Sleep disorders and vascular responsiveness in patients with rheumatoid arthritis.

BACKGROUND: Rheumatoid arthritis (RA) is the most common systemic autoimmune disease characterized by chronic systemic inflammation. Half of the deaths of patients with RA are due to cardiovascular diseases (CVD), considered to be 1.5 to -2.0-fold that in the general population. Patients with RA also experience poor sleep, which by itself is associated with endothelial dysfunction, CVD events and sudden death. Our aim was to study the mechanistic pathways and the correlations between sleep efficiency and vascular reactivity of patients with RA. METHODS AND RESULTS: A prospective study that evaluated quality of sleep using ACTi Graphs, vascular inflammation and endothelial function of 18 patients with RA. Inflammation was studied by levels of E-selectin, intercellular adhesion molecule 1 (ICAM-1) and NO in serum. Endothelial function was studied using the brachial artery plethysmography method. Eighteen RA patients (aged 57.56 ± 13.55 years; 16 women) with a long-standing active RA: Eight patients had impaired sleep efficiency and 10 had a good sleep efficiency. Those who had an impaired sleep had larger baseline diameters of the brachial artery (0.39 ± 0.08 cm vs. 0.32 ± 0.04 cm; P = 0.02). Negative correlations were found between baseline brachial artery diameter and sleep efficiency (P = 0.01), and with NO level (P = 0.04). Stepwise regression found that brachial artery diameter at baseline and NO level could predict sleep efficiency (r2  = 0.543, P = 0.01). CONCLUSION: Vascular reactivity could predict quality of sleep in patients with RA. Quality of sleep may serve as an independent CVD risk factor in patients with RA.

Detection of NRAS G12D and NRAS G13C mutant genes among apparently healthy and haematologic malignant individuals in Federal Capital Territory, Nigeria.

Rat Sarcoma gene mutations is an important aspect in the management of hematologic malignancies globally. Unfortunately, this is not the trend in West Africa, including Nigeria. This study was aimed at detecting NRAS G12D and NRAS G13C mutant genes among apparently healthy and haematologic malignant individuals, and to explore their association with some clinical and demographic factors as well as disease status and progression. A total of 200 cfDNAs, 100 each from haematologic malignant patients and blood donors, respectively, were analyzed for the presence of NRAS gene mutations in codons 12 and 13. These mutations were tested using multiplex allele-specific PCR (AS-PCR). The mutations were detected by selective amplification using mutation-specific synthetic oligonucleotides. NRAS G12D and NRAS G13C mutations were 20.0% and 10.0%, respectively. In 17.5% of the 100 haemapoietic cancer patients, NRAS G12D mutant genes were seen while 7.5% of NRAS G13C mutation was found. Both mutant genes were observed in five healthy blood donors each. This result confirms the existence of NRAS mutations in Nigerian haemapoietic cancer patients and the preponderance of G-A transitions over G-T transversions. Mutant NRAS genes were associated with the types and stages of cancer, highlighting probable connection between mutation and increased susceptibility as well as quick progression of hematologic malignancies in the population studied. The result also highlighted higher risk of susceptibility/progression associated with leukemia than other hematopoietic cancers. We recommend more studies on NRAS mutation specifically targeted at improved diagnosis and prognostic therapy. The role of RAS mutation should be explored in other aside blood cancers in the Nigerian population.

Circulating Tumour DNA Analysis for Tumour Genome Characterisation and Monitoring Disease Burden in Extramedullary Multiple Myeloma.

