'Final common pathway' of human cancer immunotherapy: targeting random somatic mutations.

Effective clinical cancer immunotherapies, such as administration of the cytokine IL-2, adoptive cell transfer (ACT) and the recent success of blockade of the checkpoint modulators CTLA-4 and PD-1, have been developed without clear identification of the immunogenic targets expressed by human cancers in vivo. Immunotherapy of patients with cancer through the use of ACT with autologous lymphocytes has provided an opportunity to directly investigate the antigen recognition of lymphocytes that mediate cancer regression in humans. High-throughput immunological testing of such lymphocytes in combination with improvements in deep sequencing of the autologous cancer have provided new insight into the molecular characterization and incidence of anti-tumor lymphocytes present in patients with cancer. Here we highlight evidence suggesting that T cells that target tumor neoantigens arising from cancer mutations are the main mediators of many effective cancer immunotherapies in humans.

Neoantigen T-Cell Receptor Gene Therapy in Pancreatic Cancer.

A patient with progressive metastatic pancreatic cancer was treated with a single infusion of 16.2×109 autologous T cells that had been genetically engineered to clonally express two allogeneic HLA-C*08:02-restricted T-cell receptors (TCRs) targeting mutant KRAS G12D expressed by the tumors. The patient had regression of visceral metastases (overall partial response of 72% according to the Response Evaluation Criteria in Solid Tumors, version 1.1); the response was ongoing at 6 months. The engineered T cells constituted more than 2% of all the circulating peripheral-blood T cells 6 months after the cell transfer. In this patient, TCR gene therapy targeting the KRAS G12D driver mutation mediated the objective regression of metastatic pancreatic cancer. (Funded by the Providence Portland Medical Foundation.).

Identification of essential genes for cancer immunotherapy.

Somatic gene mutations can alter the vulnerability of cancer cells to T-cell-based immunotherapies. Here we perturbed genes in human melanoma cells to mimic loss-of-function mutations involved in resistance to these therapies, by using a genome-scale CRISPR-Cas9 library that consisted of around 123,000 single-guide RNAs, and profiled genes whose loss in tumour cells impaired the effector function of CD8+ T cells. The genes that were most enriched in the screen have key roles in antigen presentation and interferon-γ signalling, and correlate with cytolytic activity in patient tumours from The Cancer Genome Atlas. Among the genes validated using different cancer cell lines and antigens, we identified multiple loss-of-function mutations in APLNR, encoding the apelin receptor, in patient tumours that were refractory to immunotherapy. We show that APLNR interacts with JAK1, modulating interferon-γ responses in tumours, and that its functional loss reduces the efficacy of adoptive cell transfer and checkpoint blockade immunotherapies in mouse models. Our results link the loss of essential genes for the effector function of CD8+ T cells with the resistance or non-responsiveness of cancer to immunotherapies.

High-affinity oligoclonal TCRs define effective adoptive T cell therapy targeting mutant KRAS-G12D.

Complete cancer regression occurs in a subset of patients following adoptive T cell therapy (ACT) of ex vivo expanded tumor-infiltrating lymphocytes (TILs). However, the low success rate presents a great challenge to broader clinical application. To provide insight into TIL-based immunotherapy, we studied a successful case of ACT where regression was observed against tumors carrying the hotspot mutation G12D in the KRAS oncogene. Four T cell receptors (TCRs) made up the TIL infusion and recognized two KRAS-G12D neoantigens, a nonamer and a decamer, all restricted by human leukocyte antigen (HLA) C*08:02. Three of them (TCR9a, 9b, and 9c) were nonamer-specific, while one was decamer-specific (TCR10). We show that only mutant G12D but not the wild-type peptides stabilized HLA-C*08:02 due to the formation of a critical anchor salt bridge to HLA-C. Therapeutic TCRs exhibited high affinities, ranging from nanomolar to low micromolar. Intriguingly, TCR binding affinities to HLA-C inversely correlated with their persistence in vivo, suggesting the importance of antigenic affinity in the function of therapeutic T cells. Crystal structures of TCR-HLA-C complexes revealed that TCR9a to 9c recognized G12D nonamer with multiple conserved contacts through shared CDR2ß and CDR3α. This allowed CDR3ß variation to confer different affinities via a variable HLA-C contact, generating an oligoclonal response. TCR10 recognized an induced and distinct G12D decamer conformation. Thus, this successful case of ACT included oligoclonal TCRs of high affinity recognizing distinct conformations of neoantigens. Our study revealed the potential of a structural approach to inform clinical efforts in targeting KRAS-G12D tumors by immunotherapy and has general implications for T cell-based immunotherapies.

