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Purpose: The introduction of immunotherapy in pleural mesothelioma (PM) has highlighted the need for effective outcome predictors. This study explores the role of [18F]FDG PET/CT in predicting outcomes in PM treated with immunotherapy., Methods: Patients from the NIPU trial, receiving ipilimumab and nivolumab +/- telomerase vaccine in second-line, were included. [18F]FDG PET/CT was obtained at baseline (n = 100) and at week-5 (n = 76). Metabolic tumour volume (MTV) and peak standardised uptake value (SUV peak ) were evaluated in relation to survival outcomes. Wilcoxon rank-sum test was used to assess differences in MTV, total lesion glycolysis (TLG), maximum standardised uptake value (SUV max ) and SUV peak between patients exhibiting an objective response, defined as either partial response or complete response according to the modified Response Criteria in Solid Tumours (mRECIST) and immune RECIST (iRECIST), and non-responders, defined as either stable disease or progressive disease as their best overall response., Results: Univariate Cox regression revealed significant associations of MTV with OS (HR 1.36, CI: 1.14, 1.62, p < 0.001) and PFS (HR 1.18, CI: 1.03, 1.34, p = 0.02), while multivariate analysis showed a significant association with OS only (HR 1.35, CI: 1.09, 1.68, p = 0.007). While SUV peak was not significantly associated with OS or PFS in univariate analyses, it was significantly associated with OS in multivariate analysis (HR 0.43, CI: 0.23, 0.80, p = 0.008). Objective responders had significant reductions in TLG, SUV max and SUV peak at week-5., Conclusion: MTV provides prognostic value in PM treated with immunotherapy. High SUV peak was not associated with inferior outcomes, which could be attributed to the distinct mechanisms of immunotherapy. Early reductions in PET metrics correlated with treatment response., Study Registration: The NIPU trial (NCT04300244) is registered at clinicaltrials.gov. https://classic., Clinicaltrials: gov/ct2/show/NCT04300244?cond=Pleural+Mesothelioma&cntry=NO&draw=2&rank=4., Competing Interests: Declarations Ethics approval The trial was approved by the regional ethics committee (20/47804) and each site ethics committee and was conducted in accordance with the Declaration of Helsinki of the World Medical Association and ICH E6 for Good Clinical Practice. Consent to participate All patients provided written informed consent. Consent to publish The authors affirm that human research participants provided informed consent for publication of the images in Figures 6, 7, 8. Competing Interests ST - External speaker BMSWMS – no conflict of interestEH – no conflicts of interestJBS - BMS Advisory BoardÅH - Advisory/consultancy/meeting presentation roles for AbbVie, AstraZeneca, BMS, Pfizer, Roche, Janssen, MSD, Sanofi, Bayer, Medicover, and Takeda with honoraria directed to own institution, and receiving research grants from Roche, BMS, Ultimovacs, AstraZeneca, Novartis, InCyte, Eli Lilly, Illumina, Merck, Nanopore, GlaxoSmithKline.AKN – Astra Zeneca DSMB; Research funding to institution from Astra Zeneca.ÅKÖ - no disclosures.VDH: Advisory boards: Astra Zeneca, Novartis, Lectures/text: Astra Zeneca, Pfizer, Janssen, BMS, TakedaEM – no conflictsCM – no conflict of interestLTM: No conflicts of interestRF – advisory/consultancy for AIQ SolutionsRF – advisory/consultancy for AIQ SolutionsSSN-no conflict of interestSF - Advisory/consultancy/meeting presentation MSD, Astra Zeneca, PfizerMS – no disclosures, (© 2024. The Author(s).)

Background: Radiotherapy (RT) can be used to reduce symptoms and maintain open airways for patients with non-small cell lung cancer when systemic treatment is not sufficient. For some patients, tumor control is not achieved due to radioresistance. Concurrent inhibition of epidermal growth factor receptors has been proposed as a strategy to overcome radioresistance but may increase toxicity. We performed a randomized trial to assess the efficacy, tolerance, and quality of life of concurrent erlotinib and palliative thoracic RT for patients with advanced non-small cell lung cancer., Methods: Patients were randomized 1:1 to RT alone (arm A) or in combination with erlotinib (arm B). A computed tomography (CT) scan at baseline and one at 4-12 weeks after inclusion was used to evaluate treatment response. Adverse events were registered during treatment and the subsequent 30 days. Health-related quality-of-life questionnaires were completed by the patients at baseline, weeks 2, 6, and 20., Results: A total of 114 patients were included. Of the 74 patients with CT scans available for evaluation of treatment effect, there were no significant differences in tumor size reduction between the two groups: median 14.5% reduction in the control arm A and 17.0% in the erlotinib arm B ( p = 0.68). Overall survival was not significantly different between the two treatment arms: 7.0 and 7.8 months in arm A and arm B, respectively (log-rank p = 0.32). There was no significant increase in adverse events in the experimental arm, other than what is expected from erlotinib treatment alone. Overall, patients reported similar quality of life in both treatment arms., Conclusion: Concurrent erlotinib and palliative thoracic RT for patients with advanced non-small cell lung cancer was well tolerated but did not improve the efficacy of the RT., Clinical Trial Registration: ClinicalTrials.gov, identifier NCT02714530., Competing Interests: HN has received honoraria from Astra Zeneca. HH has received honoraria for lectures or advisory boards from Astra Zeneca, Janssen, Pfizer and Roche. HE has received honorarium from Sanofi. MB has received honoraria personal/institution from Astra Zeneca, BMS and Pfizer. OB has received honoraria personal and/or institution for lectures or advisory boards from AstraZeneca, Bayer, BMS, BoehringerIngelheim, Eli Lilly, MSD, Novartis, Pfizer, Roche, Sanofi, Takeda and research grants institution from Amgen, AstraZeneca, GlaxoSmithKline, Pfizer, and Roche/Genentech. BG has received honoraria from Janssen, Pfizer, BMS, AstraZeneca, MSD, Eli Lilly and research support from Astra Zeneca og Roche. VH has received honoraria for talks and advisory boards from Astra Zeneca, Roche, Pierre Fabre, Takeda, BMS, Pfizer and Novartis and has received support for trials from the Norwegian Cancer Society and The regional health authorities in Norway. AH has given talks and advice to pharma companies, all honoraria to the hospital Astra, Roche, Janssen, Novartis, Takeda, Pfizer, BMS, EliLilly, Abbvie, Merck, Sanofi, Bayer, Medicover and received research support from AstraZeneca, Roche, Novartis, Incyte, EliLilly, BMS, Ultimovacs, GSK, the Norwegian Cancer Society, The regional health authorities in Norway, The Norwegian Research Council. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Nymoen, Alver, Horndalsveen, Eide, Bjaanæs, Brustugun, Grønberg, Haakensen and Helland.)

