Clone Detection Research Articles

Towards Program Comprehension: Knowledge Profiles for C Programmers

Abstract Following up on the issue of programmer knowledge profiles, the present article deals with the generation of profiles for C language programmers. The main idea is based on static analysis of source codes, assembling the most significant data about their author. Such a profile can point out some abilities and habits of the programmer. The process of static analysis applies methods and techniques of size metrics, complexity metrics, and clone detection. We also present an experiment focused on novice programmers. The results show that knowledge profiles provide an easy way of novice programmers’ progress tracking. The main contribution is believed to be in the area of program comprehension.

Clonal Spread and Intra- and Inter-Species Plasmid Dissemination Associated With Klebsiella pneumoniae Carbapenemase-Producing Enterobacterales During a Hospital Outbreak in Barcelona, Spain.

Objectives: The study aimed to characterize the clonal spread of resistant bacteria and dissemination of resistance plasmids among carbapenem-resistant Enterobacterales at a tertiary hospital in Catalonia, Spain.Methods: Isolates were recovered from surveillance rectal swabs and diagnostic samples. Species identification was by matrix-assisted laser desorption ionization-time time of flight mass spectrometry (MALDI-TOF MS). Molecular typing was performed by pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). Antimicrobial susceptibility was assessed by gradient-diffusion and carriage of bla genes was detected by PCR. Plasmid typing, conjugation assays, S1-PFGE studies and long-read sequencing were used to characterize resistance plasmids.Results: From July 2018 to February 2019, 125 Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacterales were recovered from 101 inpatients from surveillance (74.4%) or clinical samples (25.6%), in a tertiary hospital in Barcelona. Clonality studies identified a major clone of Klebsiella pneumoniae belonging to sequence type ST15 and additional isolates of K. pneumoniae, Escherichia coli and Enterobacter sp. from different STs. All isolates but one carried the blaKPC–2 allelic variant. The blaKPC–2 gene was located in an IncFIIk plasmid of circa 106 Kb in a non-classical Tn4401 element designated NTEKPC-pMC-2-1. Whole-genome sequencing revealed different rearrangements of the 106 Kb plasmid while the NTEKPC-pMC-2-1 module was highly conserved.Conclusion: We report a hospital outbreak caused by the clonal dissemination of KPC-producing ST15 K. pneumoniae but also the intra- and inter-species transmission of the blaKPC–2 gene associated with plasmid conjugation and/or transposon dissemination. To our knowledge, this is the first report of an outbreak caused by KPC-producing Enterobacterales isolated from human patients in Catalonia and highlights the relevance of surveillance studies in the early detection and control of antibiotic resistant high-risk clones.

BIOM-36. SERIAL ASSESSMENT OF MEASURABLE RESIDUAL DISEASE IN MEDULLOBLASTOMA LIQUID BIOPSIES

Abstract Medulloblastoma is the most common malignant brain tumor in children. Despite the use of contemporary multi-modal therapy, up to one-third of patients will succumb to disease progression. Conventional response monitoring is based on magnetic resonance imaging and cerebrospinal fluid (CSF) cytology and a marker for measurable residual disease (MRD) is lacking. In this study, we show the clinical utility of profiling CSF-derived cell-free DNA (cfDNA) as a biomarker of MRD based on a sizable cohort of children with medulloblastoma enrolled on a prospective, multi-center, risk-adapted trial (SJMB03; NCT00085202). A total of 476 CSF samples were serially collected from 123 patients by lumbar puncture at post-operative baseline, during therapy, and at regular surveillance after completion of therapy. Using low-coverage whole-genome sequencing (lcWGS), tumor-associated copy-number alterations in CSF-derived cfDNA were investigated as an MRD surrogate for correlation with clinical features and outcomes. MRD was detected in post-operative baseline CSF samples for 85% and 54% of patients with metastatic and localized disease, respectively. The number of MRD-positive patients declined with therapy, yet those with persistent MRD detected at the end of therapy (20 of 68 CSFs at this timepoint) had significantly higher risk of disease progression (hazard ratio 8.94, 95%CI 4.10-19.49; log-rank p< 0.0001). Importantly, MRD-positivity from routine surveillance samples often preceded radiographic progression by more than 3 months. Comparative analyses of copy-number variations obtained from serial CSF samples enabled identification of relapse-specific alterations and early detection of relapse-dominant clones. Overall, our findings advocate for the routine incorporation of CSF-derived liquid biopsies in future medulloblastoma trials.

Clone-advisor: recommending code tokens and clone methods with deep learning and information retrieval.

Software developers frequently reuse source code from repositories as it saves development time and effort. Code clones (similar code fragments) accumulated in these repositories represent often repeated functionalities and are candidates for reuse in an exploratory or rapid development. To facilitate code clone reuse, we previously presented DeepClone, a novel deep learning approach for modeling code clones along with non-cloned code to predict the next set of tokens (possibly a complete clone method body) based on the code written so far. The probabilistic nature of language modeling, however, can lead to code output with minor syntax or logic errors. To resolve this, we propose a novel approach called Clone-Advisor. We apply an information retrieval technique on top of DeepClone output to recommend real clone methods closely matching the predicted clone method, thus improving the original output by DeepClone. In this paper we have discussed and refined our previous work on DeepClone in much more detail. Moreover, we have quantitatively evaluated the performance and effectiveness of Clone-Advisor in clone method recommendation.