Cluster analysis unveils a severe persistent respiratory impairment phenotype 3-months after severe COVID-19.

BACKGROUND: The mid-term respiratory sequelae in survivors of severe COVID-19 appear highly heterogeneous. In addition, factors associated with respiratory sequelae are not known. In this monocentric prospective study, we performed a multidisciplinary assessment for respiratory and muscular impairment and psychological distress 3 months after severe COVID-19. We analysed factors associated with severe persistent respiratory impairment, amongst demographic, COVID-19 severity, and 3-month assessment. METHODS: Patients with severe SARS-CoV-2 pneumonia requiring ≥ 4L/min were included for a systematic 3-month visit, including respiratory assessment (symptoms, lung function, CT scan), muscular evaluation (body composition, physical function and activity, disability), psychopathological evaluation (anxiety, depression, post-traumatic stress disorder-PTSD) and quality of life. A cluster analysis was performed to identify subgroups of patients based on objective functional measurements: DLCO, total lung capacity and 6-min walking distance (6MWD). RESULTS: Sixty-two patients were analysed, 39% had dyspnea on exercise (mMRC ≥ 2), 72% had DLCO < 80%, 90% had CT-scan abnormalities; 40% had sarcopenia/pre-sarcopenia and 31% had symptoms of PTSD. Cluster analysis identified a group of patients (n = 18, 30.5%) with a severe persistent (SP) respiratory impairment (DLCO 48 ± 12%, 6MWD 299 ± 141 m). This SP cluster was characterized by older age, severe respiratory symptoms, but also sarcopenia/pre-sarcopenia, symptoms of PTSD and markedly impaired quality of life. It was not associated with initial COVID-19 severity or management. CONCLUSIONS AND CLINICAL IMPLICATION: We identified a phenotype of patients with severe persistent respiratory and muscular impairment and psychological distress 3 months after severe COVID-19. Our results highlight the need for multidisciplinary assessment and management after severe SARS-CoV-2 pneumonia. Trial registration The study was registered on ClinicalTrials.gov (May 6, 2020): NCT04376840.

A rapid and universal liquid chromatograph-mass spectrometry-based platform, refmAb-Q nSMOL, for monitoring monoclonal antibody therapeutics.

Accurate quantitation of antibodies is critical for development of monoclonal antibody therapeutics (mAbs). Therapeutic drug monitoring has been applied to measure levels of mAbs in clinics for dose adjustment for autoimmune disease. Trough levels of mAbs can be a biomarker for cancer immunotherapy. Thus, the deployment of a rapid and universal platform for mAb monitoring may benefit processes ranging from drug development to clinical practice for a wide spectrum of diseases. However, mAb monitoring often requires development and conduct of an individual ligand binding assay such as ELISA, which is impractical to scale. We streamlined quantitation of antibody therapeutics by a nano-surface and molecular-orientation limited (nSMOL) proteolysis assay using LC-MS with a universal reference antibody (refmAb-Q), for accurate multiplexed quantitation of unique signature peptides derived from mAbs. This innovative refmAb-Q nSMOL platform may provide a practical solution for quantitating an ever-increasing number of mAbs from developmental to clinical use settings.

Patient-reported outcomes and complications during head and neck cancer radiotherapy before versus during the COVID-19 pandemic.

