Abstract NG04: Mechanisms of resistance to anti-PD-1 immunotherapy through interferon pathway mutations
Authors: Zaretsky Jesse , Garcia-Diaz Angel , Shin Daniel S. , Escuin-Ordinas Helena , Hugo Willy , Hu-Lieskovan Siwen , Eroglu Zeynep , Torrejon Davis Y. , Tumeh Paul C. , Lo Roger S. , Ribas Antoni 
Published: July 2017

Abstract
Checkpoint blockade immunotherapy can produce dramatic, durable tumor regression, is now being deployed or tested in a growing list of cancer types, and has received FDA approval for the treatment of metastatic melanoma, Hodgkin’s lymphoma, and lung, renal, and head and neck carcinomas. Depending on cancer type, higher CD8 infiltration, PD-L1 expression, and mutational load have all been reported as positive prognostic factors for response. We studied response to antiprogrammed cell death 1 (PD-1) or PD-1 ligand (PD-L1) therapy in a cohort of 55 patients from centers in the US and Australia with advanced desmoplastic melanoma (a rare subtype, less than 4% of patients). Our analyses further highlight the relationship between clinical benefit from immune checkpoint blockade therapy and higher mutational load and PD-L1 expression. Objective tumor responses were noted in 39 patients (71%), with most being long lasting. Our analyses reveal that baseline tumor biopsies have significantly higher tumor parenchymal PD-L1 expression than nondesmoplastic skin melanomas that is highly correlated with CD8+ T-cell infiltration in the invasive margins, reflective of adaptive immune resistance as a result of active antitumor immune process. Desmoplastic melanoma has a known relationship to ultraviolet light radiation damage, resulting in one of the highest mutational burdens among all cancer types. Whole-exome sequencing in this cohort confirms this finding, with a median of 1315 somatic nonsynonymous mutations per tumor, as compared with a median of 485 mutations per tumor in nondesmoplastic melanomas (P = 0.01).
However, accumulating data from cutaneous melanoma suggest that primary and secondary resistance to therapy remains a major challenge. Recent pooled data from 655 patients on a clinical trial of the antiprogrammed death 1 (PD-1) therapy antibody pembrolizumab reported that ~60% of patients with metastatic melanoma had no response to therapy (primary resistance), while ~25% of those with initial objective tumor regression had progressed as much as two years later despite continuous therapy (secondary or acquired resistance). Potential mechanisms of resistance conceptually stratify into either T-cell dysfunction (lack of recognition, trafficking, intrinsic exhaustion, or extrinsic suppression) or tumor-based escape (loss of antigen presentation, resistance to killing, or upregulation of suppressive factors).
We have recently reported two genetic mechanisms of acquired resistance to anti-PD-1 therapy (1). In this study, we analyzed melanoma cases with late acquired resistance after strong initial response to anti-PD1 therapy, with the hypothesis that changes from baseline would be mechanistically meaningful in this setting, particularly with respect to immune function or mutations acquired by surviving clones. To investigate this, we performed whole exome sequencing and immunohistochemistry on four cases with paired pre/postrelapse tumor samples, followed by in vitro modeling with primary tumor cell lines.
First, we studied the local immune environment at relapse to rule out a lack of tumor-specific T-cell effectors. Immunohistochemistry revealed that CD8 T cells were present in baseline tumors, and remained present at relapse in large numbers around the tumor margins. This is a pattern that our lab and others have shown would otherwise be predictive of response to PD1 therapy. Futher studies using targeted sequencing of the T-cell receptor population in biopsies from the four reported cases showed that the top T-cell clones at baseline or during active response were detected at similar frequencies in the relapsing tumors, indicating that the local T-cell repertoire had not changed. Additionally, clones that significantly increased in frequency in the peripheral blood during active response were highly enriched among the top clones in the relapsing tumor. Together, these data indicate the T cells present at relapse are likely the same tumor-specific clones that mediated the original response. Multiplexed immunofluorescence showed that stromal cells in the tumor microenvironment at progression were strongly positive for PD-L1 expression in areas of high T-cell density. PD-L1 is often upregulated in response to the interferon gamma produced by activated T cells, raising the possibility that these T cells were currently or had recently been active.
With evidence that T cells were still present and possibly active in the resistant lesions, we investigated relapse-specific changes within the tumors themselves by whole exome sequencing. We identified new homozygous loss-of-function mutations in the interferon receptor pathway-associated kinases in the relapse biopsies of two separate cases (JAK1 Q503* and JAK2 F547_splice-site). In addition to the clinical radiographic evidence of relapse in situ, we found strong genetic evidence that the progressing lesions were directly derived from the baseline lesions by clonal selection and outgrowth. Despite up to two years between biopsies, the resistant tumor shared &gt;92% of nonsynonymous mutations originally identified at baseline. The relapsing tumors also contained the same initial chromosomal loss-of-heterozygosity (LOH) pattern as baseline, and the JAK mutations become homozygous as part of additional LOH events. Neither JAK mutation was detected pretherapy, either in the exome sequencing reads or by targeted amplicon resequencing, suggesting these represent a de novo mutation or extremely rare preexisting clone.
