In this study, we evaluated the ability of gene expression profiles to predict chemotherapy response and survival in triple-negative breast cancer (TNBC).
Gene expression and clinical–pathological data were evaluated in five independent cohorts, including three randomised clinical trials for a total of 1055 patients with TNBC, basal-like disease (BLBC) or both. Previously defined intrinsic molecular subtype and a proliferation signature were determined and tested. Each signature was tested using multivariable logistic regression models (for pCR (pathological complete response)) and Cox models (for survival). Within TNBC, interactions between each signature and the basal-like subtype (vs other subtypes) for predicting either pCR or survival were investigated.
Within TNBC, all intrinsic subtypes were identified but BLBC predominated (55–81%). Significant associations between genomic signatures and response and survival after chemotherapy were only identified within BLBC and not within TNBC as a whole. In particular, high expression of a previously identified proliferation signature, or low expression of the luminal A signature, was found independently associated with pCR and improved survival following chemotherapy across different cohorts. Significant interaction tests were only obtained between each signature and the BLBC subtype for prediction of chemotherapy response or survival.
The proliferation signature predicts response and improved survival after chemotherapy, but only within BLBC. This highlights the clinical implications of TNBC heterogeneity, and suggests that future clinical trials focused on this phenotypic subtype should consider stratifying patients as having BLBC or not.
breast cancer; genomics; subtypes; intrinsic; basal like; chemotherapy; neoadjuvant
Paclitaxel-induced neuropathy is an adverse event that often leads to therapeutic disruption and patient discomfort. We attempted to replicate a previously reported association between increased neuropathy risk and CYP2C8*3 genotype.
Patients and methods
Demographic, treatment, and toxicity data were collected for paclitaxel-treated breast cancer patients who were genotyped for the CYP2C8*3 K399R (rs10509681) variant. A log-rank test was used in the primary analysis of European-American patients. An additional independent replication was then attempted in a cohort of African-American patients, followed by modeling of the entire patient cohort with relevant covariates.
In the primary analysis of 209 European patients, there was an increased risk of paclitaxel-induced neuropathy related to CYP2C8*3 status [HR (per allele) = 1.93 (95% CI: 1.05–3.55), overall log-rank P = 0.006]. The association was replicated in direction and magnitude of effect in 107 African-American patients (P = 0.043). In the Cox model using the entire mixed-race cohort (n = 411), each CYP2C8*3 allele approximately doubled the patient's risk of grade 2+ neuropathy (P = 0.004), and non-Europeans were at higher neuropathy risk than Europeans of similar genotype (P = 0.030).
The increased risk of paclitaxel-induced neuropathy in patients who carry the CYP2C8*3 variant was replicated in two racially distinct patient cohorts.
chemotherapy-induced peripheral neuropathy; cytochrome P450 2C8*3; paclitaxel; pharmacogenetics; race
Increased plasma IL-6 levels have been associated with HIV-1 disease progression risk, yet the drivers of IL-6 production in HIV-1 infection are not known. This study was designed to explore the relationship between HIV-1 replication and IL-6 induction.
Correlations between plasma levels of IL-6 and HIV-1 RNA were examined in two clinical studies. To more directly assess the induction of IL-6 by HIV-1, several cell and tissue types that support HIV-1 replication in vivo were infected with HIV-1 and expression of IL-6 was measured.
Spearman’s rank correlations were used to examine the relationship between plasma levels of IL-6 and HIV-1 RNA. Macrophages, and colonic and lymph node histocultures were infected with HIV-1 or stimulated with bacterial products, LPS or flagellin, and IL-6 levels in supernatant were measured by ELISA or multiplex bead assay.
In the clinical studies there was weak or no correlation between plasma levels of IL-6 and HIV-1 RNA but IL-6 levels were correlated with plasma levels of the LPS coreceptor CD14. Macrophages stimulated with LPS or flagellin showed robust production of IL-6, but there was no increase in IL-6 production after HIV-1 infection. IL-6 expression was not increased in lymph node histocultures obtained from HIV-1 infected subjects nor after productive HIV-1 infection of colonic or lymph node histocultures ex vivo.
We find no evidence that HIV-1 replication is an important driver of IL-6 expression in vivo or in in vitro systems.
