Figure provided by Dr. Balakrishnan.
One of the most recent and exciting advances in cancer treatment is the development of immunotherapy to address the immune response against a target protein expressed on the surface of the tumor cells. As the targeted protein might also be expressed on normal tissues this can lead to the so-called on-target off-tumor toxicities. Another risk is that the heterogeneous intra-tumoral expression may lead to treatment escape. It is vital to understand both the uniformity of target expression on tumors and expression on normal tissues to predict the success of immunotherapies and toxicities that may occur in clinical trials.
Among these promising antigens, receptor tyrosine kinase like orphan receptor 1 (ROR1) protein was previously reported to be predominantly expressed in cancerous cells, more specifically in hematological malignancies and solid tumors, and poorly expressed in normal tissues making it an ideal target. However, previous reports have shown cytoplasmic expression of ROR1 instead of the expected surface location. This finding raises questions about the reagents being used, the validity of the reported normal ROR1 expression, as well as implications for on-target off-tumor toxicities. To tackle these doubts, Dr. Balakrishnan, a post-doctoral research fellow and Principal Investigator Dr. Riddell (Clinical Research Division) collaborated with the Experimental Histopathology core to perform an in-depth investigation of the expression pattern of ROR1 protein in various tumors and normal tissues. In this study, recently published in Clinical Cancer Research, the researchers identified organs at risk for on-target off-tumor toxicities in addition to providing information regarding treatment escape potential per tumor type based on the homogeneous or heterogeneous ROR1 expression inside the tumor.
The authors used immunohistochemistry (IHC) to detect ROR1 proteins as it provides sensitive in situ staining elucidating the protein localization and heterogeneity in the tissue of interest. In spite of the existence of several commercially available antibodies, none were able to detect ROR1 in tumors known to express high levels of ROR1. For this reason, the authors used a new monoclonal antibody developed by the Antibody Development Core. After testing several clones for specific and sensitive staining of the surface variant of ROR1, the monoclonal antibody clone 6D4 was selected to pursue the study.
ROR1 expression was analyzed in depth in tissue microarrays (TMAs) sections of epithelial tumor tissues, more specifically ovarian, breast, lung and pancreas cancers, at various stages obtained from US-Biomax and the Cooperative Human Tissue Network. Among the 159 ovarian cancers tissues that were studied, 50% demonstrated both membrane and cytoplasmic ROR1 expression and 92% of these tumors were homogenous for ROR1 expression, lowering the chance for ROR1 negative tumor cells to escape tumor treatment. ROR1 expression was highly enriched in triple negative breast cancer (TNBC) and lung adenocarcinomas subtypes, with homogenous expression in 74% of TNBCs and all lung adenocarcinomas. ROR1 has also been reported previously to promote metastasis but 40% of patients that had ROR1 positive tumors samples developed ROR1 negative metastases, suggesting that ROR1 was not involved in metastasis development in these cases. Unexpectedly, while pancreas cancer was previously associated with frequent positive ROR1 expression, only 15% of the pancreatic tumor tissues expressed ROR1 and at a low level in this study. Based on these data, and comparison with available scientific literature, it is interesting to note that some tumors that were previously thought to express ROR1 at a high level present a low expression of the antigen. These tumors may be less relevant for immunotherapy targeting ROR1. More investigation will be necessary in these cases. However, as explained by the authors “ROR1 is a promising CAR-T cell target in TNBCs and lung adenocarcinomas and clinical trials have been initiated at the Seattle Cancer Care Alliance (SCCA) targeting ROR1 in these aggressive solid tumors. The anti-ROR1 6D4 antibody is being used as a companion diagnostic to determine eligibility for this trial.”
The expression of ROR1 in normal tissues was also investigated. Previous studies had shown limited expression of surface ROR1 only on adipocytes and during B cell development. This study also revealed ROR1 in normal parathyroid, pancreatic islet cells and in various regions of the gastrointestinal tract. Importantly, ROR1 expression was not detected in the brain, lung, heart and liver. This expression pattern was confirmed on flash-frozen tissues by western blot to detect the cell surface protein and transcript by real-time PCR experiments. ROR1 expression on these non-cancerous cells was high enough to allow in vitro killing by therapeutic anti-ROR1 chimeric antigen receptor-modified (CAR) T cells and associated cytokine production, raising concerns about ROR1 CAR T cell safety in vivo.
To further understand the risks associated with ROR1 expression in normal tissues, the researchers looked into a non-human primate model. Indeed, a previous study from the same lab demonstrated the safe administration of ROR1 CAR T cells in healthy animals. In this model, ROR1 expression pattern in normal tissues is similar to the humans, and there was no evidence of toxicities, relieving concerns about CAR T cell therapy toxicities associated with ROR1 targeting. Poor trafficking of CAR T cells to these ROR1 positive non-tumoral tissues and/or an insufficient level of antigens to trigger CAR T cells attraction and activation in vivo might explain this absence of toxicity.
This study highlights the need to understand the antigen normal expression pattern to allow for control of a low risk and successful therapy, as well as anticipation of the potential toxicities issues.
This study was funded by the National Institutes of Health and the Walker Immunotherapy Research Fellowship.
Balakrishnan A, Goodpaster T, Randolph-Habecker J, Hoffstrom BG, Jalikis FG, Koch LK, Berger C, Kosasih PL, Rajan A, Sommermeyer D, Porter P, Riddell SR. 2016. Analysis of ROR1 protein expression in human cancer and normal tissues. Clinical Cancer Research.