Serial Dilution Experiments Research Articles

NOTCH4 maintains quiescent mesenchymal-like breast cancer stem cells via transcriptionally activating SLUG and GAS1 in triple-negative breast cancer.

Rationale: NOTCH4 receptor has been implicated in triple-negative breast cancer (TNBC) development and breast cancer stem cell (BCSC) regulation. However, the potential of NOTCH4 as a BCSC marker and the underlying mechanisms remain unclear.Methods: In this study, we determined the expression and activation of NOTCH4 in breast cancer cell lines and tumor samples by qRT-PCR, western blotting and immunohistochemistry. Subsequently, in vitro and in vivo serial dilution experiments were performed to demonstrate the application of NOTCH4 as an efficient mesenchymal-like (ML)-BCSC marker in TNBC. Stable overexpression of activated NOTCH4 and knockdown cell lines were established using lentivirus. RNA-seq and qRT-PCR were employed to reveal the downstream effectors of NOTCH4, followed by dual-luciferase reporter and chromatin immunoprecipitation assays to identify the genuine binding sites of NOTCH4 on SLUG and GAS1 promoters. Transwell assay, mammosphere formation and chemoresistance experiments were performed to determine the effects of SLUG, GAS1 and NOTCH4 on the mesenchymal-like characteristics of TNBC cells. Survival analysis was used to study the relation of NOTCH4, SLUG and GAS1 with prognosis of breast cancer.Results: NOTCH4 is aberrantly highly expressed and activated in TNBC, which contributes to the maintenance of ML-BCSCs. Furthermore, NOTCH4 shows significantly higher efficiency in labeling ML-BCSCs than the currently commonly used CD24-CD44+ marker. Mechanistically, NOTCH4 transcriptionally upregulates SLUG and GAS1 to promote EMT and quiescence in TNBC, respectively. The effects of NOTCH4 can be mimicked by simultaneous overexpression of SLUG and GAS1. Moreover, SLUG is also involved in harnessing GAS1, a known tumor suppressor gene, via its anti-apoptotic function.Conclusions: Our findings reveal that the NOTCH4-SLUG-GAS1 circuit serves as a potential target for tumor intervention by overcoming stemness of ML-BCSCs and by conquering the lethal chemoresistance and metastasis of TNBC.

Comparison of RNA-Based Versus DNA-Based Quantitative KIT D816V Mutation Analysis Reveals Significant Disparity in Patients with Advanced Systemic Mastocytosis

Systemic mastocytosis (SM) is characterized by activating mutations in KIT, usually KIT D816V in >90% of patients. According to the WHO classification, detection of KIT D816V is a minor diagnostic criterion. Moreover, a reduction of the RNA-based KIT D816V expressed allele burden (EAB) of ≥25% at month 6 is a favorable marker for improved survival in midostaurin-treated advanced SM (AdvSM) patients. However, only limited data exist upon the comparison between RNA-based and DNA-based quantitative KIT D816V mutation analyses. We therefore collected peripheral blood samples from 161 SM patients (indolent SM [ISM], n=40; AdvSM, n=121) at time of referral. We applied a real time reverse-transcriptase polymerase chain reaction (qRT-PCR) assay for assessment of the KIT D816V EAB and a chip-based digital PCR (dPCR) for the quantification of the DNA-based KIT D816V variant allele frequency (VAF, QuantStudio 3D dPCR System, ThermoFisher Scientific, Massachusetts, USA). The limit of detection (LOD) was assessed through serial dilution experiments with DNA from healthy individuals and DNA from a SM patient with a KIT D816V VAF of approximately 50%. All samples were analyzed in two independent dPCR runs. In an inital round-robin test for an inter-laboratory (n=4) comparison on 30 DNA samples (ISM, n=7; AdvSM, n=19), we found a very good correlation between 3 different chip-based digital PCR systems (dPCR; droplet-based dPCR, ddPCR, BioRad Laboratories, California, USA; quantitative real-time PCR, qPCR, California, USA; dPCR vs. ddPCR r=0.99, R2=0.99; dPCR vs. qPCR r=0.99, R2=0.98). In ISM patients, the comparison between EAB and VAF revealed a strong linear relationship with a correlation (r) of 0.91 and a coefficient of determination (R2) of 0.84, which was significantly inferior (r=0.71; R2=0.5) in AdvSM patients. In 45/121 (37%) and 76/121 (63%) of AdvSM patients, the EAB/VAF ratio was ≤2 (cohort A) or >2 (cohort B). To confirm the significant disparity between EAB and VAF in individual patients, serial analyses of at least 3 samples in 12 patients revealed a stable EAB/VAF ratio during follow-up. The comparison between cohort A and cohort B revealed significant differences in terms of a higher median hemoglobin level (p=0.006), a lower percentage of patients with hemoglobin <10g/dL (p=0.02), a lower median monocyte level (p=0.01), a lower median alkaline phosphatase level (p=0.03), and a lower number of patients with a high risk molecular profile (at least one gene mutation in SRSF2, ASXL1, and/or RUNX1, S/A/R, p=0.02) in cohort A. Moreover, patients of cohort A had a significantly better overall survival (OS) (median OS 12.9 versus 3.3 years; hazard ratio 2.1; 95% confidence interval 1.2-3.5; p=0.005; Figure). We conclude that a) KIT D816V EAB and VAF are significantly different in AdvSM patients but not in ISM and b) AdvSM patients with an EAB/VAF ratio >2 present with an aggressive phenotype and adverse prognosis as compared to patients with EAB/VAF ratio ≤2. We therefore recommend to routinely determine KIT D816V EAB and VAF in AdvSM patients. Disclosures Fabarius: Novartis: Research Funding. Reiter:Blueprint: Consultancy, Honoraria, Other: Travel reimbursement; Deciphera: Consultancy, Honoraria, Other: Travel reimbursement; Novartis: Consultancy, Honoraria, Other: Travel reimbursement, Research Funding.

