Hierarchical distribution of somatic variants in newly diagnosed chronic myeloid leukaemia at diagnosis and early follow-up.

There is an emerging body of evidence that patients with chronic myeloid leukaemia (CML) may carry not only breakpoint cluster region-Abelson murine leukaemia viral oncogene homologue 1 (BCR-ABL1) kinase domain mutations (BCR-ABL1 KD mutations), but also mutations in other genes. Their occurrence is highest during progression or at failure, but their impact at diagnosis is unclear. In the present study, we prospectively screened for mutations in 18 myeloid neoplasm-associated genes and BCR-ABL1 KD in the following populations: bulk leucocytes, CD34+ CD38+ progenitors and CD34+ CD38- stem cells, at diagnosis and early follow-up. In our cohort of chronic phase CML patients, nine of 49 patients harboured somatic mutations in the following genes: six ASXL1 mutations, one SETBP1, one TP53, one JAK2, but no BCR-ABL1 KD mutations. In seven of the nine patients, mutations were detected in multiple hierarchical populations including bulk leucocytes at diagnosis. The mutation dynamics reflected the BCR-ABL1 transcript decline induced by treatment in eight of the nine cases, suggesting that mutations were acquired in the Philadelphia chromosome (Ph)-positive clone. In one patient, the JAK2 V617F mutation correlated with a concomitant Ph-negative myeloproliferative neoplasm and persisted despite a 5-log reduction of the BCR-ABL1 transcript. Only two of the nine patients with mutations failed first-line therapy. No correlation was found between the mutation status and survival or response outcomes.

Novel Illumina-based next generation sequencing approach with one-round amplification provides early and reliable detection of BCR-ABL1 kinase domain mutations in chronic myeloid leukemia.

The occurrence of mutations in the BCR-ABL1 kinase domain (KD) can lead to treatment resistance in chronic myeloid leukaemia patients. Nowadays, next-generation sequencing (NGS) is an alternative method for the detection of kinase domain mutations, compared to routinely used Sanger sequencing, providing a higher sensitivity of mutation detection. However, in the protocols established so far multiple rounds of amplification limit reliable mutation detection to approximately 5% variant allele frequency. Here, we present a simplified, one-round amplification NGS protocol for the Illumina platform, which offers a robust early detection of BCR-ABL1 KD mutations with a reliable detection limit of 3% variant allele frequency.

Modification of topoisomerases in mammospheres derived from breast cancer cell line: clinical implications for combined treatments with tyrosine kinase inhibitors.

BACKGROUND: Accumulating evidences suggest that tumors are driven by a small population of cells, termed "cancer stem cells" (CSCs), which may be resistant to current therapeutic approaches. In breast carcinoma, the CSCs have been identified as a CD44+/CD24- cell population. These rare cells are able to grow as non-adherent sphere-like structures, termed "mammospheres", which enables their isolation and expansion in culture. To design efficient strategies for the complete eradication of CSCs, it is important to identify enzymes and proteins that are known as anti-cancer targets, and differ in their properties from those present in the none CSCs. Here we investigated the activity and expression of type I and type II DNA topoisomerases (topo I and topo II) in CSCs and their response to anti-topoisomerase inhibitors. METHODS: MCF7 breast cancer cells, PC3 prostate cancer cells and 4 T1-Luc-Oct3/4pG mouse mammary carcinoma cells were grown on low-attachment dishes in specific medium and allowed to form spheres. Enrichment of CSC population was verified by immunostaining, flow cytometry or fluorescent microscopy imaging. Nuclear protein extracts were prepared and topoisomerases activity and protein levels were determined. Cell viability was examined by the MTT and Neutral Red assays. RESULTS: Unlike the adherent MCF7 cell line, topo I activity is decreased and topo II activity is increased in the CSCs. However, the relative levels of the enzyme proteins were similar in both mammospheres and adherent cells. Topo I activity in mammospheres is regulated, at least in part, by PARP-1, as observed by the recovery of topo I activity after treatment with PARP-1 inhibitor 3-Aminobenzamide. Mammosphere-derived cells show reduced sensitivity to topo I inhibitor, camptothecin, and increased sensitivity to topo II inhibitor etoposide. Intact mammospheres show increased resistance to both drugs. A combined treatment of intact mammospheres with either CPT and gefitinib, or etoposide and erlotinib, increased the anti-cancer effect of both drugs. CONCLUSIONS: The data of this study suggest that the understanding of biological behavior of essential enzymes such as topoisomerases, in CSCs' progression and early stages of tumor development, is important for developing new strategies for cancer treatment as well as new therapies for advanced disease.

Digital PCR can provide improved BCR-ABL1 detection in chronic myeloid leukemia patients in deep molecular response and sensitivity of standard quantitative methods using EAC assays.

