Perceived Challenges to Routine Uptake of the Ankle Brachial Index within Primary Care Practice.

(1) Introduction: The ankle-brachial index (ABI) is the most widely used method of diagnosing peripheral arterial disease (PAD). However, the uptake of ABIs has been reported to be low in primary care settings across different various healthcare settings; however, this is yet to be investigated within the Canadian context. (2) Objective: Therefore, we sought to assess the rates of ABI usage as well as perceived barriers among primary care practitioners (PCPs) in Toronto, Canada. (3) Methods: A modified questionnaire was electronically sent to 257 PCPs in the Greater Toronto Area (GTA). Questions pertained to frequency, feasibility, utility, and barriers associated with ABI usage in clinical practice. Responses were collected and tallied. (4) Results: A total of 52 PCPs completed the questionnaire. 79% of PCPs did not routinely perform ABIs within their clinical practice, and 56% deemed ABI usage as unfeasible. Constraints in time and staff personnel, as well as complexity of ABI result interpretation, were cited as the major perceived barriers to ABI usage. The overwhelming majority of PCPs viewed alternative forms of diagnosis, such as a blood test for PAD, as being preferable to ABI, as such an approach would enhance diagnostic simplicity and efficiency. (5) Conclusion: ABI usage rates are poor within primary care practices in Toronto, Canada. Alternative approaches for diagnosing PAD may result in greater adoption rates among PCPs and therefore improve the identification of patients with PAD.

Laboratory Verification of a BRCA1 and BRCA2 Massively Parallel Sequencing Assay from Wet Bench to Bioinformatics for Germline DNA Analysis.

Introduction A robust genetic test for BRCA1 and BRCA2 genes is necessary for the diagnosis, prognosis, and treatment of patients with hereditary breast and ovarian cancer. We evaluated a commercial amplicon-based massively parallel sequencing (MPS) assay, BRCA MASTR Plus on the MiSeq platform, for germline BRCA genetic testing. Methods This study was performed on 31 DNA from cell lines and proficiency testing samples to establish the accuracy of the assay. A reference cell line DNA, NA12878 was used to determine the reproducibility of the assay. Discordant MPS result was resolved orthogonally by the current gold-standard Sanger sequencing method. Results The analytical accuracy, sensitivity, and specificity for variant detection were 93.55, 92.86, and 100.00%, respectively. Both sequencing depth and variant allele frequencies were highly reproducible by comparing the NA12878 DNA tested in three separate runs. The single discordant result, later confirmed by Sanger sequencing was due to the inability of the MASTR Reporter software to identify a 40-bp deletion in BRCA1 . Conclusion The BRCA MASTR Plus assay on the MiSeq platform is accurate and reproducible for germline BRCA genetic testing, making it suitable for use in a clinical diagnostic laboratory. However, Sanger sequencing may still serve as a confirmatory method to improve diagnostic capability of the MPS assay.

Priority Outcomes in Critically Ill Children: A Patient and Parent Perspective.

BACKGROUND: Outcomes in pediatric critical care research are typically selected by the researcher. OBJECTIVES: (1) To identify outcomes prioritized by patients and their families following a critical illness and (2) to determine the overlap between patient-centered and researcher-selected study outcomes. METHODS: An exploratory descriptive qualitative study nested within a longitudinal cohort study conducted in 2 pediatric intensive care units (PICUs). Participants were purposively sampled from the primary cohort to ensure adequate demographic representation. Qualitative descriptive approaches based on naturalistic observation were used to collect data and analyze results. Data were coded by using the International Classification of Functioning, Disability, and Health Children and Youth (ICF-CY) framework. RESULTS: Twenty-one participants were interviewed a mean of 5.1 months after PICU discharge. Outcomes fell into 2 categories: patient-centered and family-centered. In the former, diagnosis, survival, and prognosis were key priorities during the acute critical illness. Once survival appears possible, functioning (physical, cognitive, and emotional), and factors that influence recovery (ie, rehabilitation, environment, and quality of life) are prioritized. Family-centered outcomes consisted of parents' psychosocial functioning and experience of care. Patient-centered outcomes were covered well by the selected study measures of functioning, but not by the clinical outcome measures. CONCLUSION: Functioning and quality of life are key patient-centered outcomes during recovery from critical illness. These are not well captured by end points typically used in PICU studies. These results justify the importance of patient- and family-centered outcomes in PICU research and a need to determine how these outcomes can be comprehensively measured.

