Almudena Navarro-Bailón1, Cristina Jiménez1, Sara Alonso-Álvarez1, Alicia Antón1, María Carmen Chillón1,2, Miguel Alcoceba1,2, María García-Álvarez1, Isabel Prieto1, Alejandro Medina1, Verónica González-de-la-Calle1, Marcos González1,2, María Eugenia Sarasquete1,2, Ramón García-Sanz1,2
1 Hospital Universitario de Salamanca, Salamanca, Spain, 2 CIBERONC tumores hematológicos, Spain
Introduction: Hodgkin Lymphoma (HL) is a germinal centre B lymphocyte-derived neoplasia, but its genetic characterization remains complicated due to the small proportion of malignant cells in the tumour (≤1%). Exportin-1 (XPO1) is a nuclear export protein known to be overexpressed in many malignancies. Recently, a mutation hotspot in this gene (p.E571K) has been described in primary mediastinal diffuse large B-cell lymphoma (PMBL) and HL.
In this study, we assessed XPO1 E571K mutation in tumour tissue by droplet-based digital PCR (ddPCR) and investigated correlation with clinical and biological characteristics.
Methods: We evaluated samples from 89 adult HL patients at diagnosis. XPO1 E571K mutation was analyzed by ddPCR using custom Taqman primers and probes (Camus, Haematologica 2016) in a QX200 ddPCR platform (BioRad, Hercules, CA, USA). PCR reaction was performed on a C1000 Touch™Thermal Cycler (BioRad Laboratories). We defined the limit of blank/specificity (LoB) of the technique analyzing 21 wild type (WT) samples from healthy donors. Limit of detection was established using a dilution curve of mutated DNA (U-H01 cell line). Statistical analyses were carried out with IBM SPSS Statistics Software.
Results: A cutoff of 0.1% was established by the LoB of the technique (E571 mutation vs. WT), and sensitivity was 0.0085% in the dilution curve.
We detected the presence of XPO1 E571K mutation in 26 cases (29%), median mutation burden was 0.57%(range 0.1–10.4).
Characteristics of mutated and unmutated patients are summarized in Table 1. No main differences between both groups were detected, but patients with XPO1 E571K mutation were slightly older (p = 0.042) and more likely to present with abdominal involvement (p = 0.008). Of note, 70% of cases with Ann Arbor stages III-IV were mutated (p = 0.01). This was confirmed by the Hasenclever index distribution, with 42% vs. 20% mutated patients among Hasenclever ≥ 3 vs. <3, respectively (p = 0.048). Interestingly, XPO1 E571K mutation was detected in all histologic subtypes, excepting two cases with Lymphocytic Depleted subtype. No significant differences were found in response, progression free survival and overall survival.
Conclusion: -ddPCR is a sensitive and specific technique for determining XPO1 E571K mutation in biopsy samples of patients with HL.
-This mutation is present in 29% of cases.
-Presence of the mutation is correlated with advanced age, III-IV stages and abdominal involvement. No correlation was found with prognosis.