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Background: Many patients with non-small cell lung cancer (NSCLC) lack access to highly effective approved targeted therapeutics due to multiple gaps in biomarker testing. Challenges in comprehensive molecular testing include complexities associated with the need to assess the presence of multiple variants, costs of running multiple sequential assays per sample, high assay quality control (QC) failure rates, clinical need for rapid turn-around time (TAT) to initiate therapy, and insufficient tissue samples. The ASPYRE-Lung NSCLC assay addresses gaps in multiplexed testing by simultaneously analyzing DNA and RNA, detecting 114 actionable genomic variants across 11 genes, consistent with current NSCLC treatment guidelines. This study was to assess the ease of adoption and performance of ASPYRE-Lung in third-party laboratories, comparing concordance across sites and with orthogonal methods., Methods: ASPYRE-Lung was established at two academic centers with multiple operators per site. Assay concordance was evaluated across three sites using 77 patient samples [61 derived from formalin-fixed paraffin-embedded (FFPE) tissue and 16 from cytology specimens]., Results: Reproducibility for all 77 samples yielded a positive percent agreement (PPA) of 100% and negative percent agreement (NPA) of 99.99%. Concordance with next-generation sequencing (NGS)-based methods across all three sites was high with PPA of 97.2% and NPA of 99.96%., Conclusions: ASPYRE-Lung assay is a cost-effective, easy to adopt testing method requiring no specialized expertise or complicated bioinformatics, with the potential to inform genomic data on small tissue samples, thus enabling all patients with NSCLC to undergo biomarker testing in a timely manner and benefit from appropriate targeted therapies., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-24-525/coif). All authors report that this work was supported by Biofidelity Ltd., UK. S.E.H. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. She served as technical consultant for potential investors at Biofidelity (Oct 2023 to Jan 2024). She received honoraria and travel expenses from ThermoFisher to present on NSCLC at a corporate workshop at AMP 2024. C.G. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. S.J.S. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. B.A.S. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. K.A.S. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. K.F.S. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. J.M.M. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. J.M.M. is an employee of Biofidelity Ltd. and has financial interest including salary, stocks, and stock options. R.N.P. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. R.N.P. is an employee of Biofidelity Ltd. and has financial interest including salary, stocks, and stock options. C.X. reports that Biofidelity provided all materials (reagents, consumables, travel expenses to laboratories) to complete the work. C.X. is an employee of Biofidelity Ltd. and has financial interest including salary, stock, and stock options. E.G.Z. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. E.G.Z. is an employee of Biofidelity Inc. and has financial interest including salary, stock, and stock options. C.K. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. C.K. is an employee of Biofidelity Inc. and has financial interest including salary, stock, and stock options. R.T.E. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. R.T.E. is an employee of Biofidelity Inc. and has financial interest including salary, stock and stock options. A.S.G. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. A.S.G. is an employee of Biofidelity Inc. and has financial interest including salary, stock, and stock options. A.L.S. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. A.L.S. is an employee of Biofidelity Inc. or Biofidelity Ltd. and has financial interest including salary, intellectual property, stock, and stock options. M.S.J. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. M.S.J. is an employee of Biofidelity Ltd. and has financial interest including salary, intellectual property, stock, and stock options. K.v.B. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. K.v.B. is an employee of Biofidelity Ltd. and has financial interest including salary, stock, and stock options. I.T. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. I.T. is an employee of Biofidelity Ltd. and has financial interest including salary, stock, and stock options. C.H.H. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. C.H.H. is an employee of Biofidelity Ltd. and has financial interest including salary, stock, and stock options. A. Collazos reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. A. Collazos is an employee of Biofidelity Ltd. and has financial interest including salary, stock, and stock options. N.D.P. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. N.D.P. is an employee of Biofidelity Ltd. and has financial interest including salary, stock, and stock options. D.N. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. D.N. is an employee of Biofidelity Ltd. and has financial interest including salary, stock, and stock options. J.J. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. J.J. is an employee of Biofidelity Ltd. and has financial interest including salary, stock, and stock options. E.R.G. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. E.R.G. is an employee of Biofidelity Ltd. and has financial interest including salary, stock, and stock options. E.S. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. E.S. is an employee of Biofidelity Inc. and has financial interest including salary, stock, and stock options. E.S. is a former employee of Adaptive Biotechnologies, which have no interests in the work presented herein. W.J.L. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. WJL. is a consultant of Biofidelity Inc. and has financial interest including consultancy fees and work-related travel to AACR 2024 where data from this work was presented. A. Cooke reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. A. Cooke was an employee of Biofidelity Ltd. and has financial interest including salary and stock. B.W.B. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. B.W.B. reports that Biofidelity Ltd. has provided travel expenses for work-related travel to AACR 2024 where data from this work was presented. B.W.B. is an employee of Biofidelity Ltd. and has financial interest including salary, intellectual property, stock, and stock options. R.J.O. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. R.J.O. was an employee of Biofidelity Ltd., and has financial interest including salary, stock, and stock options. H.V.R. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. H.V.R. was a consultant of Biofidelity Inc. and had financial interest including consultancy fees. H.V.R. is a current employee of Belay Diagnostics, which have no interests in the work presented herein. V.M.V.D. reports that Biofidelity provided all materials (reagents, consumables, etc.) to complete the work. The authors have no other conflicts of interest to declare., (2024 AME Publishing Company. All rights reserved.)

