Breast Cancer Biomarkers with Clinical Relevance Identified by Massively-parallel DNA and RNA Sequencing

Women have a 10% lifetime risk of developing breast cancer, and the disease has surpassed lung cancer as the most frequently diagnosed type of cancer in the world. Breast cancer originates in the epithelial cells of the mammary gland and tumor cells have undergone a series of genetic and phenotypic changes that confer tumor promoting properties.Genomic rearrangement is a common phenomenon in cancer, involving breakage and dysfunctional repair of chromosomes. With the aim to characterize such variants and their progression from primary to metastatic disease, we performed whole-genome sequencing of paired primary tumors and metastases (study I) and paired contralateral breast cancers (CBC) (study II). Metastasis rearrangement profiles bore a remarkable resemblance to the respective primary tumors (median 89% shared), indicating that the rearrangements were early events in tumor development, remaining stable throughout progression. Our study on CBC (study II) subsequently allowed us to identify 1 in 10 tumor pairs that likely represented metastatic spread rather than a new primary tumor (76% of rearrangements shared). One of the risk factors for breast cancer is high exposure to estrogens; signaling via estrogen receptor (ER) α is considered the most important driver for the 75% of tumors expressing this marker. Mutations in the gene for ERα are known to be common in endocrine therapy-refractory breast cancer and confer resistance to standard anti-hormonal treatment. In study III, we interrogated RNA-seq data from 3217 primary breast tumors from the SCAN-B initiative and found that 1% of tumors were positive for one of the mutations at surgery. For those patients that received adjuvant endocrine therapy, the mutations were associated to worse overall and relapse-free survival. In study IV, we further explored the SCAN-B dataset to investigate the phenotypic properties and prognosis associated to high expression of the much less well studied ERβ. We discovered that this receptor was not abundantly expressed, with 1/3 of tumors entirely negative. Further, we saw that patients with high levels of ERβ mRNA had slightly improved overall survival and that the expression of ERβ was associated to expression of genes involved in immune cell activation.In summary, we have employed sequencing technology to study breast cancer patient material to identify and assess the validity of genomic and transcriptomic changes that may both be of value as potential biomarkers, and in elucidating biological mechanisms that drive or suppress breast cancer progression

How should we discuss genetic testing with women newly diagnosed with breast cancer? Design and implementation of a randomized controlled trial of two models of delivering education about treatment-focused genetic testing to younger women newly diagnosed with breast cancer

BACKGROUND: Germline BRCA1 and BRCA2 mutation testing offered shortly after a breast cancer diagnosis to inform women's treatment choices - treatment-focused genetic testing 'TFGT' - has entered clinical practice in specialist centers and is likely to be soon commonplace in acute breast cancer management, especially for younger women. Yet the optimal way to deliver information about TFGT to younger women newly diagnosed with breast cancer is not known, particularly for those who were not suspected of having a hereditary breast cancer syndrome prior to their cancer diagnosis. Also, little is known about the behavioral and psychosocial impact or cost effectiveness of educating patients about TFGT. This trial aims to examine the impact and efficiency of two models of educating younger women newly diagnosed with breast cancer about genetic testing in order to provide evidence for a safe and effective future clinical pathway for this service. DESIGN/METHODS: In this non-inferiority randomized controlled trial, 140 women newly diagnosed with breast cancer (aged less than 50 years) are being recruited from nine cancer centers in Australia. Eligible women with either a significant family history of breast and/or ovarian cancer or with other high risk features suggestive of a mutation detection rate of > 10% are invited by their surgeon prior to mastectomy or radiotherapy. After completing the first questionnaire, participants are randomized to receive either: (a) an educational pamphlet about genetic testing (intervention) or (b) a genetic counseling appointment at a family cancer center (standard care). Each participant is offered genetic testing for germline BRCA mutations. Decision-related and psychosocial outcomes are assessed over 12 months and include decisional conflict (primary outcome);uptake of bilateral mastectomy and/or risk-reducing salpingo-oophorectomy; cancer-specific- and general distress; family involvement in decision making; and decision regret. A process-oriented retrospective online survey will examine health professionals' attitudes toward TFGT; a health economic analysis will determine the cost effectiveness of the intervention. DISCUSSION: This trial will provide crucial information about the impact, efficiency and cost effectiveness of an educational pamphlet designed to inform younger women newly diagnosed with breast cancer about genetic testing. Issues regarding implementation of the trial are discussed

Phylogenetic analysis of metastatic progression in breast cancer using somatic mutations and copy number aberrations.

