Decoding the regulatory role and epiclonal dynamics of DNA methylation in 1482 breast tumours

Breast cancer is a clinically and molecularly heterogeneous disease displaying distinct therapeutic responses. Although recent studies have explored the genomic and transcriptomic landscapes of breast cancer, the epigenetic architecture has received less attention. To address this, an optimised Reduced Representation Bisulfite Sequencing protocol was performed on 1482 primary breast tumours (and 237 matched adjacent normal tissues). This constitutes the largest breast cancer methylome yet, and this thesis describes the bioinformatics and statistical analysis of this study. Noticeable epigenetic drift (both gain and loss of homogeneous DNA methylation patterns) was observed in breast tumours when compared to normal tissues, with markedly higher differences in late replicating genomic regions. The extent of epigenetic drift was also found to be highly heterogeneous between the breast tumours and was sharply correlated with the tumour’s mitotic index, indicating that epigenetic drift is largely a consequence of the accumulation of passive cell division related errors. A novel algorithm called DMARC (Directed Methylation Altered Regions in Cancer) was developed that utilised the tumour-specific drift rates to discriminate between methylation alterations attained as a consequence of stochastic cell division errors ($\textit{background}$) and those reflecting a more instructive biological process ($\textit{directed}$). Directed methylation alterations were significantly enriched for gene expression changes in breast cancer, compared to background alterations. Characterising these methylation aberrations with gene expression led to the identification of breast cancer subtype-specific epigenetic genes with consequences on transcription and prognosis. Cancer genes may be deregulated by multiple mechanisms. By integrating with existing copy number and gene expression profiles for these tumours, DNA methylation alterations were revealed as the predominant mechanism correlated with differentially expressed genes in breast cancer. The crucial role of DNA methylation as a mechanism to target the silencing of specific genes within copy number amplifications is also explored which led to the identification of a putative tumour suppressor gene, $\textit{THSZ2}$. Finally, the first genome-wide assessment of epigenomic evolution in breast cancer is conducted. Both, the level of intratumoural heterogeneity, and the extent of epiallelic burden were found to be prognostic, and revealed an extraordinary distinction in the role of epiclonal dynamics in different breast cancer subtypes. Collectively, the results presented in this thesis have shed light on the somatic DNA methylation basis of inter-patient as well as intra-tumour heterogeneity in breast cancer. This complements our genetic knowledge of the disease, and will help move us towards tailoring treatments to the patient's molecular profile.Wellcome Trust Cancer Research U

Computational approach to discriminate human and mouse sequences in patient-derived tumour xenografts.

Background - Patient-Derived Tumour Xenografts (PDTXs) have emerged as the pre-clinical models that best represent clinical tumour diversity and intra-tumour heterogeneity. The molecular characterization of PDTXs using High-Throughput Sequencing (HTS) is essential; however, the presence of mouse stroma is challenging for HTS data analysis. Indeed, the high homology between the two genomes results in a proportion of mouse reads being mapped as human. Results - In this study we generated Whole Exome Sequencing (WES), Reduced Representation Bisulfite Sequencing (RRBS) and RNA sequencing (RNA-seq) data from samples with known mixtures of mouse and human DNA or RNA and from a cohort of human breast cancers and their derived PDTXs. We show that using an In silico Combined human-mouse Reference Genome (ICRG) for alignment discriminates between human and mouse reads with up to 99.9% accuracy and decreases the number of false positive somatic mutations caused by misalignment by >99.9%. We also derived a model to estimate the human DNA content in independent PDTX samples. For RNA-seq and RRBS data analysis, the use of the ICRG allows dissecting computationally the transcriptome and methylome of human tumour cells and mouse stroma. In a direct comparison with previously reported approaches, our method showed similar or higher accuracy while requiring significantly less computing time. Conclusions - The computational pipeline we describe here is a valuable tool for the molecular analysis of PDTXs as well as any other mixture of DNA or RNA species

Influence of deposition temperature of thermal ALD deposited Al2O3 films on silicon surface passivation

The effect of deposition temperature (T-dep) and subsequent annealing time (t(anl)) of atomic layer deposited aluminum oxide (Al2O3) films on silicon surface passivation (in terms of surface recombination velocity, SRV) is investigated. The pristine samples (as-deposited) show presence of positive fixed charges, Q(F). The interface defect density (D-it) decreases with increase in T-dep which further decreases with tanl up to 100s. An effective surface passivation (SRV= 200 degrees C. The present investigation suggests that low thermal budget processing provides the same quality of passivation as realized by high thermal budget process (tanl between 10 to 30 min)

DNA methylation landscapes of 1538 breast cancers reveal a replication-linked clock, epigenomic instability and cis-regulation.