Background: Based on the findings of the PACIFIC trial, consolidation durvalumab following platinum-based chemoradiotherapy (CRT) is a global standard of care for patients with unresectable, stage III non-small-cell lung cancer (NSCLC). An earlier analysis from the ongoing PACIFIC-R study (NCT03798535) demonstrated the effectiveness of this regimen in terms of progression-free survival (PFS). Here, we report the first planned overall survival (OS) analysis., Patients and Methods: PACIFIC-R is an observational/non-interventional, retrospective study of patients with unresectable, stage III NSCLC who started durvalumab (10 mg/kg intravenously every 2 weeks) within an AstraZeneca-initiated early access program between September 2017 and December 2018. Primary endpoints are OS and investigator-assessed PFS, estimated using the Kaplan-Meier method., Results: By 30 November 2021, the full analysis set included 1154 participants from 10 countries (median follow-up in censored patients: 38.7 months). Median OS was not reached, and the 3-year OS rate was 63.2% (95% confidence interval 60.3% to 65.9%). Three-year OS rates were numerically higher among patients with programmed death-ligand 1 (PD-L1) expression on ≥1% versus <1% of tumor cells (TCs; 67.0% versus 54.4%) and patients who received concurrent CRT (cCRT) versus sequential CRT (sCRT) (64.8% versus 57.9%)., Conclusions: PACIFIC-R data continue to provide evidence for the effectiveness of consolidation durvalumab after CRT in a large, diverse, real-world population. Better outcomes were observed among patients with PD-L1 TCs ≥1% and patients who received cCRT. Nevertheless, encouraging outcomes were still observed among patients with TCs <1% and patients who received sCRT, supporting use of consolidation durvalumab in a broad population of patients with unresectable, stage III NSCLC., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Patients treated for lung cancer (LC) often experience locoregional failure after initial treatment. Due to technological advances, thoracic reirradiation (re-RT) has become a viable treatment option. We sought to investigate the use of thoracic re-RT in LC patients over a time period characterized by technological advances in a large, multi-center cohort., Methods and Materials: LC patients treated with thoracic re-RT in two University Hospitals from 2010-2020 were identified. Clinical variables and RT data were extracted from the medical records and treatment planning systems. Overall survival (OS) was calculated from the last day of re-RT until death or last follow up., Results: 296 patients (small cell LC n=30, non-small cell LC n=266) were included. Three-dimensional conformal radiation therapy was the RT technique used most frequently (63%), and 86% of all patients were referred for re-RT with palliative treatment intent. During the second half of the study period, the use of thoracic re-RT increased in general, more patients received curative re-RT, and there was an increased use of stereotactic body radiation therapy (SBRT). Median time between initial RT and re-RT was 18 months (range 1-213 months). Only 83/296 patients had combined treatment plans that allowed for registration of combined doses to organs at risk (OAR). Most of the combined doses to OAR were below recommendations from guidelines. Multivariate analysis showed superior OS (p<0.05) in patients treated with curative intent, SBRT or intensity modulated radiation therapy or had excellent performance status prior to re-RT., Conclusions: The use of re-RT increased in the second half of the study period, although 2020 did not follow the trend. The use of SBRT and IMRT became more frequent over the years, yet the majority received palliative re-RT. Combined dose plans were only created for one third of the patients., (Copyright © 2024. Published by Elsevier Inc.)

Purpose: The NIPU-trial investigates the effect of adding the telomerase vaccine UV1 to treatment with ipilimumab and nivolumab for patients with pleural mesothelioma (PM)., Methods: In this phase 2 open-label trial, patients with PM progressing after first-line chemotherapy were randomised to receive ipilimumab and nivolumab alone (arm B) or combined with UV1 (arm A). The primary endpoint was progression-free survival (PFS) as determined by BICR. It was estimated that 69 PFS events were needed to detect a hazard ratio (HR) of 0.60 with 80% power and a one-sided alpha level of 0.10., Results: 118 patients were randomised. The median PFS determined by blinded independent central review (BICR) was 4.2 months (95%CI 2.9-9.8) in arm A and 4.7 months (95%CI 3.9-7.0) in arm B (HR 1.01, 80%CI 0.75-1.36 P = 0.979), after a median follow-up of 12.5 months (95%CI 9.7-15.6). The investigator-determined median PFS was 4.3 months (95%CI 3.0-6.8) in arm A and 2.9 months (95%CI 2.4-5.5) in arm B (HR 0.60, 80%CI 0.45-0.81 P = 0.025). Confirmed objective response rate (ORR) by BICR was 31% in arm A and 16% in arm B (odds ratio 2.44 80%CI 1.35-4.49 P = 0.056). After a median follow-up time of 17.3 months (95%CI 15.8-22.9), the OS was 15.4 months (95%CI 11.1-22.6) in arm A and 11.1 months (95%CI 8.8-18.1) in arm B, (HR 0.73, 80%CI 0.53-1.0, P = 0.197)., Conclusion: The primary endpoint was not met. Predefined analyses of response rates are in favour of adding the vaccine., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Vilde Drageset Haakensen - Payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events: AstraZeneca, Roche, Takeda, Pfizer, BMS, MSD, Janssen. Participation on a Data Safety Monitoring Board or Advisory Board: Novartis, Astra Zeneca, BMS. Leadership or fiduciary role in other board, society, committee or advocacy group, paid or unpaid: Board member of lung cancer patient organization from 2022. Support from the South-Eastern Norway Regional Health Authorities, grant number 2021083. Maria Bjaanæs - Payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events: AstraZeneca, Pfizer, BMS. Weronika Maria Szejniuk – none declared. Solfrid Thunold - Payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events: BMS. Susana Cedres - Participation on a Data Safety Monitoring Board or Advisory Board: F. Hoffmann La Roche AG, MSD Oncology, Pfizer, Amphera, Boehringer Ingelheim, BMS Wee L Chin – none declared. Tonje Sofie Dalen – none declared. Saima Farooqi - Payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events: Merck. Oscar Grundberg – none declared. Åslaug Helland - All support for the present manuscript (e.g., funding, provision of study materials, medical writing, article processing charges, etc.): South-Eastern Norway Regional Health Authorities, grant number 202007 and 2016056, Ultimovacs, BMS. Grants or contracts from any entity: Roche, InCyte, Astra Zeneca, Merck, Novartis, Eli Lilly, GSK, Illumina, Nanopore. Payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events: AstraZeneca, Janssen, Medicovver, Bayer, Eli Lilly, MSD, BMS, Merck, Sanofi, Abbvie, Pfizer, Takeda, Novartis, Roche. Participation on a Data Safety Monitoring Board or Advisory Board: AMGEN (DSMC), AdBoard: Roche, AstraZeneca, Pfizer, Janssen, BMS, EliLilly, Abbvie, Bayer, Sanofi, Merck, Takeda. Leadership or fiduciary role in other board, society, committee or advocacy group, paid or unpaid: Board member of lung cancer patient organization until 2022 Marius Lund Iversen – none declared. Anna K Nowak - Grants or contracts from any entity: Astra Zeneca. Participation on a Data Safety Monitoring Board or Advisory Board: AstraZeneca, BMS, Douglas Pharmaceuticals Åsa Kristina Öjlert – none declared. Jens Benn Sørensen - Participation on a Data Safety Monitoring Board or Advisory Board: BMS, Merck, Astra Zeneca., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Introduction: The introduction of consolidation immunotherapy after chemoradiotherapy has improved outcome for patients with locally advanced non-small cell lung cancer. However, not all patients receive this treatment. This study identifies factors associated with failure to start durvalumab as consolidation therapy with the aim of optimizing treatment, supportive care and prehabilitation to ensure that more patients complete the planned treatment., Materials and Methods: Patients from two clinical trials and a named patient use program, were included in this study. All patients received platinum-doublet chemotherapy concomitant with radiotherapy to a total dose of 60-66 gray. Patient characteristics, cancer treatment, toxicity, performance status and laboratory data before and after chemoradiotherapy were recorded and patients who never started durvalumab were compared with those who did., Results: A total of 101 patients were included, of which 83 started treatments with durvalumab after chemoradiotherapy. The 18 patients who did not start durvalumab had significantly higher lactate dehydrogenase at baseline and a worse performance status, cumulative toxicity and higher c-reactive protein after completed chemoradiotherapy. Data also suggest that pre-treatment diabetes and reduced hemoglobin and/or diffusion capacity of the lungs for carbon monoxide contribute to the risk of treatment abruption., Conclusion: Treatment plan disruption rate was 18%. Systemic inflammation and performance status were associated with failure to receive durvalumab after chemoradiation. Further studies are needed to confirm findings and prospective trials should investigate whether prehabilitation and supportive treatment could help more patients finishing the planned treatment., Clinical Trial Registration: clinicaltrials.gov, identifier NCT03798535; NCT04392505., Competing Interests: This study has not received funding, but is based on data from trials funded completely or partially by Astra Zeneca. No funder has been involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication. ÅH has received study funding and honoraria from Astra Zeneca, but all payments have gone to the hospital and not to personal accounts. HH and VDH have received honoraria from Astra Zeneca for lectures and/or advisory boards, payments to personal accounts. The authors declare no other competing interests., (Copyright © 2023 Langberg, Horndalsveen, Helland and Haakensen.)

