DNA methylation changes in response to neoadjuvant chemotherapy are associated with breast cancer survival

Background: Locally advanced breast cancer is a heterogeneous disease with respect to response to neoadjuvant chemotherapy (NACT) and survival. It is currently not possible to accurately predict who will benefit from the specific types of NACT. DNA methylation is an epigenetic mechanism known to play an important role in regulating gene expression and may serve as a biomarker for treatment response and survival. We investigated the potential role of DNA methylation as a prognostic marker for long-term survival (> 5 years) after NACT in breast cancer. Methods: DNA methylation profiles of pre-treatment (n = 55) and post-treatment (n = 75) biopsies from 83 women with locally advanced breast cancer were investigated using the Illumina HumanMethylation450 BeadChip. The patients received neoadjuvant treatment with epirubicin and/or paclitaxel. Linear mixed models were used to associate DNA methylation to treatment response and survival based on clinical response to NACT (partial response or stable disease) and 5-year survival, respectively. LASSO regression was performed to identify a risk score based on the statistically significant methylation sites and Kaplan–Meier curve analysis was used to estimate survival probabilities using ten years of survival follow-up data. The risk score developed in our discovery cohort was validated in an independent validation cohort consisting of paired pre-treatment and post-treatment biopsies from 85 women with locally advanced breast cancer. Patients included in the validation cohort were treated with either doxorubicin or 5-FU and mitomycin NACT. Results: DNA methylation patterns changed from before to after NACT in 5-year survivors, while no significant changes were observed in non-survivors or related to treatment response. DNA methylation changes included an overall loss of methylation at CpG islands and gain of methylation in non-CpG islands, and these changes affected genes linked to transcription factor activity, cell adhesion and immune functions. A risk score was developed based on four methylation sites which successfully predicted long-term survival in our cohort (p = 0.0034) and in an independent validation cohort (p = 0.049). Conclusion: Our results demonstrate that DNA methylation patterns in breast tumors change in response to NACT. These changes in DNA methylation show potential as prognostic biomarkers for breast cancer survival.publishedVersio

Safety and efficacy of fluoxetine on functional outcome after acute stroke (AFFINITY): a randomised, double-blind, placebo-controlled trial

Background Trials of fluoxetine for recovery after stroke report conflicting results. The Assessment oF FluoxetINe In sTroke recoverY (AFFINITY) trial aimed to show if daily oral fluoxetine for 6 months after stroke improves functional outcome in an ethnically diverse population. Methods AFFINITY was a randomised, parallel-group, double-blind, placebo-controlled trial done in 43 hospital stroke units in Australia (n=29), New Zealand (four), and Vietnam (ten). Eligible patients were adults (aged ≥18 years) with a clinical diagnosis of acute stroke in the previous 2–15 days, brain imaging consistent with ischaemic or haemorrhagic stroke, and a persisting neurological deficit that produced a modified Rankin Scale (mRS) score of 1 or more. Patients were randomly assigned 1:1 via a web-based system using a minimisation algorithm to once daily, oral fluoxetine 20 mg capsules or matching placebo for 6 months. Patients, carers, investigators, and outcome assessors were masked to the treatment allocation. The primary outcome was functional status, measured by the mRS, at 6 months. The primary analysis was an ordinal logistic regression of the mRS at 6 months, adjusted for minimisation variables. Primary and safety analyses were done according to the patient's treatment allocation. The trial is registered with the Australian New Zealand Clinical Trials Registry, ACTRN12611000774921. Findings Between Jan 11, 2013, and June 30, 2019, 1280 patients were recruited in Australia (n=532), New Zealand (n=42), and Vietnam (n=706), of whom 642 were randomly assigned to fluoxetine and 638 were randomly assigned to placebo. Mean duration of trial treatment was 167 days (SD 48·1). At 6 months, mRS data were available in 624 (97%) patients in the fluoxetine group and 632 (99%) in the placebo group. The distribution of mRS categories was similar in the fluoxetine and placebo groups (adjusted common odds ratio 0·94, 95% CI 0·76–1·15; p=0·53). Compared with patients in the placebo group, patients in the fluoxetine group had more falls (20 [3%] vs seven [1%]; p=0·018), bone fractures (19 [3%] vs six [1%]; p=0·014), and epileptic seizures (ten [2%] vs two [<1%]; p=0·038) at 6 months. Interpretation Oral fluoxetine 20 mg daily for 6 months after acute stroke did not improve functional outcome and increased the risk of falls, bone fractures, and epileptic seizures. These results do not support the use of fluoxetine to improve functional outcome after stroke