Introduction: Genomic variant testing of tumors is a critical gateway for patients to access the full potential of personalized oncology therapeutics. Current methods such as next-generation sequencing are costly and challenging to interpret, while PCR assays are limited in the number of variants they can cover. We developed ASPYRE ® (Allele-Specific PYrophosphorolysis REaction) technology to address the urgent need for rapid, accessible and affordable diagnostics informing actionable genomic target variants of a given cancer. The targeted ASPYRE-Lung panel for non-small cell carcinoma covers 114 variants in 11 genes ( ALK, BRAF, EGFR, ERBB2, KRAS, RET, ROS1, MET & NTRK1/2/3 ) to robustly inform clinical management. The assay detects single nucleotide variants, insertions, deletions, and gene fusions from tissue-derived DNA and RNA simultaneously., Methods: We tested the limit of detection, specificity, analytical accuracy and analytical precision of ASPYRE-Lung using FFPE lung tissue samples from patients with non-small cell lung carcinoma, variant-negative FFPE tissue from healthy donors, and FFPE-based contrived samples with controllable variant allele fractions., Results: The sensitivity of ASPYRE-Lung was determined to be ≤ 3% variant allele fraction for single nucleotide variants and insertions or deletions, 100 copies for fusions, and 200 copies for MET exon 14 skipping. The specificity was 100% with no false positive results. The analytical accuracy test yielded no discordant calls between ASPYRE-Lung and expected results for clinical samples (via orthogonal testing) or contrived samples, and results were replicable across operators, reagent lots, runs, and real-time PCR instruments with a high degree of precision., Conclusions: The technology is simple and fast, requiring only four reagent transfer steps using standard laboratory equipment (PCR and qPCR instruments) with analysis via a cloud-based algorithm. The ASPYRE-Lung assay has the potential to be transformative in facilitating access to rapid, actionable molecular profiling of tissue for patients with non-small cell carcinoma., Competing Interests: Authors RE, EG-Z, JB, KK, CK, AG, and BB are employed by the company Biofidelity Inc. Authors A-LS, JM, RP, AT, AC, CX, IT, CH, DN, JJ, SA, NP, KB, EG, MS-J, AC and RO are employees of Biofidelity Ltd, a privately held company. All authors may hold stock or stock options. Author HR was a consultant of Biofidelity Inc at the time of the study. Biofidelity has filed patent applications on aspects of this research WO2021130494A1. The author HR declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision. The authors declare that this study received funding from Biofidelity Ltd. The following had the following involvement in the study: study design, data collection, analysis, decision to publish, and preparation of the manuscript., (Copyright © 2024 Evans, Gillon-Zhang, Brown, Knudsen, King, Green, Silva, Mordaka, Palmer, Tomassini, Collazos, Xyrafaki, Turner, Ho, Nugent, Jose, Andreazza, Potts, von Bargen, Gray, Stolarek-Januszkiewicz, Cooke, Reddi, Balmforth and Osborne.)

Background: RNA is a critical analyte for unambiguous detection of actionable mutations used to guide treatment decisions in oncology. Currently available methods for gene fusion detection include molecular or antibody-based assays, which suffer from either being limited to single-gene targeting, lack of sensitivity, or long turnaround time. The sensitivity and predictive value of next generation sequencing DNA-based assays to detect fusions by sequencing intronic regions is variable, due to the extensive size of introns. The required depth of sequencing and input nucleic acid required can be prohibitive; in addition it is not certain that predicted gene fusions are actually expressed., Results: Herein we describe a method based on pyrophosphorolysis to include detection of gene fusions from RNA, with identical assay steps and conditions to detect somatic mutations in DNA [1], permitting concurrent assessment of DNA and RNA in a single instrument run., Conclusion: The limit of detection was under 6 molecules/ 6 µL target volume. The workflow and instrumentation required are akin to PCR assays, and the entire assay from extracted nucleic acid to sample analysis can be completed within a single day., (© 2022. The Author(s).)

The lymphocyte genome is prone to many threats, including programmed mutation during differentiation 1 , antigen-driven proliferation and residency in diverse microenvironments. Here, after developing protocols for expansion of single-cell lymphocyte cultures, we sequenced whole genomes from 717 normal naive and memory B and T cells and haematopoietic stem cells. All lymphocyte subsets carried more point mutations and structural variants than haematopoietic stem cells, with higher burdens in memory cells than in naive cells, and with T cells accumulating mutations at a higher rate throughout life. Off-target effects of immunological diversification accounted for approximately half of the additional differentiation-associated mutations in lymphocytes. Memory B cells acquired, on average, 18 off-target mutations genome-wide for every on-target IGHV mutation during the germinal centre reaction. Structural variation was 16-fold higher in lymphocytes than in stem cells, with around 15% of deletions being attributable to off-target recombinase-activating gene activity. DNA damage from ultraviolet light exposure and other sporadic mutational processes generated hundreds to thousands of mutations in some memory cells. The mutation burden and signatures of normal B cells were broadly similar to those seen in many B-cell cancers, suggesting that malignant transformation of lymphocytes arises from the same mutational processes that are active across normal ontogeny. The mutational landscape of normal lymphocytes chronicles the off-target effects of programmed genome engineering during immunological diversification and the consequences of differentiation, proliferation and residency in diverse microenvironments., (© 2022. The Author(s).)

Competing Interests: Competing interests: None declared.

Objectives: Patients with gynecologic malignancies have high rates of post-operative venous thromboembolism. Currently, there is no consensus for peri-operative thromboprophylaxis specific to gynecologic oncology. We aimed to compare rates of symptomatic pulmonary embolus within 30 days post-operatively, and to identify risk factors for pulmonary embolus., Methods: The Division of Gynecologic Oncology at Sunnybrook Health Sciences Centre implemented dual thromboprophylaxis for laparotomies in December 2017. We conducted a prospective study of laparotomies for gynecologic malignancies from December 2017 to October 2018, with comparison to historical cohort from January 2016 to November 2017 using the institutional National Surgical Quality Improvement Program database (NSQIP). Pre-intervention, patients received low molecular weight heparin during admission and extended 28-day prophylaxis was continued at the surgeon's discretion. Post-intervention, all patients received both mechanical thromboprophylaxis with sequential compression devices during admission and 28-day prophylaxis with low molecular weight heparin., Results: There were 371 and 163 laparotomies pre- and post-intervention, respectively. Patient characteristics (age, body mass index, diabetes, smoking, tumor stage), rate of malignant cases, operative blood loss and duration, and length of stay were similar between groups. After implementation, pulmonary emboli rates decreased from 5.1% to 0% (p=0.001). There were more cytoreductive procedures pre-intervention (p≤0.0001) but surgical complexity scores were similar (p=0.82). Univariate analysis revealed that surgery pre-intervention (OR 4.25, 95% CI 1.04 to 17.43, p=0.04), length of stay ≥5 days (OR 11.94, 95% CI 2.65 to 53.92, p=0.002), and operative blood loss ≥500 mL (OR 2.85, 95% CI 1.05 to 7.8, p=0.04) increased risk of pulmonary embolus. On multivariable analysis, surgery pre-intervention remained associated with more pulmonary emboli (OR 4.16, 95% CI 1.03 to 16.79, p=0.045), when adjusting for operative blood loss., Conclusion: Dual thromboprophylaxis after laparotomy significantly reduced rates of pulmonary embolus in this high-risk patient population., Competing Interests: Competing interests: None declared., (© IGCS and ESGO 2022. No commercial re-use. See rights and permissions. Published by BMJ.)