Mutational characterisation in extramedullary multiple myeloma (EM-MM) patients is challenging due to inaccessible EM plasmacytomas, unsafe nature of multiple biopsies and the spatial and temporal genomic heterogeneity apparent in MM (Graphical abstract). Conventional monitoring of disease burden is through serum markers and PET-CT, however these modalities are sometimes inadequate (serum markers), not performed in a timely manner (PET-CT) and uninformative for identifying mutations driving disease progression. DNA released into the blood by tumour cells (ctDNA) contains the predominant clones derived from the multiple disease foci. Blood-derived ctDNA can, therefore, provide a holistic illustration of the major drivers of disease progression. In this report, the utility of ctDNA, as an adjunct to currently available modalities in EM-MM, is presented for a patient with EM and oligosecretory (OS) disease. Whole exome sequencing of contemporaneously acquired tumour tissue and matched ctDNA samples revealed the presence of spatial and temporal genetic heterogeneity and the identification of pathways associated with drug resistance. Longitudinal monitoring of plasma samples revealed that ctDNA can be utilised to define the dynamic clonal evolution co-existent with disease progression and as an adjunct non-invasive marker of tumour burden.

HRES-1/Rab4-mediated depletion of Drp1 impairs mitochondrial homeostasis and represents a target for treatment in SLE.

OBJECTIVE: Accumulation of mitochondria underlies T-cell dysfunction in systemic lupus erythematosus (SLE). Mitochondrial turnover involves endosomal traffic regulated by HRES-1/Rab4, a small GTPase that is overexpressed in lupus T cells. Therefore, we investigated whether (1) HRES-1/Rab4 impacts mitochondrial homeostasis and (2) Rab geranylgeranyl transferase inhibitor 3-PEHPC blocks mitochondrial accumulation in T cells, autoimmunity and disease development in lupus-prone mice. METHODS: Mitochondria were evaluated in peripheral blood lymphocytes (PBL) of 38 SLE patients and 21 healthy controls and mouse models by flow cytometry, microscopy and western blot. MRL/lpr mice were treated with 125 µg/kg 3-PEHPC or 1 mg/kg rapamycin for 10 weeks, from 4 weeks of age. Disease was monitored by antinuclear antibody (ANA) production, proteinuria, and renal histology. RESULTS: Overexpression of HRES-1/Rab4 increased the mitochondrial mass of PBL (1.4-fold; p=0.019) and Jurkat cells (2-fold; p=0.000016) and depleted the mitophagy initiator protein Drp1 both in human (-49%; p=0.01) and mouse lymphocytes (-41%; p=0.03). Drp1 protein levels were profoundly diminished in PBL of SLE patients (-86±3%; p=0.012). T cells of 4-week-old MRL/lpr mice exhibited 4.7-fold over-expression of Rab4A (p=0.0002), the murine homologue of HRES-1/Rab4, and depletion of Drp1 that preceded the accumulation of mitochondria, ANA production and nephritis. 3-PEHPC increased Drp1 (p=0.03) and reduced mitochondrial mass in T cells (p=0.02) and diminished ANA production (p=0.021), proteinuria (p=0.00004), and nephritis scores of lupus-prone mice (p<0.001). CONCLUSIONS: These data reveal a pathogenic role for HRES-1/Rab4-mediated Drp1 depletion and identify endocytic control of mitophagy as a treatment target in SLE.

Dynamin-related protein-1 controls fusion pore dynamics during platelet granule exocytosis.

OBJECTIVE: Platelet granule exocytosis serves a central role in hemostasis and thrombosis. Recently, single-cell amperometry has shown that platelet membrane fusion during granule exocytosis results in the formation of a fusion pore that subsequently expands to enable the extrusion of granule contents. However, the molecular mechanisms that control platelet fusion pore expansion and collapse are not known. METHODS AND RESULTS: We identified dynamin-related protein-1 (Drp1) in platelets and found that an inhibitor of Drp1, mdivi-1, blocked exocytosis of both platelet dense and α-granules. We used single-cell amperometry to monitor serotonin release from individual dense granules and, thereby, measured the effect of Drp1 inhibition on fusion pore dynamics. Inhibition of Drp1 increased spike width and decreased prespike foot events, indicating that Drp1 influences fusion pore formation and expansion. Platelet-mediated thrombus formation in vivo after laser-induced injury of mouse cremaster arterioles was impaired after infusion of mdivi-1. CONCLUSIONS: These results demonstrate that inhibition of Drp1 disrupts platelet fusion pore dynamics and indicate that Drp1 can be targeted to control thrombus formation in vivo.

