Specific Breakpoints Research Articles

Abstract 4867: Exome- and whole genome sequencing for clinical evaluation and precision medicine in neuroblastoma

Abstract Background. Despite major progress in treatment of pediatric cancer, aggressive neuroblastoma (NB) still constitutes a major clinical problem. Currently, the event free survival of high-risk NB is about 40% implicating that 60% either don’t go into full remission or falls into relapse. Most relapses have an acquired drug resistance meaning that conventional treatment options may not work. Therefore, further studies of this group are of great importance. Massive parallel sequencing has now become a valuable tool in both cancer research and in clinical evaluation as it provides valuable information regarding patient specific alterations that might be used in targeted therapy. Material and Methods. DNA from 54 patients in total have been subjects for massive parallel sequencing; 16 tumor/normal pairs and 27 single tumors have been analyzed using exome sequencing, 8 tumor/normal pairs with whole genome sequencing and three using both exome and whole genome sequencing. Exome sequences were mapped using BWA with GATK realignment followed by variant calling through SNPeff and copy number analysis trough ControlFreec. Whole genome data were analyzed using the CLC Genomic Workbench with annotation and filtering done in Ingenuity variant analysis software. Results. On average, 14 somatic protein changing single nucleotide variants were detected per patient (range 2-71) with recurrent alterations mainly detected mainly in ALK. Predicted deleterious germ line alterations in well-established cancer genes were detected in three patients. Structural variants included genes such as ATRX and TERT, as described by others previously, but also novel homozygous focal deletions at chromosome 19. Shortest regions of overlapping deletions of this chr19 region include seven gens whereof CIC (Capicua) might be the most interesting in a cancer context. Copy number analysis of both exome- and whole genome sequencing gives genomic profiles that are comparable to the genome profiles generated from the SNP-microarrays that current are in clinical use. Specific breakpoints for structural variants could be detected in all sample subjects for whole genome sequencing as well as for all for all MYCN amplified neuroblastomas that subjects for exome sequencing. Conclusions. Exome- and whole genome sequencing can be used for clinical evaluation of NB providing information regarding both constitutional and tumor specific alterations. The information can be used for clinical decision of patient specific therapeutic options such as ALK- or CDK4/6 inhibitors, but can also in rare cases give information regarding which treatments not to use. Furthermore, whole genome sequencing also gives the possibility to identify tumor specific structural alterations such as translocation breakpoints that can be used to monitor tumor burden in liquid biopsies. Citation Format: Angela Martinez Monleon, Susanne Reinsbach, Niloufar Javanmardi, Anna Djos, Rose-Marie Sjoberg, Per Kogner, Tommy Martinsson, Susanne Fransson. Exome- and whole genome sequencing for clinical evaluation and precision medicine in neuroblastoma [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 4867. doi:10.1158/1538-7445.AM2017-4867

Antimicrobial susceptibility monitoring of Mycoplasma hyopneumoniae and Mycoplasma bovis isolated in Europe

Mycoplasma hyopneumoniae in pigs and Mycoplasma bovis in cattle are major pathogens affecting livestock across Europe and are the focus of the MycoPath pan-European antimicrobial susceptibility monitoring programme. Fifty M. hyopneumoniae isolates from Belgium, Spain and the United Kingdom (UK), and 156 M. bovis isolates from France, Hungary, Spain and the UK that met specific criteria were tested for antimicrobial susceptibility in a central laboratory by using a microbroth dilution method. Specific isolate criteria included recovery from animals not recently treated with antimicrobials, isolates from different locations within each country and retaining only one isolate per farm. MIC50/MIC90 values were 0.031/0.5, 0.031/0.5, 0.062/0.25, ≤0.001/0.004, 0.031/0.125, 0.25/0.5 and 0.062/0.25mg/L for enrofloxacin, marbofloxacin, spiramycin, tulathromycin, tylosin, florfenicol and oxytetracycline respectively against M. hyopneumoniae and 0.25/4, 1/4, 4/16, >64/ >64, 32/ >64, 2/4 and 4/64mg/L, respectively against M. bovis. MIC50/MIC90 values for tiamulin and valnemulin against M. hyopneumoniae were 0.016/0.062 and ≤0.001/ ≤0.001mg/L respectively. The MIC50/MIC90 values of danofloxacin and gamithromycin for M. bovis were 0.25/1 and >64/ >64mg/L respectively. The highest MIC90 values for M. hyopneumoniae were found in the UK at 1.0mg/L for enrofloxacin, marbofloxacin and florfenicol. In contrast, for M. bovis the lowest MIC90 value was 1.0mg/L, but ranged to >64mg/L. Specific laboratory standards and clinical breakpoints for veterinary Mycoplasma species are required as no independently validated clinical breakpoints are specified for veterinary Mycoplasma species, which makes data interpretation and correlation to in vivo efficacy difficult.

