PURPOSE Although aromatase inhibitor (AI) treatment is effective in estrogen receptor–positive postmenopausal breast cancer, resistance is common and incompletely explained. Genomic instability, as measured by somatic copy number alterations (SCNAs), is important in breast cancer development and prognosis. SCNAs to specific genes may drive intrinsic resistance, or high genomic instability may drive tumor heterogeneity, which allows differential response across tumors and surviving cells to evolve resistance to treatment rapidly. We therefore evaluated the relationship between SCNAs and intrinsic resistance to treatment as measured by a poor antiproliferative response. PATIENTS AND METHODS SCNAs were determined by single nucleotide polymorphism array in baseline and surgery core-cuts from 73 postmenopausal patients randomly assigned to receive 2 weeks of preoperative AI or no AI in the Perioperative Endocrine Therapy—Individualizing Care (POETIC) trial. Fifty-six samples from the AI group included 28 poor responders (PrRs, less than 60% reduction in protein encoded by the MKI67 gene [Ki-67]) and 28 good responders (GdRs, greater than 75% reduction in Ki-67). Exome sequencing was available for 72 pairs of samples. RESULTS Genomic instability correlated with Ki-67 expression at both baseline (P < .001) and surgery (P < .001) and was higher in PrRs (P = .048). The SCNA with the largest difference between GdRs and PrRs was loss of heterozygosity observed at 17p (false discovery rate, 0.08), which includes TP53. Nine of 28 PrRs had loss of wild-type TP53 as a result of mutations and loss of heterozygosity compared with three of 28 GdRs. In PrRs, somatic alterations of TP53 were associated with higher genomic instability, higher baseline Ki-67, and greater resistance to AI treatment compared with wild-type TP53. CONCLUSION We observed that primary tumors with high genomic instability have an intrinsic resistance to AI treatment and do not require additional evolution to develop resistance to estrogen deprivation therapy.

Abstract Background: More than 20% of early-stage patients with estrogen positive (ER+) disease relapse. Higher levels of the proliferation marker Ki67, and lack of reduction of Ki67 in response to AI indicate poorer prognosis. Somatic mutations have been the focus of research in treatment resistance. However, recurrent somatic copy number alterations (SCNAs) are more common and affect more genes in primary breast cancer (BC) than somatic mutations. Previous studies have suggested an increased risk of recurrence for patients with high genomic instability and for patients with loss of heterozygosity (LOH) at the TP53 locus, but it is unknown if these SCNA events impact response to AI treatment. In addition, LOH and mutations at the TP53 locus had a higher risk of recurrence than LOH or mutations at TP53 alone. We hypothesised that genomic instability and SCNAs at particular loci would be increased in early BC patients with high baseline Ki67, and particularly in patients with high Ki67 despite pre-operative AI therapy. Methods: In a substudy of POETIC (UK-wide, phase III, randomised trial with 4483 women testing perioperative AI in postmenopausal women with early BC), SNParray technology was used to determine SCNAs in baseline and surgical tumour core-cuts and blood from 76 patients (59 AI-treated, 17 controls). Proliferation rate was estimated as percentage (%) of cancer cells staining for Ki67 by IHC. Poor AI responders (PR, <60% reduction in Ki67 between baseline and surgery, n=31) and good AI responders (GR, > 75% reduction in Ki67, n=28) were selected from POETIC samples. Mutation data from exome sequencing was available for tumours from 75 of the patients. Results: The fraction of the genome with SCNAs correlated with Ki67 expression in both baseline and surgical samples (baseline Spearman rho=0.5, p < 10-5; surgical Spearman rho=0.44, p < 10-3). In paired baseline vs surgical samples, 24% of samples showed discordance in SCNAs that covered > 10% of the genome. The samples showing the highest discordance were from PRs. The fraction of the genome with LOH was greater in PR (median PR 20%, GR 10%, p = .065), and the best SCNA to predict the fraction of the genome altered in a sample were segments with LOH at Chr17p13.3 (adjusted p < .001, logistic regression). There was a higher percentage of patients with LOH at Chr17p13.3 that contains the TP53 gene in the PR compared to GR group (PR 71%, GR 39%, p = .029), and integration of previously generated mutation data with SCNA showed that 9 out of 31 PRs have mutations and LOH at the TP53 locus compared to 3 out of 28 GRs (p = 0.16). Conclusions: There is discordance between the observed SCNAs in paired samples with high genomic instability and multiple biopsies may be needed to confidently assess all SCNAs. However, LOH at Chr17p13.3 is a biomarker for genomic instability and frequency of LOH is significantly greater in patients that show a poor response to AI treatment. Finally, high genomic instability is associated with high proliferation rates at baseline and surgery after 2 weeks of AI treatment suggesting de novo resistance in tumours with high instability that may lead to a higher rate of recurrence seen in these patients. Citation Format: Schuster EF, Gellert P, Segal CV, López-Knowles E, Buus R, Morden J, Robertson J, Bliss J, Smith I, Dowsett M, POETIC Trial Management Group and Trialists P. Genomic instability and poor antiproliferative response to aromatase inhibitor treatment: A POETIC study [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD5-05.

