PB2167: NGS CLONALITY TESTING FOR A DIFFERENTIAL DIAGNOSIS OF MYCOSIS FUNGOIDES

Abstract

Background: Mycosis fungoides (MF) is a primary epidermotropic lymphoma of the skin, accounting for up to 75% of all skin lymphoproliferative diseases. Diagnosis of MF in the initial stage (Ia-Ib; IIa) or its erythrodermic forms (EMF) is a difficult task. In most cases, neither histological/immunohistochemical examination, nor PCR methods for determining clonality give a definite conclusion. It is known that in the early stages, the PCR method with fragment analysis (PCR-FA) makes it possible to detect T-cell clonality in 75% of cases. On the other hand, in non-tumor dermatoses (ND), clonal products are detected in 10-15% (false-positive result). However, next-generation sequencing (NGS) of the TCRG and TCRB genes may provide more accurate identification of the T cell clone and quantification of the clonal product at the lesion site. Aims: To compare the robustness of NGS and PCR-FA methods for T-cell clonality evaluation in MF and ND. Methods: Skin biopsy specimens and peripheral blood of patients with initial stages of MF (n-7), EMF (n-3), and ND (n-17) were included in the study. Evaluation of clonality for the TCRG and TCRB genes was carried out in parallel by the PCR-FA and NGS. Data processing and T-cell clone fraction/frequency calculation were performed according to the MiXCR protocol (https://github.com/milaboratory/mixcr). Results: PCR-FA and NGS gave the same results in all samples tested. T-cell clonality was detected in 6 out of 10 patients with MF and EMF. In these patients, the fraction of the dominant TCRG clone ranged from 5.5 to 83.4% (mean 32.3%), for TCRB - 6.1 to 47.9% (mean 20.8%). In 2/7 patients with MF and 2/3 with EMF, clonality was not detected, while the fraction of the dominant T-cell clone did not exceed 4.2%. In two patients with MF and no T-cell clonality, skin samples were examined at disease progression at 6 years and 3 years from the initial biopsy, and a dominant tumor clone was found. These dominant clones were represented by only 2.2% (TCRG) and 4.2% (TCRB) in the initial biopsy for one patient, and 0.26% for the second. In 3/7 patients with the initial stages of MF, a tumor clone was detected in the blood in the amount of 0.022-0.054%. All 3 patients with the minimal disseminated disease at the onset, rapidly progressed to the advanced stages of the disease. In the control group (ND) dominant TCRG clone fractions were 0.49-4.8% (mean 1.93%), for TCRB - 0.9-6.1% (mean 3.11%). The results are shown in Figure 1. Image:Summary/Conclusion: Despite a small sample of patients, the following trends were noted: 1 - patients with the initial stages of MF with similar clinical and histological features, are diverse in the number of tumor T-lymphocytes infiltrating the skin (from 0.26% to 83.4%); 2 - the initial stage of MF with the minimal disseminated disease, might have an unfavorable outcome; 3 - comparing to PCR-FA NGS might not provide further improvement in the specificity and sensitivity of T-cell clonality evaluation for the differential diagnosis in MF.