Neogen (United States)

e15180 Background: Pancreatic cancer has apoor prognosis and limited therapeutic options. While K-RAS mutations are prevalent in this malignancy, a subset of patients harboring wild-type K-RAS presents distinct clinical behavior. Gene fusions have been discerned in pancreatic cancer and are promising candidates for administering targeted therapies. Methods: We conducted a retrospective analysis of 926 pancreatic cancer patients of which 165 (18%) carried a wild-type K-RAS gene, by next generation sequencing (NGS) or polymerase chain reaction (PCR) testing. Tumors were analyzed with a clinical-grade hybrid capture-based RNA-seq clinical fusionome test, evaluating gene fusions between 1104 genes or to other known or novel partners as well as gene expression transcript analysis. Single nucleotide variants/insertions deletions (SNV/Indels) were tested with a clinical-grade NGS assay targeting > 300 cancer genes. Results: Gene fusions were detected in 32% of K-RAS wild-type pancreatic cancers, with 75% involving known oncogenic, clinically actionable genes including FGFR (two FGFR2 and one FGFR1 fusion), two NTRK3 and one each of RAF1, RET, ROS1 and BRAF fusions. Deleterious SNVs were frequently found in both fusion-positive and fusion-negative K-RAS wild-type pancreatic cancers. Transcriptomic expression revealed additional potentially druggable targets, e.g., ROS1 and FGFR3 overexpression in a tumor harboring an NTRK3 fusion, BRAF overexpression in the context of an otherwise non-actionable ETV6 fusion, and MET overexpression in a TES::TFEC -fusion bearing cancer. Conclusions: Druggable fusions are frequent in K-RAS wild-type pancreatic cancers. Moreover, RNA fusion assays as well as transcriptomic expression testing can identify candidate personalized therapies for patients with K-RAS wild-type pancreatic cancer.

Castleman Disease (CD) is a rare lymphoproliferative disorder that can be separated into two primary forms: Unicentric Castleman disease (UCD) and multicentric Castleman disease (MCD). UCD is localized, while MCD is systemic. Though UCD generally has a favorable prognosis following surgical resection, more aggressive forms of this disease have been identified, including cases associated with dendritic and spindle cell proliferation. Genetic analysis has deepened our understanding of UCD. Despite advancements in better understanding the pathophysiology of UCD, challenges persist in the diagnosis, management, and treatment due to its rarity and heterogeneity. Here, we review current knowledge on UCD, highlighting the epidemiology, clinical presentation, diagnostic criteria, and treatment options while emphasizing the need for further research and innovation in therapeutic strategies.

The linear conformation of animals exerts an influence on health, reproduction, production, and welfare, in addition to longevity, which directly affects the profitability of milk-producing farms. The objectives of this study were (1) to perform genome-wide association studies (GWASs) of conformation traits, namely the Rump, Feet and Legs, Mammary System, Dairy Strength, and Final Classification traits, and (2) to identify genes and related pathways involved in physiological processes associated with conformation traits in Brazilian Holstein cattle. Phenotypic and genotypic data from 2339 Holstein animals distributed across the states of Rio Grande do Sul, Paraná, São Paulo, and Minas Gerais were used. The genotypic data were obtained with a 100 K SNP marker panel. The single-step genome-wide association study (ssGWAS) method was employed in the analyses. Genes close to a significant SNP were identified in an interval of 100 kb up- and downstream using the Ensembl database available in the BioMart tool. The DAVID database was used to identify the main metabolic pathways and the STRING program was employed to create the gene regulatory network. In total, 36 significant SNPs were found on 15 chromosomes; 27 of these SNPs were linked to genes that may influence the traits studied. Fourteen genes most closely related to the studied traits were identified, as well as four genes that showed interactions in important metabolic pathways such as myogenesis, adipogenesis, and angiogenesis. Among the total genes, four were associated with myogenesis (TMOD2, TMOD3, CCND2, and CTBP2), three with angiogenesis (FGF23, FGF1, and SCG3), and four with adipogenesis and body size and development (C5H12orf4, CCND2, EMILIN1, and FGF6). These results contribute to a better understanding of the biological mechanisms underlying phenotypic variability in conformation traits in Brazilian Holstein cattle.