Genomic analysis of chimeric human cytomegalovirus vaccine candidates derived from strains Towne and Toledo.

Human cytomegalovirus (HCMV) is an important opportunistic pathogen in immunocompromised patients and a major cause of congenital birth defects when acquired in utero. In the 1990s, four chimeric viruses were constructed by replacing genome segments of the high passage Towne strain with segments of the low passage Toledo strain, with the goal of obtaining live attenuated vaccine candidates that remained safe but were more immunogenic than the overly attenuated Towne vaccine. The chimeras were found to be safe when administered to HCMV-seronegative human volunteers, but to differ significantly in their ability to induce seroconversion. This suggests that chimera-specific genetic differences impacted the ability to replicate or persist in vivo and the consequent ability to induce an antibody response. To identify specific genomic breakpoints between Towne and Toledo sequences and establish whether spontaneous mutations or rearrangements had occurred during construction of the chimeras, complete genome sequences were determined. No major deletions or rearrangements were observed, although a number of unanticipated mutations were identified. However, no clear association emerged between the genetic content of the chimeras and the reported levels of vaccine-induced HCMV-specific humoral or cellular immune responses, suggesting that multiple genetic determinants are likely to impact immunogenicity. In addition to revealing the genome organization of the four vaccine candidates, this study provided an opportunity to probe the genetics of HCMV attenuation in humans. The results may be valuable in the future design of safe live or replication-defective vaccines that optimize immunogenicity and efficacy.

Transforming the canonical piecewise-linear model into a smooth-piecewise representation

A smoothed representation (based on natural exponential and logarithmic functions) for the canonical piecewise-linear model, is presented. The result is a completely differentiable formulation that exhibits interesting properties, like preserving the parameters of the original piecewise-linear model in such a way that they can be directly inherited to the smooth model in order to determine their parameters, the capability of controlling not only the smoothness grade, but also the approximation accuracy at specific breakpoint locations, a lower or equal overshooting for high order derivatives in comparison with other approaches, and the additional advantage of being expressed in a reduced mathematical form with only two types of inverse functions (logarithmic and exponential). By numerical simulation examples, this proposal is verified and well-illustrated.

Sequence Capture and Phylogenetic Utility of Genomic Ultraconserved Elements Obtained from Pinned Insect Specimens.

Obtaining sequence data from historical museum specimens has been a growing research interest, invigorated by next-generation sequencing methods that allow inputs of highly degraded DNA. We applied a target enrichment and next-generation sequencing protocol to generate ultraconserved elements (UCEs) from 51 large carpenter bee specimens (genus Xylocopa), representing 25 species with specimen ages ranging from 2–121 years. We measured the correlation between specimen age and DNA yield (pre- and post-library preparation DNA concentration) and several UCE sequence capture statistics (raw read count, UCE reads on target, UCE mean contig length and UCE locus count) with linear regression models. We performed piecewise regression to test for specific breakpoints in the relationship of specimen age and DNA yield and sequence capture variables. Additionally, we compared UCE data from newer and older specimens of the same species and reconstructed their phylogeny in order to confirm the validity of our data. We recovered 6–972 UCE loci from samples with pre-library DNA concentrations ranging from 0.06–9.8 ng/μL. All investigated DNA yield and sequence capture variables were significantly but only moderately negatively correlated with specimen age. Specimens of age 20 years or less had significantly higher pre- and post-library concentrations, UCE contig lengths, and locus counts compared to specimens older than 20 years. We found breakpoints in our data indicating a decrease of the initial detrimental effect of specimen age on pre- and post-library DNA concentration and UCE contig length starting around 21–39 years after preservation. Our phylogenetic results confirmed the integrity of our data, giving preliminary insights into relationships within Xylocopa. We consider the effect of additional factors not measured in this study on our age-related sequence capture results, such as DNA fragmentation and preservation method, and discuss the promise of the UCE approach for large-scale projects in insect phylogenomics using museum specimens.

