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Mutational analysis of circulating tumour (ct) DNA holds promise as an effective tool to predict the course of metastatic breast cancer (MBC). In the present study we used targeted next generation sequencing of ctDNA to evaluate the impact of cancer driven mutations on the prognosis of MBC. The study included 59 oestrogen receptor-positive (ER+), HER2-negative MBC patients. Sequencing analysis was performed in ESR1, PIK3CA, ERBB2, PTEN, TP53, KRAS, HRAS, NRAS, and AR. At baseline, patients started receiving either chemotherapy (34%; n = 20) or cyclin-dependent kinase 4/6 inhibitor therapy in combination with endocrine therapy (CDK4/6i+ET; 66%; n = 39). Overall, 64.4% (n = 38) of the patients carried at least one pathogenic or likely-pathogenic mutation. Number of ctDNA mutations was significantly linked with worse progression free survival (PFS; p  = 0.003) and overall survival (OS; p  = 0.007). Furthermore, ctDNA load, defined by the number of mutant ctDNA molecules per mL plasma, significantly correlated with PFS ( p  < 0.001) and OS ( p  = 0.001). Furthermore, mutational status of ESR1 and TP53 significantly predicted PFS ( p  = 0.024 and p  = 0.035, respectively) and OS ( p  < 0.001 and p  = 0.035, respectively). These results emphasizes the clinical value of ctDNA mutational analysis in the management of advanced breast cancer.

Background and Objectives: Diffuse large B-cell lymphomas (DLBCLs) are heterogeneous neoplasms. CD79B and MYD88 mutations are associated with the activated B-cell-like (ABC) subtype of DLBCL and often co-occur and lead to constitutive activation of the NF-κB pathway. Several different genetic classifications to date have recognized CD79B- and MYD88-mutated DLBCLs as a unique subtype with poor response to therapy and unfavorable survival. However, little is known about gene expression in DLBCLs with mutated CD79B (and MYD88) in comparison to their wild type counterparts. The objective of this study was to compare the gene expression in DLBCLs according to their CD79B mutational status. Methods: A total of 48 primary, treatment-naïve DLBCLs (CD79B-mutated: 35%/n = 17, CD79B-wild type: 65%/n = 31) were investigated using RNA expression profiling (770 genes), followed by immunohistochemical analysis of the up-regulated genes and survival analysis. Results: The gene expression analysis revealed that downstream of CD79B CARD11 and the NF-κB targets NFKBIZ, IL10, IL12A, PIM1 and BCL2A1 were up-regulated in CD79B-mutated DLBCLs. The strongest up-regulation was detected for ARNT2 and WNT11. Other up-regulated genes included the apoptosis-related BID and granzyme B, as well as genes of cell cycle regulation such as RUNX1, RUNX1T1 and RASGRF1. Up-regulation was also found for IL7, STAT3, MLLT4, CD14 and the HSP90B1 subunit. TP53 mutation showed an association with poorer overall survival in a secondary analysis, consistent with prior reports, while survival by CD79B/MYD88 mutation status and the differentially expressed genes showed no significant differences in this cohort. Conclusions: In conclusion, the current study identified novel up-regulated genes in CD79B-mutated DLBCLs beyond NF-κB pathway signaling, which may contribute to a better definition of potential therapeutic targets and further improves the characterization of this distinct and aggressive DLBCL subgroup.

Changes in cytoplasmic Ca2+ levels regulate a variety of fundamental cellular functions in virtually all cells. In nonexcitable cells, a major pathway of Ca2+ entry involves receptor-mediated depletion of intracellular Ca2+ stores followed by the activation of store-operated calcium channels in the plasma membrane. We have established a mouse line expressing an activating EF hand motif mutant of stromal interaction molecule 1 (Stim1), an ER receptor recently identified as the Ca2+ sensor responsible for activation of Ca2+ release-activated (CRAC) channels in T cells, whose function in mammalian physiology is not well understood. Mice expressing mutant Stim1 had macrothrombocytopenia and an associated bleeding disorder. Basal intracellular Ca2+ levels were increased in platelets, which resulted in a preactivation state, a selective unresponsiveness to immunoreceptor tyrosine activation motif-coupled agonists, and increased platelet consumption. In contrast, basal Ca2+ levels, but not receptor-mediated responses, were affected in mutant T cells. These findings identify Stim1 as a central regulator of platelet function and suggest a cell type-specific activation or composition of the CRAC complex.

