Abstract B31: Multiparameter profiling of abscopal effect (AbE) in a pediatric fibrosarcoma with EML4-NTRK3 fusion
Authors: Gartrell Robyn D. , Sims Jennifer S. , Barton Sunjay M. , Kadenhe-Chiweshe Angela , Cruz Filemon Dela , Turk Andrew T. , Kung Andrew L. , Bruce Jeffrey N. , Saenger Yvonne M. , Yamashiro Darrell J. , Connolly Eileen P. 
Published: February 2019

Abstract
Background: Abscopal effect (AbE) is a rare phenomenon in pediatric oncology, defined as regression of tumor at distal sites following radiation therapy. AbE is widely believed to result from the local activation and expansion of tumor-specific T cells in the area radiated, followed by the migration of these T cells to distant sites. We present a case of AbE in a 1-year-old with congenital fibrosarcoma (forearm) bearing a novel EML4-NTRK3 fusion who developed bilateral lung metastasis 6 months post resection of the initial forearm tumor. The patient’s lung lesions were unresponsive to chemotherapy prompting palliative, hypofractionated radiation therapy (RT) to the right lung lesion only. One month following RT, patient showed significant decrease in size of bilateral lung lesions, with no evidence of disease at 3 months post RT. Using whole-exome sequencing (WES), high-throughput sequencing of T cell receptors (TCRseq), and quantitative multiplex immunofluorescence (qmIF), we investigated the relationship between tumor progression and metastasis, the pre- and post-RT microenvironment, and the co-evolution of the tumor-infiltrating lymphocyte (TIL) populations in this patient. We believe this unusual case offers key insights into mechanisms underlying AbE.
Methods: Formalin-fixed, paraffin-embedded (FFPE) tissue specimens from the initial diagnostic lesion (forearm), pretreatment lung tumor, and bilateral post-RT lung lesions (right (RT treated) and left (outside RT field) were obtained. An FFPE slide was stained for each specimen using traditional immunohistochemistry (IHC) for CD3 and CD8 and qmIF for 7 biomarkers including DAPI, CD3, CD4, CD8, CD68, HLA-DR, and Ki67. QmIF images were taken using MANTRA and cell density analysis was performed using inForm software and R. DNA was isolated, from the tissue specimens and peripheral blood, for profiling using WES and TCRseq, generating a multiplex snapshot of each site. The TIL populations were described by the complementarity determining region 3 (CDR3) of each TCR (as identified by Adaptive), followed by analysis of the bias, diversity, and divergence of these TIL populations using entropy-based statistics.
Results: IHC and qmIF of the primary fibrosarcoma and pre-RT lung tumor showed very low immune cell infiltration. In contrast, immune infiltration was markedly increased (both CD4+ and CD8+) post-RT, particularly in the nonirradiated lung lesion (left). TIL populations were analyzed using TCRseq, revealing little similarity between those of the primary fibrosarcoma and the recurrent lung lesions. However, the TCR repertoires comparing the post-RT lung lesions, irradiated (right) and non-irradiated (left), showed substantial similarity, sharing a larger percentage of CDR3 sequences with each other (6.7% of combined total) than with the primary tumor (≤0.6% combined total) or the pre-RT lung tumor (≤1.6% combined total). Additionally, the extent of the TIL repertoire and its binding-associated divergence from the peripheral blood was greatest in the irradiated (right) lung tumor post RT, suggesting a direct link between the evolution of trans-acting immunity and antigen-driven immunogenicity at that site. Of note, comparative WES of the initial fibrosarcoma and subsequent lung tumors identified the same EML4-NTRK3 fusion, validating their metastatic relationship, although the number of coding mutations had expanded upon recurrence.
Conclusion: Using qmIF and TCRseq, we have extensively characterized immune infiltration in the tumor microenvironment prior to and following metastasis and RT, in the first reported case of AbE in a pediatric tumor. Our data show evidence of immune stimulation elicited by RT, and antitumor T-cell specificities are shared inside and outside of the radiation field. These data offer insight into AbE and how combination of radiation and immunotherapy may be useful to induce response in pediatric tumors.
Citation Format: Robyn D. Gartrell, Jennifer S. Sims, Sunjay M. Barton, Angela Kadenhe-Chiweshe, Filemon Dela Cruz, Andrew T. Turk, Andrew L. Kung, Jeffrey N. Bruce, Yvonne M. Saenger, Darrell J. Yamashiro, Eileen P. Connolly. Multiparameter profiling of abscopal effect (AbE) in a pediatric fibrosarcoma with EML4-NTRK3 fusion [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr B31.