Supplementary Data from Mutation-Specific Antibodies for the Detection of EGFR Mutations in Non–Small-Cell Lung Cancer

Signalling and post-transcriptional gene control are both critical for the regulation of pluripotency, yet how they are integrated to influence cell identity remains poorly understood. LIN28 (also known as LIN28A), a highly conserved RNA-binding protein, has emerged as a central post-transcriptional regulator of cell fate through blockade of let-7 microRNA biogenesis and direct modulation of mRNA translation. Here we show that LIN28 is phosphorylated by MAPK/ERK in pluripotent stem cells, which increases its levels via post-translational stabilization. LIN28 phosphorylation had little impact on let-7 but enhanced the effect of LIN28 on its direct mRNA targets, revealing a mechanism that uncouples LIN28's let-7-dependent and -independent activities. We have linked this mechanism to the induction of pluripotency by somatic cell reprogramming and the transition from naive to primed pluripotency. Collectively, our findings indicate that MAPK/ERK directly impacts LIN28, defining an axis that connects signalling, post-transcriptional gene control, and cell fate regulation.

Abstract Purpose: In classical Hodgkin lymphoma, the malignant Reed–Sternberg cells express the cell surface marker CD30. Brentuximab vedotin is an antibody–drug conjugate (ADC) that selectively delivers a potent cytotoxic agent, monomethyl auristatin E (MMAE), to CD30-positive cells. Although brentuximab vedotin elicits a high response rate (75%) in relapsed/refractory Hodgkin lymphoma, most patients who respond to brentuximab vedotin eventually develop resistance. Patients and Methods: We developed two brentuximab vedotin–resistant Hodgkin lymphoma cell line models using a pulsatile approach and observed that resistance to brentuximab vedotin is associated with an upregulation of multidrug resistance-1 (MDR1). We then conducted a phase I trial combining brentuximab vedotin and cyclosporine A (CsA) in patients with relapsed/refractory Hodgkin lymphoma. Results: Here, we show that competitive inhibition of MDR1 restored sensitivity to brentuximab vedotin in our brentuximab vedotin–resistant cell lines by increasing intracellular MMAE levels, and potentiated brentuximab vedotin activity in brentuximab vedotin–resistant Hodgkin lymphoma tumors in a human xenograft mouse model. In our phase I trial, the combination of brentuximab vedotin and CsA was tolerable and produced an overall and complete response rate of 75% and 42% in a population of patients who were nearly all refractory to brentuximab vedotin. Conclusions: This study may provide a new therapeutic strategy to combat brentuximab vedotin resistance in Hodgkin lymphoma. This is the first study reporting an effect of multidrug resistance modulation on the therapeutic activity of an ADC in humans. The expansion phase of the trial is ongoing and enrolling patients who are refractory to brentuximab vedotin to confirm clinical activity in this population with unmet need.

A hallmark of tumors is the ability to gain metabolic advantage that enables them to meet an increasing energy demand as part of their rapid proliferation phenotype. As part of this metabolic advantage, tumors use secreted growth factors and hormones that stimulate glucose uptake and signaling to promote glycolysis and ATP production. t‐Darpp, a truncated isoform of the dopaminergic regulator Darpp‐32, has been described as a mediator of drug resistance and enhanced growth and survival in a variety of cancer types. We now demonstrate that cells stably transfected with t‐Darpp (SK.tDp) have increased growth rate, glycolytic capacity and ATP levels relative to control SK‐Br‐3 cells. The increased growth phenotype is also conferred in a paracrine manner to neighboring SK‐Br‐3 cells grown in co‐culture with SK.tDp cells. Moreover, recombinant t‐Darpp added to the growth medium of SK‐Br‐3 cells conferred the same enhanced metabolic phenotype via a mechanism that appears to involve binding and signal transduction via IGFR1. This same effect was seen in L6 myotubes, a natural target of insulin signaling. We propose a model by which cancer cells are able to increase their metabolic capacity and provide a proliferative advantage through novel insulin‐like hormone activity linked to t‐Darpp and IGFR1 signaling.

