signatures. In an exploratory analysis, ABAT and STC2 protein expression levels had no relation with PFS after first-line tamoxifen. Conclusions: This study utilized ER+ IBC to identify a metagene including ABAT and STC2 as predictive biomarkers for endocrine therapy resistance. (C) 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Cervical cancer (CC) is the fourth most common cause of cancer deaths in women worldwide, for which prognostic and predictive biomarkers are largely lacking. RAIDs is a EU-funded project on cervical cancer that spans seven European countries. The main objective of the RAIDs project is to use this tumor type, which is easily accessible for repeated biopsies, to learn how to stratify patients into targeted therapies. The project includes: 1) a cognitive cohort study (BioRAIDs), one of the first prospective trials intended to define patient stratification for targeted therapies, 2) a targeted clinical trial using an HPV directed vaccine and 3) preclinical studies aiming at assessing new treatment strategies. Molecular analysis on quality controlled tumor and sera samples from 400 patients include Next Generation Sequencing at SeqOmics (Hungary), PIK3CA mutations detection in circulating tumor DNA at Erasmus MC (Netherlands), Reverse Phase Protein array and HPV insertion sites analyses at Institut Curie (France) and immune-microenvironment analyses at CGOA (Netherlands). In addition, 20 CC cell lines have been profiled pharmacologically using a panel of drugs which potentially synergize with “standard treatment”. The present poster will mainly focus on the Reverse Phase Protein Array (RPPA) results of BioRAIDs and how these relate to the genomic profiling and to patient outcome. More than 150 cryopreserved baseline (before treatment) samples and 23 CC cell lines have been screened by RPPA. From these, whole exome sequencing is available for 92 patient samples. Stratification of patients based on proteomics and genomics has evidenced different subgroups (clusters) of patients displaying specific molecular characteristics. Genomics data and proteomics data both demonstrate that these clusters differ notably in the pathways of oxidative phosphorylation, glycolysis and DNA repair. Patient response to treatment is assessed by the presence or absence of residual tumor at six months after treatment. The correlation of proteomics and genomics data with these clinical data is ongoing and will identify putative predictive biomarkers. Correlation of protein data with response to drugs treatment in 20 CC cell lines has identified several potential biomarkers, some of which again relate to (glucose) metabolism. In conclusion, we here present new evidence for molecular subgroups of cervical cancer that could benefit from different treatment options. Notably, our data suggest that the metabolism/glycolysis pathways are a major effector in CC and constitute a potential therapeutic target in a subset of patients. This project has received funding from the European Union’s Seventh Program for research, technological development and demonstration under grant agreement No 304810. Citation Format: Leanne de Koning, Bérengère Ouine, Aurélie Cartier, Els M. Berns, Kirsten Ruigrok-Ritstier, Corine Beaufort, Balazs Balint, Attila Kereszt, Gemma Kenter, Sanne Samuels, Ekaterina S. Jordanova, Emmanuelle Jeannot, Heiko von der Leyen, Marina Popovic, Windy Luscap-Rondof, Vonick Sibut, Choumouss Kamoun, Isabel Britto, Claudia Rincon, Philippe Hupé, Maud Kamal, Suzy M. Scholl. Rational molecular assessment and innovative drug selection (RAIDs): Paving the way to personalized medicine in cervical cancer [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 1746. doi:10.1158/1538-7445.AM2017-1746

Epithelial ovarian cancer is a highly heterogeneous disease and remains the most lethal gynaecological malignancy in the Western world. Therapeutic approaches need to account for inter-patient and intra-tumoural heterogeneity and detailed characterization of in vitro models representing the different histological and molecular ovarian cancer subtypes is critical to enable reliable preclinical testing. There are approximately 100 publicly available ovarian cancer cell lines but their cellular and molecular characteristics are largely undescribed. We have characterized 39 ovarian cancer cell lines under uniform conditions for growth characteristics, mRNA/microRNA expression, exon sequencing, drug response for clinically-relevant therapeutics and collated all available information on the original clinical features and site of origin. We tested for statistical associations between the cellular and molecular features of the lines and clinical features. Of the 39 ovarian cancer cell lines, 14 were assigned as high-grade serous, four serous-type, one low-grade serous and 20 non-serous type. Three morphological subtypes: Epithelial (n = 21), Round (n = 7) and Spindle (n = 12) were identified that showed distinct biological and molecular characteristics, including overexpression of cell movement and migration-associated genes in the Spindle subtype. Comparison with the original clinical data showed association of the spindle-like tumours with metastasis, advanced stage, suboptimal debulking and poor prognosis. In addition, the expression profiles of Spindle, Round and Epithelial morphologies clustered with the previously described C1-stromal, C5-mesenchymal and C4 ovarian subtype expression profiles respectively. Comprehensive profiling of 39 ovarian cancer cell lines under controlled, uniform conditions demonstrates clinically relevant cellular and genomic characteristics. This data provides a rational basis for selecting models to develop specific treatment approaches for histological and molecular subtypes of ovarian cancer.

