Genome sequencing and analysis of the Tasmanian devil and its transmissible cancer.

The Tasmanian devil (Sarcophilus harrisii), the largest marsupial carnivore, is endangered due to a transmissible facial cancer spread by direct transfer of living cancer cells through biting. Here we describe the sequencing, assembly, and annotation of the Tasmanian devil genome and whole-genome sequences for two geographically distant subclones of the cancer. Genomic analysis suggests that the cancer first arose from a female Tasmanian devil and that the clone has subsequently genetically diverged during its spread across Tasmania. The devil cancer genome contains more than 17,000 somatic base substitution mutations and bears the imprint of a distinct mutational process. Genotyping of somatic mutations in 104 geographically and temporally distributed Tasmanian devil tumors reveals the pattern of evolution and spread of this parasitic clonal lineage, with evidence of a selective sweep in one geographical area and persistence of parallel lineages in other populations.

Metabolomics evaluation of the photochemical impact of violet-blue light (405 nm) on ex vivo platelet concentrates.

INTRODUCTION: Microbicidal violet-blue light in the visible spectrum (405 nm) has been under evaluation for pathogen inactivation in ex vivo human plasma and platelets (PLTs) stored in plasma. Results to date have demonstrated that several blood-borne infectious disease-causing pathogens can be successfully reduced to significantly low levels in the light-treated plasma and PLTs. METHOD: In order to evaluate whether the microbicidal 405 nm light is safe for the treatment of PLT concentrates for pathogen inactivation, LC/MS-based metabolomics analyses were performed to evaluate the overall impact of 405 nm violet-blue light treatment on ex vivo PLT concentrates suspended in plasma and on plasma itself, and to identify metabolome changes in intra-platelet and extra-cellular medium (i.e., plasma). RESULTS: The metabolomics data identified that platelet activating factors (PAFs), agonists and prostaglandins, which can influence PLT basic functions such as integrity, activation, and aggregation potential were unaltered, suggesting that 405 nm light illumination is safe regarding PLT basic functions. Distinct increases in hydroxyl fatty acids and aldehydes, as well as decreases in antioxidant metabolites indicated that reactive oxygen species (ROS) were generated at high levels after only one hour of exposure to 405 nm light. Distinctly changed endogenous photosensitizer metabolites after 1 h of light exposure provided good evidence that 405 nm light was an effective microbicide acting through ROS mechanism and no external additive photosensitizers were required.

The value of cell-free DNA for molecular pathology.

Over the past decade, advances in molecular biology and genomics techniques have revolutionized the diagnosis and treatment of cancer. The technological advances in tissue profiling have also been applied to the study of cell-free nucleic acids, an area of increasing interest for molecular pathology. Cell-free nucleic acids are released from tumour cells into the surrounding body fluids and can be assayed non-invasively. The repertoire of genomic alterations in circulating tumour DNA (ctDNA) is reflective of both primary tumours and distant metastatic sites, and ctDNA can be sampled multiple times, thereby overcoming the limitations of the analysis of single biopsies. Furthermore, ctDNA can be sampled regularly to monitor response to treatment, to define the evolution of the tumour genome, and to assess the acquisition of resistance and minimal residual disease. Recently, clinical ctDNA assays have been approved for guidance of therapy, which is an exciting first step in translating cell-free nucleic acid research tests into clinical use for oncology. In this review, we discuss the advantages of cell-free nucleic acids as analytes in different body fluids, including blood plasma, urine, and cerebrospinal fluid, and their clinical applications in solid tumours and haematological malignancies. We will also discuss practical considerations for clinical deployment, such as preanalytical factors and regulatory requirements. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Sustained influence of metformin therapy on circulating glucagon-like peptide-1 levels in individuals with and without type 2 diabetes.

