Detection of MET mRNA in gastric cancer in situ. Comparison with immunohistochemistry and sandwich immunoassays.

Determination of predictive biomarkers by immunohistochemistry (IHC) relies on antibodies with high selectivity. RNA in situ hybridization (RNA ISH) may be used to confirm IHC and may potentially replace it if suitable antibodies are not available or are insufficiently selective to discriminate closely related protein isoforms. We validated RNA ISH as specificity control for IHC and as a potential alternative method for selecting patients for treatment with MET inhibitors. MET, the HGF receptor, is encoded by the MET proto-oncogene that may be activated by mutation or amplification. MET expression and activity were tested in a panel of control cell lines. MET could be detected in formalin fixed paraffin, embedded (FFPE) samples by IHC and RNA ISH, and this was confirmed by sandwich immunoassays of fresh frozen samples. Gastric cancer cell lines with high MET expression and phosphorylation of tyrosine-1349 respond to the MET inhibitor, BAY-853474. High expression and phosphorylation of MET is a predictive biomarker for response to MET inhibitors. We then analyzed MET expression and activity in a matched set of FFPE vs. fresh frozen tumor samples consisting of 20 cases of gastric cancer. Two of 20 clinical samples investigated exhibited high MET expression with RNA ISH and IHC. Both cases were shown by sandwich immunoassays to exhibits strong functional activity. Expression levels and functional activity in these two cases were in a range that predicted response to treatment. Our findings indicate that owing to its high selectivity, RNA ISH can be used to confirm findings obtained by IHC and potentially may replace IHC for certain targets if no suitable antibodies are available. RNA ISH is a valid platform for testing predictive biomarkers for patient selection.

Solubilization and partial characterization of a tumor-associated transplantation antigen of the guinea pig L2C leukemia.

A tumor-associated transplantation antigen (TATA) from guinea pig L2C leukemia cells was solubilized by different methods. It was found that the 3 M KCl extraction yielded the most immunogenic TATA of L2C cells. Immunization of normal strain 2 guinea pigs with this extract in complete Freund's adjuvant gave complete protection against a subsequent challenge with tumor cells. Further fractionation of the KCl extract of L2C cells by Sephadex G-200 chromatography suggested that the immunogenic activity was present in the fraction containing materials with estimated m.w. of less than 20,000 daltons.

Mutant lines of guinea pig L2C leukemia. III. The reaction of an alloantiserum detecting idiotypic determinants on a clonally derived guinea pig B cell leukemia with IgM and Ia molecules.

An alloantiserum was prepared in a strain 13 guinea pig against the GH line of the strain 2 guinea pig L2C leukemia. This serum contained antibodies to both IgM and Ia molecules. After absorption with normal spleen cells from a strain 2 guinea pig, this antiserum no longer reacted with strain 2 cells, but detected idiotypes on the IgM molecules of the L2C leukemia. These idiotypes were on the same IgM molecules detected by a xenogeneic sheep anti-L2C Fab mu antiserum. As assayed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the idiotype-bearing IgM molecules were synthesized by the cell, composed of normal sized mu and light chains, appeared on the cell surface as monomeric IgM, and were the only immunoglobulin molecules present on the cell. Although the alloantiserum potentially contained antibodies to unique Ia idiotypic determinants, none were found. Furthermore, the anti-IgM idiotype antisera did not react with any Ia-like molecules.