Delphine Vincent

Plasma circulating tumor DNA as an alternative to metastatic biopsies for mutational analysis in breast cancer

BACKGROUND Molecular screening programs use next-generation sequencing (NGS) of cancer gene panels to analyze metastatic biopsies. We interrogated whether plasma could be used as an alternative to metastatic biopsies. PATIENTS AND METHODS The Ion AmpliSeq™ Cancer Hotspot Panel v2 (Ion Torrent), covering 2800 COSMIC mutations from 50 cancer genes was used to analyze 69 tumor (primary/metastases) and 31 plasma samples from 17 metastatic breast cancer patients. The targeted coverage for tumor DNA was ×1000 and for plasma cell-free DNA ×25 000. Whole blood normal DNA was used to exclude germline variants. The Illumina technology was used to confirm observed mutations. RESULTS Evaluable NGS results were obtained for 60 tumor and 31 plasma samples from 17 patients. When tumor samples were analyzed, 12 of 17 (71%, 95% confidence interval (CI) 44% to 90%) patients had ≥1 mutation (median 1 mutation per patient, range 0-2 mutations) in either p53, PIK3CA, PTEN, AKT1 or IDH2 gene. When plasma samples were analyzed, 12 of 17 (71%, 95% CI: 44-90%) patients had ≥1 mutation (median 1 mutation per patient, range 0-2 mutations) in either p53, PIK3CA, PTEN, AKT1, IDH2 and SMAD4. All mutations were confirmed. When we focused on tumor and plasma samples collected at the same time-point, we observed that, in four patients, no mutation was identified in either tumor or plasma; in nine patients, the same mutations was identified in tumor and plasma; in two patients, a mutation was identified in tumor but not in plasma; in two patients, a mutation was identified in plasma but not in tumor. Thus, in 13 of 17 (76%, 95% CI 50% to 93%) patients, tumor and plasma provided concordant results whereas in 4 of 17 (24%, 95% CI 7% to 50%) patients, the results were discordant, providing complementary information. CONCLUSION Plasma can be prospectively tested as an alternative to metastatic biopsies in molecular screening programs.

Genomic Characterization of Primary Invasive Lobular Breast Cancer

PURPOSE Invasive lobular breast cancer (ILBC) is the second most common histologic subtype after invasive ductal breast cancer (IDBC). Despite clinical and pathologic differences, ILBC is still treated as IDBC. We aimed to identify genomic alterations in ILBC with potential clinical implications. METHODS From an initial 630 ILBC primary tumors, we interrogated oncogenic substitutions and insertions and deletions of 360 cancer genes and genome-wide copy number aberrations in 413 and 170 ILBC samples, respectively, and correlated those findings with clinicopathologic and outcome features. RESULTS Besides the high mutation frequency of CDH1 in 65% of tumors, alterations in one of the three key genes of the phosphatidylinositol 3-kinase pathway, PIK3CA, PTEN, and AKT1, were present in more than one-half of the cases. HER2 and HER3 were mutated in 5.1% and 3.6% of the tumors, with most of these mutations having a proven role in activating the human epidermal growth factor receptor/ERBB pathway. Mutations in FOXA1 and ESR1 copy number gains were detected in 9% and 25% of the samples. All these alterations were more frequent in ILBC than in IDBC. The histologic diversity of ILBC was associated with specific alterations, such as enrichment for HER2 mutations in the mixed, nonclassic, and ESR1 gains in the solid subtype. Survival analyses revealed that chromosome 1q and 11p gains showed independent prognostic value in ILBC and that HER2 and AKT1 mutations were associated with increased risk of early relapse. CONCLUSION This study demonstrates that we can now begin to individualize the treatment of ILBC, with HER2, HER3, and AKT1 mutations representing high-prevalence therapeutic targets and FOXA1 mutations and ESR1 gains deserving urgent dedicated clinical investigation, especially in the context of endocrine treatment.