Item does not contain fulltext PIK3CA mutations occur frequently in breast cancer, predominantly in exons 9 and 20. The aim of this retrospective study is to evaluate the PIK3CA mutation status for its relationship with prognosis and first-line endocrine therapy outcome. PIK3CA exon 9 and 20 were evaluated for mutations in 1,352 primary breast cancer specimens by SnaPshot multiplex analyses. The mutation status was studied for their relationship with metastasis-free survival (MFS) in 342 untreated lymph node-negative (LNN) patients and to time to progression (TTP) in estrogen receptor (ER)-positive patients with metastatic disease treated with first-line tamoxifen (N = 447) or aromatase inhibitors (AIs; N = 84). We detected in 423 patients hotspot mutations for PIK3CA (31 %). Mutations in exon 20 were detected in 251 patients (59 %), with H1047L and H1047R mutations in 37 (15 %) and 214 (85 %) cases, respectively. Mutations in PIK3CA exon 9 were discovered in 173 patients (41 %), with E542K and E545K mutations in 57 (32 %) and 104 (60 %) cases as most prevalent ones. Evaluation of the untreated LNN patients for prognosis showed no relationship between MFS and PIK3CA mutations, neither for exon 9 [HR = 1.04 (95 % CI 0.57-1.89), P = 0.90] nor for exon 20 [HR = 0.98 (95 % CI 0.63-1.54); P = 0.94] when compared to wild-type. The PIK3CA mutation status was also not associated with treatment outcome after first-line tamoxifen. On the other hand, patients treated with first-line AIs showed a longer TTP when having a PIK3CA mutation in exon 9 [HR = 0.40 (95 % CI 0.17-0.95); P = 0.038] or exon 20 [HR = 0.50 (95 % CI 0.27-0.91); P = 0.024] compared to wild-types, both significant in uni- and multivariate analysis including traditional predictive factors. All results remained when only HER2-negative patients were evaluated for each cohort. PIK3CA mutations in ER-positive tumors were significantly associated with a favorable outcome after first-line AIs, which needs further confirmation in other datasets. Mutations were not associated with prognosis in untreated LNN patients nor predictive outcome after first-line tamoxifen therapy in advanced disease patients.

To understand the biology of low-risk breast cancer alleles, and to investigate whether these loci also contribute to disease progression that was once established, we examined the association of SNPs tagging the low-risk breast cancer loci in or near FGFR2, LSP1, MAP3K1, H19, TOX3, POU5F1P1, MYC, and 2q35, with clinical, pathological characteristics, prognosis, and mRNA expression of the nearest genes. Tumor DNA samples of 2,480 breast cancer patients were available. Out of this cohort, 1,290 patients with lymph-node negative disease who did not receive adjuvant systemic therapy, the SNP status was associated with metastasis-free survival (MFS). In 1,401 patients, the mRNA expression levels of FGFR2, LSP1, MAP3K1, H19, TOX3, POU5F1P1, and MYC were determined and correlated with SNP genotypes. The SNP rs2981582 in FGFR2 was significantly associated with positive ER and PgR status (P