IL-6; HIV-1 RNA; histocultures; macrophages; LPS; flagellin
The attribution of incentive salience to reward associated cues is critical for motivation and the pursuit of rewards. Disruptions in the integrity of the neural systems controlling these processes can lead to avolition and anhedonia, symptoms that cross the diagnostic boundaries of many neuropsychiatric illnesses. Here, we consider whether the habenula (Hb), a region recently demonstrated to encode negatively valenced events, also modulates the attribution of incentive salience to a neutral cue predicting a food reward. The Pavlovian autoshaping paradigm was used in the rat as an investigative tool to dissociate Pavlovian learning processes imparting strictly predictive value from learning that attributes incentive motivational value. Electrolytic lesions of the fasciculus retroflexus (fr), the sole pathway through which descending Hb efferents are conveyed, significantly increased incentive salience as measured by conditioned approaches to a cue light predictive of reward. Conversely, generation of a fictive Hb signal via fr stimulation during CS+ presentation significantly decreased the incentive salience of the predictive cue. Neither manipulation altered the reward predictive value of the cue as measured by conditioned approach to the food. Our results provide new evidence supporting a significant role for the Hb in governing the attribution of incentive motivational salience to reward predictive cues and further imply that pathological changes in Hb activity could contribute to the aberrant pursuit of debilitating goals or avolition and depression-like symptoms.
sign-tracking; goal-tracking; mesolimbic; dopamine; autoshape; motivation
Pseudomonas aeruginosa is a major cause of blindness and visual impairment in the United States and worldwide. Using a murine model of keratitis in which abraded corneas are infected with P. aeruginosa parent and ΔfliC (aflagellar) strains 19660 and PAO1, we found that F4/80+ macrophages were the predominant cell type in the cornea expressing TLR2, TLR4, and TLR5. Depletion of macrophages and dendritic cells using transgenic Mafia mice, in which Fas ligand is selectively activated in these cells, resulted in diminished cytokine production and cellular infiltration to the corneal stroma and unimpaired bacterial growth. TLR4−/− mice showed a similar phenotype postinfection with ΔfliC strains, whereas TLR4/5−/− mice were susceptible to corneal infection with parent strains. Bone marrow-derived macrophages stimulated with ΔfliC bacteria induced Toll/IL-1R intracellular domain (TIR)-containing adaptor inducing IFN-β (TRIF)-dependent phosphorylation of IFN regulatory factor 3 in addition to TIR-containing adaptor protein/MyD88-dependent phosphorylation of IκB and nuclear translocation of the p65 subunit of NFκB. Furthermore, TRIF−/− mice showed a similar phenotype as TLR4−/− mice in regulating only ΔfliC bacteria, whereas MyD88−/− mice were unable to clear parent or ΔfliC bacteria. Finally, IL-1R1−/− and IL-1α/β−/− mice were highly susceptible to infection. Taken together, these findings indicate that P. aeruginosa activates TLR4/5 on resident corneal macrophages, which signal through TRIF and TIR-containing adaptor protein/MyD88 pathways, leading to NF-κB translocation to the nucleus, transcription of CXCL1 and other CXC chemokines, recruitment of neutrophils to the corneal stroma, and subsequent bacterial killing and tissue damage. IL-1α and IL-1β are also produced, which activate an IL-1R1/MyD88-positive feedback loop in macrophages and IL-1R on other resident cells in the cornea.
ER-positive (ER+ ) breast cancer includes all of the intrinsic molecular subtypes, although the luminal A and B subtypes predominate. In this study, we evaluated the ability of six clinically relevant genomic signatures to predict relapse in patients with ER+ tumors treated with adjuvant tamoxifen only.
Four microarray datasets were combined and research-based versions of PAM50 intrinsic subtyping and risk of relapse (PAM50-ROR) score, 21-gene recurrence score (OncotypeDX), Mammaprint, Rotterdam 76 gene, index of sensitivity to endocrine therapy (SET) and an estrogen-induced gene set were evaluated. Distant relapse-free survival (DRFS) was estimated by Kaplan–Meier and log-rank tests, and multivariable analyses were done using Cox regression analysis. Harrell's C-index was also used to estimate performance.
All signatures were prognostic in patients with ER+ node-negative tumors, whereas most were prognostic in ER+ node-positive disease. Among the signatures evaluated, PAM50-ROR, OncotypeDX, Mammaprint and SET were consistently found to be independent predictors of relapse. A combination of all signatures significantly increased the performance prediction. Importantly, low-risk tumors (>90% DRFS at 8.5 years) were identified by the majority of signatures only within node-negative disease, and these tumors were mostly luminal A (78%–100%).