AIMS: To investigate, in the Carotid Atherosclerosis: Metformin for Insulin Resistance (CAMERA) trial (NCT00723307), whether the influence of metformin on the glucagon-like peptide (GLP)-1 axis in individuals with and without type 2 diabetes (T2DM) is sustained and related to changes in glycaemia or weight, and to investigate basal and post-meal GLP-1 levels in patients with T2DM in the cross-sectional Diabetes Research on Patient Stratification (DIRECT) study. MATERIALS AND METHODS: CAMERA was a double-blind randomized placebo-controlled trial of metformin in 173 participants without diabetes. Using 6-monthly fasted total GLP-1 levels over 18 months, we evaluated metformin's effect on total GLP-1 with repeated-measures analysis and analysis of covariance. In the DIRECT study, we examined active and total fasting and 60-minute post-meal GLP-1 levels in 775 people recently diagnosed with T2DM treated with metformin or diet, using Student's t-tests and linear regression. RESULTS: In CAMERA, metformin increased total GLP-1 at 6 (+20.7%, 95% confidence interval [CI] 4.7-39.0), 12 (+26.7%, 95% CI 10.3-45.6) and 18 months (+18.7%, 95% CI 3.8-35.7), an overall increase of 23.4% (95% CI 11.2-36.9; P < .0001) vs placebo. Adjustment for changes in glycaemia and adiposity, individually or combined, did not attenuate this effect. In the DIRECT study, metformin was associated with higher fasting active (39.1%, 95% CI 21.3-56.4) and total GLP-1 (14.1%, 95% CI 1.2-25.9) but not post-meal incremental GLP-1. These changes were independent of potential confounders including age, sex, adiposity and glycated haemoglobin. CONCLUSIONS: In people without diabetes, metformin increases total GLP-1 in a sustained manner and independently of changes in weight or glycaemia. Metformin-treated patients with T2DM also have higher fasted GLP-1 levels, independently of weight and glycaemia.

Microbial reduction of prebagged human plasma using 405 nm light and its effects on coagulation factors.

Bacterial contamination is the most prevalent infectious complication of blood transfusion in the developed world. To mitigate this, several ultraviolet light-based pathogen reduction technologies (PRTs), some of which require photo-chemicals, have been developed to minimize infection transmission. Relative to UV light, visible 405-nm light is safer and has shown potential to be developed as a PRT for the in situ treatment of ex vivo human plasma and platelet concentrates, without the need for photo-chemicals. This study investigates the effect of 405-nm light on human plasma, with focus on the compatibility of antimicrobial light doses with essential plasma clotting factors. To determine an effective antimicrobial dose that is compatible with plasma, prebagged human plasma (up to 300 mL) was seeded with common microbial contaminants and treated with increasing doses of 405-nm light (16 mW cm-2; ≤ 403 J cm-2). Post-exposure plasma protein integrity was investigated using an AOPP assay, in vitro coagulation tests, and ELISA-based measurement of fibrinogen and Protein S. Microbial contamination in 300 mL prebagged human plasma was significantly reduced (P ≤ 0.05) after exposure to ≤ 288 J cm-2, with microbial loads reduced by > 96.2%. This dose did not significantly affect the plasma protein quality parameters tested (P > 0.05). Increased doses (≥ 345 J cm-2) resulted in a 4.3% increase in clot times with no statistically significant change in protein activity or levels. Overall, this study has demonstrated that the effective microbicidal 405 light dose shows little to no negative effect on plasma quality.

The Preclinical Validation of 405 nm Light Parasiticidal Efficacy on Leishmania donovani in Ex Vivo Platelets in a Rag2-/- Mouse Model.

Violet-blue light of 405 nm in the visible spectrum at a dose of 270 J/cm2 alone has been shown to be an effective microbicidal tool for inactivating several bacteria, HIV-1, and Trypanosoma cruzi in ex vivo plasma and platelets. Unlike chemical- and ultraviolet (UV)-based pathogen inactivation methods for plasma and platelet safety, 405 nm light is shown to be less toxic to host cells at light doses that are microbicidal. In this report, we evaluated the parasiticidal activity of a 405 nm light treatment on platelets spiked with the Leishmania donovani parasite. Following the light treatment, parasite viability was observed to be near zero in both low- and high-titer-spiked platelets relative to controls. Furthermore, to test the residual infectivity after inactivation in vivo, the light-treated low-titer L. donovani-spiked platelets were evaluated in an immunodeficient Rag2-/- mouse model and monitored for 9 weeks. The parasiticidal efficacy of 405 nm light was evident from the lack of a presence of parasites in the mice spleens. Parasiticidal activity was confirmed to be mediated through 405 nm light-induced reactive oxygen species (ROS), as quantitatively measured by a 2',7'-Dichlorodihydrofluorescein diacetate (H2DCFDA)-based assay. Overall, these results confirm the complete inactivation of L. donovani spiked in ex vivo platelets by 405 nm light treatment and exemplify the utility of the Rag2-/- mouse infection model for the preclinical validation of the parasiticidal efficacy of 405 nm light and this light-based technology as a potential PRT for ex vivo platelets.