Background Several thousand breast cancer patients develop resistance to aromatase inhibitors (AIs) each year in the UK. Rational treatment requires an improved molecular characterisation of resistant disease. Materials and methods The mutational landscape of 198 regions in 16 key breast cancer genes and RNA expression of 209 genes covering key pathways was evaluated in paired biopsies before AI treatment and at progression on AI from 48 patients. Validity of findings was assessed in another five ESR1-mutated tumours progressing on AI. Results Eighty-nine mutations were identified in 41 matched pairs ( PIK3CA in 27%; CDH1 in 20%). ESR1 ( n = 5), ERBB2 ( n = 1) and MAP2K4 ( n = 1) had mutations in the secondary sample only. There was very high heterogeneity in gene expression between AI-resistant tumours with few patterns apparent. However, in the ESR1 -mutated AI-resistant tumours, expression of four classical oestrogen-regulated genes (ERGs) was sevenfold higher than in ESR1 wild-type tumours, a finding confirmed in the second set of ESR1 -mutated tumours. In ESR1 wild-type AI-resistant tumours ERG expression remained suppressed and was uncoupled from the recovery seen in proliferation. Conclusions Major genotypic and phenotypic heterogeneity exists between AI-resistant disease. ESR1 mutations appear to drive oestrogen-regulated processes in resistant tumours.

Background: The adult mammalian heart has little regenerative capacity after myocardial infarction (MI), whereas neonatal mouse heart regenerates without scarring or dysfunction. However, the underlying pathways are poorly defined. We sought to derive insights into the pathways regulating neonatal development of the mouse heart and cardiac regeneration post-MI. Methods and Results: Total RNA-seq of mouse heart through the first 10 days of postnatal life (referred to as P3, P5, P10) revealed a previously unobserved transition in microRNA (miRNA) expression between P3 and P5 associated specifically with altered expression of protein-coding genes on the focal adhesion pathway and cessation of cardiomyocyte cell division. We found profound changes in the coding and noncoding transcriptome after neonatal MI, with evidence of essentially complete healing by P10. Over two-thirds of each of the messenger RNAs, long noncoding RNAs, and miRNAs that were differentially expressed in the post-MI heart were differentially expressed during normal postnatal development, suggesting a common regulatory pathway for normal cardiac development and post-MI cardiac regeneration. We selected exemplars of miRNAs implicated in our data set as regulators of cardiomyocyte proliferation. Several of these showed evidence of a functional influence on mouse cardiomyocyte cell division. In addition, a subset of these miRNAs, miR-144-3p, miR-195a-5p, miR-451a, and miR-6240 showed evidence of functional conservation in human cardiomyocytes. Conclusions: The sets of messenger RNAs, miRNAs, and long noncoding RNAs that we report here merit further investigation as gatekeepers of cell division in the postnatal heart and as targets for extension of the period of cardiac regeneration beyond the neonatal period.

