Kyung Young Kim

Sandwich Enzyme-Linked Immunosorbent Assays (Selisas) Quantitate and Distinguish Two Forms of Transforming Growth Factor-Beta (TGF-β1 and TGF-β2) in Complex Biological Fluids

We have developed sandwich enzyme-linked immunosorbent assays (SELISAs) for TGF-beta 1 and TGF-beta 2 using both turkey and rabbit neutralizing polyclonal antibodies against native TGF-beta s. Each assay is based on the binding of two different antibodies to distinct epitopes of a single TGF-beta molecule. With these assays, TGF-beta types 1 and 2 can each be specifically quantitated in complex biological fluids, with detection limits of 2-5 pg. TGF-beta 3 and TGF-beta 5 either do not cross-react or cross-react very poorly in these assays. TGF-beta 1.2 heterodimer, although 50-80% neutralized by either TGF-beta 1 or TGF-beta 2 antibodies, shows only a 1.5 and 3.7% cross-reactivity in the TGF-beta 1 and TGF-beta 2 SELISAs, respectively. The SELISAs reported here represent the most specific, rapid, and precise assays for TGF-beta 1 and TGF-beta 2 reported thus far.

Overexpression of transforming growth factor-. beta. in transgenic mice carrying the human T-cell lymphotropic virus type I tax gene

Human T-cell lymphotropic virus type I (HTLV-I) has been associated with an adult form of T-cell leukemia as well as tropical spastic paraparesis, a neurodegenerative disease. Adult T-cell leukemia patients express high levels of the type 1 isoform of transforming growth factor-beta (TGF-beta 1), which is mediated by the effects of the HTLV-I Tax transactivator protein on the TGF-beta 1 promoter. To understand further the regulation of TGF-beta 1 expression by Tax, we examined its expression in transgenic mice carrying the HTLV-I tax gene. We show that tumors from these mice and other tissues, such as submaxillary glands and skeletal muscle, which express high levels of tax mRNA selectively express high levels of TGF-beta 1 mRNA and protein. Moreover, TGF-beta 1 significantly stimulated the incorporation of tritiated thymidine into one of three cell lines derived from neurofibromas of tax-transgenic mice, which suggests that the excessive production of TGF-beta 1 may play a role in tumorigenesis and that these mice may serve as a useful model for studying the biological effects of TGF-beta in vivo.

Evidence for Differential Regulation of TGFβ1 and TGFβ2 Expression in Vivo by Sandwich Enzyme‐linked Immunosorbent Assays

We have developed sandwich enzyme-linked immunosorbent assays (SELISAs) for TGFPl and TGFp2 using both turkey and rabbit neutralizing polyclonal antibodies against native TGFps. Each assay is based on the binding of two different antibodies to distinct epitopes of a single TGFD molecule. For these assays, Nunc Maxisorb (Nunc, Denmark) microtiter plates are first coated with either affinity-purified anti-TGFo1 or anti-TGFP2 rabbit antibodies, and the TGFps are then added and allowed to be anchored to wells by the primary antibodies. The exposed epitopes of the immobilized TCFp1 and TGFP2 serve as binding sites for secondary affinity-purified anti-TGFpl and anti-TGFp2 antibodies, respectively. The bound secondary antibody is then detected by phosphatase activity following the binding of phosphatase-linked anti-turkey IgG. With these assays, TGFP types 1 and 2 can each be specifically quantitated in complex biological fluids with detection limits of 2 to 5 pg. Conditioned medium can be assayed after acid activation, and tissue and serum can be assayed after extraction with acid-ethanol. Various steroid hormones and peptide growth modulators do not interfere in these SELISAs. Because each of these SELISAs are based on the recognition of two independent epitopes, they exhibit much greater ligand specificity relative to immunoassays that are based on the recognition of only a single epitope. Such enhanced specificity is clearly demonstrated by our observation that only 1.5 and 3.7% of TCFpl.2 heterodimer is scored in the TGFpl and TGF(32 SELISAs, respectively, in contrast to 82 and 50% neutralization of biological activity by anti-TCFpl and antiTGFp2, respectively. Chicken TGFP3 and frog TCFp5 do not crossreact in these SELISAs, further supporting the high specificity of these SELISAs. Therefore, the many favorable features of these SELISAs make them the method of choice for rapid, accurate, and precise measurement of mFD1 and E F P 2 in complex biological fluids. The levels of E F p l and TGFp2 in sera from various animals were measured by these SELISAs (TABLE 1). Our results from this study demonstrate that TGFpl is the

Prospective Feasibility Study for Using Cell-Free Circulating Tumor DNA–Guided Therapy in Refractory Metastatic Solid Cancers: An Interim Analysis

Purpose Retrospective studies have demonstrated that cell-free circulating tumor DNA (ctDNA) hotspot testing predicts matched therapy response to first- and second-line therapies in patients with advanced non–small-cell lung cancer (NSCLC). However, no prospective outcomes studies have evaluated ctDNA-guided matched therapy decision making on the basis of comprehensive plasma genomic testing including all four major classes of alterations. Here, we report the clinical utility of this approach in advanced solid tumor cancers. Patients and Methods We conducted a multiple parallel cohort, open-label, clinical trial using ctDNA-guided matched therapy when tissue was insufficient or unobtainable for next-generation sequencing. Plasma-based digital sequencing identified point mutations in 70 genes and indels, fusions, and copy number amplifications in selected genes. Patients with prespecified targetable alterations in metastatic NSCLC, gastric cancer (GC), and other cancers were matched to several independent targeted agent trials at a tertiary academic center. Results Somatic alterations were detected in 59 patients with GC (78%), and 25 patients (33%) had targetable alterations (ERBB2, n = 11; MET, n = 5; FGFR2, n = 3; PIK3CA, n = 6). In NSCLC, 62 patients (85%) had somatic alterations, and 34 (47%) had targetable alterations (EGFR, n = 29; ALK, n = 2; RET, n = 1; ERBB2, n = 2). After confirmation of ctDNA findings on tissue (to meet trial eligibility criteria), 10 patients with GC and 17 patients with NSCLC received molecularly matched therapy. Response rate and disease control rate were 67% and 100%, respectively, in GC and 87% and 100%, respectively, in NSCLC. Response was independent of targeted alteration variant allele fraction in NSCLC (P = .63). Conclusion To our knowledge, this is the first prospective feasibility study of comprehensive ctDNA-guided treatment in advanced GC and lung cancers. Response rates in this interim analysis are similar to those in tissue-based targeted therapy studies.