The introduction of immune checkpoint inhibitors has transformed the treatment landscape of metastatic non-small cell lung cancer. However, challenges remain to increase the fraction of patients achieving durable clinical responses to these drugs and to help monitor the treatment effect. In this phase II trial, we investigated the toxicity, systemic responses and circulating tumour DNA responses in patients (n = 21) with advanced non-small-cell lung cancer treated with atezolizumab and stereotactic body radiotherapy in the second or later line. We found the combined treatment to be safe with grade 3 toxicity reported in three patients. As the best overall response, four patients had a partial response, eight had stable disease and five had progressive disease. Median overall survival time was still not reached after a median follow-up of 26.5 months and 10/15 patients with programmed death-ligand 1 negative tumours were alive >18 months after the start of the study treatment. ctDNA was detectable at baseline in 11 patients. A rapid decline in ctDNA to <30% of baseline levels was seen in three patients, two of which were radiographic responders and one was considered clinically benefiting from therapy for almost 1 year., (© 2022 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Introduction: The phase 3 PACIFIC trial established consolidation therapy with durvalumab as standard of care for patients with unresectable, stage III NSCLC and no disease progression after definitive chemoradiotherapy (CRT). The observational PACIFIC-R study assesses the real-world effectiveness of durvalumab in patients from an early access program. Here, we report treatment characteristics and a preplanned analysis of real-world progression-free survival (rwPFS)., Methods: PACIFIC-R (NCT03798535) is an ongoing, international, retrospective study of patients who started durvalumab (intravenously; 10 mg/kg every 2 wk) within an early access program between September 2017 and December 2018. The primary end points are investigator-assessed rwPFS and overall survival (analyzed by Kaplan-Meier method)., Results: As of November 30, 2020, the full analysis set comprised 1399 patients from 11 countries (median follow-up duration, 23.5 mo). Patients received durvalumab for a median of 11.0 months. Median rwPFS was 21.7 months (95% confidence interval: 19.1-24.5). RwPFS was numerically longer among patients who received concurrent versus sequential CRT (median, 23.7 versus 19.3 mo) and among patients with programmed cell death-ligand 1 expression greater than or equal to 1% versus less than 1% (22.4 versus 15.6 mo). Overall, 16.5% of the patients had adverse events leading to treatment discontinuation; 9.5% of all patients discontinued because of pneumonitis or interstitial lung disease., Conclusions: Consolidation durvalumab after definitive CRT was well tolerated and effective in this large, real-world cohort study of patients with unresectable, stage III NSCLC. As expected, rwPFS was longer among patients who received concurrent versus sequential CRT and patients with higher programmed cell death-ligand 1 expression. Nevertheless, favorable rwPFS outcomes were observed regardless of these factors., (Copyright © 2022 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.)

Immunological checkpoint inhibitors have been revolutionary in the treatment of cancer. A rare but serious adverse effect is the development of heart muscle inflammation (myocarditis). The prevalence of this type of myocarditis is increasing as more cancer patients receive treatment with immune checkpoint inhibitors. Knowledge of immune checkpoint inhibitor-induced myocarditis is important to enable early diagnosis and initiation of treatment. In this article we provide a clinical review of this.

Lung cancer is a common disease with a poor prognosis. Genomic alterations involving the KRAS gene are common in lung carcinomas, although much is unknown about how different mutations, deletions, and expressions influence the disease course. The first approval of a KRAS -directed inhibitor was recently approved by the FDA. Mutations in the KRAS gene have been associated with poor prognosis for lung adenocarcinomas, but implications of the loss of heterozygosity (LOH) of KRAS have not been investigated. In this study, we have assessed the LOH of KRAS in early-stage lung adenocarcinoma by analyzing DNA copy number profiles and have investigated the effect on patient outcome in association with mRNA expression and somatic hotspot mutations. KRAS mutation was present in 36% of cases and was associated with elevated mRNA expression. LOH in KRAS was associated with a favorable prognosis, more prominently in KRAS mutated than in wild-type patients. The presence of both LOH and mutation in KRAS conferred a better prognosis than KRAS mutation alone. For wild-type tumors, no difference in prognosis was observed between patients with and without LOH in KRAS . Our study indicates that LOH in KRAS is an independent prognostic factor that may refine the existing prognostic groups of lung adenocarcinomas., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Khadse, Haakensen, Silwal-Pandit, Hamfjord, Micke, Botling, Brustugun, Lingjærde, Helland and Kure.)