Computational cytometer based on magnetically modulated coherent imaging and deep learning

Detecting rare cells within blood has numerous applications in disease diagnostics. Existing rare cell detection techniques are typically hindered by their high cost and low throughput. Here, we present a computational cytometer based on magnetically modulated lensless speckle imaging, which introduces oscillatory motion to the magnetic-bead-conjugated rare cells of interest through a periodic magnetic force and uses lensless time-resolved holographic speckle imaging to rapidly detect the target cells in three dimensions (3D). In addition to using cell-specific antibodies to magnetically label target cells, detection specificity is further enhanced through a deep-learning-based classifier that is based on a densely connected pseudo-3D convolutional neural network (P3D CNN), which automatically detects rare cells of interest based on their spatio-temporal features under a controlled magnetic force. To demonstrate the performance of this technique, we built a high-throughput, compact and cost-effective prototype for detecting MCF7 cancer cells spiked in whole blood samples. Through serial dilution experiments, we quantified the limit of detection (LoD) as 10 cells per millilitre of whole blood, which could be further improved through multiplexing parallel imaging channels within the same instrument. This compact, cost-effective and high-throughput computational cytometer can potentially be used for rare cell detection and quantification in bodily fluids for a variety of biomedical applications.

New Blood Based M-Protein Quantification Method 3,000 Times More Sensitive Than Standard SPEP

Summary of WorkThe amino acid sequence of the M-protein for multiple myeloma (MM) is unique compared to the polyclonal antibodies in patients' blood. In this study we utilize this uniqueness to develop an ultra-sensitive M-protein detection method with mass spectrometry (MS). The method involves the de novo sequencing of the amino acid sequence of the M-protein from the baseline blood sample collected at the time of diagnosis, and a targeted MS assay to detect and quantify the unique M-protein sequence in the follow-up blood samples. This non-invasive method is purely blood based and is 3,000 and 300 times more sensitive than SPEP and IFE, respectively.De Novo Protein SequencingThe M-protein sequencing is carried out on an MS based platform (REmAb). The effectiveness of de novo protein sequencing has been proven with the sequencing of hundreds of monoclonal antibodies (McDonald et al., Poster 294737, ASMS, 2018). In the current study the M-protein from 4 MM patients' baseline blood samples were successfully sequenced, demonstrating robustness of the method in the presence of the polyclonal background. As little as 50ul of the serum was required for the de novo sequencing.Sensitivity of M-protein QuantificationThe lower limit of quantification (LLOQ) was studied with a serial dilution experiment. The baseline blood sample of one patient was sequentially diluted with a healthy donor's serum. The peptide sequence unique to the M-protein was monitored with mass spectrometry to detect and quantify the M-protein in the serial dilution. The M-protein could still be detected and quantified when the dilution ratio was 1:10,000 (the amount of M-protein relative to background polyclonal serum IgG). In a separate experiment, synthesized heavy labelled proteotypic peptides were used to estimate the LLOQ in IgG enriched serum at 60 ug/dL, over 3,000 times more sensitive than SPEP (0.2g/dL) (Bergen et al., Clin Chem 62: 1 243-251, 2016) and 300 times more sensitive than IFE (0.02g/dL) (IMWG, British Journal of Haematology, 121, 749-757, 2003). As little as 30ul of serum was required in these experiments for monitoring of M-protein levels.Case StudyA targeted MS based assay was developed to monitor the M-protein levels of a serial patient sample set (73-year-old male, treatment: Elotuzumab/Lenalidomide/Dexamethasone, progression free survival 32 months). M-protein levels were quantified in a total of 10 serial samples from partial remission, through complete remission (CR), until relapse over a period of 2 years and 3 months (Figure 1). M-protein could be detected and quantified in all samples collected during the CR period with estimated M-protein levels never below 10mg/dL. Notably, a 2-fold increase in M-protein levels (in comparison to the lowest historic level) could be detected 320 days prior to the timepoint of relapse. The upward trend continued in the next 3 serial CR samples preceding relapse. Except for one sample (see Figure 1, ‘CR Before Relapse 12-14-2016‘), CR was based on the absence of M-protein in the IFE result in the relevant clinical data.ConclusionThe work represents a first step in the application of de novo sequencing and MS based detection for the sensitive monitoring of M-protein levels in serum. It shows a much-improved sensitivity over current standard approaches and has the great potential to provide non-invasive assessment of MRD for multiple myeloma. The preliminary data warrant further development of this MS based non-invasive and highly sensitive M-protein quantification method. [Display omitted] DisclosuresMcDonald:Rapid Novor Inc: Employment, Equity Ownership. Liu:Rapid Novor Inc: Employment, Equity Ownership. Taylor:Rapid Novor Inc: Employment, Equity Ownership. Yang:Rapid Novor Inc: Employment, Equity Ownership. Ma:Rapid Novor Inc: Equity Ownership.