The role of ultrasound and mitofusin-2 levels to predict pregnancy outcomes in patients with severe preeclampsia: a case-control study.

OBJECTIVE: The aim of this study was to evaluate mitofusin-2 levels and fetal Doppler ultrasonography effects in patients with severe preeclampsia. METHODS: This single-center case-control study was conducted in the gynecology service of the university hospital in Van. A total of 90 pregnant women aged 18-40 years were included in the study. Of these, 30 are normal, 30 have mild preeclampsia, and 30 are pregnant with severe preeclampsia. In this study, especially in severe preeclampsia patients, serum mitofusin-2 levels and important fetal Doppler flows such as uterine arterial pressure, umbilical arterial pressure, and 1st and 5th minute Apgar scores, birth weight, and the relationship between postnatal outcomes such as week of birth and the number of patients in the neonatal intensive care unit were investigated. RESULTS: There was a significant difference between the three groups in terms of mitofusin-2 levels, which was the highest in the group (p<0.05). Maternal serum mitofusin-2 levels were positively correlated with uterine arterial pressure (r=0.543, p=0.007), umbilical arterial pressure (r=0.238, p=0.008), diastolic blood pressure, and systolic blood pressure (p<0.001). Receiver operating characteristic curve of mitofusin-2 in predicting preeclampsia is as follows: optimal cutoff 1.6 ng/mL; area under the curve: 0.861; 95%CI: 0.786-0.917; sensitivity: 83.9%; and specificity: 70.0%, (p≤0.001). A one-unit increase in mitofusin-2 resulted in a statistically significant 4.21-fold increase in preeclampsia risk. CONCLUSION: This study recommends the use of mitofusin-2 together with fetal Doppler ultrasound findings as a reliable indicator of preeclampsia severity.

Mitochondrial fission proteins in peripheral blood lymphocytes are potential biomarkers for Alzheimer's disease.

BACKGROUND AND PURPOSE: Expression of the mitochondrial fission proteins dynamin-related protein 1 (Drp1), S-nitrosylated Drp1 (SNO-Drp1), and Fis1 has been found to be altered in brain tissues and skin fibroblasts from patients with Alzheimer's disease (AD). The aim of this study was to determine whether these proteins are also changed in peripheral blood lymphocytes (PBL) of AD patients and whether these changes are specific and sensitive enough for AD diagnosis. METHODS: Western blot analysis and enzyme-linked immunosorbent assay (ELISA) were employed to quantify relative levels of Drp1, SNO-Drp1, and Fis1 in PBL obtained from 91 controls, 82 AD, 26 mild cognitive impairment (MCI), 12 Parkinson's disease (PD), and 36 vascular dementia (VaD) patients. Logistic regression and receiver operating characteristic (ROC) curve analysis were used to measure diagnostic accuracy of these proteins. RESULTS: Compared with controls, SNO-Drp1 and Fis1 levels were remarkably increased in PBL of AD and MCI patients, and Drp1 was significantly decreased in AD, MCI, and PD. None of these proteins were changed in VaD patients. Disease severity or duration had no major effects on levels of these proteins in AD PBL. ROC curve analysis showed that the specificity and sensitivity were 81% and 73% for Drp1, 84% and 82% for SNO-Drp1, and 89% and 80% for Fis1 in identifying AD patients from control subjects. CONCLUSIONS: Altered mitochondrial fission proteins Drp1, SNO-Drp1, and Fis1 in PBL were relatively sensitive and specific in identifying AD patients and could be serving as a biomarker in the procedure of diagnosis.

Mutation spectrum of the OPA1 gene in a large cohort of patients with suspected dominant optic atrophy: Identification and classification of 48 novel variants.