PURPOSE: This study compares patient-reported outcomes and treatment-related complications during radiotherapy before (August 2019-January 2020) versus during (March-Sept 2020) the COVID-19 pandemic. MATERIALS AND METHODS: The MD Anderson Symptom Inventory-head and neck module was used to assess curative intent in H&N cancer patients' symptoms during radiotherapy. RESULTS: There were 158 patients in the pre-pandemic cohort and 137 patients in the pandemic cohort. There was no significant difference in enteral feeding requirements between the cohorts (21% versus 30%, p = 0.07). Weight loss was higher during the pandemic (mean - 5.6% versus 6.8%, p = 0.03). On multivariate analysis, treatment during the pandemic was associated with higher symptom scores for coughing/choking while eating (2.7 versus 2.1, p = 0.013). CONCLUSIONS: Complication rates during H&N radiotherapy during the COVID-19 pandemic were similar at our institution relative to the pre-pandemic era, although weight loss was greater and patients reported more severe choking/coughing while eating.

Outcomes of Simplified Lung Ultrasound Exam in COVID-19: Implications for Self-Imaging.

OBJECTIVES: Lung ultrasound B-lines represent interstitial thickening or edema and relate to mortality in COVID-19. As B-lines can be detected with minimal training using point-of-care ultrasound (POCUS), we examined the frequency, clinical associations, and outcomes of B-lines when found using a simplified POCUS method in acutely ill patients with COVID-19. METHODS: In this retrospective cohort study, hospital data from COVID-19 patients who had undergone lung imaging during standard echocardiography or POCUS were reviewed for an ultrasound lung comet (ULC) sign, defined as the presence of ≥3 B-lines from images of only the antero-apex of either lung (ULC+). Clinical risk factors, oximetry and radiographic results, and disease severity were analyzed for associations with ULC+. Clinical risk factors and ULC+ were analyzed for associations with hospital mortality or the need for intensive care in multivariable models. RESULTS: Of N = 160 patients, age (mean ± standard deviation) was 64.8 ± 15.5 years, and 46 (29%) died. ULC+ was present in 100/160 (62%) of patients overall, in 81/103 (79%) of severe-or-greater disease versus 19/57 (33%) of moderate-or-less disease (P < .0001) and was associated with mortality (odds ratio [OR] = 2.4 [95% confidence interval [CI]: 1.1-5.4], P = .02) and the need for intensive care (OR = 5.23 [95% CI: 2.42-12.40], P < .0001). In the multivariable models, symptom duration and severe-or-greater disease were associated with ULC+, and ULC+, diabetes, and symptom duration were associated with the need for intensive care. CONCLUSIONS: B-lines in the upper chest were common and related to disease severity, intensive care, and hospital mortality in COVID-19. Validation of a simplified lung POCUS exam could provide the evidence basis for a self-imaging application during the pandemic.

[Management of suicidal crisis in general practice: evaluation of care practices according to patient age]. / Crise suicidaire, évaluation des pratiques en médecine générale selon l'âge du patient.

Nearly 800,000 people die by suicide each year worldwide. Up to 75% of suicidal patients consulted their general practitioner in the months preceding their attempt. A study, conducted among 167 practitioners in Champagne-Ardenne in 2016-2017, aims to evaluate the practices of general practitioners in the management of suicidal crisis, particularly according to the age of the patient. It provides elements for reflection on their role in suicide prevention.

Patient-reported outcome measures in patients undergoing radiotherapy for head and neck cancer.

