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BACKGROUND The molecular breast cancer subgroups comprise diverse immune biology; for human epidermal growth factor receptor 2-positive (HER2+) breast cancer (BC) and triple-negative breast cancer (TNBC) abundance of tumour infiltrating lymphocytes (TILs) have been shown to indicate good prognosis and could predict pathological complete response after neoadjuvant chemotherapy. In contrast, the clinical relevance of TILs in estrogen receptor-positive/HER2-negative (ER+/HER2-) tumours is not settled. We primary aimed to analyse the prognostic effect of TILs on BC-free interval (BCFi) in premenopausal patients, stratified by molecular subgroups. The secondary aim was to investigate the predictive value of TILs on tamoxifen benefit. METHODS Archival tissue from primary tumours was collected from patients included in the SBII:2pre trial. In this study, 564 premenopausal women were randomised between two years of adjuvant tamoxifen or no systemic treatment regardless of hormone-receptor status. TILs were scored on whole tissue sections and the tumours were divided into ER+/HER2- (n = 255), HER2+ (n = 65) and TNBC (n = 95) molecular subgroups by immunohistochemistry and in situ hybridisation (ISH). The prognostic value of TILs was evaluated in patients allocated to no systemic therapy, whose tumours were successfully scored for TILs and had IHC/ISH data for generating molecular subgroups (n = 221). All patients with ER+ tumours and successfully annotated TILs were considered for prediction of tamoxifen benefit (n = 321). The median follow-up was 28 years and the prognostic and predictive analyses were performed by cumulative incidence curves and Cox regression analyses. RESULTS TILs were successfully scored in 477 tumours with available ER status and the proportion of low ( CONCLUSIONS We demonstrate a long-term favorable prognostic value of levels of TILs in a cohort of premenopausal BC patients. This effect seems to be extended to the ER+/HER2- subgroup. A beneficial effect of tamoxifen in ER+ patients was observed in patients with tumours of low TILs infiltration as compared to patients with tumours of high immune infiltration, but we could not confirm a treatment predictive effect. Table 1. Uni- and multivariable Cox regression analyses of BCFi and OS (control arm)Univariable*MultivariableEndpointBCFiOSBCFiOSHR (95% CI); P valueTILs, category***All molecular subgroups(n = 221)(n = 213)Low (Ref.)1.001.001.001.00Intermediate1.10 (0.78-1.54); 0.611.26 (0.88-1.80); 0.210.61 (0.40-0.93); 0.020.65 (0.41-1.02); 0.06High0.40 (0.22-0.71); 0.0020.52 (0.29-0.95); 0.030.22 (0.11-0.43); 20 mm vs. ≤20), NHG (1 vs. 2 vs. 3), ER (positive vs. negative), PR (positive vs. negative), HER2 (positive vs. negative), LVI (present vs. absent) and TILs (high vs. intermediate vs. low). Stratified by Region.*** TILs were categorised as low: Citation Format: Christine Lundgren, Pär-Ola Bendahl, Maria Ekholm, Mårten Fernö, Carina Forsare, Ute Krüger, Bo Nordenskjöld, Olle Stål, Lisa Rydén. Tumour infiltrating lymphocytes of prognostic value in different molecular breast cancer subgroups and as a suggestive predictive factor for adjuvant tamoxifen benefit in premenopausal patients after 30 years follow-up [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS5-09.

Background: Use of menopausal hormone therapy (MHT) is associated with increased risk for breast cancer. However, the relevant mechanisms and its interaction with genetic variants are not fully understood. Methods: We conducted a genome-wide interaction analysis between MHT use and genetic variants for breast cancer risk in 27,585 cases and 34,785 controls from 26 observational studies. All women were post-menopausal and of European ancestry. Multivariable logistic regression models were used to test for multiplicative interactions between genetic variants and current MHT use. We considered interaction p-values-8 as genome-wide significant, and p-values-5 as suggestive. Linkage disequilibrium (LD)-based clumping was performed to identify independent candidate variants.Results: None of the 9.7 million genetic variants tested for interactions with MHT use reached genome-wide significance. Only 213 variants, representing 18 independent loci, had p-values5. The strongest evidence was found for rs4674019 (p-value=2.27x10-7), which showed genome-wide significant interaction (p-value=3.8x10-8) with current MHT use when analysis was restricted to population-based studies only. Limiting the analyses to combined estrogen-progesterone MHT use only or to estrogen receptor (ER) positive cases did not identify any genome-wide significant evidence of interactions. Conclusions: In this large genome-wide SNP-MHT interaction study of breast cancer, we found no strong support for common genetic variants modifying the effect of MHT on breast cancer risk. These results suggest that common genetic variation has limited impact on the observed MHT–breast cancer risk association.

