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Aims Development of novel targeted therapies directed against hepatocyte growth factor (HGF) or its receptor (MET) necessitates the availability of quality diagnostics to facilitate their safe and effective use. Limitations of some commercially available anti‐MET antibodies have prompted development of the highly sensitive and specific clone A2H2‐3. Here we report its analytical properties when applied by an automated immunohistochemistry method. Methods and results Excellent antibody specificity was demonstrated by immunoblot, ELISA, and IHC evaluation of characterised cell lines including NIH3T3 overexpressing the related kinase MST1R (RON). Sensitivity was confirmed by measurements of MET in cell lines or characterised tissues. IHC correlated well with FISH and quantitative RT‐PCR assessments of MET (P < 0.001). Good total agreement (89%) was observed with the anti‐MET antibody clone SP44 using whole‐tissue sections, but poor positive agreement (21–47%) was seen in tissue microarray cores. Multiple lots displayed appropriate reproducibility (R2 > 0.9). Prevalence of MET positivity by IHC was higher in non‐squamous cell NSCLC, MET or EGFR amplified cases, and in tumours harbouring abnormalities in EGFR exon 19 or 21. Conclusions The anti‐MET antibody clone A2H2‐3 displays excellent specificity and sensitivity. These properties make it suitable for clinical trial investigations and development as a potential companion diagnostic.

Context.— RET gene fusions are oncogenic drivers in nonsmall cell lung cancer and nonmedullary thyroid cancer. Selpercatinib (RETEVMO), a targeted inhibitor of RET, was approved by the US Food and Drug Administration for the treatment of RET fusion–positive nonsmall cell lung cancer and nonmedullary thyroid cancer emphasizing the need for rapid and accurate diagnosis of RET fusions. Fluorescence in situ hybridization (FISH) has been used to detect gene rearrangements, but its performance detecting RET rearrangements is understudied. Objective.— To validate and describe the performance of Abbott Molecular RET break-apart FISH probes for detecting RET rearrangements. Design.— A training set with RET fusion–positive (13) and RET fusion–negative nonsmall cell lung cancer and nonmedullary thyroid cancer samples (12) was used to establish criteria for FISH scoring. The scoring criteria was then applied to a larger validation set of samples (96). Results.— A cutoff of 19% or more positive nuclei by FISH was established in the training set and determined by the mean ±3 SD. The validation set was tested using Abbott Molecular RET break-apart FISH compared with sequencing. With this cutoff, a sensitivity of 86% (12 of 14) and specificity of 99% (81 of 82) was achieved. Bootstrapping showed sensitivity could be optimized by using a greater than 13% cutoff with indeterminate samples of 13% to 18% abnormal nuclei requiring confirmation by an orthogonal method. Using this 3-tier scoring system sensitivity increased to 100% (14 of 14) and specificity was 96% (79 of 82). Conclusions.— Abbott Molecular break-apart FISH probes can be used to detect RET fusions. Laboratories can optimize cutoffs and/or testing algorithms to maximize sensitivity and specificity to ensure appropriate patients receive effective, timely therapy.

An indirect immunoperoxidase technique was used to study by light microscopy the binding of serum from experimental autoimmune myasthenia gravis (EAMG) rabbits to junctionally and extrajunctionally located acetylcholine receptors (AChRs) in human and rat muscles. Binding was restricted to junctional AChR. Alpha bungarotoxin (a-BGT) partially blocked the binding of EAMG serum, while myasthenia gravis serum, carbamylcholine, decamethonium, and tubocurarine did not. A radioimmunoassay showed significant binding of antibodies in EAMG sera to 125l AChR. This binding was not inhibited by a-BGT, nor by carbamylcholine, decamethonium, or tubocurarine. Sera from 10 myasthenia gravis patients did not contain antibodies binding to the 125l AChR. We suggest that EAMG in rabbits induced by Torpedo AChR differs serologically from myasthenia gravis in patients, probably owing to antigenic differences between Torpedo and human AChR, and that antigenic differences also exist between junctional and extrajunctional receptors.

