Current Status of Staphylococcal Cassette Chromosome mec (SCCmec)

Staphylococcal cassette chromosome mec (SCCmec) typing was established in the 2000s and has been employed as a tool for the molecular epidemiology of methicillin-resistant Staphylococcus aureus, as well as the evolution investigation of Staphylococcus species. Molecular cloning and the conventional sequencing of SCCmec have been adopted to verify the presence and structure of a novel SCCmec type, while convenient PCR-based SCCmec identification methods have been used in practical settings for many years. In addition, whole-genome sequencing has been widely used, and various SCCmec and similar structures have been recently identified in various species. The current status of the SCCmec types, SCCmec subtypes, rules for nomenclature, and multiple methods for identifying SCCmec types and subtypes were summarized in this review, according to the perspective of the International Working Group on the Classification of Staphylococcal Cassette Chromosome Elements.

Paired-end Mappability of Transposable Elements in the Human Genome

Though transposable elements make up around half of the human genome, the repetitive nature of their sequences makes it difficult to accurately align conventional sequencing reads. However, in light of new advances in sequencing technology, such as increased read length and paired-end libraries, these repetitive regions are now becoming easier to align to. This study investigates the mappability of transposable elements with 50bp, 76bp and 100bp paired-end read libraries. With respect to those read lengths and allowing for 3 mismatches during alignment, over 68%, 85%, and 88% of all transposable elements in the RepeatMasker database are uniquely mappable, suggesting that accurate locus-specific mapping of older transposable elements is well within reach.

RNA Sequencing Based Whole Transcriptome Analysis Detected Precisely All Fusion Transcripts in Leukemias

Introduction: Fusion transcript is a chimeric RNA encoded by a fusion gene or by two different genes by subsequent trans-splicing. Detection of fusion transcripts is an integral part of routine diagnostics of hematological malignancies. However, most of previous analytical methods couldn't detect all fusion transcripts in leukemia. In this study, we developed accurate fusion transcript detection methood using whole transcriptome sequencing, fusion gene detection software and expression analysis. Methods: RNA sequencing (RNA-seq) for whole transcriptome was performed in 11 patients with hematological malignancies (4 AML, 2 APL, 2 ALL, and 3 CML) having fusion transcripts detected by multiplex RT-PCR (HemaVision, DNA Diagnostic, Risskov, Denmark). Library were prepared with 1 ug of total RNA for each sample by TruSeq mRNA Sample Prep kit (Illumina, San Diego, USA). The libraries were quantified using qPCR according to the qPCR Quantification Protocol Guide (KAPA Library Quantificatoin kits for Illumina Sequecing platforms) and qualified using the TapeStation D1000 ScreenTape (Agilent Technologies, Santa Clara, USA). Indexed libraries were then sequenced using the HiSeq2500 platform (Illumina). The data obtained from the sequencing was analyzed using STAR-Fusion (v1.2.0). Novel fusion transcripts were confirmed by conventional sequencing. Results: Using STAR-Fusion, average number of fusion candidates per sample was 949.8 (range, 286-1752). To exclude false positive results and obtain true positive results, we developed the following filtering algorithm. First filtering criterion is to have more than 5 junction reads, the second is to detect more than one number of spanning reads, and the third criterion is to be in-frame fusion, which type of fusion can actually synthesize intact protein. Fusion candidates remaining after applying the above three filtering criteria were 1-3 per sample. All known fusion transcripts (PML--RARA, RUNX--RUNX1T1, CBFB--MYH11, KMT2A--MLLT3, BCR--ABL1, DEK--NUP214, ETV6--RUNX1) by multiplex RT-PCR were also detected in RNA-seq. In addition, 10 novel fusion transcripts (IGKV4-1--IGKC, IGLV1-47--IGLC2, HBA2--HBB, DEFA3--MBNL1, HBB--HBA2, MPO--HBA2, HBS1L--AHI1, HBB--HBA2, IGKV4-1--IGKC, SS18L1--ADRM1) were detected and among them, 6 fusions were confirmed by conventional sequencing. Conclusions: Whole transcriptome sequencing and optimized filtering algorithms successfully detected all known fusion transcripts and various novel fusions. Disclosures No relevant conflicts of interest to declare.