PAM50 intrinsic subtyping and risk of recurrence (ROR) score are approved for risk profiling in postmenopausal women. We aimed to examine their long-term prognostic value in terms of breast cancer-free interval (BCFi) and overall survival (OS) (n = 437) in premenopausal women randomised to 2 years of tamoxifen versus no systemic treatment irrespective of hormone-receptor status. Intrinsic subtyping added independent prognostic information in patients with oestrogen receptor-positive/human epidermal growth factor 2-negative tumours for BCFi and OS after maximum follow-up (overall P-value 0.02 and 0.006, respectively) and those with high versus low ROR had worse prognosis (maximum follow-up: hazard ratio (HR)BCFi: 1.70, P = 0.04). The prognostic information by ROR was similar regarding OS and in multivariable analysis. These results support that PAM50 subtyping and ROR score provide long-term prognostic information in premenopausal women. Moreover, tamoxifen reduced the incidence of breast cancer events only in patients with Luminal APAM50 tumours (0–10 years: HRBCFi(Luminal A): 0.41, HRBCFi(Luminal B): 1.19, Pinteraction = 0.02).Trial registration: This trial is registered in the ISRCTN database, trial ID: ISRCTN12474687.

Background Tumour-infiltrating lymphocytes (TILs) are of important prognostic and predictive value in human epidermal growth factor receptor 2-positive (HER2+) breast cancer (BC) and triple-negative breast cancer (TNBC), but their clinical relevance in oestrogen receptor-positive/HER2-negative (ER+/HER2−) remains unknown. The primary study aim was to analyse the prognostic effect of TILs on the BC-free interval (BCFi) in premenopausal patients stratified by BC subtypes. The secondary aim was to investigate if TILs are predictive of tamoxifen (TAM) benefit. Methods Archival tissues from primary breast tumours were collected from patients from the SBII:2pre trial, in which 564 premenopausal women were randomised to 2 years of adjuvant TAM or no systemic treatment, regardless of hormone receptor status. TILs were scored on whole tissue sections from 447 patients with available ER status. Tumours were divided into ER+/HER2−, HER2+ and TNBC subtypes by immunohistochemistry and in situ hybridisation. The prognostic value of TILs was analysed in systemically untreated patients (n = 221); the predictive information was investigated in the ER+ subgroup (n = 321) by cumulative incidence curves and Cox regression analyses. The median follow-up was 28 years. Results High (≥ 50%) infiltration of TILs was a favourable prognostic factor in terms of BCFi (univariable analysis: hazard ratioBCFi (HRBCFi) 0.40; 95% confidence interval (CI) 0.22–0.71; P = 0.002). Similar effects were observed across all BC subtypes. The effect of adjuvant TAM was stronger in patients with ER+ tumours and TILs BCFi 0.63; 95% CI 0.47–0.84; P = 0.002) than in patients with high immune infiltration (≥ 50%) (HRBCFi 0.84; 95% CI (0.24–2.86); P = 0.77). However, evidence for differential effects of TAM in categories of TILs, i.e. interaction, was weak. Conclusions We demonstrate a long-term favourable prognostic value of high infiltration of TILs in a cohort of premenopausal BC patients and the positive prognostic effect was extended to the ER+/HER2− subgroup. A beneficial effect of TAM in ER+ patients was observed in patients with tumours of low TIL infiltration, but evidence for a treatment predictive effect was weak. Trial registration This trial is registered in the ISRCTN database, trial ID: ISRCTN12474687.