Deep learning application on code clone detection: A review of current knowledge

Bad smells in code are indications of low code quality representing potential threats to the maintainability and reusability of software. Code clone is a type of bad smells caused by code fragments that have the same functional semantics with syntactic variations. In the recent years, the research on duplicate code has been dramatically geared up by deep learning techniques powered by advances in computing power. However, there exists little work studying the current state-of-art and future prospects in the area of applying deep learning to code clone detection. In this paper, we present a systematic review of the literature on the application of deep learning on code clone detection. We aim to find and study the most recent work on the subject, discuss their limitations and challenges, and provide insights on the future work.

Fully Automated Workflows Quantify and Report Key T-Cell and B-Cell Receptor Biomarkers Relevant to Immuno-Oncology and Heme-Oncology Research

BackgroundT-cell and B-cell repertoire analysis is used in oncology research, to understand the etiology of complex disease phenotypes, for the identification of biomarkers predictive of disease burden, outcome, and response to treatment, and for research in diagnosis and recurrence monitoring. Key predictors include secondary and tertiary repertoire features not reported by existing sequencing software solutions. For example, due to ongoing somatic hypermutation in mature B-cell receptors, the underlying sequence of a given clone can accumulate base differences and appear as several distinct clones with smaller frequencies, thereby hampering the ability of analysis software to detect its presence as a single dominant clone with the highest frequency. This has particularly detrimental implications for research in disorders such as follicular lymphoma and may require clonal lineage analysis for proper mitigation. Therefore, to aid the downstream analytics of biomarker identification and the study of complex disease, we developed fully automated analysis solutions that directly compute and report several key features (clonal lineage, amongst several others described below) pertinent to this area of research.ResultsWe developed the Oncomine™ TCR Beta-SR, TCR Gamma-SR, BCR IGH-SR and BCR IGKL-SR workflows on Ion Reporter™ to characterize T-cell (β, γ chains) and B-cell (heavy and light (κ, δ) chains) repertoires. These workflows generate output tables and visualizations for primary repertoire features such as detected clones (viz., unique rearrangements in the receptor DNA sequence), their frequencies, as well as their somatic hypermutation levels in the case of B-cells (Figure 1a & 1b) for clonality assessment and rare clone detection. The software also quantifies and reports several secondary and tertiary repertoire features in a sample, such as clonal diversity, evenness of the clonal population, and B-cell lineage groupings useful in identifying related sub-clones. It includes spectratyping format plots to simultaneously assess the above features as a function of v-gene usage and CDR3 length combinations (Figure 1c & 1d), thereby providing users a complete snapshot of the repertoire, and also the capability to quickly determine CDR3 lengths and V-gene usage of highly expanded or mutated clones. A separate CDR3 lengths histogram is included, as well as a heatmap that depicts the distributions/intensity of Variable-Joining gene combinations (Figure 1e & 1f).Furthermore, the TCR workflows also report (i) convergence frequencies (fraction of clones with different nucleotide sequences, but identical amino acid sequences), and (ii) haplotype grouping for an analyzed sample, based on V-gene allele genotyping and clustering (Figure 1g). In addition, the long read Oncomine™ BCR IGH-LR workflow uniquely reports the isotype class for every detected clone, and includes a visualization of total reads, clones and lineages in the sample represented by isotype (Figure 1h).ConclusionThe Oncomine™ immune repertoire workflows for T-cell and B-cell receptor sequencing were designed to be of high utility in distinct areas of malignancy research, and we expect them to greatly simplify complex downstream analyses. The unique capabilities of the workflows to automatically report secondary and tertiary repertoire features such as (i) clonal lineages for improved dominant clone detection in blood cancers, (ii) TCR clone convergence for prediction of response to immune checkpoint inhibitors [1,2], (iii) TCR haplotype grouping for evaluation of risk factors for autoimmunity and immune-related adverse events [3], and (iv) isotype classification in BCRs for studying pan-cancer immune evasion mechanisms, demonstrate the clear advantages of using these automated workflows over other existing solutions.For research use only.References1) Looney TJ et al. (2020) TCR Convergence in Individuals Treated With Immune Checkpoint Inhibition for Cancer. Front. Immunol. 10:2985.2) Naidus et al. (2021) Early changes in the circulating T cells are associated with clinical outcomes after PD-L1 blockade by durvalumab in advanced NSCLC patients. Cancer Immunology, Immunotherapy 70:2095-21023) Looney TJ et al. (2019) Haplotype Analysis of the T-Cell Receptor Beta (TCRB) Locus by Long-amplicon TCRB Repertoire Sequencing. Journal of Immunotherapy and Precision Oncology. 2 (4): 137-143. [Display omitted] DisclosuresSarda: Thermo Fisher Scientific: Current Employment. Lowman: Thermo Fisher Scientific: Current Employment. Toro: Thermo Fisher Scientific: Current Employment. Pickle: Thermo Fisher Scientific: Current Employment. Looney: Thermo Fisher Scientific: Ended employment in the past 24 months; Singular Genomics: Current Employment. Hyland: Thermo Fisher Scientific: Current Employment.