Worldwide, breast cancer is the most frequently occurring malignancy in women. Early age at full term pregnancy has a protective effect against breast cancer. Evidence coming from a rat breast cancer model suggests a possible role for the pregnancy hormone hCG, a ligand of the LH receptor, as a mediator for this effect. in a previous study, we found that a common polymorphism in the LH receptor associates with tumor progression in premenopausal breast cancer patients, as carriers of the variant receptor showed a shorter disease free survival compared to non-carriers. How hCG and its receptor exert their effects on breast cancer, however, is unclear. One possibility is that these effects take place through LH receptors present in the ovaries, thereby influencing steroid hormone production. Another possibility is that the effects take place through LH receptors present in breast tumor cells itself, as some studies have detected the receptor in both normal and neoplastic breast tissues and in breast cancer cell lines. To investigate whether a direct effect of LH signaling in breast cancer is likely, we measured LH receptor mRNA expression levels in 1551 breast tumors and 42 different human breast cancer cell lines using a qRT-PCR with a wide dynamic range. In addition, associations between LH receptor expression and clinico-pathologic factors were investigated. Assay validation showed that as little as similar to 10 copies per reaction volume of LH receptor cDNA could still be detected by our assay. We show that LH receptors are undetectable in 62% of breast tumor samples and 41 of 42 breast cancer cell lines. For the remaining samples we found expression levels to be very low. Although low, expression of the LH receptor appears to be associated with normal breast cells, favorable tumor characteristics and low tumor percentage. Since expression of the LH receptor in breast cancer cells is very low, it almost excludes the possibility of direct signaling effects. We therefore conclude that signaling effects of the LH receptor on breast cancer most likely take place by an indirect pathway through the ovaries. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Purpose In our microarray analysis we observed that Seven-in-Absentia Homolog 2 (SIAH2) levels were low in estrogen receptor (ER) positive breast tumors of patients resistant to first-line tamoxifen therapy. The aim of this study was to evaluate SIAH2 for its (a) predictive/prognostic value, and (b) functional role in endocrine therapy resistance. Patients and methods SIAH2 expression was measured with quantitative Real-Time-PCR (qRT-PCR) in 1205 primary breast tumor specimens and related to disease outcome. The functional role of SIAH2 was determined in human breast cancer cell lines ZR-75-1, ZR/HERc, and MCF7. Cell lines were treated with estrogen (E2), anti-estrogen ICI164.384 or epidermal growth factor (EGF). Moreover, MCF7 was treated with ICI164.384 after silencing SIAH2 expression. Results SIAH2 was not prognostic in 603 lymph node negative patients who had not received adjuvant systemic therapy. In multivariate analysis of ER-positive tumors of 235 patients with recurrent disease, SIAH2 as continuous variable, significantly predicted first-line tamoxifen treatment failure (OR = 1.48; P = 0.05) and progression-free survival (PFS) (HR = 0.79; P = 0.007). Furthermore, in primary breast cancer patients treated with adjuvant tamoxifen, SIAH2 predicted metastasis-free survival (MFS) (HR = 0.73; P = 0.005). In vitro experiments showed that SIAH2 silencing in MCF7 cells resulted in resistance to ICI164.384-treatment when compared with mock silenced cells (P = 0.008). Interestingly, in ZR cells transfected with EGFR (ZR/HERc), SIAH2 expression was induced by E2 but downregulated by EGF. Conclusion In primary breast tumor specimens as well as in vitro low SIAH2 levels associated with resistance to endocrine therapy. Moreover, SIAH2 expression showed an opposite regulation by E2 and EGF.