Abstract 2700: Transcriptome analyses reveal fusion transcripts and transcript variants that are recurrent across sample series of testicular germ cell tumors

Abstract Testicular germ cell tumors (TGCT) are the most frequently diagnosed solid tumors in young men. Embryonal carcinoma (EC) comprises a histological subgroup of TGCT that exhibits pluripotent characteristics similar to embryonic stem (ES) cells. The genetic drivers underlying malignant transformation of TGCT, including ECs, are unknown. To elucidate the abnormal genetic events potentially contributing to TGCT malignancy, such as the formation of fusion genes or expression of aberrant transcript variants, we have previously performed RNA sequencing of EC cell lines and their non-malignant ES cell line counterparts. From this dataset, we identified and characterized eight novel fusion transcripts and one gene with alternative promoter usage, ETV6. These were the first fusion genes to be shown in TGCT (Hoff et al., Cancer Research, in press). To search for recurrence of the originally nominated TGCT specific fusion transcripts and other transcript variants in an external sample series, we have here parsed raw RNA sequencing data from 102 primary TGCTs obtained from The Cancer Genome Atlas (TCGA; unpublished). In this process, we have evaluated multiple fusion finder algorithms and approaches to determine the optimal method, on the basis of sensitivity and specificity, to query specific transcript variants and breakpoints in RNA sequencing datasets. We were able to detect the recurrent fusion transcripts; RCC1-ABHD12B, RCC1-HENMT1, CLEC6A-CLEC4D and the transcript variant of ETV6, which was previously detected from our in-house RNA sequencing dataset from EC cell lines. We also demonstrate that expression of several fusion transcripts correlate significantly with the histological subtype. In conclusion, the novel fusion transcripts and ETV6 transcript variant are expressed from a high fraction of primary TGCTs with low differentiation grade, as detected across independent sample series. Citation Format: Andreas M. Hoff, Sen Zhao, Bjarne Johannessen, Rolf I. Skotheim. Transcriptome analyses reveal fusion transcripts and transcript variants that are recurrent across sample series of testicular germ cell tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2700.

Abstract 104: Investigation of genetic architecture of multiple myeloma by next generation sequencing

