A Fab of trastuzumab to treat HER2 overexpressing breast cancer brain metastases.

Despite major therapeutic advances for two decades, including the most recently approved anti-HER2 drugs, brain metastatic localizations remain the major cause of death for women with metastatic HER2 breast cancer. The main reason is the limited drug passage of the blood-brain barrier after intravenous injection and the significant efflux of drugs, including monoclocal antibodies, after administration into the cerebrospinal fluid. We hypothesized that this efflux was linked to the presence of a FcRn receptor in the blood-brain barrier. To overcome this efflux, we engineered two Fab fragments of trastuzumab, an anti-HER2 monoclonal antibody, and did a thorough preclinical development for therapeutic translational purpose. We demonstrated the safety and equal efficacy of the Fabs with trastuzumab in vitro, and in vivo using a patient-derived xenograft model of HER2 overexpressing breast cancer. For the pharmacokinetic studies of intra-cerebrospinal fluid administration, we implemented original rat models with catheter implanted into the cisterna magna. After intraventricular administration in rats, we demonstrated that the brain-to-blood efflux of Fab was up to 10 times lower than for trastuzumab, associated with a two-fold higher brain penetration compared to trastuzumab. This Fab, capable of significantly reducing brain-to-blood efflux and enhancing brain penetration after intra-cerebrospinal fluid injection, could thus be a new and original effective drug in the treatment of HER2 breast cancer brain metastases, which will be demonstrated by a phase I clinical trial dedicated to women in resort situations.

A Sub-Group of Kidney-Transplant Recipients with Highly Aggressive Squamous Cell Carcinoma Expressing Phosphorylated Serine392p53.

Cutaneous squamous cell carcinomas in kidney-transplant recipients are frequent, with an increasing incidence linked to long immunosuppression durations and exposure to ultraviolet radiation. p53 is at the cornerstone of ultraviolet-induced DNA damage, but the role of p53 post-translational modifications in this context is not yet deciphered. Here, we investigated the phosphorylation status of p53 at Serine 392 in 25 cutaneous squamous cell carcinomas in kidney-transplant recipients, compared with 22 non-transplanted patients. Cutaneous squamous cell carcinomas in transplanted patients occurred after a median period of 19 years of immunosuppression, with a median number of 15 cutaneous squamous cell carcinomas and more aggressive histological and clinical characteristics. There was no significant difference between Ki67, p53, and pSer392p53 expression in the two groups. Using principal component analysis, we identified a cluster of exclusively transplanted patients with a median of 23 years of immunosuppression duration, significantly more aggressive biological characteristics, and higher pSer392p53 expression. pSer392p53 was expressed in the whole tumor, suggesting an early carcinogenic event in the course of prolonged immunosuppression. This high, diffuse pSer392p53 expression, corresponding to a high level of DNA damage, might be useful to identify aggressive cutaneous squamous cell carcinomas in kidney-transplant recipients to treat them more aggressively.

Mitapivat reprograms the RBC metabolome and improves anemia in a mouse model of hereditary spherocytosis.

Hereditary spherocytosis (HS) is the most common, nonimmune, hereditary, chronic hemolytic anemia after hemoglobinopathies. The genetic defects in membrane function causing HS lead to perturbation of the RBC metabolome, with altered glycolysis. In mice genetically lacking protein 4.2 (4.2-/-; Epb42), a murine model of HS, we showed increased expression of pyruvate kinase (PK) isoforms in whole and fractioned RBCs in conjunction with abnormalities in the glycolytic pathway and in the glutathione (GSH) system. Mitapivat, a PK activator, metabolically reprogrammed 4.2-/- mouse RBCs with amelioration of glycolysis and the GSH cycle. This resulted in improved osmotic fragility, reduced phosphatidylserine positivity, amelioration of RBC cation content, reduction of Na/K/Cl cotransport and Na/H-exchange overactivation, and decrease in erythroid vesicles release in vitro. Mitapivat treatment significantly decreased erythrophagocytosis and beneficially affected iron homeostasis. In mild-to-moderate HS, the beneficial effect of splenectomy is still controversial. Here, we showed that splenectomy improves anemia in 4.2-/- mice and that mitapivat is noninferior to splenectomy. An additional benefit of mitapivat treatment was lower expression of markers of inflammatory vasculopathy in 4.2-/- mice with or without splenectomy, indicating a multisystemic action of mitapivat. These findings support the notion that mitapivat treatment should be considered for symptomatic HS.