PURPOSE The Luminal intrinsic subtypes and Luminal-like surrogate subtypes of breast cancer are based on gene expression and pathological markers, respectively. We aimed to study the prognostic value of PAM50 intrinsic subtypes and risk of recurrence (ROR) score in premenopausal women with early breast cancer. Prognostic differences of Luminal PAM50 vs. surrogate subtyping, and the predictive effect of Luminal PAM50 subtypes on tamoxifen benefit were further evaluated. METHODS PAM50 intrinsic subtypes and ROR score (n = 437) were determined by gene expression analyses (NanoString’s Breast Cancer 360 TM assay (BC360)) after RNA isolation of breast cancer tissues from patients included in the randomised SBII:2pre trial. Premenopausal women were randomised between two years of adjuvant tamoxifen vs. control. Surrogate subtyping was performed according to the St. Gallen 2013 classification. The endpoints breast cancer-free interval (BCFi) and overall survival (OS) (median follow-up: 28 and 33 years, respectively) were evaluated using the maximum follow-up and the two disjoint time intervals 0-10 years and >10 years. RESULTS After maximum follow-up, patients with Luminal B tumours by PAM50 subtyping had worse prognosis as compared to those with Luminal A tumours (Hazard ratio (HR)BCfi 1.60; 95% confidence interval (CI) 1.17-2.18; P = 0.004; Table 1). For this time interval, high vs. low ROR score was associated with higher incidence of breast cancer events (HRBCFi 1.70; 95% CI 1.01-2.85; P = 0.04), with a similar trend seen in node-negative patients. In total, 58% of tumours classified as Luminal B-like by surrogate subtyping were re-classified into Luminal A by PAM50 subtyping. At 10 years follow-up, patients whose tumours were classified as Luminal B-like/Luminal A, had a better prognosis as compared to those uniformly classified as Luminal B-like/Luminal B (HRBCFi 0.52; 95% CI 0.33-0.83; P = 0.006) but the effect weakened with longer follow-up (Table 1). According to 10 years data, the incidence of breast cancer events was reduced by two thirds by tamoxifen in patients with PAM50 Luminal A tumours, while no effect was seen in those with Luminal B tumours (HRBCFi 0.41 and HRBCFi 1.19, respectively, (Pinteraction = 0.02); Table 2). CONCLUSIONS PAM50 and ROR score provided prognostic information also in premenopausal women. More than 50% of patients with Luminal B-like tumours defined by surrogate subtyping were re-classified as Luminal A according to PAM50 subtyping. These patients had an improved prognosis at 10 years of follow-up, but the effect attenuated with time. At 10 years, PAM50 Luminal A, but not Luminal B, intrinsic subtype was associated with tamoxifen benefit. Table 1.Prognostic effect of PAM50 intrinsic subtypes and St. Gallen 2013 surrogate subtyping regarding BCFi and OS for different time intervals (uni- and multivariable analyses)UnivariableMultivariableaBCFiOSBCFiOSHR (95% CI); P valuePAM50 intrinsic subtype0–10 years(n = 437, n = 218 events)(n = 437, n = 176 events)(n = 411, n = 203 events)(n = 411, n = 166 events)LumA (Ref.)1.001.001.001.00LumB1.88 (1.31–2.71); 0.0012.33 (1.52–3.58); 10 yearsb(n = 210, n = 68 events)(n = 261, n = 124 events)(n = 199, n = 66 events)(n = 245, n = 119 events)LumA (Ref.)1.001.001.001.00LumB1.05 (0.55–2.00); 0.890.85 (0.52–1.39), 0.501.92 (0.92–4.01); 0.081.16 (0.66–2.04); 0.62HER2-E0.51 (0.20–1.29); 0.150.70 (0.38–1.29); 0.251.27 (0.33–4.90): 0.731.77 (0.76–4.14); 0.19Basal-like0.95 (0.50–1.82); 0.880.69 (0.40–1.17); 0.172.58 (0.94–7.08); 0.071.90 (0.86–4.18); 0.11Maximum follow-up timec(n = 437, n = 286 events)(n = 437, n = 300 events)(n = 411, n = 269 events)(n = 411, n = 285 events)LumA (Ref.)1.001.001.001.00LumB1.60 (1.17–2.18); 0.0041.41 (1.03–1.93); 0.031.66 (1.17–2.37), 0.0051.48 (1.04–2.10); 0.03HER2-E1.84 (1.32–2.56); 10 yearsb(n = 117, n = 42 events)(n = 147, n = 70 events)(n = 115, n = 41 events)(n = 145, n = 70 events)LumB-like/LumB (Ref.)1.001.001.001.00LumB-like/LumA1.30 (0.62–2.69); 0.491.11 (0.62–1.98); 0.720.64 (0.26–1.60); 0.340.59 (0.30–1.15); 0.12LumA-like/LumB----LumA-like/LumA0.45 (0.16–1.23); 0.120.81 (0.41–1.59); 0.540.26 (0.08–0.80); 0.020.39 (0.18–0.84); 0.02Maximum follow-up timec(n = 207, n = 129 events)(n = 207, n = 130 events)(n = 205, n = 128 events)(n = 205, n = 130 events)LumB-like/LumB (Ref.)1.001.001.001.00LumB-like/LumA0.70 (0.47–1.02); 0.060.65 (0.44–0.96); 0.030.58 (0.37–0.90); 0.020.50 (0.32–0.79); 0.003LumA-like/LumB0.34 (0.08–1.41); 0.140.37 (0.09–1.52); 0.170.53 (0.12–2.24); 0.380.48 (0.11–2.05): 0.32LumA-like/LumA0.39 (0.23–0.67); 0.0010.51 (0.32–0.83); 0.0070.39 (0.24–0.70); 0.0020.43 (0.25–0.74); 0.003Abbreviations: BCFi, breast cancer free-interval; CI, confidence interval; HR, hazard ratio; Lum, Luminal; NHG, Nottingham histological grade; OS, overall survival; TAM, tamoxifenaAll analyses were stratified by study region and adjusted for age (continuous), tumour size (>20 mm vs. ≤20), NHG (1 vs. 2 vs. 3), nodal status (N0 vs. N1 vs. N2) and treatment arm in addition to surrogate/molecular subtype b10 years–maximum follow-up time, starting at year 10c32 and 36 years regarding BCFi and OS, respectively Table 2.Predictive value (univariable analyses) of Lum PAM50 intrinsic subtypes for TAM response with respect to BCFi and OS (ER-positive/HER2-negative cohort)BCFiOSHR (95% CI); P value0-10 years(n = 217, n = 92 events)(n = 217, n = 64 events)TAM vs. control in Lum A0.41 (0.23-0.74); 0.0030.61 (0.30-1.26); 0.18TAM vs. control in Lum B1.19 (0.63-2.27); 0.591.76 (0.85-3.63); 0.13Interaction Lum PAM50 subtype x TAM (HR ratio)0.34 (0.14-0.83); 0.020.35 (0.13-0.97); 0.04>10 yearsa(n = 121, n = 43 events)(n = 153, n = 74 events)TAM vs. control in Lum A0.69 (0.35-1.37); 0.290.74 (0.44-1.25); 0.26TAM vs. control in Lum B0.17 (0.04-0.80); 0.030.25 (0.08-0.77); 0.02Interaction Lum PAM50 subtype x TAM (HR ratio)4.05 (0.74-22.1); 0.112.95 (0.85-10.2); 0.09Maximum follow-up timeb(n = 217, n = 135 events)(n = 217, n = 138 events)TAM vs. control in Lum A0.52 (0.34-0.81); 0.0040.71 (0.46-1.08); 0.11TAM vs. control in Lum B0.80 (0.45-1.41); 0.440.87 (0.49-1.54); 0.63Interaction Lum PAM50 subtype x TAM (HR ratio)0.65 (0.32-1.34); 0.240.82 (0.40-1.65); 0.57Abbreviations: BCFi, breast cancer-free interval; CI, confidence interval; ER, oestrogen receptor; HER2, human epidermal growth factor receptor 2; HR, hazard ratio; Lum, Luminal; TAM, tamoxifenAll analyses were stratified by study regiona10 years-maximum follow-up time, starting at year 10b32 and 36 years regarding BCFi and OS, respectively Citation Format: Christine Lundgren, Pär-Ola Bendahl, Maria Ekholm, Mårten Fernö, Carina Forsare, Ute Krüger, Bo Nordenskjöld, Olle Stål, Lisa Rydén. Prognostic and tamoxifen-predictive effect of PAM50 and ROR score in premenopausal women included in the randomised SBII:2 trial [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD9-05.