OBJECTIVES: Head and neck (H&N) cancer patients experience significant acute side effects from treatment. This study evaluates prospectively collected patient-reported outcomes (PROs) in H&N patients undergoing radiotherapy (RT) to assess feasibility of electronically collecting PROs and to objectively document symptom acuity and trajectory during RT. MATERIALS AND METHODS: H&N patients undergoing radical RT at our multicentre institution completed a 12-item partial survey of the Vanderbilt Head & Neck Symptom Survey 2.0 prior to RT and weekly on RT. Between October 2016 and October 2018, 318 of 333 patients completed a baseline survey and at least one weekly survey. RESULTS: The average number of weekly questionnaires completed was 5 (range 1-8). The mean maximum symptom scores were highest for dysgeusia (5.8/10), pain (5.4/10), mucositis (4.8/10), weight loss due to swallowing (4.5/10) and mucus causing choking/gagging (4.3/10). On multivariate analysis, female gender, sinonasal, nasopharynx and oropharynx primaries were associated with a greater risk of moderate-severe pain (p < 0.05). Sinonasal, nasopharynx, oral cavity, oropharynx and thyroid primaries were associated with a greater risk of moderate-severe mucositis during radiation (p < 0.0001). Salivary gland, sinonasal, nasopharynx and oropharynx primaries and higher radiation dose were associated with a greater risk of moderate-severe dysgeusia (all p < 0.05). CONCLUSIONS: Electronic PRO collection during H&N cancer RT is feasible. H&N cancer patients experience significant symptoms during RT, and the most severe symptoms reported were dysgeusia, pain and mucositis. Oropharynx cancer patients reported the highest symptom scores during RT.

Treatment de-escalation for HPV-associated oropharyngeal squamous cell carcinoma with radiotherapy vs. trans-oral surgery (ORATOR2): study protocol for a randomized phase II trial.

BACKGROUND: Patients with human papillomavirus-positive (HPV+) oropharyngeal squamous cell carcinoma (OPC) have substantially better treatment response and overall survival (OS) than patients with HPV-negative disease. Treatment options for HPV+ OPC can involve either a primary radiotherapy (RT) approach (± concomitant chemotherapy) or a primary surgical approach (± adjuvant radiation) with transoral surgery (TOS). These two treatment paradigms have different spectrums of toxicity. The goals of this study are to assess the OS of two de-escalation approaches (primary radiotherapy and primary TOS) compared to historical control, and to compare survival, toxicity and quality of life (QOL) profiles between the two approaches. METHODS: This is a multicenter phase II study randomizing one hundred and forty patients with T1-2 N0-2 HPV+ OPC in a 1:1 ratio between de-escalated primary radiotherapy (60 Gy) ± concomitant chemotherapy and TOS ± de-escalated adjuvant radiotherapy (50-60 Gy based on risk factors). Patients will be stratified based on smoking status (< 10 vs. ≥ 10 pack-years). The primary endpoint is OS of each arm compared to historical control; we hypothesize that a 2-year OS of 85% or greater will be achieved. Secondary endpoints include progression free survival, QOL and toxicity. DISCUSSION: This study will provide an assessment of two de-escalation approaches to the treatment of HPV+ OPC on oncologic outcomes, QOL and toxicity. Results will inform the design of future definitive phase III trials. TRIAL REGISTRATION: Clinicaltrials.gov identifier: NCT03210103. Date of registration: July 6, 2017, Current version: 1.3 on March 15, 2019.

Websites, Websites Everywhere: How Thyroid Cancer Patients Use the Internet.

Thyroid cancer incidence rates and Internet use are both increasing. Thyroid cancer is common in young patients, who are likely to use the Internet. This study aims to characterize thyroid cancer patient Internet use and search patterns, usability of online resources, and effects on clinical care. From May to December 2017, inclusive, patients with thyroid cancer attending two tertiary cancer centers were invited to complete a survey about Internet use. Thirty-nine of 72 questionnaires were returned (54%). Ninety-seven percent of participants used the Internet, and 87% had looked for thyroid cancer information. The majority (94%) searched on Google. Patients most often looked for information about treatment (94%) and symptom management (76%). Many patients evaluated content quality by comparing several resources (71%), discussing with a physician (56%) or using a credible academic or government site (53%). Online information was somewhat hard to understand for 32%, but 91% found it useful. Over half (60%) of treatment decisions were affected by web resources, and information helped 50% of patients make decisions with their physicians. Respondents highlighted a lack of resources on survivorship and uncommon tumors such as medullary or anaplastic cancer. Physicians should recognize that patients overwhelmingly access online information, which often impacts patients' decision-making. Clinicians can guide thyroid cancer patients through abundant web-based information and assist in interpreting this information. Educators can use this information to guide resource development, tailoring content and design to thyroid cancer patients' needs.

