Optimization of magnetic bead-based nucleic acid extraction for SARS-CoV-2 testing using readily available reagents.

The COVID-19 pandemic has highlighted the need for generic reagents and flexible systems in diagnostic testing. Magnetic bead-based nucleic acid extraction protocols using 96-well plates on open liquid handlers are readily amenable to meet this need. Here, one such approach is rigorously optimized to minimize cross-well contamination while maintaining sensitivity.

The contrasting role of nasopharyngeal angiotensin converting enzyme 2 (ACE2) transcription in SARS-CoV-2 infection: A cross-sectional study of people tested for COVID-19 in British Columbia, Canada.

BACKGROUND: Angiotensin converting enzyme 2 (ACE2) protein serves as the host receptor for SARS-CoV-2, with a critical role in viral infection. We aim to understand population level variation of nasopharyngeal ACE2 transcription in people tested for COVID-19 and the relationship between ACE2 transcription and SARS-CoV-2 viral load, while adjusting for expression of: (i) the complementary protease, Transmembrane serine protease 2 (TMPRSS2), (ii) soluble ACE2, (iii) age, and (iv) biological sex. The ACE2 gene was targeted to measure expression of transmembrane and soluble transcripts. METHODS: A cross-sectional study of n = 424 "participants" aged 1-104 years referred for COVID-19 testing was performed in British Columbia, Canada. Patients who tested positive for COVID-19 were matched by age and biological sex to patients who tested negative. Viral load and host gene expression were assessed by quantitative reverse-transcriptase polymerase chain reaction. Bivariate analysis and multiple linear regression were performed to understand the role of nasopharyngeal ACE2 expression in SARS-CoV-2 infection. FINDINGS: Analysis showed no association between age and nasopharyngeal ACE2 transcription in those who tested negative for COVID-19 (P = 0•092). Mean relative transcription of transmembrane (P = 0•00012) and soluble (P<0•0001) ACE2 isoforms, as well as TMPRSS2 (P<0•0001) was higher in COVID-19-negative participants than COVID--19 positive ones, yielding a negative correlation between targeted host gene expression and positive COVID-19 diagnosis. In bivariate analysis of COVID-19-positive participants, transcription of transmembrane ACE2 positively correlated with SARS-CoV-2 viral RNA load (B = 0•49, R2=0•14, P<0•0001), transcription of soluble ACE2 negatively correlated (B= -0•85, R2= 0•26, P<0•0001), and no correlation was found with TMPRSS2 transcription (B= -0•042, R2=<0•10, P = 0•69). Multivariable analysis showed that the greatest viral RNA loads were observed in participants with high transmembrane ACE2 transcription (Β= 0•89, 95%CI: [0•59 to 1•18]), while transcription of the soluble isoform appears to protect against high viral RNA load in the upper respiratory tract (Β= -0•099, 95%CI: [-0•18 to -0•022]). INTERPRETATION: Nasopharyngeal ACE2 transcription plays a dual, contrasting role in SARS-CoV-2 infection of the upper respiratory tract. Transcription of the transmembrane ACE2 isoform positively correlates, while transcription of the soluble isoform negatively correlates with viral RNA load after adjusting for age, biological sex, and transcription of TMPRSS2. FUNDING: This project (COV-55) was funded by Genome British Columbia as part of their COVID-19 rapid response initiative.

Predictors of sustained research involvement among MD/PhD programme graduates.

