Advancing Science - A Virtual Oncology Conference

Targeted therapies for cancer are increasingly being developed as an alternative for, and in addition to, immunotherapy. Altered tumor signaling can be exploited to single-out cancer cells for destruction while leaving normal cells intact. However, as we continue to learn, a therapeutic strategy that treats the biology of the tumor by itself in a vacuum without the context of the immune response and the surrounding microenvironment can lead to reduced efficacy. The new nCounter Tumor Signaling 360 gene expression panel homes in on signaling pathways involved in the sustained growth and maintenance of tumor cells while providing a holistic view of the immune response and microenvironment that can be complicit in the development and progression of cancer. Using the foundation of the PanCancer IO 360 and Breast Cancer 360 panels as a model, the Tumor Signaling 360 panel was designed to include 770 genes across 40+ pathways integral for tumor biology, immune evasion, and microenvironment remodeling. The panel offers the same direct, digital detection benefits of other NanoString gene expression panels and includes the 18-gene Tumor Inflammation Signature (TIS) as well as 14 immune cell type signatures for profiling the peripheral immune response.

Rational: Pancreatic Ductal Adenocarcinoma (PDAC) is a highly aggressive disease associated with poor outcome. Most PDAC patients die within the first two years, and only a small subgroup of patients exceeds this survival rate. So far, factors and pathways underlying long-term survivorship in PDAC are unknown. In a previous study we showed that high CD8/FoxP3 ratio is associated with a favorable outcome. Therefore, we aimed to reveal the immune-related key players that drive the long-term survivorship in PDAC patients.

Methods: The immune-related gene expression profiles of 10 PDAC patients survived and free of recurrence for ≥ 5 years, was compared to 10 PDAC patients in whom recurrence and death occurred within 6 months after surgery. Samples were profiled using the PanCancer Immune Profiling Panel of nanoString technology. Subsequently, a subgroup of samples was measured by the GeoMx Digital Spatial Profiler (DSP) to determine the spatial location of the immune cells.

Results: The immune microenvironment in long-term survivors was altered differentially than that of the short-term survivors. B cells were found to be highly expressed in long-term survivors by gene expression profile. The presence of B cells was confirmed by performing a high-plex proteomic analysis with spatial resolution (GeoMx DSP). B cells infiltrated to the tumor stroma together with T cells and antigen presenting cells in long-term survivors.

Conclusion: This is the first comprehensive study that compares the expression and the spatial infiltration of immune cells in PDAC patients with different survival rates. Our data demonstrate that long-term survivorship of PDAC patients is influenced by the presence of B cells in the tumor microenvironment. The role of B cells in PDAC disease is controversial. However, our findings illustrate that B cells interact with other compartments of the immune system and drive long-term survivorship in PDAC patients.

Hosein Aziz1, Lawlaw Saida1, Jasper Dumas2,4, Andrew Stubbs2,3, Yunlei Li2,3, Casper van Eijck1,4 and Dana A. Mustafa2,4

1 Department of Surgery, 2 Department of Pathology, 3 Division of Clinical Bioinformatics, 4 The Tumor Immuno-Pathology (TIP) Laboratory, Erasmus University Medical Center, Rotterdam, the Netherlands.

Background: Pathologists have long recognized that the interaction between immune and tumour cells is critical in the development and progression of breast cancer. Studies have demonstrated the presence of tumour-infiltrating lymphocytes (TILs) correlates with improved clinical outcome in breast cancer especially in the triple negative and HER-2 positive subtypes. TILs predict for improved response to certain therapies including chemotherapy and trastuzumab. The predictive value of TILs in ER positive tumours is less clear. It has been demonstrated the higher presence of the immune microenvironment is associated with a better prognosis and as a result a higher likelihood of benefit from chemotherapy and possibly from immunotherapy, whereas cold immune microenvironment carries greater risk of relapse and lower benefit from chemotherapy and possibly immuno-therapies. In this study we examined the immune landscape in early breast cancer patients prior to anthracycline treatment.

Methods: NanoString’s DSP platform was used to analysis 256 patient samples from the BR9601 clinical trial. A panel of 56 antibodies incorporating the Immune Profile Core; 22 targets, IO Drug Target, Immune Activation Status, Immune Cell Typing, and Pan Tumour were examined and 2 fluorescent markers (pan-cytokeratin (pan-CK) and CD45) were used to select regions of interest (ROI).

Results: Proteomic analysis was successfully performed on 256 cores from the BR9601 cohort. Multivariate analysis demonstrated 27 proteins were associated with distant relapse free survival. A T-cell score was generated using the average expression of CD3, CD4 and CD8. The T-cell score was examined in both the tumour and TME. Using the T cell score it was apparent that the cohort had a range of immune “hot” and immune “cold” tumours. It was demonstrated that immune “hot” TME doesn’t not always correlate with immune “hot” tumour expression. Proteins that were most associated with T-cell exclusion (p<0.01) in the TME were Fibronectin, B7-H3, PTEN, ER-α, TGFB1, FAPα and CD34. This would indicate that these proteins are causal inhibitors of T-cell invasion.

