Prospective Breast Cancer Biobanking
Prospective Breast Cancer Biobanking
Prospective Breast Cancer Biobanking study (PBCB) will apply advanced monitoring in liquid
 biopsies of early staged breast cancer ration in order to facilitate A. Early detection of
 systemic relapses B.Improve adherence and drug monitoring av tamoxifen treatment C. Tumour
 microenvironment in breast cancer - adipose stromal immune infiltration and interaction with
 tumour at the growth zone D.Monitor side effects, QoL, depression, fatigue and work life
 participation
A. Early detection of systemic relapses in breast cancer.

 WP 1. Analysis of primary tumor.

 Analysis of primary tumor and metastases (Pathology group). In this work package both DNA and
 RNA from the primary tumors of 125 high-risk (Luminal-B) patients will be analyzed by
 next-generation sequencing (NGS). Furthermore, the investigators will also analyze DNA and
 RNA from the available metastatic lesions (currently material available for 3/8) that have
 appeared, and will appear during the study period.

 MiRNA and mRNA will be profiled using PureLink™ miRNA Isolation Kit (Invitrogen), Dynabeads
 mRNA direct micro kit (Ambion),) and Ion total RNA-seq kit V2 (Life Technologies) on our Ion
 Proton NGS instrument. Bioinformatic analyses of paired mRNA and microRNA expression profiles
 will be used to reveal differentially expressed microRNAs between patients with and without
 recurrences under treatment. These data will be linked to our previous studies and used to
 identify target candidates for microRNA analysis in exosomes and TEPs.

 In addition, NGS will also be performed on DNA from the primary tumors and metastases to
 verify DNA mutations especially in ER and other known oncogenes, including those examined in
 WP3. The DNA sequencing will be performed using the Oncomine Comprehensive Assay. This is an
 amplicon based approach analyzing hotspot SNVs, indels, and CNVs. These analyses will provide
 us with a biological understanding and background knowledge for each of the individual tumor,
 and these datasets can also be compared to the recently published data.

 WP 2. Circulating tumor celles (CTC).

 Circulating tumor cells (Oncology group) Circulating tumor cells are being enriched from
 peripheral blood samples by density centrifugation and subsequent immunomagnetic depletion of
 leucocytes. The oncology group has recently developed and published a new depletion method,
 termed MINDEC (Multi- marker Immuno- magnetic Negative Depletion Enrichment of CTCs), which
 has superior recovery and enrichment rates. RNA and DNA are isolated simultaneously from the
 enriched fraction, to allow CTC detection by both RNA and DNA-based approaches. Specific mRNA
 markers, with high levels in tumor cells and low levels in normal leukocytes, are being
 pre-amplified and quantified by real-time PCR as surrogate markers for CTCs. Both
 epithelial-specific markers and markers related to epithelial mesenchymal transition are part
 of the marker panel. Until now, the investigatorshave analyzed 170 consecutive blood samples
 from the larger PBCB cohort, enriched by the MINDEC procedure and found evidence for CTCs in
 about 20% of the samples (from both low- and high-risk patients), a finding that encourages
 us to continue this project. The investigators have also analyzed blood samples from 30
 healthy female volunteers for comparison. All collected blood samples from 125 high-risk
 patients will be analyzed (around 575 samples). The presence or level of CTCs in the analyzed
 samples will later be compared to known prognostic factors, treatment effect and disease
 outcome.

 WP 3. circulating tumor DNA (ctDNA).

 Tumor-specific mutations can be utilized as markers for ctDNA because they are not present in
 normal cells and normal plasma DNA. The oncology group has recently demonstrated the clinical
 relevance of ctDNA measurements in pancreatic cancer. The investigators will now measure
 ctDNA levels in plasma samples from high-risk breast cancer patients by targeted
 next-generation sequencing. The recently released "Oncomine breast cfDNA assay" (Thermo
 Fisher) will be used to detect mutations in a panel of ten genes that are frequently mutated
 in breast cancer. The assay is based on molecular barcoding of templates and allows
 reproducible detection of mutations down to 0,1% control samples. Taking the low
 concentration of cell free DNA (=cfDNA) in plasma into account (typically 10 ng per ml
 blood), a sensitivity of 0,1% is considered sufficient. The sequencing will be performed on
 our Ion Proton NGS instrument (Life Technologies). The pre-operative blood sample and yearly
 follow-up samples from the 125 high-risk patients will be analyzed in this WP. Blood samples
 from 30 healthy female volunteers have already been collected and will be analyzed for
 comparison.

