Colorimetric Assessment of BCR-ABL1 Transcripts in Clinical Samples Via Gold Nanoprobes

Gold nanoparticles functionalized with thiolated oligonucleotides (Au-nanoprobes) have been used in a range of applications for the detection of bioanalytes of interest, from ions to proteins and DNA targets. These detection strategies are based on the unique optical properties of gold nanoparticles, in particular, the intense color that is subject to modulation by modification of the medium dieletric. Au-nanoprobes have been applied for the detection and characterization of specific DNA sequences of interest, namely pathogens and disease biomarkers. Nevertheless, despite its relevance, only a few reports exist on the detection of RNA targets. Among these strategies, the colorimetric detection of DNA has been proven to work for several different targets in controlled samples but demonstration in real clinical bioanalysis has been elusive. Here, we used a colorimetric method based on Au-nanoprobes for the direct detection of the e14a2 BCR-ABL fusion transcript in myeloid leukemia patient samples without the need for retro-transcription. Au-nanoprobes directly assessed total RNA from 38 clinical samples, and results were validated against reverse transcription-nested polymerase chain reaction (RT-nested PCR) and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The colorimetric Au-nanoprobe assay is a simple yet reliable strategy to scrutinize myeloid leukemia patients at diagnosis and evaluate progression, with obvious advantages in terms of time and cost, particularly in low- to medium-income countries where molecular screening is not routinely feasible. Graphical abstract Gold nanoprobe for colorimetric detection of BCR-ABL1 fusion transcripts originating from the Philadelphia chromosome.

Stromal upregulation of lateral epithelial adhesions : gene expression analysis of signalling pathways in prostate epithelium

BACKGROUND: Stromal signalling increases the lateral cell adhesions of prostate epithelial cells grown in 3D culture. The aim of this study was to use microarray analysis to identify significant epithelial signalling pathways and genes in this process. METHODS: Microarray analysis was used to identify genes that were differentially expressed when epithelial cells were grown in 3D Matrigel culture with stromal co-culture compared to without stroma. Two culture models were employed: primary epithelial cells (ten samples) and an epithelial cell line (three experiments). A separate microarray analysis was performed on each model system and then compared to identify tissue-relevant genes in a cell line model. RESULTS: TGF beta signalling was significantly ranked for both model systems and in both models the TGF beta signalling gene SOX4 was significantly down regulated. Analysis of all differentially expressed genes to identify genes that were common to both models found several morphology related gene clusters; actin binding (DIAPH2, FHOD3, ABLIM1, TMOD4, MYH10), GTPase activator activity (BCR, MYH10), cytoskeleton (MAP2, MYH10, TMOD4, FHOD3), protein binding (ITGA6, CD44), proteinaceous extracellular matrix (NID2, CILP2), ion channel/ ion transporter activity (CACNA1C, CACNB2, KCNH2, SLC8A1, SLC39A9) and genes associated with developmental pathways (POFUT1, FZD2, HOXA5, IRX2, FGF11, SOX4, SMARCC1). CONCLUSIONS: In 3D prostate cultures, stromal cells increase lateral epithelial cell adhesions. We show that this morphological effect is associated with gene expression changes to TGF beta signalling, cytoskeleton and anion activity.

A randomized controlled pilot study comparing aqueous cream with a beeswax and herbal oil cream in the provision of relief from postburn pruritis

Postburn itch is reported to affect up to 87% of the burn population. Although treatments for postburn itch are multimodal, they remain consistently ineffective. However, recent anecdotal evidence from several outpatients at a tertiary referral hospital suggests that a cream combining beeswax and several herbal oils may be effective in the minimization of postburn itch. The aim of this study was to test the efficacy of beeswax and herbal oil cream against the standard treatment of aqueous cream in the provision of relief from the symptoms of postburn itch. A randomized controlled trial compared two groups using a visual analog scale, frequency of cream application, itch recurrence after cream application, use of antipruritic medications, and sleep disturbance to determine the effect of itch severity and duration. Fifty-two participants were enrolled in the study (84% male) with a mean age of 35 years (SD = 16) and mean burn TBSA of 7.2% (SD = 7.7). Study results found that the beeswax and herbal oil cream reduce itch after application more frequently than aqueous cream (P = .001). In addition, when managed with beeswax and herbal oil cream, participants found that their itch recurred later (P ≤ .001) and their use of antipruritic medications was lower (P = .023). Findings of this study suggest beeswax and herbal oil cream to be more effective in the minimization of postburn itch than aqueous cream. Given this, a larger study examining the efficacy of beeswax and herbal oil cream appears warranted.