Allergic asthma is a chronic inflammatory disease dominated by a CD4+ T helper 2 (Th2) cell signature. The immune response amplifies in self-enforcing loops, promoting Th2-driven cellular immunity and leaving the host unable to terminate inflammation. Posttranscriptional mechanisms, including microRNAs (miRs), are pivotal in maintaining immune homeostasis. Since an altered expression of various miRs has been associated with T cell-driven diseases, including asthma, we hypothesized that miRs control mechanisms ensuring Th2 stability and maintenance in the lung. We isolated murine CD4+ Th2 cells from allergic inflamed lungs and profiled gene and miR expression. Instead of focusing on the magnitude of miR differential expression, here we addressed the secondary consequences for the set of molecular interactions in the cell, the interactome. We developed the Impact of Differential Expression Across Layers, a network-based algorithm to prioritize disease-relevant miRs based on the central role of their targets in the molecular interactome. This method identified 5 Th2-related miRs (mir27b, mir206, mir106b, mir203, and mir23b) whose antagonization led to a sharp reduction of the Th2 phenotype. Overall, a systems biology tool was developed and validated, highlighting the role of miRs in Th2-driven immune response. This result offers potentially novel approaches for therapeutic interventions.

Myogenesis is a complex process required for skeletal muscle formation during embryonic development and for regeneration and growth of myofibers in adults. Accumulating evidence suggests that long non-coding RNAs (lncRNAs) play key roles in regulating cell fate decision and function in various tissues. However, the role of lncRNAs in the regulation of myogenesis remains poorly understood. In this study, we identified a novel muscle-enriched lncRNA called 'Myolinc (AK142388)', which we functionally characterized in the C2C12 myoblast cell line. Myolinc is predominately localized in the nucleus, and its levels increase upon induction of the differentiation. Knockdown of Myolinc impairs the expression of myogenic regulatory factors and formation of multi-nucleated myotubes in cultured myoblasts. Myolinc also regulates the expression of Filip1 in a cis-manner. Similar to Myolinc, knockdown of Filip1 inhibits myogenic differentiation. Furthermore, Myolinc binds to TAR DNA-binding protein 43 (TDP-43), a DNA/RNA-binding protein that regulates the expression of muscle genes (e.g. Acta1 and MyoD). Knockdown of TDP-43 inhibits myogenic differentiation. We also show that Myolinc-TDP-43 interaction is essential for the binding of TDP-43 to the promoter regions of muscle marker genes. Finally, we show that silencing of Myolinc inhibits skeletal muscle regeneration in adult mice. Altogether, our study identifies a novel lncRNA that controls key regulatory networks of myogenesis.

Resistance to endocrine therapy remains a major clinical problem in breast cancer. Genetic studies highlight the potential role of estrogen receptor-α ( ESR1 ) mutations, which show increased prevalence in the metastatic, endocrine-resistant setting. No naturally occurring ESR1 mutations have been reported in in vitro models of BC either before or after the acquisition of endocrine resistance making functional consequences difficult to study. We report the first discovery of naturally occurring ESR1 Y537C and ESR1 Y537S mutations in MCF7 and SUM44 ESR1-positive cell lines after acquisition of resistance to long-term-estrogen-deprivation (LTED) and subsequent resistance to fulvestrant (ICIR). Mutations were enriched with time, impacted on ESR1 binding to the genome and altered the ESR1 interactome. The results highlight the importance and functional consequence of these mutations and provide an important resource for studying endocrine resistance.