We have recently shown that cancer cells of various origins take up extracellular citrate through the plasma membrane citrate carrier (pmCiC), a specific plasma membrane citrate transporter. Extracellular citrate is required to support cancer cell metabolism, in particular fatty acid synthesis, mitochondrial activity, protein synthesis and histone acetylation. In addition, cancer cells tend to acquire a metastatic phenotype in the presence of extracellular citrate. Our recent study also showed that cancer-associated stromal cells synthesise and release citrate and that this process is controlled by cancer cells. In the present study, we evaluated the expression of pmCiC, fibroblast activation protein-α (FAP) and the angiogenesis marker cluster of differentiation 31 (CD31) in human cancer tissues of different origins. In the cohort studied, we found no correlation between disease stage and the expression of FAP or CD31. However, we have identified a clear correlation between pmCiC expression in cancer cells and cancer-associated stroma with tumour stage. It can be concluded that pmCiC is increased in cancer cells and in cancer-supporting cells in the tumour microenvironment at the later stages of cancer development, particularly at the metastatic sites. Therefore, pmCiC expression has the potential to serve as a prognostic marker, although further studies are needed.

It is shown that the formation of amyloid-β oligomers, one of the histopathological signatures of Alzheimer’s disease, can be triggered by small quantities of a specifically truncated and post-translationally modified version of amyloid-β. Hypertoxic amyloid variants Here it is demonstrated that the formation of hypertoxic amyloid-β (Aβ) oligomers can be triggered by small quantities of a specifically truncated and post-translationally modified (pyroglutamylated) version of Aβ, called pEAβ. Previous studies have shown that pE modification of Aβ enhances its aggregation kinetics, toxicity and resistance to degradation, but a mechanistic explanation for these observations was lacking. This study shows that pEAβ causes template-induced misfolding of Aβ 1–42 into small hypertoxic structurally distinct oligomers that propagate through a prion-like mechanism. Tau expression is required for the cytotoxicity of these oligomers, and similar molecules can be isolated from the brains of people with Alzheimer's disease. Extracellular plaques of amyloid-β and intraneuronal neurofibrillary tangles made from tau are the histopathological signatures of Alzheimer’s disease. Plaques comprise amyloid-β fibrils that assemble from monomeric and oligomeric intermediates, and are prognostic indicators of Alzheimer’s disease. Despite the importance of plaques to Alzheimer’s disease, oligomers are considered to be the principal toxic forms of amyloid-β 1 , 2 . Interestingly, many adverse responses to amyloid-β, such as cytotoxicity 3 , microtubule loss 4 , impaired memory and learning 5 , and neuritic degeneration 6 , are greatly amplified by tau expression. Amino-terminally truncated, pyroglutamylated (pE) forms of amyloid-β 7 , 8 are strongly associated with Alzheimer’s disease, are more toxic than amyloid-β, residues 1–42 (Aβ 1–42 ) and Aβ 1–40 , and have been proposed as initiators of Alzheimer’s disease pathogenesis 9 , 10 . Here we report a mechanism by which pE-Aβ may trigger Alzheimer’s disease. Aβ 3(pE)–42 co-oligomerizes with excess Aβ 1–42 to form metastable low- n oligomers (LNOs) that are structurally distinct and far more cytotoxic to cultured neurons than comparable LNOs made from Aβ 1–42 alone. Tau is required for cytotoxicity, and LNOs comprising 5% Aβ 3(pE)–42 plus 95% Aβ 1–42 (5% pE-Aβ) seed new cytotoxic LNOs through multiple serial dilutions into Aβ 1–42 monomers in the absence of additional Aβ 3(pE)–42 . LNOs isolated from human Alzheimer’s disease brain contained Aβ 3(pE)–42 , and enhanced Aβ 3(pE)–42 formation in mice triggered neuron loss and gliosis at 3 months, but not in a tau-null background. We conclude that Aβ 3(pE)–42 confers tau-dependent neuronal death and causes template-induced misfolding of Aβ 1–42 into structurally distinct LNOs that propagate by a prion-like mechanism. Our results raise the possibility that Aβ 3(pE)–42 acts similarly at a primary step in Alzheimer’s disease pathogenesis.