Somatic mutations drive the development of cancer and may contribute to ageing and other diseases 1,2 . Despite their importance, the difficulty of detecting mutations that are only present in single cells or small clones has limited our knowledge of somatic mutagenesis to a minority of tissues. Here, to overcome these limitations, we developed nanorate sequencing (NanoSeq), a duplex sequencing protocol with error rates of less than five errors per billion base pairs in single DNA molecules from cell populations. This rate is two orders of magnitude lower than typical somatic mutation loads, enabling the study of somatic mutations in any tissue independently of clonality. We used this single-molecule sensitivity to study somatic mutations in non-dividing cells across several tissues, comparing stem cells to differentiated cells and studying mutagenesis in the absence of cell division. Differentiated cells in blood and colon displayed remarkably similar mutation loads and signatures to their corresponding stem cells, despite mature blood cells having undergone considerably more divisions. We then characterized the mutational landscape of post-mitotic neurons and polyclonal smooth muscle, confirming that neurons accumulate somatic mutations at a constant rate throughout life without cell division, with similar rates to mitotically active tissues. Together, our results suggest that mutational processes that are independent of cell division are important contributors to somatic mutagenesis. We anticipate that the ability to reliably detect mutations in single DNA molecules could transform our understanding of somatic mutagenesis and enable non-invasive studies on large-scale cohorts.

Introduction: Low risk gestational trophoblastic neoplasia, WHO prognostic score of 0 to 6, is highly curable. There is no consensus on the optimal chemotherapy. Common regimens are q2wk actinomycin-D (ACT-D), weekly intramuscular methotrexate (MTX) or multi-day MTX. Combination MTX/ACT-D is rarely used., Methods: A four centre, retrospective cohort study was carried out comparing commonly used regimens: weekly MTX, q2weekly ACT-D and q2 weekly MTX and ACT-D., Results: 412 patients - 196 MTX/ACT-D, 107 MTX, 109 ACT-D - were treated between October 1994 and January 2019. Initial regimen failure (secondary to resistance or toxicity) occurred in 37% (MTX), 21% (ACT-D) and 5% (MTX/ACT-D). Relapse after completion of primary therapy (initial plus switch to another therapy if needed) was rare (0-5%). All eventually were cured. Mean number of cycles required to achieve remission were 10.1 (MTX), 7 (ACT-D) and 5.6 (MTX/ACT-D) with corresponding mean treatment durations of 3.12, 2.9 and 2.26 months. Dosage reductions occurred in 3% (MTX), 0% (ACT-D) and 29% (MTX/ACT-D). Higher failure rates occurred with WHO prognostic scores of 5 to 6 and HCG levels ≥10,000., Summary: Initial regimen failure ie the need to switch to an alternative treatment was more common with MTX. ACT-D and MTX/ACT-D were similar within prognostic score 0-4 or HCG < 10,000. ACT-D then appears the better initial choice with its superior convenience. Above these levels primary failure rates are less with MTX/ACT-D, making it a better choice., Competing Interests: Conflict of interest statement Hoskins: advisory boards- Astra Zeneca, Pfizer, GSK, Janssen; educational grant Roche Kumar: advisory boards –Roche, Astra Zeneca, Pfizer, Novartis Le, Kim, Pina, Sabourin, Osborne: no conflict of interest, (Copyright © 2020 Elsevier Inc. All rights reserved.)

The evolution and progression of multiple myeloma and its precursors over time is poorly understood. Here, we investigate the landscape and timing of mutational processes shaping multiple myeloma evolution in a large cohort of 89 whole genomes and 973 exomes. We identify eight processes, including a mutational signature caused by exposure to melphalan. Reconstructing the chronological activity of each mutational signature, we estimate that the initial transformation of a germinal center B-cell usually occurred during the first 2 nd -3 rd decades of life. We define four main patterns of activation-induced deaminase (AID) and apolipoprotein B mRNA editing catalytic polypeptide-like (APOBEC) mutagenesis over time, including a subset of patients with evidence of prolonged AID activity during the pre-malignant phase, indicating antigen-responsiveness and germinal center reentry. Our findings provide a framework to study the etiology of multiple myeloma and explore strategies for prevention and early detection.

The colorectal adenoma-carcinoma sequence has provided a paradigmatic framework for understanding the successive somatic genetic changes and consequent clonal expansions that lead to cancer 1 . However, our understanding of the earliest phases of colorectal neoplastic changes-which may occur in morphologically normal tissue-is comparatively limited, as for most cancer types. Here we use whole-genome sequencing to analyse hundreds of normal crypts from 42 individuals. Signatures of multiple mutational processes were revealed; some of these were ubiquitous and continuous, whereas others were only found in some individuals, in some crypts or during certain periods of life. Probable driver mutations were present in around 1% of normal colorectal crypts in middle-aged individuals, indicating that adenomas and carcinomas are rare outcomes of a pervasive process of neoplastic change across morphologically normal colorectal epithelium. Colorectal cancers exhibit substantially increased mutational burdens relative to normal cells. Sequencing normal colorectal cells provides quantitative insights into the genomic and clonal evolution of cancer.

