ESR1 amplification is rare in breast cancer and is associated with high grade and high proliferation: a multiplex ligation-dependent probe amplification study.
ABSTRACT: BACKGROUND: Expression of estrogen receptor alpha (ER?) is predictive for endocrine therapy response and an important prognostic factor in breast cancer. Overexpression of ER? can be caused by estrogen receptor 1 (ESR1) gene amplification and was originally reported to be a frequent event associated with a significantly longer survival for ER-positive women treated with adjuvant tamoxifen monotherapy, which was however questioned by subsequent studies. METHODS: This study aimed to reanalyze the frequency of ESR1 amplification by multiplex ligation-dependent probe amplification (MLPA) and fluorescence in situ hybridisation (FISH), and to assess clinicopathologic correlations. MLPA was performed in a group of 135 breast cancer patients, and gains/amplifications were subjected to FISH. RESULTS: True ESR1 amplification by MLPA was rare (2%) and only 6% more patients showed a modest gain of ESR1. All MLPA-detected ESR1 amplifications and nearly all ESR1 gains were also FISH amplified and gained, but not all FISH amplifications/gains were MLPA amplified/gained, leading to an overall concordance of only 60% between both techniques. All 3 MLPA and FISH ESR1 amplified cases had high ER? expression, but there was no obvious correlation between ESR1 gain and ER status by IHC. ESR1 gains/amplifications were not associated with HER2 gain/amplification, but seemed to be associated with older age. Surprisingly, ESR1 gain/amplification was not associated with low grade as reported previously, but correlated with high grade and high proliferation. Furthermore, ESR1 gain/amplification by MLPA was not associated with nodal status or tumor size (pT status). CONCLUSIONS: ESR1 amplification as detected by MLPA is rare in breast cancer, and seems to be associated with high ER? expression, high age, high grade and high proliferation. This study confirms previous studies that showed differences in the ESR1 amplification frequencies detected by different techniques.
Project description:Prevalence of ESR1 amplification in breast cancer is highly disputed and discrepancies have been related to different technical protocols and different scoring approaches. In addition, pre-mRNA artifacts have been proposed to influence outcome of ESR1 FISH analysis. We analyzed ESR1 gene copy number status combining an improved RNase FISH protocol with multiplex ligation-dependent probe amplification (MLPA) after laser microdissection. FISH showed a high prevalence of ESR1 gains and amplifications despite RNase treatment but MLPA did not confirm ESR1 copy number increases detected by FISH in more than half of cases. We suggest that the combination of the ESR1-specific intra-tumor heterogeneity and low-level copy number increase accounts for these discrepancies.
Project description:DNA sequencing has identified a limited number of driver mutations in metastatic breast cancer beyond single base-pair mutations in the estrogen receptor (ESR1). However, our previous studies and others have observed that structural variants, such as ESR1 fusions, may also play a role. Therefore, we expanded upon these observations by performing a comprehensive and highly sensitive characterization of copy-number (CN) alterations in a large clinical cohort of metastatic specimens. NanoString DNA hybridization was utilized to measure CN gains, amplifications, and deletions of 67 genes in 108 breast cancer metastases, and in 26 cases, the patient-matched primary tumor. For ESR1, a copyshift algorithm was applied to identify CN imbalances at exon-specific resolution and queried large data sets (>15,000 tumors) that had previously undergone next-generation sequencing (NGS). Interestingly, a subset of ER+ tumors showed increased ESR1 CN (11/82, 13%); three had CN amplifications (4%) and eight had gains (10%). Increased ESR1 CN was enriched in metastatic specimens versus primary tumors, and this was orthogonally confirmed in a large NGS data set. ESR1-amplified tumors showed a site-specific enrichment for bone metastases and worse outcomes than nonamplified tumors. No ESR1 CN amplifications and only one gain was identified in ER- tumors. ESR1 copyshift was present in 5 of the 11 ESR1-amplified tumors. Other frequent amplifications included ERBB2, GRB7, and cell-cycle pathway members CCND1 and CDK4/6, which showed mutually exclusivity with deletions of CDKN2A, CDKN2B, and CDKN1B. IMPLICATIONS: Copy-number alterations of ESR1 and key CDK pathway genes are frequent in metastatic breast cancers, and their clinical relevance should be tested further.