Laboratory plasticware – Use at your own risk: Suitability of microcentrifuge tubes for spores’ analysis of Clostridium difficile

Clostridium difficile is a Gram-positive spore forming rod-shaped bacterium which causes mild to severe diarrhea. Spores play a key role in transmission of C. difficile in hospital environment. To investigate ability of spores to stay on fomites and to assess levels of contamination it is essential to prevent loss of spores collected for the analysis. Working with C. difficile spores we noticed a significant loss after vortexing of spore suspensions and investigated if it can be prevented by using a specific brand or type of microcentrifuge tubes. 7 types of microcentrifuge tubes from 3 manufacturers were tested. Spores of three types of C. difficile, NAP1/027, NAP4/014 and NAP7/078 (clinical isolates) were used. C. difficile was grown on Brucella Supplemented Agar for 9 days, spores were collected, washed and density of 3 suspensions was normalized to optical density (OD) 550 0.1 or 0.05. These suspensions (OD 0.1) were used in serial dilutions with 3 experimental conditions – pipetting, vortexing or vortexing in 3% albumin solution and in vortexing experiment when 150 μl were vortexed for 1 min at 1500 rpm per tube and loss of spores was measured by a decrease in dipicolinic acid (DPA) concentration measured by time-delayed terbium fluorescence. Inner surface of the tubes was visualized with microscopy to observe adhered spores. In serial dilution experiment, initial concentration of spores would be underestimated by up to 18X in case of vortexing for NAP1 strain and 9X for NAP4 strain. Presence of 3% albumin significantly decreases this effect but does not eliminate it completely.Comparison of 7 types of tubes shows that a single vortexing for 1 min of diluted spore suspension at concentrations of 1.8 × 107 spores per ml leads to a loss of up to 90% of spores in some tubes. Degree of spores' adhesion varied between brands and types of tubes and between tubes of the same type. In some brands there was a significant variability in adhesion between tubes from the same batch. Microscopy after vortexing shows a film of spores attached to the tube's wall. Adherence could be affected by the type of plastic, additives (plasticizers) used in manufacturing and quality of moulds (e.g.“diamond polished”). To identify the most appropriate type of tubes for the experiment it is essential to test it beforehand as not every brand is suitable for this purpose. Using tubes with a high degree of adherence could significantly affect measurement of spores' concentration in serial dilutions, e.g. when quantifying spores production by a specific strain or when limits of detection are measured. Also, sensitivity of commercial tests for detection of C. difficile in clinical specimens can be decreased if an unsuitable type of plastic containers and tubes is used.

Abstract PD3-15: Novel cFlip inhibitor suppresses chemotherapy-induced breast cancer stem cell activity through blocking HIF1-α

Abstract Background: The emergence of the cancer stem cell (CSC) hypothesis has helped to explain previously poorly understood clinical concepts such as metastases, late tumour recurrence and resistance to chemotherapy. Triple Negative Breast Cancer (TNBC) has the worst prognosis of all types of breast cancer with a more frequent relapse rate and reduced length of survival in metastatic disease. It has been shown to contain a higher proportion of CSCs than other types of breast cancer. Paclitaxel, a taxane in widespread use in breast cancer, induces apoptosis in a ligand-independent manner through the extrinsic apoptosis pathway. cFLIP is both an antagonist of this apoptosis pathway and has the ability to form aggregates that interfere with the ubiquitylation and subsequent degradation of both HIF1α and β-catenin- two molecules involved in CSC-signalling. Using a novel compound targeted against cFLIP, we wanted to assess whether its combination with paclitaxel effectively targeted CSCs, particularly in TNBC. Methods: In vitro experiments were used to assess the effect on cell viability, mammosphere formation and ALDH+ of both chemotherapy and our novel agent, OH14. We then used siRNA-mediated knockdown of cFLIP to confirm an on-target effect. Annexin V assays were used to assess apoptosis and both Western blotting and qPCR was used to examine the effect of paclitaxel and OH14 on protein levels and gene expression of CSC-signalling pathways. Athymic mice were used to in serial dilution experiments and for in vivo treatmenst with paclitaxel and OH14 on a TNBC cell line. Results: We established an in vitro model demonstrating that a wide range of chemotherapeutic agents (FEC, Paclitaxel and Docetaxel) increased CSC-like behaviour and the ALDH+ population in a broad range of breast cancer cell lines. A mathematical model demonstrated that chemotherapy increased the absolute number of CSCs after treatment suggesting that CSC-like signaling was being induced. OH14, our novel compound, sensitised Triple Negative Breast Cancer (TNBC) cell lines to paclitaxel by increasing its apoptotic effect but had a more profound effect on mammosphere formation and ALDH positivity. This suggests that it preferentially targets CSCs. Serial dilution experiments demonstrated that chemotherapy increased the tumour forming potential of a TNBC cell line in vivo and that this effect was abrogated with OH14. Treatment of the same cell line in vivo demonstrated that paclitaxel +/- OH14 successfully targeted tumours but that the paclitaxel tumours recurred whereas those subject to combined treatment remained undetectable. Further experiments examining CSC signalling showed that HIF1α-mediated signalling was increased by paclitaxel and abrogated by the addition of OH14. Conclusion: cFLIP has a dual effect in both increasing apoptosis and targeting signaling in TNBC CSCs. In a breast cancer subtype in desperate need of novel therapeutic strategies, targeting cFLIP warrants further investigation and progression towards clinical trials. Citation Format: Robinson T, Turnham D, Gruca A, Piggott L, Clarkson R. Novel cFlip inhibitor suppresses chemotherapy-induced breast cancer stem cell activity through blocking HIF1-α [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD3-15.