Logarithmic molecular sampling for next-generation sequencing

Next-generation sequencing enables measurement of chemical and biological signals at high throughput and falling cost. Conventional sequencing requires increasing sampling depth to improve signal to noise discrimination, a costly procedure that is also impossible when biological material is limiting. We introduce a new general sampling theory, Molecular Entropy encodinG (MEG), which uses biophysical principles to functionally encode molecular abundance before sampling. SeQUential DepletIon and enriCHment (SQUICH) is a specific example of MEG that, in theory and simulation, enables sampling at a logarithmic or better rate to achieve the same precision as attained with conventional sequencing. In proof-of-principle experiments, SQUICH reduces sequencing depth by a factor of 10. MEG is a general solution to a fundamental problem in molecular sampling and enables a new generation of efficient, precise molecular measurement at logarithmic or better sampling depth.

Clinical Relevance of Low Burden BCR-ABL1 Mutations Detectable By Amplicon Deep Sequencing (DS) in Philadelphia-Positive (Ph+) Acute Lymphoblastic Leukemia (ALL) Patients (pts): The Type of Mutation Matters

Background and Aims- In Ph+ ALL pts treated with tyrosine kinase inhibitors (TKIs), the likelihood of acquiring TKI-insensitive mutations and the striking incidence of highly resistant T315I and compound mutants underscore the importance of BCR-ABL1 kinase domain (KD) sequence surveillance for timely and rational therapeutic reassessment. We used an amplicon DS strategy of the BCR-ABL1 KD to assess the following issues: i) whether DS allows earlier detection of emerging TKI-insensitive mutations in pts undergoing BCR-ABL1 KD mutation screening for minimal residual disease (MRD) persistence; ii) whether TKI-insensitive low burden mutations can be identified in relapsed pts with negative conventional sequencing results; iii) whether TKI-insensitive low burden mutations are necessary and sufficient to predict for treatment failure in all cases. Methods- This study was conducted in a total of 56 Ph+ ALL pts who received TKI-based therapies at our or collaborating institutions and were referred to our laboratory for MRD follow-up monitoring by RQ-PCR and for BCR-ABL1 KD mutation analysis in case of MRD positivity. These pts were divided into three different cohorts: i) 10 de novo and 24 advanced Ph+ ALL pts who relapsed and developed BCR-ABL1 KD mutations on TKI-based therapy administered 1st-line or for recurrent disease, respectively. To reconstruct the dynamics of mutation emergence, longitudinal re-analysis of monthly-collected samples from the time of hematologic relapse backwards was performed by DS. Whenever samples were available, the analysis was done back to the time of diagnosis (n=10/10) or back to the time of first or former relapse (n=15/24), respectively. Two to 6 samples were analyzed for each pt, for a total of 109 samples. ii) 14 Ph+ ALL pts who were known to be negative for mutations at the time of hematologic relapse as assessed by conventional sequencing. Relapse samples were reanalyzed by DS. iii) 8 Ph+ ALL pts with long-term relapse-free survival despite persistent or intermittent MRD positivity at multiple timepoints. Up to 5 samples were analyzed for each pt, for a total of 28 samples. DS was performed on a Roche GS Junior. Lower mutation detection limit of DS was 1%. Results- In the 34 de novo or advanced Ph+ ALL pts who were known to have acquired TKI-insensitive mutations at the time of relapse on tyrosine kinase inhibitor (TKI) therapy, longitudinal retrospective reanalysis by DS allowed mutation backtracking in 13 (41%) cases. One patient was found to harbour a low burden Y253H at diagnosis. In 3 imatinib (IM)-resistant pts who switched to dasatinib (DAS), a low burden T315I mutation was already detectable at baseline. In the 14 pts with no mutations detectable by conventional sequencing at the time of relapse on IM or DAS, low burden TKI-insensitive mutations were detected by DS in 6 (43%) cases. In 2 cases who had relapsed on DAS, a T315I and an F317L mutation, respectively, were present just below the lower detection limit of conventional sequencing (15.9% and 12.4%, respectively); in the remaining 4 pts, DS identified multiple (2 to 3) low burden mutations, all of which known to confer a moderate to high degree of insensitivity to the ongoing TKI. In the 8 pts with persistently or transiently detectable BCR-ABL1 transcripts at multiple timepoints despite stable hematologic remission, DS detected low burden mutations in 9 samples from 4 pts. However, no mutation known to be truly insensitive to the ongoing TKI could be recognized. Conclusions MRD persistence in Ph+ ALL pts may hide emerging TKI-insensitive BCR-ABL1 KD mutations that DS may identify earlier than conventional sequencing - allowing a greater leeway before overt hematologic relapse occurs; polyclonal resistance sustained by multiple TKI-insensitive low burden mutations may explain relapse in a proportion of cases with unmutated BCR-ABL1 KD sequences as assessed by conventional sequencing; the type of mutation matters: detection of low burden mutations insensitive to the ongoing TKI was always found to predict/correlate with treatment failure. Detection of low burden mutations with low/unknown IC50 might explain low level MRD but does not predict for an impending relapse; MRD-triggered, BCR-ABL1 KD mutation screening by DS may be precious for earlier and more effective use of preemptive rescue therapies. Supported by ELN, AIL, AIRC, FP7 NGS-PTL project, Progetto Regione-Università 2010-12 (L. Bolondi) Disclosures Soverini: Ariad: Consultancy; Bristol-Myers Squibb: Consultancy; Novartis: Consultancy. Abruzzese:BMS, Novartis, Pfizer, Ariad: Consultancy. Baccarani:ARIAD Pharmaceuticals, Inc.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; PFIZER: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; NOVARTIS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Cavo:Onyx: Honoraria; BMS: Honoraria; Novartis: Consultancy, Honoraria; Millenium Pharmaceuticals: Honoraria; Celgene: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Jansenn: Consultancy, Honoraria. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Martinelli:Pfizer: Consultancy; BMS: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; ROCHE: Consultancy; AMGEN: Consultancy; Ariad: Consultancy; MSD: Consultancy.