Autosomal dominant optic atrophy is one of the most common inherited optic neuropathies. This disease is genetically heterogeneous, but most cases are due to pathogenic variants in the OPA1 gene: depending on the population studied, 32-90% of cases harbor pathogenic variants in this gene. The aim of this study was to provide a comprehensive overview of the entire spectrum of likely pathogenic variants in the OPA1 gene in a large cohort of patients. Over a period of 20 years, 755 unrelated probands with a diagnosis of bilateral optic atrophy were referred to our laboratory for molecular genetic investigation. Genetic testing of the OPA1 gene was initially performed by a combined analysis using either single-strand conformation polymorphism or denaturing high performance liquid chromatography followed by Sanger sequencing to validate aberrant bands or melting profiles. The presence of copy number variations was assessed using multiplex ligation-dependent probe amplification. Since 2012, genetic testing was based on next-generation sequencing platforms. Genetic screening of the OPA1 gene revealed putatively pathogenic variants in 278 unrelated probands which represent 36.8% of the entire cohort. A total of 156 unique variants were identified, 78% of which can be considered null alleles. Variant c.2708_2711del/p.(V903Gfs*3) was found to constitute 14% of all disease-causing alleles. Special emphasis was placed on the validation of splice variants either by analyzing cDNA derived from patients´ blood samples or by heterologous splice assays using minigenes. Splicing analysis revealed different aberrant splicing events, including exon skipping, activation of exonic or intronic cryptic splice sites, and the inclusion of pseudoexons. Forty-eight variants that we identified were novel. Nine of them were classified as pathogenic, 34 as likely pathogenic and five as variant of uncertain significance. Our study adds a significant number of novel variants to the mutation spectrum of the OPA1 gene and will thereby facilitate genetic diagnostics of patients with suspected dominant optic atrophy.

Serum Mitochondrial Quality Control Related Biomarker Levels are Associated with Organ Dysfunction in Septic Patients.

BACKGROUND: To investigate the feasibility and the value of using mitochondrial quality control (MQC)-related proteins as biomarkers in septic patients. METHODS: The enrolled subjects were divided into four groups: healthy control group (n = 30), intensive care unit (ICU) control group (n = 62), septic nonshock group (n = 40), and septic shock group (n = 94). Serum levels of peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), fission protein 1 (Fis1), mitofusin2 (Mfn2), and Parkin were measured by enzyme-linked immunosorbent assay at the time of enrollment for all groups. Clinical parameters and laboratory test results were also collected. RESULTS: The levels of MQC-related biomarkers between any two of the four groups were significantly different (P < 0.001 for all). The serum levels of PGC-1α, Mfn2, and Parkin were lowest in healthy individuals; the levels were dramatically higher in the ICU control group compared with the others, and they decreased progressively from the septic nonshock group to the septic shock group. However, the pattern for Fis1 was inverse; the more severe the condition was, the higher the level of Fis1. Moreover, there was moderate correlation between MQC-related biomarkers and the SOFA score (PGC-1α, r = -0.662; Fis1, r = 0.609; Mfn2, r = -0.677; Parkin, r = 0.-0.674, P < 0.001 for all). CONCLUSIONS: The serum levels of PGC-1α, Fis1, Mfn2, and Parkin were significantly correlated with organ dysfunction and reflected the disease progression and severity. The dynamic surveillance of these four biomarkers could be beneficial to predict outcome and guide treatment.

Construction of a reference material panel for detecting KRAS/NRAS/EGFR/BRAF/MET mutations in plasma ctDNA.