Abstract Myeloma is a genetically heterogeneous disease and is sub-classified based on the presence of structural variants and genetic mutations. Structural variants/copy number changes are historically identified by traditional methods such as karyotyping and fluorescence in situ hybridization (FISH). Although microarray based genome wide analyses greatly improve the resolution of structural variation, they may be limited by probe density. Consequently, identification of structural variation may be insensitive to specific disrupted gene(s), neglecting the sequence complexity that might underlie these rearrangements. Determination of the specific breakpoints of structural variants at the nucleotide level is required for a better understanding of the genetic causes and to enhance the development of therapeutics for patients. The emergence of Next-Generation Sequencing (NGS) technology has led to the identification of structural variants in the genome at a higher resolution relative to currently used cytogenetic methods. We analyzed DNA extracted from a set of patients with multiple myeloma, who had Affymetrix SNP array (∼2.7 million probes) data, by whole exome sequencing (WES) at 100X coverage on the Illumina HiSeq platform to identify the full spectrum of associated genomic aberrations. Sequence data was mapped to the hg19 reference sequence and analyzed by various in-house developed and open source data analytic tools. Additionally, a custom sequence analysis pipeline was written to interrogate chromosomal deletions and translocations in these samples. Our analysis showed that ∼43% (6/14) of patients have deletions in chr17p and/or chr13q. We further confirmed structural variants using the Integrative Genomics Viewer (IGV). These data indicate the efficacy of WES for the precise determination of translocation and inversion breakpoints. In addition, we were able to identify single nucleotide variants (SNVs) and insertions/deletions (indels) in these samples. We then used the Ingenuity Variant Analysis (IGV) program to identify clinically actionable variants. These datasets are being further analyzed by various pathway analysis tools to define possible pathogenic mechanisms in multiple myeloma. Citation Format: Kerry Deutsch, Fang Yin Lo, Claire Olson, Sharon Austin, Kellie Howard, Amanda Leonti, Lindsey Maassel, Christopher Subia, Tuuli Saloranta, Nicole Christopherson, Kathryn Shiji, Shradha Patil, Stuart Schwartz, Peter Papenhausen, Steven Anderson, Anup Madan. Investigation of genetic architecture of multiple myeloma by next generation sequencing. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 104.

Abstract 1386: Clinical research results for a NGS-based kit for targeted detection of clinically relevant gene rearrangements in lung tumor samples

Abstract In recent years, advances in next-generation sequencing (NGS) technologies have enabled faster and cheaper methods for uncovering the genetic basis of disease. For cancer, NGS based screening for known tumor subtypes may inform diagnosis and allow the clinician to tailor a specific therapeutic approach in the future. Here, we present the testing results of one such NGS based kit used to detect specific chromosomal translocations in retrospective non-small cell lung cancer (NSCLC) samples by targeting specific breakpoints in known fusion transcripts. The included panel tested consists of a single primer pool containing amplicon designs to simultaneously screen for over 75 specific rearrangements involving the receptor tyrosine kinase (RTK) genes ALK, RET and ROS1 as well as NTRK1. The panel was compatible with formalin-fixed paraffin-embedded (FFPE) lung tumor research samples and achieved high-sensitivity down to 10 ng of RNA input. In addition, amplicon assays designed at the 5’ and 3’ ends the RTK genes provide non-specific evidence that a translocation exists in a sample by comparing expression imbalance between the two ends. Testing was carried out at three external clinical research laboratories. In addition to positive and negative control samples, each site contributed FFPE lung tumor research samples for which ALK fusion status was known prior to NGS library preparation carried out using the Ion AmpliSeq™ workflow. For site-specific samples (n = 144, 16 samples per sequencing run), high concordance, sensitivity and specificity were measured at 97.2%, 90.5% and 98.4%, respectively. Citation Format: Jeoffrey J. Schageman, José Luis Costa, Orla Sheils, John E. Glassco, David Chi, Jon Sherlock, John Bishop, Rosella P. Petraroli, Kelli S. Bramlett. Clinical research results for a NGS-based kit for targeted detection of clinically relevant gene rearrangements in lung tumor samples. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1386.

Highly personalized detection of minimal Ewing sarcoma disease burden from plasma tumor DNA.