Project description:The Estrogen Receptor (ER) is an established predictive marker for the selection of adjuvant endocrine treatment in early breast cancer. During the 1990s Immunohistochemistry (IHC) replaced cytosol based assays for determination of ER status. This study examined the association between ER protein level determined by two different methods and ESR1 gene copy number. From 289 primary high-risk breast cancer patients, randomized in the Danish Breast Cancer Cooperative Group (DBCG) 77C trial, results from cytosolic ER levels were available from ligand binding assays. Archival tumor tissue was retrieved from 257 patients. ESR1/CEN-6 ratio was analyzed successfully by Fluorescence In Situ Hybridization (FISH) in 220 (86%) patients. ESR1 amplification (ESR1/CEN-6 ≥ 2.00) was observed in 23% of the patients and ESR1 deletion (ESR1/CEN-6 < 0.80) was observed in 32%. Further, we identified ESR1 gain (ratio ESR1/CEN-6 from 1.30 to 1.99) in 19% of the patients. A positive correlation of ESR1 FISH with both ER-cytosol and ER IHC was found (p < 0.0001). Amplification and gain of the ESR1 gene are associated with higher ER protein content measured by ligand binding assay and a more intense nuclear staining by IHC compared to tumors with normal ESR1 gene status. Major variations in ER measured by ligand binding assay and IHC are observed within all ESR1 copy number subgroups and other mechanisms than gene copy number seem to contribute to the ER protein content in the tumors.
Project description:Invasive lobular carcinoma (ILC) is the second most common histological subtype of breast cancer following invasive ductal carcinoma (IDC). To identify potential genetic drivers of ILC progression, we used NanoString nCounter technology to investigate the DNA copy number (CN) in 70 well-curated primary ILC samples. We confirmed prior observations of frequent amplification of CCND1 (33%), and MYC (17%) in ILC, but additionally identified a substantial subset of ILCs with ESR1 and ERBB2 (19%) amplifications. Of interest, tumors with ESR1 CN gains (14%) and amplification (10%) were more likely to recur compared to those with normal CN. Finally, we observed that MDM4 (MDMX) was amplified in 17% of ILC samples. MDM4 knockdown in TP53 wild-type ILC cell lines caused increased apoptosis, decreased proliferation associated with cell cycle arrest, and concomitant activation of TP53 target genes. Similar effects were seen in TP53 mutant cells, indicting a TP53-independent role for MDM4 in ILC. To conclude, amplification of ESR1 and MDM4 are potential genetic drivers of ILC. These amplifications may represent actionable, targetable tumor dependencies, and thus have potential clinical implications and warrant further study.
Project description:BACKGROUND:Estrogen Receptor 1 (ESR1) aberrations may be associated with expression of estrogen receptor (ER) or progesterone receptor (PgR), human epidermal growth factor receptor-2 (HER2) or Ki-67 labeling index and prognosis. PATIENTS AND METHODS:ESR1 was assessed in 1129 (81%) of 1396 postmenopausal Danish women with early breast cancer randomly assigned to receive 5 years of letrozole, tamoxifen or a sequence of these agents in the Breast International Group 1-98 trial and who had ER ? 1% after central review. RESULTS:By FISH, 13.6% of patients had an ESR1-to-Centromere-6 (CEN-6) ratio ? 2 (amplified), and 4.2% had ESR1-to-CEN-6 ratio <0.8 (deleted). Deletion of ESR1 was associated with significantly lower levels of ER (P < 0.0001) and PgR (P = 0.02) and more frequent HER2 amplification. ESR1 deletion or amplification was associated with higher-Ki-67 than ESR1-normal tumors. Overall, there was no evidence of heterogeneity of disease-free survival (DFS) or in treatment effect according to ESR1 status. However, significant differences in DFS were observed for subsets based on a combination of ESR1 and HER2 status (P = 0.02). CONCLUSIONS:ESR1 aberrations were associated with HER2 status, Ki-67 labeling index and ER and PgR levels. When combined with HER2, ESR1 may be prognostic but should not be used for endocrine treatment selection in postmenopausal women with endocrine-responsive early breast cancer.