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

MR metabolic characterization of locally advanced breast cancer: – treatment effects and prognosis

Brystkreft er den hyppigste kreftsykdommen blant kvinner. Lokalavansert brystkreft utgjør omtrent 10% av alle brystkrefttilfeller og omfatter en heterogen pasientgruppe med ulike prognoser. Pasienter med lokalavansert brystkreft får ofte kjemoterapi før kirurgisk fjerning av tumor, såkalt neoadjuvant kjemoterapi, for å redusere størrelsen på tumoren. Det er stor variasjon i behandlingsrespons for denne pasientgruppen, og det er derfor behov for å utvikle målrettet og individualisert behandling, samt metoder for oppfølging av behandlingsrespons. Metabolomics er en systematisk analyse av småmolekylære forbindelser (metabolitter) i biologiske prøver. Den metabolske profilen til brystkreftvev er vist å korrelere med viktige kliniske parametere, for eksempel tumorgrad, hormonreseptorstatus og lymfeknutespredning. Magnetisk resonans (MR) spektroskopi er en metode som kan gi en detaljert beskrivelse av metabolittprofilen i vevet. En fordel med denne metoden er at vevsprøven er intakt etter analysen slik at den samme vevsprøven kan analyseres videre med andre metoder, for eksempel immunohistokjemi, genuttrykk- eller proteinanalyse. En sentral gruppe metabolitter innenfor brystkreftforskning er kolinforbindelser. Disse metabolittene er viktig for celledeling, signaloverføring, lipidmetabolisme og cellens membranstruktur. Laktat er en annen viktig metabolitt som inngår i energimetabolismen. I dette arbeidet ble vevsprøver fra pasienter med lokalavansert brystkreft analysert ved bruk av MR spektroskopi for prediksjon av behandlingsrespons og overlevelse. I tillegg undersøkte vi rollen til glycerophosphodiester phosphodiesterase (GDPD) i regulering av kolinforbindelser Alle pasienter hadde en effekt av behandlingen som ble gitt, og nesten alle fikk en reduksjon i tumorstørrelse etter behandling. Resultatene viste ingen metabolske forskjeller mellom pasienter med klinisk god eller dårlig behandlingsrespons. Resultatene viser derimot at de metabolske forandringene som skjer under neoadjuvant kjemoterapi er forskjellig i pasienter som overlever mer enn fem år og de som dør før fem år. Høyere nivå av laktat, glycine og kolinforbindelser etter behandling var forbundet med dårlig prognose. Analysene av GDPD5 tyder på at enzymet er involvert i reguleringen av kolinmetabolismen, men dets rolle for bruk i målrettet terapi er fortsatt uklart og nye studier må til for å undersøke dette. MR metabolomics kan brukes til å undersøke metabolske forandringer under neoadjuvant kjemoterapi behandling og kan identifisere viktige metabolitter for prediksjon av overlevelse hos pasienter med lokalavansert brystkreft