PurposeProtein kinase C (PKC) plays a pivotal role in malignant cell proliferation, apoptosis, invasiveness and migration. However, its exploitation as therapeutic target in breast cancer has been merely explored. Here were evaluated the AEB071 (Sotrastaurin™) treatment efficiency of breast cancer cell lines derived from estrogen receptor positive (T-47D), estrogen/HER2 receptor positive (BT474), and triple negative (HCC1806) breast cancer cells under 2D (monolayer) and 3D (multicellular tumor spheroids) culture conditions. Additionally, spheroid cocultures of BC and N1 fibroblasts were analyzed.MethodsWe quantitatively assessed the proliferation capacity of breast cancer cells and fibroblasts as a function of AEB071 treatment using flow cytometry. The activities of PKC isoforms, substrates, and key molecules of the PKC signaling known to be involved in the regulation of tumor cell proliferation and cellular survival were additionally evaluated. Moreover, a multigene expression analysis (PanCancer Pathways assay) using the nanoString™ technology was applied.ResultsAll breast cancer cell lines subjected to this study were sensitive to AEB071 treatment, whereby cell proliferation in 2D culture was considerably (BT474) or moderately (HCC1806) retarded in G0/G1 or in G2/M phase (T-47D) of the cell cycle. Regardless of the breast cancer subtype the efficiency of AEB071 treatment was significantly lower in the presence of N1 fibroblast cells. Subtype specific driver molecules, namely IL19, c-myb, and NGFR were mostly affected by the AEB071 treatment.ConclusionA combined targeting of PKC and a subtype specific driver molecule might complement specified breast cancer treatment.

Some Aβ peptides contain pyroglutamate modifications that affect the aggregation properties of these peptides. The authors find that the enzyme glutaminyl cyclase is responsible for this pyroglutamate modification. When they inhibit the enzyme in Alzheimer's model mice, fewer plaques form in the brain, and some measures of learning and memory are improved. Because of their abundance, resistance to proteolysis, rapid aggregation and neurotoxicity, N-terminally truncated and, in particular, pyroglutamate (pE)-modified Aβ peptides have been suggested as being important in the initiation of pathological cascades resulting in the development of Alzheimer's disease 1 , 2 , 3 , 4 , 5 , 6 . We found that the N-terminal pE-formation is catalyzed by glutaminyl cyclase in vivo . Glutaminyl cyclase expression was upregulated in the cortices of individuals with Alzheimer's disease and correlated with the appearance of pE-modified Aβ. Oral application of a glutaminyl cyclase inhibitor resulted in reduced Aβ 3(pE)–42 burden in two different transgenic mouse models of Alzheimer's disease and in a new Drosophila model. Treatment of mice was accompanied by reductions in Aβ x–40/42 , diminished plaque formation and gliosis and improved performance in context memory and spatial learning tests. These observations are consistent with the hypothesis that Aβ 3(pE)–42 acts as a seed for Aβ aggregation by self-aggregation and co-aggregation with Aβ 1–40/42 . Therefore, Aβ 3(pE)–40/42 peptides seem to represent Aβ forms with exceptional potency for disturbing neuronal function. The reduction of brain pE-Aβ by inhibition of glutaminyl cyclase offers a new therapeutic option for the treatment of Alzheimer's disease and provides implications for other amyloidoses, such as familial Danish dementia.