The recent success of vitamin D and its analogues in the treatment of psoriasis has generated extensive research into the role of vitamin D and calcium in this hyperproliferative skin disease. The ohjective of this study was to evaluate calcium and phosphorus levels in patients admitted to our dermatology service for treatment of severe psoriasis. The charts of 15 patients from 21 to 80 years of age were reviewed and admission lahoratory data values recorded. Retrospective analysis of laboratory data from patients admitted with psoriasis demonstrated no abnormalities of calcium metabolism when assessed by calcium and phosphorus levels. Although vitamin D and its analogues appear to have a role in the treatment of psoriasis, this study suggests that a systemic aberration of calcium metabolism probably does not occur.

Background: MET amplification (amp) is a resistance mechanism to EGFR TKI treatment. Emibetuzumab, a bivalent MET antibody (Ab) blocks HGF binding to MET and internalizes the receptor. Combination of emibetuzumab with EGFR TKIs (erlotinib, AZD9291, CO1686) or EGFR Ab (necitumumab, cetuximab) was evaluated in 3 ER xenograft models. Methods: Model 1: ER cell line HCC827ERL with high focal MET amp, high pMET, EGFR ex19 del (no T790M) was created from parental HCC827 NSCLC (EGFR ex19 del, EGFR amp, no MET amp) by increasing concentration of erlotinib in vitro over 7 months. Model 2: ER cell line HCC827-A8 was derived from HCC827 parental xenograft tumor serially passed in vivo with long term treatment of gefitinib and erlotinib. HCC827-A8 cells express high focal MET amp, high pMET/AXL (Western blot) while retaining EGFR ex19 del (no T790M). Model 3: LU0858 was an ER patient-derived NSCLC xenograft tumor, with focal MET amp, EGFR L858R (no T790M). MET amp and EGFRmt was determined by FISH and LNA-PCR sequencing respectively. Compound dosing: emibetuzumab 20 mg/kg qw; necitumumab 4 mg/kg or 20 mg/kg biw; cetuximab 4 mg/kg biw; erlotinib 25 mg/kg qd; 5 mg/kg AZD9291 qd; 30 mg/kg CO1686 bid. Results: EGFR inhibitors, but not emibetuzumab showed significant single agent anti-tumor effect in xenograft tumors derived from non-MET amp HCC827 parental cells. In MET amp ER models, single agent emibetuzumab resulted in tumor growth inhibition in Model 1 (T/C= 51.7%-61.0%, p&lt;0.05) and 3 (T/C=2.8%, p&lt;0.05)] but no tumor regression, and no anti-tumor effect in Model 2. Where evaluated, EGFR inhibitors showed no anti-tumor effect in the 3 ER models as monotherapy, except necitumumab (20 mg/kg) in Model 1 (T/C = 36.2%, p&lt;0.05). However, combination of emibetuzumab with AZD9291, CO1686, necitumumab (20 mg/kg), or erlotinib resulted in 80.4%, 58.2%, 44.4%, 69.1% tumor regression respectively (p&lt;0.001) in Model 1, while emibetuzumab + cetuximab (4 mg/kg) resulted in tumor stasis (T/C=0.2%, p&lt;0.05). In Model 2, emibetuzumab + AZD9291 resulted in tumor stasis (T/C = 12.9%, p&lt;0.05). In Model 3, emibetuzumab + necitumumab (20 mg/kg) resulted in 80.1% tumor regression (p&lt;0.001). Conclusion: The three erlotinib resistant models with MET amp and retaining sensitizing EGFRmt (ex19 del or L858R), and no acquired T790M were found resistant to other EGFR inhibitors (Abs and TKIs). Emibetuzumab in combination with either EGFR TKI or Ab showed anti-tumor activity in MET amp ER xenograft models including tumor regression in 2 out of 3 models. The combination of emibetuzumab with erlotinib is being evaluated in NSCLC patients with EGFR activating mutation (NCT01897480). Citation Format: Suzane L. Um, Victoria L. Peek, Jennifer R. Stephens, Jessica A. Baker, Holly K. Cannon, Joel D. Cook, Isabella H. Wulur, Roger Agyei, Sudhakar Chintharlapalli, Robert J. Evans, William J. Feaver, Lysiane Huber, Linda N. Lee, Ling Liu, Liandong Ma, Ruslan Novosiadly, Volker Wacheck, Sau-Chi Betty Yan. Antitumor activity of MET antibody emibetuzumab (LY2875358) in combination with EGFR inhibitors in erlotinib resistant (ER) xenograft mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 519. doi:10.1158/1538-7445.AM2017-519