Human platelet concentrates treated with microbicidal 405 nm light retain hemostasis activity.

Chemical and UV light-based pathogen reduction technologies are currently in use for human platelet concentrates (PCs) to enhance safety from transfusion-transmitted infections. Relative to UV light, 405 nm violet-blue light in the visible spectrum is known to be less harmful. Hence, in this report for the first time, we have assessed the global hemostasis activity of PCs stored in plasma and the activities of six plasma coagulation factors (CFs) as a measure of in vitro hemostatic activity following exposure to the microbicidal 405 nm light. Apheresis PC samples collected from each screened human donor (n = 22) were used for testing of PCs and platelet poor plasma (PPP). Both PCs and PPPs were treated for 5 h with 405 nm light to achieve a previously established microbicidal light dose of 270 J/cm2. Activated partial thromboplastin time and prothrombin time-based potency assays using a coagulation analyzer and hemostatic capacity via Thromboelastography were analyzed. Thromboelastography analysis of the light-treated PCs and plasma present in the PCs showed little difference between the treated and untreated samples. Further, plasma present in the PCs during the light treatment demonstrated a better stability in potency assays for several coagulation factors compared to the plasma alone prepared from PCs first and subjected to the light treatment separately. Overall, PCs stored in plasma treated with 405 nm violet-blue light retain activity for hemostasis.

Antimicrobial 405 nm violet-blue light treatment of ex vivo human platelets leads to mitochondrial metabolic reprogramming and potential alteration of Phospho-proteome.

Continued efforts to reduce the risk of transfusion-transmitted infections (TTIs) through blood and blood components led to the development of ultraviolet (UV) light irradiation technologies known as pathogen reduction technologies (PRT) to enhance blood safety. While these PRTs demonstrate germicidal efficiency, it is generally accepted that these photoinactivation techniques have limitations as they employ treatment conditions shown to compromise the quality of the blood components. During ex vivo storage, platelets having mitochondria for energy production suffer most from the consequences of UV irradiation. Recently, application of visible violet-blue light in the 400-470 nm wavelength range has been identified as a relatively more compatible alternative to UV light. Hence, in this report, we evaluated 405 nm light-treated platelets to assess alterations in energy utilization by measuring different mitochondrial bioenergetic parameters, glycolytic flux, and reactive oxygen species (ROS). Furthermore, we employed untargeted data-independent acquisition mass spectrometry to characterize platelet proteomic differences in protein regulation after the light treatment. Overall, our analyses demonstrate that ex vivo treatment of human platelets with antimicrobial 405 nm violet-blue light leads to mitochondrial metabolic reprogramming to survive the treatment, and alters a fraction of platelet proteome.

Integration of whole transcriptome spatial profiling with protein markers.

Spatial transcriptomics and proteomics provide complementary information that independently transformed our understanding of complex biological processes. However, experimental integration of these modalities is limited. To overcome this, we developed Spatial PrOtein and Transcriptome Sequencing (SPOTS) for high-throughput simultaneous spatial transcriptomics and protein profiling. Compared with unimodal measurements, SPOTS substantially improves signal resolution and cell clustering and enhances the discovery power in differential gene expression analysis across tissue regions.

Breast Cancer Macrophage Heterogeneity and Self-renewal are Determined by Spatial Localization.

Tumor-infiltrating macrophages support critical steps in tumor progression, and their accumulation in the tumor microenvironment (TME) is associated with adverse outcomes and therapeutic resistance across human cancers. In the TME, macrophages adopt diverse phenotypic alterations, giving rise to heterogeneous immune activation states and induction of cell cycle. While the transcriptional profiles of these activation states are well-annotated across human cancers, the underlying signals that regulate macrophage heterogeneity and accumulation remain incompletely understood. Here, we leveraged a novel ex vivo organotypic TME (oTME) model of breast cancer, in vivo murine models, and human samples to map the determinants of functional heterogeneity of TME macrophages. We identified a subset of F4/80highSca-1+ self-renewing macrophages maintained by type-I interferon (IFN) signaling and requiring physical contact with cancer-associated fibroblasts. We discovered that the contact-dependent self-renewal of TME macrophages is mediated via Notch4, and its inhibition abrogated tumor growth of breast and ovarian carcinomas in vivo, as well as lung dissemination in a PDX model of triple-negative breast cancer (TNBC). Through spatial multi-omic profiling of protein markers and transcriptomes, we found that the localization of macrophages further dictates functionally distinct but reversible phenotypes, regardless of their ontogeny. Whereas immune-stimulatory macrophages (CD11C+CD86+) populated the tumor epithelial nests, the stroma-associated macrophages (SAMs) were proliferative, immunosuppressive (Sca-1+CD206+PD-L1+), resistant to CSF-1R depletion, and associated with worse patient outcomes. Notably, following cessation of CSF-1R depletion, macrophages rebounded primarily to the SAM phenotype, which was associated with accelerated growth of mammary tumors. Our work reveals the spatial determinants of macrophage heterogeneity in breast cancer and highlights the disruption of macrophage self-renewal as a potential new therapeutic strategy.