 The presence and level of tumor-specific mutations will be analyzed in relation to treatment
 effect and disease outcome. The mutational profile in the primary tumor biopsy and the plasma
 samples will be compared in order to reveal potential heterogeneity. In addition,
 longitudinal changes in the mutation profile of ctDNA will be compared with disease
 development to potentially shed some light on the biological mechanisms causing treatment
 resistance or late disease relapses.

 WP 4. microRNA (miRNA)

 Circulating microRNA from exosomes and TEPs (Pathology group) Total RNA will be isolated from
 exosomes and TEPs from the blood samples taken before and during treatment. The investigators
 have recently established methods for isolation of total RNA from exosomes (using exoRNeasy
 serum Plasma kit (Qiagen) and miRCURY RNA isolation kit (Exiqon)) and TEPs. Using the
 mentioned protocol, total RNA from exosomes has already been isolated from all the 125 high
 risk patients first visit/before treatment, additionally from the last blood samples
 collected will from these patients, total RNA will be isolated from both exosomes and TEPs.
 Furthermore, microRNA profile will be performed on the isolated RNA using the pipeline and
 platform already established for our Ion Proton instrument. These profiles will be compared
 with the bioinformatic analysis of mRNA-miRNA profiles from the primary tumor (WP1) and the
 samples taken before treatment. MicroRNA that are not present in the tissue sample and/or in
 the blood sample before treatment, but do appear in blood right before a recurrence appears
 can then be retrospectively traced in previous blood samples in order to see how sensitive
 these microRNAs are in predicting treatment resistance. MicroRNA profile from TEPs will be
 compared to the microRNA profile from exosomes and to the mRNA-miRNA profiles from the
 primary tumor (WP1, to see if TEPs reflects the tumor and if they have the potential to
 predict relapse.

 WP 5. Metabolomics

 Metabolomics is the study of small molecules comprising substrates, intermediates and end
 products of cellular metabolism, such as amino acids, sugars and small organic acids. The
 metabolic state of cancer cells is substantially altered compared to normal cells, a fact
 that can be utilized for diagnostic purposes. Specific metabolic signatures from tumor tissue
 provide additional information for determination of breast cancer subtypes and prediction of
 outcome1. For example, increased tumor lactate and glycine levels are related to poor
 prognosis in patients with estrogen receptor (ER) positive cancer. Metabolomic analyses of
 primary tumors have also demonstrated predictive value in relation to neoadjuvant treatment
 of patients with locally advanced disease.

 Circulating metabolites have also been shown to provide prognostic information in operable
 breast cancer and further stratify risk within existing genetically determined risk
 categories. Importantly, metabolomic alterations may arise directly or indirectly from
 micrometastatic disease, rather than primary tumor. Recently, the investigators have shown
 that systemic lactate and pyruvate levels predict inferior outcome in patients with operable
 ER-positive breast cancers. Tumor-adjacent tissue and immunological responses may also
 contribute to an altered metabolomic profile. There is also evidence that metabolic profiling
 can be used for patient monitoring in some cancers, although such evidence is still lacking
 in breast cancer. Therefore, the investigators intend to investigate whether postoperative
 monitoring by means of metabolic profiling in blood is useful for early detection of breast
 cancer recurrence.