Metabolic characterization of triple negative breast cancer

Background The aims of this study were to characterize the metabolite profiles of triple negative breast cancer (TNBC) and to investigate the metabolite profiles associated with human epidermal growth factor receptor-2/neu (HER-2) overexpression using ex vivo high resolution magic angle spinning magnetic resonance spectroscopy (HR MAS MRS). Metabolic alterations caused by the different estrogen receptor (ER), progesterone receptor (PgR) and HER-2 receptor statuses were also examined. To investigate the metabolic differences between two distinct receptor groups, TNBC tumors were compared to tumors with ERpos/PgRpos/HER-2pos status which for the sake of simplicity is called triple positive breast cancer (TPBC). Methods The study included 75 breast cancer patients without known distant metastases. HR MAS MRS was performed for identification and quantification of the metabolite content in the tumors. Multivariate partial least squares discriminant analysis (PLS-DA) modeling and relative metabolite quantification were used to analyze the MR data. Results Choline levels were found to be higher in TNBC compared to TPBC tumors, possibly related to cell proliferation and oncogenic signaling. In addition, TNBC tumors contain a lower level of Glutamine and a higher level of Glutamate compared to TPBC tumors, which indicate an increase in glutaminolysis metabolism. The development of glutamine dependent cell growth or “Glutamine addiction” has been suggested as a new therapeutic target in cancer. Our results show that the metabolite profiles associated with HER-2 overexpression may affect the metabolic characterization of TNBC. High Glycine levels were found in HER-2pos tumors, which support Glycine as potential marker for tumor aggressiveness. Conclusions Metabolic alterations caused by the individual and combined receptors involved in breast cancer progression can provide a better understanding of the biochemical changes underlying the different breast cancer subtypes. Studies are needed to validate the potential of metabolic markers as targets for personalized treatment of breast cancer subtypes

Historical Biobanks in Breast Cancer Metabolomics — Challenges and Opportunities

Background: Metabolomic characterization of tumours can potentially improve prediction of cancer prognosis and treatment response. Here, we describe efforts to validate previous metabolomic findings using a historical cohort of breast cancer patients and discuss challenges with using older biobanks collected with non-standardized sampling procedures. Methods: In total, 100 primary breast cancer samples were analysed by high-resolution magic angle spinning magnetic resonance spectroscopy (HR MAS MRS) and subsequently examined by histology. Metabolomic profiles were related to the presence of cancer tissue, hormone receptor status, T-stage, N-stage, and survival. RNA integrity number (RIN) and metabolomic profiles were compared with an ongoing breast cancer biobank. Results: The 100 samples had a median RIN of 4.3, while the ongoing biobank had a significantly higher median RIN of 6.3 (p = 5.86 × 10−7). A low RIN was associated with changes in choline-containing metabolites and creatine, and the samples in the older biobank showed metabolic differences previously associated with tissue degradation. The association between metabolomic profile and oestrogen receptor status was in accordance with previous findings, however, with a lower classification accuracy. Conclusions: Our findings highlight the importance of standardized biobanking procedures in breast cancer metabolomics studies

DNA methylation changes in response to neoadjuvant chemotherapy are associated with breast cancer survival

Background Locally advanced breast cancer is a heterogeneous disease with respect to response to neoadjuvant chemotherapy (NACT) and survival. It is currently not possible to accurately predict who will benefit from the specific types of NACT. DNA methylation is an epigenetic mechanism known to play an important role in regulating gene expression and may serve as a biomarker for treatment response and survival. We investigated the potential role of DNA methylation as a prognostic marker for long-term survival (> 5 years) after NACT in breast cancer. Methods DNA methylation profiles of pre-treatment (n = 55) and post-treatment (n = 75) biopsies from 83 women with locally advanced breast cancer were investigated using the Illumina HumanMethylation450 BeadChip. The patients received neoadjuvant treatment with epirubicin and/or paclitaxel. Linear mixed models were used to associate DNA methylation to treatment response and survival based on clinical response to NACT (partial response or stable disease) and 5-year survival, respectively. LASSO regression was performed to identify a risk score based on the statistically significant methylation sites and Kaplan–Meier curve analysis was used to estimate survival probabilities using ten years of survival follow-up data. The risk score developed in our discovery cohort was validated in an independent validation cohort consisting of paired pre-treatment and post-treatment biopsies from 85 women with locally advanced breast cancer. Patients included in the validation cohort were treated with either doxorubicin or 5-FU and mitomycin NACT. Results DNA methylation patterns changed from before to after NACT in 5-year survivors, while no significant changes were observed in non-survivors or related to treatment response. DNA methylation changes included an overall loss of methylation at CpG islands and gain of methylation in non-CpG islands, and these changes affected genes linked to transcription factor activity, cell adhesion and immune functions. A risk score was developed based on four methylation sites which successfully predicted long-term survival in our cohort (p = 0.0034) and in an independent validation cohort (p = 0.049). Conclusion Our results demonstrate that DNA methylation patterns in breast tumors change in response to NACT. These changes in DNA methylation show potential as prognostic biomarkers for breast cancer survival