Clinical Importance of Clonal Hematopoiesis in Metastatic Gastrointestinal Tract Cancers.

Importance: Clonal hematopoiesis (CH) has been associated with development of atherosclerosis and leukemia and worse survival among patients with cancer; however, the association with cancer therapy efficacy, in particular immune checkpoint blockade (ICB), and toxicity has not yet been established. Given the widespread use of ICB and the critical role hematopoietic stem cell-derived lymphocytes play in mediating antitumor responses, CH may be associated with therapeutic efficacy and hematologic toxicity. Objective: To determine the association between CH and outcomes, hematologic toxicity, and therapeutic efficacy in patients with metastatic gastrointestinal tract cancers being treated with systemic therapy, both in the first-line metastatic treatment setting and in ICB. Design, Setting, and Participants: This retrospective cohort study included 633 patients with stage IV colorectal (CRC) and esophagogastric (EGC) cancer who were treated with first-line chemotherapy and/or ICB at Memorial Sloan Kettering Cancer Center. Patients underwent matched tumor and peripheral blood DNA sequencing using the Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets next-generation sequencing assay between January 1, 2006, and December 31, 2020. Exposures: Clonal hematopoiesis-related genetic alterations were identified by next-generation sequencing of patients' tumor and normal blood buffy coat samples, with a subset of these CH alterations annotated as likely putative drivers (CH-PD) based upon previously established criteria. Main Outcomes and Measures: Patients with CH and CH-PD in peripheral blood samples were identified, and these findings were correlated with survival outcomes (progression-free survival [PFS] and overall survival [OS]) during first-line chemotherapy and ICB, as well as baseline white blood cell levels and the need for granulocyte colony-stimulating factor (G-CSF) support. Results: Among the 633 patients included in the study (390 men [61.6%]; median age, 58 [IQR, 48-66] years), the median age was 52 (IQR, 45-63) years in the CRC group and 61 (IQR, 53-69) years in the EGC group. In the CRC group, 161 of 301 patients (53.5%) were men, compared with 229 of 332 patients (69.0%) in the EGC group. Overall, 62 patients (9.8%) were Asian, 45 (7.1%) were Black or African American, 482 (76.1%) were White, and 44 (7.0%) were of unknown race or ethnicity. Presence of CH was identified in 115 patients with EGC (34.6%) and 83 with CRC (27.6%), with approximately half of these patients harboring CH-PD (CRC group, 44 of 83 [53.0%]; EGC group, 55 of 115 [47.8%]). Patients with EGC and CH-PD exhibited a significantly worse median OS of 16.0 (95% CI, 11.6-22.3) months compared with 21.6 (95% CI, 19.6-24.3) months for those without CH-PD (P = .01). For patients with CRC and EGC, CH and CH-PD were not associated with PFS differences in patients undergoing ICB or first-line chemotherapy. Neither CH nor CH-PD were correlated with baseline leukocyte levels or increased need for G-CSF support. Conclusions and Relevance: These findings suggest CH and CH-PD are not directly associated with the treatment course of patients with metastatic gastrointestinal tract cancer receiving cancer-directed therapy.

Novel case of takotsubo cardiomyopathy following COVID-19 vaccination.