Background: Malignant pleural mesothelioma (MPM) is a rare and aggressive tumour. For patients with inoperable disease, few treatment options are available after first line chemotherapy. The combination of ipilimumab and nivolumab has recently shown increased survival compared to standard chemotherapy, but most patients do not respond and improvements are called for. Telomerase is expressed in mesothelioma cells, but only sparsely in normal tissues and is therefore an attractive target for therapeutic vaccination. Vaccination against telomerase is tolerable and has shown to induce immune responses associated with increased survival in other cancer types. There is a well-founded scientific rationale for the combination of a telomerase vaccine and checkpoint inhibition to improve treatment response in MPM patients., Methods: NIPU is a randomized, multi-centre, open-label, phase II study comparing the efficacy and safety of nivolumab and ipilimumab with or without telomerase vaccine in patients with inoperable malignant pleural mesothelioma after first-line platinum-based chemotherapy. Participants (n = 118) are randomized 1:1 into two treatment arms. All participants receive treatment with nivolumab (240 mg every 2 weeks) and ipilimumab (1 mg/kg every 6 weeks) until disease progression, unacceptable toxicity or for a maximum of 2 years. Patients randomised to the experimental arm receive 8 intradermal injections of UV1 vaccine during the first three months of treatment. Tumour tissue, blood, urine, faeces and imaging will be collected for biomarker analyses and exploration of mechanisms for response and resistance to therapy., Discussion: Checkpoint inhibition is used for treatment of mesothelioma, but many patients still do not respond. Increasing therapy response to immunotherapy is an important goal. Possible approaches include combination with chemotherapy, radiotherapy, targeted therapy and other immunotherapeutic agents. Predictive biomarkers are necessary to ensure optimal treatment for each patient and to prevent unnecessary side effects. This trial seeks to improve treatment response by combining checkpoint inhibition with a telomerase vaccine and also to explore mechanisms for treatment response and resistance. Knowledge gained in the NIPU study may be transferred to the first line setting and to other cancers with limited benefit from immunotherapy., Trial Registration: ClinicalTrials.gov: NCT04300244, registered March 8th, 2020, https://clinicaltrials.gov/ct2/show/NCT04300244?term=NIPU&draw=2&rank=1 .

Anatomical changes during chemoradiation for lung cancer may decrease dose to the target or increase dose to organs at risk. To assess our ability to identify clinically significant anatomical alterations, we followed 67 lung cancer patients by daily cone-beam CT scans to ensure correct patient positioning and observe anatomical alterations. We also re-calculated the original dose distribution on a planned control CT scan obtained halfway during the treatment course to identify anatomical changes that potentially affected doses to the target or organs at risk. Of 66 patients who completed the treatment, 12 patients needed adaptation, two patients were adapted twice. We conclude that daily cone-beam CT and routines at the treatment machine discover relevant anatomical changes during curative radiotherapy for patients with lung cancer without additional imaging., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Author(s).)

Despite significant advancements in breast cancer (BC) research, clinicians lack robust serological protein markers for accurate diagnostics and tumor stratification. Tumor interstitial fluid (TIF) accumulates aberrantly externalized proteins within the local tumor space, which can potentially gain access to the circulatory system. As such, TIF may represent a valuable starting point for identifying relevant tumor-specific serological biomarkers. The aim of the study was to perform comprehensive proteomic profiling of TIF to identify proteins associated with BC tumor status and subtype. A liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of 35 TIFs of three main subtypes: luminal (19), Her2 (4), and triple-negative (TNBC) (12) resulted in the identification of > 8800 proteins. Unsupervised hierarchical clustering segregated the TIF proteome into two major clusters, luminal and TNBC/Her2 subgroups. High-grade tumors enriched with tumor infiltrating lymphocytes (TILs) were also stratified from low-grade tumors. A consensus analysis approach, including differential abundance analysis, selection operator regression, and random forest returned a minimal set of 24 proteins associated with BC subtypes, receptor status, and TIL scoring. Among them, a panel of 10 proteins, AGR3, BCAM, CELSR1, MIEN1, NAT1, PIP4K2B, SEC23B, THTPA, TMEM51, and ULBP2, was found to stratify the tumor subtype-specific TIFs. In particular, upregulation of BCAM and CELSR1 differentiates luminal subtypes, while upregulation of MIEN1 differentiates Her2 subtypes. Immunohistochemistry analysis showed a direct correlation between protein abundance in TIFs and intratumor expression levels for all 10 proteins. Sensitivity and specificity were estimated for this protein panel by using an independent, comprehensive breast tumor proteome dataset. The results of this analysis strongly support our data, with eight of the proteins potentially representing biomarkers for stratification of BC subtypes. Five of the most representative proteomics databases currently available were also used to estimate the potential for these selected proteins to serve as putative serological markers., (© 2020 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Personalised cancer treatment depends on identification of therapeutically relevant biological subgroups of patients for assessing effect of treatment and to discover new therapeutic options. By analyses in heterogeneous patient populations, the effects may be lost in noise. Squamous cell carcinoma of the lung is a major killer worldwide. Despite recent advances, mortality is high and response to therapies varies greatly from patient to patient. Target search in biologically relevant subgroups may identify treatment options not so far discovered. A total of 198 patients undergoing surgery for squamous cell carcinomas of the lung were included in the study. The tumours were analysed for copy number alterations (n = 152) and gene expression from tumour (n = 188) and normal lung (n = 21), with both data levels present in 140 patients. We studied alterations in tumours harbouring mutations in TP53 and in previously published gene expression subtypes. Genes with consistent alterations in both genomic levels were identified as putative biomarkers. Results were validated in TCGA. The most convincing biomarker in TP53 mutated squamous cell carcinomas of the lung was BIRC5 with amplification in 36% of mutated samples, 5% in wild-type samples and a 17%-fold change of expression between TP53 mutated tumours and normal lung tissue. BIRC5 was significantly altered in the classical and primitive subtypes. We suggest BIRC5 as a putative predictive biomarker and putative druggable target in squamous cell lung carcinomas harbouring TP53 mutation or classified as classical and primitive subtypes., (© 2020 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

Background: Studies on tumor-secreted microRNAs point to a functional role of these in cellular communication and reprogramming of the tumor microenvironment. Uptake of tumor-secreted microRNAs by neighboring cells may result in the silencing of mRNA targets and, in turn, modulation of the transcriptome. Studying miRNAs externalized from tumors could improve cancer patient diagnosis and disease monitoring and help to pinpoint which miRNA-gene interactions are central for tumor properties such as invasiveness and metastasis., Methods: Using a bioinformatics approach, we analyzed the profiles of secreted tumor and normal interstitial fluid (IF) microRNAs, from women with breast cancer (BC). We carried out differential abundance analysis (DAA), to obtain miRNAs, which were enriched or depleted in IFs, from patients with different clinical traits. Subsequently, miRNA family enrichment analysis was performed to assess whether any families were over-represented in the specific sets. We identified dysregulated genes in tumor tissues from the same cohort of patients and constructed weighted gene co-expression networks, to extract sets of co-expressed genes and co-abundant miRNAs. Lastly, we integrated miRNAs and mRNAs to obtain interaction networks and supported our findings using prediction tools and cancer gene databases., Results: Network analysis showed co-expressed genes and miRNA regulators, associated with tumor lymphocyte infiltration. All of the genes were involved in immune system processes, and many had previously been associated with cancer immunity. A subset of these, BTLA, CXCL13, IL7R, LAMP3, and LTB, was linked to the presence of tertiary lymphoid structures and high endothelial venules within tumors. Co-abundant tumor interstitial fluid miRNAs within this network, including miR-146a and miR-494, were annotated as negative regulators of immune-stimulatory responses. One co-expression network encompassed differences between BC subtypes. Genes differentially co-expressed between luminal B and triple-negative breast cancer (TNBC) were connected with sphingolipid metabolism and predicted to be co-regulated by miR-23a. Co-expressed genes and TIF miRNAs associated with tumor grade were BTRC, CHST1, miR-10a/b, miR-107, miR-301a, and miR-454., Conclusion: Integration of IF miRNAs and mRNAs unveiled networks associated with patient clinicopathological traits, and underlined molecular mechanisms, specific to BC sub-groups. Our results highlight the benefits of an integrative approach to biomarker discovery, placing secreted miRNAs within a biological context.