 WP 6. Integrative molecular monitoring for recurrence detection

 New technology has revolutionized the level of biological information that can be obtained
 from clinical samples, represented by the new "omics" terms genomics, transcriptomics,
 metabolomics, etc. The availability of such big datasets, even in the public domain, has
 encouraged the development of integrative methods that can extract vital information from
 multiple combined data sources. Surprising new connections between omics-datasets have been
 revealed by such approaches, exemplified by a link between cell-free DNA fragmentation and
 gene expression46. Accordingly, our knowledge about breast cancer has also been extended by
 integrative approaches, resulting in a more comprehensive understanding of the disease. The
 prognostic subclassification of breast cancers has for instance been refined and novel
 tumor-specific antigens identified. Integrated molecular data have even been shown to have a
 higher prognostic power than separate molecular levels in breast cancer. Thus, a combined
 analysis of the genetic (ctDNA), transcriptomic (miRNA) and metabolomic data levels in
 peripheral blood samples is therefore planned in the current project to maximize their joint
 biomarker potential in operable breast cancer.

 Sample size calculations.

 The investigators performed sample size calculations using SPSS Sample Power software. These
 calculations demonstrated that 125 high-risk patients should be sufficient to give a log rank
 test power of 80%, when testing the prognostic value of ctDNA/CTC detection before surgery.
 The sample size calculations were based on the qualified assumption that the average 5-year
 survival rate in the high-risk group is 90%, whereas it is 75% and 95% in the ctDNA/CTC
 positive and negative subgroups, respectively. Of the estimated 125 patients, 30 were assumed
 positive for ctDNA/CTC and 95 negative.

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 B. Improve drug monitoring of tamoxifen in breast cancer

 About 75% of all breast cancer belong to the luminal subtypes, which express the hormonal
 receptors Estrogen Receptor and Progesterone Receptor. These patients are treated with the
 anti estrogenic drugs; tamoxifen and/or aromatase inhibitors. The two most active tamoxifen
 metabolites are Z-4OHtam and Z-4OHNDtam (Z-endoxifen) have 30-100 times higher affinity for
 ER than tamoxifen. These metabolites ultimately constitute the blocking effect at the
 ER-level aiming to eradicate micro metastatic disease and are responsible for the improved
 survival following the establishment of this adjuvant systemic treatment. Direct measurement
 of these metabolites bypasses all disturbances from the diversity of CYP2D6 activity i.e.
 alternative metabolic pathways, adherence to the drug and inhibiting drug interactions. Our
 novel LC / MS-MS methodology takes into account all of the above-mentioned variables and
 provides a functional read-out report of the serum level of the active tamoxifen metabolites
 in the individual patient. This method can also distinguish between the inactive and active
 isomers of Endoxifen and 4-OHtam, which are the most active ER-blocking metabolites of
 tamoxifen. In collaboration with the Oslo Breast Cancer Research Group, the investigators
 have recently shown in a retrospective observational study that patients with low serum
 concentrations of Z-4OHtam < 3.26 nM or Z-Endoxifen < 9.00 nM (about 12% of all patients)
 have significant worse breast cancer specific and total survival than patients with serum
 concentrations above these thresholds(adjusted HR = 4.3; CI95 = 1.9-13.6) (red curves in
 Figure 4). Patients with a very high level of these metabolites (approximately 12% of
 patients) had no breast-specific endpoints Now, the investigators need to validate this
 discovery in independent patient materials. If validated, this will be of direct clinical
 benefit for 25% of ER + breast cancer patients planning adjuvant tamoxifen treatment. Such
 therapeutic drug monitoring (TDM) could identify risk patients with inadequate levels of
 active tamoxifen metabolites. This could lead to a dose increase or switch to an alternative
 endocrine treatment form in these patients. Patients with very high metabolite levels can
 continue on tamoxifen and do not need to switch to an aromatase inhibitor.

 Thus, TDM may turn out to be a paradigmatic shift in endocrine tam treatment of ER-positive
 breast cancer. Importantly, the distance from "bench to bed" in this study is very short due
 to our recent findings, a feasible method and over 30 years of experience with tamoxifen in
 the clinical setting.

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 C. Tumour microenvironment in breast cancer - adipose stromal immune infiltration and
 interaction with tumour at the growth zone

 Background: Breast cancer is still the most common cancer type among women in the Western
 World, Norway included. Yearly, around 3500 new Norwegian breast cancer patients are
 diagnosed. During the last 50 years, the incidence of breast cancer in Norway has more than
 doubled. Interestingly, during the same time frame over-weight and obesity have increased
 alarmingly. Molecular subtyping of breast cancer tumours has improved the understanding of
 its intrinsic biology. This has opened up for a more personalized treatment approach and
 improved the outcome for breast cancer patients over the last 20 years .