Even though virtually all patients with Ewing sarcoma achieve a radiographic complete response, up to 30% of patients who present with localized disease and up to 90% of those who present with metastases experience a metastatic disease recurrence, highlighting the inability to identify patients with residual disease at the end of therapy. Up to 95% of Ewing sarcomas carry a driving EWS-ETS translocation that has an intronic breakpoint that is specific to each tumor, and the authors developed a system to quantitatively detect the specific breakpoint DNA fragment in patient plasma. The authors used a long-range multiplex polymerase chain reaction (PCR) technique to identify tumor-specific EWS-ETS breakpoints in Ewing sarcoma cell lines, patient-derived xenografts, and patient tumors, and this sequence was used to design tumor-specific primer sets to detect plasma tumor DNA (ptDNA) by droplet digital PCR in xenograft-bearing mice and patients. Tumor-specific breakpoint DNA fragments were detected in the plasma of xenograft-bearing mice, and the signal correlated with tumor burden during primary tumor growth, after surgical resection, and at the time of metastatic disease recurrence. Furthermore, the authors were able to detect the specific breakpoint in plasma DNA obtained from 3 patients with Ewing sarcoma and in 2 patients the authors were able to detect ptDNA when there was radiographically undetectable disease present. The use of droplet digital PCR to detect tumor-specific EWS-ETS fusion gene breakpoint ptDNA fragments can be developed into a highly personalized biomarker of disease recurrence that can be optimized in animal studies for ultimate use in patients. Cancer 2016;122:3015-3023. © 2016 American Cancer Society.

Resistance of Campylobacter jejuni Isolated from Layer Farms in Northern Jordan Using Microbroth Dilution and Disc Diffusion Techniques.

Campylobacter jejuni is an important pathogen of significant public health importance. This pathogen is associated with human infection and has been isolated from mammals and birds. Ninety-two cloacal C. jejuni isolates were identified from 35 layer farms in Northern Jordan. Antimicrobial susceptibility was determined using minimal inhibitory concentration (MIC) and disc diffusion techniques with variable suggested breakpoints. Using MIC and EUCAST cut-off values, the study revealed a significantly high resistance level (100%) among the layers' isolates against ciprofloxacin and tetracycline. A relatively high resistance (41%) toward gentamicin and amoxicillin and low resistance to nalidixic acid (21%), erythromycin (14%), and florfenicol (6.5%) were also found. This high level of resistance may indicate abuses in the handling of antibiotics, which may require stricter control in the local layer industry. A good agreement between the 2 techniques used was demonstrated and the disc diffusion technique could be used as a rapid screening test for antimicrobial susceptibility of C. jejuni to many antibiotics using specific Campylobacter breakpoints.

Antimicrobial susceptibility monitoring of bacterial pathogens isolated from respiratory tract infections in dogs and cats across Europe: ComPath results

ComPath is a pan-European resistance monitoring programme collecting bacterial pathogens from dogs and cats. We present data for respiratory tract infection (RTI) isolates collected between 2008 and 2010. Antimicrobial minimal inhibitory concentrations (MICs) were determined and susceptibility calculated following Clinical Laboratory Standards Institute (CLSI) standards for veterinary medicine. The main pathogen from dogs was Staphylococcus intermedius Group (49/215, 22.8%) which was >90% susceptible to most antimicrobials (including oxacillin − 93.9%; 3 isolates confirmed mecA-positive) but only 59.2%, 73.5% and 87.8% susceptible to tetracycline, chloramphenicol and penicillin. Bordetella bronchiseptica (48/215, 22.3%), streptococci (36/215, 16.7%), Escherichia coli (24/215, 11.2%) and Pasteurella multocida (23/215, 10.7%) were also found in dog RTI. There are no breakpoints for Bordetella bronchiseptica. Most streptococci were penicillin- chloramphenicol-, ampicillin- and pradofloxacin-susceptible. None were enrofloxacin-resistant but 6 isolates (16.7%) were of intermediate susceptibility. The least active agent against streptococci was tetracycline (47.2% susceptible). For E. coli, 37.5% were ampicillin-susceptible but 83.3% were amoxicillin/clavulanic acid-susceptible. Only chloramphenicol showed susceptibility>90% against E. coli, with 66.7% tetracycline-susceptible and 79.2% to 87.5% susceptibility to enrofloxacin, trimethoprim-sulfamethoxazole or pradofloxacin. P. multocida were susceptible to pradofloxacin (no other breakpoints are available). The main pathogen from cats was P. multocida (82/186, 44.1%), where only pradofloxacin has breakpoints (100% susceptible). Streptococci were also collected from cats (25/186, 13.4%) and were >90% susceptible to all antimicrobials except tetracycline (36% susceptible). Most susceptibility was calculated with human-derived breakpoints and some antimicrobials had no breakpoints. Therefore predictions of clinical utility for dog and cat RTI will remain problematical unless specific breakpoints are set.

