Efficacy of Combinatorial Treatment Approaches in CTCL

BACKGROUND. Mycosis fungoides/Sezary syndrome (MF/SS) are the most common subtypes of cutaneous T cell lymphoma. The patients (pts) with early-stage MF usually have an indolent course and are managed with skin directed therapies. In contrast, pts with advanced-stage disease, have poor prognosis and a median overall survival between 2 and 5 years. Over the past 20 years only six novel biological/targeted agents (bexarotene, denileukin diftitox, vorinostat, romidepsin, brentuximab vedotin, mogamulizumab) have been FDA approved for systemic therapy of relapsed/refractory MF/SS however, none render curative approach. Overall response rate across all agents is 30% with CR rate of only 10%. Classical chemotherapy has no advantage over less toxic novel targeted agents in MF/SS. The head-to-head comparison of active therapeutic agents in prospective randomized trials is mostly absent and systemic therapy sequencing strategies are usually derived from personal experience, expert opinion, or retrospective reviews. The biomarkers directing the selection of targeted agents for individual patients are not available or reliable. In this study, we tested sensitivity of autologous lymphoma cells obtained from peripheral blood to various therapeutic agents and combinations ex vivo, in order to predict response to tested agents in vivo and develop more sophisticated, personalized, cost-effective, and clinical approach for treatment of pts with advanced stage MF/SS.

METHODS. A 384-well plate is coated with poly-L-ornithine to help anchor the cells and the CTCL cells collected from the patients at Moffitt Cancer Center are thawed and incubated over-night at 37C. The next day, isolated CTCL cells are stained with CytoLight Rapid Green Reagent from Essen Bioscience. Once the dye is neutralized and washed out from the cells CytoxRed from Essen Bioscience is added to the media with cells. The cells are then distributed in a 384-well plate. Next, the drugs are diluted and added to the plate. There is a 5-time serial dilution of the drug and if it is a drug combination, one drug is serial diluted and the second drug is constant. After plating, the cells are incubated at 37C for 30min to let them rest and equilibrate. The EVOS M7000 (Life Technologies) is used using the GFP and Texas Red cubes to detect live (green) and dead (red) cells at 0h, 6h, 12h, 18h and 24h. When analyzing the data, the Celleste 5 (Life Technologies) program is used, specifically the Live/Dead application. The intensity threshold is kept constant throughout the entire experiment. The total number of live cells are used to calculate the viability of the cells at each time point. All the time points are done in triplicates and media with DMSO is used as the control.

RESULTS. Out of 11 MF/SS pts samples treated with single agent and/or combinatorial drugs, 4 pts samples had no appreciable responses to single or combination treatments. In this study, response to treatment is deemed favorable if there is a decrease of 50% or more in viable cells in the course of the experiment. Seven pts samples did have responses against either a single drug or a combination of drugs. Out of the 7 pts that did respond, the most prevailing treatment was the combination of Venetoclax and Duvelisib with 6 out of 7 pts samples having a response to this combination. The next noteworthy response was seen with Romidepsin alone in 4 out of 7 pts.

CONCLUSION. Our observations on MF/SS pts samples thus far demonstrates various sensitivities to treatments for individual pts. This may account for genetic and epigenetic changes which highlight the unmet need for personalization of treatment and sensitivity screening for each patient, with the decisive goal to offer a personalized and efficacious treatment modalities.

Sokol:Kyowa-Kirin: Honoraria, Research Funding; Dren Bio: Consultancy. Pinilla Ibarz:AbbVie: Consultancy; Pharmacyclics: Consultancy; SecuraBio: Research Funding; AstraZeneca: Consultancy; Janssen Pharmaceuticals: Consultancy.