Haematopoietic stem cells drive blood production, but their population size and lifetime dynamics have not been quantified directly in humans. Here we identified 129,582 spontaneous, genome-wide somatic mutations in 140 single-cell-derived haematopoietic stem and progenitor colonies from a healthy 59-year-old man and applied population-genetics approaches to reconstruct clonal dynamics. Cell divisions from early embryogenesis were evident in the phylogenetic tree; all blood cells were derived from a common ancestor that preceded gastrulation. The size of the stem cell population grew steadily in early life, reaching a stable plateau by adolescence. We estimate the numbers of haematopoietic stem cells that are actively making white blood cells at any one time to be in the range of 50,000-200,000. We observed adult haematopoietic stem cell clones that generate multilineage outputs, including granulocytes and B lymphocytes. Harnessing naturally occurring mutations to report the clonal architecture of an organ enables the high-resolution reconstruction of somatic cell dynamics in humans.

Objective: To evaluate the efficacy and safety of second uterine curettage in lieu of chemotherapy for patients with low-risk, nonmetastatic gestational trophoblastic neoplasia (GTN) and to evaluate whether response to second curettage is independent of patient age, World Health Organization (WHO) risk score, registration human chorionic gonadotropin (hCG) level, lesion size, and depth of myometrial invasion measured on ultrasound examination., Methods: This was a cooperative group multicenter prospective phase II study. Prestudy testing included quantitative hCG level, pelvic ultrasonography, and chest radiography. Patients were categorized according to WHO risk scoring criteria (low risk with a score of 0-6)., Results: Sixty-four women with newly diagnosed low-risk, nonmetastatic GTN were enrolled. Four patients were excluded. Twenty-four patients (40%) (lower 95% confidence limit 27.6%) were cured after second curettage. An additional two patients (3%) achieved a complete response but did not complete follow-up. Overall, 26 of 60 patients were able to avoid chemotherapy. Surgical failure was observed in 34 women (59%) and was more common in women 19 years old or younger or 40 years old or older. One case of grade 1 uterine perforation was successfully managed by observation. Four grade 1 and one grade 3 uterine hemorrhages were reported. New metastatic disease (lung) was identified in one of these women after second curettage. In three patients (surgical failures), the second curettage pathology was placental site trophoblastic tumor, and it was placental nodule in one additional patient., Conclusion: Second uterine curettage as initial treatment for low-risk, nonmetastatic GTN cures 40% of patients without significant morbidity., Clinical Trial Registration: ClinicalTrials.gov, https://clinicaltrials.gov/, NCT00521118.

Rationale: Cytomegalovirus (CMV), which is one of the most common infections after lung transplantation, is associated with chronic lung allograft dysfunction and worse post-transplantation survival. Current approaches for at-risk patients include a fixed duration of antiviral prophylaxis despite the associated cost and side effects., Objectives: We sought to identify a specific immunologic signature that predicted protection from subsequent CMV., Methods: CMV-seropositive lung transplantation recipients were included in the discovery (n = 43) and validation (n = 28) cohorts. Polyfunctional CMV-specific immunity was assessed by stimulating peripheral blood mononuclear cells with CMV pp65 or IE-1 peptide pools and then by measuring T-cell expression of CD107a, IFN-γ, tumor necrosis factor-α (TNF-α), and IL-2. Recipients were prospectively monitored for subsequent viremia. A Cox proportional hazards regression model that considered cytokine responses individually and in combination was used to create a predictive model for protection from CMV reactivation. This model was then applied to the validation cohort., Measurements and Main Results: Using the discovery cohort, we identified a specific combination of polyfunctional T-cell subsets to pp65 that predicted protection from subsequent CMV viremia (concordance index 0.88 [SE, 0.087]). The model included both protective (CD107a(-)/IFN-γ(+)/IL-2(+)/TNF-α(+) CD4(+) T cells, CD107a(-)/IFN-γ(+)/IL-2(+)/TNF-α(+) CD8(+) T cells) and detrimental (CD107a(+)/IFN-γ(+)/IL-2(-)/TNF-α(-) CD8(+) T cells) subsets. The model was robust in the validation cohort (concordance index 0.81 [SE, 0.103])., Conclusions: We identified and validated a specific T-cell polyfunctional response to CMV antigen stimulation that provides a clinically useful prediction of subsequent cytomegalovirus risk. This novel diagnostic approach could inform the optimal duration of individual prophylaxis.

Objective: To develop RNA splicing biomarkers of disease severity and therapeutic response in myotonic dystrophy type 1 (DM1) and type 2 (DM2)., Methods: In a discovery cohort, we used microarrays to perform global analysis of alternative splicing in DM1 and DM2. The newly identified splicing changes were combined with previous data to create a panel of 50 putative splicing defects. In a validation cohort of 50 DM1 subjects, we measured the strength of ankle dorsiflexion (ADF) and then obtained a needle biopsy of tibialis anterior (TA) to analyze splice events in muscle RNA. The specificity of DM-associated splicing defects was assessed in disease controls. The CTG expansion size in muscle tissue was determined by Southern blot. The reversibility of splicing defects was assessed in transgenic mice by using antisense oligonucleotides to reduce levels of toxic RNA., Results: Forty-two splicing defects were confirmed in TA muscle in the validation cohort. Among these, 20 events showed graded changes that correlated with ADF weakness. Five other splice events were strongly affected in DM1 subjects with normal ADF strength. Comparison to disease controls and mouse models indicated that splicing changes were DM-specific, mainly attributable to MBNL1 sequestration, and reversible in mice by targeted knockdown of toxic RNA. Splicing defects and weakness were not correlated with CTG expansion size in muscle tissue., Interpretation: Alternative splicing changes in skeletal muscle may serve as biomarkers of disease severity and therapeutic response in myotonic dystrophy., (© 2013 American Neurological Association.)

We present an intramolecular reaction, Reflex™, to derive shorter, sequencer-ready, daughter polymerase chain reaction products from a pooled population of barcoded long-range polymerase chain reaction products, whilst still preserving the cognate DNA barcodes. Our Reflex workflow needs only a small number of primer extension steps to rapidly enable uniform sequence coverage of long contiguous sequence targets in large numbers of samples at low cost on desktop next-generation sequencers.