A novel genomic inversion in Wiskott-Aldrich–associated autoinflammation

A novel genomic inversion in Wiskott-Aldrich–associated autoinflammation

Antimicrobial susceptibility of Mycoplasma bovis isolates from veal calves and dairy cattle in the Netherlands

Control of Mycoplasma bovis infections depends on good husbandry practices and antibiotic treatment. To allow more prudent use of antimicrobial drugs, there is a need for information on the susceptibility profile of this pathogen. The objective of the present study was to analyse the in vitro antimicrobial susceptibility of clinical M. bovis isolates in the Netherlands. The collection comprised 95 bovine isolates, originating from lungs (n=56), mastitis milk (n=27), and synovial fluid (n=12), collected between 2008 and 2014. Minimal inhibitory concentrations (MICs) were assessed by broth microdilution, both by using in-house prepared MIC plates and by using commercially available MIC plates. For each antimicrobial agent, the range of MIC results, the MIC50, and MIC90 values were calculated. M. bovis strains recently isolated in the Netherlands appeared to be characterized by relatively high MIC values for antimicrobial agents that, until now, have been recommended by the Dutch Association of Veterinarians for treating pneumonia caused by Mycoplasma species. Fluoroquinolones appeared to be the most efficacious in inhibiting M. bovis growth, followed by tulathromycin and oxytetracycline. The highest MIC values were obtained for erythromycin, tilmicosin, and tylosin. Future studies should be done on determining M. bovis specific clinical breakpoints, standardization of methods to determine MIC values as well as molecular studies on detection of antimicrobial resistance mechanisms of M. bovis isolates to develop PCR assays for determining resistance.

Fusion genes in malignant neoplastic disorders of haematopoietic system

Objectives: The new World Health Organization's (WHO) classification of haematopoietic and lymphoid tissue neoplasms incorporating the recurrent fusion genes as the defining criteria for different haematopoietic malignant phenotypes is reviewed. The recurrent fusion genes incorporated in the new WHO's classification and other chromosomal rearrangements of haematopoietic and lymphoid tissue neoplasms are reviewed.Methodology: Cytokines and transcription factors in haematopoiesis and leukaemic mechanisms are described. Genetic features and clinical implications due to the encoded chimeric neoproteins causing malignant haematopoietic disorders are reviewed.Results and discussion: Multiple translocation partner genes are well known for leukaemia such as MYC, MLL, RARA, ALK, and RUNX1. With the advent of more sophisticated diagnostic tools and bioinformatics algorithms, an exponential growth in fusion genes discoveries is likely to increase.Conclusion: Demonstration of fusion genes and their specific translocation breakpoints in malignant haematological disorders are crucial for understanding the molecular pathogenesis and clinical phenotype of cancer, determining prognostic indexes and therapeutic responses, and monitoring residual disease and relapse status.

Critical junctures and institution-building: regional cooperation on free trade and food security in East Asia

This study examines conditions under which states in East Asia engage in the development of regional institutions. It assumes that crucial external events and shocks, which produce specific historical breakpoints – critical junctures – constitute a significant breakpoint at which the regional states willingly elevated a path to develop regional institutions to a new level. The analysis of the development of regional institutions for a free trade area and food stock for emergency revealed that regional states in East Asia changed their views on the evolving reality created by external shocks and such changes led to the creation of new regional institutions.

Genetic screening and evaluation for chromosomal abnormalities of infertile males in Jilin Province, China.