While the JAK kinases are known to associate with growth factor, cytokine, and interferon receptors, in the context of immune resistance we focused on their role in interferon sensitivity. Interferons cause phosphorylation of the STAT transcription factors, and upregulate expression of chemokines, antigen presentation machinery, and PD-L1. In vitro western blot, RNA (Nanostring), and flow cytometry studies using primary cell lines derived from pre- and postprogression biopsies from the JAK2 mutated case showed that the JAK2 mutated cells were entirely insensitive to interferon gamma. Other melanoma cell lines with JAK1 or JAK2 deleted by CRISPR-Cas9 approach produced similar results. We also found the JAK mutant cell lines to be resistant to interferon-gamma induced growth inhibition, while the baseline and JAK wild-type cell lines remained sensitive. Notably, the JAK2 mutant cell lines were still sensitive to growth inhibition by interferon-alpha/beta or a STING agonist (all of which are JAK2-independent), showing the lack of growth arrest was specific to interferon gamma resistance and not a generalized phenomenon.
In a third case, we also identified a new truncating mutation in the antigen presentation gene beta-2 microglobulin (B2M), which led to loss of surface expression of major histocompatibility complex (MHC) class I. This mechanism has been previously implicated in immune escape. Since the publication of this work we have gathered seven additional biopsies of late secondary progression from patients with advanced melanoma (four of them with a paired baseline) in order to assess the frequency of acquired genetic mutations in the interferon gamma and antigen presentation pathways, as well as define other potential mechanisms.
Our studies of primary resistance to anti-PD-1 therapy provide further support that loss of function mutations in JAK1 or JAK2 are of high relevance for resistance to antiprogrammed death protein 1 (PD-1) therapy. Immune selection may lead to similar mutations in previously untreated patients, which would result in lack of adaptive PD-L1 response by cancer cells to a T-cell infiltrate, and result in primary resistance to anti-PD-1 therapy. Whole-exome sequencing of metastatic melanoma biopsies from 23 patients treated with the anti-PD1 revealed a JAK1 missense mutation with chromosomal amplification in the tumor from a nonresponding patient who otherwise had the highest mutational load in the cohort. Similarly, among 16 patients with mismatch repair deficient colon cancer treated with PD-1 blockade, we again found a homozygous JAK1 loss-of-function mutation in the tumor of a nonresponding patient. Analysis of genomic and proteomic data from the TCGA suggests that JAK1/2 loss-of-function alterations confer adverse outcomes in patients, which may result from an immune-editing process. Therefore, we propose that JAK1/2 loss-of-function mutations are positively selected for by immune-editing processes, leading to primary as well as secondary resistance to PD-1 blockade therapy.
The relevance of genetic alterations in the interferon gamma receptor pathway is highlighted by the results published by another group (2). In their study, knockdown of the interferon gamma receptor in a mouse model of melanoma showed impaired tumor rejection in response to anti-CTLA4 therapy, and they identified increased frequency of mutations in interferon-related genes in patients who failed to respond to the anti-CTLA-4 antibody ipilimumab. While the final frequency of interferon signaling or B2M defects and their contribution to primary versus secondary resistance remains to be seen, cataloguing these and other mechanisms will be important for determining who will benefit from immunotherapy and designing rational next-generation drug combinations to improve outcomes with these therapies.
References
1. Zaretsky JM, Garcia-Diaz A, Shin DS et al. Mutations associated with acquired resistance to PD-1 blockade in melanoma. N Engl J Med 2016;375(9):819-29. PMID: 27433842.
2. Gao J, Shi LZ, Zhao et al. Loss of IFN-pathway genes in tumor cells as a mechanism of resistance to anti-CTLA4 therapy. Cell 2016;167(2):397-404. PMID: 27667683.
Citation Format: Jesse Zaretsky, Angel Garcia-Diaz, Daniel S. Shin, Helena Escuin-Ordinas, Willy Hugo, Siwen Hu-Lieskovan, Zeynep Eroglu, Davis Y. Torrejon, Paul C. Tumeh, Roger S. Lo, Antoni Ribas. Mechanisms of resistance to anti-PD-1 immunotherapy through interferon pathway mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr NG04. doi:10.1158/1538-7445.AM2017-NG04