Uncovering the genomic heterogeneity of multifocal breast cancer

Multifocal breast cancer (MFBC), defined as multiple synchronous unilateral lesions of invasive breast cancer, is relatively frequent and has been associated with more aggressive features than unifocal cancer. Here, we aimed to investigate the genomic heterogeneity between MFBC lesions sharing similar histopathological parameters. Characterization of different lesions from 36 patients with ductal MFBC involved the identification of non‐silent coding mutations in 360 protein‐coding genes (171 tumour and 36 matched normal samples). We selected only patients with lesions presenting the same grade, ER, and HER2 status. Mutations were classified as ‘oncogenic’ in the case of recurrent substitutions reported in COSMIC or truncating mutations affecting tumour suppressor genes. All mutations identified in a given patient were further interrogated in all samples from that patient through deep resequencing using an orthogonal platform. Whole‐genome rearrangement screen was further conducted in 8/36 patients. Twenty‐four patients (67%) had substitutions/indels shared by all their lesions, of which 11 carried the same mutations in all lesions, and 13 had lesions with both common and private mutations. Three‐quarters of those 24 patients shared oncogenic variants. The remaining 12 patients (33%) did not share any substitution/indels, with inter‐lesion heterogeneity observed for oncogenic mutation(s) in genes such as PIK3CA, TP53, GATA3, and PTEN. Genomically heterogeneous lesions tended to be further apart in the mammary gland than homogeneous lesions. Genome‐wide analyses of a limited number of patients identified a common somatic background in all studied MFBCs, including those with no mutation in common between the lesions. To conclude, as the number of molecular targeted therapies increases and trials driven by genomic screening are ongoing, our findings highlight the presence of genomic inter‐lesion heterogeneity in one‐third, despite similar pathological features. This implies that deeper molecular characterization of all MFBC lesions is warranted for the adequate management of those cancers.

Constitutive phosphorylated STAT3-associated gene signature is predictive for trastuzumab resistance in primary HER2-positive breast cancer

BackgroundThe likelihood of recurrence in patients with breast cancer who have HER2-positive tumors is relatively high, although trastuzumab is a remarkably effective drug in this setting. Signal transducer and activator of transcription 3 protein (STAT3), a transcription factor that is persistently tyrosine-705 phosphorylated (pSTAT3) in response to numerous oncogenic signaling pathways, activates downstream proliferative and anti-apoptotic pathways. We hypothesized that pSTAT3 expression in HER2-positive breast cancer will confer trastuzumab resistance.MethodsWe integrated reverse phase protein array (RPPA) and gene expression data from patients with HER2-positive breast cancer treated with trastuzumab in the adjuvant setting.ResultsWe show that a pSTAT3-associated gene signature (pSTAT3-GS) is able to predict pSTAT3 status in an independent dataset (TCGA; AUC = 0.77, P = 0.02). This suggests that STAT3 induces a characteristic set of gene expression changes in HER2-positive cancers. Tumors characterized as high pSTAT3-GS were associated with trastuzumab resistance (log rank P = 0.049). These results were confirmed using data from the prospective, randomized controlled FinHer study, where the effect was especially prominent in HER2-positive estrogen receptor (ER)-negative tumors (interaction test P = 0.02). Of interest, constitutively activated pSTAT3 tumors were associated with loss of PTEN, elevated IL6, and stromal reactivation.ConclusionsThis study provides compelling evidence for a link between pSTAT3 and trastuzumab resistance in HER2-positive primary breast cancers. Our results suggest that it may be valuable to add agents targeting the STAT3 pathway to trastuzumab for treatment of HER2-positive breast cancer.

Integrative proteomic and gene expression analysis identify potential biomarkers for adjuvant trastuzumab resistance: analysis from the Fin-her phase III randomized trial.

Trastuzumab is a remarkably effective therapy for patients with human epidermal growth factor receptor 2 (HER2) - positive breast cancer (BC). However, not all women with high levels of HER2 benefit from trastuzumab. By integrating mRNA and protein expression data from Reverse-Phase Protein Array Analysis (RPPA) in HER2-positive BC, we developed gene expression metagenes that reflect pathway activation levels. Next we assessed the ability of these metagenes to predict resistance to adjuvant trastuzumab using gene expression data from two independent datasets. 10 metagenes passed external validation (false discovery rate [fdr] < 0.05) and showed biological relevance with their pathway of origin. These metagenes were further screened for their association with trastuzumab resistance. An association with trastuzumab resistance was observed and validated only for the AnnexinA1 metagene (ANXA1). In the randomised phase III Fin-her study, tumours with low levels of the ANXA1 metagene showed a benefit from trastuzumab (multivariate: hazard ratio [HR] for distant recurrence = 0.16[95%CI 0.05–0.5]; p = 0.002; fdr = 0.03), while high expression levels of the ANXA1 metagene were associated with a lack of benefit to trastuzmab (HR = 1.29[95%CI 0.55–3.02]; p = 0.56). The association of ANXA1 with trastuzumab resistance was successfully validated in an independent series of subjects who had received trastuzumab with chemotherapy (Log Rank; p = 0.01). In conclusion, in HER2-positive BC, some proteins are associated with distinct gene expression profiles. Our findings identify the ANXA1metagene as a novel biomarker for trastuzumab resistance.