Amplification by polymerase chain reaction is often used in the preparation of template DNA molecules for next-generation sequencing. Amplification increases the number of available molecules for sequencing but changes the representation of the template molecules in the amplified product and introduces random errors. Such changes in representation hinder applications requiring accurate quantification of template molecules, such as allele calling or estimation of microbial diversity. We present a simple method to count the number of template molecules using degenerate bases and show that it improves genotyping accuracy and removes noise from PCR amplification. This method can be easily added to existing DNA library preparation techniques and can improve the accuracy of variant calling.

Purpose: There is no consensus on the best regimen for the primary treatment of low-risk gestational trophoblastic neoplasia (GTN)., Patients and Methods: Two commonly used single-drug regimens were compared with respect to the proportion of patients meeting the criteria for a complete response (CR) in a randomized phase III trial conducted by the Gynecologic Oncology Group. Eligibility was purposefully broad to maximize the generalizability of the results and included patients with a WHO risk score of 0 to 6 and patients with metastatic disease (limited to lung lesions < 2 cm, adnexa, or vagina) or choriocarcinoma., Results: Two hundred forty women were enrolled, and 216 were deemed eligible. Biweekly intravenous dactinomycin 1.25 mg/m² was statistically superior to weekly intramuscular (IM) methotrexate 30 mg/m² (CR: 70% v 53%; P = .01). Similarly, in patients with low-risk GTN as defined before the 2002 WHO risk score revisions (risk score of 0 to 4 and excluding choriocarcinoma), response was 58% and 73% in the methotrexate and dactinomycin arms, respectively (P = .03). Both regimens were less effective if the WHO risk score was 5 or 6 or if the diagnosis was choriocarcinoma (CR: 9% and 42%, respectively). There were two potential recurrences; one at 4 months (dactinomycin) and one at 22 months (methotrexate). Not all patients completed follow-up. Both regimens were well tolerated., Conclusion: The biweekly dactinomycin regimen has a higher CR rate than the weekly IM methotrexate regimen in low-risk GTN, a generally curable disease.

Bone morphogenetic protein (BMP) signaling regulates a range of cellular processes and plays an important role in the specification and patterning of the early embryo. However, due to the functional redundancy of BMP ligands and receptors in tissues where they are coexpressed, relatively little is known about the role of individual BMP ligands in human disease. Here we report heterozygous variations in BMP7, including a frameshift, missense, and Kozak sequence mutation, in individuals with developmental eye anomalies and a range of systemic abnormalities, including developmental delay, deafness, scoliosis, and cleft palate. We determined that BMP7 is expressed in the developing eye, brain, and ear in human embryos in a manner consistent with the phenotype seen in our mutation cases. These data establish BMP7 as an important gene in human eye development, and suggest that BMP7 should be considered during clinical evaluation of individuals with developmental eye anomalies.

The common form of myotonic dystrophy (DM1) is associated with the expression of expanded CTG DNA repeats as RNA (CUG(exp) RNA). To test whether CUG(exp) RNA creates a global splicing defect, we compared the skeletal muscle of two mouse models of DM1, one expressing a CTG(exp) transgene and another homozygous for a defective muscleblind 1 (Mbnl1) gene. Strong correlation in splicing changes for approximately 100 new Mbnl1-regulated exons indicates that loss of Mbnl1 explains >80% of the splicing pathology due to CUG(exp) RNA. In contrast, only about half of mRNA-level changes can be attributed to loss of Mbnl1, indicating that CUG(exp) RNA has Mbnl1-independent effects, particularly on mRNAs for extracellular matrix proteins. We propose that CUG(exp) RNA causes two separate effects: loss of Mbnl1 function (disrupting splicing) and loss of another function that disrupts extracellular matrix mRNA regulation, possibly mediated by Mbnl2. These findings reveal unanticipated similarities between DM1 and other muscular dystrophies.

Mutations in the transcription factor encoding TFAP2A gene underlie branchio-oculo-facial syndrome (BOFS), a rare dominant disorder characterized by distinctive craniofacial, ocular, ectodermal and renal anomalies. To elucidate the range of ocular phenotypes caused by mutations in TFAP2A, we took three approaches. First, we screened a cohort of 37 highly selected individuals with severe ocular anomalies plus variable defects associated with BOFS for mutations or deletions in TFAP2A. We identified one individual with a de novo TFAP2A four amino acid deletion, a second individual with two non-synonymous variations in an alternative splice isoform TFAP2A2, and a sibling-pair with a paternally inherited whole gene deletion with variable phenotypic expression. Second, we determined that TFAP2A is expressed in the lens, neural retina, nasal process, and epithelial lining of the oral cavity and palatal shelves of human and mouse embryos--sites consistent with the phenotype observed in patients with BOFS. Third, we used zebrafish to examine how partial abrogation of the fish ortholog of TFAP2A affects the penetrance and expressivity of ocular phenotypes due to mutations in genes encoding bmp4 or tcf7l1a. In both cases, we observed synthetic, enhanced ocular phenotypes including coloboma and anophthalmia when tfap2a is knocked down in embryos with bmp4 or tcf7l1a mutations. These results reveal that mutations in TFAP2A are associated with a wide range of eye phenotypes and that hypomorphic tfap2a mutations can increase the risk of developmental defects arising from mutations at other loci.

