Abstract 4248: Multispectral immunofluorescence identifies pS6 as a biomarker of intrinsic resistance to ruxolitinib in patients with relapsed/refractory T-cell lymphomas
Authors: Ghione Paola , Moskowitz Alison , Kumar Priadarshini , Knezevic Andrea , Seshan Venkatraman , Jacobsen Eric , Ruan Jia , Schatz Johnathan , Noor Sarah , Myskowski Patricia , Hancock Helen , Davey Theresa , Obadi Obadi , Onwasigwe Carlissa , Ganesan Nivetha , Pomerantz Lauren , Jarjies Christine , Sigler Allison , Geyer Mark , Noy Ariela , Straus David , Galasso Natasha , Inghirami Giorgio , Horwitz Steven , Weinstock David , Hollmann Travis , Dogan Ahmet 
Published: September 2020

Abstract
Introduction: Peripheral and cutaneous T cell lymphomas (TCL) are highly heterogeneous diseases that in the relapsed/refractory (R/R) stage have poor outcome. JAK/STAT, Pi3K/AKT and MAP kinase signaling are commonly involved in the pathogenesis of these lymphomas, and the efficacy of small molecule inhibitors targeting these pathways is being assessed in phase II trials. However, biomarkers to define patients most likely to benefit from specific inhibitors are lacking and mechanisms of acquired resistance in TCLs have not been reported.
Methods: We are conducting two early phase trials in R/R TCL: 1) NCT02974647 targets JAK1/JAK2 with ruxolitinib (RUX, a JAK 1-2 inhibitor) and has enrolled 53 patients and 2) NCT02783625 targets Pi3K/AKT with duvelisib (DUV, a Pi3K gamma-delta inhibitor) in combination with either romidepsin or bortezomib and has enrolled 92 patients. We analyzed pre-, on and post- treatment biopsies with multiplex immunofluorescence (mIF) using the Vectra platform, and analyzed images for marker expression and cellular location using HALO software. We designed multiple mIF panels with 6 markers per slide, 3 identifying TCL cells based on disease-specific phenotypes (e.g. CD4+PD1+CD8- for angioimmunoblastic TCL), pSTAT3,5, pS6, and a macrophage marker (CD68). Areas of TCL involvement within biopsy slides were manually defined during image analysis based on mIF and comparison to clinical immunohistochemistry. Differences in marker expressions were analyzed with the Wilcoxon test using the program R. Results: We analyzed 53 biopsies from 39 patients with high-quality mIF images, of which 17 were from lymph nodes and 36 were from extranodal sites. Of 39 patients, 13 were treated with RUX (9 biopsies pre-, 7 on, 4 post- treatment) and 26 with DUV (20 biopsies pre-, 6 on, 10 post- treatment). Median number of total TCL cells in pre-treatment biopsies was 1041 (range, 97-6463) and median TCL cell fraction within involved areas was 27.86% (range, 5.12-88.02%). Median macrophage fraction within involved areas was 5.92% (range, 2.89-11.88%). For either agent, response to treatment was not associated with: 1) quantity of macrophages (p=0.1 for responders vs non-responders to DUV, p=0.53 for RUX), 2) average distance between TCL cells and closest macrophage, 3) TCL cell fraction within involved areas, or 4) fraction of TCL cells expressing pSTAT3/5 (Figure). In contrast, pS6 expression in &lt;25% of TCL cells within pre-treatment biopsy completely distinguished responders from non-responders (Figure; p=0.02). Neither agent induced consistent changes in expression of pSTAT3/5 or pS6 at the on or post-treatment timepoints.
Conclusions: mIF is a feasible platform for biomarker discovery and translation in TCL, a set of lymphomas with heterogeneous immunophenotypes. pS6 expression in &lt;25% of TCL cells may predict response to ruxolitinib, suggesting that activation of S6 kinase downstream of PI3K or MAP kinase, may overcome dependence on JAK/STAT signaling. This is also the first data suggesting that ruxolitinib responses involve a TCL cell-intrinsic mechanism of action. Based on these data, we are assessing the use of pS6 IHC as a clinically-validated biomarker in patients with TCL prior to ruxolitinib.
Citation Format: Paola Ghione, Alison Moskowitz, Priadarshini Kumar, Andrea Knezevic, Venkatraman Seshan, Eric Jacobsen, Jia Ruan, Johnathan Schatz, Sarah Noor, Patricia Myskowski, Helen Hancock, Theresa Davey, Obadi Obadi, Carlissa Onwasigwe, Nivetha Ganesan, Lauren Pomerantz, Christine Jarjies, Allison Sigler, Mark Geyer, Ariela Noy, David Straus, Natasha Galasso, Giorgio Inghirami, Steven Horwitz, David Weinstock, Travis Hollmann, Ahmet Dogan. Multispectral immunofluorescence identifies pS6 as a biomarker of intrinsic resistance to ruxolitinib in patients with relapsed/refractory T-cell lymphomas [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4248.