Background: The presence of bacteria and fungi in medicinal or recreational Cannabis poses a potential threat to consumers if those microbes include pathogenic or toxigenic species. This study evaluated two widely used culture-based platforms for total yeast and mold (TYM) testing marketed by 3M Corporation and Biomérieux, in comparison with a quantitative PCR (qPCR) approach marketed by Medicinal Genomics Corporation. Methods: A set of 15 medicinal Cannabis samples were analyzed using 3M and Biomérieux culture-based platforms and by qPCR to quantify microbial DNA. All samples were then subjected to next-generation sequencing and metagenomics analysis to enumerate the bacteria and fungi present before and after growth on culture-based media. Results: Several pathogenic or toxigenic bacterial and fungal species were identified in proportions of >5% of classified reads on the samples, including Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa, Ralstonia pickettii, Salmonella enterica, Stenotrophomonas maltophilia, Aspergillus ostianus, Aspergillus sydowii, Penicillium citrinum and Penicillium steckii. Samples subjected to culture showed substantial shifts in the number and diversity of species present, including the failure of Aspergillus species to grow well on either platform. Substantial growth of Clostridium botulinum and other bacteria were frequently observed on one or both of the culture-based TYM platforms. The presence of plant growth promoting (beneficial) fungal species further influenced the differential growth of species in the microbiome of each sample. Conclusions: These findings have important implications for the Cannabis and food safety testing industries.

The Center for Disease Control estimates 128,000 people in the U.S. are hospitalized annually due to food borne illnesses. This has created a demand for food safety testing targeting the detection of pathogenic mold and bacteria on agricultural products. This risk extends to medical Cannabis and is of particular concern with inhaled, vaporized and even concentrated Cannabis products . As a result, third party microbial testing has become a regulatory requirement in the medical and recreational Cannabis markets, yet knowledge of the Cannabis microbiome is limited. Here we describe the first next generation sequencing survey of the fungal communities found in dispensary based Cannabis flowers by ITS2 sequencing, and demonstrate the sensitive detection of several toxigenic Penicillium and Aspergillus species, including P. citrinum and P. paxilli, that were not detected by one or more culture-based methods currently in use for safety testing.

Rare genetic variants in the core endocannabinoid system genes CNR1, CNR2, DAGLA, MGLL and FAAH were identified in molecular testing data from 6,032 patients with a broad spectrum of neurological disorders. The variants were evaluated for association with phenotypes similar to those observed in the orthologous gene knockouts in mice. Heterozygous rare coding variants in CNR1, which encodes the type 1 cannabinoid receptor (CB1), were found to be significantly associated with pain sensitivity (especially migraine), sleep and memory disorders—alone or in combination with anxiety—compared to a set of controls without such CNR1 variants. Similarly, heterozygous rare variants in DAGLA, which encodes diacylglycerol lipase alpha, were found to be significantly associated with seizures and neurodevelopmental disorders, including autism and abnormalities of brain morphology, compared to controls. Rare variants in MGLL, FAAH and CNR2 were not associated with any neurological phenotypes in the patients tested. Diacylglycerol lipase alpha synthesizes the endocannabinoid 2-AG in the brain, which interacts with CB1 receptors. The phenotypes associated with rare CNR1 variants are reminiscent of those implicated in the theory of clinical endocannabinoid deficiency syndrome. The severe phenotypes associated with rare DAGLA variants underscore the critical role of rapid 2-AG synthesis and the endocannabinoid system in regulating neurological function and development. Mapping of the variants to the 3D structure of the type 1 cannabinoid receptor, or primary structure of diacylglycerol lipase alpha, reveals clustering of variants in certain structural regions and is consistent with impacts to function.