BACKGROUND: The absence of high-quality next-generation sequencing (NGS) reference material (RM) has impeded the clinical use of liquid biopsies with plasma cell-free DNA (cfDNA) in China. OBJECTIVE: This study aimed to develop a national RM panel for external quality assessment and performance evaluation during kit registration of non-small-cell lung cancer (NSCLC)-related Kirsten rat sarcoma viral oncogene (KRAS)/neuroblastoma ras oncogene (NRAS)/epidermal growth factor receptor (EGFR)/B-type Raf kinase (BRAF)/mesenchymal-epithelial transition factor (MET) genetic assays using plasma circulating tumor DNA (ctDNA). METHODS: Mutation cell lines detected by NGS and validated by Sanger sequencing were selected to establish the RM. Cell line genomic DNA was sheared and used to spike basal plasma cfDNA at 10% concentration. Then, the calibration accuracy was determined by four sequencing platforms. Average values were adopted and diluted to 0.1%, 0.3%, 1% and 3% concentrations with basal plasma as the RM panel. Then, five manufacturers were invited to evaluate the performance of the RM panel. RESULTS: 20 cell lines with 23 clinically important mutations were selected, including six mutations in KRAS, two mutations in NRAS, three in BRAF, four in phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), six in EGFR, one EGFR Gain (4-5 copy) and one MET Gain (2-5 copy). The RM panel consisted of 87 samples, including these 21 mutations at four concentrations (0.1%, 0.3%, 1% and 3%), one MET gain, one EGFR gain and one wild type. The detection rate was 100% for the 3%, 1% and 0.3% samples at all five companies. For the 0.1% concentration, 15 samples had inconsistent results, but at least three companies had correct results for each mutation. CONCLUSION: RM for a KRAS/NRAS/EGFR/BRAF/MET mutation panel for plasma ctDNA was developed, which will be essential for quality control of the performance of independent laboratories.

Rac1 and Rac2 GTPases are necessary for early erythropoietic expansion in the bone marrow but not in the spleen.

BACKGROUND: The small Rho GTPases Rac1 and Rac2 have both overlapping and distinct roles in actin organization, cell survival, and proliferation in various hematopoietic cell lineages. The role of these Rac GTPases in erythropoiesis has not yet been fully elucidated. DESIGN AND METHODS: Cre-recombinase-induced deletion of Rac1 genomic sequence was accomplished on a Rac2-null genetic background, in mouse hematopoietic cells in vivo. The erythroid progenitors and precursors in the bone marrow and spleen of these genetically engineered animals were evaluated by colony assays and flow cytometry. Apoptosis and proliferation of the different stages of erythroid progenitors and precursors were evaluated by flow cytometry. RESULTS: Erythropoiesis in Rac1(-/-);Rac2(-/-) mice is characterized by abnormal burst-forming unit-erythroid colony morphology and decreased numbers of megakaryocyte-erythrocyte progenitors, erythroid colony-forming units, and erythroblasts in the bone marrow. In contrast, splenic erythropoiesis is increased. Combined Rac1 and Rac2 deficiency compromises proliferation of the megakaryocyte-erythrocyte progenitor population in the bone marrow, while it allows increased survival and proliferation of megakaryocyte-erythrocyte progenitors in the spleen. Conclusions These data suggest that Rac1 and Rac2 GTPases are essential for normal bone marrow erythropoiesis but that they are dispensable for erythropoiesis in the spleen, implying different signaling pathways for homeostatic and stress erythropoiesis.

Statins have beneficial effects on platelet free radical activity and intracellular distribution of GTPases in hyperlipidaemia.

In addition to lowering cholesterol, statins may alter endothelial release of the vasodilator NO and harmful superoxide free radicals. Statins also reduce cholesterol intermediates including isoprenoids. These are important for post-translational modification of substances including the GTPases Rho and Rac. By altering the membrane association of these molecules, statins affect intracellular positioning and hence activity of a multitude of substances. These include eNOS(endothelial NO synthase), which produces NO (inhibited by Rho), and NADPH oxidase, which produces superoxide (dependent on Rac). Statins may improve endothelial function by enhancing production of NO while decreasing superoxide production. A total of 40 hypercholesterolaemic patients were randomized to treatment with either atorvastatin or placebo; 20 normolipidaemic patients were also studied. Platelet nitrite, NO and superoxide were examined as was the cellular distribution of the GTPases Rho and Rac at baseline and after 8 weeks of treatment.Following atorvastatin therapy, platelet NO was increased (3.2 pmol/10(8) platelets) and superoxide output was attenuated [-3.4 pmol min(-1) (10(8) platelets)(-1)] when compared with placebo. The detection of both Rho and Rac was significantly reduced in the membranes of platelets, implying reduced activity. In conclusion, the results of the present study show altered NO/superoxide production following statin therapy. A potential mechanism for this is the change in the distribution of intracellular GTPases, which was considered to be secondary to decreases in isoprenoid intermediates, suggesting that the activity of the former had been affected by atorvastatin.