BCR-ABL1 molecular detection using quantitative PCR (qPCR) methods is the golden standard of chronic myeloid leukemia (CML) monitoring. However, due to variable sensitivity of qPCR assays across laboratories, alternative methods are tested. Digital PCR (dPCR) has been suggested as a robust and reproducible option. Here we present a comparison of droplet dPCR with routinely used reverse-transcription qPCR (RT-qPCR) and automated GeneXpert systems. Detection limit of dPCR was above 3 BCR-ABL1 copies, although due to background amplification the resulting sensitivity was 0.01% BCR-ABL1 (MR4.0). Nevertheless, in comparison with GeneXpert, dPCR categorized more than 50% of the patients into different MR groups, showing a potential for improved BCR-ABL1 detection.

Effect of feeding status on adjuvant arthritis severity, cachexia, and insulin sensitivity in male Lewis rats.

We studied the effect of food restriction, overfeeding, and normofeeding on cachexia, inflammatory and metabolic parameters, and insulin sensitivity in chronic adjuvant arthritis (AA) in rats. Food restriction during AA increased circulating ghrelin, corticosterone, decreased leptin, and ameliorated arthrogram score and systemic inflammation compared to normofeeding. Overfeeding worsened arthrogram score and systemic inflammation, and led to lipid accumulation in the liver, but not to alterations of adipokine and ghrelin plasma levels relative to normofeeding. Independently of feeding status, AA induced cachexia, in which modulation of mRNA expressions for appetite-regulating neuropeptides (NPY, AgRP, POMC, CART) in the arcuate nucleus (ARC) does not play a primary role. The overexpression of IL-1ß mRNA in the ARC suggests its role in the mechanisms of impaired energy balance during AA under all feeding conditions. Normal HOMA index in all arthritic groups does not indicate the development of insulin resistance by feeding interventions in these rats.

Autoimmune diabetes mellitus with adult onset and type 1 diabetes mellitus in children have different genetic predispositions.

Type 1 diabetes with manifestation after 35 years of age is defined by CP <200 pmol/L and institution of insulin therapy within 6 months after diagnosis. Latent autoimmune diabetes mellitus in adults (LADA) manifesting after 35 years of age is defined by minimum 6 months after diagnosis without insulin therapy and C peptide (CP) >200 pmol/L and antiGAD > 50 ng/mL. We aimed to find a possible genetic discrimination among different types of autoimmune diabetes. To accomplish this goal, we analyzed DNA samples from 31 LADA patients, 75 patients with adult-onset type 1 diabetes mellitus, 188 type 1 diabetic children, and 153 healthy adult individuals. We studied five genetic loci on chromosomes 6, 11, 4, and 14: HLA DRB1 and DQB1 alleles, major histocompatibility complex (MHC) class I-related gene-A (MIC-A) microsatellite polymorphism, interleukin (IL)-18 single nucleotide polymorphism, the microsatellite polymorphism of nuclear factor kappa B gene (NF-kappaB1), and the single nucleotide polymorphism of a gene for its inhibitor (NF-kappaBIA). HLA-DR3 was detected as the predisposition allele for LADA (OR = 4.94, P < 0.0001). Further we found a statistically significant increase of NF-kappaBIA AA genotype (OR = 2.68, P < 0.01). On the other hand, DRB1*04, which is linked with DQB1*0302, was observed as a risk factor in patients with type 1 diabetes mellitus (T1DM) onset after 35 years of age (OR = 10.47, P < 0.0001 and OR = 9.49, P < 0.0001, respectively). There was also an association with MIC-A5.1 (OR = 2.14, P < 0.01). Statistically significant difference was found in the distribution of IL-18 promoter -607 (C/A) polymorphism between LADA and T1DM in adults (P < 0.01). We conclude that all subgroups of autoimmune diabetes have partly different immunogenetic predisposition.

Carnosine as a protective factor in diabetic nephropathy: association with a leucine repeat of the carnosinase gene CNDP1.

The risk of diabetic nephropathy is partially genetically determined. Diabetic nephropathy is linked to a gene locus on chromosome 18q22.3-q23. We aimed to identify the causative gene on chromosome 18 and to study the mechanism by which the product of this gene could be involved in the development of diabetic nephropathy. DNA polymorphisms were determined in 135 case (diabetic nephropathy) and 107 control (diabetes without nephropathy) subjects. The effect of carnosine on the production of extracellular matrix components and transforming growth factor-beta (TGF-beta) after exposure to 5 and 25 mmol/l d-glucose was studied in cultured human podocytes and mesangial cells, respectively. A trinucleotide repeat in exon 2 of the CNDP1 gene, coding for a leucine repeat in the leader peptide of the carnosinase-1 precursor, was associated with nephropathy. The shortest allelic form (CNDP1 Mannheim) was more common in the absence of nephropathy (P = 0.0028, odds ratio 2.56 [95% CI 1.36-4.84]) and was associated with lower serum carnosinase levels. Carnosine inhibited the increased production of fibronectin and collagen type VI in podocytes and the increased production of TGF-beta in mesangial cells induced by 25 mmol/l glucose. Diabetic patients with the CNDP1 Mannheim variant are less susceptible for nephropathy. Carnosine protects against the adverse effects of high glucose levels on renal cells.