Rare genetic variants in the core endocannabinoid system genes CNR1, CNR2, DAGLA, MGLL and FAAH were identified in molecular testing data from up to 6.032 patients with a broad spectrum of neurological disorders. The variants were evaluated for association with phenotypes similar to those observed in the orthologous gene knockouts in mice. Heterozygous rare coding variants in CNR1, which encodes the type 1 cannabinoid receptor (CB1), were found to be significantly associated with pain sensitivity (especially migraine), sleep and memory disorders -alone or in combination with anxiety -compared to a set of controls without such CNR1 variants. Similarly, heterozygous rare variants in DAGLA, which encodes diacylglycerol lipase alpha, were found to be significantly associated with seizures and developmental disorders, including abnormalities of brain morphology, compared to controls. Rare variants in MGLL, FAAH and CNR2 were not associated with any neurological phenotypes in the patients tested. Diacylglycerol lipase alpha synthesizes the endocannabinoid 2-AG in the brain, which interacts with CB1 receptors. The phenotypes associated with rare CNR1 variants are reminiscent of those implicated in the theory of clinical endocannabinoid deficiency syndrome. The severe phenotypes associated with rare DAGLA variants underscore the critical role of rapid 2-AG synthesis and the endocannabinoid system in regulating neurological function and development. Mapping of the variants to the 3D structure of the type 1 cannabinoid receptor, or primary structure of diacylglycerol lipase alpha, reveals clustering of variants in certain structural regions and is consistent with impacts to function.

The Center for Disease Control estimates 128,000 people in the U.S. are hospitalized annually due to food borne illnesses. This has created a demand for food safety testing targeting the detection of pathogenic mold and bacteria on agricultural products. This risk extends to medical Cannabis and is of particular concern with inhaled, vaporized and even concentrated Cannabis products. As a result, third party microbial testing has become a regulatory requirement in the medical and recreational Cannabis markets, yet knowledge of the Cannabis microbiome is limited. Here we describe the first next generation sequencing survey of the microbial communities found in dispensary based Cannabis flowers and demonstrate the limitations in the culture-based regulations that are being superimposed from the food industry.

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A297diagnostic odyssey (i.e., a prolonged and complex diagnostic journey), informing prognosis, treatment selection and patient management. Physicians surveyed indicated prolonged diagnostic odyssey prior to making a definitive diagnosis of genetic etiology may result in uncontrolled disease for longer than necessary, suboptimal or potentially harmful treatments, undue healthcare resource use and, in some cases, adverse impact on development and cognition, as well as increased risk of physical injury such as from falls, aspiration, and anoxic brain damage. ConClusions: Optimized use of multigene sequencing panels could allow for earlier and more efficient diagnosis of underlying causes of seizure disorders. Information provided facilitates clinical management decisions, potentially improving clinical outcomes and quality-of-life, and reducing healthcare resource utilization for pediatric patients. Multigene sequencing panel testing offers a more cost-efficient method to obtain an earlier diagnosis for patients with epilepsy.

9042 Background: The administration of a lung cancer-specific immunotherapy with 22 tumor-associated antigens (ADXS-503, A503), has been evaluated as an add-on therapy for patients (pts) with metastatic non-small-cell lung cancer (NSCLC) who have progressed on pembrolizumab (pembro) as last therapy [Haigentz M et al. ASCO 2021]. The present study explores the immunogenicity and potential reversal of immune resistance with A503 when added-on to pembro at the time of progressive disease (PD). Methods: A phase 2 study of A503 + pembro is being conducted in pts with metastatic squamous or non-squamous NSCLC. In Part B of the study, A503 was added-on to pembro within 12 weeks after the first scan showing disease progression following pembro therapy (per RECIST criteria v1.1). Both A503 (1x108 CFU) and pembro (200 mg) were infused by IV every 3 weeks until disease progression or dose-limiting toxicity. Immunogenicity assays included serum cytokine and chemokine levels; flow cytometry; and in-vitro stimulation FluoroSpot assay with 4 different antigen-pools represented in A503 [i.e., hot spot mutations, heteroclitic/wild-type tumor-associated antigens and other antigens not included in the A503 construct (antigen spreading)]. Results: A total of 14 pts have been treated in Part B, of which 13 are clinically evaluable and up to 11 have immune assessments. Combination therapy was well tolerated with transient increased secretion of cytokines for several hours after infusion of A503 consistent with the expected immune activation and transient ‘flu-like’ syndrome. The objective response rate (16%) and disease control rate (46%) were encouraging with 2 partial responses (PR), 4 stable diseases (SD) and 7 pts with PD. Pts with disease control, in particular, generated CD8+ T cells reactive to neoantigens in 1 or more of the 4 antigen pools tested in FluoroSpot. Also, activation of NK cells and of cytotoxic- and memory-CD8+ T cells was mainly observed in pts with PR or SD, but not in those with PD, as shown in the table. Conclusions: Adding A503 to pembro after PD appears to induce innate and adaptive immune responses that may restore or enhance sensitivity to checkpoint inhibitors in pts with clinical benefit. Clinical trial information: NCT03847519. [Table: see text]