Abstract PD3-7: Plasma circulating tumor DNA as an alternative to metastatic biopsies for mutational analyses in breast cancer

Background: Molecular screening programs are using next-generation sequencing (NGS) for cancer gene panels in metastatic biopsies. We interrogated whether plasma can be used as an alternative to metastatic biopsies. Patients and methods: The Ion AmpliSeqTM Cancer Hotspot Panel v2 (Ion Torrent), covering approximately 2,800 COSMIC mutations from 50 cancer genes was used to analyze 70 primary and/or metastases and 29 plasma samples from 17 metastatic breast cancer patients. The targeted coverage for tissue DNA was 1000x and for plasma circulating DNA 25000x. Whole blood normal DNA was used to exclude germline variants. The Illumina technology was used for independent validation. Results: Twelve patients had estrogen receptor (ER)+/ human epidermal growth factor receptor 2 (HER2)-, 1 ER+/HER2+, 2 ER-/HER2+ and 2 ER-/HER2- tumors. Evaluable NGS results were obtained for 61 primary/metastases and 29 plasma samples from 17 patients. When primary/metastases were analyzed, 12 of 17 patients had at least 1 mutation (median 1 mutation per patient, range 0-2) in either p53, PIK3CA, PTEN, AKT1 or IDH2 gene. When plasma was analyzed, 11 of 17 patients had at least 1 mutation (median 1 mutation per patient, range 0-2) in either p53, PIK3CA, PTEN, AKT1, IDH2 and SMAD4. All primary/metastases/plasma mutations were independently validated using the illumina technology. When we focused on metastases and plasma samples collected at the same time point, we observed that in 4 patients, no mutation was identified in either metastases or plasma, in 9 patients the same mutations were identified in metastases and plasma, in 2 patients a mutation was identified in metastases but not in plasma and in 2 patients a mutation was identified in plasma but not in metastases (Table 1). Thus, in 13 of 17 (76%) patients, metastases and plasma analysis provided concordant results whereas in 4 of 17 (24%) demonstated discordant results providing complementary information (Table 1). Conclusion: Plasma can be tested as an alternative tissue source in molecular screening programs. Citation Format: Michail Ignatiadis, Francoise Rothe, Jean-Francois Laes, Diether Lambrechts, Dominiek Smeets, Delphine Vincent, Marion Maetens, Debora Fumagalli, Stefan Michiels, Stylianos Drisis, Carine Moerman, Jean-Pol Detiffe, Denis Larsimont, Ahmad Awada, Martine Piccart, Christos Sotiriou. Plasma circulating tumor DNA as an alternative to metastatic biopsies for mutational analyses in 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 PD3-7.

Abstract S2-05: Characterization and clinical relevance of the genomic alterations defining lobular breast cancer