FOXE3 is a lens-specific transcription factor with a highly conserved forkhead domain previously implicated in congenital primary aphakia and anterior segment dysgenesis. Here, we identify new recessive FOXE3 mutations causative for microphthalmia, sclerocornea, primary aphakia, and glaucoma in two extended consanguineous families by SNP array genotyping followed by a candidate gene approach. Following an additional screen of 236 subjects with developmental eye anomalies, we report two further novel heterozygous mutations segregating in a dominant fashion in two different families. Although the dominant mutations were penetrant, they gave rise to highly variable phenotypes including iris and chorioretinal colobomas, Peters' anomaly, and isolated cataract (cerulean type and early onset adult nuclear and cortical cataract). Using in situ hybridization in human embryos, we demonstrate expression of FOXE3 restricted to lens tissue, predominantly in the anterior epithelium, suggesting that the extralenticular phenotypes caused by FOXE3 mutations are most likely to be secondary to abnormal lens formation. Our findings suggest that mutations in FOXE3 can give rise to a broad spectrum of eye anomalies, largely, but not exclusively related to lens development, and that both dominant and recessive inheritance patterns can be represented. We suggest including FOXE3 in the diagnostic genetic screening for these anomalies.

Genomic expansions of simple tandem repeats can give rise to toxic RNAs that contain expanded repeats. In myotonic dystrophy, the expression of expanded CUG repeats (CUGexp) causes abnormal regulation of alternative splicing and neuromuscular dysfunction. We used a transgenic mouse model to show that derangements of myotonic dystrophy are reversed by a morpholino antisense oligonucleotide, CAG25, that binds to CUGexp RNA and blocks its interaction with muscleblind-like 1 (MBNL1), a CUGexp-binding protein. CAG25 disperses nuclear foci of CUGexp RNA and reduces the overall burden of this toxic RNA. As MBNL1 is released from sequestration, the defect of alternative splicing regulation is corrected, thereby restoring ion channel function. These findings suggest an alternative use of antisense methods, to inhibit deleterious interactions of proteins with pathogenic RNAs.

Myotonic dystrophy type 1 (DM1) is an RNA dominant disease in which mutant transcripts containing an expanded CUG repeat (CUG(exp)) cause muscle dysfunction by interfering with biogenesis of other mRNAs. The toxic effects of mutant RNA are mediated partly through sequestration of splicing regulator Muscleblind-like 1 (Mbnl1), a protein that binds to CUG(exp) RNA. A gene that is prominently affected encodes chloride channel 1 (Clcn1), resulting in hyperexcitability of muscle (myotonia). To identify DM1-affected genes and study mechanisms for dysregulation, we performed global mRNA profiling in transgenic mice that express CUG(exp) RNA, when compared with Mbnl1 knockout and Clcn1 null mice. We found that the majority of changes induced by CUG(exp) RNA in skeletal muscle can be explained by reduced activity of Mbnl1, including many changes that are secondary to myotonia. The pathway most affected comprises genes involved in calcium signaling and homeostasis. Some effects of CUG(exp) RNA on gene expression are caused by abnormal alternative splicing or downregulation of Mbnl1-interacting mRNAs. However, several of the most highly dysregulated genes showed altered transcription, as indicated by parallel changes of the corresponding pre-mRNAs. These results support the idea that trans-dominant effects of CUG(exp) RNA on gene expression in this transgenic model may occur at the level of transcription, RNA processing and mRNA decay, and are mediated mainly but not entirely through sequestration of Mbnl1.

Severe ocular malformations, including anophthalmia-microphthalmia (AM), are responsible for around 25% of severe visual impairment in childhood. Recurrent interstitial deletions of 14q22-23 are associated with AM and a wide range of extra-ocular phenotypes including brain anomalies. The homeobox gene OTX2 is located at 14q22.3 and has recently been identified as mutated in AM patients. Eight human OTX2 mutations have been reported in subjects with severe eye malformations, including AM, and variable developmental delay. We screened a novel AM cohort for mutations and deletions in OTX2, and identified four new mutations in six individuals and two cases of whole gene deletions. Our data suggest that OTX2 mutations and deletions account for 2-3% of AM cases., ((c) 2008 Wiley-Liss, Inc.)

We describe conditions for producing uninterrupted expanded CTG repeats consisting of up to 2000 repeats using 29 DNA polymerase. Previously, generation of such repeats was hindered by CTG repeat instability in plasmid vectors maintained in Escherichia coli and poor in vitro ligation of CTG repeat concatemers due to strand slippage. Instead, we used a combination of in vitro ligation and 29 DNA polymerase to amplify DNA. Correctly ligated products generating a dimerized repeat tract formed substrates for rolling circle amplification (RCA). In the presence of two non-complementary primers, hybridizing to either strand of DNA, ligations can be amplified to generate microgram quantities of repeat containing DNA. Additionally, expanded repeats generated by rolling circle amplification can be produced in vectors for expression of expanded CUG (CUG(exp)) RNA capable of sequestering MBNL1 protein in cell culture. Amplification of dimerized expanded repeats (ADER) opens new possibilities for studies of repeat instability and pathogenesis in myotonic dystrophy, a neurological disorder caused by an expanded CTG repeat.

Transmembrane chloride ion conductance in skeletal muscle increases during early postnatal development. A transgenic mouse model of myotonic dystrophy type 1 (DM1) displays decreased sarcolemmal chloride conductance. Both effects result from modulation of chloride channel 1 (CLCN1) expression, but the respective contributions of transcriptional vs. posttranscriptional regulation are unknown. Here we show that alternative splicing of CLCN1 undergoes a physiological splicing transition during the first 3 wk of postnatal life in mice. During this interval, there is a switch to production of CLCN1 splice products having an intact reading frame, an upregulation of CLCN1 mRNA encoding full-length channel protein, and an increase of CLCN1 function, as determined by patch-clamp analysis of single muscle fibers. In a transgenic mouse model of DM1, however, the splicing transition does not occur, CLCN1 channel function remains low throughout the postnatal interval, and muscle fibers display myotonic discharges. Thus alternative splicing is a posttranscriptional mechanism regulating chloride conductance during muscle development, and the chloride channelopathy in a transgenic mouse model of DM1 results from a failure to execute a splicing transition for CLCN1.