The use of the Panda-Nerve Block pain app in single-shot peripheral nerve block patients: a feasibility study.

PURPOSE: Peripheral nerve blocks (PNBs) provide excellent perioperative analgesia but can increase the risk of severe postoperative pain once the block wears off. Poor adherence to discharge instructions may increase this risk. Panda-Nerve Block (Panda) is an app that alerts the patient to assess their PNB, score their pain, and take scheduled pain medication. We assessed the usability and feasibility of Panda for assisting patients after receiving a PNB. METHODS: Twenty-nine patients tested Panda in three rounds, for two to seven days, postoperatively to assess and manage their pain and PNB. Feedback was provided via phone interview and the Computer System Usability Questionnaire (CSUQ). Additionally, each user's usage log was analyzed for parameters such as alert response times. Feasibility was determined by alert responses that occurred before the next alert, with a goal of greater than 50%. User adherence was measured as percentage compliance with alerts within one hour; usability and user satisfaction were determined from the CSUQ and interviews. RESULTS: A median [interquartile range (IQR)] of 68 [34-93]% responded before the next alert during the first 48 hr of app use, and 83 [54-92]% responded before the next alert with 87 [75-96]% of these within one hour. There were no significant differences in usage between rounds. Ninety-three percent of patients reported Panda to be easy to use and helpful, and 79% of patients would use Panda again. Critical themes included changes to the layout and appearance, clarification of the language of the PNB check, and requests for dynamic adjustments to the medication schedule based on user responses. CONCLUSION: Panda-Nerve Block is a feasible method for PNB patients to manage postoperative pain with a high response rate. Future work should include providing two-way communication for patients and clinicians and assessing its effect on pain outcomes. TRIAL REGISTRATION: www.clinicaltrials.gov (NCT03369392); registered 5 December 2017.

RéSUMé: OBJECTIF: Les blocs nerveux périphériques (BNP) procurent une excellente analgésie périopératoire mais peuvent augmenter le risque de douleur postopératoire élevée une fois que le bloc disparait. Un mauvais respect des instructions de congé pourrait augmenter ce risque. L'application Panda (Panda-Nerve Block) avertit le patient afin qu'il évalue son BNP, quantifie sa douleur, et prenne ses médicaments analgésiques prescrits. Nous avons évalué la facilité d'utilisation et la faisabilité de l'application Panda pour aider les patients ayant reçu un BNP. MéTHODE: Vingt-neuf patients ont testé l'application Panda en trois itérations de deux à sept jours après leur opération afin d'évaluer et de prendre en charge leur douleur et le BNP. Les rétroactions étaient partagées par entretien téléphonique et via le Questionnaire sur la convivialité du système informatique (CSUQ - Computer System Usability Questionnaire). En outre, le journal d'utilisation de chaque utilisateur a été analysé pour en étudier certains paramètres tels que les temps de réponse aux alertes. La faisabilité était déterminée par les réponses aux alertes survenant avant la prochaine alerte, avec un objectif de plus de 50 %. L'observance des utilisateurs était mesurée en tant que pourcentage de conformité aux alertes dans l'heure suivante; la facilité d'utilisation et la satisfaction des utilisateurs étaient déterminées à partir du CSUQ et des entretiens. RéSULTATS: En moyenne [écart interquartile (ÉIQ)], 68 [34­93] % des patients ont répondu avant la prochaine alerte au cours des premières 48 h d'utilisation de l'application, et 83 [54­92] % ont répondu avant la prochaine alerte, avec 87 [75­96] % de ces patients dans l'heure qui suivait. Il n'y a pas eu de différence significative dans l'utilisation entre les itérations. Quatre-vingt-treize pour cent des patients ont rapporté qu'ils trouvaient l'application Panda conviviale et utile, et 79 % l'utiliseraient à nouveau. Les critiques comprenaient des modifications de la disposition et de l'apparence de l'application, la clarification du langage lors des vérifications du BNP, et des demandes pour des ajustements dynamiques du traitement selon les réponses des utilisateurs. CONCLUSION: L'application Panda constitue une méthode possible de prise en charge de la douleur postopératoire pour les patients ayant reçu un BNP, avec un taux de réponse élevé. Les travaux futurs devraient inclure la fourniture d'une communication bidirectionnelle pour les patients et les cliniciens et l'évaluation de l'effet de l'utilisation de l'application sur des devenirs de douleur. ENREGISTREMENT DE L'éTUDE: www.clinicaltrials.gov (NCT03369392); enregistrée le 5 décembre 2017.