 In addition to the molecular subtyping, the diagnostics of breast cancer today consists of
 staging (through the TNM-classification system) and microscopic examination by a pathologist.
 There is increasing evidence for the prognostic value of evaluating tumour microenvironment
 (e.g. tumour infiltrating lymphocytes (TILs)) as a part of diagnostics in breast cancer, but
 as of yet this is not implemented in clinical practice. High numbers of TILs are strongly
 associated with good prognosis in patients with triple-negative breast cancer, but have not
 shown any such correlation in luminal breast cancer. Macrophages in breast adipose tissue
 differentiate into two distinct phenotypes, often classified as M1 and M2 with an increase of
 the latter in breast cancer. M1 attack and phagocytize cancer cells, while M2 have
 anti-inflammatory properties and have been shown to have tumour-promoting functions.

 The present project will increase our knowledge of the interaction between the primary breast
 tumour and the immune cell infiltration in the juxta-tumoral adipose tissue. Our findings
 will be correlated to molecular breast cancer subtypes, proximity to tumour, markers of
 proliferation, and ultimately to clinical outcome The researcers reason that this knowledge
 will provide novel predictive targets for immune-modulating adjuvant cancer therapy. This PhD
 project has three interrelated work packages (WP) with the ultimate goal of identifying
 markers of inflammation in juxta-tumoral adipose tissue that are relevant in clinical
 assessment of adjuvant therapy of breast cancer. In WP 1 and 2, the phenotypical and genomic
 landscape of juxta-tumoral adipose tissue will be characterized, and in WP 3 the researches
 will investigate whether this knowledge can predict clinical outcome.

 The project is based on a strong regional collaboration between the Haukeland University
 Hospital (HUH) and Stavanger University Hospital (SUH). For the current project, the
 researches have in a systematic manner collected adipose and tumour tissue from 30
 consecutive breast cancer patients undergoing mastectomies at SUH. Surgical specimens of the
 breast are without delay forwarded to the pathologist who, under morphological control,
 sample relevant tissue from the invasive front and the core (0,5x1x1 cm) of the primary
 tumour. Controls from normal breast tissue (2000 mg) from the distant contralateral quadrant
 will also be collected and verified morphologically by the pathologist. The tissue is
 collected in duplicates pairs. One of the samples is immediately snap-frozen in liquid
 nitrogen for preservation, while the other half of each pair is preserved in formalin and
 fixated in paraffin (FFPE). In addition, three pairs of samples of adipose tissue (each of
 1x1x1 cm) with increasing distance from the tumour border will make up a gradient of adipose
 tissue from the invasive front of the tumour area. The gradients collected are also
 immediately snap-frozen in liquid nitrogen and one half of each pair stored as FFPE. The
 researches have performed a pilot study confirming a sufficient RNA yield from the frozen
 tissue samples for RNA sequencing.

 The tissue is collected as part of the general research biobank, Prospective Breast Cancer
 Biobank (PBCB) (REK# 2010/1957), an ongoing biobank-project.

 The retrospective Stavanger cohort consist of all breast cancer patients diagnosed with first
 onset invasive breast cancer at the department of Pathology, Stavanger University Hospital,
 between 1993 - 2004. As SUH is the only hospital in the region this is a true population
 based biobank, already included are clinicopathological data like: grading, stage, TNM,
 treatment and proliferation markers (Ki-67, PPH3/MAI); last clinical follow-up took place in
 2016.

 The researches have four main hypotheses related to inflammation and immune cell infiltration
 in breast cancer:

 1. The juxta-tumoral adipose tissue close to the tumour border shows an increased level of
 inflammation and immune cell infiltration.

 2. The inflammation and immune cell infiltration attenuate with increased distance from the
 tumour border.

 3. There are differences in the inflammation and immune cell infiltration between different
 subtypes of breast cancer (Luminal A, Luminal B and Basal like).

 4. Increased inflammation and immune cell infiltration are correlated to adverse clinical
 outcome (i.e. relapse of disease).