Radiotherapy versus transoral robotic surgery and neck dissection for oropharyngeal squamous cell carcinoma (ORATOR): an open-label, phase 2, randomised trial.

BACKGROUND: Transoral robotic surgery (TORS) with concurrent neck dissection has supplanted radiotherapy in the USA as the most common treatment for oropharyngeal squamous cell carcinoma (OPSCC), yet no randomised trials have compared these modalities. We aimed to evaluate differences in quality of life (QOL) 1 year after treatment. METHODS: The ORATOR trial was an investigator-initiated, multicentre, international, open-label, parallel-group, phase 2, randomised study. Patients were enrolled at six hospitals in Canada and Australia. We randomly assigned (1:1) patients aged 18 years or older, with Eastern Cooperative Oncology Group scores of 0-2, and with T1-T2, N0-2 (≤4 cm) OPSCC tumour types to radiotherapy (70 Gy, with chemotherapy if N1-2) or TORS plus neck dissection (with or without adjuvant chemoradiotherapy, based on pathology). Following stratification by p16 status, patients were randomly assigned using a computer-generated randomisation list with permuted blocks of four. The primary endpoint was swallowing-related QOL at 1 year as established using the MD Anderson Dysphagia Inventory (MDADI) score, powered to detect a 10-point improvement (a clinically meaningful change) in the TORS plus neck dissection group. All analyses were done by intention to treat. This study is registered with ClinicalTrials.gov (NCT01590355) and is active, but not currently recruiting. FINDINGS: 68 patients were randomly assigned (34 per group) between Aug 10, 2012, and June 9, 2017. Median follow-up was 25 months (IQR 20-33) for the radiotherapy group and 29 months (23-43) for the TORS plus neck dissection group. MDADI total scores at 1 year were mean 86·9 (SD 11·4) in the radiotherapy group versus 80·1 (13·0) in the TORS plus neck dissection group (p=0·042). There were more cases of neutropenia (six [18%] of 34 patients vs none of 34), hearing loss (13 [38%] vs five [15%]), and tinnitus (12 [35%] vs two [6%]) reported in the radiotherapy group than in the TORS plus neck dissection group, and more cases of trismus in the TORS plus neck dissection group (nine [26%] vs one [3%]). The most common adverse events in the radiotherapy group were dysphagia (n=6), hearing loss (n=6), and mucositis (n=4), all grade 3, and in the TORS plus neck dissection group, dysphagia (n=9, all grade 3) and there was one death caused by bleeding after TORS. INTERPRETATION: Patients treated with radiotherapy showed superior swallowing-related QOL scores 1 year after treatment, although the difference did not represent a clinically meaningful change. Toxicity patterns differed between the groups. Patients with OPSCC should be informed about both treatment options. FUNDING: Canadian Cancer Society Research Institute Grant (#701842), Ontario Institute for Cancer Research Clinician-Scientist research grant, and the Wolfe Surgical Research Professorship in the Biology of Head and Neck Cancers grant.

T-Cell Transfer Therapy Targeting Mutant KRAS in Cancer.

We identified a polyclonal CD8+ T-cell response against mutant KRAS G12D in tumor-infiltrating lymphocytes obtained from a patient with metastatic colorectal cancer. We observed objective regression of all seven lung metastases after the infusion of approximately 1.11×1011 HLA-C*08:02-restricted tumor-infiltrating lymphocytes that were composed of four different T-cell clonotypes that specifically targeted KRAS G12D. However, one of these lesions had progressed on evaluation 9 months after therapy. The lesion was resected and found to have lost the chromosome 6 haplotype encoding the HLA-C*08:02 class I major histocompatibility complex (MHC) molecule. The loss of expression of this molecule provided a direct mechanism of tumor immune evasion. Thus, the infusion of CD8+ cells targeting mutant KRAS mediated effective antitumor immunotherapy against a cancer that expressed mutant KRAS G12D and HLA-C*08:02.