Background: We examined the associations between germline variants and breast cancer mortality using a large meta-analysis of women of European ancestry. Methods: Meta-analyses included summary estimates based on Cox models of twelve datasets using ~10.4 million variants for 96,661 women with breast cancer and 7697 events (breast cancer-specific deaths). Oestrogen receptor (ER)-specific analyses were based on 64,171 ER-positive (4116) and 16,172 ER-negative (2125) patients. We evaluated the probability of a signal to be a true positive using the Bayesian false discovery probability (BFDP). Results: We did not find any variant associated with breast cancer-specific mortality at P < 5 × 10 −8 . For ER-positive disease, the most significantly associated variant was chr7:rs4717568 (BFDP = 7%, P = 1.28 × 10 −7 , hazard ratio [HR] = 0.88, 95% confidence interval [CI] = 0.84–0.92); the closest gene is AUTS2. For ER-negative disease, the most significant variant was chr7:rs67918676 (BFDP = 11%, P = 1.38 × 10 −7 , HR = 1.27, 95% CI = 1.16–1.39); located within a long intergenic non-coding RNA gene (AC004009.3), close to the HOXA gene cluster. Conclusions: We uncovered germline variants on chromosome 7 at BFDP < 15% close to genes for which there is biological evidence related to breast cancer outcome. However, the paucity of variants associated with mortality at genome-wide significance underpins the challenge in providing genetic-based individualised prognostic information for breast cancer patients.