 4.1 STUDY DESIGN, METHODS AND ANALYSES

 The researches have four main hypotheses related to inflammation and immune cell infiltration
 in breast cancer:

 1. The juxta-tumoral adipose tissue close to the tumour border shows an increased level of
 inflammation and immune cell infiltration.

 2. The inflammation and immune cell infiltration attenuate with increased distance from the
 tumour border.

 3. There are differences in the inflammation and immune cell infiltration between different
 subtypes of breast cancer (Luminal A, Luminal B and Basal like).

 4. Increased inflammation and immune cell infiltration are correlated to adverse clinical
 outcome (i.e. relapse of disease).

 WP 1: MORPHOLOGIC AND GENOMIC MAPPING OF IMMUNE CELL INFILTRATION IN ADIPOSE TISSUE
 SURROUNDING BREAST CANCER

 Background: Inflammation is an important facilitator for tumour development (16). Adipose
 tissue near breast cancer have been shown to have increased immune cell infiltration, but it
 is not yet known how this relates to molecular subtypes and/or proximity to tumour. The
 researches will use IHC and RNA-sequencing to evaluate the level of inflammation,
 infiltration of immune cells and immune gene expression signatures in the adipose tissue
 surrounding breast cancer.

 Design: Prospective explorative study Patients, material and power calculation: Adipose
 tissue is collected from the prospectively collected mastectomy specimens. Tissue samples are
 collected from three different molecular subgroups (Luminal A, Luminal B and Basal like
 breast cancer), 10 in each group. Core biopsies are collected in a gradient with increasing
 distance from tumour border (fig. 1). The Norwegian Genomics Consortium (NGC) has confirmed
 that the sample sizes that are based on the selection in specific breast cancer subtypes are
 sufficient for statistical power.

 Methods: Slides of formalin fixated tissue from tumour, invasive front and adipose tissue
 (fig. 1) will be stained using IHC and antibodies against immune cells (total immune cells,
 CD45; T cells, CD3, CD8, FoxP3; myeloid cells, CD68, HLA-DR). The slides will be evaluated by
 microscopy and the results will be correlated to molecular subgroups of breast cancer,
 localization in relation to tumour and proliferation markers (i.e. Ki67, PPH3 and MAI). In
 parallel, the researches will perform RNA-sequencing on frozen tissue collected from the same
 tissue samples as described above. Several analysis approaches will be used on the
 RNAseq-data. Except for analysing the data exploratory, specific analysis algorithms
 including deconvoluting immune cells (ABIS, CIBERSORTx) and more specifically looking at
 tumour immune signatures (TIP analysis), among others.

 Expected outcome: The researches expect to find increased inflammation and immune cell
 infiltration in the juxta-tumoral adipose tissue and altered expression of inflammatory gene
 signatures that the researches can further investigate in WP 2.

 WP 2: ASSESSMENT OF INFLAMMATORY REACTION IN JUXTA-TUMOURAL ADIPOSE TISSUE IN BREAST CANCER
 USING IMMUNOHISTOCHEMISTRY AND IMAGING MASS CYTOMETRY.

 Background: Breast cancer cells interact with the surrounding tissue and causes inflammation
 and immune cell infiltration in the tumour and the adipose tissue. Imaging mass cytometry has
 shown that immune cell infiltration in tumours correlates with prognosis, but as of yet there
 are no studies examining whether the adipose tissue surrounding the tumour also show
 structural patterns of immune cell infiltration. Therefore, the researches will examine the
 adipose tissue surrounding breast cancer tumours using Hyperion™ Imaging Mass Cytometry.

 Design: Prospective explorative study Patients, material and power calculation: Tissue
 samples are collected from 30 prospectively obtained mastectomy specimens with three
 different molecular subgroups (Luminal A, Luminal B and Basal like breast cancer), 10 in each
 group. Adipose tissue is collected in a gradient away from tumour and from the contralateral
 quadrant as a control sample (fig. 2). A sample size of 30 patients is deemed to be
 sufficient for an explorative study of this sort.