Background: Facioscapulohumeral muscular dystrophy (FSHD) is caused by deletions within a tandem array of D4Z4 repeats on chromosome 4q35. In addition to muscle degeneration, most patients with FSHD develop abnormalities of the retinal vasculature. Previous work has suggested that muscle degeneration in FSHD results from increased expression of genes proximal to the deletion, including FRG1., Objectives: To reexamine this mechanism and identify pathways that are abnormally regulated early in the disease process., Methods: We prospectively studied gene expression in skeletal muscle in patients with FSHD (n = 19) vs healthy individuals (n = 30) and patients with myotonic dystrophy type 1 (n = 12). We used oligonucleotide microarrays for global analysis of gene expression and reverse transcriptase-PCR (RT-PCR) to assess expression or alternative splicing for particular genes., Results: Expression of FRG1 was not increased in patients with FSHD, either by microarray analysis or quantitative RT-PCR. Among genes on 4q35, only LRP2BP showed upregulation that was specific to FSHD. However, neither LRP2BP nor FRG1 showed imbalance of allelic expression by RT-PCR. After filtering out genes that showed similar dysregulation in other forms of muscular dystrophy, only 44 genes were specifically upregulated early in FSHD. Among these, 34 genes were characterized or partially characterized, of which 11 (32%) had a role in vascular smooth muscle or endothelial cells., Conclusion: Expression of genes on chromosome 4q35 was normally regulated in the early stages of facioscapulohumeral muscular dystrophy. Our results support a possible link between muscular dystrophy and retinal vasculopathy in facioscapulohumeral muscular dystrophy.

Several examples have come to light in which mutations in non-protein-coding regions give rise to a deleterious gain-of-function by non-coding RNA. Expression of the toxic RNA is associated with formation of nuclear inclusions and late-onset degenerative changes in brain, heart or skeletal muscle. In the best studied example, myotonic dystrophy, it appears that the main pathogenic effect of the toxic RNA is to sequester binding proteins and compromise the regulation of alternative splicing. This review describes some of the recent advances in understanding the pathophysiology of RNA-dominant diseases.

Liposomal doxorubicin (Caelyx/Doxil) has been shown to be active in around 20% of recurrent ovarian cancers. As yet, there is little clinical data on combinations of existing agents with liposomal doxorubicin, despite considerable clinical experience with soluble doxorubicin in combination. In this study, we have used an ATP-based tumor chemosensitivity assay to determine the relative efficacy of high concentrations of doxorubicin tested in combination with cisplatin, treosulfan, 5-fluorouracil (5-FU) or vinorelbine against cells obtained from recurrent ovarian tumor tissue. The results show little enhancement of the efficacy of high concentrations of doxorubicin by 5-FU, cisplatin, or treosulfan. However, vinorelbine+liposomal doxorubicin showed additive inhibition, and this combination is worthy of further testing in clinical trials.

This paper reports on a study designed to measure the value attached to second-line, palliative chemotherapy by people with advanced cancer, compared with the value healthy people assume they would attach if they had cancer and to assess reasons for accepting or declining treatment. Subjects comprised 92 people with cancer, 76 healthy control subjects, 60 medical oncologists, 128 clinical oncologists, 72 palliative care physicians, 58 general practitioners and 59 qualified nurses working within oncology. Using a questionnaire presenting two typical but imaginary treatment scenarios, subjects indicated whether treatment would be acceptable to them and, if so, what minimum chance and duration of benefit would make treatment worthwhile. Demographic and social data were also collected. Patients accepted a lower chance of benefit than all other groups. Although the minimum worthwhile duration of benefit was more evenly spread, patients choosing lower time values were over-represented. These results were consistent even when treatment involved greater toxicity. The conclusion drawn from this study is that people with advanced cancer are more willing to accept second-line chemotherapy with a lower chance and shorter duration of benefit than others may imagine. Health care professionals must recognize this when discussing treatment options with patients., (Copyright 2001 John Wiley & Sons, Ltd.)

We report here a new framework map that integrates data from 143 studies on the loss of heterozygosity/allelic imbalance in breast cancer. Full details of these loss of heterozygosity maps can be found at the web site (http://www.nottingham.ac.uk/pdzmgh/loh/) that accompanies this report. By combining results from these data, we have also been able to highlight and identify minimum commonly deleted regions on each chromosome. In addition to finding commonly deleted regions at both the BRCA1 and BRCA2 loci, which confirmed the power of the technique, 24 other regions were identified on 16 different chromosomes.

The genome of Heliothis armigera entomopoxvirus (HaEPV) has been mapped with four restriction endonuclease enzymes (BamHI, HindIII, PstI and XhoI), and its length estimated at 233 kbp. An EcoRI-generated HaEPV genomic fragment hybridized to all fragments identified as genomic termini, providing the first experimental evidence for the presence of terminal repeat elements in an EPV genome. The HaEPV spheroidin and nucleoside triphosphate phosphohydrolase I (NPHI) genes have been cloned and sequenced, and their deduced products shown to possess high levels of identity with homologues from other Genus B entomopoxviruses (EPVs). The genomic locations of these and other HaEPV genes and open reading frames have been determined; comparison of their locations with those of homologues in the Amsacta moorei EPV genome largely supports an hypothesis that the Genus B EPVs share a conserved genomic organization which differs from that of chordopoxviruses. It is proposed that genes of EPVs can be assigned to five actual or predicted homology-based groups, a categorization which is useful for directing and interpreting investigations of EPV gene functions and relationships.

The Heliothis armigera entomopoxvirus (HaEPV) genome encodes a predicted 68 kDa polypeptide related to the 'rifampicin resistance' protein of vaccinia virus (with 30 % identity), and an homologous swinepox virus protein (27% identity). We were unable to isolate an HaEPV genotypic variant encoding a predicted C-terminal truncated form of the protein, suggesting that the C terminus of the molecule may be essential to protein function, and, in turn, that this function may be essential to viral replication. HaEPV replication was substantially reduced in host cells exposed to rifampicin, but the observed cytotoxic properties of the drug made it impossible to determine the specific cause of that inhibition. We suggest that possession of a gene encoding a member of this polypeptide family might represent a defining molecular characteristic of the Poxviridae.