High-resolution loss of heterozygosity screening implicates PTPRJ as a potential tumor suppressor gene that affects susceptibility to non-Hodgkin's Lymphoma

We employed a Hidden-Markov-Model (HMM) algorithm in loss of heterozygosity (LOH) analysis of high-density single nucleotide polymorphism (SNP) array data from Non-Hodgkin’s lymphoma (NHL) entities, follicular lymphoma (FL), and diffuse large B-cell lymphoma (DLBCL). This revealed a high frequency of LOH over the chromosomal region 11p11.2, containing the gene encoding the protein tyrosine phosphatase receptor type J (PTPRJ). Although PTPRJ regulates components of key survival pathways in B-cells (i.e., BCR, MAPK, and PI3K signaling), its role in B-cell development is poorly understood. LOH of PTPRJ has been described in several types of cancer but not in any hematological malignancy. Interestingly, FL cases with LOH exhibited down-regulation of PTPRJ, in contrast no significant variation of expression was shown in DLBCLs. In addition, sequence screening in Exons 5 and 13 of PTPRJ identified the G973A (rs2270993), T1054C (rs2270992), A1182C (rs1566734), and G2971C (rs4752904) coding SNPs (cSNPs). The A1182 allele was significantly more frequent in FLs and in NHLs with LOH. Significant over-representation of the C1054 (rs2270992) and the C2971 (rs4752904) alleles were also observed in LOH cases. A haplotype analysis also revealed a significant lower frequency of haplotype GTCG in NHL cases, but it was only detected in cases with retention. Conversely, haplotype GCAC was over-representated in cases with LOH. Altogether, these results indicate that the inactivation of PTPRJ may be a common lymphomagenic mechanism in these NHL subtypes and that haplotypes in PTPRJ gene may play a role in susceptibility to NHL, by affecting activation of PTPRJ in these B-cell lymphomas.

An audit of first-aid treatment of pediatric burns patients and their clinical outcome

This study describes the first aid used and clinical outcomes of all patients who presented to the Royal Children's Hospital, Brisbane, Australia in 2005 with an acute burn injury. A retrospective audit was performed with the charts of 459 patients and information concerning burn injury, first-aid treatment, and clinical outcomes was collected. First aid was used on 86.1% of patients, with 8.7% receiving no first aid and unknown treatment in 5.2% of cases. A majority of patients had cold water as first aid (80.2%), however, only 12.1% applied the cold water for the recommended 20 minutes or longer. Recommended first aid (cold water for >or=20 minutes) was associated with significantly reduced reepithelialization time for children with contact injuries (P=.011). Superficial depth burns were significantly more likely to be associated with the use of recommended first aid (P=.03). Suboptimal treatment was more common for children younger than 3.5 years (P<.001) and for children with friction burns. This report is one of the few publications to relate first-aid treatment to clinical outcomes. Some positive clinical outcomes were associated with recommended first-aid use; however, wound outcomes were more strongly associated with burn depth and mechanism of injury. There is also a need for more public awareness of recommended first-aid treatment.

Silver absorption on burns after the application of Acticoat : data from pediatric patients and a porcine burn model