Metastatic clear-cell renal cell carcinoma: a frequent NOTCH1 mutation predictive of response to anti-NOTCH1 CB-103 treatment.

BACKGROUND: Clear-cell renal cell carcinomas (ccRCCs) are malignant tumors with high metastatic potential and resistance to treatments occurs almost constantly. Compared to primary tumors, there are still limited genomic data that has been obtained from metastatic samples. METHODS: We aimed to characterize metastatic ccRCC by way of whole-genome analyses of metastatic formalin-fixed samples, using OncoScan® technology. We identified a frequent, unexpected pL1575P NOTCH1 mutation which we set out to characterize for translational purposes. We thus implemented patient-derived xenografts from metastatic samples of human ccRCC to explore its clinical significance. RESULTS: We showed that pL1575P NOTCH1 mutation was an activating mutation, leading to the expression of NOTCH1-intracellular domain-active fragments in both cancer cells and tumor endothelial cells, suggesting a trans-differentiation of cancer cells into tumor micro-vessels. We demonstrated that this mutation could be used as a predictive biomarker of response to CB-103, a specific NOTCH1-intracellular domain inhibitor. One striking result was the considerable anti-angiogenic effect, coherent with the presence of NOTCH1 mutation in tumor micro-vessels. CONCLUSIONS: We identified a frequent, unexpected pL1575P_c4724T_C NOTCH1 mutation as a new biomarker for ccRCC metastases, predictive of response to the CB103 NOTCH1-intracellular domain inhibitor.

In Humanized Sickle Cell Mice, Imatinib Protects Against Sickle Cell-Related Injury.

Drug repurposing is a valuable strategy for rare diseases. Sickle cell disease (SCD) is a rare hereditary hemolytic anemia accompanied by acute and chronic painful episodes, most often in the context of vaso-occlusive crisis (VOC). Although progress in the knowledge of pathophysiology of SCD have allowed the development of new therapeutic options, a large fraction of patients still exhibits unmet therapeutic needs, with persistence of VOCs and chronic disease progression. Here, we show that imatinib, an oral tyrosine kinase inhibitor developed for the treatment of chronic myelogenous leukemia, acts as multimodal therapy targeting signal transduction pathways involved in the pathogenesis of both anemia and inflammatory vasculopathy of humanized murine model for SCD. In addition, imatinib inhibits the platelet-derived growth factor-B-dependent pathway, interfering with the profibrotic response to hypoxia/reperfusion injury, used to mimic acute VOCs. Our data indicate that imatinib might be considered as possible new therapeutic tool for chronic treatment of SCD.

Ultrasound assessment is linked to histological vein wall thickness in chronic venous disease.