e15186 Background: Gene fusions have important implications for therapeutic selection and patient quality of care and many fusions are targeted effectively in a tumor type agnostic manner. While most assays detect hundreds of fusions at once, typically the results are masked and analyzed according to medical requisition. Unfortunately, patients in the community setting most often are only tested for NTRK fusions, which have a prevalence of ~ 1%, and often following a positive IHC screen result. Here we compared side by side the clinical actionability gap in the community when using panTRK IHC testing and NGS fusion panels for NTRKs or for additional fusions, with both NGS panels selected presenting identical assay performance characteristics and medicare coverage. Methods: Clinical samples (n = 8307) from 33 tumor types were tested for RNA fusions using a hybridization capture RNA-seq based fusionome assay for solid tumors in our clinical laboratory, targeting RNA breakpoints at 1104 genes. Deidentified results were analyzed for either the 19 actionable fusion genes (ALK, BRAF, FGFR1-4, MET, NOTCH1/2, NRG1, NTRK1-3, PDGFB, PDGFRA/B, RAF1, RET and ROS1) reportable under Solid Tumor NGS fusion Panel, or only for NTRK fusions. Pan-TRK IHC results on fusion positive patients were analyzed. All the Deidentified patient data presented, was analyzed according to an IRB-approved protocol. Results: Targeted therapy -associated fusions were present in 422 (5.1%) patients, where 53% of patients were female with a median age of 68 years old, and 47% were male with a median age of 70 years old. However, when filtering only for NTRK fusions, just 104 (1.25%) patients had an actionable fusion (38 NTRK1, 10 NTRK2, 56 NTRK3). The additional 318 (3.8%) patients had other therapy candidate fusions: ALK (0.6%), BRAF 0.3%, FGFRs 0.9%, MET 0.4%, NOTCH2 0.2%, NRG1 0.2%, PDGFRA 0.1%, RAF1 0.2%, RET 0.5%, ROS1 0.2%, excluding NTRK positive cases. There were only 2 (0.02%) cases that were positive for both an NTRK fusion and an additional Targeted Panel fusion gene. Interestingly, we found that IHC testing was able to identify 75% of NTRK1 and 55% NTRK2 positive samples but only 20% of NTRK3 fusion positive patients. Moreover only 54% of the panTRK IHC positive patients expressed and NTRK fusion while 46% expressed one of the other 16 druggable fusions, with FGFRs on 20%, and ALK and ROS1 with 4% each. Conclusions: The data demonstrates that up to 4 -fold more patients can have a fusion matched therapy when testing for these 19 druggable fusions at once as compared to testing only for NTRK fusions or following IHC testing. Moreover, IHC testing as a screening method as very poor sensitivity for NTRK fusions, specially for NTRK3. Changes on the molecular testing paradigm in the community setting is needed to provide more patients with a therapeutic opportunity, when assay performance and financial considerations are equal to increase cancer care access.

Pathogens such as porcine epidemic diarrhea virus (PEDV), porcine reproductive and respiratory syndrome (PRRSV), and E. coli are known to spread by contaminated vehicles and equipment. Pork producers have adopted trailer wash policies where each trailer is washed, disinfected, and dried before it can return to a farm. Cleanliness of livestock trailers after washing is determined by visual inspection rather than any objective method. Adenosine triphosphate (ATP) bioluminescence is used in many industries to provide real-time feedback on surface cleanliness through the detection of ATP from organic sources. That same technology may provide trailer wash facilities a way of objectively characterizing a livestock trailer's suitability to return to a farm after washing. Two ATP luminometers (3M Clean-Trace and Neogen AccuPoint) were used to estimate the correlation between ATP bioluminescence readings and aerobic bacterial plate counts (APCs) from sampled surfaces and to determine locations within a livestock trailer that can accurately estimate surface cleanliness. Five locations in livestock trailers were evaluated. Those locations included the nose access door (NAD), back door flush gate, rear side access door (RSAD), belly flush gate (BFG), and belly side access door (BSAD). There was a positive log-log association between the two luminometers (r = 0.59, P < 0.01). Every log unit increase in one unit, resulted in a 0.42 log increase (P < 0.01) in the other unit. ATP can come from bacteria, yeasts, molds, and manure. There was a poor association (r ≥ 0.10, P ≥ 0.02) between APCs and the ATP luminometers. Still, an increase in relative light units (RLUs) resulted in a corresponding increase in colony-forming units. The greatest area of surface contamination measured by APC was the NAD. RLUs were also greater in the NAD compared to the RSAD, the BFG, and the BSAD (P ≤ 0.01). Because APCs and luminometer RLUs provided similar outcomes, statistical process control charts were developed to determine control limits for RLUs. This provides real-time feedback to trailer wash workers in determining cleanliness outcomes for livestock trailers. These data suggest that ATP bioluminescence can be a reliable method to monitor cleaning effectiveness in livestock trailers. Bioluminescence is a monitoring tool that should be used in conjunction with microbial methods to monitor procedures for cleaning and disinfection.