Maternal serum mitofusin-2 levels in patients with preeclampsia: the possible role of mitochondrial dysfunction in preeclampsia.

Purpose: Hypoxia alters mitochondria function and our aim was to measure mitochondrial fusion protein mitofusin-2 (Mfn2) in patients with preeclampsia.Materials and methods: This cross-sectional study was conducted including 82 pregnant women, 27 with normal pregnancy and 55 with preeclampsia (27 with early-onset preeclampsia and 28 with late-onset preeclampsia). Maternal serum levels of Mfn2 were measured by using enzyme-linked immunosorbent assay kits.Results: The mean serum mitofusin-2 levels were higher in women with preeclampsia than in the control group (68.02 ± 8.7 pg/mL vs. 99.72 ± 37.27 pg/mL, p < .0001). The mean serum mitofusin-2 level was found to be the highest in the early-onset preeclampsia (EOPE) group (EOPE: 101.6 ± 38.5 pg/mL). Maternal serum mitofusin-2 levels correlated with both systolic and diastolic blood pressures as well as uterine artery pulsatility index. The optimal cutoff value of Mfn2 for determining preeclampsia was 75.3 pg/mL.Conclusion: Mfn2 has regulatory roles in stress response. Maternal serum Mfn2 is higher in patients with preeclampsia suggesting that Mfn2 increases in the maternal system as a stress response against hypoxia and endothelial dysfunction.What do the results of this study add? Hypoxia causes mitochondrial dysfunction that has been linked to the etiology of many diseases including preeclampsia. Mitofusin-2 is a mitochondrial fusion protein, and the levels can be altered in preeclampsia. For the first time, we showed that maternal levels of mitofusin-2 are higher in patients with preeclampsia. Further, we reported the correlation of mitofusin-2 with blood pressures and uterine artery pulsatility index. These findings will open up other avenues for researchers to investigate other mitochondrial molecules while under stress.

Development and Clinical Validation of Discriminatory Multitarget Digital Droplet PCR Assays for the Detection of Hot Spot KRAS and NRAS Mutations in Cell-Free DNA.

Detection and quantification of tumor-derived KRAS and NRAS mutations in plasma cell-free DNA (cfDNA) holds great potential for cancer diagnostics and treatment response monitoring. Because of high sensitivity, specificity, robustness, and affordability, digital droplet PCR (ddPCR) is ideally suited for this application but requires discriminatory multiplexing when used as screening assay. We therefore designed, optimized, and clinically validated mutation-specific locked nucleic acid-based ddPCR assays for 14 commonly occurring KRAS and NRAS mutations and assembled these assays into seven discriminatory multitarget screening assays covering two to six single-nucleotide variants each. Limit of detection, limit of blank, and interassay accuracy were determined. Assay performance and suitability for screening in cfDNA were validated with plasma samples from a clinically fully characterized cohort of pancreatic cancer patients and healthy controls. Limits of detection for single-target assays were between 0.0015% and 0.069% variant allele fraction, and between 0.022% and 0.16% for multitarget assays. Dilution linearity and interassay accuracy were excellent throughout (r2 > 0.99). Multitarget assay screening of cfDNA extracted from pancreatic cancer patients with unknown KRAS mutational status correctly identified single-nucleotide variants in 45 of 45 (100%) of tumor-derived cell-free DNA-positive samples. In summary, we herein present and clinically validate generic single-target and discriminatory multitarget ddPCR assays for KRAS and NRAS hot spot mutations with broad applicability for clinical and translational research.

Tracking myeloma tumor DNA in peripheral blood.