Gene expression profiling of tumours is an important source of information for cancer patient stratification. Detecting subtle alterations of gene expression remains a challenge, however. Here, we propose a novel tool for high-sensitivity detection of differential pathway activity in tumours. For a pathway defined by a collection of genes, the samples are projected onto a low-dimensional manifold in the subspace spanned by those genes. For each sample, a score is next found by calculating the distance between each projected sample and the projection of a subgroup of reference samples. Depending on the aim of the analysis and the available data, the reference samples may represent e.g. normal tissue or tumour samples with a particular genotype or phenotype. The proposed tool, PathTracer, is demonstrated on gene expression data from 1952 invasive breast cancer samples, 10 DCIS, 9 benign samples and 144 tumour adjacent normal breast tissue samples. PathTracer scores are shown to predict survival, clinical subtypes, cellular proliferation and genomic instability. Furthermore, predictions are shown to outperform those obtained with other comparable methods.

High mammographic density (MD) is associated with a 4-6 times increase in breast cancer risk. For post-menopausal women, MD often decreases over time, but little is known about the underlying biological mechanisms. MD reflects breast tissue composition, and may be associated with microenvironment subtypes previously identified in tumor-adjacent normal tissue. Currently, these subtypes have not been explored in normal breast tissue. We obtained biopsies from breasts of healthy women at two different time points several years apart and performed microarray gene expression analysis. At time point 1, 65 samples with both MD and gene expression were available. At time point 2, gene expression and MD data were available from 17 women, of which 11 also had gene expression data available from the first time point. We validated findings from our previous study; negative correlation between RBL1 and MD in post-menopausal women, indicating involvement of the TGFβ pathway. We also found that breast tissue samples from women with a large decrease in MD sustained higher expression of genes in the histone family H4. In addition, we explored the previously defined active and inactive microenvironment subtypes and demonstrated that normal breast samples of the active subtype had characteristics similar to the claudin-low breast cancer subtype. Breast biopsies from healthy women are challenging to obtain, but despite a limited sample size, we have identified possible mechanisms relevant for changes in breast biology and MD over time that may be of importance for breast cancer risk and tumor initiation.

Cancers elicit an immune response by modifying the microenvironment. The immune system plays a pivotal role in cancer recognition and eradication. While the potential clinical value of infiltrating lymphocytes at the tumor site has been assessed in breast cancer, circulating cytokines - the molecules coordinating and fine-tuning immune response - are still poorly characterized. Using two breast cancer cohorts (MicMa, n = 131, DCTB, n = 28) and the multiplex Luminex platform, we measured the levels of 27 cytokines in the serum of breast cancer patients prior to treatment. We investigated the cytokine levels in relation to clinicopathological characteristics and in perspective of the tumor infiltrating immune cells predicted from the bulk mRNA expression data. Unsupervised clustering analysis of the serum cytokine levels in the MicMa cohort identified a cluster of pro-inflammatory, pro-angiogenic, and Th2-related cytokines which was associated with poor prognosis. Notably high levels of platelet derived growth factor BB (PDGF) reflected a more aggressive tumor phenotype and larger tumor size. A significant positive correlation between serum levels of interferon gamma-induced protein 10 (IP10) and its mRNA expression at the tumor site suggested that tumor-IP10-production may outflow to the bloodstream. High IP10 serum levels were associated with a worse prognosis. Finally, we found serum levels of both PDGF and IP10 associated with enrichment scores of specific tumor infiltrating immune cells. Our study suggests that monitoring cytokine circulating levels in breast cancer could be used to characterize breast cancers and the immune composition of their microenvironment through readily available biological material.

Particular N-glycan structures are known to be associated with breast malignancies by coordinating various regulatory events within the tumor and corresponding microenvironment, thus implying that N-glycan patterns may be used for cancer stratification and as predictive or prognostic biomarkers. However, the association between N-glycans secreted by breast tumor and corresponding clinical relevance remain to be elucidated. We profiled N-glycans by HILIC UPLC across a discovery dataset composed of tumor interstitial fluids (TIF, n = 85), paired normal interstitial fluids (NIF, n = 54) and serum samples (n = 28) followed by independent evaluation, with the ultimate goal of identifying tumor-related N-glycan patterns in blood of patients with breast cancer. The segregation of N-linked oligosaccharides revealed 33 compositions, which exhibited differential abundances between TIF and NIF. TIFs were depleted of bisecting N-glycans, which are known to play essential roles in tumor suppression. An increased level of simple high mannose N-glycans in TIF strongly correlated with the presence of tumor infiltrating lymphocytes within tumor. At the same time, a low level of highly complex N-glycans in TIF inversely correlated with the presence of infiltrating lymphocytes within tumor. Survival analysis showed that patients exhibiting increased TIF abundance of GP24 had better outcomes, whereas low levels of GP10, GP23, GP38, and coreF were associated with poor prognosis. Levels of GP1, GP8, GP9, GP14, GP23, GP28, GP37, GP38, and coreF were significantly correlated between TIF and paired serum samples. Cross-validation analysis using an independent serum dataset supported the observed correlation between TIF and serum, for five of nine N-glycan groups: GP8, GP9, GP14, GP23, and coreF. Collectively, our results imply that profiling of N-glycans from proximal breast tumor fluids is a promising strategy for determining tumor-derived glyco-signature(s) in the blood. N-glycans structures validated in our study may serve as novel biomarkers to improve the diagnostic and prognostic stratification of patients with breast cancer., (© 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

Using our recently developed high-throughput automated platform, N-glycans from all serum glycoproteins from patients with breast cancer were analysed at diagnosis, after neoadjuvant chemotherapy, surgery, radiotherapy and up to 3 years after surgery. Surprisingly, alterations in the serum N-glycome after chemotherapy were pro-inflammatory with an increase in glycan structures associated with cancer. Surgery, on the other hand, induced anti-inflammatory changes in the serum N-glycome, towards a noncancerous phenotype. At the time of first follow-up, glycosylation in patients with affected lymph nodes changed towards a malignant phenotype. C-reactive protein showed a different pattern, increasing after first line of neoadjuvant chemotherapy, then decreasing throughout treatment until 1 year after surgery. This may reflect a switch from acute to chronic inflammation, where chronic inflammation is reflected in the serum after the acute phase response subsides. In conclusion, we here present the first time-course serum N-glycome profiling of patients with breast cancer during and after treatment. We identify significant glycosylation changes with chemotherapy, surgery and follow-up, reflecting the host response to therapy and tumour removal., (© 2017 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