INTRODUCTION:Physical activity (PA) leads to improved survival in women following the diagnosis of breast cancer, but it is less clear whether PA has equally positive effects regardless of age at diagnosis. The purpose of our study was to evaluate the association between post-diagnosis PA and survival in women aged below or over 55 years at diagnosis.METHODS:From a prospective population-based cohort of Swedish women, we included 847 women, aged 34-84 years, who were diagnosed with breast cancer from 1992 to 2012. A PA score was calculated based on three different questions regarding self-reported PA. Cox proportional hazard model was used to estimate the association between PA and mortality.RESULTS:A significant association between PA score and all-cause mortality was observed, in a dose-response manner (ptrend = 0.01). The mortality was clearly lower in the most active compared to the least active group (hazard ratio 0.29, 95% confidence intervals 0.09-0.90). A subgroup analysis showed that the improved survival was only seen in women over 55 years of age at diagnosis.CONCLUSION:Physical activity, which is a modifiable lifestyle factor, should be encouraged after breast cancer diagnosis, especially in women with post-menopausal breast cancer.

Breast cancer risk is influenced by rare coding variants in susceptibility genes, such as BRCA1, and many common, mostly non-coding variants. However, much of the genetic contribution to breast cancer risk remains unknown. Here we report the results of a genome-wide association study of breast cancer in 122,977 cases and 105,974 controls of European ancestry and 14,068 cases and 13,104 controls of East Asian ancestry. We identified 65 new loci that are associated with overall breast cancer risk at P