Most established genomic signatures were successful in outcome predictions in ER+ breast cancer and provided statistically independent information. From a clinical perspective, multiple signatures combined together most accurately predicted outcome, but a common finding was that each signature identified a subset of luminal A patients with node-negative disease who might be considered suitable candidates for adjuvant endocrine therapy alone.
breast cancer; genomics; luminal; mammaprint; oncotype; PAM50
The neonatal immune system is believed to be biased towards T helper type 2 (Th2) immunity, but under certain conditions neonates can also develop Th1 immune responses. Neonatal Th2 immunity to myelin antigens is not pathogenic and can prevent induction of experimental autoimmune encephalomyelitis (EAE) in adulthood, but the consequences of neonatally induced Th1 immunity to self antigens have remained unresolved.
Here, we show that neonatal injection of mice with myelin antigens emulsified in complete Freunds’ adjuvant (CFA) induced vigorous production of IFN-γ and IL-17, but not IL-5, consistent with myelin-specific Th1/Th17 immunity. Importantly, the myelin-specific Th1/Th17 cells persisted in the mice until adulthood without causing symptoms of EAE. Intraperitoneal, but not subcutaneous injection of neonates with myelin antigens protected against induction of EAE as adults. Intraperitoneally injected neonates showed a substantial decrease of the number and avidity of myelin-reactive Th17 cells, suggesting a decrease in IL-17 producing precursor cells as the mechanism of protection from EAE upon reinjection with myelin antigens as adults. The results could provide a rationale for the presence of autoreactive T cells found in healthy human individuals without autoimmune disease.
Multiple sclerosis; neonates; Th1; Th2; Th17
Immunization with vaccinia virus causes long-term immunity. Efforts have been made to characterize the T cells responsible for this protection. Recently, T cell subsets were described that not only co-express multiple cytokines, but also show increased per cell cytokine productivity. These highly productive cells are often considered to be the most protective. We used ELISPOT assays to measure per cell IFN-γ productivity of vaccinia specific T cells in childhood immunized adults immediately before and at different time points after vaccinia re-vaccination. Apart from an increase in frequency, we found a marked increase of IFN-γ productivity following vaccinia re-vaccination. However, these changes were short-lived as both parameters quickly returned to baseline values within 22 days after re-vaccination. Therefore, increased per cell IFN-γ productivity seems to be a sign of recent in vivo T cell activation rather than a stable marker of a distinct T cell subset responsible for long-term immune protection.
Vaccinia; T cell memory; Interferon gamma; Cytokine productivity; ELISPOT
For immune diagnostic purposes it would be critical to be able to distinguish between ongoing immune processes, such as active infections, and long-term immune memory, for example imprinted by infections that have been cleared a long time ago or by vaccinations. We tested the hypothesis that the secretion of Granzyme B, as detected in ex vivo ELISPOT assays, permits this distinction. We studied EBV-, flu- and CMV-specific CD8+ cells in healthy individuals, Vaccinia virus-reactive CD8+ cells in the course of vaccination, and HIV-specific CD8+ cells in HIV-infected individuals. Antigen-specific ex vivo GzB production was detected only transiently after Vaccinia immunization, and in HIV-infected individuals. Our data suggest that ex vivo ELISPOT measurements of granzyme B permit the identification of actively ongoing CD8+ cell responses – a notion that is pertinent to the immune diagnostic of infections, transplantation, allergies, autoimmune diseases, tumors, and vaccine development.
