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BACKGROUND: Alterations in the tumor microenvironment (TME) can result in a “T-cell inflamed” TME, which is characterized by activation of the PD1/PD-L1 pathway, immunosuppression, and production of anergic, “exhausted” CD8(+)-T-cells. Similar alterations in the TME of Mycosis Fungoides (MF) have been linked to disease progression. However, the relationship between progression and the TME in MF remains incompletely understood. We aimed to further study the TME in MF using the PanCancer IO 360™ 750-gene expression panel and nCounter® platform (Nanostring; Seattle, WA). DESIGN: Clinical and laboratory data from the electronic medical record were reviewed in combination with microscopic review of biopsies from 27 patients to confirm diagnosis of MF and assign samples as “low-grade” (patch/plaque; n=8) or “high-grade” (tumor/large cell transformation, n=19). Patients were also classified as having “limited stage” (TNMB1 1B, n=19) disease. All patients received prior, heterogenous treatment, but no patients received prior anti-PD1 therapy. RNA was extracted from formalin-fixed, paraffin-embedded (FFPE), punch biopsy sections and analyzed per manufacturer’s instructions. Gene expression scores (log2) and a “tissue inflammation score” (TIS) were calculated for each sample. A TIS of >5 has been associated with the presence of a “T-cell inflamed” microenvironment. Samples scores were compared by histologic group and disease stage as described above. RESULTS: Twenty-nine of 30 samples had sufficient RNA integrity for analysis. TIS was >5 in 26/30 samples (median, 7.6; range, 4.3-9.3). No significant differences in TIS were identified between low-grade/limited-stage and high-grade/advanced-stage samples. High-grade samples had significantly increased expression of PD1 and higher amounts of exhausted CD8(+)-cells than low-grade samples (p= 0.015, 0.027). Low-grade samples showed significantly higher expression of ARG1, B7-H3, and genes associated with tissue hypoxia. Expression of genes associated with tumor proliferation was higher in high-grade lesions (p= 0.0019). Limited stage samples showed higher levels of endothelial cells, mast cells, and T-regulatory cells (p=0.033, 0.024, 0.004,) than advanced stage samples. Proliferation, PD1, and exhausted CD8 scores trended higher in advanced stage disease but did not reach statistical significance. CONCLUSION: Our findings indicate that the IO 360™ panel provides comprehensive analysis of the TME in MF. Most samples showed a TIS of >5, suggesting a T-cell inflamed TME in MF. Increased PD1 and exhausted CD8(+) cells were found in high-grade lesions, supporting a role for this pathway in progression. Increased expression of factors associated with angiogenesis (endothelial cells, ARG1) and immunosuppression (B7-H3, tissue hypoxia, mast cells, T-regulatory cells) suggest these mechanisms may play a role in early stages of disease. These findings highlight the complex and dynamic nature of the TME during MF progression, and the need for further study. Citation Format: Stanton Miller, Akshat Patel, Kiran A Kumar, Farrukh Awan, Heather W Goff, Travis Vandergriff, Marisa Juntilla, Franklin Fuda, Weina Chen, Jesse M Jaso. Evaluation of the tumor microenvironment in mycosis fungoides using the PanCancer IO 360™ assay and nCounter® platform [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr A40.

e15549 Background: Liquid biopsy is a candidate for detection of minimal residual disease (MRD) in early colorectal cancer. Detection of circulating tumor (ct)DNA in early stage cancer is challenging given the low abundance of ctDNA molecules. We developed a tissue agnostic MRD Test to optimize sensitivity and specificity of ctDNA detection by integrating tumor-specific genomic mutation and DNA methylation signatures coupled to noise-reduction of non-tumor background cfDNA signals. Methods: This MRD Test was developed following CLIA, Nex-StoCT Working Group, and AMP/CAP guidance and validation principles. Using a single input plasma sample, data from somatic and epigenomic targeted capture panels are integrated to generate a final test result: ctDNA detected or ctDNA not detected. ctDNA detected is defined by the presence of mutation or epigenomic classifier inputs exceeding a defined threshold. Quality control (QC) measures were implemented in both process and final sequencing results. Analytical specificity, sensitivity, and accuracy were determined using 130 samples. Results: The MRD Test specificity was 95% (70/74) using colonoscopy screened negative samples.A retrospective review of the clinical histories of these false positive samples showed high risk clinical features such as smoking, family history of CRC, and sub-optimal bowel prep prior to the colonoscopy. Analytical sensitivity (LoD) was established by diluting late stage CRC samples at two clinically relevant cfDNA inputs at three dilutions (30ng or 10ng, ranging 0.1% to 0.75% tumor fraction). The 95% LoD was determined to be 0.3% for 10ng and < 0.1% for 30ng, the lowest level tested for each input. Accuracy was determined by diluting advanced CRC samples to 0.5-0.75% MAF tested at 30ng or 10ng cfDNA with a detection result of 100% (38/38). Conclusions: We present a CLIA validated assay with performance characteristics sufficient to detect residual disease in Stage II/III CRC post-curative intent therapy. This assay is currently being used in multiple interventional clinical trials.