FISH and WGS in Newly Diagnosed and Relapsed/Refractory Multiple Myeloma - WGS Will Affect Future Treatment Decisions

Background:FISH is the gold standard for genetic characterization and risk stratification of multiple myeloma (MM) at diagnosis. Retrospective studies have shown the potential of whole genome sequencing (WGS) to gain new insights into MM genetics. With respect to clinical application, we prospectively performed FISH and WGS in parallel in patients (pts) with newly diagnosed MM (NDMM) and relapsed/refractory MM (RRMM) to evaluate advantages and limitations.Patients and methods:From bone marrow (BM) samples of 280 MM pts CD138+ cells were enriched by MACS. In 100 pts (30 NDMM, 70 RRMM) FISH (del(1p32), del(13q), del(17p), t(4;14), t(11;14), t(14;16), t(14;20), t(6;14), 1q gain, MYC rearrangement (MYCr) and trisomies 3, 5, 9) and WGS were performed. WGS: 150bp paired-end sequences were generated on NovaSeq 6000 (Illumina); median coverage 104x. Single nucleotide variants (SNV) were called with Strelka software, copy number variations (CNV) with GATK4 and structural variants (SV) with Manta. Technical artefacts and germline calls were reduced by a tumor/unmatched normal workflow with genomic DNA from a mix of anonymous donors. Variants with global population frequency >0.5% (gnomAD) were excluded.Results:In 100/280 (36%) MM pts WGS could be performed in parallel to FISH in this cohort with a low median BM plasma cell infiltration (PCI) of 6% (range 1-95%). Likelihood of DNA yields sufficient for WGS increased with higher PCI (23% (1-95%) vs 3% (1-50%) in 100 pts with WGS vs 180 pts without sufficient DNA).Comparison of FISH and WGS results for 100 pts showed 100% concordance for recurrent SV (table 1): t(11;14) in 29%, t(4;14) in 17% and t(14;16) in 5%. No pts harbored a t(14;20) but in one case FISH indicated another alteration of MAFB and WGS revealed MAFB rearranged to FAM46C resulting in an elevated MAFB expression.Due to heterogeneous breakpoints FISH is less reliable for the detection of MYCr than WGS (24/42 (57%) detected by FISH vs 40/42 (95%) by WGS). Partners of MYC included IGH (n=10), FAM46C (n=4), IGK (n=3), IGL (n=3), FOXO3 (n=2) and IRF4 (n=1). Rare IGH rearrangements with partner genes such as IRF1, IRF4, ZBTB38 and ZFP36L1 were observed in 11 pts all identified by WGS, but only 8 (73%) were detected by FISH.Total frequencies of CNV were del(13q) in 50%, del(17p) in 29%, del(1p32) in 22%, 1q gains in 61% and hyperdiploidy in 36%. WGS detected 95% of CNV (details table 1). CNV that were missed by WGS included one del(17p) and were all identified in small subclones with a median clone size of 10% (6-25%) in FISH.Tetraploid MM, potentially harboring adverse prognostic implication, was identified in 11 pts by FISH but not in WGS.However, in 97 pts WGS depicted additional SV and CNV compared to FISH. This includes aberrations of prognostic significance such as a TP53 deletion that was located adjacent to a 451kb gain on 17p. The FISH probe encompassing TP53 confirmed the 17p gain but masked the TP53 deletion. Also 1p deletions, which have been described to be a negative prognostic factor, were found more frequently by WGS with 24 pts harboring a del(1p) other than del(1p32) (locus evaluated by FISH) which was detected in 22 pts by both methods. Furthermore, as WGS cannot only detect SV and CNV but also SNV, WGS allows screening for potentially clinically exploitable drug targets, e.g. BRAF V600E mutation (5 pts in our cohort) or resistance associated mutations such as in cereblon for immunomodulatory compounds (CRBN mutation in 3 pts) as well as biallelic events in TP53 (del(17p)+TP53 mutation), detected in 15 of our pts suggesting worst prognosis for affected pts. WGS in RRMM can shed light on the genetic background of relapses. Of note, homozygous del(16p) inducing loss of the treatment target were first described using our dataset in two patients with acquired resistance toward anti-BCMA CAR T cells or bispecific antibody therapy.Conclusions:FISH is the preferred approach in pts with BM PCI <10% and for the detection of clones <15% or variant ploidy levels. WGS is superior to FISH regarding the identification of biallelic events and rearrangements with heterogeneous breakpoints and rare partners. But even more, WGS can address various questions in follow up in terms of increase of complexity, target specific therapy, therapy failure and relapse. Thus, WGS allows a holistic insight into the genetics of MM and provides crucial information for clinical decision making. [Display omitted] DisclosuresKern: MLL Munich Leukemia Laboratory: Other: Part ownership. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Einsele: Janssen, Celgene/BMS, Amgen, GSK, Sanofi: Consultancy, Honoraria, Research Funding. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership.