Background: Invasive lobular breast cancer (ILC) is the second most common histological subtype of breast cancer, is mostly estrogen receptor-positive (ER+) and has distinct clinico-pathological features when compared to ER+ invasive ductal carcinoma (IDC). In this study, we aimed to characterize the genomic alterations defining ILC in a large cohort of ILC patients with long-term follow up (FU). Material & methods: In 499 centrally histologically confirmed ILC patients (median FU= 9.8 years) we analyzed mutational data gathered from targeted sequencing of 360 cancer genes at an average coverage of 106X (alignment done with BWA, substitutions and indels –further referred to as mutations- called with Caveman and Pindel). Matched normal DNA was available for 242 patients. Genome-wide copy number (CN) data were available for 178 patients. E-cadherin (CDH1) and beta-catenin (CTNNB1) stains were carried out using the DakoTM antibodies. Invasive disease free survival (IDFS) was considered as primary survival endpoint. Results: A median of 6 [range:0-38] non-silent mutations was identified across the primary tumors of all patients. The most frequently mutated genes (>3%) are listed in Table 1. Of those, CDH1, PIK3CA, TBX3, FOXA1 and the chromatin-related genes MLL2, MLL3, ARID1A and ARID1B were more frequently mutated in our ER+/HER2- ILC (n= 451) compared to the ER+/HER2- IDC (n=266) from The Cancer Genome Atlas, whereas GATA3, TP53 and MAP3K1 were less frequently mutated. Samples with a CDH1 mutation were associated with changes at the protein level: 97.5% displayed a complete loss of the protein, associated with cytoplasmic staining for CTNNB1, compared to only 63% of the CDH1 non-mutated tumors. CDH1 mutated tumors were further characterized by increased mutational frequencies of the ERBB-genes: 15.4% for the CDH1 mutated tumors versus 3% in the CDH1 non-mutated tumors, most of those mutations being described in the literature as activating the pathway. Almost all tumors (97%) with CN data had a heterozygous loss of CDH1. The special alveolar, solid and trabecular lobular histotypes were associated with specific CN alterations and mutations. Tumors with mutated ARID1A or ATM were associated with worse IDFS at the univariate level and ARID1A remained significant in a multivariate analysis including standard parameters (HR =2.07, p=0.003). At the CN level, ATM and ARID1A losses, as well as HER2 and VEGFA gains/amplifications were associated with decreased IDFS, all but ARID1A holding significance at the multivariate level (HR_HER2=2.41, HR_VEGFA= 1.99, HR_ATM= 1.79, all p Conclusion: This is the first and largest study to our knowledge to report genomic alterations present in ILC and their association with survival. This work therefore opens new avenues for a better understanding of the disease and its clinical management. Citation Format: Christine Desmedt, Gunes Gundem, Gabriele Zoppoli, Giancarlo Pruneri, Elia Biganzoli, Marco Fornili, Debora Fumagalli, Francoise Rothe, David Brown, Peter Van Loo, Sylvain Brohee, Delphine Vincent, Naima Keddoumi, Samira Majjaj, Ghizlane Rouas, Thomas Van Brussel, Diether Lambrechts, Otto Metzger, Christine Galant, Francois Bertucci, Martine Piccart, Denis Larsimont, Giuseppe Viale, Peter J Campbell, Christos Sotiriou. Characterization and clinical relevance of the genomic alterations defining lobular 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 S2-05.

Abstract P1-07-08: Effect of sample preservation method and transportation duration on tumor gene expression profiling in breast cancer.

Background: Translational research studies in the context of international clinical trials may incur prolonged transportation time of tumor specimens. The effect of prolonged transportation time and sample preservation method on RNA quality and on reliability of derived gene expression profiling (GEP) is unknown. Methods: Tumor material from surgical specimens of patients with primary breast cancer (BC) was obtained with cold ischemia time (CIT) recorded for each patient. Each sample was divided into 8 aliquots 4 that were placed in RNAlater and 4 that were frozen. To simulate prolonged transportation duration, samples were kept 1 week in a bucket of dry ice replenished daily, with temperature monitoring. The bucket was stored at room temperature. RNA extraction and GEP using Affymetrix HG-U133 Plus2 were performed at baseline and at day 2, 4 and 7. Linear mixed effect models were used to explore effects of transportation time and preservation method on RNA quality (RNA Integrity Number-RIN). Pre-defined single genes (n = 12), gene modules (n = 25) and molecular subtypes (PAM50) were evaluated. P Results: Samples were collected from 13 patients with an average CIT of 37 minutes (range: 20–85). During the simulation period, the mean temperature at which samples were stored was −77.8°C (SD+/−1.6°C). RIN values decreased significantly over time (1-day change −0.08, CI = −0.12 to −0.04, p AURKA, PTEN , CASP3 and WOUND (p ESR1 and ERBB2 was unaffected. Those that were significantly influenced by sample preservation method, adjusted for time, were genes ESR1 , PLAU, VEGF, PIK3CA, PTEN and gene modules GENE21 and GENE70 was (P Conclusions: In this study, frozen samples were significantly less degraded over time compared with samples stored in RNAlater. Preservation method had a stronger influence on genes and gene modules values compared with prolonged transport duration. In international clinical trials we recommend freezing samples to assure more stable gene expression analysis and limit transport duration to less than 3 days. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-07-08.