We present an unusual case of takotsubo cardiomyopathy (TTC) following administration of the second dose of the DNA ChadOX1 nCOV-19 (AZD122) vaccination. This woman in her early 50s presented to the emergency department 8 days following her vaccine with central chest pain. Initial investigations revealed a raised troponin and evolving T wave inversion on ECG. Acute coronary syndrome management was commenced. Further investigations revealed non-obstructive coronary arteries on coronary angiography and imaging revealed hypokinesia of the anterior and anterior-septal walls in the apex and midcavity level, myocardial oedema and no infarction, all in keeping with TTC. Given the large-scale roll out of vaccinations during the COVID-19 pandemic better understanding of potential adverse events is essential. This is the first case report of TTC following a second dose of the DNA ChadOX1 nCOV-19 (AZD122) vaccination.

Visible 405 nm Violet-Blue Light Successfully Inactivates HIV-1 in Human Plasma.

Despite significant advances in ensuring the safety of the blood supply, there is continued risk of transfusion transmitted infections (TTIs) from newly emerging or re-emerging infections. Globally, several pathogen reduction technologies (PRTs) for blood safety have been in development as an alternative to traditional treatment methods. Despite broad spectrum antimicrobial efficacy, some of the approved ultraviolet (UV) light-based PRTs, understandably due to UV light-associated toxicities, fall short in preserving the full functional spectrum of the treated blood components. As a safer alternative to the UV-based microbicidal technologies, investigations into the use of violet-blue light in the region of 405 nm have been on the rise as these wavelengths do not impair the treated product at doses that demonstrate microbicidal activity. Recently, we have demonstrated that a 405 nm violet-blue light dose of 270 J/cm2 was sufficient for reducing bacteria and the parasite in plasma and platelets suspended in plasma while preserving the quality of the treated blood product stored for transfusion. Drawn from the previous experience, here we evaluated the virucidal potential of 405 nm violet-blue light dose of 270 J/cm2 on an important blood-borne enveloped virus, the human immunodeficiency virus 1 (HIV-1), in human plasma. Both test plasma (HIV-1 spiked and treated with various doses of 405 nm light) and control plasma (HIV-1 spiked, but not treated with the light) samples were cultured with HIV-1 permissive H9 cell line for up to 21 days to estimate the viral titers. Quantitative HIV-1 p24 antigen (HIV-1 p24) levels reflective of HIV-1 titers were measured for each light dose to assess virus infectivity. Our results demonstrate that a 405 nm light dose of 270 J/cm2 is also capable of 4-5 log HIV-1 reduction in plasma under the conditions tested. Overall, this study provides the first proof-of-concept that 405 nm violet-blue light successfully inactivates HIV-1 present in human plasma, thereby demonstrating its potential towards being an effective PRT for this blood component safety.

Violet-blue 405-nm Light-based Photoinactivation for Pathogen Reduction of Human Plasma Provides Broad Antibacterial Efficacy Without Visible Degradation of Plasma Proteins.

In transfusion medicine, bacterial contamination can occur in ex vivo stored blood plasma, and there are continued efforts to improve blood safety and reduce the risk of transfusion-transmitted infections. Visible 405-nm violet-blue light has demonstrated potential for in situ pathogen reduction in ex vivo stored plasma and platelet concentrates. This study investigates the broad-spectrum antibacterial efficacy and compatibility potential of 405-nm light for treatment of blood plasma. Human plasma seeded with bacteria at a range of densities (101 -103 , 104 -106 , 107 -108 CFU mL-1 ) was exposed to 360 J cm-2 405-nm light (1 h at 0.1 W cm-2 ), with this fixed dose selected based on the initial analysis of inactivation kinetics. One-dimensional protein mobility analysis and measurement of advanced oxidation protein products (AOPP) was conducted to evaluate compatibility of the antimicrobial dose with plasma proteins and, identify upper levels at which protein degradation can be detected. Broad-spectrum antibacterial efficacy was observed with a fixed treatment of 360 J cm-2 , with 98.9-100% inactivation achieved across all seeding densities for all organisms, except E. coli, which achieved 95.1-100% inactivation. At this dose (360 J cm-2 ), no signs of protein degradation occurred. Overall, 405-nm light shows promise for broad-spectrum bacterial inactivation in blood plasma, while preserving plasma protein integrity.

Genome-wide mutational signatures in low-coverage whole genome sequencing of cell-free DNA.