Conclusion: In conclusion, this study highlights the significance of assessing the entire tumour since TILs, tumour and stromal cells collectively engage in a complex interplay that contributes to disease development and progression. NanoString’s GeoMx DSP is a promising technology for multiplexed analysis. TILs, whether measured using automated software, or estimation by protein profiling, are predictive of chemotherapy benefit.

The majority of patients with advanced NSCLC do not respond to monotherapy with PD-1 axis inhibition, and more robust predictive biomarkers are needed. In this study, we assessed tumor samples from 67 immunotherapy-treated NSCLC cases represented in a tissue microarray, 53 of whom had pre-treatment samples and received monotherapy. Using GeoMx Digital Spatial Profiling (DSP) system (NanoString), we quantified 39 immune parameters simultaneously in four tissue compartments defined by fluorescence co-localization (tumor [panCK+], leucocytes [CD45+], macrophages [CD68+], and non-immune stroma). In the univariate unadjusted analysis, we found 18 markers associated with outcome in spatial context, 5 of which remained significant after multiplicity adjustment and after controlling for clinical confounding factors. In the multivariate analysis, high expression of CD56 and CD4 in the CD45 compartment were significantly associated with all favorable clinical outcomes, whereas high levels of VISTA and CD127 in the tumor compartment were markers associated with immunotherapy resistance. We also validated the DSP finding that high CD56+ immune cell counts in the stroma were predictive for PFS and OS in the same set of patients using multiplex immunofluorescence, strengthening the relevance of NK/NKT cells

Background: Growing evidence supports the critical role of preexisting immune response in triple negative breast cancer (TNBC). However, there are limitations with current evaluation approaches: inability to functionally assess the type of immune infiltration with traditional pathologic evaluation and loss of spatial distribution in conventional high-plex gene or protein expression analyses from the whole tumor section. Here, we report the initial analysis of immune protein expression as a function of spatial distribution and clinical outcomes in TNBC samples.

Methods: NanoString GeoMx® Digital Spatial Profiling (DSP) was used to quantify 39 immunerelated proteins in stromal and tumor segments from 44 TNBC samples from the FinXX trial. Samples were matched for patient characteristics, treatment arm (capecitabine vs. 5- fluorouracil), and outcome based on recurrence-free survival (RFS) with 22 samples from patients who recurred and 22 samples from patients with durable RFS. Regions of interest (ROIs) were selected based on expression of cytokeratin (tumor), CD45 (leukocytes), or CD68 (macrophages). Each ROI was segmented into tumor (pancytokeratin-positive area) and stroma (pancytokeratin-negative/nuclear SYTO13-positive area). The general linear model was used for statistical analysis of differential expression with RFS as a categorical variable (recur yes or no).

Results: A total of 950 tumor and stroma segments were included in this initial analysis. In both tumor and stroma segments, over-expression of T cell activation markers (CD137, GITR) was associated with better outcome, whereas T cell markers (CD3, CD4, CD8) were not significantly associated with outcome. In tumor segments alone, improved outcome was significantly associated with increased protein expression [> 2-fold change (FC) at p2.0, p2.0, p
In CD45-enriched stroma, improved outcome was associated with elevated expression (FC>2.0, p2.0, p
whereas durable RFS was associated with elevated PD-L2 and IDO1 expression in CD68-enriched stroma. In contrast, macrophage/dendritic cell markers CD68 and CD11c were not associated with outcome. In tumor cells adjacent to CD45-enriched stroma, durable RFS was associated with increased abundance (FC>2.0, p
Among 39 immune function proteins, only elevated CTLA4 expression in CD68-enriched stroma was associated with recurrence (FC=0.55, p whereas tumor-segment CTLA4 was associated with RFS.

Conclusions: Using an in-depth analysis to precisely quantify the abundance of multiple immune function proteins in a spatially defined manner, we observed that PD-L2, IDO1, and T cell activation markers were robustly associated with durable RFS in both tumor and stromal segments. In contrast, MHC components (HLA-DR, beta-2-microglobulin), B cell markers (CD20), and NK cell markers (CD56) were strongly associated with favorable outcome in tumor but not in stromal segments. Our study highlights the conclusion that the immune landscape of TNBC is far too complex to be encompassed by any single molecular marker, and more detailed analyses of the DSP data reported here are ongoing with a view towards using quantitative multiplex analyses to refine our understanding of how therapeutic outcome is influenced by interactions among immune cells and between immune and tumor cells.