Reveal® 3-D for Gluten is an immunochromatographic assay for the qualitative detection of gluten in environmental samples. The test uses monoclonal antibodies reactive to prolamins in wheat. The objective of the study was to validate the Reveal 3-D test for detection of gluten in clean-in-place rinse and swabs from a stainless steel surface. Elements of the study included food selectivity and interference testing, matrix testing, an assay robustness study, and reagent stability/lot-to-lot consistency testing. Wheat flour was used as the spiking material for all matrixes. In selectivity and interference testing, nine target matrixes all tested positive and 36 of 39 non-target matrixes tested negative. Almond flour, sesame flour, and cornstarch produced positive results as 100% commodities; reactivity can be eliminated with dilution or by testing without use of food extraction buffer, which is not a standard part of the environmental testing method. With a gluten spike at 9.3 mg/kg, chestnut flour, guar gum, and xanthan gum as 100% commodities inhibited the ability of the assay to detect gluten when tested without dilution. In quaternary ammonium clean-in-place rinse and swabs from stainless steel, 100% positive results were obtained at levels of 2.8 mg/kg and 4.7 µg/100 cm2, respectively. Results of independent laboratory testing of swabs from stainless steel supported those of internal trials. Robustness testing showed that introducing variations to three operating parameters simultaneously had no adverse effect on assay performance. In the reagent stability study, data supported kit expiration dating of 11 months. Results of the current study show that the Reveal test is an accurate and reliable method for qualitative detection of gluten in select clean-in-place rinse and environmental samples. The Reveal test was able to detect gluten at levels of 2.8 ppm in clean-in-place rinse and 4.7 µg/100 cm2 in swabs from stainless steel.

An 8-wk-old, male, mixed-breed puppy was adopted from a rescue organization. From thetime of adoption, the puppy suffered episodes of illness affecting various organ systems,which resolved with supportive therapy but relapsed once medical therapy was discontinued.Review of the hematologic data revealed cyclic fluctuations in circulating blood cells.Cyclicity was most prominent in neutrophils, with recurrent severe neutropenia.Neutropenic episodes lasted 5–6 d, with regular cycles of 11–14 d between nadir neutrophilcounts. Genetic testing determined that the patient was homozygous mutant for theframeshift mutation in the adaptor protein complex 3 β-subunit (AP3B1)gene, originally identified in gray collies with cyclic hematopoiesis (CH). Pedigreeinformation was not available, but the patient’s features were phenotypically distinctfrom those of collies. We describe here a case of the AP3B1 mutation in amixed-breed dog that did not resemble a collie, undescribed previously, to our knowledge.Our findings indicate that the AP3B1 mutation and CH are present withinthe general canine population and are not restricted to collies.

Soleris® Direct Yeast and Mold (DYM) is a growth-based, automated method for detection of yeast and mold in select foods and other sample types including nutraceuticals and cosmetics. The Soleris method is used in a "dilute-to-specification" or threshold manner in which a result is scored as positive or negative around a predetermined cutoff (in CFU/g) established by the dilution and volume of sample homogenate tested. The objective of this study was to validate the method for testing of dried cannabis flower. The validation was conducted under the Emergency Response Validation program of the AOAC Research Institute. The study included inclusivity and exclusivity testing, in particular testing of yeast and mold species associated with cannabis, and a matrix study in which Soleris method presumptive results were compared with Soleris confirmed results using Dichloran Rose-Bengal Chloramphenicol (DRBC) agar for confirmation. Samples at four different levels of natural yeast and mold contamination were tested at two test thresholds. In inclusivity testing, all 63 yeast and mold strains tested produced positive results within the specified test duration of 72 h. In exclusivity testing, 36 of 37 strains tested produced no detection within 72 h. In matrix testing, there were no significant differences between Soleris presumptive and confirmed results for any contamination level or test threshold as determined by probability of detection analysis. Results indicate that the Soleris method is an effective procedure for detection of yeast and mold in dried cannabis flower. With the Soleris method, results are available within 72 h compared with the 5-7 days required for microbiological culture methods.