High-throughput combinatorial screening reveals interactions between signaling molecules that regulate adult neural stem cell fate.

Advancing our knowledge of how neural stem cell (NSC) behavior in the adult hippocampus is regulated has implications for elucidating basic mechanisms of learning and memory as well as for neurodegenerative disease therapy. To date, numerous biochemical cues from the endogenous hippocampal NSC niche have been identified as modulators of NSC quiescence, proliferation, and differentiation; however, the complex repertoire of signaling factors within stem cell niches raises the question of how cues act in combination with one another to influence NSC physiology. To help overcome experimental bottlenecks in studying this question, we adapted a high-throughput microculture system, with over 500 distinct microenvironments, to conduct a systematic combinatorial screen of key signaling cues and collect high-content phenotype data on endpoint NSC populations. This novel application of the platform consumed only 0.2% of reagent volumes used in conventional 96-well plates, and resulted in the discovery of numerous statistically significant interactions among key endogenous signals. Antagonistic relationships between fibroblast growth factor 2, transforming growth factor ß (TGF-ß), and Wnt-3a were found to impact NSC proliferation and differentiation, whereas a synergistic relationship between Wnt-3a and Ephrin-B2 on neuronal differentiation and maturation was found. Furthermore, TGF-ß and bone morphogenetic protein 4 combined with Wnt-3a and Ephrin-B2 resulted in a coordinated effect on neuronal differentiation and maturation. Overall, this study offers candidates for further elucidation of significant mechanisms guiding NSC fate choice and contributes strategies for enhancing control over stem cell-based therapies for neurodegenerative diseases.

Documentation and incidence of late effects and screening recommendations for adolescent and young adult head and neck cancer survivors treated with radiotherapy.

PURPOSE: A retrospective review of adolescent and young adult (AYA) head and neck cancer (HNC) patients treated with radiation therapy (RT) at British Columbia Cancer  was performed to determine the incidence of late toxicities, the documented late side effects discussed and the screening recommendations provided at the time of transfer of care to primary care providers (PCPs). METHODS: Charts (n = 162) were reviewed for all patients 15 to 35 years at diagnosis with HNC treated with RT from 1960 to 2010 who survived > 5 years after diagnosis. RESULTS: A discussion regarding the risk of long-term side effects was documented in the initial consultation for 85% of patients. The majority of patients (78%) developed > 1 documented late effect. The most common were xerostomia (44%), skin changes (28%), neck fibrosis (22%), nasal crusting (16%), epistaxis (16%), and dental decay (14%). In all, 20% were currently followed or were followed until they died. Of the 80% transferred to their PCP, 14% had a formal discharge summary. For those discharged from British Columbia Cancer, documented recommendations included regular dental care (34%) and screening for hypothyroidism (5%) and second malignancy (4%). CONCLUSIONS: The majority of AYA HNC patients treated with RT developed late side effects, and most PCPs were not sent a discharge summary outlining screening recommendations for delayed late effects. IMPLICATIONS FOR CANCER SURVIVORS: AYA HNC survivors treated with RT are at high risk for late effects and would benefit from a survivorship care plan outlining these risks and screening recommendations.

An Efficient Single-Cell RNA-Seq Approach to Identify Neoantigen-Specific T Cell Receptors.