cytokines; cytotoxicity; CD8+ T cells; ELISPOT; granzyme B; interferon-γ; immunodeficiency; memory; vaccination; viral infections
Elevated circulating levels of chemokines have been reported in patients with dengue fever and are proposed to contribute to the pathogenesis of dengue disease. To establish in vitro models for chemokine induction by dengue 2 virus (DEN2V), we studied a variety of human cell lines and primary cells. DEN2V infection of HepG2 and primary dendritic cells induced the production of interleukin-8 (IL-8), RANTES, MIP-1α, and MIP-1β, whereas only IL-8 and RANTES were induced following dengue virus infection of HEK293 cells. Chemokine secretion was accompanied by an increase in steady-state mRNA levels. No chemokine induction was observed in HEK293 cells treated with poly(I:C) or alpha interferon, suggesting a direct effect of virus infection. To determine the mechanism(s) involved in the induction of chemokine production by DEN2V, individual dengue virus genes were cloned into plasmids and expressed in HEK293 cells. Transfection of a plasmid expressing NS5 or a dengue virus replicon induced IL-8 gene expression and secretion. RANTES expression was not induced under these conditions, however. Reporter assays showed that IL-8 induction by NS5 was principally through CAAT/enhancer binding protein, whereas DEN2V infection also induced NF-κB. These results indicate a role for the dengue virus NS5 protein in the induction of IL-8 by DEN2V infection. Recruitment and activation of potential target cells to sites of DEN2V replication by virus-induced chemokine production may contribute to viral replication as well as to the inflammatory components of dengue virus disease.
Antiviral roles of natural killer (NK) cell subsets were examined in C57BL/6 mice infected with murine cytomegalovirus (MCMV) and other viruses, including lymphocytic choriomeningitis virus (LCMV), vaccinia virus (VV), and mouse hepatitis virus (MHV). Each virus vigorously induced an NK cell infiltrate into the peritoneal cavity and liver, causing some redistributions of NK cell subsets defined by monoclonal antibody (mAb) directed against Ly49A, C/I, D, and G2. Striking results were seen with a mAb (1F8) reactive with the positively signaling molecule Ly49H, present in MCMV-resistant C57BL/6 mice. mAb 1F8 also stains Ly49 C and I, but exclusion of those reactivities with mAb 5E6, which recognizes Ly49 C and I, indicated that Ly49H+ cells infiltrated the peritoneal cavity and liver and were particularly effective at synthesizing interferon γ. Depletion of 1F8+ but not 5E6+ cells in vivo by mAb injections enhanced MCMV titers by 20-1,000-fold in the spleen and approximately fivefold in the liver. Titers of LCMV or VV were not enhanced. These anti-MCMV effects were attributed to prototypical NK1.1+CD3− NK cells and not to NK1.1+CD3+ “NK/T” cells. This is the first evidence that control of a virus infection in vivo is mediated by a distinct NK cell subset.
NK cells; NK receptors; Ly49; murine cytomegalovirus; IFN-γ
Escherichia coli Fis is a small DNA binding and bending protein that has been implicated in a variety of biological processes. A minimal promoter sequence consisting of 43 bp is sufficient to generate its characteristic growth phase-dependent expression pattern and is also subject to negative regulation by stringent control. However, information about the precise identification of nucleotides contributing to basal promoter activity and its regulation has been scant. In this work, 72 independent mutations were generated in the fis promoter (fis P) region from −108 to +78 using both random and site-directed PCR mutagenesis. β-Galactosidase activities from mutant promoters fused to the (trp-lac)W200 fusion on a plasmid were used to conclusively identify the sequences TTTCAT and TAATAT as the −35 and −10 regions, respectively, which are optimally separated by 17 bp. We found that four consecutive substitutions within the GC-rich sequence just upstream of +1 and mutations in the −35 region, but not in the −10 region, significantly reduced the response to stringent control. Analysis of the effects of mutations on growth phase-dependent regulation showed that replacing the predominant transcription initiation nucleotide +1C with a preferred nucleotide (A or G) profoundly altered expression such that high levels of fis P mRNA were detected during late logarithmic and early stationary phases. A less dramatic effect was seen with improvements in the −10 and −35 consensus sequences. These results suggest that the acute growth phase-dependent regulation pattern observed with this promoter requires an inefficient transcription initiation process that is achieved with promoter sequences deviating from the −10 and −35 consensus sequences and, more importantly, a dependence upon the availability of the least favored transcription initiation nucleotide, CTP.