CONTEXT: MD/PhD programmes provide structured paths for physician-scientist training. However, considerable proportions of graduates of these programmes do not pursue careers in research consistent with their training. OBJECTIVES: We sought to identify factors associated with sustained involvement in research after completion of all postgraduate training. METHODS: Anonymised data from a national survey of Canadian MD/PhD programme graduates who had completed all physician-scientist training (n = 70) were analysed. Multivariable logistic regression was used to measure the associations between characteristics of graduates and five indicators of sustained research involvement following postgraduate training: (i) protected research time in the current appointment; (ii) percentage of time dedicated to research; (iii) planned future involvement in research; (iv) role as a principal investigator on a recent funded project, and (v) receipt of funding from a federal granting agency since graduation. RESULTS: The majority of graduates were significantly involved in research on the basis of at least one outcome. Completion of a research fellowship, number of first-authored or co-authored manuscripts published during MD/PhD training, and duration of MD/PhD training were positively associated with continued research involvement. Completion of a Masters degree prior to MD/PhD training, female gender, debt greater than CAD$50 000 at completion of training, and pursuit of a clinical specialty other than internal medicine, paediatrics, neurology, pathology and the surgical specialties were negatively associated with sustained research involvement. CONCLUSIONS: Most MD/PhD programme graduates remain significantly involved in research, but this involvement often does not correspond to traditional physician-scientist roles, in which a majority of time is dedicated to research. To minimise loss of investment in physician-scientist training, MD/PhD programmes should prioritise research productivity during training and the pursuit of additional research training during residency, and policymakers should establish stable sources of funding to reduce debt among graduates. Our data suggest further study is warranted to identify interventions to reduce attrition among female MD/PhD programme graduates.

The pathobiology of primary testicular diffuse large B-cell lymphoma: Implications for novel therapies.

Primary testicular lymphomas (PTL) are the most prevalent type of testicular cancer arising in men over the age of 60. PTL accounts for approximately 1-2% of all non-Hodgkin lymphomas and most present with localized disease but despite this, outcome is poor. The majority of cases represent an extranodal manifestation of diffuse large B-cell lymphoma (DLBCL), known as primary testicular DLBCL (PT-DLBCL). Gene expression profiling has established that over 75% of PT-DLBCLs resemble the activated B-cell-like (ABC) or non-germinal center subtype of nodal DLBCL. In distilling the specific mutational landscape and immunophenotypic profiles, immune-escape and sustained signalling emerge as prominent features of PT-DLBCL. These include genomic alterations arising within the core components of antigen presentation (CIITA, B2M, and HLA loci) and structural rearrangements of programmed death ligands 1 (CD274) and 2 (PDCD1LG2). Enrichment for somatic mutations within NF-κB pathway genes (MYD88, CD79B, NFKBIZ, BCL10, and MALT1) also feature prominently in PT-DLBCL. Taken together, the unique molecular and clinical characteristics of PT-DLBCL have informed on aspects of the distinct disease biology of this organotypic lymphoma that may guide rational therapeutic strategies.

Cross-sectional-derived determinants of satisfaction with physician-scientist training among Canadian MD/PhD graduates.

Although MD/PhD programs require considerable commitment on behalf of students and learning institutions, they serve as an integral means of training future physician-scientists; individuals who engage in translational medicine. As attrition from these programs has longstanding effects on the community of translational medicine and comes at substantial cost to MD/PhD programs, we aimed to identify determinants that were associated with satisfaction among MD/PhD graduates, a feature that might inform on limiting program attrition. Anonymized data from a national survey of 139 Canadian MD/PhD alumni was analyzed. Factor analysis was conducted to evaluate the reliability of three questions that measured satisfaction and logistic regression was used to assess the association of outcomes with 17 independent determinants. Eighty-one percent of graduates were satisfied with MD/PhD training. Factor analysis confirmed the reliability of the questions measuring satisfaction. Determinants of self-reported satisfaction with physician-scientist training included co-authorship of more than six manuscripts during MD/PhD training. Additionally, protected research time at the place of current appointment was strongly associated with agreement that MD/PhD training had helped career progression. Demographic variables were not associated with any satisfaction indicator. Taken together, the majority of Canadian MD/PhD graduates are satisfied with their physician-scientist training. Project collaboration leading to co-authorships and protected research time were strongly associated with training satisfaction among graduates. If the value of collaboration can be realized among current and future physician-scientist trainees who are dissatisfied with their training, this might ultimately reduce program attrition.

Characteristics and outcomes of Canadian MD/PhD program graduates: a cross-sectional survey.