DNA methylation is aberrant in cancer, but the dynamics, regulatory role and clinical implications of such epigenetic changes are still poorly understood. Here, reduced representation bisulfite sequencing (RRBS) profiles of 1538 breast tumors and 244 normal breast tissues from the METABRIC cohort are reported, facilitating detailed analysis of DNA methylation within a rich context of genomic, transcriptional, and clinical data. Tumor methylation from immune and stromal signatures are deconvoluted leading to the discovery of a tumor replication-linked clock with genome-wide methylation loss in non-CpG island sites. Unexpectedly, methylation in most tumor CpG islands follows two replication-independent processes of gain (MG) or loss (ML) that we term epigenomic instability. Epigenomic instability is correlated with tumor grade and stage, TP53 mutations and poorer prognosis. After controlling for these global trans-acting trends, as well as for X-linked dosage compensation effects, cis-specific methylation and expression correlations are uncovered at hundreds of promoters and over a thousand distal elements. Some of these targeted known tumor suppressors and oncogenes. In conclusion, this study demonstrates that global epigenetic instability can erode cancer methylomes and expose them to localized methylation aberrations in-cis resulting in transcriptional changes seen in tumors

A Biobank of Breast Cancer Explants with Preserved Intra-tumor Heterogeneity to Screen Anticancer Compounds.

The inter- and intra-tumor heterogeneity of breast cancer needs to be adequately captured in pre-clinical models. We have created a large collection of breast cancer patient-derived tumor xenografts (PDTXs), in which the morphological and molecular characteristics of the originating tumor are preserved through passaging in the mouse. An integrated platform combining in vivo maintenance of these PDTXs along with short-term cultures of PDTX-derived tumor cells (PDTCs) was optimized. Remarkably, the intra-tumor genomic clonal architecture present in the originating breast cancers was mostly preserved upon serial passaging in xenografts and in short-term cultured PDTCs. We assessed drug responses in PDTCs on a high-throughput platform and validated several ex vivo responses in vivo. The biobank represents a powerful resource for pre-clinical breast cancer pharmacogenomic studies (http://caldaslab.cruk.cam.ac.uk/bcape), including identification of biomarkers of response or resistance.This research was supported with funding from Cancer Research UK and from the European Union to the EUROCAN Network of Excellence (FP7; grant numnumber 260791). M.C. has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Sk1odowska-Curie grant agreement no. 660060 and was supported by the Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy. R.N.B. is supported by the Wellcome Trust PhD Programme in Mathematical Genomics and Medicine. S-J.S. is supported by the Wellcome Trust PhD Programme for Clinicians in Cambridge. A.Bruna, O.M.R., E.M., V.S., and C.C. are members of the EurOPDX Consortium. Weare very grateful for the generosity of all the patients that donated samples for implantation. We are also deeply indebted to all the staff (surgeons, pathologists, oncologists, theatre staff, and other ancillary personnel) at the Cambridge Breast Unit, Cambridge University Hospital NHS Foundation Trust, for facilitating the timely collection of samples. We thank the Cancer Research UK Cambridge Institute Genomics, Bioinformatics, Histopathology, Flow Cytometry, Biological Resource, and Bio-repository Core Facilities for support during the execution of this project.This is the final version of the article. It first appeared from Elsevier at http://dx.doi.org/10.1016/j.cell.2016.08.041

Dynamics of breast-cancer relapse reveal late-recurring ER-positive genomic subgroups.