The evaluation of smartphone versions of the visual analogue scale and numeric rating scale as postoperative pain assessment tools: a prospective randomized trial.

PURPOSE: The Pain assessment using a novel digital application (Panda) is a smartphone application that contains the digital versions of the visual analogue scale (VAS-100) and numeric rating scale (NRS-11). This study aimed to investigate if the Panda versions of these two pain scales are equivalent to the paper versions in adult patients. METHODS: This was a prospective, randomized, cross-over-controlled trial of subjects aged 19-75 yr undergoing procedures with anticipated post-surgical pain. Each subject used both the Panda and paper versions of VAS-100 or NRS-11 pain scores after emergence from anesthesia and after meeting postanesthesia care unit (PACU) discharge criteria. Correlations between the two tools were analyzed, and Bland-Altman agreement was calculated. The smartphone and paper versions were considered equivalent at each time point if the differences (and their 95% confidence interval [CI]) between them were less than 20 points for the VAS-100 and 2.1 for NRS-11. RESULTS: The two versions of the VAS-100 correlated strongly after emergence (Pearson's r = 0.93; P < 0.001) and upon meeting discharge criteria (r = 0.94; P < 0.001); the mean (standard deviation [SD]) Panda score after emergence was 35 (27) compared with the paper score of 37 (26) (mean difference, - 2; 95% CI, - 22 to 19). The mean (SD) VAS-100 Panda score upon meeting discharge criteria was 21 (20) compared with the paper score of 23 (21) (mean difference, - 2; 95% CI, - 17 to 13). For the NRS-11, Panda again correlated strongly with the original tool scores after emergence (r = 0.93; P < 0.001) and upon meeting discharge criteria (r = 0.96; P < 0.001); the mean (SD) Panda and paper scores after emergence were both 4 (3) (mean difference, 0.05; 95% CI, - 1.87 to 1.96). The mean (SD) NRS-11 Panda and paper scores upon meeting PACU discharge criteria were both 3 (2) (mean difference, - 0.08; 95% CI, - 1.41 to 1.26). CONCLUSION: Following emergence from anesthesia in adult patients, the digital Panda version of the NRS-11, but not the VAS-100, is equivalent to the validated paper version. In those who are ready for discharge from the PACU, the digital Panda versions of both the VAS-100 and NRS-11 agreed adequately and can be used in place of the original paper versions.

BRAF mutation in papillary thyroid cancer-Prevalence and clinical correlation in a South-East Asian cohort.

OBJECTIVE: BRAF mutation is the commonest mutation seen in papillary thyroid cancer (PTC), but its prevalence and clinical significance vary across countries. We aim to evaluate the prevalence and clinico-pathological correlation of BRAF mutation in PTC patients at our centre. STUDY DESIGN: Retrospective cohort study of 75 consecutive archival thyroid specimens, whereby BRAF mutation was detected using a polymerase chain reaction (PCR) technique and correlated with clinical and pathological features and outcomes. SETTING: Tertiary university hospital in Singapore. PARTICIPANTS: A total of 75 consecutive histologically proven archival thyroid specimens from patients who underwent thyroidectomy for PTC were accrued for this study. MAIN OUTCOME MEASURES: Main outcome is to determine the prevalence of the BRAF mutation in our South-East Asian population. Secondary aim is to correlate the mutational status with adverse pathological features like histological variants, multi-focality, lymphovascular invasion and extra-thyroidal extension, clinical features like demographics, TNM stage, recurrence and survival, as well as treatment details like type of surgery performed and radioiodine doses. RESULTS: BRAF mutation was detected in 56% (42/75) of PTC. All but one BRAF-mutated PTC had the BRAFV600E mutation. BRAF-mutated tumours were associated with an advanced T-stage (P = 0.049) and were more likely to have a central neck dissection (P = 0.036). There was no significant correlation between BRAF mutation status and clinical outcomes. CONCLUSION: The prevalence of BRAF mutation is 56%. BRAF mutation-positive tumours were associated with locally advanced disease, but not poorer survival.

The National Palliative Care Research Center: Ten Years of Promoting and Developing Research in Palliative Care.

