P-095: Normal human tissue expression of G-protein coupled receptor 5D (GPRC5D), a promising novel target for Multiple Myeloma, is restricted to plasma cells and hard keratinized tissues

Background GPRC5D is an attractive target for treatment of multiple myeloma (MM) due to its high expression on MM cells, and favorable normal tissue expression profile that suggests a low risk for on-target/off-tumor toxicity. However, current literature and public databases report conflicting data for normal GPRC5D RNA and protein expression. For example, cerebellum is reported to have very low levels of GPRC5D mRNA by some sources whereas others report its absence. Likewise, the GTEx database has high maximum expression in the skin, but very low median expression; this is also reflected in the high variability of GPRC5D mRNA levels in skin assessed by RT-PCR reported in the literature. The protein expression data in Human Protein Atlas cannot be used to verify RNA expression because it was obtained using an unvalidated, polyclonal antibody with low specificity by protein array. Other sources of protein expression are incomplete and did not assess critical tissues. Clarification of GPRC5D expression in normal tissues is critical to understanding potential on-target/off-tumor risks to patients treated with GPRC5D-targeted therapies. Methods Key human tissues of interest for protein expression studies were identified through literature search and analysis of an internal RNAseq database. Further elucidation of normal GPRC5D expression in those tissues of interest was done using IHC and ISH on FFPE human tissue samples, including: skin, lung, tongue, tonsil, parotid gland, salivary gland, sublingual gland, submandibular gland, choroid plexus, cerebellum, and brainstem-medulla (inferior olivary nucleus, ION). The primary antibody used was a mouse anti-GPRC5D monoclonal antibody (Abcam, Ab55044, clone 6D9) qualified for its sensitivity and specificity in positive and negative control FFPE human cell pellets. The RNA-ISH GPRC5D probe for human was from ACD (Hs-GPCR5D, cat#489699). All test samples underwent QC screens for IHC and ISH, and only samples that passed were included in the definitive experiments. Stained slides were examined by a pathologist for presence and localization of IHC and ISH signals. Results By IHC and ISH, GPRC5D is expressed in epithelial cells of the hair follicles in the skin (the number of hair follicles in skin samples explains the variability in RNA expression), and at the base of the epithelial columns supporting the filiform papillae (keratinized structures in the tongue). In all other tissues assessed, GPRC5D protein was observed only in interstitial plasma cells. Mild GPRC5D RNA expression was detected by ISH in the motor neurons of the ION (without IHC correlate); its close connection with the cerebellum may explain the very low levels of GPRC5D mRNA expression that have been reported in the literature. Conclusion These data support that GPRC5D is not broadly expressed in normal tissues beyond resident plasma cell populations and hard keratinized tissues. GPRC5D is an attractive target for treatment of multiple myeloma (MM) due to its high expression on MM cells, and favorable normal tissue expression profile that suggests a low risk for on-target/off-tumor toxicity. However, current literature and public databases report conflicting data for normal GPRC5D RNA and protein expression. For example, cerebellum is reported to have very low levels of GPRC5D mRNA by some sources whereas others report its absence. Likewise, the GTEx database has high maximum expression in the skin, but very low median expression; this is also reflected in the high variability of GPRC5D mRNA levels in skin assessed by RT-PCR reported in the literature. The protein expression data in Human Protein Atlas cannot be used to verify RNA expression because it was obtained using an unvalidated, polyclonal antibody with low specificity by protein array. Other sources of protein expression are incomplete and did not assess critical tissues. Clarification of GPRC5D expression in normal tissues is critical to understanding potential on-target/off-tumor risks to patients treated with GPRC5D-targeted therapies. Key human tissues of interest for protein expression studies were identified through literature search and analysis of an internal RNAseq database. Further elucidation of normal GPRC5D expression in those tissues of interest was done using IHC and ISH on FFPE human tissue samples, including: skin, lung, tongue, tonsil, parotid gland, salivary gland, sublingual gland, submandibular gland, choroid plexus, cerebellum, and brainstem-medulla (inferior olivary nucleus, ION). The primary antibody used was a mouse anti-GPRC5D monoclonal antibody (Abcam, Ab55044, clone 6D9) qualified for its sensitivity and specificity in positive and negative control FFPE human cell pellets. The RNA-ISH GPRC5D probe for human was from ACD (Hs-GPCR5D, cat#489699). All test samples underwent QC screens for IHC and ISH, and only samples that passed were included in the definitive experiments. Stained slides were examined by a pathologist for presence and localization of IHC and ISH signals. By IHC and ISH, GPRC5D is expressed in epithelial cells of the hair follicles in the skin (the number of hair follicles in skin samples explains the variability in RNA expression), and at the base of the epithelial columns supporting the filiform papillae (keratinized structures in the tongue). In all other tissues assessed, GPRC5D protein was observed only in interstitial plasma cells. Mild GPRC5D RNA expression was detected by ISH in the motor neurons of the ION (without IHC correlate); its close connection with the cerebellum may explain the very low levels of GPRC5D mRNA expression that have been reported in the literature. These data support that GPRC5D is not broadly expressed in normal tissues beyond resident plasma cell populations and hard keratinized tissues.