A comparison of the effects of romidepsin treatment on gene expression in a leukemic patient with Sezary syndrome using two different methods for blood collection
Authors:   , Billouin Shere Shievonne , Spotila James Robert 
Published: July 2021

Background: Sezary Syndrome (SS), an aggressive, leukemic form of Cutaneous T-Cell Lymphoma, is resistant to conventional treatments with chemotherapy. Although SS is treated with several modalities, these therapies have poor response rates and are not curative. Histone Deacetylase Inhibitors (HDACi) are newer treatments that have been shown to be an effective treatment option for SS. Although the response of SS patients to HDACi has been encouraging, the response mechanisms are not understood. This project examined the effect of HDACi treatment on gene expression patterns using a novel HDACi known as Romidepsin. As a first step, two different blood sample collection methods were compared. Blood samples were collected from a SS patient using the BD Vacutainer(R) CPT(TM) Cell Preparation tubes that are routinely used for peripheral blood mononuclear cells (PBMC) isolation, and with a newer method for whole blood sample collection in PreAnalytiX PAXgene tubes. PAXgene tubes do not require PBMC purification for RNA isolation, but the RNA is immediately stabilized at the time of collection into the PAXgene reagent. Validation of the PAXgene method would standardize sample collection for blood based microarray studies and eliminate the need to rapidly process CPT samples to maintain mRNA profiles. Previous research has shown that varying blood Background: Sezary Syndrome (SS), an aggressive, leukemic form of Cutaneous T-Cell Lymphoma, is resistant to conventional treatments with chemotherapy. Although SS is treated with several modalities, these therapies have poor response rates and are not curative. Histone Deacetylase Inhibitors (HDACi) are newer treatments that have been shown to be an effective treatment option for SS. Although the response of SS patients to HDACi has been encouraging, the response mechanisms are not understood. This project examined the effect of HDACi treatment on gene expression patterns using a novel HDACi known as Romidepsin. As a first step, two different blood sample collection methods were compared. Blood samples were collected from a SS patient using the BD Vacutainer(R) CPT(TM) Cell Preparation tubes that are routinely used for peripheral blood mononuclear cells (PBMC) isolation, and with a newer method for whole blood sample collection in PreAnalytiX PAXgene tubes. PAXgene tubes do not require PBMC purification for RNA isolation, but the RNA is immediately stabilized at the time of collection into the PAXgene reagent. Validation of the PAXgene method would standardize sample collection for blood based microarray studies and eliminate the need to rapidly process CPT samples to maintain mRNA profiles. Previous research has shown that varying blood collection methods and processing times for RNA purification significantly impacts steady state gene expression profiles, and affects the conclusions of these studies, such as a measure of the effectiveness of a treatment. Objectives: This research examined the effects of progressive Romidepsin treatment on gene expression in blood samples collected pre- and 4 hours post- treatment over 6 months. Samples were collected in CPT and PAXgene tubes. CPT tubes require rapid processing and yield only purified PBMCs for downstream RNA projects, whereas PAXgene tubes, which can be stored before processing, yield immediately stabilized RNA from mononuclear cells and granulocytes. Gene expression patterns from both collection systems were compared to determine whether the PAXgene method, which includes cell types lost in the PBMC purification, will yield new and important information as compared to the CPT method. Methods: Peripheral blood samples from a SS patient treated with Romidepsin were collected in CPT and PAXgene tubes. PBMC were purified from the CPT samples within 2 hours of collection. In one case, the CPT sample was processed 24 hrs after collection. RNA was then extracted from the PBMC samples and RNA was also purified from e PAXgene samples after several months of storage at -80°C. The RNA samples were amplified utilizing the Illumina® Total PrepTM RNA Amplification kit followed by hybridization to the Illumina® Sentrix Human Expression BeadChips for microarray analysis. Data analysis was performed and validation of some genes using quantitative Real Time-Polymerase Chain Reaction.