Project description:Treatments for lung cancer include therapies targeting aberrant oncoproteins, but there remains a high medical need for novel therapies. Our previous studies showed that gene amplification/high-level polysomy of AKT1/2 occurs in more than 10% of lung carcinomas. Here, we describe multiplex ligation-dependent probe amplification analysis (MLPA) as a high-throughput method to evaluate copy number increases (CNIs) of AKT1/2 in lung carcinomas. The performance of MLPA using custom-made probes in formalin-fixed paraffin-embedded tissue was evaluated by comparing it to immunohistochemistry and fluorescence in situ hybridization analysis (FISH). By MLPA, we found 4 out of 30 samples harboring gene "gain" when the conventional cutoff value (> 1.3) was used. Two samples with gene amplification by FISH had MLPA values of 1.85 and 1.75, which were lower than the conventional cutoff for "amplification" (> 2.0). Moreover, samples with CNIs due to polysomy by FISH gave MLPA values between 1.13 and 1.47, so some samples had lower values than 1.3. The reasons appeared to be stromal contamination and the presence of carcinoma cells without CNIs. However, when we changed the cutoff for "gain" to the "average+2xstandard error", we detected CNIs in 10 samples, with only one each of false-positive and false-negative results. The sensitivity was 90% and the specificity was 98%. Consistently, all cases exhibiting CNI by this criteria revealed Akt activation. In conclusion, MLPA implemented with custom-made probes and an optimized cutoff value is a feasible screening method to semi-quantitatively detect oncogene aberrations, and may contribute to the design of individualized, molecularly targeted therapies against lung carcinoma.
Project description:BACKGROUND: Discrepant data have been published on the incidence and prognostic significance of ESR1 gene amplification in early breast cancer. PATIENTS AND METHODS: Formalin-fixed paraffin-embedded tumor blocks were collected from women with early breast cancer participating in two HeCOG adjuvant trials. Messenger RNA was studied by quantitative PCR, ER protein expression was centrally assessed using immunohistochemistry (IHC) and ESR1 gene copy number by dual fluorescent in situ hybridization probes. RESULTS: In a total of 1010 women with resected node-positive early breast adenocarcinoma, the tumoral ESR1/CEP6 gene ratio was suggestive of deletion in 159 (15.7%), gene gain in 551 (54.6%) and amplification in 42 cases (4.2%), with only 30 tumors (3%) harboring five or more ESR1 copies. Gene copy number ratio showed a significant, though weak correlation to mRNA and protein expression (Spearman's Rho <0.23, p?=?0.01). ESR1 clusters were observed in 9.5% (57 gain, 38 amplification) of cases. In contrast to mRNA and protein expression, which were favorable prognosticators, gene copy number changes did not obtain prognostic significance. When ESR1/CEP6 gene ratio was combined with function (as defined by ER protein and mRNA expression) in a molecular classifier, the Gene Functional profile, it was functional status that impacted on prognosis. In univariate analysis, patients with functional tumors (positive ER protein expression and gene ratio normal or gain/amplification) fared better than those with non-functional tumors with ESR1 gain (HR for relapse or death 0.49-0.64, p?=?0.003). Significant interactions were observed between gene gain/amplification and paclitaxel therapy (trend for DFS benefit from paclitaxel only in patients with ESR1 gain/amplification, p?=?0.066) and Gene Functional profile with HER2 amplification (Gene Functional profile prognostic only in HER2-normal cases, p?=?0.029). CONCLUSIONS: ESR1 gene deletion and amplification do not constitute per se prognostic markers, instead they can be classified to distinct prognostic groups according to their protein-mediated functional status.
Project description:BACKGROUND: Oncogene amplification and overexpression occur in tumor cells. Amplification status may provide diagnostic and prognostic information and may lead to new treatment strategies. Chromosomal regions 8p12, 8q24, 11q13, 17q12 and 20q13 are recurrently amplified in breast cancers. METHODS: To assess the frequencies and clinical impact of amplifications, we analyzed 547 invasive breast tumors organized in a tissue microarray (TMA) by fluorescence in situ hybridization (FISH) and calculated correlations with histoclinical features and prognosis. BAC probes were designed for: (i) two 8p12 subregions centered on RAB11FIP1 and FGFR1 loci, respectively; (ii) 11q13 region centered on CCND1; (iii) 12p13 region spanning NOL1; and (iv) three 20q13 subregions centered on MYBL2, ZNF217 and AURKA, respectively. Regions 8q24 and 17q12 were analyzed with MYC and ERBB2 commercial probes, respectively. RESULTS: We observed amplification of 8p12 (amplified at RAB11FIP1 and/or FGFR1) in 22.8%, 8q24 in 6.1%, 11q13 in 19.6%, 12p13 in 4.1%, 17q12 in 9.9%, 20q13Z (amplified at ZNF217 only) in 9.9%, and 20q13Co (co-amplification of two or three 20q13 loci) in 8.5% of cases. The 8q24, 12p13, and 17q12 amplifications were correlated with high grade. The most frequent single amplifications were 8p12 (9.8%), 8q24 (3.3%) and 12p13 (3.3%), 20q13Z and 20q13Co (1.6%) regions. The 17q12 and 11q13 regions were never found amplified alone. The most frequent co-amplification was 8p12/11q13. Amplifications of 8p12 and 17q12 were associated with poor outcome. Amplification of 12p13 was associated with basal molecular subtype. CONCLUSION: Our results establish the frequencies, prognostic impacts and subtype associations of various amplifications and co-amplifications in breast cancers.