Background: Enhancer of Zeste Homolog 2 (EZH2) is a member of the polycomb complex 2 and serves as a histone methyltransferase. Through trimethylation of histone 3 of lysine 27, EZH2 regulates ‘genome wide’ gene transcription silencing. Downregulation of EZH2 is associated with increased expression of the estrogen receptor (ER) and favorable outcome to tamoxifen in metastatic breast cancer. The binding of ER to its target genes is enhanced by Forkhead Box A1 (FOXA1), a pioneer factor that binds to and opens chromatized DNA. The aim of this study is to investigate the potential association between EZH2 and FOXA1 and their relation with treatment outcome in patients with advanced breast cancer. Experimental design: EZH2 and FOXA1 mRNA levels were analysed using available gene expression profile-data of 344 primary breast cancer specimens of lymph-node-negative (LNN) patients and in 109 ER-positive (ER+) tumors of patients with metastatic disease treated with first-line tamoxifen. To functionally analyze EZH2, cell lines were generated with different knockdown-constructs for EZH2, followed by evaluation of mRNA and protein expression levels and chromatin immunoprecipitation (ChIP) experiments combined with qPCR and/or next generation sequencing (NGS; ChIPseq). Results: In the 344 LNN breast tumors, EZH2 was highly expressed in breast tumors of the basal subtype. In contrast, FOXA1 was hardly expressed in this subtype, but highly in the luminal A and B subtypes. FOXA1 was related to a prolonged time to progression (TTP) after tamoxifen (HR = 0.51 [0.35–0.74], P < 0.001) in 59 LNN patients with ER+ primary tumors and advanced disease. This positive association between FOXA1 and TTP could be confirmed in an independent set of 109 ER+ primary tumors of patients with metastatic disease treated with tamoxifen (HR = 0.56 [0.37–0.85] p = 0.006). We obtained 50–70% EZH2 knockdown in the breast cancer cell lines MCF7 (ER+/PR+/FOXA1+), SUM52PE (ER+/PR−/FOXA1+), and MM231 (ER−/PR−/FOXA1−). ChIP followed by qPCR demonstrated higher levels of H3K27 trimethylation at the ER-locus in the parental cell lines compared to the EZH2-knockdowns for MM231 and at the PR-locus for SUM52PE and MM231. ChIPseq evaluation in the EZH2 knockdown of MM231 identified 200 loci with a significant decrease in read numbers for H3K27me3 compared to the parental. Interestingly, amongst these 200 loci the FOXA1-locus was identified. These results suggest FOXA1 as an EZH2-target since its expression was not seen in the parental MM231. Conclusion: High EZH2 and low FOXA1 mRNA levels are associated with poor clinical outcome for tamoxifen therapy of advanced breast cancer. Functional studies indicate that EZH2 might regulate the expression of FOXA1. These results suggest that EZH2 and/or FOXA1 could be used as a predictive marker to identify patients at risk for tamoxifen therapy failure and possibly as a target for therapy. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-04-08.

Epithelial ovarian cancer is the most lethal gynecological malignancy in the Western world. Up to 70% of the patients do not survive the first 5 years after diagnosis, mostly due to acquired chemotherapy resistance. With the goal to stratify a heterogeneous group of patients, recently, a number of studies have identified molecular subtypes within ovarian carcinomas. However, the relation with response to chemotherapeutics is unknown. An extensive model system in which the response to different chemotherapeutics can be related to molecular subtypes is therefore imperative to know how to individualize treatment based on these molecular subtypes. We have an (increasing) collection of so far 40 ovarian cancer cell lines. The large number and extensive characterization of the cell lines makes this a unique model. We have data of the growth and morphological properties, exon-based gene expression (22.000 genes), microRNA expression (400 miRNAs), mutation analysis (PIK3CA, RAF & RAS genes), microsatellite instability status (MSI) and response curves for eight chemotherapeutics used in first- and second-line treatment. Unsupervised absolute clustering using the most variable expressed genes and microRNAs revealed two even clusters of cell lines with significantly different characteristics. The cell lines in the first cluster compared to the second: -show more often a round and less often an epithelial morphology (cell lines with spindle morphology are divided over both clusters), -show more often no Epithelial Cell Adhesion Molecule (EpCAM) and high CD44 membrane expression -are highly proliferative -are more often PI3K/Ras mutated and MSI Interestingly, the cell lines within the first cluster are significantly more sensitive to six of the eight tested chemotherapeutics compared to the second cluster BRB class comparison analysis revealed that 287 genes were differentially expressed between the two clusters (permutation p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4238. doi:1538-7445.AM2012-4238