The tumor microenvironment is now widely recognized for its role in tumor progression, treatment response, and clinical outcome. The intratumoral immunological landscape, in particular, has been shown to exert both pro-tumorigenic and anti-tumorigenic effects. Identifying immunologically active or silent tumors may be an important indication for administration of therapy, and detecting early infiltration patterns may uncover factors that contribute to early risk. Thus far, direct detailed studies of the cell composition of tumor infiltration have been limited; with some studies giving approximate quantifications using immunohistochemistry and other small studies obtaining detailed measurements by isolating cells from excised tumors and sorting them using flow cytometry. Herein we utilize a machine learning based approach to identify lymphocyte markers with which we can quantify the presence of B cells, cytotoxic T-lymphocytes, T-helper 1, and T-helper 2 cells in any gene expression data set and apply it to studies of breast tissue. By leveraging over 2,100 samples from existing large scale studies, we are able to find an inherent cell heterogeneity in clinically characterized immune infiltrates, a strong link between estrogen receptor activity and infiltration in normal and tumor tissues, changes with genomic complexity, and identify characteristic differences in lymphocyte expression among molecular groupings. With our extendable methodology for capturing cell type specific signal we systematically studied immune infiltration in breast cancer, finding an inverse correlation between beneficial lymphocyte infiltration and estrogen receptor activity in normal breast tissue and reduced infiltration in estrogen receptor negative tumors with high genomic complexity., Competing Interests: CONFLICTS OF INTEREST The authors declare that they have no competing interests.

It has been hypothesized based on accumulated data that a class of small noncoding RNAs, termed microRNAs, are key factors in intercellular communication. Here, microRNAs present in interstitial breast tumor fluids have been analyzed to identify relevant markers for a diagnosis of breast cancer and to elucidate the cross-talk that exists among cells in a tumor microenvironment. Matched tumor interstitial fluid samples (TIF, n = 60), normal interstitial fluid samples (NIF, n = 51), corresponding tumor tissue specimens (n = 54), and serum samples (n = 27) were collected from patients with breast cancer, and detectable microRNAs were analyzed and compared. In addition, serum data from 32 patients with breast cancer and 22 healthy controls were obtained for a validation study. To identify potential serum biomarkers of breast cancer, first the microRNA profiles of TIF and NIF samples were compared. A total of 266 microRNAs were present at higher level in the TIF samples as compared to normal counterparts. Sixty-one of these microRNAs were present in > 75% of the serum samples and were subsequently tested in a validation set. Seven of the 61 microRNAs were associated with poor survival, while 23 were associated with the presence of immune cells and adipocytes. To our knowledge, these data demonstrate for the first time that profiling of microRNAs in TIF can identify novel biomarkers for the prognostic classification and detection of breast cancer. In addition, the present findings demonstrate that microRNAs may represent the cross-talk that occurs between tumor cells and their surrounding stroma., (© 2016 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

Development of lung cancer is closely related to smoking in a majority of patients. Most smokers, however, do not develop lung cancer in spite of a high mutational load accumulating in the lung tissue. Here we investigate whether a cancer-specific footprint can be revealed by investigating circulating inflammatory markers in patients with non-small cell lung cancer (NSCLC) compared with patients with chronic obstructive pulmonary disease (COPD), both cohorts characterised by similar smoking history. Serum concentrations of 57 cytokines and matrix metalloproteinases (MMPs) from 43 patients with advanced NSCLC were evaluated by multiplex immunoassays and compared with serum samples from 35 patients with COPD. Unsupervised hierarchical clustering and non-parametric analyses were performed. False discovery rate was used to adjust for multiple testing. Clustering of cytokine and MMP concentrations in the serum revealed a distinct separation of the NSCLC patients from the COPD group. Individual concentrations of thymus and activation-regulated cytokine (C-C motif chemokine ligand 17), Gro-b (C-X-C motif chemokine ligand 2 (CXCL2)), CXCL13, interleukin (IL)-1ra, IL-6, IL-8 (CXCL8), IL-16, IL-17A, macrophage migration inhibitory factor (MIF), granulocyte colony-stimulating factor, platelet-derived growth factor subunit B, MMP-2, MMP-8 and MMP-12 were significantly different in serum from NSCLC and COPD patients. Moreover, the interferon-γ/IL-10 ratio was lower in cancer patients compared with COPD patients, consistent with a cytokine milieu favouring tumour tolerance. Our results suggest that NSCLC is characterised by a distinct inflammatory signature in serum. The different cytokine profiles in NSCLC and COPD patients may represent tumour-promoting and tumour-suppressing immune responses developing in response to mucosal inflammation and mutations induced by smoking.

Robust markers of invasiveness may help reduce the overtreatment of in situ carcinomas. Breast cancer is a heterogeneous disease and biological mechanisms for carcinogenesis vary between subtypes. Stratification by subtype is therefore necessary to identify relevant and robust signatures of invasive disease. We have identified microRNA (miRNA) alterations during breast cancer progression in two separate datasets and used stratification and external validation to strengthen the findings. We analyzed two separate datasets (METABRIC and AHUS) consisting of a total of 186 normal breast tissue samples, 18 ductal carcinoma in situ (DCIS) and 1,338 invasive breast carcinomas. Validation in a separate dataset and stratification by molecular subtypes based on immunohistochemistry, PAM50 and integrated cluster classifications were performed. We propose subtype-specific miRNA signatures of invasive carcinoma and a validated signature of DCIS. miRNAs included in the invasive signatures include downregulation of miR-139-5p in aggressive subtypes and upregulation of miR-29c-5p expression in the luminal subtypes. No miRNAs were differentially expressed in the transition from DCIS to invasive carcinomas on the whole, indicating the need for subtype stratification. A total of 27 miRNAs were included in our proposed DCIS signature. Significant alterations of expression included upregulation of miR-21-5p and the miR-200 family and downregulation of let-7 family members in DCIS samples. The signatures proposed here can form the basis for studies exploring DCIS samples with increased invasive potential and serum biomarkers for in situ and invasive breast cancer., (© 2016 UICC.)