In Chronic Myeloid Leukemia Patients on 2nd-Line Tyrosine Kinase Inhibitor Therapy, Deep Sequencing at the Time of Warning May Allow Sensitive Detection of Emerging BCR-ABL1 Mutants

Background and Aims: Next generation amplicon-based deep sequencing (DS) on the Roche, Illumina or Ion Torrent instruments is becoming accessible to a wider and wider number of diagnostic laboratories. Although conventional sequencing is still the gold standard, DS has been hailed by many as the future of diagnostic BCR-ABL1 kinase domain (KD) mutation screening. BCR-ABL1 KD mutations are infrequent in newly diagnosed chronic myeloid leukemia (CML) patients (pts) receiving 1st-line TKI therapy, but remain a challenge in relapsed pts, who usually display a greater genetic instability. Indeed, pts already harboring BCR-ABL1 KD mutations have a higher likelihood of developing additional, dasatinib (DAS)- or nilotinib (NIL)-resistant mutations – which is defined as a ‘failure’ by the 2013 European LeukemiaNet (ELN) recommendations. Taking advantage of a next-generation amplicon sequencing design and protocol set up and validated in the framework of the IRON-II international study, we aimed to assess whether DS may allow a larger window of detection of emerging BCR-ABL1 KD mutants predicting for an impending relapse. Methods: among the imatinib (IM)-resistant CML pts who switched to 2nd-line TKI therapy and were referred to our laboratory for routine BCR-ABL1 transcript level monitoring and KD mutation screening by conventional sequencing, 51 acquired DAS- or NIL-resistant mutations after a median of 9 months (range, 3-27 months) of therapy and had leftover cDNA available at previous timepoints. To reconstruct the dynamics of mutation emergence, resequencing on a Roche GS Junior instrument was performed from the time of failure and mutation detection by conventional sequencing backwards. Runs were designed to achieve high sequencing depth, allowing reliable detection of variants down to 1% abundance. BCR-ABL1/ABL1%IS transcript levels and/or cytogenetic response, whichever available, were used to define whether the patient had an ‘optimal response’, ‘warning’ or ‘failure’ at the time of first mutation detection by DS. Results: baseline mutation status, as assessed by conventional sequencing, was available for all the 51 CML pts included in this retrospective study; 29/51 pts were positive for BCR-ABL1 KD mutations, with switch to NIL or DAS selected accordingly. Twenty-six pts were later found to have acquired DAS-resistant mutations (T315I, n=13; F317L/V, n=10; V299L, n=3) and 25 pts were later found to have acquired NIL-resistant mutations (T315I, n=4; F359V/I/C, n=7; Y253H, n=6; E255K, n=9; one patient acquired two mutations). DS was able to backtrack the DAS- or NIL-resistant mutations to the previous sample(s) in 23/51 (45%) pts. Median mutation burden at the time of first detection by DS was 5% (range, 1-17%); median interval between detection by DS and detection by conventional sequencing was 3 months (range, 3-9 months). In 5 cases, the mutations were traceable at baseline; in the remaining cases, correlation with response at the time mutations were first detected by DS revealed a ‘warning’ according to the 2013 ELN definitions of response to 2nd-line therapy in 13 cases; an ‘optimal response’ in one case; a ‘failure’ in 4 cases. As a control, we used DS to explore BCR-ABL1 KD mutation status in 10 randomly selected pts with ‘warning’ at various timepoints, that later turned into optimal responses; no DAS- or NIL-resistant mutations were detected. Conclusions: the 2011 ELN recommendations for mutation analysis suggest BCR-ABL1 KD to be screened by conventional sequencing in case of ‘failure’ of 2nd-line TKI therapy – according to the provisional definitions available at the time. Earlier detection of emerging BCR-ABL1 KD mutations allows a greater leeway in tackling drug resistance and enhancing therapeutic efficacy. Data presented herein indicate that: 1) DS may reliably pick TKI-resistant mutations earlier than conventional sequencing in a proportion of pts, and that 2) the recently introduced definitions of ‘warning’ may provide a rational trigger, besides ‘failure’, for DS-based BCR-ABL1 KD mutation screening in CML pts on 2nd-line TKI therapy. A prospective cost-benefits evaluation of using DS in this and other settings is warranted, and will contribute useful information to the revision of the ELN recommendations for BCR-ABL1 KD mutation analysis. Supported by: European LeukemiaNet, AIL, AIRC, FP7 NGS-PTL project, Progetto Regione-Università 2010-12 (L. Bolondi). Disclosures Soverini: Novartis: Consultancy; Bristol-Meyers Squibb: Consultancy; Ariad: Consultancy. Castagnetti:Novartis Farma: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria; Pfizer: Consultancy. Gugliotta:Novartis: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria. Bonifacio:Amgen Inc.: Consultancy. Rosti:Novartis: Consultancy; Bristol-Myers Squibb: Consultancy. Baccarani:Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; Ariad: Consultancy; Pfizer: Consultancy. Martinelli:NOVARTIS: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy.