OBJECTIVES: Few studies compared both ultrasound and histological approaches for the same series of patients with chronic venous disease (CVD). We aimed to assess the diagnostic performances of duplex ultrasound assessment (US) of Vein Wall Thickness (VWT) among patients with CVD. METHODS: 38 adults with primary varicose veins having undergone Great Saphenous Vein thermal ablation with phlebectomy, and agreeing to biopsy of the Posterior Accessory Great Saphenous Vein (PASV) were consecutively included in a two-center prospective study. VWT assessment of the PASV was performed using both US, and microscope examination. High values for microscope-assessed VWT were defined at > 0.5 mm. RESULTS: The mean age was 53.0 ± 13.1 years, 71% were women. Maximization of US performances was obtained with a threshold of 0.6 mm: Sensitivity (Se) = 92.9%, Specificity (Sp) = 91.7%, positive (86.7%) and negative predictive value (NPV) (95.7%), positive (11.1) and negative likelihood ratio (NLR) (0.07). CONCLUSIONS: US assessment of VWT could be a non-invasive tool for diagnosis and follow-up in CVD, and an interesting in vivo parameter complementing diameter and reflux measures, with a view to optimizing treatment. It could help to determine i) the energy level necessary in case of endovenous laser ablation, and ii) the sclerosing agent concentration in case of chemical ablation.

BCL-2 Inhibitor ABT-737 Effectively Targets Leukemia-Initiating Cells with Differential Regulation of Relevant Genes Leading to Extended Survival in a NRAS/BCL-2 Mouse Model of High Risk-Myelodysplastic Syndrome.

During transformation, myelodysplastic syndromes (MDS) are characterized by reducing apoptosis of bone marrow (BM) precursors. Mouse models of high risk (HR)-MDS and acute myelogenous leukemia (AML) post-MDS using mutant NRAS and overexpression of human BCL-2, known to be poor prognostic indicators of the human diseases, were created. We have reported the efficacy of the BCL-2 inhibitor, ABT-737, on the AML post-MDS model; here, we report that this BCL-2 inhibitor also significantly extended survival of the HR-MDS mouse model, with reductions of BM blasts and lineage negative/Sca1+/KIT+ (LSK) cells. Secondary transplants showed increased survival in treated compared to untreated mice. Unlike the AML model, BCL-2 expression and RAS activity decreased following treatment and the RAS:BCL-2 complex remained in the plasma membrane. Exon-specific gene expression profiling (GEP) of HR-MDS mice showed 1952 differentially regulated genes upon treatment, including genes important for the regulation of stem cells, differentiation, proliferation, oxidative phosphorylation, mitochondrial function, and apoptosis; relevant in human disease. Spliceosome genes, found to be abnormal in MDS patients and downregulated in our HR-MDS model, such as Rsrc1 and Wbp4, were upregulated by the treatment, as were genes involved in epigenetic regulation, such as DNMT3A and B, upregulated upon disease progression and downregulated upon treatment.

The pyruvate kinase activator mitapivat reduces hemolysis and improves anemia in a ß-thalassemia mouse model.

Anemia in ß-thalassemia is related to ineffective erythropoiesis and reduced red cell survival. Excess free heme and accumulation of unpaired α-globin chains impose substantial oxidative stress on ß-thalassemic erythroblasts and erythrocytes, impacting cell metabolism. We hypothesized that increased pyruvate kinase activity induced by mitapivat (AG-348) in the Hbbth3/+ mouse model for ß-thalassemia would reduce chronic hemolysis and ineffective erythropoiesis through stimulation of red cell glycolytic metabolism. Oral mitapivat administration ameliorated ineffective erythropoiesis and anemia in Hbbth3/+ mice. Increased ATP, reduced reactive oxygen species production, and reduced markers of mitochondrial dysfunction associated with improved mitochondrial clearance suggested enhanced metabolism following mitapivat administration in ß-thalassemia. The amelioration of responsiveness to erythropoietin resulted in reduced soluble erythroferrone, increased liver Hamp expression, and diminished liver iron overload. Mitapivat reduced duodenal Dmt1 expression potentially by activating the pyruvate kinase M2-HIF2α axis, representing a mechanism additional to Hamp in controlling iron absorption and preventing ß-thalassemia-related liver iron overload. In ex vivo studies on erythroid precursors from patients with ß-thalassemia, mitapivat enhanced erythropoiesis, promoted erythroid maturation, and decreased apoptosis. Overall, pyruvate kinase activation as a treatment modality for ß-thalassemia in preclinical model systems had multiple beneficial effects in the erythropoietic compartment and beyond, providing a strong scientific basis for further clinical trials.