Glycosylation and related processes play important roles in cancer development and progression, including metastasis. Several studies have shown that N-glycans have potential diagnostic value as cancer serum biomarkers. We have explored the significance of the abundance of particular serum N-glycan structures as important features of breast tumour biology by studying the serum glycome and tumour transcriptome (mRNA and miRNA) of 104 breast cancer patients. Integration of these types of molecular data allows us to study the relationship between serum glycans and transcripts representing functional pathways, such as metabolic pathways or DNA damage response. We identified tri antennary trigalactosylated trisialylated glycans in serum as being associated with lower levels of tumour transcripts involved in focal adhesion and integrin-mediated cell adhesion. These glycan structures were also linked to poor prognosis in patients with ER negative tumours. High abundance of simple monoantennary glycan structures were associated with increased survival, particularly in the basal-like subgroup. The presence of circulating tumour cells was found to be significantly associated with several serum glycome structures like bi and triantennary, di- and trigalactosylated, di- and trisialylated. The link between tumour miRNA expression levels and N-glycan production is also examined., (Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Alterations in glycan structures are early signs of malignancy and have recently been proposed to be in part a driving force behind malignant transformation. Here, we explore whether differences in expression of genes related to the process of glycosylation exist between breast carcinoma subtypes - and look for their association to clinical parameters. Five expression datasets of 454 invasive breast carcinomas, 31 ductal carcinomas in situ (DCIS), and 79 non-malignant breast tissue samples were analysed. Results were validated in 1960 breast carcinomas. 419 genes encoding glycosylation-related proteins were selected. The DCIS samples appeared expression-wise similar to carcinomas, showing altered gene expression related to glycosaminoglycans (GAGs) and N-glycans when compared to non-malignant samples. In-situ lesions with different aggressiveness potentials demonstrated changes in glycosaminoglycan sulfation and adhesion proteins. Subtype-specific expression patterns revealed down-regulation of genes encoding glycan-binding proteins in the luminal A and B subtypes. Clustering basal-like samples using a consensus list of genes differentially expressed across discovery datasets produced two clusters with significantly differing prognosis in the validation dataset. Finally, our analyses suggest that glycolipids may play an important role in carcinogenesis of breast tumors - as demonstrated by association of B3GNT5 and UGCG genes to patient survival. In conclusion, most glycan-specific changes occur early in the carcinogenic process. We have identified glycan-related alterations specific to breast cancer subtypes including a prognostic signature for two basal-like subgroups. Future research in this area may potentially lead to markers for better prognostication and treatment stratification of breast cancer patients., (Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

An improved separation of the human serum N-glycome using hydrophilic interaction chromatography technology with UPLC is described, where more than 140 N-glycans were assigned. Using this technique, serum samples from 107 healthy controls and 62 newly diagnosed breast cancer patients were profiled. The most statistically significant alterations were observed in cancer patients compared with healthy controls: an increase in sialylation, branching, and outer-arm fucosylation and a decrease in high-mannosylated and biantennary core-fucosylated glycans. In the controls and cases combined systemic features were analyzed; serum estradiol was associated with increase in digalactosylated glycans, and higher mammographic density was associated with increase in biantennary digalactosylated glycans and with decrease in trisialylated and in outer-arm fucosylated glycans. Furthermore, particular glycans were altered in some features of the breast carcinomas; bisected biantennary nonfucosylated glycans were decreased in patients with progesterone receptor positive tumors, and core-fucosylated biantennary bisected monogalactosylated glycans were decreased in patients with the TP53 mutation. Systemic features show more significant associations with the serum N-glycome than do the features of the breast carcinomas. In conclusion, the UPLC-based glycan analysis technique described here reveals highly significant differences between healthy women and breast cancer patients. Significant associations with breast carcinoma and systemic features are described.

Background: Ductal carcinoma in situ (DCIS) of the breast is a precursor of invasive breast carcinoma. DNA methylation alterations are thought to be an early event in progression of cancer, and may prove valuable as a tool in clinical decision making and for understanding neoplastic development., Results: We generate genome-wide DNA methylation profiles of 285 breast tissue samples representing progression of cancer, and validate methylation changes between normal and DCIS in an independent dataset of 15 normal and 40 DCIS samples. We also validate a prognostic signature on 583 breast cancer samples from The Cancer Genome Atlas. Our analysis reveals that DNA methylation profiles of DCIS are radically altered compared to normal breast tissue, involving more than 5,000 genes. Changes between DCIS and invasive breast carcinoma involve around 1,000 genes. In tumors, DNA methylation is associated with gene expression of almost 3,000 genes, including both negative and positive correlations. A prognostic signature based on methylation level of 18 CpGs is associated with survival of breast cancer patients with invasive tumors, as well as with survival of patients with DCIS and mixed lesions of DCIS and invasive breast carcinoma., Conclusions: This work demonstrates that changes in the epigenome occur early in the neoplastic progression, provides evidence for the possible utilization of DNA methylation-based markers of progression in the clinic, and highlights the importance of epigenetic changes in carcinogenesis.

Early full-term pregnancy is one of the most effective natural protections against breast cancer. To investigate this effect, we have characterized the global gene expression and epigenetic profiles of multiple cell types from normal breast tissue of nulliparous and parous women and carriers of BRCA1 or BRCA2 mutations. We found significant differences in CD44(+) progenitor cells, where the levels of many stem cell-related genes and pathways, including the cell-cycle regulator p27, are lower in parous women without BRCA1/BRCA2 mutations. We also noted a significant reduction in the frequency of CD44(+)p27(+) cells in parous women and showed, using explant cultures, that parity-related signaling pathways play a role in regulating the number of p27(+) cells and their proliferation. Our results suggest that pathways controlling p27(+) mammary epithelial cells and the numbers of these cells relate to breast cancer risk and can be explored for cancer risk assessment and prevention., (Copyright © 2013 Elsevier Inc. All rights reserved.)

We use an integrated approach to understand breast cancer heterogeneity by modeling mRNA, copy number alterations, microRNAs, and methylation in a pathway context utilizing the pathway recognition algorithm using data integration on genomic models (PARADIGM). We demonstrate that combining mRNA expression and DNA copy number classified the patients in groups that provide the best predictive value with respect to prognosis and identified key molecular and stromal signatures. A chronic inflammatory signature, which promotes the development and/or progression of various epithelial tumors, is uniformly present in all breast cancers. We further demonstrate that within the adaptive immune lineage, the strongest predictor of good outcome is the acquisition of a gene signature that favors a high T-helper 1 (Th1)/cytotoxic T-lymphocyte response at the expense of Th2-driven humoral immunity. Patients who have breast cancer with a basal HER2-negative molecular profile (PDGM2) are characterized by high expression of protumorigenic Th2/humoral-related genes (24-38%) and a low Th1/Th2 ratio. The luminal molecular subtypes are again differentiated by low or high FOXM1 and ERBB4 signaling. We show that the interleukin signaling profiles observed in invasive cancers are absent or weakly expressed in healthy tissue but already prominent in ductal carcinoma in situ, together with ECM and cell-cell adhesion regulating pathways. The most prominent difference between low and high mammographic density in healthy breast tissue by PARADIGM was that of STAT4 signaling. In conclusion, by means of a pathway-based modeling methodology (PARADIGM) integrating different layers of molecular data from whole-tumor samples, we demonstrate that we can stratify immune signatures that predict patient survival.