Over the past years, the emergence of liquid biopsy technologies has dramatically expanded our ability to assess multiple myeloma without the need for invasive sampling. Interrogation of cell-free DNA from the peripheral blood recapitulates the mutational landscape at excellent concordance with matching bone marrow aspirates. It can quantify disease burden and identify previously undetected resistance mechanisms which may inform clinical management in real-time. The convenience of sample acquisition and storage provides strong procedural benefits over currently available testing. Further investigations will have to define the role of cell-free DNA as a diagnostic measure by determining clinically relevant tumor thresholds in comparison to existing routine parameters. This review presents an overview of currently available assays and discusses the clinical value, potential and limitations of cell-free DNA technologies for the assessment of this challenging disease.

Longitudinal Monitoring of ctDNA in Patients with Melanoma and Brain Metastases Treated with Immune Checkpoint Inhibitors.

PURPOSE: Brain involvement occurs in the majority of patients with metastatic melanoma. The potential of circulating tumor DNA (ctDNA) for surveillance and monitoring systemic therapy response in patients with melanoma brain metastases merits investigation. EXPERIMENTAL DESIGN: This study examined circulating BRAF, NRAS, and c-KIT mutations in patients with melanoma with active brain metastases receiving PD-1 inhibitor-based therapy. Intracranial and extracranial disease volumes were measured using the sum of product of diameters, and response assessment performed using RECIST. Longitudinal plasma samples were analyzed for ctDNA over the first 12 weeks of treatment (threshold 2.5 copies/mL plasma). RESULTS: Of a total of 72 patients, 13 patients had intracranial metastases only and 59 patients had concurrent intracranial and extracranial metastases. ctDNA detectability was 0% and 64%, respectively, and detectability was associated with extracranial disease volume (P < 0.01). Undetectable ctDNA on-therapy was associated with extracranial response (P < 0.01) but not intracranial response. The median overall survival in patients with undetectable (n = 34) versus detectable (n = 38) ctDNA at baseline was 39.2 versus 10.6 months [HR, 0.51; 95% confidence interval (CI), 0.28-0.94; P = 0.03] and on-therapy was 39.2 versus 9.2 months (HR, 0.32; 95% CI, 0.16-0.63; P < 0.01). CONCLUSIONS: ctDNA remains a strong prognostic biomarker in patients with melanoma with brain metastases, especially in patients with concurrent extracranial disease. However, ctDNA was not able to detect or monitor intracranial disease activity, and we recommend against using ctDNA as a sole test during surveillance and therapeutic monitoring in patients with melanoma.

[Free circulating DNA based colorectal cancer screening from peripheral blood: the possibility of the methylated septin 9 gene marker]. / Szabad DNS-alapú vastagbéldaganat-szurés perifériás vérbol: a metilált szeptin-9 génmarker lehetoségei.

DNA methylation acts in early tumorigenesis. Its detection is possible either from tissue, stool or peripheral blood. Septin 9 is a sensitive methylation marker, which has been studied in several cancers such as breast and ovarian tumors and in neurological or hematological diseases. Septin proteins have an important role from cytoskeleton organisation to development of embryonal pattern. Nowadays intensive researches are going on about the relation between the septin 9 gene hypermethylation and colorectal cancer development.

Phase 2 Trial of Nivolumab Combined With Stereotactic Body Radiation Therapy in Patients With Metastatic or Locally Advanced Inoperable Melanoma.