Immune Repertoire Analysis of Multiple Myeloma Research Samples Using NGS Characterization of Multiple B Cell Receptors in a Single Reaction

IntroductionB cell repertoire analysis by next-generation sequencing (NGS) is at the forefront of leukemia and lymphoma research. Some advantages provided by NGS-based techniques include a lower limit-of-detection and simpler paths to standardization compared to other methods. Importantly, in research of post-germinal B cell disorders, such as multiple myeloma (MM), NGS methods allow for the study of clonal lineage based on somatic hypermuation patterns. Current targeted NGS assays require multiple libraries to survey each B cell receptor chain (IGH, IgK, IgL), and this fact is highlighted when initial clonality detection fails due to mutations under primer binding sites. This issue can be especially true in MM which has a high rate of SHM. To address these issues, we have developed an assay for B cell analysis, based on Ion AmpliSeq™ technology, which enables efficient detection of IGH, IgK, and IgL chain rearrangements in a single reaction.MethodsThe B cell pan-clonality panel (Oncomine™ BCR Pan-Clonality Assay) targets the framework 3 (FR3) portion of the variable gene and the joining gene region of heavy- and light-chain loci (IGH, IgK, IgL) for all alleles found within the IMGT database, enabling readout of the complementary-determining region 3 (CDR3) sequence of each immunoglobulin chain. To maximize sensitivity, we included primers to amplify IgK loci rearrangements involving Kappa deletion element and the constant region intron. To evaluate assay performance, we conducted reproducibility studies and clonality assessment using gDNA from a total of 45 MM research samples. All MM cases examined in this work were confirmed clonal previously by light chain restriction via flow cytometry or IHC/ISH in tissue sections - 16 of the 45 MM samples were identified as lambda light chain restricted. For comparison, a small cohort of 12 B-ALL samples were also included in the study. Sequencing and repertoire analyses were performed using the Ion GeneStudio S5 System and Ion Reporter 5.16 analysis software.ResultsClonality assessment of MM clinical research samples show an 93% overall positive detection rate by an assay which combines the IGH, IgK, and IgL chains in a single reaction using published guidelines for clonality assignment. Thirty-four of 45 samples show positive detection of an IGH rearrangement, while 41 of 45 showed positive detection of at least one light chain receptor. In total, 42 of 45 samples were deemed clonal by the single tube assay based on detection for one or more receptor. Clonality results for this sample set are well correlated with orthogonal data from flow, IHC/ISH, or alternate NGS assays. A clonal lambda light chain was identified in 14 of 16 samples determined to be lambda restricted by flow cytometry. In two of the lambda restricted samples only a clonal lambda rearrangement was identified, showing the benefit of including primers targeting both the kappa and lambda light chains in a pan-clonality NGS assay. Both the MM and B-ALL cohorts were evaluated for biased IGHV gene usage. IGHV3-11 was observed in 5 of 45 MM and 5 of 12 B-ALL samples. IGHV4-34, typically linked to autoreactive antibodies and underrepresented in germinal center and memory B-cells, was nonetheless found in 5 of 45 MM samples surveyed. Estimates of somatic hypermutation rates were calculated using the BCR pan-clonality assay. Most MM samples, as expected, contained some somatic hypermutation with 6 of 45 samples showing greater than 10% mutation rates. Automated lineage analysis, based on somatic hypermuation signatures within each sample, identified 8 of 45 MM samples which contained 5 or more clones in the primary clonal lineage, with one case containing a lineage with 23 clones. Two MM samples showed no somatic hypermutation as measured using the FR3 primers contained in the BCR pan-clonality assay. These samples were also evaluated using an FR1-J targeted NGS assay, which confirmed relatively low mutation rates for these MM samples at 0.44% and 1.3%, respectively.ConclusionsThese results demonstrate the utility of a novel assay for combined repertoire analysis of B cell receptor heavy and light chains in a single library preparation reaction. We expect this assay to simplify laboratory workflows and including analysis tools such as automated somatic hypermutation rate calculation and clonal lineage identification may open new paths for research in lymphoid cell disorders.For research use only. DisclosuresLowman: Thermo Fisher Scientific: Current Employment. Toro: Thermo Fisher Scientific: Current Employment. Pickle: Thermo Fisher Scientific: Current Employment. Ostresh: Thermo Fisher Scientific: Current Employment. Sarda: Thermo Fisher Scientific: Current Employment. Yang: Thermo Fisher Scientific: Current Employment.

Phylogenetic Analysis and Genome-Wide Association Study Applied to an Italian Listeria monocytogenes Outbreak.