A novel lncRNA TCLlnc1 promotes peripheral T cell lymphoma progression through acting as a modular scaffold of HNRNPD and YBX1 complexes.

Long noncoding RNAs (lncRNAs) play an essential role in tumor progression. Few researches focused on the clinical and biological relevance of lncRNAs in peripheral T cell lymphoma (PTCL). In this research, a novel lncRNA (ENST00000503502) was identified overexpressed in the main subtypes of PTCL, and designated as T cell lymphoma-associated lncRNA1 (TCLlnc1). Serum TCLlnc1 was associated with extranodal involvement, high-risk International Prognostic Index, and poor prognosis of the patients. Both in vitro and in vivo, overexpression of TCLlnc1 promoted T-lymphoma cell proliferation and migration, both of which were counteracted by the knockdown of TCLlnc1 using small interfering RNAs. As the mechanism of action, TCLlnc1 directly interacted with transcription activator heterogeneous nuclear ribonucleoprotein D (HNRNPD) and Y-box binding protein-1 (YBX1) by acting as a modular scaffold. TCLlnc1/HNRNPD/YBX1 complex upregulated transcription of TGFB2 and TGFBR1 genes, activated the tumor growth factor-ß signaling pathway, resulting in lymphoma progression, and might be a potential target in PTCL.

A novel prognostic model based on four circulating miRNA in diffuse large B-cell lymphoma: implications for the roles of MDSC and Th17 cells in lymphoma progression.

MicroRNA (miRNA) have been emerged as prognostic biomarkers in diffuse large B-cell lymphoma (DLBCL). To understand the potential underlying mechanisms and translate these findings into clinical prediction on lymphoma progression, large patient cohorts should be evaluated. Here, using miRNA PCR array, we analyzed the miRNA expression profiles in serum samples of 20 DLBCL patients at diagnosis, remission and relapse. Four candidate miRNA were identified and subsequently evaluated for their ability to predict relapse and survival. A prognostic model based on four circulating miRNA (miR21, miR130b, miR155 and miR28) was established and tested in a training cohort of 279 patients and in a validation cohort of 225 patients (NCT01852435). The prognostic value of the 4-circulating miRNA model was assessed by univariate and multivariate analyses. The novel 4-circulating miRNA prognostic model significantly predicted clinical outcome of DLBCL, independent of International Prognostic Index in the training cohort [hazard ratio (HR) = 2.83, 95% CI 2.14-3.51, P < 0.001] and in the validation cohort (HR = 2.71, 95% CI 1.91-3.50, P < 0.001). Moreover, DNA- and RNA-sequencing was performed on tumor samples to detect genetic mutations and signaling pathway dysregulation. DNA-sequencing data showed no significant difference of tumor mutation burden between the low-risk and the high-risk groups of the 4-circulating miRNA model. RNA-sequencing revealed a correlation between the 4-circulating miRNA model and aberrant Ras protein signaling transduction. The impact of the miRNA signature on oncogenic signaling and tumor microenvironment was analyzed in vitro and in vivo. In B-lymphoma cells, modulation of the miRNA regulated IGF1 and JUN expression, thereby altering MDSC and Th17 cells. In DLBCL patients, the high-risk group presented Ras signaling activation, increased MDSC and Th17 cells, and immunosuppressive status compared with the low-risk group. In conclusion, the easy-to-use 4-circulating miRNA prognostic model effectively predicted relapse and survival in DLBCL. Moreover, the tumor microenvironment contributes to the role of the 4-circulating miRNA model in DLBCL progression.

Immunometabolism at the cornerstone of inflammaging, immunosenescence, and autoimmunity in COVID-19.