PURPOSE: Nivolumab improves survival in patients with metastatic melanoma. Unfortunately, most patients do not respond to this treatment. Preclinical data indicate that radiation therapy could work synergistically with nivolumab and improve response rates. METHODS AND MATERIALS: We conducted a phase 2 trial in 20 patients with inoperable or metastatic melanoma with ≥2 measurable lesions (Response Evaluation Criteria in Solid Tumors v1.1). Stereotactic body radiation therapy (SBRT) of 3 × 8 Gy to the largest lesion was delivered before the second nivolumab cycle. The primary endpoint was overall response rate (ORR) in the nonirradiated lesions (Response Evaluation Criteria in Solid Tumors v1.1). Secondary endpoints included toxicity. An exploratory endpoint was mutant BRAF and NRAS circulating tumor DNA (ctDNA) on serial blood samples. RESULTS: An ORR of 45% was noted with 3 complete and 6 partial responses. Three patients experienced stable disease and 7 had progressive disease as best response. All patients with a complete response in the nonirradiated lesions exhibited a local complete response in the irradiated lesion. Grade 1 to 2 treatment-related adverse events (AEs) occurred in 17 patients; 3 patients experienced grade 3 AEs (lymphopenia, gastroenteritis, and bullous pemphigoid). No grade 4 to 5 AEs occurred. ctDNA was detected in 8 patients, and changes corresponded to clinical response and suggested that a subset of patients with a low programmed death ligand-1 score only started responding after SBRT. CONCLUSIONS: We conclude that the combination treatment was well tolerated and led to an ORR of 45% in patients with metastatic or inoperable melanoma, similar to historical response rates of nivolumab monotherapy. Although underpowered, our data therefore do not indicate a substantial abscopal response. Nonetheless, serial ctDNA analyses suggest that a subset of patients responded only after the addition of SBRT.

Illustrative cases for monitoring by quantitative analysis of BRAF/NRAS ctDNA mutations in liquid biopsies of metastatic melanoma patients who gained clinical benefits from anti-PD1 antibody therapy.

Anti-programmed death 1 (PD-1) monoclonal antibodies improve the survival of metastatic melanoma patients. Predictive or monitoring biomarkers for response to this therapy could improve the clinical management of these patients. To date, no established biomarkers are available for monitoring the response to immunotherapy. Tumor- specific mutations in circulating tumor DNA (ctDNA) such as BRAF and NRAS mutations for melanoma patients have been proposed for monitoring of immunotherapy response. We present seven illustrative cases for the use of ctDNA BRAF and NRAS mutations' monitoring in plasma. The cases described exemplify four distinct clinical benefit patterns: rapid and durable complete response (CR), early progression, followed by CR, CR followed by early progression after interrupting treatment and long-term disease stabilization. These representative cases suggest that comprehensive BRAF/NRAS ctDNA monitoring during anti-PD1 therapy is informative and can be of added value for the monitoring of melanoma patients gaining clinical benefit on anti-PD1 treatment. An important advantage of our approach is that using the cartridge system on the Idylla platform for mutation analysis, the results become available the same day 2 h after plasma collection. Therefore, in the future, the ctDNA level can be an element in the clinical management of the patients.

Biochemical characterization of baculovirus-expressed rap1A/Krev-1 and its regulation by GTPase-activating proteins.

Normal human rap1A and 35A rap1A (which encodes a protein with a Thr-35----Ala mutation) were cloned into a baculovirus transfer vector and expressed in Sf9 insect cells. The resulting proteins were purified, and their nucleotide binding, GTPase activities, and responsiveness to GTPase-activating proteins (GAPs) were characterized and compared with those of Rap1 purified from human neutrophils. Recombinant wild-type Rap1A bound GTP gamma S, GTP, and GDP with affinities similar to those observed for neutrophil Rap1 protein. The rate of exchange of GTP by Rap1 without Mg2+ was much slower than that by Ras. The basal GTPase activities by both recombinant proteins were lower than that observed with the neutrophil Rap1, but the GTPase activity of the neutrophil and wild-type recombinant Rap1 proteins could be stimulated to similar levels by Rap-GAP activity in neutrophil cytosol. In contrast to wild-type Rap1A, the GTPase activity of 35A Rap was unresponsive to Rap-GAP stimulation. Neither recombinant Rap1A nor neutrophil Rap1 protein GTPase activity could be stimulated by recombinant Ras-GAP at a concentration 25-fold higher than that required to hydrolyze 50% of H-Ras-bound GTP under similar conditions. These results suggest that the putative effector domains (amino acids 32 to 40) shared between Rap1 and Ras are functionally similar and interact with their respective GAPs. However, although Rap1 and Ras are identical in this region, secondary structure or additional regions must confer the ability to respond to GAPs.