From May 2015 to March 2016, a severe outbreak due to Listeria monocytogenes ST7 strain occurred in Central Italy and caused 24 confirmed clinical cases. The epidemic strain was deeply investigated using whole-genome sequencing (WGS) analysis. In the interested area, the foodborne outbreak investigation identified a meat food-producing plant contaminated by the outbreak strain, carried by pork-ready-to-eat products. In the same region, in March 2018, the epidemic strain reemerged causing one listeriosis case in a 10-month-old child. The aim of this study was to investigate the phylogeny of the epidemic and reemergent strains over time and to compare them with a closer ST7 clone, detected during the outbreak and with different pulsed-field gel electrophoresis (PFGE) profiles, in order to identify genomic features linked to the persistence and the reemergence of the outbreak. An approach combining phylogenetic analysis and genome-wide association study (GWAS) revealed that the epidemic and reemergent clones were genetically closer to the ST7 clone with different PFGE profiles and strictly associated with the pork production chain. The repeated detection of both clones was probably correlated with (i) the presence of truly persistent clones and the repeated introduction of new ones and (ii) the contribution of prophage genes in promoting the persistence of the epidemic clones. Despite that no significant genomic differences were detected between the outbreak and the reemergent strain, the two related clones detected during the outbreak can be differentiated by transcriptional factor and phage genes associated with the phage LP-114.

CloneRetriever: An Automated Algorithm to Identify Clonal B and T Cell Gene Rearrangements by Next-Generation Sequencing for the Diagnosis of Lymphoid Malignancies.

Clonal immunoglobulin and T-cell receptor rearrangements serve as tumor-specific markers that have become mainstays of the diagnosis and monitoring of lymphoid malignancy. Next-generation sequencing (NGS) techniques targeting these loci have been successfully applied to lymphoblastic leukemia and multiple myeloma for minimal residual disease detection. However, adoption of NGS for primary diagnosis remains limited. We addressed the bioinformatics challenges associated with immune cell sequencing and clone detection by designing a novel web tool, CloneRetriever (CR), which uses machine-learning principles to generate clone classification schemes that are customizable, and can be applied to large datasets. CR has 2 applications-a "validation" mode to derive a clonality classifier, and a "live" mode to screen for clones by applying a validated and/or customized classifier. In this study, CR-generated multiple classifiers using 2 datasets comprising 106 annotated patient samples. A custom classifier was then applied to 36 unannotated samples. The optimal classifier for clonality required clonal dominance ≥4.5× above background, read representation ≥8% of all reads, and technical replicate agreement. Depending on the dataset and analysis step, the optimal algorithm yielded sensitivities of 81%-90%, specificities of 97%-100%, areas under the curve of 91%-94%, positive predictive values of 92-100%, and negative predictive values of 88%-98%. Customization of the algorithms yielded 95%-100% concordance with gold-standard clonality determination, including rescue of indeterminate samples. Application to a set of unknowns showed concordance rates of 83%-96%. CR is an out-of-the-box ready and user-friendly software designed to identify clonal rearrangements in large NGS datasets for the diagnosis of lymphoid malignancies.

Improved Detectability of Plasmodium falciparum Clones with Repeated Sampling in Incident and Chronic Infections in Burkina Faso.

ABSTRACT.We evaluated the detectability of Plasmodium falciparum clones when assessed on 3 consecutive days in incident and chronic infections in naturally exposed children living in an area of intense malaria transmission in Burkina Faso. The median number of clones by merozoite surface protein 2 (MSP2) genotyping was 3 (interquartile range [IQR] 2–5) in incident infections compared with 6 (IQR 4–8) in chronic infections (P < 0.0001). When all clones detected on days 1-3 were considered as true complexity of infection, sampling on day 1 detected only 69.4% (109/157) or 68.3% (228/334) of all clones in incident and chronic infections, respectively. Our findings demonstrate that a large proportion of clones are missed by single time-point sampling. In addition, because of the high complexity of infection early in incident infections, our data suggest many infections may be caused by genetically complex inocula.

IoT-based Cloud Service for Secured Android Markets using PDG-based Deep Learning Classification

Software piracy is an act of illegal stealing and distributing commercial software either for revenue or identify theft. Pirated applications on Android app stores are harming developers and their users by clone scammers. The scammers usually generate pirated versions of the same applications and publish them in different open-source app stores. There is no centralized system between these app stores to prevent scammers from publishing pirated applications. As most of the app stores are hosted on cloud storage, therefore a cloud-based interaction system can prevent scammers from publishing pirated applications. In this paper, we proposed IoT-based cloud architecture for clone detection using program dependency analysis. First, the newly submitted APK and possible original files are selected from app stores. The APK Extractor and JDEX decompiler extract APK and DEX files for Java source code analysis. The dependency graphs of Java files are generated to extract a set of weighted features. The Stacked-Long Short-Term Memory (S-LSTM) deep learning model is designed to predict possible clones. Experimental results have shown that the proposed approach can achieve an average accuracy of 95.48% among clones from different application stores.

