Correlation of novel ALKATI with ALK immunohistochemistry and clinical outcomes in metastatic melanoma.

AIMS: Recently, a novel isoform of anaplastic lymphoma kinase, with alternative transcription initiation (ALKATI ), has been described in melanoma and is susceptible to targeted ALK-inhibitor therapy. Clinical outcomes of patients with ALKATI mutated melanoma as well as correlation with immunohistochemical (IHC) methods have not yet been described. METHODS AND RESULTS: Clinicopathological characteristics were abstracted for 324 patients with metastatic melanoma (MM). IHC, fluorescence in-situ hybridisation and RNA-based digital molecular analysis assays were performed on archival tissue from 173 stage III and 192 stage IV tumours. ALKATI was identified in 12.7 and 4.8% stage III and IV tumours, respectively. Discrete presentations of the ALKATI are seen: isolated ALKATI (n = 20) and mixed ALKATI (combined ALKATI and ALKWT ; n = 7). Isolated ALKWT expression (n = 4) was seen with no ALK fusions. Stage III patients showed improved survival with ALKATI expression compared to those with ALKWT or no expression [5-year survival 80, 95% confidence interval (CI) = 57-100% versus 43%, 95% CI = 34-55%, P = 0.013]. Clinicopathological characteristics were not statistically significant. Strong diffuse cytoplasmic staining of ALK IHC (n = 12) has a sensitivity of 52.2%, specificity 100%, PPV of 100% and NPV of 92.5% of detecting isolated ALKATI . CONCLUSION: Presence of ALKATI is a good prognostic indicator in MM. ALK IHC and digital molecular analysis can be incorporated into MM evaluation to identify patients with ALKATI for targeted therapy.

Generation of An Endogenous FGFR2-BICC1 Gene Fusion/58 Megabase Inversion Using Single-Plasmid CRISPR/Cas9 Editing in Biliary Cells.

Fibroblast growth factor receptor 2 (FGFR2) gene fusions are bona fide oncogenic drivers in 10-15% of intrahepatic cholangiocarcinoma (CCA), yet currently there are no cell lines publically available to study endogenous FGFR2 gene fusions. The ability of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 to generate large yet precise chromosomal rearrangements has presented the possibility of engineering endogenous gene fusions for downstream studies. In this technical report, we describe the generation of an endogenous FGFR2-Bicaudal family RNA binding protein 1 (BICC1) fusion in multiple independent cholangiocarcinoma and immortalized liver cell lines using CRISPR. BICC1 is the most common FGFR2 fusion partner in CCA, and the fusion arises as a consequence of a 58-megabase-sized inversion on chromosome 10. We replicated this inversion to generate a fusion product that is identical to that seen in many human CCA. Our results demonstrate the feasibility of generating large megabase-scale inversions that faithfully reproduce human cancer aberrations.

Enterohemorrhagic E. coli alters murine intestinal epithelial tight junction protein expression and barrier function in a Shiga toxin independent manner.

Shiga toxin (Stx) is implicated in the development of hemorrhagic colitis and hemolytic-uremic syndrome, but early symptoms of enterohemorrhagic Escherichia coli (EHEC) infection such as nonbloody diarrhea may be Stx independent. In this study, we defined the effects of EHEC, in the absence of Stx, on the intestinal epithelium using a murine model. EHEC colonization of intestines from two groups of antibiotic-free and streptomycin-treated C57Bl/6J mice were characterized and compared. EHEC colonized the cecum and colon more efficiently than the ileum in both groups; however, greater amounts of tissue-associated EHEC were detected in streptomycin-pretreated mice. Imaging of intestinal tissues of mice infected with bioluminescent EHEC further confirmed tight association of the bacteria with the cecum and colon. Greater numbers of EHEC were also cultured from stool samples obtained from streptomycin-pretreated mice, as compared with those that received no antibiotics. Transmission electron microscopy shows that EHEC infection leads to microvillous effacement of mouse colonocytes. Hematoxylin and eosin staining of the colonic tissues of infected mice revealed a slight increase in the number of lamina propria polymorphonuclear leukocytes. Transmucosal electrical resistance, a measure of epithelial barrier function, was reduced in the colonic tissues of infected animals. Increased mucosal permeability to 4- kDa FITC-dextran was also observed in the colonic tissues of infected mice. Immunofluorescence microscopy showed that EHEC infection resulted in redistribution of the tight junction (TJ) proteins occludin and claudin-3 and increased the expression of claudin-2, whereas ZO-1 localization remained unaltered. Quantitative real-time PCR showed that EHEC altered mRNA transcription of OCLN, CLDN2, and CLDN3. Most notably, claudin-2 expression was significantly increased and correlated with increased intestinal permeability. Our data indicate that C57Bl/6J mice serve as an in vivo model to study the physiological effects of EHEC infection on the intestinal epithelium and suggest that altered transcription of TJ proteins has a role in the increase in intestinal permeability.

Whole Exome Sequencing and Molecular Modeling of a Missense Variant in TNFAIP3 That Segregates with Disease in a Family with Chronic Urticaria and Angioedema.

Chronic urticaria is a common condition characterized by recurrent hives lasting several weeks or months and is usually idiopathic. Approximately half of the individuals with chronic urticaria will present with episodes of angioedema that can be severe and debilitating. In this report, we describe a 47-year-old Hispanic male who presented initially for an evaluation of chronic hives following hospitalization due to hive-induced anaphylaxis. The individual had a history significant for urticaria and angioedema beginning in his early 30s. Interestingly, both the individual's 41-year-old sister and 12-year-old daughter were also affected with chronic urticaria and severe angioedema. Whole exome sequencing of the proband and several family members revealed a heterozygous variant of uncertain significance in exon 2 of TNFAIP3, denoted as c.65G>A (p.R22Q), in all affected members. Variants in TNFAIP3 have been associated with multiple autoimmune diseases, susceptibility to allergy and asthma, and periodic fever syndromes, suggesting that this variant could potentially play a role in disease.