Mutational signatures accumulate in somatic cells as an admixture of endogenous and exogenous processes that occur during an individual's lifetime. Since dividing cells release cell-free DNA (cfDNA) fragments into the circulation, we hypothesize that plasma cfDNA might reflect mutational signatures. Point mutations in plasma whole genome sequencing (WGS) are challenging to identify through conventional mutation calling due to low sequencing coverage and low mutant allele fractions. In this proof of concept study of plasma WGS at 0.3-1.5x coverage from 215 patients and 227 healthy individuals, we show that both pathological and physiological mutational signatures may be identified in plasma. By applying machine learning to mutation profiles, patients with stage I-IV cancer can be distinguished from healthy individuals with an Area Under the Curve of 0.96. Interrogating mutational processes in plasma may enable earlier cancer detection, and might enable the assessment of cancer risk and etiology.

Phase I/Ib Study of the Efficacy and Safety of Buparlisib and Ibrutinib Therapy in MCL, FL, and DLBCL with Serial Cell-Free DNA Monitoring.

PURPOSE: Activation of Bruton tyrosine kinase (BTK) and phosphatidylinositol-3-kinase (PI3K) represent parallel, synergistic pathways in lymphoma pathogenesis. As predominant PI3Kδ inhibition is a possible mechanism of tumor escape, we proposed a clinical trial of dual BTK and pan-PI3K inhibition. PATIENTS AND METHODS: We conducted a single-center phase I/Ib trial combining a BTK inhibitor (ibrutinib) and a pan-PI3K inhibitor (buparlisib) in 37 patients with relapsed/refractory (R/R) B-cell lymphoma. Buparlisib and ibrutinib were administered orally, once daily in 28-day cycles until progression or unacceptable toxicity. The clinical trial is registered with clinicaltrials.gov, NCT02756247. RESULTS: Patients with mantle cell lymphoma (MCL) receiving the combination had a 94% overall response rate (ORR) and 33-month median progression-free survival; ORR of 31% and 20% were observed in patients with diffuse large B-cell lymphoma and follicular lymphoma, respectively. The maximum tolerated dose was ibrutinib 560 mg plus buparlisib 100 mg and the recommended phase II dose was ibrutinib 560 mg plus buparlisib 80 mg. The most common grade 3 adverse events were rash/pruritis/dermatitis (19%), diarrhea (11%), hyperglycemia (11%), and hypertension (11%). All grade mood disturbances ranging from anxiety, depression, to agitation were observed in 22% of patients. Results from serial monitoring of cell-free DNA samples corresponded to radiographic resolution of disease and tracked the emergence of mutations known to promote BTK inhibitor resistance. CONCLUSIONS: BTK and pan-PI3K inhibition in mantle cell lymphoma demonstrates a promising efficacy signal. Addition of BCL2 inhibitors to a BTK and pan-PI3K combination remain suitable for further development in mantle cell lymphoma.

Complete Inactivation of Blood Borne Pathogen Trypanosoma cruzi in Stored Human Platelet Concentrates and Plasma Treated With 405 nm Violet-Blue Light.

The introduction of pathogen reduction technologies (PRTs) to inactivate bacteria, viruses and parasites in donated blood components stored for transfusion adds to the existing arsenal toward reducing the risk of transfusion-transmitted infectious diseases (TTIDs). We have previously demonstrated that 405 nm violet-blue light effectively reduces blood-borne bacteria in stored human plasma and platelet concentrates. In this report, we investigated the microbicidal effect of 405 nm light on one important bloodborne parasite Trypanosoma cruzi that causes Chagas disease in humans. Our results demonstrated that a light irradiance at 15 mWcm-2 for 5 h, equivalent to 270 Jcm-2, effectively inactivated T. cruzi by over 9.0 Log10, in plasma and platelets that were evaluated by a MK2 cell infectivity assay. Giemsa stained T. cruzi infected MK2 cells showed that the light-treated parasites in plasma and platelets were deficient in infecting MK2 cells and did not differentiate further into intracellular amastigotes unlike the untreated parasites. The light-treated and untreated parasite samples were then evaluated for any residual infectivity by injecting the treated parasites into Swiss Webster mice, which did not develop infection even after the animals were immunosuppressed, further demonstrating that the light treatment was completely effective for inactivation of the parasite; the light-treated platelets had similar in vitro metabolic and biochemical indices to that of untreated platelets. Overall, these results provide a proof of concept toward developing 405 nm light treatment as a pathogen reduction technology (PRT) to enhance the safety of stored human plasma and platelet concentrates from bloodborne T. cruzi, which causes Chagas disease.

Cell-free DNA profiling in retinoblastoma patients with advanced intraocular disease: An MSKCC experience.