Multiplexing T- and B-Cell FLUOROSPOT Assays: Experimental Validation of the Multi-Color ImmunoSpot® Software Based on Center of Mass Distance Algorithm.

Over the past decade, ELISPOT has become a highly implemented mainstream assay in immunological research, immune monitoring, and vaccine development. Unique single cell resolution along with high throughput potential sets ELISPOT apart from flow cytometry, ELISA, microarray- and bead-based multiplex assays. The necessity to unambiguously identify individual T and B cells that do, or do not co-express certain analytes, including polyfunctional cytokine producing T cells has stimulated the development of multi-color ELISPOT assays. The success of these assays has also been driven by limited sample/cell availability and resource constraints with reagents and labor. There are few commercially available test kits and instruments available at present for multi-color FLUOROSPOT. Beyond commercial descriptions of competing systems, little is known about their accuracy in experimental settings detecting individual cells that secrete multiple analytes vs. random overlays of spots. Here, we present a theoretical and experimental validation study for three and four color T- and B-cell FLUOROSPOT data analysis. The ImmunoSpot® Fluoro-X™ analysis system we used includes an automatic image acquisition unit that generates individual color images free of spectral overlaps and multi-color spot counting software based on the maximal allowed distance between centers of spots of different colors or Center of Mass Distance (COMD). Using four color B-cell FLUOROSPOT for IgM, IgA, IgG1, IgG3; and three/four color T-cell FLUOROSPOT for IL-2, IFN-γ, TNF-α, and GzB, in serial dilution experiments, we demonstrate the validity and accuracy of Fluoro-X™ multi-color spot counting algorithms. Statistical predictions based on the Poisson spatial distribution, coupled with scrambled image counting, permit objective correction of true multi-color spot counts to exclude randomly overlaid spots.

Utility of CD157 as a Common Leukocytes Marker for Paroxysmal Nocturnal Hemoglobinuria Screening in a Single Tube Five Color Combination

To study the utility and advantage of CD157 in the paroxysmal nocturnal hemoglobinuria (PNH) screening along with its ability to replace CD24 and CD14. This was a confirmatory study to analyse the role and advantage of CD157 in a single tube five color combination to identify the PNH clones. A serial tenfold dilution experiments was carried out for sensitivity assessment. Reproducibility was checked in the intra-assay and inter-assay experiments. The results obtained with CD157 based assay were compared with the routinely used single tube six color CD24/CD14 based assay. CD157 showed a high degree of sensitivity at the level of 10-4. PNH positive clone sizes were precise with CVs of inter-assay and intra-assay precision analysis for polymorphs/monocytes ranging from 2.94 to 4.31/2.52 to 8.93, and 0.91 to 3.23/1.65 to 5.33%; respectively. The results were similar to those obtained from CD24/CD14 based assay (R2>0.993). There was no false positive or false negative result. CD157 was found better in delineating the type II clones. CD157 can be used as a common PNH leucocyte marker with high degree of sensitivity and precision. It can replace CD24 and CD14 from the currently used assays and thus bring down the cost of PNH screening.