BACKGROUND: The evidence base to support high-quality clinical care and number of scientists available to develop this evidence base are inadequate. OBJECTIVE: To describe the first 10 years of the National Palliative Care Research Center's (NPCRC) programs and their outcomes. DESIGN: Established in 2005, NPCRC was created in direct response to the recommendations of the Institute of Medicine. Specifically, NPCRC was created to expand the palliative care evidence-based needed for both health policy and clinical practice by supporting research scientists, stimulating research and innovation, and creating a community of researchers focused on the needs of persons with serious illness and their families. MEASUREMENTS: Subsequent grant funding following NPCRC investment (web searches of NIH Research Portfolio Online Reporting Tools [RePORT], Veterans Administration and Patient Centered Outcomes Research Institute [PCORI] grant databases, grantee on-line surveys, and grantee annual reports) promotions (grantee on-line surveys and annual reports), publications (PubMed searches), and NPCRC participant satisfaction (grantee questionnaires). RESULTS: As of July 2017, NPCRC has funded 47 junior investigators representing over 10 disciplines. These investigators have leveraged NPCRC's $7.8 million investment into 52 federal grants totaling $74.8 million dollars and 69 foundation grants totaling $16 million. Thirty-five grants ($5.8 million) have been awarded to experienced investigators, resulting in additional grant funding of $104.5 million dollars ($78.5 million federal, $26 million nonfederal). Satisfaction with NPCRC's program has been uniformly high and policy efforts have resulted in enhanced federal funding opportunities in palliative care research. CONCLUSIONS: NPCRC's focus on people and infrastructure in conjunction with a top-down bottom-up strategy has been critical in improving the palliative care evidence base.

Preleukemic and second-hit mutational events in an acute myeloid leukemia patient with a novel germline RUNX1 mutation.

BACKGROUND: Germline mutations in the RUNX1 transcription factor give rise to a rare autosomal dominant genetic condition classified under the entity: Familial Platelet Disorders with predisposition to Acute Myeloid Leukaemia (FPD/AML). While several studies have identified a myriad of germline RUNX1 mutations implicated in this disorder, second-hit mutational events are necessary for patients with hereditary thrombocytopenia to develop full-blown AML. The molecular picture behind this process remains unclear. We describe a patient of Malay descent with an unreported 7-bp germline RUNX1 frameshift deletion, who developed second-hit mutations that could have brought about the leukaemic transformation from a pre-leukaemic state. These mutations were charted through the course of the treatment and stem cell transplant, showing a clear correlation between her clinical presentation and the mutations present. CASE PRESENTATION: The patient was a 27-year-old Malay woman who presented with AML on the background of hereditary thrombocytopenia affecting her father and 3 brothers. Initial molecular testing revealed the same novel RUNX1 mutation in all 5 individuals. The patient received standard induction, consolidation chemotherapy, and a haploidentical stem cell transplant from her mother with normal RUNX1 profile. Comprehensive genomic analyses were performed at diagnosis, post-chemotherapy and post-transplant. A total of 8 mutations (RUNX1, GATA2, DNMT3A, BCORL1, BCOR, 2 PHF6 and CDKN2A) were identified in the pre-induction sample, of which 5 remained (RUNX1, DNMT3A, BCORL1, BCOR and 1 out of 2 PHF6) in the post-treatment sample and none were present post-transplant. In brief, the 3 mutations which were lost along with the leukemic cells at complete morphological remission were most likely acquired leukemic driver mutations that were responsible for the AML transformation from a pre-leukemic germline RUNX1-mutated state. On the contrary, the 5 mutations that persisted post-treatment, including the germline RUNX1 mutation, were likely to be part of the preleukemic clone. CONCLUSION: Further studies are necessary to assess the prevalence of these preleukemic and secondary mutations in the larger FPD/AML patient cohort and establish their prognostic significance. Given the molecular heterogeneity of FPD/AML and other AML subtypes, a better understanding of mutational classes and their involvement in AML pathogenesis can improve risk stratification of patients for more effective and targeted therapy.

Evaluation of the Luminex ARIES HSV 1&2 Assay and Comparison with the FTD Neuro 9 and In-house Real-Time PCR Assays for Detecting Herpes Simplex Viruses.