Silver dressings have been widely used to successfully prevent burn wound infection and sepsis. However, a few case studies have reported the functional abnormality and failure of vital organs, possibly caused by silver deposits. The aim of this study was to investigate the serum silver level in the pediatric burn population and also in several internal organs in a porcine burn model after the application of Acticoat. A total of 125 blood samples were collected from 46 pediatric burn patients. Thirty-six patients with a mean of 13.4% TBSA burns had a mean peak serum silver level of 114 microg/L, whereas 10 patients with a mean of 1.85% TBSA burns had an undetectable level of silver (<5.4 microg/L). Overall, serum silver levels were closely related to burn sizes. However, the highest serum silver was 735 microg/L in a 15-month-old toddler with 10% TBSA burns and the second highest was 367 microg/L in a 3-year old with 28% TBSA burns. In a porcine model with 2% TBSA burns, the mean peak silver level was 38 microg/L at 2 to 3 weeks after application of Acticoat and was then significantly reduced to an almost undetectable level at 6 weeks. Of a total of four pigs, silver was detected in all four livers (1.413 microg/g) and all four hearts (0.342 microg/g), three of four kidneys (1.113 microg/g), and two of four brains (0.402 microg/g). This result demonstrated that although variable, the level of serum silver was positively associated with the size of burns, and significant amounts of silver were deposited in internal organs in pigs with only 2% TBSA burns, after application of Acticoat.

Nilotinib and MEK inhibitors induce synthetic lethality through paradoxical activation of RAF in drug-resistant chronic myeloid leukemia

We show that imatinib, nilotinib, and dasatinib possess weak off-target activity against RAF and, therefore, drive paradoxical activation of BRAF and CRAF in a RAS-dependent manner. Critically, because RAS is activated by BCR-ABL, in drug-resistant chronic myeloid leukemia (CML) cells, RAS activity persists in the presence of these drugs, driving paradoxical activation of BRAF, CRAF, MEK, and ERK, and leading to an unexpected dependency on the pathway. Consequently, nilotinib synergizes with MEK inhibitors to kill drug-resistant CML cells and block tumor growth in mice. Thus, we show that imatinib, nilotinib, and dasatinib drive paradoxical RAF/MEK/ERK pathway activation and have uncovered a synthetic lethal interaction that can be used to kill drug-resistant CML cells in vitro and in vivo.

Unswitch-ABL drugs overcome resistance in chronic myeloid leukemia

ABL inhibitors have revolutionized the clinical management of chronic myeloid leukemia, but the BCR-ABLT315I mutation confers resistance to currently approved drugs. Chan et al. show, in this issue of Cancer Cell, that " switch-control" inhibitors block BCR-ABLT315I activity by preventing ABL from switching from the inactive to active conformation.

Detection of DNA mutations using novel SERS (Surface-Enhanced Raman Spectroscopy) diagnostic platform

This article describes the detection of DNA mutations using novel Au-Ag coated GaN substrate as SERS (surface-enhanced Raman spectroscopy) diagnostic platform. Oligonucleotide sequences corresponding to the BCR-ABL (breakpoint cluster region-Abelson) gene responsible for development of chronic myelogenous leukemia were used as a model system to demonstrate the discrimination between the wild type and Met244Val mutations. The thiolated ssDNA (single-strand DNA) was immobilized on the SERS-active surface and then hybridized to a labeled target sequence from solution. An intense SERS signal of the reporter molecule MGITC was detected from the complementary target due to formation of double helix. The SERS signal was either not observed, or decreased dramatically for a negative control sample consisting of labeled DNA that was not complementary to the DNA probe. The results indicate that our SERS substrate offers an opportunity for the development of novel diagnostic assays.