Project description:Primitive neuroectodermal tumors of the central nervous system (CNS-PNETs) are a rare group of neoplasms occurring in the CNS that includes supratentorial CNS-PNETs, medulloepitheliomas, and ependymoblastomas. While ependymoblastomas frequently carry chromosome 19q13.41 amplification and show aggressive clinical behavior, the biological mechanisms and molecular alterations contributing to the pathogenesis of supratentorial CNS-PNETs remain poorly understood. Moreover, genetic alterations suitable for molecular risk stratification are undefined to date.In order to identify possible molecular markers, we performed multiplex ligation-dependent probe amplification (MLPA) and molecular inversion probe (MIP) analysis on DNA samples of 25 supratentorial CNS-PNETs (median age, 5.35 years; range, 2.41–17.28 years). Tumors with ependymoblastic rosettes (ependymoblastoma/ETANTR) and LIN28A positivity were excluded.MLPA and MIP analysis revealed large losses of genomic material of chromosomes 3, 4, 5, and 13, while frequent gains affected chromosomes 1, 17, 19, 20, and 22. High copy number gains (amplifications) were found in particular at chromosomes 2p24.3 (MYCN, n = 6 cases) and 4q12 (n = 2 cases). Patients with tumors harboring 2p gain or MYCN amplification showed unfavorable overall survival (P = .003 and P = .001, respectively).These markers were independent of the presence of metastases, which was indeed a clinical factor associated with poor overall survival (P = .01) in this series.In the era of the personalized neuro-oncology, the identification of these molecular prognostic markers associated with patient outcome may represent a significant step towards improved patient stratification and risk-adapted therapeutic strategies for patients suffering from supratentorial CNS-PNETs.
Project description:BACKGROUND: Around 20% of breast cancers (BC) show ERBB2 gene amplification and overexpression of the ERBB2 tyrosine kinase receptor. They are associated with a poor prognosis but can benefit from targeted therapy. A better knowledge of these BCs, genomically and biologically heterogeneous, may help understand their behavior and design new therapeutic strategies. METHODS: We defined the high resolution genome and gene expression profiles of 54 ERBB2-amplified BCs using 244K oligonucleotide array-comparative genomic hybridization and whole-genome DNA microarrays. Expression of ERBB2, phosphorylated ERBB2, EGFR, IGF1R and FOXA1 proteins was assessed by immunohistochemistry to evaluate the functional ERBB2 status and identify co-expressions. RESULTS: First, we identified the ERBB2-C17orf37-GRB7 genomic segment as the minimal common 17q12-q21 amplicon, and CRKRS and IKZF3 as the most frequent centromeric and telomeric amplicon borders, respectively. Second, GISTIC analysis identified 17 other genome regions affected by copy number aberration (CNA) (amplifications, gains, losses). The expression of 37 genes of these regions was deregulated. Third, two types of heterogeneity were observed in ERBB2-amplified BCs. The genomic profiles of estrogen receptor-positive (ER+) and negative (ER-) ERBB2-amplified BCs were different. The WNT/?-catenin signaling pathway was involved in ER- ERBB2-amplified BCs, and PVT1 and TRPS1 were candidate oncogenes associated with ER+ ERBB2-amplified BCs. The size of the ERBB2 amplicon was different in inflammatory (IBC) and non-inflammatory BCs. ERBB2-amplified IBCs were characterized by the downregulated and upregulated mRNA expression of ten and two genes in proportion to CNA, respectively. IHC results showed (i) a linear relationship between ERBB2 gene amplification and its gene and protein expressions with a good correlation between ERBB2 expression and phosphorylation status; (ii) a potential signaling cross-talk between EGFR or IGF1R and ERBB2, which could influence response of ERBB2-positive BCs to inhibitors. FOXA1 was frequently coexpressed with ERBB2 but its expression did not impact on the outcome of patients with ERBB2-amplified tumors. CONCLUSION: We have shown that ER+ and ER- ERBB2-amplified BCs are different, distinguished ERBB2 amplicons in IBC and non-IBC, and identified genomic features that may be useful in the design of alternative therapeutical strategies.