BACKGROUND: Combined MD/PhD programs provide a structured path for physician-scientist training, but assessment of their success within Canada is limited by a lack of quantitative data. We collected outcomes data for graduates of Canadian MD/PhD programs. METHODS: We developed and implemented a Web-based survey consisting of 41 questions designed to collect outcomes data for Canadian MD/PhD program alumni from 8 Canadian universities who had graduated before September 2015. Respondents were categorized into 2 groups according to whether they had or had not completed all training. RESULTS: Of the 186 eligible alumni of MD/PhD programs, 139 (74.7%) completed the survey. A total of 136/138 respondents (98.6%) had completed or were currently completing residency training, and 66/80 (82%) had completed at least 1 postgraduate fellowship. Most (58 [83%]) of the 70 respondents who had completed all training were appointed as faculty at academic institutions, and 37 (53%) had been principal investigators on at least 1 recent funded project. Among the 58 respondents appointed at academic institutions, 44/57 (77%) dedicated at least 20% of their time to research, and 25/57 (44%) dedicated at least 50% to research. During their combined degree, 102/136 respondents (75.0%) published 3 or more first-author papers, and 133/136 (97.8%) matched with their first choice of specialty. The median length of physician-scientist training was 13.5 years. Most respondents graduated with debt despite having been supported by Canadian Institutes of Health Research MD/PhD studentships. INTERPRETATION: Most Canadian MD/PhD program alumni pursued careers consistent with their physician-scientist training, which indicates that these programs are meeting their primary objective. Nevertheless, our findings highlight that a minority of these positions are research intensive; this finding warrants further study. Our data provide a baseline for future monitoring of the output of Canadian MD/PhD programs.

Comprehensive characterization of programmed death ligand structural rearrangements in B-cell non-Hodgkin lymphomas.

Programmed death ligands (PDLs) are immune-regulatory molecules that are frequently affected by chromosomal alterations in B-cell lymphomas. Although PDL copy-number variations are well characterized, a detailed and comprehensive analysis of structural rearrangements (SRs) and associated phenotypic consequences is largely lacking. Here, we used oligonucleotide capture sequencing of 67 formalin-fixed paraffin-embedded tissues derived from primary B-cell lymphomas and 1 cell line to detect and characterize, at base-pair resolution, SRs of the PDL locus (9p24.1; harboring PDL1/CD274 and PDL2/PDCD1LG2). We describe 36 novel PDL SRs, including 17 intrachromosomal events (inversions, duplications, deletions) and 19 translocations involving BZRAP-AS1, CD44, GET4, IL4R, KIAA0226L, MID1, RCC1, PTPN1 and segments of the immunoglobulin loci. Moreover, analysis of the precise chromosomal breakpoints reveals 2 distinct cluster breakpoint regions (CBRs) within either CD274 (CBR1) or PDCD1LG2 (CBR2). To determine the phenotypic consequences of these SRs, we performed immunohistochemistry for CD274 and PDCD1LG2 on primary pretreatment biopsies and found that PDL SRs are significantly associated with PDL protein expression. Finally, stable ectopic expression of wild-type PDCD1LG2 and the PDCD1LG2-IGHV7-81 fusion showed, in coculture, significantly reduced T-cell activation. Taken together, our data demonstrate the complementary utility of fluorescence in situ hybridization and capture sequencing approaches and provide a classification scheme for PDL SRs with implications for future studies using PDL immune-checkpoint inhibitors in B-cell lymphomas.

The Canadian clinician-scientist training program must be reinstated.

Clinical investigators within the Canadian and international communities were shocked when the Canadian Institutes of Health Research (CIHR) announced that their funding for the MD/PhD program would be terminated after the 2015-2016 academic year. The program has trained Canadian clinician-scientists for more than two decades. The cancellation of the program is at odds with the CIHR's mandate, which stresses the translation of new knowledge into improved health for Canadians, as well as with a series of internal reports that have recommended expanding the program. Although substantial evidence supports the analogous Medical Scientist Training Program in the United States, no parallel analysis of the MD/PhD program has been performed in Canada. Here, we highlight the long-term consequences of the program's cancellation in the context of increased emphasis on translational research. We argue that alternative funding sources cannot ensure continuous support for students in clinician-scientist training programs and that platform funding of the MD/PhD program is necessary to ensure leadership in translational research.