Novel prognostic factors in chronic myeloid leukemia

Chronic myeloid leukemia (CML) is a malignant clonal blood disease that originates from a pluripotent hematopoietic stem cell. The cytogenetic hallmark of CML, the Philadelphia chromosome (Ph), is formed as a result of reciprocal translocation between chromosomes 9 and 22, which leads to a formation of a chimeric BCR-ABL fusion gene. The BCR-ABL protein is a constitutively active tyrosine kinase that changes the adhesion properties of cells, constitutively activates mitogenic signaling, enhances cell proliferation and reduces apoptosis. This results in leukemic growth and the clinical disease, CML. With the advent of targeted therapies against the BCR-ABL fusion protein, the treatment of CML has changed considerably during the recent decade. In this thesis, the clinical significance of different diagnostic methods and new prognostic factors in CML have been assessed. First, the association between two different methods for measuring CML disease burden (the RQ-PCR and the high mitotic index metaphase FISH) was assessed in bone marrow and peripheral blood samples. The correlation between positive RQ-PCR and metaphase FISH samples was high. However, RQ-PCR was more sensitive and yielded measurable transcripts in 40% of the samples that were negative by metaphase FISH. The study established a laboratory-specific conversion factor for setting up the International Scale when standardizing RQ-PCR measurements. Secondly, the amount of minimal residual disease (MRD) after allogeneic hematopoietic stem cell transplantation (alloHSCT) was determined. For this, metaphase FISH was done for the bone marrow samples of 102 CML patients. Most (68%), had no residual cells during the entire follow-up time. Some (12 %) patients had minor (<1%) MRD which decreased even further with time, whereas 19% had a progressive rise in MRD that exceeded 1% or had more than 1% residual cells when first detected. Residual cells did not become eradicated spontaneously if the frequency of Ph+ cells exceeded 1% during follow-up. Next, the impact of deletions in the derivative chromosome 9, was examined. Deletions were observed in 15% of the CML patients who later received alloHSCT. After alloHSCT, there was no difference in the total relapse rate in patients with or without deletions. Nor did the estimates of overall survival, transplant-related mortality, leukemia-free survival and relapse-free time show any difference between these groups. When conventional treatment regimens are used, the der(9) status could be an important criterion, in conjunction with other prognostic factors, when allogeneic transplantation is considered. The significance of der(9) deletions for patients treated with tyrosine kinase inhibitors is not clear and requires further investigation. In addition to the der(9) status of the patient, the significance of bone marrow lymphocytosis as a prognostic factor in CML was assessed. Bone marrow lymphocytosis during imatinib therapy was a positive predictive factor and heralded optimal response. When combined with major cytogenetic response at three months of treatment, bone marrow lymphocytosis predicted a prognostically important major molecular response at 18 months of imatinib treatment. Although the validation of these findings is warranted, the determination of the bone marrow lymphocyte count could be included in the evaluation of early response to imatinib treatment already now. Finally, BCR-ABL kinase domain mutations were studied in CML patients resistant against imatinib treatment. Point mutations detected in the kinase domain were the same as previously reported, but other sequence variants, e.g. deletions or exon splicing, were also found. The clinical significance of the other variations remains to be determined.

NK-and T-cell function in chronic myeloid leukemia patients treated with interferon-? monotherapy

Chronic myeloid leukemia (CML) is one of the most studied human malignancies. It is caused by an autonomously active tyrosine kinase BCR-ABL, which is a result from a translocation between chromosomes 9 and 22 in the hematopoietic stem cell. As an outcome, a Philadelphia (Ph) chromosome is formed. BCR-ABL causes disturbed cell proliferation among other things. Although targeted tyrosine kinase inhibitor therapy has been developed in the beginning of the millenium and the survival rate has increased significantly, it is still not known why some patients benefit more from the treatment than others. Furthermore, the therapy is not considered to be curative. Before the era of tyrosine kinase inhibitors, the first-line treatment for CML was interferon-? (IFN-?). However, only a small proportion of patients benefitted from the treatment. Of these patients, a few were able to discontinue the treatment without renewal of the disease. The mechanism of IFN-? is not completely understood, but it is believed that differences in the immune system can be one of the reasons why some patients have better therapy response. Kreutzman, Rohon et al. have recently discovered that patients who have been able to stop IFN-? treatment have an increased number of NK- and T-cells. They also have a unique clonal T-cell population and more cytotoxic CD8+ T-cells and less CD4+ T-cells. The aim of this master’s thesis was to study the function of T- and NK-cells in IFN-? treated patients. Although it was shown earlier that IFN-? treated patients have increased NK-cell count, the function of these cells was unknown. Therefore, we have now investigated the killing potential of patients’ NK-cells, their activation status and cell surface antigen expression. In addition, we have also studied the activation status of patients’ T-cells and their cytotoxic properties. We observed that NK-cells from patients treated with IFN-? are unable to kill leukemic cells (K562) than NK-cells from healthy controls. In addition, patients on IFN-? treatment have more active T-cells and their NK-cells have an undifferentiated immunoregulatory phenotype. Patients that have been able to stop the treatment have anergic T-and NK-cells. As a conclusion our results suggest that IFN-? therapy induces increased NK-cell count, NK-cell immunoregulatory functions and more active T-cells. After stopping IFN-? therapy, NK- and T-cells from CML patients restore anergy typical for CML.