Background: There is great interest in the mutational landscape of metastatic breast cancer (BC) for personalised medicine. Knowledge about how this landscape differs in recurrent lesions from primary BC as a result of metastatic selection or the impact of treatment will extend our knowledge of mechanisms of endocrine resistance and determine the manner in which diagnostics and treatment are integrated. A small number of limited series have reported an increased presence of ESR1 mutations in metastatic lesions after endocrine treatments including aromatase inhibitors (AIs) which are the most common agents for ER+ postmenopausal BC. Aim: To determine the change in mutational profile of 16 genes affected by driver mutations in patients with metastatic BC at the time of progression after an AI. Methods: We conducted targeted sequencing with a custom AmpliSeq panel in 48 matched pairs of FFPE archival blocks of pre-treatment diagnostic and recurrent lesions. 24 of these were metastatic, 24 local recurrences. The genes were AKT1, BRAF, CDH1, ERBB2, ESR1, GATA3, KIT, KRAS, MAP2K4, MAP3K1, PIC3CA, PIK3R1, PTEN, RUNX, SF3B1 and TP53. Only mutations with high depth (u003e250x) were retained. Germline mutations were identified by 1000 Genomes Project data. C:Gu003eT:A transitions with low frequency ( Results: Median coverage was 782-fold. Good quality data was available on 42 pairs. At least one mutation was found in 34 pairs. Three cases had lost IHC expression of ER in the recurrence. A total of 115 mutations in coding regions or splice sites were identified, the largest number in PIK3CA (in 20 samples), CHD1 (18), MAP3K1 (12), TP53 (12) and PTEN (9). In 47 instances the mutation was private to one or other sample. There was no difference in the number of mutations between the presentation and recurrent lesions. Six ESR1 mutations were found, all private to 5 recurrent lesions (all IHC ER+). Four of these mutations have been previously reported (2x E380Q, 2x D538G), one was novel (D484G) and also in the ligand-binding domain. One had an additional previously reported mutation (H524L). Four of these lesions were metastatic but one in a local recurrence suggesting that the metastatic process is more likely, but not essential in selecting the emergence of ESR1 mutations. HER2 mutations were identified in 3 patients; in two cases private in the presentation tumour while in the other case the mutation in the recurrent lesion (L755S) differed from that in the primary. L755S has been reported to be sensitive to neratinib in experimental systems. CDH1 (e-cadherin) mutations were numerically higher than reported in other series. The mutations were across the gene and private in 4 pretreatment and 2 recurrent lesions (6 had identical mutations). They were not associated with lobular phenotype. Conclusions: These data confirm that recurrences after AI but not primary ER+ tumours often contain ESR1 mutations that could influence clinical decision making. The high number of CDH1 mutations at diagnosis may be at least in part because of the selection of recurrent ER+ cases. The data stress the individuality of mutational profiles in recurrent BC and the need for individual interpretation of the data. Citation Format: Gellert P, Ribas R, Pancholi S, Lopez-Knowles E, Yeo B, Garcia-Murillas I, Pearson A, Smith I, Turner N, Dowsett M, Martin L-A. Occurrence of natural ESR1 mutations during acquisition of endocrine resistance in breast cancers and widely used ER+ cell lines. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr S6-02.

Abstract Background: There is great interest in the mutational landscape of metastatic breast cancer (BC) for personalised medicine. Knowledge about how this landscape differs in recurrent lesions from primary BC as a result of metastatic selection or the impact of treatment will extend our knowledge of mechanisms of endocrine resistance and determine the manner in which diagnostics and treatment are integrated. A small number of limited series have reported an increased presence of ESR1 mutations in metastatic lesions after endocrine treatments including aromatase inhibitors (AIs) which are the most common agents for ER+ postmenopausal BC. Aim: To determine the change in mutational profile of 16 genes affected by driver mutations in patients with metastatic BC at the time of progression after an AI. Methods: We conducted targeted sequencing with a custom AmpliSeq panel in 48 matched pairs of FFPE archival blocks of pre-treatment diagnostic and recurrent lesions. 24 of these were metastatic, 24 local recurrences. The genes were AKT1, BRAF, CDH1, ERBB2, ESR1, GATA3, KIT, KRAS, MAP2K4, MAP3K1, PIC3CA, PIK3R1, PTEN, RUNX, SF3B1 and TP53. Only mutations with high depth (>250x) were retained. Germline mutations were identified by 1000 Genomes Project data. C:G>T:A transitions with low frequency (<10%) were removed as artefacts from formalin cross-linking. Results: Median coverage was 782-fold. Good quality data was available on 42 pairs. At least one mutation was found in 34 pairs. Three cases had lost IHC expression of ER in the recurrence. A total of 115 mutations in coding regions or splice sites were identified, the largest number in PIK3CA (in 20 samples), CHD1 (18), MAP3K1 (12), TP53 (12) and PTEN (9). In 47 instances the mutation was private to one or other sample. There was no difference in the number of mutations between the presentation and recurrent lesions. Six ESR1 mutations were found, all private to 5 recurrent lesions (all IHC ER+). Four of these mutations have been previously reported (2x E380Q, 2x D538G), one was novel (D484G) and also in the ligand-binding domain. One had an additional previously reported mutation (H524L). Four of these lesions were metastatic but one in a local recurrence suggesting that the metastatic process is more likely, but not essential in selecting the emergence of ESR1 mutations. HER2 mutations were identified in 3 patients; in two cases private in the presentation tumour while in the other case the mutation in the recurrent lesion (L755S) differed from that in the primary. L755S has been reported to be sensitive to neratinib in experimental systems. CDH1 (e-cadherin) mutations were numerically higher than reported in other series. The mutations were across the gene and private in 4 pretreatment and 2 recurrent lesions (6 had identical mutations). They were not associated with lobular phenotype. Conclusions: These data confirm that recurrences after AI but not primary ER+ tumours often contain ESR1 mutations that could influence clinical decision making. The high number of CDH1 mutations at diagnosis may be at least in part because of the selection of recurrent ER+ cases. The data stress the individuality of mutational profiles in recurrent BC and the need for individual interpretation of the data. Citation Format: Gellert P, Ribas R, Pancholi S, Lopez-Knowles E, Yeo B, Garcia-Murillas I, Pearson A, Smith I, Turner N, Dowsett M, Martin L-A. Occurrence of natural ESR1 mutations during acquisition of endocrine resistance in breast cancers and widely used ER+ cell lines. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr S6-02.