Inflammaging constitutes the common factor for comorbidities predisposing to severe COVID-19. Inflammaging leads to T-cell senescence, and immunosenescence is linked to autoimmune manifestations in COVID-19. As in SLE, metabolic dysregulation occurs in T-cells. Targeting this T-cell dysfunction opens the field for new therapeutic strategies to prevent severe COVID-19. Immunometabolism-mediated approaches such as rapamycin, metformin and dimethyl fumarate, may optimize COVID-19 treatment of the elderly and patients at risk for severe disease.

Influence of oncogenic mutations and tumor microenvironment alterations on extranodal invasion in diffuse large B-cell lymphoma.

BACKGROUND: Diffuse large B-cell lymphoma (DLBCL) is an aggressive subtype of lymphoma, and multiple extranodal involvement (ENI) indicates adverse clinical outcomes. The aim of this study was to investigate the influence of oncogenic mutations and tumor microenvironment alterations on ENI in DLBCL. METHODS: The clinical features of 1960 patients with newly diagnosed DLBCL were analyzed, and DNA and RNA sequencing was performed on 670 and 349 patients, respectively. Oncogenic mutations and tumor microenvironment alterations were compared according to ENI and evaluated in zebrafish patient-derived tumor xenograft models. RESULTS: Multiple ENI was significantly associated with poor performance status, advanced stage, elevated serum lactate dehydrogenase, low response rate, and inferior prognosis. Lymphoma invasion of the bones, spleen, bone marrow, liver, and central nervous system were independent unfavorable prognostic factors. MYD88 was frequently mutated in patients with multiple ENI, co-occurred with mutations in CD79B, PIM1, TBL1XR1, BTG1, MPEG1, and PRDM1, and correlated with invasion of the bones, kidney/adrenal glands, breasts, testes, skin, and uterus/ovaries. For tumor microenvironment alterations, patients with multiple ENI showed higher regulatory T-cell (Treg)-recruiting activity, but lower extracellular matrix-encoding gene expression, than those without ENI and with single ENI. Elevated Treg-recruiting activity was related to mutations in B2M, SGK1, FOXO1, HIST1H1E, and ARID1A, and correlated with invasion of the bone marrow and thyroid. Additionally, mutations in MYD88, PIM1, TBL1XR1, SGK1, FOXO1, HIST1H1E, and ARID1A were associated with decreased major histocompatibility complex class I expression. Zebrafish models further revealed relationships between MYD88 mutations and invasion of the kidneys and gonads, as well as B2M mutations and invasion of the bone marrow. Increased CXCR4 expression is linked to bone marrow invasion in an organotropic way. CONCLUSIONS: Our findings thus contribute to an improved understanding of the biological behavior of multiple ENI and provide a clinical rationale for targeting ENI in DLBCL.

Assessment of Inflammation and Calcification in Pseudoxanthoma Elasticum Arteries and Skin with 18F-FluroDeoxyGlucose and 18F-Sodium Fluoride Positron Emission Tomography/Computed Tomography Imaging: The GOCAPXE Trial.