Several studies using genome-wide molecular techniques have reported various degrees of genetic heterogeneity between primary tumours and their distant metastases. However, it has been difficult to discern patterns of dissemination owing to the limited number of patients and available metastases. Here, we use phylogenetic techniques on data generated using whole-exome sequencing and copy number profiling of primary and multiple-matched metastatic tumours from ten autopsied patients to infer the evolutionary history of breast cancer progression. We observed two modes of disease progression. In some patients, all distant metastases cluster on a branch separate from their primary lesion. Clonal frequency analyses of somatic mutations show that the metastases have a monoclonal origin and descend from a common 'metastatic precursor'. Alternatively, multiple metastatic lesions are seeded from different clones present within the primary tumour. We further show that a metastasis can be horizontally cross-seeded. These findings provide insights into breast cancer dissemination

Morbidity in neurofibromatosis 1: Epidemiological perspectives on breast cancer and diabetes

Neurofibromatosis type 1 (NF1) is a dominantly inherited rare disorder caused by pathogenic variants of the NF1 tumor suppressor gene. The syndrome can be diagnosed based on its clinical manifestations, such as café-au-lait macules, skinfold freckling, and benign cutaneous neurofibroma tumors. NF1 affects several organ systems, yet it is best known as a tumor predisposition syndrome. In this thesis, a Finnish cohort of 1,476 individuals with NF1 was used to study the prevalence of NF1 and the risks for breast cancer and diabetes among individuals with NF1. For a more precise assessment of the risk for contralateral breast cancer in NF1, the Finnish NF1 cohort was analyzed together with four other European NF1 cohorts. Moreover, the effect of NF1 on the concentration of circulating free plasma DNA (cfDNA) was assessed in a small-scale clinical study. Breast cancer diagnoses of individuals with NF1 were obtained from the Finnish Cancer Registry. Diagnoses of diabetes were inferred from drug purchases and hospital visits and hospital stays. The characteristics of NF1-related breast cancer were also analyzed. The results demonstrate that the overall prevalence of NF1 may be as high as 1/2,052. NF1 is associated with increased mortality throughout the lifetime, and the age-specific prevalence of NF1 declines in older age groups. Women with NF1 face a marked risk for breast cancer, and the risk for being diagnosed with breast cancer is 7.8% by 50 years of age. The survival after NF1-related breast cancer is worse compared to breast cancer in the general population. Breast cancers diagnosed in individuals with NF1 also exhibit poor prognostic factors, such as hormone receptor negativity. Moreover, women with NF1 and breast cancer have a 16% risk for contralateral breast cancer within 20 years. In contrast, the risk for diabetes and type 2 diabetes, in particular, is decreased among individuals with NF1. The NF1 syndrome, as such, may not significantly alter plasma cfDNA concentration. The results highlight the need for identifying all individuals with NF1 in order to provide them surveillance for NF1-related complications, such as breast cancer. The findings demonstrate the role of the NF1 gene in the pathogenesis of two common diseases, namely breast cancer and diabetes. Sairastavuus tyypin 1 neurofibromatoosissa: epidemiologisia näkökulmia rintasyöpään ja diabetekseen Tyypin 1 neurofibromatoosi (NF1) on vallitsevasti periytyvä harvinaissairaus, joka aiheutuu muutoksista NF1-kasvunrajoitegeenissä. Sairaus voidaan diagnosoida kliinisten oireiden, kuten ihon maitokahviläiskien, taivealueiden kesakoiden ja hyvänlaatuisten neurofibrooma-ihokasvainten perusteella. NF1 vaikuttaa moniin elinjärjestelmiin, mutta parhaiten se tunnetaan kasvainalttiusoireyhtymänä. Tässä tutkimuksessa tarkasteltiin NF1:n yleisyyttä sekä potilaiden alttiutta sairastua rintasyöpään ja diabetekseen. Tutkimuksessa käytettiin 1476 henkilön suomalaista NF1-kohorttia. Vastakkaisen rinnan rintasyövän riskiä tutkittaessa hyödynnettiin tietoja myös neljästä muusta eurooppalaisesta NF1-kohortista. Lisäksi tarkasteltiin NF1:n vaikutusta plasman vapaiden nukleiinihappojen pitoisuuteen. NF1-potilaiden rintasyöpädiagnoosit haettiin Suomen Syöpärekisteristä. Diabetesdiagnoosit pääteltiin lääkeostotiedoista sekä sairaalakäynneistä ja -jaksoista. Lisäksi tarkasteltiin NF1:een liittyvän rintasyövän ominaisuuksia. Tulokset osoittavat, että NF1:n vallitsevuus on jopa 1/2052. NF1 aiheuttaa lisääntynyttä kuolleisuutta kaikissa ikäryhmissä, minkä vuoksi NF1:n vallitsevuus laskee vanhemmissa ikäryhmissä. NF1:een liittyy merkittävä rintasyöpäriski, ja NF1:tä sairastavilla naisilla on 7,8 %:n todennäköisyys sairastua rintasyöpään ennen 50 vuoden ikää. NF1 huonontaa rintasyövän ennustetta, ja NF1-naisten rintasyövät ovat usein hormonireseptorinegatiivisia. Lisäksi rintasyöpään sairastuneilla NF1- naisilla on 16 %:n riski sairastua vastakkaisen rinnan rintasyöpään 20 vuoden kuluessa. Sen sijaan erityisesti tyypin 2 diabeteksen riski on NF1:ssä pienempi kuin vertailuväestössä. NF1-sairaus itsessään ei vaikuta merkittävästi muuttavan plasman vapaiden nukleiinihappojen pitoisuutta. Tulokset korostavat tarvetta tunnistaa kaikki NF1:tä sairastavat henkilöt, jotta he pääsevät taudin edellyttämän seurannan piiriin. Tulosten perusteella NF1-geeni vaikuttaa kahden yleisen sairauden, rintasyövän ja diabeteksen, kehityksee