Continuously replicating cell lines have been established from embryonic tissue and circulating hemolymph cells of the melolonthine Antitrogus parvulus Britton. Isozyme analyses demonstrated that cell lines from both tissue sources expressed essentially the same isoforms of enzymes as A. parvulus larvae and thus confirmed the species of their origin. Karyotype analyses showed that cells from both tissue sources had accumulated changes in chromosome number and morphology during culture. Availability of melolonthine-derived cells should assist in vitro studies of the pathogens of this important group of beetles.

Purpose: To determine the maximum-tolerated dose (MTD) and toxicity of PSC 833 infusion administered with etoposide for 5 days in patients with cancer, and to determine the effect of PSC 833 on etoposide pharmacokinetics., Patients and Methods: Thirty-five patients were entered onto the study, one of whom was ineligible. Etoposide was delivered from day 1 as a 2-hour infusion over 5 consecutive days at a dose of 75 to 100 mg/m2/d. PSC 833 was administered from day 2 as a 2-hour loading dose and as a 5-day continuous infusion. Doses were escalated from 1 to 2 mg/kg (loading dose) and 1 to 15 mg/kg/d (continuous infusion)., Results: Thirty-four patients were treated with 53 cycles of PSC 833 and etoposide. Steady-state blood PSC 833 levels more than 1,000 ng/mL were achieved in all patients treated at PSC 833 doses > or = 6.6 mg/kg/d by continuous infusion. Myelosuppression was the most common toxicity. The major dose-related toxicity of PSC 833 was reversible hyperbilirubinemia, which occurred in 83% of cycles. The dose-limiting toxicity of PSC 833 was severe ataxia, which occurred in two of nine patients treated at 12 mg/kg/d and in both of the single patients treated at 13.5 and 15 mg/kg/d. PSC 833 concentrations more than 2,000 ng/mL resulted in an increase in etoposide area under the curve (AUC) of 89%, a decrease in etoposide clearance (Cl) of 45%, a decrease in volume of steady-state distribution (Vss) of 41%, and an insignificant increase in alpha half-life (t 1/2 alpha) and significant increase of beta half-life (t 1/2 beta) of 19% and 77%, respectively., Conclusion: PSC 833 can be administered in combination with etoposide with acceptable toxicity. The recommended continuous infusion dose of PSC 833 for this schedule is 10 mg/kg/d over 5 days. PSC 833 results in an increase in etoposide exposure and etoposide doses should be reduced in patients receiving PSC 833.

Chromosome 3p allele loss is frequent in ovarian and testicular tumours. The von Hippel-Lindau (VHL) disease tumour suppressor gene maps to chromosome 3p25. Gonadal tumours may occur in patients with VHL disease, so somatic VHL gene mutations might be involved in the pathogenesis of sporadic gonadal tumours. To investigate this hypothesis, we screened 60 gonadal tumours (36 ovarian and 24 testicular) for VHL gene mutations and chromosome 3p allele loss. Although 38% (10/26) of informative ovarian and 54% (7/13) of testicular tumours demonstrated 3p allele loss, no somatic VHL gene mutations were detected in the 60 gonadal tumours analysed. This suggested that chromosome 3p tumour suppressor gene(s) other than VHL are involved in gonadal tumorigenesis.

Background: Many patients with small cell lung cancer are reported to lose weight, but the mechanism of this effect is unclear., Patients and Methods: Measurements of resting energy expenditure (REE), using indirect calorimetry and body composition (fat, fat-free mass and organ mass), using dual energy X-ray absorptiometry (DXA) and abdominal CT scans were measured in 38 patients with newly-diagnosed small cell lung cancer. Twenty-eight patients were restudied at the end of treatment., Results: In those who responded to treatment there was no change in body weight, but a decrease in REE of 15.7 +/- 11.7 kJ/kg fat free mass/day, whilst in the non-responders body weight decreased 4.33 +/- 5.4 kg, but REE was unchanged., Conclusions: This study provides evidence for tumour-induced hypermetabolism which is independent of changes in gross body composition, although the absolute increase is small, approximately 0.8 MJ/day. However since body weight was maintained in those patients who responded to treatment either total energy expenditure was decreased, implying decreases in physical activity, or energy intake was increased.

Purpose: Etoposide is a schedule-dependent drug, as demonstrated by the superiority of 5 consecutive daily infusions over a continuous 24-hour infusion in patients with small-cell lung cancer. A randomized trial has therefore been conducted to compare an extended 8-day regimen with the 5-day schedule., Patients and Methods: Ninety-four patients with small-cell lung cancer (35 limited disease, 59 extensive disease) were randomized to receive single-agent etoposide 500 mg/m2, either as 5 daily 2-hour infusions of 100 mg/m2 or as 8 daily 75-minute infusions of 62.5 mg/m2, both repeated every 3 weeks for six cycles. Single-agent carboplatin was administered at relapse in both arms of the study. Patients were stratified at randomization according to extent of disease and Karnofsky performance status (KPS)., Results: The overall response rate was 81% in the 5-day arm and 87% in the 8-day arm, with median survival durations of 7.1 and 9.4 months, respectively (no significant differences). The time over which plasma etoposide exceeded low plasma concentrations was significantly longer in patients who responded compared with patients who did not respond. This was most significant for time at concentrations greater than 1, 1.5, and 2 micrograms/mL. Hematologic toxicity was significantly worse in the 5-day arm of the study (cycle no. 1 nadir neutrophil count, 0.8 x 10(9)/L v 1.7 x 10(9)/L). Stepwise regression analysis found duration of exposure to plasma etoposide greater than 3 micrograms/mL to be predictive of nadir neutrophil count and duration of exposure to plasma etoposide greater than 2 micrograms/mL to be predictive of nadir WBC count., Conclusion: The 5-day and 8-day regimens had equivalent activity in small-cell lung cancer. A pharmacokinetic association between concentrations of etoposide and response and toxicity was found. Antitumor activity was associated with the maintenance of lower levels of etoposide than found to be associated with hematologic toxicity. This supports the hypothesis that the schedule of etoposide administration may affect efficacy and toxicity, and that prolonged exposure to low concentrations of etoposide may improve the therapeutic ratio for this drug.