Chromosomal abnormality is the most common genetic cause of male infertility, particularly in cases of azoospermia, oligozoospermia, and recurrent spontaneous abortion. Chromosomal rearrangement may interrupt an important gene or exert position effects. The functionality of genes at specific breakpoints, perhaps with a specific role in spermatogenesis, may be altered by such rearrangements. Structural chromosome abnormalities are furthermore known to increase the risk of pregnancy loss. In this study, we aimed to assess chromosomal defects in infertile men from Jilin Province, China, by genetic screening and to evaluate the relationship between structural chromosome abnormalities and male infertility. The prevalence of chromosomal abnormalities among the study participants (receiving genetic counseling in Jilin Province, China) was 10.55%. The most common chromosome abnormality was Klinefelter syndrome, and the study findings suggested that azoospermia and oligospermia may result from structural chromosomal abnormalities. Chromosome 1 was shown to be most commonly involved in male infertility and balanced chromosomal translocation was identified as one of the causes of recurrent spontaneous abortion. Chromosomes 4, 7, and 10 were the most commonly involved chromosomes in male partners of women experiencing repeated abortion.

The Sub-Clonal Complexity of STIL-TAL1 T-ALL

IntroductionThe STIL-TAL1 fusion is found in 16% cases of paediatric and adolescent T-ALL, making it one of the most common T-ALL subgroups. Our study considers this leukaemia subtype in the context of a complex ecosystem that is diverse, evolving and subject to selective pressures. We used single cell methods to understand the order of co-operating mutational events and the clonal evolution of mutations in genes that are re-iteratively targeted, such as PTEN.MethodsDiagnostic DNA from five STIL-TAL1 positive T-ALL cases was exome sequenced using Agilent SureSelect Human all Exon kit plus Illumina paired end sequencing. Driver copy number alterations and NOTCH1/PTEN exon 7 mutation status had been identified in a previous study and candidate driver mutations for inclusion in single cell experiments were validated by sequencing or Q-PCR using custom assays. Where more than one mutation was present within the same exon of a candidate driver gene, cloning experiments were carried out to verify the independent mutation sequences. Material from xenograft transplants was available in three of the five cases to assess their clonal heterogeneity in the leukaemia initiating cell compartment. Single cell multiplex Q-PCR was used to examine the single cell genetics of the pre-defined mutation events. Briefly, single cells were sorted and lysed prior to multiplex specific (DNA) target amplification and Q-PCR using the 96.96 dynamic microfluidic array and the BioMark HD (Fluidigm, UK). Copy number assays for the 1p33 deletion and custom assays for the patient specific STIL-TAL1 fusion breakpoints were used to confirm that the 1p33 deletion leading to this gene fusion was a clonal event.ResultsThe only aberrant events common to all five samples were CKDN2A copy number loss and the 1p33 deletion that results in the STIL-TAL1 fusion. Exome sequencing revealed further mutations in known T-ALL drivers including NOTCH1, PTEN and PHF6 as well as candidate driver mutations in FREM2, PIK3CD, RPL14, BMPR1A and CDH18. Both NOTCH1 and PTEN demonstrated re-iterative inactivation and this was investigated in detail for PTEN. Case 1 had multiple PTEN exon 7 mutations and sub-clonal copy number loss. Case 2 had parallel frameshift mutations in PTEN exons 5 and 7. Case 3 contained an exon 8 mutation and multiple PTEN exon 7 mutations. In this case the three most frequent PTEN exon 7 indels were validated and tracked in a single cell multiplex Q-PCR experiment. This revealed a branching sub-clonal genetic architecture (see figure 1) in which all malignant cells at the proposed apex of the branching architecture harboured the STIL-TAL1 fusion and CDKN2A deletion with copy number losses of 4p, 6q and FREM2 and PTEN mutations occurring as sub-clonal events. PTEN indels 2 and 3 were found co-localised in the same sub-clone. Preliminary analysis of the paired mouse xenograft bone marrow did not detect PTEN exon 7 indels 1 – 3 in 84 single cells. However, bulk Sanger Sequencing analysis did identify the PTEN exon 8 mutation in the mouse. Ongoing work is in progress to determine whether single cells of the xenograft carry alternative PTEN exon 7 mutations detected in the diagnostic sample exome data and to characterise in which diagnostic sub-clone the PTEN exon 8 mutation resides.ConclusionsThis study demonstrates how exome sequencing and single cell multiplex Q-PCR can be used as complementary tools to understand the sub-clonal complexity of STIL-TAL1 T-ALL. PTEN inactivation is sub-clonal by single cell analysis, demonstrating the parallel evolution of multiple independent PTEN inactivated sub-clones, highlighting PTEN inactivation as a key event in this T-ALL subgroup. In a wider cohort of 20 patients collected by our group at least 50% had PTEN inactivation as assessed by sequencing of exon 7 and copy number data alone. Results indicate a strong evolutionary pressure selecting for mutational events that result in inactivation of the PTEN-PI3Kinase pathway. These events occur via multiple mechanisms, including copy number loss and truncating mutations, which are not limited to the known T-ALL hotspot in exon 7. Current work is focussing on using a similar approach to examine the clonal evolution of NOTCH1 mutations in STIL-TAL1 T-ALL samples in diagnostic and xenograft samples of cases 4 and 5. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Surface Aesthetics in Tip Rhinoplasty: A Step-by-Step Guide.