NFkappaB and its inhibitor IkappaB in relation to type 2 diabetes and its microvascular and atherosclerotic complications.

Nuclear factor kappa B (NFkappaB) is an important transcription factor that together with its inhibitor (IkappaB) participates in the activation of genes involved in immune responses. We examined the CA repeat polymorphism of the NFKB1 gene (encoding for NFkappaB) and A/G point variation in the 3'UTR region of the nuclear factor kappa B inhibitor alpha (NFKBIA) gene (encoding for IkappaB) in Czech and German patients with type 2 diabetes. The sample consisted of 211 patients, both with and without kidney complications, and 159 controls. Additionally, 152 patients with systemic lupus erythematosus (SLE) were genotyped for NFKBIA polymorphism. We observed a significant increase in the homozygous AA genotype of the NFKBIA gene when compared with the control group (the highest value was in diabetics without diabetic nephropathy [p(c)* = 0.0015, odds ratio = 3.59]). No differences were seen between the SLE and control groups. With regard to the polymorphism of the NFKB1 gene, we did not observe any significant differences between the groups. Since the AA genotype of the NFKBIA gene presents a risk for type 2 diabetes development but not for diabetic nephropathy alone, we believe that the NFkappaB gene polymorphism can influence the pathogenesis of diabetes mellitus and affect its complications. Negative findings relative to other inflammatory autoimmune diseases, such as SLE, suggest a specific relationship between NFkappaB and type 2 diabetes mellitus.

Unique Versus Redundant Functions of IL-1α and IL-1ß in the Tumor Microenvironment.

Interleukin-1 (IL-1) is a major "alarm" upstream pro-inflammatory cytokine that also affects immunity and hematopoiesis by inducing cytokine cascades. In the tumor arena, IL-1 is produced by malignant or microenvironmental cells. As a pleiotropic cytokine, IL-1 is involved in tumorigenesis and tumor invasiveness but also in the control of anti-tumor immunity. IL-1α and IL-1ß are the major agonists of IL-1, while IL-1Ra is a physiological inhibitor of pre-formed IL-1. In their secreted form, IL-1α and IL-1ß bind to the same receptors and induce the same biological functions, but IL-1α and IL-1ß differ in their compartmentalization within the producing cell or the microenvironment. IL-1ß is only active in its processed, secreted form, and mediates inflammation, which promotes carcinogenesis, tumor invasiveness, and immunosuppression, whereas IL-1α is mainly cell-associated and in the tumor context, when expressed on the cell membrane, it stimulates anti-tumor cell immunity manifested by tumor regression. In the tumor milieu, extracellular levels of IL-1α are usually low and do not stimulate broad inflammation that promotes progression. Immunosuppression induced by IL-1ß in the tumor microenvironment, mainly through MDSC induction, usually inhibits or masks anti-tumor cell immunity induced by cell-associated IL-1α. However, in different tumor systems, redundant or unique patterns of IL-1α and IL-1ß expression and function have been observed. Recent breakthroughs in inflammasome biology and IL-1ß processing/secretion have spurred the development of novel anti-IL-1 agents, which are being used in clinical trials in patients with diverse inflammatory diseases. Better understanding of the integrative role of IL-1α and IL-1ß in distinct malignancies will facilitate the application of novel IL-1 modulation approaches at the bedside, in cancer patients with minimal residual disease (MRD), as an adjunct to conventional approaches to reduce the tumor burden.

The transcription of the alarmin cytokine interleukin-1 alpha is controlled by hypoxia inducible factors 1 and 2 alpha in hypoxic cells.

During hypoxia, cells undergo transcriptional changes to adjust to metabolic stress, to promote cell survival, and to induce pro-angiogenic factors. Hypoxia-induced factors (HIFs) regulate these transcriptional alterations. Failure to restore oxygen levels results in cell death by necrosis. IL-1α is one of the most important mediators of sterile inflammation following hypoxia-mediated necrosis. During hypoxia, IL-1α is up-regulated and released from necrotic cells, promoting the initiation of sterile inflammation. This study examined the role of IL-1α transcription in initiation of hypoxic stress and the correlation between IL-1α transcription and HIFα factors. In an epithelial cell line cultured under hypoxic conditions, IL-1α transcription was up-regulated in a process mediated and promoted by HIFα factors. IL-1α transcription was also up-regulated in hypoxia in a fibroblast cell line, however, in these cells, HIFα factors inhibited the elevation of transcription. These data suggest that HIFα factors play a significant role in initiating sterile inflammation by controlling IL-1α transcription during hypoxia in a differential manner, depending on the cell type.