Clone detection through srcClone: A program slicing based approach

Software clone detection is an often used approach to understand and maintain software systems. One category of clones that is challenging to detect but very useful is semantic clones, which are similar in semantics but differ in syntax significantly. Semantic clone detectors have trouble scaling to larger systems and sometimes struggle with recall and precision. To address this, we developed a slice-based scalable approach that detects both syntactic and semantic code clones, srcClone. srcClone ascertains code segment similarity by assessing the similarity of their corresponding program slices. We employ a lightweight, publicly-available, scalable program slicer within our clone detection approach. Using dependency analysis to detect and assess cloned components, we discover insights about code components that can be affected by a clone pair or set. These elements are critical in impact analysis. It can also be used by program analysts to run on non-compilable and incomplete source code, which serves comprehension and maintenance tasks very well. We first evaluate srcClone by comparing it to six state-of-the-art tools and two additional semantic clone detectors in performance, recall, and precision. We use the BigCloneBench real clones benchmark to facilitate comparison. We use an additional 16 established benchmark scenarios to perform a qualitative comparison between srcClone and 44 clone detection approaches in their capabilities to detect these scenarios. To further measure scalability, we evaluate srcClone on 191 versions of Linux kernel, containing approximately 87 MLOC. In our evaluations, we illustrate our approach is both relatively scalable and accurate. While its precision is slightly less than some other techniques, it makes up for it in higher recall including semantic clones unable to be found by any existing techniques. We contend our approach is an important advancement in software cloning that it is both demonstrably scalable and can detect more types of clones than existing work, thus providing developers greater information into their software.

Ig Gene Clonality Analysis Using Next-Generation Sequencing for Improved Minimal Residual Disease Detection with Significant Prognostic Value in Multiple Myeloma Patients

Next-generation sequencing (NGS) of rearranged Ig genes is an effective technology for identifying pathologic clonal cells in multiple myeloma (MM) and tracking minimal residual disease. The clinical effect of implementing NGS in Ig gene clonality analysis was evaluated via a retrospective chart review. A total of 312 patients diagnosed with MM were enrolled in the study. Ig gene clonality was determined by fragment analysis using BIOMED-2 multiplex PCR assays and by NGS using the LymphoTrack IGH FR1 Assay and LymphoTrack IGK Assay. The clonality detection rates in diagnostic samples obtained using fragment analysis and NGS were 96.7% and 95.4%, respectively (statistically nonsignificant difference; P=0.772). Among samples of patients in complete remission, the clonality detection rates obtained using fragment analysis and NGS were 33.3% and 60.3%, respectively (statistically significant difference; P=0.034). Progression-free survival was significantly longer in negative than positive patients by NGS analysis (P=0.03). Clonality detection by NGS-based methods using IGH FR1 and IGK assays in routine clinical practice is feasible, provides good clonality detection rates in diagnostic samples, and allows monitoring of samples in MM patients with significant prognostic value.

Interlaboratory Analytical Validation of a Next-Generation Sequencing Strategy for Clonotypic Assessment and Minimal Residual Disease Monitoring in Multiple Myeloma.

Minimal residual disease (MRD) is a major prognostic factor in multiple myeloma, although validated technologies are limited. To standardize the performance of the LymphoTrack next-generation sequencing (NGS) assays (Invivoscribe), targeting clonal immunoglobulin rearrangements, in order to reproduce the detection of tumor clonotypes and MRD quantitation in myeloma. The quantification ability of the assay was evaluated through serial dilution experiments. Paired samples from 101 patients were tested by LymphoTrack, using Sanger sequencing and EuroFlow's next-generation flow (NGF) assay as validated references for diagnostic and follow-up evaluation, respectively. MRD studies using LymphoTrack were performed in parallel at 2 laboratories to evaluate reproducibility. Sensitivity was set as 1.3 tumor cells per total number of input cells. Clonality was confirmed in 99% and 100% of cases with Sanger and NGS, respectively, showing great concordance (97.9%), although several samples had minor discordances in the nucleotide sequence of rearrangements. Parallel NGS was performed in 82 follow-up cases, achieving a median sensitivity of 0.001%, while for NGF, median sensitivity was 0.0002%. Reproducibility of LymphoTrack-based MRD studies (85.4%) and correlation with NGF (R2 > 0.800) were high. Bland-Altman tests showed highly significant levels of agreement between flow and sequencing. Taken together, we have shown that LymphoTrack is a suitable strategy for clonality detection and MRD evaluation, with results comparable to gold standard procedures.

Decluttering the problem list in electronic health records.