PURPOSE: The enucleation rate for retinoblastoma has dropped from over 95% to under 10% in the past 10 years as a result of improvements in therapy. This reduces access to tumor tissue for molecular profiling, especially in unilateral retinoblastoma, and hinders the confirmation of somatic RB1 mutations necessary for genetic counseling. Plasma cell-free DNA (cfDNA) has provided a platform for noninvasive molecular profiling in cancer, but its applicability in low tumor burden retinoblastoma has not been shown. We analyzed cfDNA collected from 10 patients with available tumor tissue to determine whether sufficient tumorderived cfDNA is shed in plasma from retinoblastoma tumors to enable noninvasive RB1 mutation detection. METHODS: Tumor tissue was collected from eye enucleations in 10 patients diagnosed with advanced intra-ocular unilateral retinoblastoma, three of which went on to develop metastatic disease. Tumor RB1 mutation status was determined using an FDA-cleared tumor sequencing assay, MSK-IMPACT. Plasma samples were collected before eye enucleation and analyzed with a customized panel targeting all exons of RB1. RESULTS: Tumor-guided genotyping detected 10 of the 13 expected somatic RB1 mutations in plasma cfDNA in 8 of 10 patients (average variant allele frequency 3.78%). Without referring to RB1 status in the tumor, de novo mutation calling identified 7 of the 13 expected RB1 mutations (in 6 of 10 patients) with high confidence. CONCLUSION: Plasma cfDNA can detect somatic RB1 mutations in patients with unilateral retinoblastoma. Since intraocular biopsies are avoided in these patients because of concern about spreading tumor, cfDNA can potentially offer a noninvasive platform to guide clinical decisions about treatment, follow-up schemes, and risk of metastasis.

Non-ionizing 405 nm Light as a Potential Bactericidal Technology for Platelet Safety: Evaluation of in vitro Bacterial Inactivation and in vivo Platelet Recovery in Severe Combined Immunodeficient Mice.

Bacterial contamination of ex vivo stored platelets is a cause of transfusion-transmitted infection. Violet-blue 405 nm light has recently demonstrated efficacy in reducing the bacterial burden in blood plasma, and its operational benefits such as non-ionizing nature, penetrability, and non-requirement for photosensitizing agents, provide a unique opportunity to develop this treatment for in situ treatment of ex vivo stored platelets as a tool for bacterial reduction. Sealed bags of platelet concentrates, seeded with low-level Staphylococcus aureus contamination, were 405 nm light-treated (3-10 mWcm-2) up to 8 h. Antimicrobial efficacy and dose efficiency was evaluated by quantification of the post-treatment surviving bacterial contamination levels. Platelets treated with 10 mWcm-2 for 8 h were further evaluated for survival and recovery in severe combined immunodeficient (SCID) mice. Significant inactivation of bacteria in platelet concentrates was achieved using all irradiance levels, with 99.6-100% inactivation achieved by 8 h (P < 0.05). Analysis of applied dose demonstrated that lower irradiance levels generally resulted in significant decontamination at lower doses: 180 Jcm-2/10 mWcm-2 (P = 0.008) compared to 43.2 Jcm-2/3 mWcm-2 (P = 0.002). Additionally, the recovery of light-treated platelets, compared to non-treated platelets, in the murine model showed no significant differences (P = >0.05). This report paves the way for further comprehensive studies to test 405 nm light treatment as a bactericidal technology for stored platelets.

Circulating cell-free DNA for non-invasive cancer management.

Cell-free DNA (cfDNA) was first identified in human plasma in 1948 and is thought to be released from cells throughout the body into the circulatory system. In cancer, a portion of the cfDNA originates from tumour cells, referred to as circulating-tumour DNA (ctDNA), and can contain mutations corresponding to the patient's tumour, for instance specific TP53 alleles. Profiling of cfDNA has recently become an area of increasing clinical relevance in oncology, in particular due to advances in the sensitivity of molecular biology techniques and development of next generation sequencing technologies, as this allows tumour mutations to be identified and tracked non-invasively. This has opened up new possibilities for monitoring tumour evolution and acquisition of resistance, as well as for guiding treatment decisions when tumour biopsy tissue is insufficient or unavailable. In this review, we will discuss the biology of cell-free nucleic acids, methods of analysis, and the potential clinical uses of these techniques, as well as the on-going clinical development of ctDNA assays.