New insights into genotype and phenotype of VWD

Recent advances in VWD research have improved our understanding of the genotype and phenotype of VWD. The VWF gene is highly polymorphic, with a large number of sequence variations reported in healthy individuals. This can lead to some difficulty when attempting to discern genotype–phenotype correlations because sequence variations may not represent disease. In type 1 VWD, mutations can be found throughout the VWF gene, but likely pathogenic sequence variations are found in only ∼2/3 of type 1 VWD patients. Sequence variations in type 2 VWD are located in the region corresponding to the defect in the VWF protein found in each type 2 variant. In type 3 VWD, sequence variations are not confined to a specific region of the VWF gene and also include large deletions that may not be picked up using conventional sequencing techniques. Use of genetic testing may be most helpful in diagnosis of type 2 VWD, in which a larger number of known, well characterized mutations are present and demonstration of one of these may help to confirm the diagnosis. Bleeding symptoms in general are more severe with decreasing VWF levels and more severe in type 2 and type 3 VWD compared with type 1 VWD. Prediction of phenotype for an individual patient, however, is still difficult, and the addition of genetic data will be most helpful in ascertaining the correct diagnosis for VWD patients.

New insights into genotype and phenotype of VWD

Recent advances in VWD research have improved our understanding of the genotype and phenotype of VWD. The VWF gene is highly polymorphic, with a large number of sequence variations reported in healthy individuals. This can lead to some difficulty when attempting to discern genotype–phenotype correlations because sequence variations may not represent disease. In type 1 VWD, mutations can be found throughout the VWF gene, but likely pathogenic sequence variations are found in only ∼2/3 of type 1 VWD patients. Sequence variations in type 2 VWD are located in the region corresponding to the defect in the VWF protein found in each type 2 variant. In type 3 VWD, sequence variations are not confined to a specific region of the VWF gene and also include large deletions that may not be picked up using conventional sequencing techniques. Use of genetic testing may be most helpful in diagnosis of type 2 VWD, in which a larger number of known, well characterized mutations are present and demonstration of one of these may help to confirm the diagnosis. Bleeding symptoms in general are more severe with decreasing VWF levels and more severe in type 2 and type 3 VWD compared with type 1 VWD. Prediction of phenotype for an individual patient, however, is still difficult, and the addition of genetic data will be most helpful in ascertaining the correct diagnosis for VWD patients.