BACKGROUND: Pseudoxanthoma elasticum (PXE) is an inherited metabolic disease characterized by elastic fiber fragmentation and ectopic calcification. There is growing evidence that vascular calcification is associated with inflammatory status and is enhanced by inflammatory cytokines. Since PXE has never been considered as an inflammatory condition, no incidence of chronic inflammation leading to calcification in PXE has been reported and should be investigated. In atherosclerosis and aortic stenosis, positron emission tomography combined with computed tomographic (PET-CT) imaging has demonstrated a correlation between inflammation and calcification. The purpose of this study was to assess skin/artery inflammation and calcification in PXE patients. Methods: 18F-FluroDeoxyGlucose (18F-FDG) and 18F-Sodium Fluoride (18F-NaF) PET-CT, CT-imaging and Pulse wave velocity (PWV) were used to determine skin/vascular inflammation, tissue calcification, arterial calcium score (CS) and stiffness, respectively. In addition, inorganic pyrophosphate, high-sensitive C-reactive protein and cytokines plasma levels were monitored. RESULTS: In 23 PXE patients, assessment of inflammation revealed significant 18F-FDG uptake in diseased skin areas contrary to normal regions, and exclusively in the proximal aorta contrary to the popliteal arteries. There was no correlation between 18F-FDG uptake and PWV in the aortic wall. Assessment of calcification demonstrated significant 18F-NaF uptake in diseased skin regions and in the proximal aorta and femoral arteries. 18F-NaF wall uptake correlated with CS in the femoral arteries, and aortic wall PWV. Multivariate analysis indicated that aortic wall 18F-NaF uptake is associated with diastolic blood pressure. There was no significant correlation between 18F-FDG and 18F-NaF uptake in any of the artery walls. CONCLUSION: In the present cross-sectional study, inflammation and calcification were not correlated. PXE would appear to more closely resemble a chronic disease model of ectopic calcification than an inflammatory condition. To assess early ectopic calcification in PXE patients, 18F-NaF-PET-CT may be more relevant than CT imaging. It potentially constitutes a biomarker for disease-modifying anti-calcifying drug assessment in PXE.

Expression of angiopoietin-like 4 fibrinogen-like domain (cANGPTL4) increases risk of brain metastases in women with breast cancer.

BACKGROUND: Brain metastases challenge daily clinical practice, and the mechanisms by which cancer cells cross the blood-brain barrier remain largely undeciphered. Angiopoietin-like 4 (ANGPTL4) proteolytic fragments have controversial biological effects on endothelium permeability. Here, we studied the link between ANGPTL4 and the risk of brain metastasis in cancer patients. MATERIALS AND METHODS: From June 2015 to June 2016, serum samples from 113 cancer patients were prospectively collected, and ANGPTL4 concentrations were assessed. Using a murine model of brain metastases, we investigated the roles of nANGPTL4 and cANGPTL4, the two cleaved fragments of ANGPTL4, in the occurrence of brain metastases. RESULTS: An ANGPTL4 serum concentration over 0.1 ng/mL was associated with decreased overall-survival. Multivariate analyses found that only breast cancer brain metastases were significantly associated with elevated ANGPTL4 serum concentrations. 4T1 murine breast cancer cells were transfected with either nANGPTL4- or cANGPTL4-encoding cDNAs. Compared to mice injected with wild-type 4T1 cells, mice injected with nANGPTL4 cells had shorter median survival (p < 0.05), while mice injected with cANGPTL4 had longer survival (p < 0.01). On tissue sections, compared to wild-type mice, mice injected with nANGPTL4 cells had significantly larger surface areas of lung metastases (p < 0.01), and mice injected with cANGPTL4 had significantly larger surface areas of brain metastases (p < 0.01). CONCLUSIONS: In this study, we showed that a higher expression of Angiopoietin-like 4 Fibrinogen-Like Domain (cANGPTL4) was associated with an increased risk of brain metastases in women with breast cancer.

Advances in targeted therapy for malignant lymphoma.

The incidence of lymphoma has gradually increased over previous decades, and it ranks among the ten most prevalent cancers worldwide. With the development of targeted therapeutic strategies, though a subset of lymphoma patients has become curable, the treatment of refractory and relapsed diseases remains challenging. Many efforts have been made to explore new targets and to develop corresponding therapies. In addition to novel antibodies targeting surface antigens and small molecular inhibitors targeting oncogenic signaling pathways and tumor suppressors, immune checkpoint inhibitors and chimeric antigen receptor T-cells have been rapidly developed to target the tumor microenvironment. Although these targeted agents have shown great success in treating lymphoma patients, adverse events should be noted. The selection of the most suitable candidates, optimal dosage, and effective combinations warrant further investigation. In this review, we systematically outlined the advances in targeted therapy for malignant lymphoma, providing a clinical rationale for mechanism-based lymphoma treatment in the era of precision medicine.