Recurrent genomic rearrangements in primary testicular lymphoma.

Primary testicular diffuse large B cell lymphoma (PTL) is an aggressive malignancy that occurs in the immune-privileged anatomical site of the testis. We have previously shown that structural genomic rearrangements involving the MHC class II transactivator CIITA and programmed death ligands (PDLs) 1 and 2 are frequent across multiple B cell lymphoma entities. Specifically in PTL, we found rearrangements in the PDL locus by fluorescence in situ hybridization (FISH). However, breakpoint anatomy and rearrangement partners were undetermined, while CIITA rearrangements had not been reported previously in PTL. Here, we performed bacterial artificial chromosome capture sequencing on three archival, formalin-fixed, paraffin-embedded tissue biopsies, interrogating 20 known rearrangement hotspots in B cell lymphomas. We report novel CIITA, FOXP1 and PDL rearrangements involving IGHG4, FLJ45248, RFX3, SMARCA2 and SNX29. Moreover, we present immunohistochemistry data supporting the association between PDL rearrangements and increased protein expression. Finally, using FISH, we show that CIITA (8/82; 10%) and FOXP1 (5/74; 7%) rearrangements are recurrent in PTL. In summary, we describe rearrangement frequencies and novel rearrangement partners of the CIITA, FOXP1 and PDL loci at base-pair resolution in a rare, aggressive lymphoma. Our data suggest immune-checkpoint inhibitor therapy as a promising intervention for PTL patients harbouring PDL rearrangements.

Structural genomic alterations in primary mediastinal large B-cell lymphoma.

Primary mediastinal large B-cell lymphoma (PMBCL) is an aggressive non-Hodgkin lymphoma that displays phenotypic and genotypic similarity to Hodgkin lymphoma and diffuse large B-cell lymphoma. Studies using genome-wide discovery tools have revealed specific, recurrent structural aberrations as critical somatic events in the pathogenesis of PMBCL. These structural alterations prominently include transcript and protein altering rearrangements and copy number variations of the programmed death ligands 1 (CD274) and 2 (PDCD1LG2), CIITA, JAK2 and REL. Importantly, evidence is emerging that these acquired structural genomic changes, in synergy with other somatic alterations, contribute to PMBCL pathogenesis by influencing tumor microenvironment interactions that favor malignant B-cell growth. The means by which these rearrangements arise are not well understood. However, analysis of breakpoint junctions at base-pair resolution provides preliminary insight into putative rearrangement mechanisms. As the field also anticipates predictive value and therapeutic targeting of structural changes involving programmed death ligands and JAK2, a review of therapies that will likely shape future lymphoma treatment is needed.

Genomic rearrangements involving programmed death ligands are recurrent in primary mediastinal large B-cell lymphoma.

The pathogenesis of primary mediastinal large B-cell lymphoma (PMBCL) is incompletely understood. Recently, specific genotypic and phenotypic features have been linked to tumor cell immune escape mechanisms in PMBCL. We studied 571 B-cell lymphomas with a focus on PMBCL. Using fluorescence in situ hybridization here, we report that the programmed death ligand (PDL) locus (9p24.1) is frequently and specifically rearranged in PMBCL (20%) as compared with diffuse large B-cell lymphoma, follicular lymphoma, and Hodgkin lymphoma. Rearrangement was significantly correlated with overexpression of PDL transcripts. Utilizing high-throughput sequencing techniques, we characterized novel translocations and chimeric fusion transcripts involving PDLs at base-pair resolution. Our data suggest that recurrent genomic rearrangement events underlie an immune privilege phenotype in a subset of B-cell lymphomas.