Tip rhinoplasty is a key component of aesthetic rhinoplasty. An understanding of the correlation between tip surface aesthetics and the underlying anatomic structures enables proper identification and correction of tip abnormalities. Surface aesthetics of the attractive nose are created by certain lines, shadows, and highlights with specific proportions and breakpoints. In this Featured Operative Technique, the authors describe a stepwise process for tip rhinoplasty that conceptualizes aesthetic subunits as geometric polygons to define the existing deformity, the operative plan, and the aesthetic goals. Tip rhinoplasty is described in detail, from initial markings through incisions and dissection. The autorim graft concept is explained, and lateral crural steal and footplate setback techniques are described for the attainment of symmetric domes with correct lateral crural resting angles. Methods in columellar reconstruction are described, including creating the columella (C') breakpoint and the infralobular caudal contour graft. The principal author (B.Ç.) has applied these techniques to 257 consecutive "polygon rhinoplasties" over the past 3 years.

Determining structural breaks in central bank reaction functions of the financial crisis

Determining breakpoints in central bank reaction functions is crucial to monitor the decisions of the governing councils correctly. This is especially true during the recent financial crisis. However, reaction functions do not necessarily change at the beginning of a crisis and so the specific breakpoints need to be estimated rather than being set exogenously. While central bank reaction functions typically include more than one exogenous variable it is possible that the breakpoints in the reaction coefficients change from one independent variable to the other. We develop an estimation strategy using real-time data that account explicitly for variable breakpoints in the different independent variables. Using Taylor reaction functions for the ECB and the Fed we show that there are indeed different breakpoints with respect to the lagged interest rate, the inflation rate and the output gap within the financial crisis, in which the reaction coefficients tend to be significantly lower. Out-of-sample forecasts underline the result of a less volatile reaction of central banks during the crisis period.

Real-time process dynamics monitoring in Anammox reactors.

Process dynamics in Anammox systems were evaluated through continuous monitoring of pH, oxidation reduction potential (ORP) and conductivity in two separate newly started-up sequencing batch reactors, one seeded with an enriched Anammox sludge and the other seeded with mixed activated sludge. The pH and ORP profiles exhibited characteristic patterns depending on the process dynamics during early start-up, start-up and enrichment phases of the operational period of 410 days. The simultaneously continuing processes of the start-up period showed apparent indicative trend lines in pH and ORP profiles. Conductivity profiles were consistent with the process dynamics in all phases. During the enrichment phase, conductivity decreases could quantitatively be related to process removal efficiencies and all real-time profiles exhibited specific break-points which coincided with the end of Anammox in each cycle. The end of Anammox was observed as an 'apex' on pH profiles and a 'valley' on ORP profiles. The 'apex' and 'valley' points exactly coincided with the end point of the linear decrease in the conductivity profiles. The overall findings suggested a great potential in using real-time pH, ORP and conductivity measurements for quick and reliable monitoring of Anammox systems during start-up and enrichment periods.