Performance Characteristics of Oncomine Focus Assay for Theranostic Analysis of Solid Tumors, A (21-Months) Real-Life Study.

Next generation sequencing analysis is crucial for therapeutic decision in various solid tumor contexts. The sequencing method must remain accurate and robust throughout the instrument lifespan allowing the biological validation of patients' results. This study aims to evaluate the long-term sequencing performances of the Oncomine Focus assay kit allowing theranostic DNA and RNA variants detection on the Ion S5XL instrument. We evaluated the sequencing performances of 73 consecutive chips over a 21-month period and detailed the sequencing data obtained from both quality controls and clinical samples. The metrics describing sequencing quality remained stable throughout the study. We showed that an average of 11 × 106 (±0.3 × 106) reads were obtained using a 520 chip leading to an average of 6.0 × 105 (±2.6 × 105) mapped reads per sample. Of 400 consecutive samples, 95.8 ± 16% of amplicons reached the depth threshold of 500X. Slight modifications of the bioinformatics workflow improved DNA analytical sensitivity and allowed the systematic detection of expected SNV, indel, CNV, and RNA alterations in quality controls samples. The low inter-run variability of DNA and RNA-even at low variant allelic fraction, amplification factor, or reads counts-indicated that our method was adapted to clinical practice. The analysis of 429 clinical DNA samples indicated that the modified bioinformatics workflow allowed detection of 353 DNA variants and 88 gene amplifications. RNA analysis of 55 clinical samples revealed 7 alterations. This is the first study showing the long-term robustness of the Oncomine Focus assay in clinical routine practice.

The Affinity of Hemoglobin for Oxygen Is Not Altered During COVID-19.

Background: A computational proteomic analysis suggested that SARS-CoV-2 might bind to hemoglobin (Hb). The authors hypothesized that this phenomenon could result in a decreased oxygen (O2) binding and lead to hemolytic anemia as well. The aim of this work was to investigate whether the affinity of Hb for O2 was altered during COVID-19. Methods: In this retrospective, observational, single-center study, the blood gas analyses of 100 COVID-19 patients were compared to those of 100 non-COVID-19 patients. Fifty-five patients with carboxyhemoglobin (HbCO) ≥8% and 30 with sickle cell disease (SCD) were also included ("positive controls" with abnormal Hb affinity). P50 was corrected for body temperature, pH, and PCO2. Results: Patients did not differ statistically for age or sex ratio in COVID-19 and non-COVID-19 groups. Median P50 at baseline was 26 mmHg [25.2-26.8] vs. 25.9 mmHg [24-27.3], respectively (p = 0.42). As expected, P50 was 22.5 mmHg [21.6-23.8] in the high HbCO group and 29.3 mmHg [27-31.5] in the SCD group (p < 0.0001). Whatever the disease severity, samples from COVID-19 to non-COVID-19 groups were distributed on the standard O2-Hb dissociation curve. When considering the time-course of P50 between days 1 and 18 in both groups, no significant difference was observed. Median Hb concentration at baseline was 14 g.dl-1 [12.6-15.2] in the COVID-19 group vs. 13.2 g.dl-1 [11.4-14.7] in the non-COVID-19 group (p = 0.006). Among the 24 COVID-19 patients displaying anemia, none of them exhibited obvious biological hemolysis. Conclusion: There was no biological argument to support the hypothesis that SARS-CoV-2 could alter O2 binding to Hb.

Cellular response to alkylating agent MNNG is impaired in STAT1-deficients cells.

The SN 1 alkylating agents activate the mismatch repair system leading to delayed G2 /M cell cycle arrest and DNA repair with subsequent survival or cell death. STAT1, an anti-proliferative and pro-apoptotic transcription factor is known to potentiate p53 and to affect DNA-damage cellular response. We studied whether STAT1 may modulate cell fate following activation of the mismatch repair system upon exposure to the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Using STAT1-proficient or -deficient cell lines, we found that STAT1 is required for: (i) reduction in the extent of DNA lesions, (ii) rapid phosphorylation of T68-CHK2 and of S15-p53, (iii) progression through the G2 /M checkpoint and (iv) long-term survival following treatment with MNNG. Presence of STAT1 is critical for the formation of a p53-DNA complex comprising: STAT1, c-Abl and MLH1 following exposure to MNNG. Importantly, presence of STAT1 allows recruitment of c-Abl to p53-DNA complex and links c-Abl tyrosine kinase activity to MNNG-toxicity. Thus, our data highlight the important modulatory role of STAT1 in the signalling pathway activated by the mismatch repair system. This ability of STAT1 to favour resistance to MNNG indicates the targeting of STAT1 pathway as a therapeutic option for enhancing the efficacy of SN1 alkylating agent-based chemotherapy.