The adoptive transfer of neoantigen-reactive tumor-infiltrating lymphocytes (TILs) can result in tumor regression in patients with metastatic cancer. To improve the efficacy of adoptive T cell therapy targeting these tumor-specific mutations, we have proposed a new therapeutic strategy, which involves the genetic modification of autologous T cells with neoantigen-specific T cell receptors (TCRs) and the transfer of these modified T cells back to cancer patients. However, the current techniques to isolate neoantigen-specific TCRs are labor intensive, time consuming, and technically challenging, not suitable for clinical applications. To facilitate this process, a new approach was developed, which included the co-culture of TILs with tandem minigene (TMG)-transfected or peptide-pulsed autologous antigen-presenting cells (APCs) and the single-cell RNA sequencing (RNA-seq) analysis of T cells to identify paired TCR sequences associated with cells expressing high levels of interferon-γ (IFN-γ) and interleukin-2 (IL-2). Following this new approach, multiple TCRs were identified, synthesized, cloned into a retroviral vector, and then transduced into donor T cells. These transduced T cells were shown to specifically recognize the neoantigens presented by autologous APCs. In conclusion, this approach provides an efficient procedure to isolate neoantigen-specific TCRs for clinical applications, as well as for basic and translational research.

Profiling grapevine trunk pathogens in planta: a case for community-targeted DNA metabarcoding.

BACKGROUND: DNA metabarcoding, commonly used in exploratory microbial ecology studies, is a promising method for the simultaneous in planta-detection of multiple pathogens associated with disease complexes, such as the grapevine trunk diseases. Profiling of pathogen communities associated with grapevine trunk diseases is particularly challenging, due to the presence within an individual wood lesion of multiple co-infecting trunk pathogens and other wood-colonizing fungi, which span a broad range of taxa in the fungal kingdom. As such, we designed metabarcoding primers, using as template the ribosomal internal transcribed spacer of grapevine trunk-associated ascomycete fungi (GTAA) and compared them to two universal primer widely used in microbial ecology. RESULTS: We first performed in silico simulations and then tested the primers by high-throughput amplicon sequencing of (i) multiple combinations of mock communities, (ii) time-course experiments with controlled inoculations, and (iii) diseased field samples from vineyards under natural levels of infection. All analyses showed that GTAA had greater affinity and sensitivity, compared to those of the universal primers. Importantly, with GTAA, profiling of mock communities and comparisons with shotgun-sequencing metagenomics of field samples gave an accurate representation of genera of important trunk pathogens, namely Phaeomoniella, Phaeoacremonium, and Eutypa, the abundances of which were over- or under-estimated with universal primers. CONCLUSIONS: Overall, our findings not only demonstrate that DNA metabarcoding gives qualitatively and quantitatively accurate results when applied to grapevine trunk diseases, but also that primer customization and testing are crucial to ensure the validity of DNA metabarcoding results.

Stable, Nonviral Expression of Mutated Tumor Neoantigen-specific T-cell Receptors Using the Sleeping Beauty Transposon/Transposase System.

Neoantigens unique to each patient's tumor can be recognized by autologous T cells through their T-cell receptor (TCR) but the low frequency and/or terminal differentiation of mutation-specific T cells in tumors can limit their utility as adoptive T-cell therapies. Transfer of TCR genes into younger T cells from peripheral blood with a high proliferative potential could obviate this problem. We generated a rapid, cost-effective strategy to genetically engineer cancer patient T cells with TCRs using the clinical Sleeping Beauty transposon/transposase system. Patient-specific TCRs reactive against HLA-A*0201-restriced neoantigens AHNAK(S2580F) or ERBB2(H473Y) or the HLA-DQB*0601-restricted neoantigen ERBB2IP(E805G) were assembled with murine constant chains and cloned into Sleeping Beauty transposons. Patient peripheral blood lymphocytes were coelectroporated with SB11 transposase and Sleeping Beauty transposon, and transposed T cells were enriched by sorting on murine TCRß (mTCRß) expression. Rapid expansion of mTCRß(+) T cells with irradiated allogeneic peripheral blood lymphocytes feeders, OKT3, interleukin-2 (IL-2), IL-15, and IL-21 resulted in a preponderance of effector (CD27(-)CD45RA(-)) and less-differentiated (CD27(+)CD45RA(+)) T cells. Transposed T cells specifically mounted a polyfunctional response against cognate mutated neoantigens and tumor cell lines. Thus, Sleeping Beauty transposition of mutation-specific TCRs can facilitate the use of personalized T-cell therapy targeting unique neoantigens.