The rates and routes of lethal systemic spread in breast cancer are poorly understood owing to a lack of molecularly characterized patient cohorts with long-term, detailed follow-up data. Long-term follow-up is especially important for those with oestrogen-receptor (ER)-positive breast cancers, which can recur up to two decades after initial diagnosis1-6. It is therefore essential to identify patients who have a high risk of late relapse7-9. Here we present a statistical framework that models distinct disease stages (locoregional recurrence, distant recurrence, breast-cancer-related death and death from other causes) and competing risks of mortality from breast cancer, while yielding individual risk-of-recurrence predictions. We apply this model to 3,240 patients with breast cancer, including 1,980 for whom molecular data are available, and delineate spatiotemporal patterns of relapse across different categories of molecular information (namely immunohistochemical subtypes; PAM50 subtypes, which are based on gene-expression patterns10,11; and integrative or IntClust subtypes, which are based on patterns of genomic copy-number alterations and gene expression12,13). We identify four late-recurring integrative subtypes, comprising about one quarter (26%) of tumours that are both positive for ER and negative for human epidermal growth factor receptor 2, each with characteristic tumour-driving alterations in genomic copy number and a high risk of recurrence (mean 47-62%) up to 20 years after diagnosis. We also define a subgroup of triple-negative breast cancers in which cancer rarely recurs after five years, and a separate subgroup in which patients remain at risk. Use of the integrative subtypes improves the prediction of late, distant relapse beyond what is possible with clinical covariates (nodal status, tumour size, tumour grade and immunohistochemical subtype). These findings highlight opportunities for improved patient stratification and biomarker-driven clinical trials.Cancer Research UK (CRUK) travel grant (SWAH/047) 282 to visit Prof. Curtis’ Lab. C.R. is supported by award MTM2015-71217-R. Ca.C. is 283 supported by CRUK, ECMC and NIHR. C.C. is supported by the National Institutes 284 of Health through the NIH Director’s Pioneer Award (DP1-CA238296) and the Breast 285 Cancer Research Foundation

Case Reports1. A Late Presentation of Loeys-Dietz Syndrome: Beware of TGFβ Receptor Mutations in Benign Joint Hypermobility

Background: Thoracic aortic aneurysms (TAA) and dissections are not uncommon causes of sudden death in young adults. Loeys-Dietz syndrome (LDS) is a rare, recently described, autosomal dominant, connective tissue disease characterized by aggressive arterial aneurysms, resulting from mutations in the transforming growth factor beta (TGFβ) receptor genes TGFBR1 and TGFBR2. Mean age at death is 26.1 years, most often due to aortic dissection. We report an unusually late presentation of LDS, diagnosed following elective surgery in a female with a long history of joint hypermobility. Methods: A 51-year-old Caucasian lady complained of chest pain and headache following a dural leak from spinal anaesthesia for an elective ankle arthroscopy. CT scan and echocardiography demonstrated a dilated aortic root and significant aortic regurgitation. MRA demonstrated aortic tortuosity, an infrarenal aortic aneurysm and aneurysms in the left renal and right internal mammary arteries. She underwent aortic root repair and aortic valve replacement. She had a background of long-standing joint pains secondary to hypermobility, easy bruising, unusual fracture susceptibility and mild bronchiectasis. She had one healthy child age 32, after which she suffered a uterine prolapse. Examination revealed mild Marfanoid features. Uvula, skin and ophthalmological examination was normal. Results: Fibrillin-1 testing for Marfan syndrome (MFS) was negative. Detection of a c.1270G > C (p.Gly424Arg) TGFBR2 mutation confirmed the diagnosis of LDS. Losartan was started for vascular protection. Conclusions: LDS is a severe inherited vasculopathy that usually presents in childhood. It is characterized by aortic root dilatation and ascending aneurysms. There is a higher risk of aortic dissection compared with MFS. Clinical features overlap with MFS and Ehlers Danlos syndrome Type IV, but differentiating dysmorphogenic features include ocular hypertelorism, bifid uvula and cleft palate. Echocardiography and MRA or CT scanning from head to pelvis is recommended to establish the extent of vascular involvement. Management involves early surgical intervention, including early valve-sparing aortic root replacement, genetic counselling and close monitoring in pregnancy. Despite being caused by loss of function mutations in either TGFβ receptor, paradoxical activation of TGFβ signalling is seen, suggesting that TGFβ antagonism may confer disease modifying effects similar to those observed in MFS. TGFβ antagonism can be achieved with angiotensin antagonists, such as Losartan, which is able to delay aortic aneurysm development in preclinical models and in patients with MFS. Our case emphasizes the importance of timely recognition of vasculopathy syndromes in patients with hypermobility and the need for early surgical intervention. It also highlights their heterogeneity and the potential for late presentation. Disclosures: The authors have declared no conflicts of interes