Heat shock proteins (hsp's) isolated from murine cancer cells can elicit protective immunity and specific cytotoxic T lymphocytes (CTLs) by channeling tumor-derived peptides bound to hsp's to the major histocompatibility class I antigen presentation pathway. Here we have investigated if hsp70 can be used in a novel peptide vaccine for the induction of protective antiviral immunity and memory CTLs. A CTL epitope from the well-defined lymphocytic choriomeningitis virus (LCMV) system was mixed with recombinant hsp70 in vitro under conditions that optimize peptide binding to hsp70. Mice were immunized with the hsp70–peptide mixture and challenged with LCMV. Virus titers were reduced 10–100-fold in these mice compared to control mice. Immunization with the hsp70–peptide mixture resulted in the development of CTL memory cells that could be reactivated during LCMV infection, and that in a 51Cr-release assay could lyse cells pulsed with the same peptide, but not cells pulsed with another LCMV peptide. These results show that hsp70 can be used with CTL epitopes to induce efficient protective antiviral immunity and the generation of peptide-specific CTLs. The results also demonstrate the usefulness of hsp70 as an alternative to adjuvants and DNA vectors for the delivery of CTL epitopes to antigen-presenting cells.
Human sera contain high levels of natural antibody (Ab) to Galα1-3Gal, a terminal glycosidic structure expressed on the surface of cells of mammals other than Old World primates. Incorporation of this determinant onto retroviral membranes by passage of viruses in cells encoding α-1-3-galactosyltransferase (GT) renders retroviruses sensitive to lysis by natural Ab and complement in normal human serum (NHS). Plasma membrane-budding viruses representing four additional virus groups were examined for their sensitivities to serum inactivation after passage through human cell lines that lack a functional GT or human cells expressing recombinant porcine GT. The inactivation of lymphocytic choriomeningitis virus (LCMV) by NHS directly correlated with host modification of the virus via expression of Galα1-3Gal and was blocked by incorporation of soluble Galα1-3Gal disaccharide into the inactivation assay. GT-deficient mice immunized to make high levels of Ab to Galα1-3Gal (anti-Gal Ab) were tested for resistance to LCMV passaged in GT-expressing cells. Resistance was not observed, but in vitro analyses of the mouse immune sera revealed that the antiviral activity of the sera was insufficient to eliminate LCMV infectivity on its natural targets of infection, macrophages, which express receptors for Ab and complement. Newcastle disease virus and vesicular stomatitis virus (VSV) were inactivated by NHS regardless of cell passage history, whereas Sindbis virus (SV) passaged in human cells resisted inactivation. Both VSV and SV passaged in Galα1-3Gal-expressing human cells incorporated this sugar moiety onto their major envelope glycoproteins. SV passaged in mouse cells expressing Galα1-3Gal was moderately sensitive to inactivation by NHS. These results indicate that enveloped viruses expressing Galα1-3Gal differ in their sensitivities to NHS and that a potent complement source, such as that in NHS, is required for efficient inactivation of sensitive viruses in vitro and in vivo.
OBJECTIVE: To determine the effectiveness of a health check and assess any particular benefits resulting from feedback of plasma cholesterol concentration or coronary risk score, or both. DESIGN: Randomised controlled trial in two Glasgow work sites. SUBJECTS: 1,632 employees (89% male) aged 20 to 65 years. INTERVENTIONS: At the larger work site, (a) health education; (b) health education and feedback on cholesterol concentration; (c) health education and feedback on risk score; (d) health education with feedback on cholesterol concentration and risk score (full health check); (e) no health intervention (internal control). At the other work site there was no health intervention (external control). MAIN OUTCOME MEASURES: Changes in Dundee risk score, plasma cholesterol concentration, diastolic blood pressure, body mass index and self-reported behaviours (smoking, exercise, alcohol intake, and diet) in comparison with internal and external control groups. RESULTS: Comparisons between the full health check and the internal control groups showed a small difference (0.13 mmol/l) in the change in mean cholesterol concentration (95% confidence interval 0.02 to 0.22, P = 0.02) but no significant differences for changes in Dundee risk score (P = 0.21), diastolic blood pressure (P = 0.71), body mass index (P = 0.16), smoking (P = 1.00) or exercise (P = 0.41). Significant differences between the two groups were detected for changes in self-reported consumption of alcohol (41% in group with full health check v 17% in internal control group, P = 0.001) fruit and vegetables (24% v 12%, P < 0.001), and fat (30% v 9%, P < 0.001). Comparison of all groups showed no advantage from feedback of cholesterol concentration or risk score, or both. CONCLUSIONS: The health check only had a small effect on reversible coronary risk. It was effective in influencing self reported alcohol consumption and diet. Feedback on cholesterol concentration and on risk score did not provide additional motivation for a change in behaviour.