Abstract 2736: A digital droplet PCR assay for the quantitation of androgen receptor and splice variant expression in CTCs from metastatic castration resistant prostate cancer patients

Abstract Prostate cancer (PC) is the second leading cause of cancer death in men in the US. The aberrant functioning of androgen receptor signaling is the central driving force behind prostatic tumorigenesis and its transition into metastatic castration resistant disease. Hence, androgen deprivation therapy (ADT) is the first line of treatment for PC patients. However, many patients progress becoming resistant to ADT therapy, due to the expression of AR splice variants (AR-Vs), which lack the ligand binding domain and are constitutively active in the nucleus. Expression of the AR splice variant, AR-v7, in circulating tumor cells (CTCs) isolated from the blood of PC patients was correlated with resistance to enzalutamide and abiraterone, which are the next generation AR signaling inhibitors in CRPC. Further, there is evidence that AR-Vs may convey cross-resistance, not only to enzalutamide and abiraterone, but also to taxanes, highlighting that their assessment in the clinic may have clinical utility. We developed a novel, specific and highly sensitive assay to measure mRNA expression of the AR full length (AR-FL) and the splice variants ARv7 and ARv567es, by using Droplet Digital PCR in CTCs isolated from CRPC patients. The analytical specificity of the assay was determined by transfecting cells with plasmids encoding AR-FL, AR-v7 and AR-v567 and showed that each probe detected signal only in cells expressing the respective transcript. No signal was detected against genomic DNA, indicating lack of non-specific binding. Also, the assay detected endogenous expression of AR-FL and AR-v7 in VCAP or 22RV1 cells, while no variant expression was detected in healthy donor blood. The analytical sensitivity of the assay was determined in a series of serial dilution experiments that showed sensitivity down to single cell. We then used this assay to determine the clinical prevalence and expression pattern of each of these variants in CTCs from about 200 mCRPC patient samples and blood from 40 healthy donors. CTCs were enriched by EpCAM- or PSMA-based positive selection or CD45 negative depletion in an antigen-agnostic manner. AR-FL was detected in ~80% of mCRPC samples irrespective of CTC-enrichment technology. AR-v7 was expressed in 65% of the samples in which in CTCs were enriched either by PSMA-positive selection or by negative depletion. In contrast, EpCAM-based CTC enrichment showed lower AR-v7 expression both in terms of expression levels and prevalence. In addition, CTC enrichment following negative depletion showed that 30% of the samples had higher AR-v7 expression levels as compared to AR-FL. This expression pattern was not observed in the samples using EpCAM-based selection. Collectively, these data suggest distinct CTC subpopulations are present in CRPC patient samples, with differential expression of AR-Vs that could have important predictive and prognostic implications. Citation Format: Ada Gjyrezi, Giuseppe Galletti, Areti Strati, Seaho Kim, Evi Lianidou, David M. Nanus, Jun Luo, Emmanuel Antonarakis, Scott T. Tagawa, Andrew Armstrong, Paraskevi Giannakakou. A digital droplet PCR assay for the quantitation of androgen receptor and splice variant expression in CTCs from metastatic castration resistant prostate cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2736. doi:10.1158/1538-7445.AM2017-2736

Multiplex Ultrasensitive Genotyping of Patients with Non-Small Cell Lung Cancer for Epidermal Growth Factor Receptor (EGFR) Mutations by Means of Picodroplet Digital PCR

Epidermal growth factor receptor (EGFR) mutations have been used as the strongest predictor of effectiveness of treatment with EGFR tyrosine kinase inhibitors (TKIs). Three most common EGFR mutations (L858R, exon 19 deletion, and T790M) are known to be major selection markers for EGFR-TKIs therapy. Here, we developed a multiplex picodroplet digital PCR (ddPCR) assay to detect 3 common EGFR mutations in 1 reaction. Serial-dilution experiments with genomic DNA harboring EGFR mutations revealed linear performance, with analytical sensitivity ~0.01% for each mutation. All 33 EGFR-activating mutations detected in formalin-fixed paraffin-embedded (FFPE) tissue samples by the conventional method were also detected by this multiplex assay. Owing to the higher sensitivity, an additional mutation (T790M; including an ultra-low-level mutation, <0.1%) was detected in the same reaction. Regression analysis of the duplex assay and multiplex assay showed a correlation coefficient (R2) of 0.9986 for L858R, 0.9844 for an exon 19 deletion, and 0.9959 for T790M. Using ddPCR, we designed a multiplex ultrasensitive genotyping platform for 3 common EGFR mutations. Results of this proof-of-principle study on clinical samples indicate clinical utility of multiplex ddPCR for screening for multiple EGFR mutations concurrently with an ultra-rare pretreatment mutation (T790M).

Single-Tube 10-Fluorochrome Analysis for Efficient Flow Cytometric Evaluation of Minimal Residual Disease in Plasma Cell Myeloma.

Widespread adoption of recent recommendations for minimal residual disease (MRD) detection in myeloma has partly been impeded by a paucity of studies detailing multiparameter flow cytometry (MPF) assay validation. In response, we have validated a novel and efficient single-tube 10-color assay for MRD detection that incorporates the recently recommended plasma cell markers. Aspirate samples from 53 patients with plasma cell disorder were analyzed using a novel single-tube 10-color method. The limit of detection, precision of measurement, and linearity of measurement of our new assay were determined using serial dilution experiments. The stability of the new antibody cocktail and the viability/specificity of stained samples were evaluated using serial time course measurements. There was a high degree of quantitative agreement between our new 10-color method and an established eight-color method. Four positive samples detected by the 10-color assay were below or at the limit of detection of the eight-color assay, confirming its high sensitivity. In two cases, subsequent revision of the International Myeloma Working Group Uniform Response Criteria was necessary. Adoption of our validated 10-color assay would enable clinical laboratories to satisfy current MRD recommendations without significantly increasing the demands on current workflow practices.