 Methods: A Hyperion™ IMC panel of up to 50 unique antibodies will be developed based on an
 already existing and validated panel available at the UoB core facility which the researches
 have previously successfully used in other projects. The available panel will be further
 extended based on findings from WP 1 and first validated on "non-essential" breast tissue
 samples before used on the above-described study samples. Using Hyperion™ IMC the researches
 intend to analyse the levels of inflammatory reaction in the juxta-tumoural adipose tissue.
 IMC data will be analysed with histoCAT+, MAV and FlowJo (typically used for flow cytometry
 data analysis but also highly suitable for high-parameter imaging anslysis). Furthermore, the
 researches will be able to use gene expression data of known and novel factors, identified in
 WP 1, and visualize whether the expression is generalized or compartmentalized in the adipose
 tissue. Normal adipose breast tissue from the contralateral quadrant will be used as control.
 In order to examine the influence of BC on the inflammation of juxta-tumoural adipose tissue
 of the various molecular subtypes in more detail, the researches also intend to stain tissue
 from three tissue segments extending from the tumour in order to determine whether the levels
 depend on the distance from tumour or the molecular subtype (Fig. 1). The inflammation in the
 juxta-tumoural adipose tissue will also be assessed morphologically by using IHC. This will
 allow us to select regions of interest to investigate using IMC.

 Expected outcome: The researches expect to find increased inflammation and immune cell
 infiltration in the adipose tissue near the tumour and to identify novel biomarkers in the
 adipose tissue allowing for investigation of the clinical significance in WP 3.

 WP 3: INFLAMMATORY REACTION IN JUXTA-TUMOURAL ADIPOSE TISSUE IN BREAST CANCER AND CLINICAL
 OUTCOME.

 Background:

 Immunohistochemistry is a cornerstone in breast cancer diagnostics and serves as the main
 modality for identifying molecular subgroups of breast cancer and proliferation markers such
 as Ki-67. This is essential in determining what treatment regimen is best suited for the
 particular patient. Heterogeneity in the tumour tissue (both tumour cells and infiltrating
 immune cells) have been linked to difference in clinical outcome , but it is not yet known if
 the same applies for the adipose tissue surrounding the tumour. Thus, the researches will
 examine juxta-tumoral adipose tissue for inflammation and immune cell infiltration and
 correlate this with clinical outcome in breast cancer patients.

 Design: Clinical observational study

 Patients and Methods: A number of 3500 breast cancer patients are accrued in a biobank with a
 median follow-up time of 15 years. A large selection of tissue samples from these patients
 will be assessed with respect to immune cells in the adipose tissue and biomarkers discovered
 in WP 1 and 2 using IHC. The samples will be stratified into three groups according to
 molecular subgroups (i.e. Luminal A, Luminal B and Basal like breast cancer). The tissue
 slides will be analysed and the results correlated with clinical outcome from the patient
 journals (access to journals is already approved by REK). Using Cox proportional hazard ratio
 and log-rank test, the researches will evaluate the effect increased inflammation in adipose
 tissue has on patient survivability.

 Power calculation: The researches estimated the test power of the difference in recurrence
 free survival between patients positive and negative for juxta tumoral inflammation as
 assessed by IHC and IMC. The effect measure is the hazard rate (HR) as calculated by the Cox
 regression method. For example: 1000 patients with a recurrence rate of 15% will have created
 at least 150 endpoints. If the inflammation rate is 20% the researches will have an 80% power
 to detect a survival difference with a hazard ratio (HR) of 1.80 or greater.

 Expected outcome: This last work package is designed to give proof of principle that it is
 feasible to incorporate histological examination of juxta-tumoural adipose tissue as a part
 of breast cancer diagnostics. In the longer perspective the researches aim to identify new
 specific biomarkers of immune infiltration in adipose tissue that correlate to adverse
 outcome in breast cancer patients. Such biomarkers may serve as predictive tools for
 identifying patients eligible for adjuvant anti-inflammatory or immune-modulating treatment.