Catalase polymorphisms and metabolic diseases.

PURPOSE OF REVIEW: This review summarizes our current understanding of the implication of catalase polymorphisms in the occurrence, control and comorbidities of metabolic diseases. RECENT FINDINGS: Whatever impaired glucose tolerance, insulin resistance on diabetes and whatever their occurrence or implications, the studies taken together converge toward the hypothesis that catalase polymorphisms play a role in glucose disorders. -262C/T and -844A>G single nucleotide polymorphisms are associated to hypertension susceptibility and/or onset. Concerning dyslipidemia, very recent studies requiring confirmation report a -262C/T implication. Finally, a role of catalase polymorphisms in bone metabolism is described. SUMMARY: Plethora of studies on catalase SNPs and their link with diseases exist. It is now clear that genetic variations in the catalase gene and its promoter are putative risk factors for metabolic disease. The question of how these polymorphisms actively play a role in various metabolisms remains unanswered. Further functional studies are required in order to gain a deeper insight into the direct role of catalase.

STAT1-dependent IgG cell-surface expression in a human B cell line derived from a STAT1-deficient patient.

STAT1 is a key effector of cytokines involved in the resistance to pathogens; its identified transcriptional targets mediate the innate immune response involved in the defense against viruses and bacteria. Little is known about the role of STAT1 in adaptive immunity, including its impact on BCR or surface Ig expression. Analysis of this point is difficult in humans, as STAT1 deficiency is extremely rare. SD patients die early in childhood from a severe immunodeficiency. Herein, a SD B cell line obtained from a SD patient was compared with a B cell line from a STAT1-proficient subject in search of differences in surface Ig expression. In this SD B cell line, a complete absence of surface IgG was noted. The mRNA encoding the surface form of IgG was detected only in STAT1-proficient B cells; the mRNAs encoding the secreted and the surface forms were detected in SD and STAT1-proficient B cells. Re-expression of STAT1 in SD B cells restored surface IgG expression and a functional BCR. Conversely, shRNA silencing of STAT1 in B cells reduced considerably the expression of the surface IgG. Although limited to one B cell line, these results suggest that STAT1 may play an essential role in surface IgG expression in human B cells. Possible mechanisms involve regulation of mRNA splicing, transcription, or both. These observations extend the role of STAT1 further in adaptive immunity, including the regulation of BCR expression.

Interest and limits of glomerular filtration rate (GFR) estimation with formulae using creatinine or cystatin C in the malnourished elderly population.

Renal function is often altered in elderly patients. A lot of formulae are proposed to estimate GFR to adjust drug posology. French guidelines recommend the Cockcroft-Gault formula corrected with the body surface area (cCG), but the initially described unadjusted Cockcroft-Gault equation (CG) is mainly used in geriatric clinical practice. International recommendations have proposed the modification of diet in renal disease (MDRD) formula, since several authors recommended the Rule formula using cystatin C (cystC) in particular population. To appreciate the most accurate GFR estimation for posology adaptation in an elderly polypathological population, a cross-sectional study with prospective inclusion was carried out in Charles Foix Hospital. Plasma glucose levels (PGL), creatinine (CREA) levels and serum cystC, albumin (ALB), transthyretin (TTR), C-reactive protein (CRP), orosomucoid (ORO) total cholesterol (tCHOL) levels were determined among 193 elderly patients aged 70 and older. The results showed that in a malnourished, inflamed old population, CG, MDRD and Rule formulae resulted in different estimations of GFR, depending on nutritional and inflammatory parameters. Only cCG estimation was shown to be independent from these parameters. To conclude, cCG seems to be the most accurate and appropriate formula in a polypathological elderly population to evaluate renal function in order to adapt drug posology.

Gene polymorphisms of oxidative stress enzymes: prediction of elderly renutrition.