The ability to cross the blood–cerebrospinal fluid barrier is a generic property of acute lymphoblastic leukemia blasts

AbstractPrevention of central nervous system (CNS) relapse is critical for cure of childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Despite this, mechanisms of CNS infiltration are poorly understood, and the timing, frequency, and properties of BCP-ALL blasts entering the CNS compartment are unknown. We investigated the CNS-engrafting potential of BCP-ALL cells xenotransplanted into immunodeficient NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ mice. CNS engraftment was seen in 23 of 29 diagnostic samples (79%): 2 of 2 from patients with overt CNS disease and 21 of 27 from patients thought to be CNS negative by diagnostic lumbar puncture. Histologic findings mimic human pathology and demonstrate that leukemic cells transit the blood–cerebrospinal fluid barrier situated close to the dural sinuses, the site of recently discovered CNS lymphatics. Retrieval of blasts from the CNS showed no evidence for chemokine receptor–mediated selective trafficking. The high frequency of infiltration and lack of selective trafficking led us to postulate that CNS tropism is a generic property of leukemic cells. To test this, we performed serial dilution experiments which showed CNS engraftment in 5 of 6 mice after transplant of as few as 10 leukemic cells. Clonal tracking techniques confirmed the polyclonal nature of CNS-infiltrating cells, with multiple clones engrafting in both the CNS and periphery. Overall, these findings suggest that subclinical seeding of the CNS is likely to be present in most BCP-ALL patients at original diagnosis, and efforts to prevent CNS relapse should concentrate on effective eradication of disease from this site rather than targeting entry mechanisms.

An improved sequencing-based strategy to estimate locus-specific DNA methylation

BackgroundDNA methylation is an important epigenetic mechanism of transcriptional control that plays an essential role in several cellular functions. Aberrant DNA methylation in cancer has been frequently associated with downregulation of microRNAs and protein coding genes, such as miR-200c/miR-141 cluster and E-cadherin. Current strategies to assess DNA methylation, including bisulfite treatment-based assays, tend to be time-consuming and may be quite expensive when a precise appraisal is required. The Sanger-sequencing of the amplified bisulfite-treated DNA (BSP) might represent a practical option to measure DNA methylation at single CpG resolution. However, this strategy often produces noisy data, which affects accurate quantification. Here we propose an improved, reliable and cost-effective BSP-based protocol that allows proper DNA methylation assessment.MethodsOur strategy, named normalized-BSP (NBSP), takes advantage of tailed C-balanced primers and a normalization procedure based on C/T ratio to overcome BSP-associated noise problems and nucleotide signal unbalance. NBSP was applied to estimate miR-200c/miR-141 locus methylation in serial dilution experiments and was compared to conventional methods. Besides, it was applied in the analysis of FFPE breast cancer samples and further validated in the context of the E-cadherin promoter.ResultsNBSP strategy outperformed conventional BSP in the estimate of the fraction of methylated cytosine in serial dilution experiments, providing data in agreement with the widely used but cumbersome cloning-based protocol. This held true for both miR-200c/miR-141 locus and E-cadherin promoter analyses. Moreover, the miR-200c/miR-141 locus methylation reflected the decrease in miRNA expression both in breast cancer cell lines and in the FFPE samples.ConclusionsNBSP is a rapid and economical method to estimate the extent of methylation at each CpG of a given locus. Notably, NBSP works efficiently on FFPE samples, thus disclosing the perspective of its application also in the diagnostic setting.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1646-6) contains supplementary material, which is available to authorized users.

Evolutionary engineering of a wine yeast strain revealed a key role of inositol and mannoprotein metabolism during low-temperature fermentation.

BackgroundWine produced at low temperature is often considered to improve sensory qualities. However, there are certain drawbacks to low temperature fermentations: e.g. low growth rate, long lag phase, and sluggish or stuck fermentations. Selection and development of new Saccharomyces cerevisiae strains well adapted at low temperature is interesting for future biotechnological applications. This study aimed to select and develop wine yeast strains that well adapt to ferment at low temperature through evolutionary engineering, and to decipher the process underlying the obtained phenotypes.ResultsWe used a pool of 27 commercial yeast strains and set up batch serial dilution experiments to mimic wine fermentation conditions at 12 °C. Evolutionary engineering was accomplished by using the natural yeast mutation rate and mutagenesis procedures. One strain (P5) outcompeted the others under both experimental conditions and was able to impose after 200 generations. The evolved strains showed improved growth and low-temperature fermentation performance compared to the ancestral strain. This improvement was acquired only under inositol limitation. The transcriptomic comparison between the evolved and parental strains showed the greatest up-regulation in four mannoprotein coding genes, which belong to the DAN/TIR family (DAN1, TIR1, TIR4 and TIR3). Genome sequencing of the evolved strain revealed the presence of a SNP in the GAA1 gene and the construction of a site-directed mutant (GAA1Thr108) in a derivative haploid of the ancestral strain resulted in improved fermentation performance. GAA1 encodes a GPI transamidase complex subunit that adds GPI, which is required for inositol synthesis, to newly synthesized proteins, including mannoproteins.ConclusionsIn this study we demonstrate the importance of inositol and mannoproteins in yeast adaptation at low temperature and the central role of the GAA1 gene by linking both metabolisms.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1755-2) contains supplementary material, which is available to authorized users.