Pre-surgical studies allow study of the relationship between mutations and response of oestrogen receptor-positive (ER+) breast cancer to aromatase inhibitors (AIs) but have been limited to small biopsies. Here in phase I of this study, we perform exome sequencing on baseline, surgical core-cuts and blood from 60 patients (40 AI treated, 20 controls). In poor responders (based on Ki67 change), we find significantly more somatic mutations than good responders. Subclones exclusive to baseline or surgical cores occur in ∼30% of tumours. In phase II, we combine targeted sequencing on another 28 treated patients with phase I. We find six genes frequently mutated: PIK3CA, TP53, CDH1, MLL3, ABCA13 and FLG with 71% concordance between paired cores. TP53 mutations are associated with poor response. We conclude that multiple biopsies are essential for confident mutational profiling of ER+ breast cancer and TP53 mutations are associated with resistance to oestrogen deprivation therapy.

Aims 1. To determine the variability of mutational profiles and sub-clonality in core-cut biopsies from ER+ BC and the impact of 2-weeks’ AI therapy on these. 2. To identify mutations or patterns of mutations associated with poor anti-proliferative response to AI treatment. Background DNA alterations may lead to de novo and acquired resistance to medical therapies including AIs. Assessing this requires single time-point or sequential sampling usually with core-cuts but there is little information on their ability to represent mutational profiles or sub-clonal structure. We studied this in paired biopsies from ER+ BC primaries in 60 selected postmenopausal patients from the Peri-Operative Endocrine Therapy for Individualising Care (POETIC) trial (CRUK/07/015) before and after 2-weeks’ non-steroidal AI or no AI (randomised 2:1). Methods DNA was extracted from RNAlater-preserved diagnostic and surgical 14-gauge core-cut samples and peripheral blood from 20 no AI (Control) and 40 AI-treated patients (15 poor and 25 good Ki67-responders [PR and GR, respectively]). Patients with low ER+ BC or unsuppressed estradiol on treatment were not considered. Exome sequencing (Illumina HiSeq 2000) achieved u003e60% coverage across the exome at 15x depth. Variants were validated by re-sequencing (median u003e100x) together with 79 genes of interest curated from COSMIC and selected publications. Statistically significant genes (SMGs) were determined using MuSiC. Sub-clonality was analysed by SciClone. Results Good quality exomes were obtained on 102 samples including 44 pairs (control n=14; PR n=10; GR n=20). There were 5684 mutations (including 3616 missense and 1322 silent) affecting 3261 genes. SMGs in this series were PIK3CA (35.3%), TP53 (27.5%), CDH1 (13.8%), HEATR7B2 (8.8%), GATA3 (5.9%), CENPF (5.9%), MAP3K1 (5.9%), MAP2K4 (4.9%), HTR1A (2.9%) and C22orf23 (1%). PR had more mutations than GR (median 65 vs 36, p=0.04). More PR than GR were HER2+ (5/14 vs 1/24, p=0.019) and/or TP53-mutated (5/10 vs 3/20, p=0.08) but similar proportions were PIK3CA-mutated. The correlation of diagnostic vs surgical variant allele frequencies was strong for the control (r=0.75) and treated (r=0.89) groups (for treated GR r=0.83; for treated PR r=0.65). In the treated group there were fewer mutations at surgery vs diagnosis (p Conclusion This is the largest reported study of exome reproducibility in ER+ BC for mutation profiles based on core-cut biopsy. Multiple sub-clones are identifiable in ER+ primary BC. In c.20% tumours, a single core-cut does not allow inference of all sub-clonal populations, probably due to spatial heterogeneity. TP53 mutations but not PIK3CA mutations are associated with PR. Large numbers of BC will be needed to identify any associations of lower frequency mutations with resistance. A trend to fewer mutations after just 2 weeks AI needs confirmation. Citation Format: Pascal Gellert, Corrinne V Segal, Qiong Gao, Tiandao Li, Christopher A Miller, Elaine Mardis, Lesley-Ann Martin, Christopher Holcombe, Anthony Skene, Judith Bliss, John Robertson, Ian Smith, Mitch Dowsett, POETIC Trial Management Group and Trialists. Exome sequencing of post-menopausal ER+ breast cancer (BC) treated pre-surgically with aromatase inhibitors (AIs) in the POETIC trial (CRUK/07/015) [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 S1-04.