The purpose of this study was to evaluate the current state of problem list maintenance at an academic medical center. We included problem list data for patients who had at least 2 face-to-face encounters at Vanderbilt University Medical Center or its clinics between January 1, 2018, and December 31, 2019. We used the frequency of problem list additions, resolutions, deletions, duplicate problems (exact and SNOMED CT duplicates), inconsistencies (contradicting stages of disease state), and items that could be documented elsewhere in the electronic health record as surrogate markers of problem list maintenance. Descriptive statistics were used to summarize the results. A total of 546,510 patients met inclusion criteria. There were 3,762 (0.7%) patients who had the exact same active problem listed more than once. SNOMED CT code duplications occurred in the records for 56,399 (10.5%) patients. Of the patients with asthma, 2.5% (223/8,779) had contradicting asthma stages active on their problem list, and 6.4% (950/14,950) of patients with chronic kidney disease (CKD) had contradicting CKD stages. In addition, 17,205 (3.1%) patients had 20,365 active family history problems and 39,464 (7.2%) patients had an allergy documented on their problem list. On average, there were 43.7 (95% confidence interval [CI], 14-73.4) additions, 8.7 (95% CI, 0.1-17.4) resolutions, and 2.1 (95% CI, 0-4.6) deletions of problems per 100 face-to-face encounters, inpatient or outpatient. Our study suggests areas for improvement for problem list maintenance. Further studies into semantic duplication and clinical decision support tools to encourage problem list maintenance and deduplication are needed.

ANALYSIS OF BASELINE CHARACTERISTICS, DISEASE BURDEN AND LONG-TERM FOLLOW-UP OF 167 PATIENTS WITH BRAZILIAN PAROXYSMAL NOCTURNAL HEMOGLOBINURIA - ANOTHER NATURAL HISTORY

Paroxysmal nocturnal hemoglobinuria (PNH) may occur as classical hemolytic disease or a small PNH clone found in a patient with bone marrow failure. To describe clinical features and long-term follow-up of 167 patients with PNH and demonstrate differences between disease categories. Multiparametric flow cytometry performed on 1025 patients referred from Jan/2000 to Dec/2019 found 167 (16.3%) confirmed PNH clone, 87M/70F. Clinical characteristics at first visit, and laboratory results, incidence of thrombosis, treatment and outcome during follow-up visits were considered for statistical analysis. Most patients (89.2%) had hypocellular bone marrow at diagnosis; 55(32.9%) developed hemoglobinuria, and 22(13.2%) developed thrombosis during monitoring. Mean age at aplasia diagnosis 28.4 years (range 5.6 - 71.2), and mean age at PNH clone detection 31.0 years (7.2 - 71.3 years). Clonal evolution occurred in 77 (46.1%) patients at a median of 4 years (range 6-281 months) after the onset of cytopenia. The cohort was divided into 15 classic PNH, 55 hemolytic PNH with bone marrow hypoplasia (PNH/AA), and 97 definitive bone marrow disease with minor PNH clones - subclinical PNH (sc-PNH). Subclinical patients had lower erythrocyte (2.0% vs 24.0% vs 57.8%) and granulocyte PNH clones (11.7% vs 58.8% vs 81.2%) than PNH/AA and classic PNH, respectively. LDH, reticulocytes, absolute neutrophils and bone marrow cellularity were all lower in sc-PNH than in the hemolytic group. Pearson's analysis showed a statistically significant correlation (p < 0.001) between PNH granulocyte clone and RBC clone (0.697), LDH (0.602), absolute neutrophils (0.310), platelet count (0.430), absolute reticulocytes (0.546), total bilirubin (0.330), and bone marrow biopsy (0.435). PNH granulocyte clone greater than 50% was found in 16/22 thrombotic patients. Mean granulocyte clone was 89.1% (3.4%-99.2%) versus 12.3%(0.1%-34.0%), p < 0.001, and erythrocytes 28.4% (3.4-99.8) versus 0.7% (0.1-14.0) in thrombotic versus the remaining 145 patients. Treatment was highly heterogeneous during this 20-year follow-up: immunosuppressive therapy with CSA and/or thymoglobulin was given to 72 patients in the subclinical group (73.1%) and 45 (64.3%) in the hemolytic group; 52 patients received HSCT (31.1%) of which 40 in sc-PNH and 11 in PNH/AA; a total of 19 patients received Eculizumab after 2008 and 3 patients never received treatment. Incidentally, there were 17 thrombotic events in hemolytic patients (38.6%) diagnosed by 2007, compared to only five events (19.2%) in patients diagnosed after 2008. The average follow-up period was 74 months (1 - 330 months), with 31 deaths in total (80.4% OS). Sepsis was the leading cause of sc-PNH mortality (16/18, 88.8%), and thrombosis in both hemolytic groups (11/13, 84.6%). Log rank test shows similar survival when comparing patients with diagnosis until and after 2007 (p = 0.04). this study suggests clonal evolution in the long-term follow-up of aplastic patients and confirms the observation that high levels of PNH and LDH clones are associated with life-threatening hemolysis and thrombosis, while small PNH clones and young age are associated with the subclinical form of the disease. Although without statistical significance, the cumulative incidence of thrombosis in the group diagnosed after 2008 appears to be lower than the previous period, perhaps due to the routine use of Eculizumab.