Abstract Brentuximab vedotin (BV) is an antibody–drug conjugate that specifically delivers the potent cytotoxic drug monomethyl auristatin E (MMAE) to CD30-positive cells. BV is FDA approved for treatment of relapsed/refractory Hodgkin lymphoma and anaplastic large cell lymphoma (ALCL); however, many patients do not achieve complete remission and develop BV-resistant disease. We selected for BV-resistant Hodgkin lymphoma (L428) and ALCL (Karpas-299) cell lines using either constant (ALCL) or pulsatile (Hodgkin lymphoma) exposure to BV. We confirmed drug resistance by MTS assay and analyzed CD30 expression in resistant cells by flow cytometry, qRT-PCR, and Western blotting. We also measured drug exporter expression, MMAE resistance, and intracellular MMAE concentrations in BV-resistant cells. In addition, tissue biopsy samples from 10 Hodgkin lymphoma and 5 ALCL patients who had relapsed or progressed after BV treatment were analyzed by immunohistocytochemistry for CD30 expression. The resistant ALCL cell line, but not the Hodgkin lymphoma cell line, demonstrated downregulated CD30 expression compared with the parental cell line. In contrast, the Hodgkin lymphoma cell line, but not the ALCL cell line, exhibited MMAE resistance and increased expression of the MDR1 drug exporter compared with the parental line. For both Hodgkin lymphoma and ALCL, samples from patients relapsed/resistant on BV persistently expressed CD30 by immunohistocytochemistry. One Hodgkin lymphoma patient sample expressed MDR1 by immunohistocytochemistry. Although loss of CD30 expression is a possible mode of BV resistance in ALCL in vitro models, this has not been confirmed in patients. MMAE resistance and MDR1 expression are possible modes of BV resistance for Hodgkin lymphoma both in vitro and in patients. Mol Cancer Ther; 14(6); 1376–84. ©2015 AACR.

The goal of this work was to develop and validate a pancreas tumor animal model to investigate the relationship between photodynamic therapy (PDT) effectiveness and photosensitizer drug delivery. More specifically, this work lays the foundation for investigating the utility of dynamic contrast enhanced blood perfusion imaging to be used to inform subsequent PDT. A VX2 carcinoma rabbit cell line was grown in the tail of the pancreas of three New Zealand White rabbits and approximately 3-4 weeks after implantation the rabbits were imaged on a CT scanner using a contrast enhanced perfusion protocol, providing parametric maps of blood flow, blood volume, mean transit time, and vascular permeability surface area product.

Abstract Background Brentuximab vedotin (BV) is an antibody-drug conjugate that delivers a potent cytotoxic agent, monomethyl auristatin E (MMAE), specifically to cells expressing surface CD30. In pivotal phase II trials BV demonstrated a complete response rate (CR) of 34% in patients with relapsed/refractory Hodgkin lymphoma (HL) and CR of 50% in patients with relapsed/refractory anaplastic large cell lymphoma (ALCL). However, patients who do not attain CR will eventually develop progressive disease despite active treatment with BV. We aim to select for BV resistant lymphoma cell lines and examine for alterations in surface CD30 expression in cell lines. Methods In vitro experiments used two HL cell lines (L428, KM-H2) and one ALCL cell line (Karpas 299). The selection of BV resistant cell lines was done in two different approaches (pulsatile and constant exposure). Confirmation of BV resistance was done by MTS assays and cell counting assays. Measurement of surface CD30 was performed by flow cytometry using monoclonal mouse anti-human CD 30, clone Ber-H2. Results MTS assay showed the IC50 of parental cell lines to be: L428 (24 ug/ml), KM-H2 (9 ug/ml), and Karpas (16 ng/ml). The cell lines were incubated at supra-IC50 dosage until no proliferation was seen and then rescued with BV free media in the pulsatile approach. Only L428 was able to be rescued and kept in BV free media until consistent proliferation was seen (defined as incremental growth for 3 consecutive weeks). L428 was then incubated again in 50 mg/ml of BV until consistent proliferation was seen. For the constant exposure approach, the cell lines were incubated at sub-IC50 dosages and dosages of BV were adjusted up depending on cell proliferation rates. Only KM-H2 and Karpas 299 showed consistent proliferation in the constant exposure approach. We were able to select for 1 resistant line per cell type. Cell counting assay showed that all three resistant cell lines were able to proliferate in the presence of BV at dosages twice their respective IC50s (Figure 1, only L428 is shown). MTS assays showed that all resistant cell lines had increased IC50s (Table 1). Flow cytometry showed downregulation of surface CD30 expression in Karpas 299 and KM-H2 but not L428 (Table1). For Karpas 299, both the percentage of CD30+ cells and the median CD30 intensity decreased in the resistant line. For KM-H2, only the median CD30 intensity decreased in the resistant line. Conclusion Prolonged exposure of HL and ALCL cell lines to BV led to resistant cell lines. When compared to their parental cell lines, two out of three resistant lines showed downregulation in surface CD30 expression. Disclosures: Chen: Seattle Genetics, Inc.: Consultancy, Research Funding, Speakers Bureau, Trave expenses Other.