Relevance of CO2-based IAQ indicators: Feedback from long-term monitoring of three nearly zero-energy houses

International audienceNowadays, many countries include requirements for building airtightness in their current national regulations or energy-efficiency programs, mainly for concern about reducing building energy consumption due to air leakage. Moreover, more and more countries impose a mandatory justification with an air leakage measurement at building commissioning. Therefore, the uncertainty of the measurements results has become a key concern in several countries over the past year. More specifically, the influence of wind speed has been identified as one of the major sources of error on the measurement result. The goal of this paper is to present the experimental facility we design and built to improve the uncertainty estimates and the measurement protocol based on model scale experiments in controlled laboratory conditions. We first present the similarity criteria we identified for our model scale experiment. Secondly, we present the experimental design. Finally, we characterize the wind speed inside the wind tunnel and we present the preliminary results regarding the reproduction of fan pressurization tests on the model for different leakage distributions

Twelve-Month Outcomes of the AFFINITY Trial of Fluoxetine for Functional Recovery After Acute Stroke: AFFINITY Trial Steering Committee on Behalf of the AFFINITY Trial Collaboration

Background and Purpose: The AFFINITY trial (Assessment of Fluoxetine in Stroke Recovery) reported that oral fluoxetine 20 mg daily for 6 months after acute stroke did not improve functional outcome and increased the risk of falls, bone fractures, and seizures. After trial medication was ceased at 6 months, survivors were followed to 12 months post-randomization. This preplanned secondary analysis aimed to determine any sustained or delayed effects of fluoxetine at 12 months post-randomization. Methods: AFFINITY was a randomized, parallel-group, double-blind, placebo-controlled trial in adults (n=1280) with a clinical diagnosis of stroke in the previous 2 to 15 days and persisting neurological deficit who were recruited at 43 hospital stroke units in Australia (n=29), New Zealand (4), and Vietnam (10) between 2013 and 2019. Participants were randomized to oral fluoxetine 20 mg once daily (n=642) or matching placebo (n=638) for 6 months and followed until 12 months after randomization. The primary outcome was function, measured by the modified Rankin Scale, at 6 months. Secondary outcomes for these analyses included measures of the modified Rankin Scale, mood, cognition, overall health status, fatigue, health-related quality of life, and safety at 12 months. Results: Adherence to trial medication was for a mean 167 (SD 48) days and similar between randomized groups. At 12 months, the distribution of modified Rankin Scale categories was similar in the fluoxetine and placebo groups (adjusted common odds ratio, 0.93 [95% CI, 0.76–1.14]; P =0.46). Compared with placebo, patients allocated fluoxetine had fewer recurrent ischemic strokes (14 [2.18%] versus 29 [4.55%]; P =0.02), and no longer had significantly more falls (27 [4.21%] versus 15 [2.35%]; P =0.08), bone fractures (23 [3.58%] versus 11 [1.72%]; P =0.05), or seizures (11 [1.71%] versus 8 [1.25%]; P =0.64) at 12 months. Conclusions: Fluoxetine 20 mg daily for 6 months after acute stroke had no delayed or sustained effect on functional outcome, falls, bone fractures, or seizures at 12 months poststroke. The lower rate of recurrent ischemic stroke in the fluoxetine group is most likely a chance finding. REGISTRATION: URL: http://www.anzctr.org.au/ ; Unique identifier: ACTRN12611000774921