Towards quantitative SERS detection of hydrogen cyanide at ppb level for human breath analysis

Lung infections with Pseudomonas aeruginosa (PA) is the most common cause of morbidity and mortality in cystic fibrosis (CF) patients. Due to its ready adaptation to the dehydrated mucosa of CF airways, PA infections tend to become chronic, eventually killing the patient. Hydrogen cyanide (HCN) at ppb level has been reported to be a PA biomarker. For early PA detection in CF children not yet chronically lung infected a non-invasive Surface-Enhanced Raman Spectroscopy (SERS)-based breath nanosensor is being developed. The triple bond between C and N in cyanide, with its characteristic band at ∼2133cm−1, is an excellent case for the SERS-based detection due to the infrequent occurrence of triple bonds in nature. For demonstration of direct HCN detection in the gas phase, a gold-coated silicon nanopillar substrate was exposed to 5ppm HCN in N2. Results showed that HCN adsorbed on the SERS substrate can be consistently detected under different experimental conditions and up to 9days after exposure. For detection of lower cyanide concentrations serial dilution experiments using potassium cyanide (KCN) demonstrated cyanide quantification down to 1μM in solution (corresponding to 18ppb). Lower KCN concentrations of 10 and 100nM (corresponding to 0.18 and 1.8ppb) produced SERS intensities that were relatively similar to the reference signal. Since HCN concentration in the breath of PA colonized CF children is reported to be ∼13.5ppb, the detection of cyanide is within the required range.

Clonal distribution of BCR-ABL1 mutations and splice isoforms by single-molecule long-read RNA sequencing.

BackgroundThe evolution of mutations in the BCR-ABL1 fusion gene transcript renders CML patients resistant to tyrosine kinase inhibitor (TKI) based therapy. Thus screening for BCR-ABL1 mutations is recommended particularly in patients experiencing poor response to treatment. Herein we describe a novel approach for the detection and surveillance of BCR-ABL1 mutations in CML patients.MethodsTo detect mutations in the BCR-ABL1 transcript we developed an assay based on the Pacific Biosciences (PacBio) sequencing technology, which allows for single-molecule long-read sequencing of BCR-ABL1 fusion transcript molecules. Samples from six patients with poor response to therapy were analyzed both at diagnosis and follow-up. cDNA was generated from total RNA and a 1,6 kb fragment encompassing the BCR-ABL1 transcript was amplified using long range PCR. To estimate the sensitivity of the assay, a serial dilution experiment was performed.ResultsOver 10,000 full-length BCR-ABL1 sequences were obtained for all samples studied. Through the serial dilution analysis, mutations in CML patient samples could be detected down to a level of at least 1%. Notably, the assay was determined to be sufficiently sensitive even in patients harboring a low abundance of BCR-ABL1 levels. The PacBio sequencing successfully identified all mutations seen by standard methods. Importantly, we identified several mutations that escaped detection by the clinical routine analysis. Resistance mutations were found in all but one of the patients. Due to the long reads afforded by PacBio sequencing, compound mutations present in the same molecule were readily distinguished from independent alterations arising in different molecules. Moreover, several transcript isoforms of the BCR-ABL1 transcript were identified in two of the CML patients. Finally, our assay allowed for a quick turn around time allowing samples to be reported upon within 2 days.ConclusionsIn summary the PacBio sequencing assay can be applied to detect BCR-ABL1 resistance mutations in both diagnostic and follow-up CML patient samples using a simple protocol applicable to routine diagnosis. The method besides its sensitivity, gives a complete view of the clonal distribution of mutations, which is of importance when making therapy decisions.

Rapid, targeted and culture-free viral infectivity assay in drop-based microfluidics.

A key viral property is infectivity, and its accurate measurement is crucial for the understanding of viral evolution, disease and treatment. Currently viral infectivity is measured using plaque assays, which involve prolonged culturing of host cells, and whose measurement is unable to differentiate between specific strains and is prone to low number fluctuation. We developed a rapid, targeted and culture-free infectivity assay using high-throughput drop-based microfluidics. Single infectious viruses are incubated in a large number of picoliter drops with host cells for one viral replication cycle followed by in-drop gene-specific amplification to detect infection events. Using murine noroviruses (MNV) as a model system, we measure their infectivity and determine the efficacy of a neutralizing antibody for different variants of MNV. Our results are comparable to traditional plaque-based assays and plaque reduction neutralization tests. However, the fast, low-cost, highly accurate genomic-based assay promises to be a superior method for drug screening and isolation of resistant viral strains. Moreover our technique can be adapted to measuring the infectivity of other pathogens, such as bacteria and fungi.