Cholangiocarcinoma (CCA) is an aggressive and locally invasive biliary tract malignancy with a poor prognosis and no approved drug treatment. LY2801653 is an orally bioavailable small molecule oncokinase inhibitor1 of MET, a receptor reported to be dysregulated and correlated with a poor outcome in CCA. We evaluated additional oncotargets of LY2801653 (MET/HGF, pMET, AXL/pAXL, the MKNK1/2 substrate p-eIF4E, and ROS1) in 7 CCA cell lines (EGI-1, TFK-1, SNU245, SNU478, SNU869, SNU1079, and SNU1196) and 5 CCA patient-derived mouse xenograft (PDX) tumors. Cell line expression of MET, EGFR and p-eIF4E were evaluated by western blot. Although HGF expression was not detected in any of the cell lines by ELISA, pMET was detected in 5 cell lines (not in TFK-1 or SNU1079). ROS1 fusion was not detected in the 7 cell lines using break-apart FISH probes. Very high AXL and pAXL were detected in the SNU1196 line by western blot. Despite the high levels of pAXL, further increases in pAXL were noted after addition of its ligand, Gas6. In the SNU1196 cell line, pAXL expression and cell proliferation were completely inhibited by LY2801653, but not by a MET-specific inhibitor PF4217903. HGF-induced SNU1196 cell migration was inhibited equally well by LY2801653 and PF4217903. Unlike CCA cell line derived mouse xenograft tumors, the 5 PDX tumors retained prominent desmoplastic stroma. As analyzed by immunohistochemistry (IHC), MET was highly expressed in 3 of the 5 PDX tumors, and high levels of p-eIF4E were expressed in all 5. Increased AXL expression (IHC) appeared to be associated with more poorly differentiated PDX tumors. LY2801653 demonstrated potent in vivo anti-tumor activity in several CCA xenograft models. In a xenograft mouse model with the extrahepatic CCA derived EGI-1 cell line, treatment with LY2801653 at 24 mg/kg dosed twice daily resulted anti-tumor effect of 38.5% (% treated/control). In the SNU869 extrahepatic CCA cell line-derived xenograft mouse model, combination treatment with LY2801653 (12 mg/kg twice daily) and either cisplatin (5 mg/kg once weekly) or gemcitabine (60 mg/kg once weekly) resulted in an additive anti-tumor effect as compared to LY2801653 alone. Moreover, the combination of LY2801653 with gemcitabine resulted in tumor regression in this model. In vivo studies are ongoing with LY2801653 in the extrahepatic SNU1196 CCA cell line-derived xenograft model as well as two of the PDX models. The preclinical data in this study support the ongoing phase 1 clinical evaluation of LY2801653 in cholangiocarcinoma patients (trial I3O-MC-JSBA, NCT01285037). (1 - Yan et al. Invest New Drugs 2013;31:833-44). Citation Format: Sau-Chi Betty Yan, Suzane L. Um, Victoria L. Peek, Megan N. Thobe, Kelly M. Credille, Jennifer R. Stephens, Jason R. Manro, Darryl W. Ballard, Jessica A. Baker, Joel D. Cook, Bruce W. Konicek, Jeremy R. Graff, Timothy R. Holzer, Richard A. Walgren. Preclinical evaluation of LY2801653, an orally bioavailable small molecule oncokinase inhibitor, in cholangiocarcinoma models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4403. doi:10.1158/1538-7445.AM2014-4403

Several immunological variables were examined in patients receiving high-single-dose, alternate-day prednisone therapy for neuromuscular diseases. Dose-dependent leukocytosis, lymphopenia, and monocytopenia occurred which were maximal 6 hours after prednisone administration but returned to control levels by the 24-hour point. The lymphopenia involved T-cells, B-cells, and null cells, with the T-cells most affected. Plasma cortisol levels and lymphocyte transformation in response to mitogens were also transiently and reversibly suppressed. There was a persistent decrease in serum IgG. Lymphocyte transformation was also suppressed when normal lymphocytes were incubated with treated patient sera or when treated patient lymphocytes were incubated in autologous pretreatment sera. The suppression factor was not removed from the lymphocytes by extensive washing. Patients whose disease responded to the high-single-dose, alternate-day prednisone regiment were indistinguishable from nonresponders by the immunological responses measured.