Abstract Aims 1. To determine the variability of mutational profiles and sub-clonality in core-cut biopsies from ER+ BC and the impact of 2-weeks’ AI therapy on these. 2. To identify mutations or patterns of mutations associated with poor anti-proliferative response to AI treatment. Background DNA alterations may lead to de novo and acquired resistance to medical therapies including AIs. Assessing this requires single time-point or sequential sampling usually with core-cuts but there is little information on their ability to represent mutational profiles or sub-clonal structure. We studied this in paired biopsies from ER+ BC primaries in 60 selected postmenopausal patients from the Peri-Operative Endocrine Therapy for Individualising Care (POETIC) trial (CRUK/07/015) before and after 2-weeks’ non-steroidal AI or no AI (randomised 2:1). Methods DNA was extracted from RNAlater-preserved diagnostic and surgical 14-gauge core-cut samples and peripheral blood from 20 no AI (Control) and 40 AI-treated patients (15 poor and 25 good Ki67-responders [PR and GR, respectively]). Patients with low ER+ BC or unsuppressed estradiol on treatment were not considered. Exome sequencing (Illumina HiSeq 2000) achieved >60% coverage across the exome at 15x depth. Variants were validated by re-sequencing (median >100x) together with 79 genes of interest curated from COSMIC and selected publications. Statistically significant genes (SMGs) were determined using MuSiC. Sub-clonality was analysed by SciClone. Results Good quality exomes were obtained on 102 samples including 44 pairs (control n=14; PR n=10; GR n=20). There were 5684 mutations (including 3616 missense and 1322 silent) affecting 3261 genes. SMGs in this series were PIK3CA (35.3%), TP53 (27.5%), CDH1 (13.8%), HEATR7B2 (8.8%), GATA3 (5.9%), CENPF (5.9%), MAP3K1 (5.9%), MAP2K4 (4.9%), HTR1A (2.9%) and C22orf23 (1%). PR had more mutations than GR (median 65 vs 36, p=0.04). More PR than GR were HER2+ (5/14 vs 1/24, p=0.019) and/or TP53-mutated (5/10 vs 3/20, p=0.08) but similar proportions were PIK3CA-mutated. The correlation of diagnostic vs surgical variant allele frequencies was strong for the control (r=0.75) and treated (r=0.89) groups (for treated GR r=0.83; for treated PR r=0.65). In the treated group there were fewer mutations at surgery vs diagnosis (p<0.026). PIK3CA and TP53 mutation status was identical between the paired samples in 41/44 and 40/44 cases; less frequently mutated genes showed lower concordance. SciClone plots to infer sub-clonality were possible in 37 pairs; for 8 pairs (22%) there was clear evidence of at least one sub-clone being present in only one core-cut sample. Conclusion This is the largest reported study of exome reproducibility in ER+ BC for mutation profiles based on core-cut biopsy. Multiple sub-clones are identifiable in ER+ primary BC. In c.20% tumours, a single core-cut does not allow inference of all sub-clonal populations, probably due to spatial heterogeneity. TP53 mutations but not PIK3CA mutations are associated with PR. Large numbers of BC will be needed to identify any associations of lower frequency mutations with resistance. A trend to fewer mutations after just 2 weeks AI needs confirmation. Citation Format: Pascal Gellert, Corrinne V Segal, Qiong Gao, Tiandao Li, Christopher A Miller, Elaine Mardis, Lesley-Ann Martin, Christopher Holcombe, Anthony Skene, Judith Bliss, John Robertson, Ian Smith, Mitch Dowsett, POETIC Trial Management Group and Trialists. Exome sequencing of post-menopausal ER+ breast cancer (BC) treated pre-surgically with aromatase inhibitors (AIs) in the POETIC trial (CRUK/07/015) [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 S1-04.