Gene expression studies on breast cancer have generally been performed on tissue obtained at the time of surgery. In this study, we have compared the gene expression profiles in preoperative tissue (core needle biopsies) while tumor is still in its normal milieu to postoperative tissue from the same tumor obtained during surgery. Thirteen patients were included of which eleven had undergone sentinel node diagnosis procedure before operation. Microarray gene expression analysis was performed using total RNA from all the samples. Paired significance analysis of microarrays revealed 228 differently expressed genes, including several early response stress-related genes such as members of the fos and jun families as well as genes of which the expression has previously been associated with cancer. The expression profiles found in the analyses of breast cancer tissue must be evaluated with caution. Different profiles may simply be the result of differences in the surgical trauma and timing of when samples are taken and not necessarily associated with tumor biology.

Background: Increased understanding of the variability in normal breast biology will enable us to identify mechanisms of breast cancer initiation and the origin of different subtypes, and to better predict breast cancer risk., Methods: Gene expression patterns in breast biopsies from 79 healthy women referred to breast diagnostic centers in Norway were explored by unsupervised hierarchical clustering and supervised analyses, such as gene set enrichment analysis and gene ontology analysis and comparison with previously published genelists and independent datasets., Results: Unsupervised hierarchical clustering identified two separate clusters of normal breast tissue based on gene-expression profiling, regardless of clustering algorithm and gene filtering used. Comparison of the expression profile of the two clusters with several published gene lists describing breast cells revealed that the samples in cluster 1 share characteristics with stromal cells and stem cells, and to a certain degree with mesenchymal cells and myoepithelial cells. The samples in cluster 1 also share many features with the newly identified claudin-low breast cancer intrinsic subtype, which also shows characteristics of stromal and stem cells. More women belonging to cluster 1 have a family history of breast cancer and there is a slight overrepresentation of nulliparous women in cluster 1. Similar findings were seen in a separate dataset consisting of histologically normal tissue from both breasts harboring breast cancer and from mammoplasty reductions., Conclusion: This is the first study to explore the variability of gene expression patterns in whole biopsies from normal breasts and identified distinct subtypes of normal breast tissue. Further studies are needed to determine the specific cell contribution to the variation in the biology of normal breasts, how the clusters identified relate to breast cancer risk and their possible link to the origin of the different molecular subtypes of breast cancer.

Background: High serum levels of estradiol are associated with increased risk of postmenopausal breast cancer. Little is known about the gene expression in normal breast tissue in relation to levels of circulating serum estradiol., Methods: We compared whole genome expression data of breast tissue samples with serum hormone levels using data from 79 healthy women and 64 breast cancer patients. Significance analysis of microarrays (SAM) was used to identify differentially expressed genes and multivariate linear regression was used to identify independent associations., Results: Six genes (SCGB3A1, RSPO1, TLN2, SLITRK4, DCLK1, PTGS1) were found differentially expressed according to serum estradiol levels (FDR = 0). Three of these independently predicted estradiol levels in a multivariate model, as SCGB3A1 (HIN1) and TLN2 were up-regulated and PTGS1 (COX1) was down-regulated in breast samples from women with high serum estradiol. Serum estradiol, but none of the differentially expressed genes were significantly associated with mammographic density, another strong breast cancer risk factor. In breast carcinomas, expression of GREB1 and AREG was associated with serum estradiol in all cancers and in the subgroup of estrogen receptor positive cases., Conclusions: We have identified genes associated with serum estradiol levels in normal breast tissue and in breast carcinomas. SCGB3A1 is a suggested tumor suppressor gene that inhibits cell growth and invasion and is methylated and down-regulated in many epithelial cancers. Our findings indicate this gene as an important inhibitor of breast cell proliferation in healthy women with high estradiol levels. In the breast, this gene is expressed in luminal cells only and is methylated in non-BRCA-related breast cancers. The possibility of a carcinogenic contribution of silencing of this gene for luminal, but not basal-like cancers should be further explored. PTGS1 induces prostaglandin E2 (PGE2) production which in turn stimulates aromatase expression and hence increases the local production of estradiol. This is the first report studying such associations in normal breast tissue in humans.

Glycosylation is the stepwise procedure of covalent attachment of oligosaccharide chains to proteins or lipids, and alterations in this process have been associated with malignant transformation. Simultaneous analysis of the expression of all glycan-related genes clearly gives the advantage of enabling a comprehensive view of the genetic background of the glycobiological changes in cancer cells. Studies focusing on the expression of the whole glycome have now become possible, which prompted us to review the present knowledge on glycosylation in relation to breast cancer diagnosis and progression, in the light of available expression data from tumors and breast tissue of healthy individuals. We used various data resources to select a set of 419 functionally relevant genes involved in synthesis, degradation and binding of N-linked and O-linked glycans, Lewis antigens, glycosaminoglycans (chondroitin, heparin and keratan sulfate in addition to hyaluronan) and glycosphingolipids. Such glycans are involved in a number of processes relevant to carcinogenesis, including regulation of growth factors/growth factor receptors, cell-cell adhesion and motility as well as immune system modulation. Expression analysis of these glycan-related genes revealed that mRNA levels for many of them differ significantly between normal and malignant breast tissue. An associative analysis of these genes in the context of current knowledge of their function in protein glycosylation and connection(s) to cancer indicated that synthesis, degradation and adhesion mediated by glycans may be altered drastically in mammary carcinomas. Although further analysis is needed to assess how changes in mRNA levels of glycan genes influence a cell's glycome and the precise role that such altered glycan structures play in the pathogenesis of the disease, lessons drawn from this study may help in determining directions for future research in the rapidly-developing field of glycobiology., (Copyright 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Introduction: Mammographic density (MD), as assessed from film screen mammograms, is determined by the relative content of adipose, connective and epithelial tissue in the female breast. In epidemiological studies, a high percentage of MD confers a four to six fold risk elevation of developing breast cancer, even after adjustment for other known breast cancer risk factors. However, the biologic correlates of density are little known., Methods: Gene expression analysis using whole genome arrays was performed on breast biopsies from 143 women; 79 women with no malignancy (healthy women) and 64 newly diagnosed breast cancer patients, both included from mammographic centres. Percent MD was determined using a previously validated, computerized method on scanned mammograms. Significance analysis of microarrays (SAM) was performed to identify genes influencing MD and a linear regression model was used to assess the independent contribution from different variables to MD., Results: SAM-analysis identified 24 genes differentially expressed between samples from breasts with high and low MD. These genes included three uridine 5'-diphospho-glucuronosyltransferase (UGT) genes and the oestrogen receptor gene (ESR1). These genes were down-regulated in samples with high MD compared to those with low MD. The UGT gene products, which are known to inactivate oestrogen metabolites, were also down-regulated in tumour samples compared to samples from healthy individuals. Several single nucleotide polymorphisms (SNPs) in the UGT genes associated with the expression of UGT and other genes in their vicinity were identified., Conclusions: Three UGT enzymes were lower expressed both in breast tissue biopsies from healthy women with high MD and in biopsies from newly diagnosed breast cancers. The association was strongest amongst young women and women using hormonal therapy. UGT2B10 predicts MD independently of age, hormone therapy and parity. Our results indicate that down-regulation of UGT genes in women exposed to female sex hormones is associated with high MD and might increase the risk of breast cancer.