Werner syndrome is an autosomal recessive disorder associated with premature aging and cancer predisposition caused by mutations of the WRN gene. WRN is a member of the RecQ DNA helicase family with functions in maintaining genome stability. Sir2, an NAD-dependent histone deacetylase, has been proven to extend life span in yeast and Caenorhabditis elegans. Mammalian Sir2 (SIRT1) has also been found to regulate premature cellular senescence induced by the tumor suppressors PML and p53. SIRT1 plays an important role in cell survival promoted by calorie restriction. Here we show that SIRT1 interacts with WRN both in vitro and in vivo; this interaction is enhanced after DNA damage. WRN can be acetylated by acetyltransferase CBP/p300, and SIRT1 can deacetylate WRN both in vitro and in vivo. WRN acetylation decreases its helicase and exonuclease activities, and SIRT1 can reverse this effect. WRN acetylation alters its nuclear distribution. Down-regulation of SIRT1 reduces WRN translocation from nucleoplasm to nucleoli after DNA damage. These results suggest that SIRT1 regulates WRN-mediated cellular responses to DNA damage through deacetylation of WRN.

B2 Background: NSCLC patients carrying the somatic mutation of epidermal growth factor receptor (EGFR) has been shown to be hyperresponsive to the EGFR tyrosine kinase inhibitor Gefitinib and Erlotinib. The most common NSCLC associated EGFR mutations are the in-frame deletion in exon 19 (E746_A750del) and the point mutation in exon 21 (L858R), accounting for 85-90% EGFR mutations. The ability to detect mutated gene products in cancer cells can identify patients most likely benefit from such therapies. Methods: We generated rabbit monoclonal antibodies (RmAb) against EGFR with E746-A750 deletions and L858R point mutation. We tested the antibodies by Western blot, Immunofluorescence (IF) and Immunohistochemistry (IHC). In addition, we tested these antibodies on 40 molecularly pre-typed tumors by IHC. Finally, we used a panel of four antibodies (two mutant antibodies, a total EGFR antibody, and a pan-keratin antibody) to screen 340 cases of tumor samples with unknown genotype by IHC. Results: The results from Western blot, IF, and IHC confirmed that these antibodies could specifically detect the mutant EGFR proteins. IHC data from 40 molecular pre-typed samples perfectly matched the result from DNA sequencing. In those 340 cases of tumor samples, 28 cases were stained positive by L858R antibody and 24 cases were stained positive by EGFR deletion antibody. The positive rate by both antibodies is 15.3%. The DNA from all IHC positive samples and negative adenocarcinoma samples were sent for DNA sequencing. The sensitivity of these mutant specific antibodies is 92.5% and the specificity is 95.8%. In addition, some tumor samples positive for EGFR mutation by IHC and Mass Spec based DNA sequencing were negative by direct DNA sequencing due to low percentage of cancer cells. Conclusions: The IHC combined mutant EGFR specific antibodies and total EGFR antibody can be used to detect the EGFR mutations and measure the expression level of total EGFR protein in tumors. In addition, this assay enables us to examine paraffin blocks from small biopsy samples, which are difficult to extract enough high quality DNA for sequencing. This IHC assay has the potential to be developed for use to screen lung cancer patients for the treatment with EGFR kinase inhibitors in a clinical setting.