BACKGROUND: Human herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) are responsible for a plethora of human diseases, of which cutaneous and mucocutaneous infections are the most prevalent. In its most severe form, HSV infection can cause meningitis/encephalitis. We compared the Luminex ARIES HSV 1&2 assay (Luminex Corp., Austin, TX, USA), an automated sample-to-result molecular solution, to two non-automated HSV DNA assays. METHODS: A total of 116 artificial controls were used to determine the analytical performance of the ARIES assay. Controls were prepared by spiking universal transport medium (UTM) and cerebrospinal fluid (CSF) samples from patients who tested negative for HSV by an in-house HSV-1 and -2 DNA assay with reference materials (SeraCare Life Sciences, MA, USA; ZeptoMetrix Corp., MA, USA). Another 117 clinical samples were then used to compare the clinical performance of the ARIES assay with those of an in-house assay and the FTD Neuro 9 assay (Fast Track Diagnostics, Junglinster, Luxembourg). RESULTS: The analytical sensitivity (95% limit of detection) of the ARIES assay was 318 copies/mL (UTM samples) and 935 copies/mL (CSF samples) for HSV-1 strain 96 and 253 copies/mL (UTM samples) and 821 copies/mL (CSF samples) for HSV-2 strain 09. No cross-reactivity was observed in samples spiked with 14 non-HSV microorganisms. Compared with the reference result (agreement between the in-house and FTD Neuro 9 results), the ARIES assay had overall concordance rates of 98.2% (111/113) and 100% (113/113) for HSV-1 and HSV-2, respectively. CONCLUSIONS: The ARIES assay appears to be an excellent alternative for rapid detection and differentiation of HSV in skin and genital infections, meningitis, and encephalitis.

Identification of novel fusion transcripts in multiple myeloma.

AIMS: Multiple myeloma (MM) is a heterogeneous disease characterised by genetically complex abnormalities. The classical mutational spectrum includes recurrent chromosomal aberrations and gene-level mutations. Recurrent translocations involving the IGH gene such as t(11;14), t(4;14) and t(14;16) are well known. However, the presence of complex genetic abnormalities raises the possibility that fusions other than the recurrent IGH translocations exist. We therefore employed a targeted RNA-sequencing panel to identify novel putative fusions in a local cohort of MM. METHODS: Targeted RNA-sequencing was performed on 21 patient samples using the Illumina TruSight RNA Pan-Cancer Panel (comprising 1385 genes). Fusion calls were generated from the Illumina RNA-Sequencing Alignment software (V.1.0.0). These samples had conventional cytogenetic and fluorescence in situ hybridisation data for the common recurrent chromosomal abnormalities (t(11;14), t(4;14), t(14;16) and 17p13 deletion). The MMRF CoMMpass dataset was analysed using the TopHat-fusion pipeline. RESULTS: A total of 10 novel fusions were identified by the TruSight RNA Pan-Cancer Panel. Two of these fusions, HGF/CACNA2D1 and SMC3/MXI1, were validated by reverse transcription PCR and Sanger sequencing as they involve genes that may have biological relevance in MM genesis. Four of these (MAP2K4/MAP2K4P1) are likely to be spurious secondary to misalignment of reads to a pseudogene. One record of the HGF/CACNA2D1 fusion was identified from the MMRF CoMMpass dataset. CONCLUSIONS: The identification of novel fusions offers insights into the biology of MM and might have clinical relevance. Further functional studies are required to determine the biological and clinical relevance of these novel fusions.

Comparison of cobas HCV GT against Versant HCV Genotype 2.0 (LiPA) with confirmation by Sanger sequencing.

BACKGROUND: Correct identification of infecting hepatitis C virus (HCV) genotype is helpful for targeted antiviral therapy. OBJECTIVES: Here, we compared the HCV genotyping performance of the cobas HCV GT assay against the Versant HCV Genotype 2.0 (LiPA) assay, using 97 archived serum samples. STUDY DESIGN: In the event of discrepant or indeterminate results produced by either assay, the core and NS5B regions were sequenced. RESULTS: Of the 97 samples tested by the cobas, 25 (26%) were deemed indeterminate. Sequencing analyses confirmed 21 (84%) of the 25 samples as genotype 6 viruses with either subtype 6m, 6n, 6v, 6xa, or unknown subtype. Of the 97 samples tested by the LiPA, thirteen (13%) were deemed indeterminate. Seven (7%) were assigned with genotype 1, with unavailable/inconclusive results from the core region of the LiPA. Notably, the 7 samples were later found to be either genotype 3 or 6 by sequencing analyses. Moreover, 1 sample by the LiPA was assigned as genotypes 4 (cobas: indeterminate) but were later found to be genotype 3 by sequencing analyses, highlighting its limitation in assigning the correct genotype. CONCLUSIONS: The cobas showed similar or slightly higher accuracy (100%; 95% CI 94-100%) compared to the LiPA (99%; 95% CI 92-100%). Twenty-six percent of the 97 samples tested by the cobas had indeterminate results, mainly due to its limitation in identifying genotype 6 other than subtypes 6a and 6b. This presents a significant assay limitation in Southeast Asia, where genotype 6 infection is highly prevalent.