The aim of the present population-based cohort study was to analyze the association between the prevalence of 32 types of human papilloma virus (HPV) in 615 female patients with abnormal cervical cytopathology findings. In total, 32 HPV types were screened by DNA array technology. HPV infection was detected in 470 women (76.42%), 419 of whom (89.15%) were infected with ≥1 high-risk (HR)-HPV type. HPV16, which is recognized as the main HR-HPV type responsible for the development of cervical cancer, was observed in 32.98% of HPV+ participants, followed by HPV42 (18.09%), HPV31 (17.66%), HPV51 (13.83%), HPV56 (10.00%), HPV53 (8.72%) and HPV66 (8.72%). The prevalence of HR-HPV types, which may be suppressed directly (in the case of HPV16 and 18), or possibly via cross-protection (in the case of HPV31) following vaccination, was considerably lower in participants ≤22 years of age (HPV16, 28.57%; HPV18, 2.04%; HPV31, 6.12%), compared with participants 23-29 years of age (HPV16, 45.71%; HPV18, 7.86%; HPV31, 22.86%), who were less likely to be vaccinated. Consequently, the present study hypothesizes that there may be a continuous shift in the prevalence of HPV types as a result of vaccination. Furthermore, the percentage of non-vaccine HR-HPV types was higher than expected, considering that eight HPV types formerly classified as 'low-risk' or 'probably high-risk' are in fact HR-HPV types. Therefore, it may be important to monitor non-vaccine HPV types in future studies, and an investigation concerning several HR-HPV types as risk factors for the development of cervical cancer is required.

Citrate is important for lipid synthesis and epigenetic regulation in addition to ATP production. We have previously reported that cancer cells import extracellular citrate via the pmCiC transporter to support their metabolism. Here, we show for the first time that citrate is supplied to cancer by cancer-associated stroma (CAS) and also that citrate synthesis and release is one of the latter’s major metabolic tasks. Citrate release from CAS is controlled by cancer cells through cross-cellular communication. The availability of citrate from CAS regulated the cytokine profile, metabolism and features of cellular invasion. Moreover, citrate released by CAS is involved in inducing cancer progression especially enhancing invasiveness and organ colonisation. In line with the in vitro observations, we show that depriving cancer cells of citrate using gluconate, a specific inhibitor of pmCiC, significantly reduced the growth and metastatic spread of human pancreatic cancer cells in vivo and muted stromal activation and angiogenesis. We conclude that citrate is supplied to tumour cells by CAS and citrate uptake plays a significant role in cancer metastatic progression.

Acute and chronic inflammatory disorders are characterized by detrimental cytokine and chemokine expression. Frequently, the chemotactic activity of cytokines depends on a modified N‐terminus of the polypeptide. Among those, the N‐terminus of monocyte chemoattractant protein 1 (CCL2 and MCP‐1) is modified to a pyroglutamate (pE‐) residue protecting against degradation in vivo . Here, we show that the N‐terminal pE‐formation depends on glutaminyl cyclase activity. The pE‐residue increases stability against N‐terminal degradation by aminopeptidases and improves receptor activation and signal transduction in vitro . Genetic ablation of the glutaminyl cyclase iso‐enzymes QC ( QPCT ) or isoQC ( QPCTL ) revealed a major role of isoQC for pE 1 ‐CCL2 formation and monocyte infiltration. Consistently, administration of QC‐inhibitors in inflammatory models, such as thioglycollate‐induced peritonitis reduced monocyte infiltration. The pharmacologic efficacy of QC/isoQC‐inhibition was assessed in accelerated atherosclerosis in ApoE3*Leiden mice, showing attenuated atherosclerotic pathology following chronic oral treatment. Current strategies targeting CCL2 are mainly based on antibodies or spiegelmers. The application of small, orally available inhibitors of glutaminyl cyclases represents an alternative therapeutic strategy to treat CCL2‐driven disorders such as atherosclerosis/restenosis and fibrosis. →See accompanying article http://dx.doi.org/10.1002/emmm.201100161