Background: In breast cancer we have recently discovered that decreased EZH2 expression levels are associated with a favorable outcome to tamoxifen in advanced disease. Furthermore, EZH2 knockdown in the breast cancer cell line MCF7 resulted in estrogen receptor (ER) upregulation and increased sensitivity to anti-estrogens. Interestingly, EZH2 has been identified as target of miRNA-26a and miRNA-101. Objective: The main objective of this study is to investigate miRNA-26a and miRNA-101 for: A) their association with EZH2 and B) their predictive value in breast cancer patients treated with first-line tamoxifen monotherapy. Materials & Methods: Expression levels of miRNA-26a, miRNA-101, EZH2 and references were measured using quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) in 235 ER-positive primary breast cancer specimens from patients with advanced disease. The levels of expression were related to clinicopathologic factors and disease outcome. Computations were performed with STATA and P-values Results: Expression levels of miRNA-26a and miRNA-101 were not correlated with age, tumor grade, tumor size, and nodal status. The miRNA-26a levels were significantly associated with progesterone (PgR) levels, HER2-status and EGFR levels, whereas miRNA-101 levels showed significant relations with PgR expression and menopausal status. The miRNA-26a and miRNA-101 levels showed an inverse relation with EZH2 mRNA levels (Spearman Rank Correlation of −0.21 and −0.15, respectively, P Conclusion & Discussion: The miRNA-26a and miRNA-101 levels have an inverse relation with EZH2, but only miRNA-26a has predictive value in advanced breast cancer. High levels of miRNA-26a and low EZH2 levels are associated with a favorable outcome to tamoxifen therapy. Interestingly, estrogens repress miRNA-26a expression suggesting that ER-positive tumors have less miRNA-26a than ER-negative tumors. This contradicts, however, with the lower EZH2 levels observed in ER-positive compared to ER-negative breast cancers. To clarify this paradox, future (functional) studies will focus on miRNA-26a and EZH2 in different breast cancer subtypes and determine their activated pathways in relation with response to anti-estrogens. Citation Information: Clin Cancer Res 2010;16(7 Suppl):A15

Background: We have previously identified a gene signature for resistance to first-line tamoxifen therapy in advanced breast cancer. One of these genes is Enhancer of Zeste Homolog 1 (EZH1), a member of the EZH family of which EZH2 has been identified as being prognostic. Both genes are involved in transcriptional control and epigenetic memory maintenance and act as polycomb repressors. The aim of this study is to investigate these genes for their predictive value and, if associated with clinical outcome, to evaluate the functional role in treatment response in vitro.Experimental design: Expression levels of EZH1 and EZH2 expression were measured using quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) in primary breast cancer specimens and related to clinicopathologic factors and disease outcome. Functional studies were done in MCF7, a human estrogen sensitive breast cancer cell line. Expression levels of EZH2 were downregulated with siRNAs, and changes in cell numbers were determined treating with (the anti-estrogen) ICI164.384. In addition, alterations in expression levels of putative downstream genes, including the estrogen receptor (ESR1), were measured.Results: When analysed as continuous variable in univariate analysis, only EZH2 was significantly associated with therapy resistance and a shorter Progression Free Survival (PFS) in 278 patients with advanced disease treated with first-line tamoxifen monotherapy. In univariate analysis the tertile with highest EZH2 levels was associated with clinical benefit (OR=0.48, 95%CI: 0.26-0.89; P=0.02) and with PFS (HR=1.80, 95%CI: 1.32-2.46;P Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5131.

Abstract #6127 Background: In prostate and breast cancer high levels of Enhancer of Zeste Homolog 2 (EZH2) are associated with tumor progression. EZH2, a histone H3 methyl transferase, is part of the polycomb complex. Together with histone deacetylases (HDACs) EZH2 regulates “genome wide” gene transcription silencing. The aims of this study are a) to correlate EZH2 expression with endocrine therapy response in patients with recurrent disease treated with first-line tamoxifen monotherapy and b) to determine the role of EZH2 in endocrine therapy response using in vitro cell line models. Material and Methods: EZH2 mRNA levels were measured with quantitative real-time PCR (qRT-PCR) in 297 retrospectively collected hormone receptor positive (HR+) primary breast tumor specimens of patients with recurrent disease who did respond (N=110) or were resistant (N=187) to first-line tamoxifen monotherapy. In vitro, EZH2 and estrogen receptor (ESR1) expression was downregulated with siRNAs in the human breast cancer cell line MCF7 and assessed for their sensitivity to the selective estrogen receptor degrader ICI164.384 after 96hrs treatment (N=3). To establish therapy response in vitro, cell number counts were determined. All p-values are two-sided and significant if P Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 6127.