Effect of low thermal budget annealing on surface passivation of silicon by ALD based aluminum oxide films

Thermal ALD deposited Al2O3 films on silicon show a marked difference in surface passivation quality as a function of annealing time (using a rapid thermal process). An effective and quality passivation is realized in short anneal duration (similar to 100 s) in nitrogen ambient which is reflected in the low surface recombination velocity (SRV <10 cm s(-1)). The deduced values are close to the best reported SRV obtained by the high thermal budget process (with annealing time between 10-30 min), conventionally used for improved surface passivation. Both as-deposited and low thermal budget annealed films show the presence of positive fixed charges and this is never been reported in the literature before. The role of field and chemical passivation is investigated in terms of fixed charge and interface defect densities. Further, the importance of the annealing step sequence in the MIS structure fabrication protocol is also investigated from the view point of its effect on the nature of fixed charges

Treatment of hepatitis C virus infection in patients of northern India

Background and Aim: The purpose of the present study was to evaluate the therapeutic response of north Indian patients with chronic hepatitis C (CH-C) to two different treatment regimens of interferon and ribavirin. Methods: Consecutive patients with a diagnosis of CH-C attending the Liver Clinic at the All India Institute of Medical Sciences, New Delhi between April 1999 and April 2002 were included in the study. A competitive reverse transcription-polymerase chain reaction (RT-PCR) method developed in the authors' laboratory was used for quantification of hepatitis C virus (HCV)-RNA. Genotyping of HCV was also determined. The clinical, biochemical, virological and histological parameters were used to assess the therapeutic response among a clinical cohort of patients with chronic hepatitis C. They were treated with two different protocols (interferon [IFN]-&#945; -2b, 3 million units daily and ribavirin 10.6 mg/kg daily in two divided doses for 6 months or IFN-&#945;-2b, 3 million units thrice weekly and ribavirin 10.6 mg/kg daily for 6 months). Results: Sixty-five patients with CH-C were included in the study. Blood transfusion (n = 28, 43%) and community-acquired (n = 23, 35%) HCV infections were the commonest. The mean HCV load was high (24.14 &#177; 12.5 × 108 copies/mL). Genotype 2 and 3 were prevalent in 80% (41/51) of the patients. Forty-five patients received 3 million units of IFN thrice weekly and 20 received the same dose daily. All received the same dose of ribavirin. A sustained virological response (SVR) of 95% (19/20) was achieved among patients receiving daily IFN, whereas 64.4% (29/45) of those who received IFN thrice weekly had SVR. The virological relapse was significantly lower among patients who received daily IFN than in those treated with thrice weekly IFN (n = 1/20, 5% vs 10/39, 25.6%; P = 0.015). The proportion of patients receiving daily IFN among those achieving SVR (19/48, 40%) was significantly higher than the proportion of patients receiving similar therapy among patients without SVR (1/17, 6%; P = 0.02). Conclusions: Transfusion and community-acquired HCV infection were the major causes of CH-C. Genotype 2 and 3 HCV were most prevalent among these patients. Despite high viral load, these patients responded well to a combination of daily IFN-&#945; -2b and ribavirin

Silicon surface passivation using thin HfO2 films by atomic layer deposition

Hafnium oxide (HfO2) is a potential material for equivalent oxide thickness (EDT) scaling in microelectronics; however, its surface passivation properties particularly on silicon are not well explored. This paper reports investigation on passivation properties of thermally deposited thin HfO2 films by atomic layer deposition system (ALD) on silicon surface. As-deposited pristine film (similar to 8 nm) shows better passivation with <100 cm/s surface recombination velocity (SRV) vis-a-vis thicker films. Further improvement in passivation quality is achieved with annealing at 400 degrees C for 10 min where the SRV reduces to similar to 20 cm/s. Conductance measurements show that the interface defect density (D-it) increases with film thickness whereas its value decreases after annealing. XRR data corroborate with the observations made by FTIR and SRV data