Epigenetic marks such as cytosine methylation are important determinants of cellular and whole-body phenotypes. However, the extent of, and reasons for inter-individual differences in cytosine methylation, and their association with phenotypic variation are poorly characterised. Here we present the first genome-wide study of cytosine methylation at single-nucleotide resolution in an animal model of human disease. We used whole-genome bisulfite sequencing in the spontaneously hypertensive rat (SHR), a model of cardiovascular disease, and the Brown Norway (BN) control strain, to define the genetic architecture of cytosine methylation in the mammalian heart and to test for association between methylation and pathophysiological phenotypes. Analysis of 10.6 million CpG dinucleotides identified 77,088 CpGs that were differentially methylated between the strains. In F1 hybrids we found 38,152 CpGs showing allele-specific methylation and 145 regions with parent-of-origin effects on methylation. Cis-linkage explained almost 60% of inter-strain variation in methylation at a subset of loci tested for linkage in a panel of recombinant inbred (RI) strains. Methylation analysis in isolated cardiomyocytes showed that in the majority of cases methylation differences in cardiomyocytes and non-cardiomyocytes were strain-dependent, confirming a strong genetic component for cytosine methylation. We observed preferential nucleotide usage associated with increased and decreased methylation that is remarkably conserved across species, suggesting a common mechanism for germline control of inter-individual variation in CpG methylation. In the RI strain panel, we found significant correlation of CpG methylation and levels of serum chromogranin B (CgB), a proposed biomarker of heart failure, which is evidence for a link between germline DNA sequence variation, CpG methylation differences and pathophysiological phenotypes in the SHR strain. Together, these results will stimulate further investigation of the molecular basis of locally regulated variation in CpG methylation and provide a starting point for understanding the relationship between the genetic control of CpG methylation and disease phenotypes.

Introduction: Congenital heart defects (CHD) are the most common birth defects in humans with an incidence of 8 per 1,000 live births. Hypoplastic left heart syndrome (HLHS) is characterized by atresia or stenosis of the aortic and mitral valves, as well as variable hypoplasia of the left ventricle and the ascending aorta. It accounts for < 1% of all CHD but continues to be a leading cause of infant death due to cardiovascular malformation and is associated with significant long-term morbidity. Despite notable advances in diagnosis and management, little is known about the etiology of HLHS, but current models suggest a complex genetic basis.

Background: Bronchial asthma is a heterogeneous chronic inflammatory disease, which is orchestrated by antigen-specific TH cells. By secreting cytokines, these cells shape the augmented immune response, which finally leads to structural changes of chronic inflamed airways. MicroRNA (miRNA), as small non-coding RNAs, are recognized as key regulatory elements in gene expression and have been linked with a number of complex human chronic inflammatory diseases. Yet, their implication in augmented TH cell activity in asthma has not been thoroughly investigated.