Abstract 220: Disease monitoring of mycosis fungoides via high throughput sequencing of CDR3
Authors: Wang Kevin Y. , Wang Tao , Liu Yuehua , Lian Christine 
Published: September 2020

Abstract
Background
High-throughput T-cell receptor sequencing (HTS) has been shown to be both sensitive and specific at diagnosing Mycosis Fungoides (MF); a sensitivity of 85% was achieved with superiority to TCRγ; PCR in detecting monoclonality and a specificity of 99.89% was achieved in detecting minimal residual disease, specifically Sezary Syndrome cells with superiority to flow cytometry. In this study, we look at the potential application of HTS as a method for monitoring treatment response in patients with MF.
Methods
We sequenced the CDR3 region of the T-cell receptor (TCR) subunit of T cells in the skin lesional tissue, as well as in pre- and post-treatment blood samples of 7 patients with late stage Mycosis Fungoides (stage IIB-IV). DNA and RNA were extracted from peripheral blood and skin samples. Library preparation followed the manufacturer's instructions (Illumina). The template DNA fragments of the constructed libraries were hybridized to the surface of Nimblegen sequence Capture chip and detected with Hiseq2000 platform. Raw data was analyzed and verified to obtain aberrantly expressed genes. A multiplex PCR system was used to amplify the specific CDR3 region. Distinct TCR CDR3 sequences were plotted on the basis of their frequency of appearance to generate the TCR “repertoire”. The malignant clonal TCR signature was determined by the highest frequency CDR3 sequence of the tumor cells from skin biopsy specimens and confirmed by pathological diagnosis. This malignant sequence was identified in pre-treatment blood and monitored in post-treatment blood. Further analysis of each patient's TCR repertoire allowed us to calculate a diversity index (Simpson's diversity index) for each patient's skin sample. Results of diversity index calculation was analyzed with unpaired T-test using GraphPad Prism version 7 (GraphPad Software, La Jolla, CA). All p-values were two-tailed, with a p&lt;0.05 considered statistically significant.
Results
Frequency of the identified malignant TCR clone remained high in the post-treatment blood samples of the patients with poor treatment response, while the number of TCR signature clones diminished in the post-treatment blood samples of the patients with good treatment response. Higher levels of diversity of TCR was also seen in the skin pre-treatment for good responders to treatment.
Conclusion
This study used HTS to identify malignant TCR signatures from skin biopsy specimens and to monitor the changes in blood samples in MF patients. The findings suggest that HTS is a potential ancillary blood test to monitor treatment response in MF patients by comparing the levels of the TCR signature before and after therapy. A greater diversity of T-cells in the skin may also predict a better treatment response in MF, as the presence of tumor infiltrating lymphocytes has been correlated with better outcomes in many different cancers.
Citation Format: Kevin Y. Wang, Tao Wang, Yuehua Liu, Christine Lian. Disease monitoring of mycosis fungoides via high throughput sequencing of CDR3 [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 220.