To characterize the mechanisms of complement activation by human immunodeficiency virus type 1 (HIV-1)-infected cells, Cl-4 cells stably expressing the envelope glycoproteins of HIV-1 and the parent African green monkey cell line CV-1 were tested for C1q binding and complement activation. While the parent cell line CV-1 only showed a weak spontaneous activation of the alternative pathway, Cl-4 cells additionally triggered the classical pathway of complement activation independent of anti-HIV antibodies by direct C1q binding. Earlier studies had shown different complement activating potential of cells infected with various HIV isolates. Recombinant soluble CD4-induced shedding of gp120 from the surface of HIV-1-infected cells converted a weak activator isolate (MVP-899) into a strong complement activator. The increase in complement activation was paralleled by the concomitant unmasking of a previously hidden gp41 epitope comprising the major complement-activating domain of gp41 (aa. 601-613). Our results strongly suggest that the transmembrane protein gp41 induces the activation of complement on the surface of infected cells as has been described previously for purified HIV-1 virions. Furthermore, we present evidence that the different potential of HIV isolates to activate the complement system on the cell surface is caused by different degrees of spontaneous gp120 shedding by various HIV isolates.

In order to mimic the histidine binding motives of naturally occurring histones as DNA complexing proteins, hyperbranched poly(ethylene imine) and polyglycerol were functionalized with imidazole or 3-dimethylamino propyl groups. These new polycationic polymers were tested for interaction with dye-labelled oligonucleotide and DNA using UV and fluorescence spectroscopy and gel electrophoresis. Formation of stable complexes was observed above N/P ratios of 4 for unfunctionalized and 8 for functionalized PEIs. No stable complexes were formed with polyglycerol-based polyamines up to N/P 16. Cytotoxicity determined by MTT assay of all functionalized PEI polymers was found to be significantly lower than for unfunctionalized PEI. PG-based polymers showed no toxicity in the tested concentration range. Dynamic light scattering showed that only for PEI(21)-Imidaz polyplexes hydrodynamic diameters below 250 nm could be reached.The influence of functionalization and polymer type on transfection efficiency was evaluated in L929, NIH/3T3 and HeLa cells. Only imidazole-functionalized PEIs reached similar transfection efficiencies as unfunctionalized PEIs, while 3-dimethylamino propyl modification resulted in lower transfection efficiencies. We also demonstrated that the polymer plays an important role for transfection properties since, regardless of the modifications of polyglycerol, only low transfection efficiencies were observed at functionalization levels below 50%.

Polycationic dendrimers are interesting nonviral vectors for in vitro DNA delivery. We describe a simple approach to the synthesis of dendritic polyamines with different molecular weights and adjustable flexibility (degrees of branching; DB). Both parameters influence the transfection efficiency and the cell toxicity of the polymer. Functionalization of hyperbranched polyethylenimine (PEI) by a two-step procedure generated fully branched pseudodendrimers (analogues of polypropylenimine (PPI) and polyamidoamine (PAMAM) dendrimers). The DNA transfection efficiencies observed for these polymers depended on the cell line investigated. The highest efficiencies were observed for polymers whose unfunctionalized PEI cores had molecular weights in the range M(w)=6000-25 000 g mol(-1). The cytotoxicity of the dendrimers generally rises with increasing core size. The data collected for NIH/3T3 and COS-7 cells indicate a maximum transfection efficiency at around 60 % branching for the PPI analogues, and at a PEI-core molecular weight of M(w)=25 000 g mol(-1). PAMAM functionalization of PEI (M(w)=5000 and 21 000 g mol(-1)) leads to polymers with little or no cytotoxity in the cell lines investigated.

To gain more insight into the processes leading to HIV-1 Env-induced cell death, we aim to coexpress stably wild-type and relevant mutant variants of both HIV-1 Env and human CD4 in lymphocyte cell lines. Here we report on the generation and characterization of several cell lines inducibly or constitutively expressing wild-type or cleavage-defective HIV-1 glycoproteins and human CD4 either singly or in combination. Coexpression of CD4 and wild-type Env led to the formation of multinucleated syncytia, to growth arrest and cell death, effects that all could be prevented by cultivation in the presence of monoclonal antibodies that inhibit cell surface membrane fusion. Cell lines coexpressing CD4 and mutated, noncleavable Env, detectable at the cell surface and still retaining CD4-binding capacity, were not retarded in their growth and cytolysis did not occur. These results indicate that cell lysis requires cell surface interaction of CD4 and gp120/41 and cleavage of gp160 to gp120 and gp41.