Mammary gland-specific ablation of focal adhesion kinase reduces the incidence of p53-mediated mammary tumour formation.

BACKGROUND Elevated expression of focal adhesion kinase (FAK) occurs in numerous human cancers including colon-, cervix- and breast cancer. Although several studies have implicated FAK in mammary tumour formation induced by ectopic oncogene expression, evidence supporting a role for FAK in spontaneous mammary tumour development caused by loss of tumour suppressor genes such as p53 is lacking. Alterations in the tumour suppressor gene p53 have been implicated in over 50% of human breast cancers. Given that elevated FAK expression highly correlates with p53 mutation status in human breast cancer, we set out to investigate the importance of FAK in p53-mediated spontaneous mammary tumour development. METHODS To directly assess the role of FAK, we generated mice with conditional inactivation of FAK and p53. We generated female p53(lox/lox)/FAK(+/+)/WapCre, p53(lox/lox)/FAK(flox/+)/WapCre and p53(lox/lox)/FAK(flox/-)/WapCre mice, and mice with WapCre-mediated conditional expression of p53(R270H), the mouse equivalent of human p53(R273H) hot spot mutation, together with conditional deletion of FAK, P53(R270H/+)/FAK(lox/+)/WapCre and p53(R270H/+)/FAK(flox/-)/WapCre mice. All mice were subjected to one pregnancy to induce WapCre-mediated deletion of p53 or expression of p53 R270H, and Fak genes flanked by two loxP sites, and subsequently followed the development of mammary tumours. RESULTS Using this approach, we show that FAK is important for p53-induced mammary tumour development. In addition, mice with the mammary gland-specific conditional expression of p53 point mutation R270H, the mouse equivalent to human R273H, in combination with conditional deletion of Fak showed reduced incidence of p53(R270H)-induced mammary tumours. In both models these effects of FAK were related to reduced proliferation in preneoplastic lesions in the mammary gland ductal structures. CONCLUSIONS Mammary gland-specific ablation of FAK hampers p53-regulated spontaneous mammary tumour formation. Focal adhesion kinase deletion reduced proliferative capacity of p53 null and p53(R270H) mammary epithelial cells but did not lead to increased apoptosis in vivo. Our data identify FAK as an important regulator in mammary epithelial cell proliferation in p53-mediated and p53(R270H)-induced mammary tumour development

Getting the gist of it: An investigation of gist processing and the learning of novel gist categories