CEBPA mutational analysis in acute myeloid leukaemia by a laboratory-developed next-generation sequencing assay.

AIM: The presence of biallelic CEBPA mutations is a favourable prognostic feature in acute myeloid leukaemia (AML). CEBPA mutations are currently identified through conventional capillary sequencing (CCS). With the increasing adoption of next-generation sequencing (NGS) platforms, challenges with regard to amplification efficiency of CEBPA due to the high GC content may be encountered, potentially resulting in suboptimal coverage. Here, the performance of an amplicon-based NGS method using a laboratory-developed CEBPA-specific Nextera XT (CEBNX) was evaluated. METHODS: Mutational analyses of the CEBPA gene of 137 AML bone marrow or peripheral blood retrospective specimens were performed by the amplification of the CEBPA gene using the Expand Long Range dNTPack and the amplicons processed by CCS and NGS. CEBPA-specific libraries were then constructed using the Nextera XT V.2 kit. All FASTQ files were then processed with the MiSeq Reporter V.2.6.2.3 using the PCR Amplicon workflow via the customised CEBPA-specific manifest file. The variant calling format files were analysed using the Illumina Variant Studio V.2.2. RESULTS: A coverage per base of 3631X to 28184X was achieved. 22 samples (16.1%) were found to contain CEBPA mutations, with variant allele frequencies (VAF) ranging from 3.8% to 58.2%. Taking CCS as the 'gold standard', sensitivity and specificity of 97% and 97% was achieved. For the transactivation domain 2 polymorphism (c.584_589dupACCCGC/p.His195_Pro196dup), the CEBNX achieved 100% sensitivity and 100% specificity relative to CCS. CONCLUSIONS: Our laboratory-developed CEBNX workflow shows high coverage and thus overcomes the challenges associated with amplification efficiency and low coverage of CEBPA. Therefore, our assay is suitable for deployment in the clinical laboratory.

Performance evaluation of Cepheid Xpert Norovirus kit with a user-modified protocol.

The Cepheid Xpert® Norovirus kit automates sample processing, nucleic acid extraction, and real-time reverse transcription polymerase chain reactions (RT-PCRs) to detect norovirus genogroups I (GI) and II (GII). Eighty-five stool samples collected between February 2015 and May 2017 were used to compare the performance of a user-modified Xpert assay against a clinically validated laboratory-developed test (LDT). Of the 85 samples, 54 were previously archived in -80°C freezer. The remaining 31 were fresh samples tested concurrently with the LDT. The results of all samples tested using the Xpert kit and LDT were found to be concordant, including 12 GI- and 42 GII-positive samples, 1 GI and GII coinfection, and 30 negative samples. Comparison of the assays showed perfect concordance with a kappa coefficient score of 1.00 (95%CI from 1.00 to 1.00). Of the 30 negative stool samples tested, three samples were positive for rotavirus detected using an immunochromatographic assay, with no cross-reactivity shown in both LDT and Xpert assays. In-run sample processing control of the Xpert assay for all negative samples tested showed no/minor inhibition. Compared to the LDT, the Xpert assay produced similar or better Ct values for detection. It also showed better mitigation of PCR inhibition in stool sample testing.

Identifying large indels in targeted next generation sequencing assays for myeloid neoplasms: a cautionary tale of the ZRSR1 pseudogene.

Targeted next generation sequencing platforms have been increasingly utilised for identification of novel mutations in myeloid neoplasms, such as acute myeloid leukaemia (AML), and hold great promise for use in routine clinical diagnostics. In this study, we evaluated the utility of an open source variant caller in detecting large indels in a targeted sequencing of AML samples. While we found that this bioinformatics pipeline has the potential to accurately capture large indels (>20 bp) in patient samples, we highlighted the pitfall of a confounding ZRSR1 pseudogene that led to an erroneous ZRSR2 variant call. We further discuss possible clinical implications of the ZRSR1 pseudogene in myeloid neoplasms based on its molecular features. Knowledge of the confounding ZRSR1 pseudogene in ZRSR2 sequencing assays could be particularly important in AML diagnostics because the detection of ZRSR2 in AML patients is highly specific for an s-AML diagnosis.