Bio-P-01 - Prognostic value of the clonal rearrangement of T-cell receptor genes detection in the bone marrow in patients with mycosis fungoides

<h3>Introduction:</h3> Mycosis fungoides constitutes more than 60% of primary cutaneous T-cell lymphomas. The course of disease is progradient: the gradual changes of macular, plaque and tumor stages are noted. The evaluation of the spreading process is critically important for the prognosis determination and therapy choosing. T-cell clonality detection by rearranging genes of the gamma chain of the T-cell receptor in the blood and bone marrow aspirates can be detected at the early stages. It is known to be a prognostically unfavorable factor for diagnostics in peripheral blood, while the value of the rearranging presence of T-cell receptor gene in the bone marrow aspirates has not been fully studied. <h3>Aim:</h3> To evaluate the frequency and prognostic value of the T-cell clonality detection by the gamma chain of the T-cell receptor in the bone marrow in patients with mycosis fungoides/Sézary syndrome (MF/SS). <h3>Materials and methods:</h3> The patients with mycosis fungoides/Sézary syndrome were observed at the National Research Centre of Hematology during the period from 2000 to 2015. <h3>Results:</h3> 45 patients diagnosed with mycosis fungoides/Sézary syndrome (MF/SS) corresponded to inclusion criteria. Men/women 1:1. The age of the participants is varied from 20 to 81 years. The diagnosis of mycosis fungoides was made in 36 cases (80%) and the diagnosis of Sézary syndrome was made in 9 cases (20%) respectively. The early stages were diagnosed in 13 patients (29%), the later stages were diagnosed in 32 patients. T-cell clonality by the gamma chain rearrangement of the T-cell receptor was detected in the bone marrow in 15 out of 45 patients (33%). 13 cases were the patients with the late disease stages and clonality at the early Ib stage was detected only in 2 cases. The morphological bone marrow involvement was identified in 5 patients, but in two cases T-cell clonality was not detected. The median survival of patients with morphologically confirmed bone marrow damage was 3 months. The median survival of patients with defined T-cell clonality in the bone marrow was 21 months. The median survival with no molecular damages of bone marrow was 41 months. <h3>Conclusions:</h3> T-cell clonality by gamma chain rearranging of the T-cell receptor in the bone marrow in patients with mycosis fungoides/Sézary syndrome is determined in a third of patients mainly at the late MF stages. This is an unfavorable prognostic factor.

Analysis of baseline characteristics, disease burden and long-term follow-up of 167 patients with Paroxysmal Nocturnal Hemoglobinuria at a single center in Brazil

Paroxysmal nocturnal hemoglobinuria (PNH) can occur as a hemolytic form or small PNH clone found in a patient with bone marrow failure. MethodsDescribe Brazilian retrospective PNH cohort and identify the impact of disease burden on long-term follow-up. Results167 patients, mean age at diagnosis 28.4 (7.1–71.2 years), four years mean interval between onset of cytopenia/aplasia diagnosis and PNH clone detection. Patients were divided into 15 Classic PNH, 55 Hemolytic PNH with bone marrow hypoplasia (PNH/AA), and 97 Subclinical PNH (sc-PNH). Hypocellular bone marrow was found in 89.2%; 55 had hemoglobinuria and 22 thrombosis during monitoring. WBC PNH clone correlated with RBC PNH clone, LDH and cytopenia. Subclinical patients had lower median lower RBC clone (2.0% vs 24.0% vs 57.8%) and WBC clone (11.7% vs 58.8% vs 81.2%) than PNH/AA and Classic PNH, respectively. PNH granulocyte clone was 89.1% in thrombotic patients. Ten-year overall survival 80.4% and mortality in transplanted patients 9.6%. Sepsis was mortality cause in subclinical PNH (16/18, 88.8%), and thrombosis in hemolytic PNH (11/13, 84.6%). ConclusionLarge PNH clones and LDH burden were associated with increased hemolysis and thrombosis risks, while young patients were associated with small PNH clones and subclinical form of the disease. Knowledge of the patient profile, the low risk associated with HSCT, and the use of long-term IST may be instrumental in the clinical management of PNH in restricted-resources countries.

RepliComment: Identifying clones in code comments

Code comments are the primary means to document implementation and facilitate program comprehension. Thus, their quality should be a primary concern to improve program maintenance. While much effort has been dedicated to detecting bad smells, such as clones in code, little work has focused on comments. In this paper we present our solution to detect clones in comments that developers should fix. RepliComment can automatically analyze Java projects and report instances of copy-and-paste errors in comments, and can point developers to which comments should be fixed. Moreover, it can report when clones are signs of poorly written comments. Developers should fix these instances too in order to improve the quality of the code documentation. Our evaluation of 10 well-known open source Java projects identified over 11K instances of comment clones, and over 1,300 of them are potentially critical. We improve on our own previous work Blasi and Gorla (2018), which could only find 36 issues in the same dataset. Our manual inspection of 412 issues reported by RepliComment reveals that it achieves a precision of 79% in reporting critical comment clones. The manual inspection of 200 additional comment clones that RepliComment filters out as being legitimate, could not evince any false negative.