BACKGROUND: The free radical theory of aging suggests that damage caused by oxidative stress leads to impaired physiologic functions. This damage is stemmed by an adequate antioxidant status, which minimizes the occurrence of infection, thus potentially playing a role in improving nutritional status. The role played by genetic factors remains unknown. OBJECTIVE: The aim of this study was to investigate whether a single nucleotide polymorphism (SNP) of a gene coding for endogenous antioxidant enzymes could influence either nutritional status or renutrition process in an elderly population. DESIGN: Nutritional and inflammatory status were studied in 77 elderly outpatients and in 99 malnourished elderly inpatients over 6 wk of health care treatment. Renutrition efficiency was evaluated with use of the ratio between initial transthyretinemia and 6-wk variation. A genetic study was performed on superoxide dismutase (Ala-9Val), glutathione peroxidase (Pro197Leu), and catalase (from promoter to the first intron). RESULTS: Among the SNPs studied, the G-844A, A-89T, and C-20T catalase SNPs could each be markers predicting renutrition efficiency. These catalase mutant alleles were associated with a lower efficiency of renutrition in malnourished elderly subjects, regardless of initial nutritional and inflammatory status. Genotyping one of these catalase SNPs could make it possible to identify a high-risk subpopulation of mutant allele carriers within the elderly polypathological population. CONCLUSION: In a malnutrition setting, this subpopulation should be given personalized health care, including a strengthened refeeding program. Thus, catalase genotyping could enable earlier recovery of satisfactory nutritional status and thus avoid the consequences of malnutrition, which are especially deleterious in the elderly.

Careful adjustment of Epo non-viral gene therapy for beta-thalassemic anaemia treatment.

BACKGROUND: In situ production of a secreted therapeutic protein is one of the major gene therapy applications. Nevertheless, the plasmatic secretion peak of transgenic protein may be deleterious in many gene therapy applications including Epo gene therapy. Epo gene transfer appears to be a promising alternative to recombinant Epo therapy for severe anaemia treatment despite polycythemia was reached in many previous studies. Therefore, an accurate level of transgene expression is required for Epo application safety. The aim of this study was to adapt posology and administration schedule of a chosen therapeutic gene to avoid this potentially toxic plasmatic peak and maintain treatment efficiency. The therapeutic potential of repeated muscular electrotransfer of light Epo-plasmid doses was evaluated for anaemia treatment in beta-thalassemic mice. METHODS: Muscular electrotransfer of 1 microg, 1.5 microg, 2 microg, 4 microg or 6 microg of Epo-plasmid was performed in beta-thalassemic mice. Electrotransfer was repeated first after 3.5 or 5 weeks first as a initiating dose and then according to hematocrit evolution. RESULTS: Muscular electrotransfer of the 1.5 microg Epo-plasmid dose repeated first after 5 weeks and then every 3 months was sufficient to restore a subnormal hematrocrit in beta-thalassemic mice for more than 9 months. CONCLUSION: This strategy led to efficient, long-lasting and non-toxic treatment of beta-thalassemic mouse anaemia avoiding the deleterious initial hematocrit peak and maintaining a normal hematocrit with small fluctuation amplitude. This repeat delivery protocol of light doses of therapeutic gene could be applied to a wide variety of candidate genes as it leads to therapeutic effect reiterations and increases safety by allowing careful therapeutic adjustments.

Comparison of promoter region constructs for in vivo intramuscular expression.

BACKGROUND: High transgene expression is generally expected after gene transfer. However, different level, kinetics and localization of expression might be needed for relevant therapeutic applications. Former studies have compared various promoter regions driving gene expression leading to conflicting results. In the present work, two promoter families have been compared using the efficient in vivo intramuscular electrotransfer technique. METHODS: Three promoter regions were constructed by associating the strong ubiquitous cytomegalovirus (CMV) enhancer-promoter to its homologous intron A or to a heterologous intron, or to a hybrid intron. Promoter regions derived from the muscle creatine kinase (MCK) promoter were also studied. The expression of the same transgene (SeAP or neurotrophin-3) under control of these different promoters was compared after plasmid electrotransfer in mouse tibialis-cranialis skeletal muscle. RESULTS: Heterologous intron association to the CMV promoter did not modify gene expression kinetics nor increase gene expression level. Usefulness of intron A or hybrid intron association to the CMV promoter depended on the gene. The various MCK promoters drove efficient gene expression but lower than that obtained with the CMV promoter. Furthermore, peak value was reached earlier with MCK promoter regions (14 days). CONCLUSION: For applications of gene transfer restricted to skeletal muscle, the MCK promoter or a MCK promoter variant would be a promising alternative to the CMV promoter. Indeed, it has been demonstrated that the use of MCK promoter limits humoral and cell-mediated immune responses. Furthermore, the MCK promoter decreases the initial expression peak that may be detrimental, drives a sustained gene expression, and improves gene transfer safety.