Targeting the cyclin dependent kinase and retinoblastoma axis overcomes standard of care resistance in BRAF V600E -mutant melanoma.

Patient-derived tumor xenograft (PDTX) mouse models were used to discover new therapies for naïve and drug resistant BRAFV600E -mutant melanoma. Tumor histology, oncogenic protein expression, and antitumor activity were comparable between patient and PDTX-matched models thereby validating PDTXs as predictive preclinical models of therapeutic response in patients. PDTX models responsive and non-responsive to BRAF/MEK standard of care (SOC) therapy were used to identify efficacious combination therapies. One such combination includes a CDK4/6 inhibitor that blocks cell cycle progression. The rationale for this is that the retinoblastoma protein (pRb) is 95% wildtype in BRAF mutant melanoma. We discovered that 77/77 stage IV metastatic melanoma tissues were positive for inactive phosphorylated pRb (pRb-Ser780). Rb is hyperphosphorylated and inactivated by CDK4/6:cyclin D1 and when restored to its hypophosphorylated active form blocks cell cycle progression. The addition of a CDK4/6 inhibitor to SOC therapy was superior to SOC. Importantly, triple therapy in an upfront treatment and salvage therapy setting provided sustained durable response. We also showed that CDK4/6 blockade resensitized drug resistant melanoma to SOC therapy. Durable response was associated with sustained suppression of pRb-Ser780. Thus, reactivation of pRb may prove to be a clinical biomarker of response and the mechanism responsible for durable response. In light of recent clinical trial data using this triple therapy against BRAFV600E -mutant melanoma, our findings demonstrating superior and prolonged durable response in PDTX models portend use of this therapeutic strategy against naïve and SOC resistant BRAFV600E -mutant metastatic melanoma coupled with pRB-Ser780 as a biomarker of response.

Patient-derived tumor xenograft models for melanoma drug discovery.

INTRODUCTION: Cutaneous metastatic melanoma (MM) is an aggressive form of skin cancer, with treatment providing cures to a minority of patients. The multiple risk factors that contribute to MM development suggest that cutaneous melanomas embody a repertoire of altered genetic events requiring studies to better understand its biology in order to develop novel therapies. AREAS COVERED: Patient-derived tumor xenograft (PDTX) mouse models are noted to be superior for novel drug discovery and tumor biology studies due to their ability to maintain tumor heterogeneity and their use as real-time individualized patient models. In this review, the authors highlight the utility of PDTX models in advancing treatment options for patients with MM by creating invaluable preclinical models that exhibit patient-relevant treatment outcomes. EXPERT OPINION: There is a strong necessity to reassess current approaches in which preclinical experiments are designed and executed in order to minimize unwarranted clinical trials. With rigorously performed preclinical studies, PDTX models have the capability to effectively confirm or deny drug effective outcomes. The ability to do this, however, will demand better aids to guide experimental design, the redefining of preclinical efficacy, and the understanding that these models should be viewed as complementary to other drug prediction and efficacy tools.

Human papillomavirus vaccination: a case study in translational science.

Each year 610,000 cases of anogenital and oropharyngeal cancers caused by human papillomavirus (HPV) occur worldwide. HPV vaccination represents a promising opportunity to prevent cancer on a global scale. The vaccine's story dates back to discoveries in chickens at the beginning of the 20th century with evidence that a cell-free filtrate could transmit the propensity to grow cancers. Later, studies with similarly derived filtrates from mammalian tumors showed that hosts could develop immunity to subsequent exposures. Epidemiologic studies linked cervical cancer to members of a family of viruses that cause papillomatosis and common warts. This led to work with DNA hybridization demonstrating a causal relationship. The formation of virus-like particles from viral capsid proteins led to the development of models for safe and effective vaccines. While much work remains with the acceptance of universal vaccination, the HPV vaccines Gardasil and Cervarix thus represent a century of successful translational research.

Determination of spatial and temporal colonization of enteropathogenic E. coli and enterohemorrhagic E. coli in mice using bioluminescent in vivo imaging.

Infectious diarrhea is a major contributor of child morbidity and mortality in developing nations. Murine models to study the pathogenesis of infectious diarrhea caused by organisms such as enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC) are not fully characterized. More emphasis has been placed on infection of mice with the murine specific pathogen Citrobacter rodentium. While these three organisms are genetically related they are not identical. Our goal was to better characterize the murine model of EPEC and EHEC infection by using bioluminescent bacteria to determine temporal and spatial colonization of these two human pathogens. EPEC and EHEC were transformed with a bacterial luciferase expression plasmid containing the constitutive OmpC promoter. C57BL/6 mice were orally inoculated with bioluminescent EPEC or EHEC and bacterial localization in the intestine was monitored ex vivo and in vivo by IVIS. At 3 days after infection, EPEC, EHEC and Citrobacter rodentium were all localized in the cecum and colon. EPEC colonization peaked at day 2-3 and was undetectable by day 7. The bioluminescent EPEC adheres to the cecum and colon of the mouse intestine. However, when EPEC infected mice were administered xylazine/ketamine for in vivo live imaging, the EPEC persisted at high densities for up to 31 days. This is the first report of a bioluminescent imaging of luciferase expressing EPEC in a mouse model.