Single-cell genomic profiling of acute myeloid leukemia for clinical use: A pilot study.

Although bulk high-throughput genomic profiling studies have led to a significant increase in the understanding of cancer biology, there is increasing awareness that bulk profiling approaches do not completely elucidate tumor heterogeneity. Single-cell genomic profiling enables the distinction of tumor heterogeneity, and may improve clinical diagnosis through the identification and characterization of putative subclonal populations. In the present study, the challenges associated with a single-cell genomics profiling workflow for clinical diagnostics were investigated. Single-cell RNA-sequencing (RNA-seq) was performed on 20 cells from an acute myeloid leukemia bone marrow sample. Putative blasts were identified based on their gene expression profiles and principal component analysis was performed to identify outlier cells. Variant calling was performed on the single-cell RNA-seq data. The present pilot study demonstrates a proof of concept for clinical single-cell genomic profiling. The recognized limitations include significant stochastic RNA loss and the relatively low throughput of the current proposed platform. Although the results of the present study are promising, further technological advances and protocol optimization are necessary for single-cell genomic profiling to be clinically viable.

Comparison of Luminex NxTAG Respiratory Pathogen Panel and xTAG Respiratory Viral Panel FAST Version 2 for the Detection of Respiratory Viruses.

Owing to advancements in molecular diagnostics, recent years have seen an increasing number of laboratories adopting respiratory viral panels to detect respiratory pathogens. In December 2015, the NxTAG respiratory pathogen panel (NxTAG RPP) was approved by the United States Food and Drug Administration. We compared the clinical performance of this new assay with that of the xTAG respiratory viral panel (xTAG RVP) FAST v2 using 142 clinical samples and 12 external quality assessment samples. Discordant results were resolved by using a laboratory-developed respiratory viral panel. The NxTAG RPP achieved 100% concordant negative results and 86.6% concordant positive results. It detected one coronavirus 229E and eight influenza A/H3N2 viruses that were missed by the xTAG RVP FAST v2. On the other hand, the NxTAG RPP missed one enterovirus/rhinovirus and one metapneumovirus that were detected by FAST v2. Both panels correctly identified all the pathogens in the 12 external quality assessment samples. Overall, the NxTAG RPP demonstrated good diagnostic performance. Of note, it was better able to subtype the influenza A/H3N2 viruses compared with the xTAG RVP FAST v2.

Clinical implications of DNMT3A mutations in a Southeast Asian cohort of acute myeloid leukaemia patients.

AIMS: In recent years, genomic technologies have enabled the identification of mutations in acute myeloid leukaemia (AML). DNMT3A is a recurrently mutated epigenetic modifier gene in AML. To date, the prognostic significance of DNMT3A mutations has not been studied in a Southeast Asian AML population. We sought to investigate the clinical implications of DNMT3A mutations in a Southeast Asian cohort of AML patients. METHODS: DNMT3A mutations were identified using a targeted next-generation sequencing panel in 157 AML patients. We studied the molecular and clinical features of patients with DNMT3A mutations and assessed the prognostic impact of DNMT3A mutations. RESULTS: DNMT3A mutations were found in 33 of 157 (21.0%) AML patients. 114 patients were included for statistical analysis. Pretreatment data revealed that patients with DNMT3A mutations were older (≥60 years old), had a higher white blood cell count at diagnosis, had more adverse cytogenetic risk profiles and were more often associated with NPM1 mutations compared with patients with wild-type DNMT3A. Survival analysis showed that DNMT3A mutations were associated with poorer clinical outcomes. This was especially when associated with NPM1 and FLT3-ITD mutations (AML NPM1/FLT3/DNMT3A ), which are common. The AML NPM1/FLT3/DNMT3A subtype was an independent predictor for poorer overall survival (OS). Other independent risk factors for poorer OS include advanced age at diagnosis and adverse cytogenetic risk stratification. CONCLUSIONS: DNMT3A mutations are associated with an unfavourable clinical outcome in our Southeast Asian AML patient cohort. In particular, AML NPM1/FLT3/DNMT3A patients had the poorest prognosis.