Gist extraction rapidly processes global structural regularities to provide access to the general meaning and global categorizations of our visual environment – the gist. Medical experts can also extract gist information from mammograms to categorize them as normal or abnormal. However, the visual properties influencing the gist of medical abnormality are largely unknown. It is also not known how medical experts, or any observer for that matter, learned to recognise the gist of new categories. This thesis investigated the processing and acquisition of the gist of abnormality. Chapter 2 observed no significant differences in performance between 500 ms and unlimited viewing time, suggesting that the gist of abnormality is fully accessible after 500 ms and remains available during further visual processing. Next, chapter 3 demonstrated that certain high-pass filters enhanced gist signals in mammograms at risk of future cancer, without affecting overall performance. These filters could be used to enhance mammograms for gist risk-factor scoring. Chapter 4’s multi-session training showed that perceptual exposure with global feedback is sufficient to induce learning of a new gist categorisation. However, learning was affected by individual differences and was not significantly retained after 7-10 days, suggesting that prolonged perceptual exposure might be needed for consolidation. Chapter 5 observed evidence for the neural signature of gist extraction in medical experts across a network of regions, where neural activity patterns showed clear individual differences. Overall, the findings of this thesis confirm the gist extraction of medical abnormality as a rapid, global process that is sensitive to spatial structural regularities. Additionally, it was shown that a gist category can be learned via global feedback, but this learning is hard to retain and is affected by individual differences. Similarly, individual differences were observed in the neural signature of gist extraction by medical experts

Computational Logic for Biomedicine and Neurosciences

We advocate here the use of computational logic for systems biology, as a \emph{unified and safe} framework well suited for both modeling the dynamic behaviour of biological systems, expressing properties of them, and verifying these properties. The potential candidate logics should have a traditional proof theoretic pedigree (including either induction, or a sequent calculus presentation enjoying cut-elimination and focusing), and should come with certified proof tools. Beyond providing a reliable framework, this allows the correct encodings of our biological systems. % For systems biology in general and biomedicine in particular, we have so far, for the modeling part, three candidate logics: all based on linear logic. The studied properties and their proofs are formalized in a very expressive (non linear) inductive logic: the Calculus of Inductive Constructions (CIC). The examples we have considered so far are relatively simple ones; however, all coming with formal semi-automatic proofs in the Coq system, which implements CIC. In neuroscience, we are directly using CIC and Coq, to model neurons and some simple neuronal circuits and prove some of their dynamic properties. % In biomedicine, the study of multi omic pathway interactions, together with clinical and electronic health record data should help in drug discovery and disease diagnosis. Future work includes using more automatic provers. This should enable us to specify and study more realistic examples, and in the long term to provide a system for disease diagnosis and therapy prognosis

Breast cancer risk associated with changes in mammographic density.

PhD ThesisBreast cancer is the most common cancer in the UK, and mammographic density (‘density’) is one of its strongest known risk factors. At present, most research focuses on static measures of density to determine population effects. The central hypothesis of this thesis is that repeated measures of density are more valuable for personalised breast cancer prevention. This hypothesis was tested through the following research. Study-I investigated within-women associations between body mass index (BMI) and density, to assess whether density (visual/Cumulus/volumetric ‘Stepwedge’) acts as a mediator for breast cancer risk reduction during a premenopausal weight-loss intervention (n=65). Study-II evaluated the benefit of using a woman’s longitudinal history of (BI-RADS) density to improve breast cancer risk estimation (n=132,439). Study-III was a Cochrane systematic review investigating the association between endocrine therapy-induced density reduction and breast cancer risk and mortality. Studies-IV and V (n=575) evaluated visually-assessed density reduction with prophylactic anastrozole during the International Breast Cancer Intervention Study-II, and its use as a biomarker for concurrent breast cancer risk reduction, respectively. In Study-I, change in BMI was associated with change in breast fat but not dense tissue, negating density reduction as a biomarker for risk reduction with weight-loss. In Study-II, longitudinal density provided approximately a quarter more statistical information than most recent density and improved discriminatory accuracy. Study-III found evidence that density reduction may be a biomarker for reduction in risk and mortality with tamoxifen, but the level of evidence was limited by some study quality issues. Study-IV indicated that preventive anastrozole might marginally reduce density, but statistical significance was not obtained. In Study-V, sample size was too small to draw definitive conclusions. Overall, changes in density were useful for the study of breast cancer risk and should be considered for personalised breast cancer prevention strategies