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 D.Monitor side effects, QoL, depression, fatigue and work life participation Background

 Treatment of breast cancer employs all surgery, chemotherapy, radiation therapy and various
 targeted therapy options like antiestrogen and anti HER-2 therapy.These treatment options
 create bothersome side effects both in a short term and long term perspective. Importantly,
 more complex relationships between oncological therapy modalities and subjective health
 complaints in breast cancer patients are recently discovered. Fatigue, anxiety and depression
 scores are probably functional read outs of the total treatment burden. The anti cancer
 treatment has also a substantial impact on Quality of Life and social aspects for the cancer
 survivors which extends to work life participation. Sick leave (SL) five years after the
 breast cancer diagnosis is more dependent on social factors than on illness (2). Hence,
 breast cancer hits the women twice; first the disease burden then the sosio-economic worries.
 Breast cancer survivors have almost 3 x higher risk of receiving disability pension (DP)
 compared to cancer -free patients in a retrospective register based study (3). However, this
 study was based on mostly abandoned oncological treatment regimens.

 Aims

 The aim of this study is to identify risk factors for becoming a long term SL, WAA or DP
 receiver by an in-depth qualitative analysis and mixed-method approach by triangulating PROM
 data, clinical/biological data and the NAV-data. Use of NAV-data as endpoints will provide
 valuable information to clinicians and general practitioners who may optimize the follow-up
 also in regards to work-life participation. Hence, the investigators have a robust study
 design to identy fisk factors for high SL, WAA and DP rates. Ultimately, the investigators
 aim to lower the financial expenses breast cancer generates to society.

 Study design / material & methods.

 The study design is an observational study where the investigators will in the present study
 map the rates of and denty fisk factors for high SL, WAA and DP among breast cancer survivors
 with the up-to date treatment schedules. The investigators will follow a 4-step translational
 approach:

 1. The investigators will first conduct an exploratory qualitative study using document
 analysis of health and work life policy in the Norwegian context, combined with
 empirical data from semi- structured interviews and focus group with 20 high risk
 patients) to be analyzed thematically. The theoretical framework for this qualitative
 project is derived from symbolic interactionism in which uncovering and understanding
 meaning in specific contexts is a goal. Since meaningful social and professional
 interactions are important for psychosocial rehabilitation of breast cancer individuals
 symbolic interactionism is an appropriate methodological framework. This qualitative
 approach will create the bases of the approach in the PROM based and NAV-database
 oriented studies below.

 2. NAV- data

 The investigators currently collaborate with vice director Anneline Christine Teigen and
 senior consultant Günter Olsborg in NAV-Rogaland and the Mikrodata section at Statistics
 Norway (SSB). The aim for this collaboration is gain more knowledge about the use of
 social security services among breast cancer patients together with the NAV- Rogaland
 experts and together with SSB use the FD-trygd database to obtain reliable prospective
 sociodemographic data on SLs (both short & long term), Work Assessment Allowance (WAA)
 (Arbeidsavklaringspenger) and DPs (both time limited

 & life long) for each patients in the present PerMoBreCan study.

 3. Patient reported outcome measure (PROM) data is obtained at baseline and thereafter
 yearly from all PerMoBreCan patients. The PROM data consists of 1.HRQoL instruments
 (EORTC QLQ-C30,EORTC QLQ-BR23 and FACT B), 2.Hospital Anxiety and Depression Scale, HAD,
 3. Fatigue instruments Fatique Impact Scale, FIS; Fatugue Severity Scale (FSS) and
 VAS-fatigue, 4.Side effects questionnaires, 5. Joint- pain questionnaire, 6. The Mishel
 Uncertainty in Illness Scale, MUIS; and 7. Food habits questionnaire and the 8.ROMA III
 questionnaire of bowel complaints (IBD and IBS).

 4. Biomarker identification.

 All patients attending this study will be screened on circulating biomarkers releated to
 fatigue. These biomarkers are on the protein level, genetic expression level and also on the
 epigenetic level. Our long-established pleasant cooperation with prof. Roald Omdal at
 Stavanger University Hospital, who is a clinical immuologist and transaltional fatigue
 researcher, is committed to conduct these analyses in his research lab.
Inclusion Criteria:

 -  Early stage Breast Cancer

 -  DCIS gr III

 Exclusion Criteria:

 -  Not able to read Norwegian

 -  Not able to communicate in Norwegian

 -  Previously diagnosed with cancer

 -  Dementia