The Tomographic Gamma Scanning (TGS) technique is a relatively new method in the field of non-destructive assay (NDA) of radioactive waste. The TGS technique combines High Resolution Gamma Spectrometry (HRGS) with Three-Dimensional (3-D) low spatial resolution transmission and emission image reconstruction techniques to achieve assay goals. When compared to the traditional methods such as Segmented Gamma Scanning (SGS), the TGS technique can yield better accuracies for cases where the radionuclide is distributed non-uniformly in a heterogeneous matrix. The TGS technique is ideally suited for low-to-moderate density waste matrices, say 1.0gcm−3 or below for 55 US gal. drums, although it can be extended to higher densities by using alternative approaches to the design or analyses. Recently Canberra Industries designed, built and characterized four such TGS systems for nuclear power plant applications. Many of the design features and the end application itself set these TGS systems apart from the others that had been built previously. The four TGS systems are the first commercial grade systems that could quantify radionuclides contained in nuclear power plant waste, using the TGS technique. The TGS systems featured two different combinations of collimator and source–detector distance; a “near” geometry with a collimator aperture of 50.8mm and a “Far” geometry with a narrower collimator aperture of 40.6mm. For assaying drums with matrix densities greater than 1.0gcm−3 and/or dose rates greater than 6mSvh−1 the system could be configured as a SGS. In the SGS mode, five different assay geometries could be configured using different collimator, source-to-detector distance and absorber combinations. During operation, the appropriate assay geometry was selected automatically based on the drum weight (density) and dose rate measurements. The characterization and performance of the one of the TGS systems are discussed in detail for both TGS and SGS modes of operation. Quantitative results are presented for point source and rod source nuclide distributions. Transmission and emission images obtained in the TGS assays will be presented.

The insulin-signaling network regulates blood glucose levels, controls metabolism, and when dysregulated, may lead to the development of type 2 diabetes. Although the role of tyrosine phosphorylation in this network is clear, only a limited number of insulin-induced tyrosine phosphorylation sites have been identified. To address this issue and establish temporal response, we have, for the first time, carried out an extensive, quantitative, mass spectrometry-based analysis of tyrosine phosphorylation in response to insulin. The study was performed with 3T3-L1 adipocytes stimulated with insulin for 0, 5, 15, and 45 min. It has resulted in the identification and relative temporal quantification of 122 tyrosine phosphorylation sites on 89 proteins. Insulin treatment caused a change of at least 1.3-fold in tyrosine phosphorylation on 89 of these sites. Among the responsive sites, 20 were previously known to be tyrosine phosphorylated with insulin treatment, including sites on the insulin receptor and insulin receptor substrate-1. The remaining 69 responsive sites have not previously been shown to be altered by insulin treatment. They were on proteins with a wide variety of functions, including components of the trafficking machinery for the insulin-responsive glucose transporter GLUT4. These results show that insulin-elicited tyrosine phosphorylation is extensive and implicate a number of hitherto unrecognized proteins in insulin action.

P. Attema, G. Burgers, E. Van Joolen, M. Van Leusen, and B. Mater (Eds), New Developments in Italian Landscape Archaeology: Theory and Methodology of Field Survey, Land Evaluation and Landscape Perception, Pottery Production and Distribution (Proceedings of a three-day conference held at the University of Groningen, April 13–15, 2000) (British Archaeological Reports International series 1091). Oxford: Archaeopress, 2002. Pp. 265, 4 pls, illus. ISBN 1-8417-1469-0. £35.00. - Volume 95

AS160 is a newly described substrate for the protein kinase Akt that links insulin signaling and GLUT4 trafficking. In this study, we determined the expression of and in vivo insulin action on AS160 in human skeletal muscle. In addition, we compared the effect of physiological hyperinsulinemia on AS160 phosphorylation in 10 lean-to-moderately obese type 2 diabetic and 9 healthy subjects. Insulin infusion increased the phosphorylation of several proteins reacting with a phospho-Akt substrate antibody. We focused on AS160, as this Akt substrate has been linked to glucose transport. A 160-kDa phosphorylated protein was identified as AS160 by immunoblot analysis with an AS160-specific antibody. Physiological hyperinsulinemia increased AS160 phosphorylation 2.9-fold in skeletal muscle of control subjects (P < 0.001). Insulin-stimulated AS160 phosphorylation was reduced 39% (P < 0.05) in type 2 diabetic patients. AS160 protein expression was similar in type 2 diabetic and control subjects. Impaired AS160 phosphorylation was related to aberrant Akt signaling; insulin action on Akt Ser(473) phosphorylation was not significantly reduced in type 2 diabetic compared with control subjects, whereas Thr(308) phosphorylation was impaired 51% (P < 0.05). In conclusion, physiological hyperinsulinemia increases AS160 phosphorylation in human skeletal muscle. Moreover, defects in insulin action on AS160 may impair GLUT4 trafficking in type 2 diabetes.