Single-tube, highly parallel mutation enrichment in cancer gene panels by use of temperature-tolerant COLD-PCR.

Multiplexed detection of low-level mutations presents a technical challenge for many technologies, including cancer gene panels used for targeted-resequencing. Analysis of mutations below approximately 2%-5% abundance in tumors with heterogeneity, samples with stromal contamination, or biofluids is problematic owing to increased noise from sequencing errors. Technologies that reduce noise via deep sequencing unavoidably reduce throughput and increase cost. Here we provide proof of principle that coamplification at lower denaturation temperature (COLD)-PCR technology enables multiplex low-level mutation detection in cancer gene panels while retaining throughput. We have developed a multiplex temperature-tolerant COLD-PCR (fast-TT-COLD-PCR) approach that uses cancer gene panels developed for massively parallel sequencing. After multiplex preamplification from genomic DNA, we attach tails to all amplicons and perform fast-TT-COLD-PCR. This approach gradually increases denaturation temperatures in a step-wise fashion, such that all possible denaturation temperatures are encompassed. By introducing modified nucleotides, fast-COLD-PCR is adapted to enrich for melting temperature (Tm)-increasing mutations over all amplicons, in a single tube. Therefore, in separate reactions, both Tm-decreasing and Tm-increasing mutations are enriched. Using custom-made and commercial gene panels containing 8, 50, 190, or 16 000 amplicons, we demonstrate that fast-TT-COLD-PCR enriches mutations on all examined targets simultaneously. Incorporation of deoxyinosine triphosphate (dITP)/2,6-diaminopurine triphosphate (dDTP) in place of deoxyguanosine triphosphate (dGTP)/deoxyadenosine triphosphate (dATP) enables enrichment of Tm-increasing mutations. Serial dilution experiments demonstrate a limit of detection of approximately 0.01%-0.1% mutation abundance by use of Ion-Torrent and 0.1%-0.3% by use of Sanger sequencing. Fast-TT-COLD-PCR improves the limit of detection of cancer gene panels by enabling mutation enrichment in multiplex, single-tube reactions. This novel adaptation of COLD-PCR converts subclonal mutations to clonal, thereby facilitating detection and subsequent mutation sequencing.

Detection of hepatitis B virus A1762T/G1764A mutant by amplification refractory mutation system

ObjectiveTo study the role of hepatitis B virus with A1762T/G1764A double mutation in liver cirrhosis and hepatocellular carcinoma, and create a sensitive, fast, accurate assay for detection of A1762T/G1764A double mutation. MethodsWe developed an accurate and fast real-time amplification refractory mutation system to detect A1762T/G1764A double mutation. Cloned hepatitis B virus genome was used as a control. Assay sensitivity was determined by serial dilution and mixed template experiments. Specificity was determined by cross experiments with wild and mutant hepatitis B virus. Fifty clinical samples were tested by the real-time amplification refractory mutation system and the results were compared with sequencing. ResultsThe real-time amplification refractory mutation system had a sensitivity of 100 copies of virus with these mutations, and 0.1% weak population virus with double mutation could be found in mixtures. A total of 50 randomly collected clinical samples were detected by real-time amplification refractory mutation system, and the results were consistent with those by DNA sequencing. Hepatitis B virus genotype C was more prevalent in 39 of 50 samples than genotype B (11 samples), and about 75% of genotype C carried a double mutation compared to 45% of genotype B. However, the percentage of A1762T/G1764A double mutation in hepatitis B e antigen-negative (58.3%) samples was almost the same as in hepatitis B e antigen-positive (61%) samples. ConclusionThe real-time amplification refractory mutation system is sensitive and specific for detection of hepatitis B virus double mutation.

Rapid detection of IDH2 (R140Q and R172K) mutations in acute myeloid leukemia

NADP-dependent enzyme isocitrate dehydrogenase (IDH) mutations, IDH1 and IDH2, have been described in acute myeloid leukemia (AML) using next generation sequencing approaches. IDH2 mutations are heterozygous; they alter a single arginine residue at position 140 or 172 and have distinct prognostic significance. The current detection methods of IDH2 mutations are laborious and time consuming as they require DNA sequencing. Herein, we report a new allele-specific oligonucleotide-polymerase chain reaction (ASO-PCR) method to detect the IDH2 mutations. Analysis of leukemic DNA samples from 120 AML patients enabled to identify IDH2 mutations in 22 cases which were confirmed by direct DNA sequencing. Of these, 17 harbored IDH2 (R140Q) and 5 IDH2 (R172K) mutations. Serial dilution experiments showed that the assay enable to detect mutations in 10⁻³ dilutions. Our ASO-PCR method appears useful for routine diagnostic screening of these prognostically relevant alterations in AML and may be conveniently included in the diagnostic workup.