Research Article: Microsatellite Alterations Are also Present in the Less Aggressive Types of Adult T-Cell Leukemia-Lymphoma

Date Published: January 15, 2015

Publisher: Public Library of Science

Author(s): Marcelo Magalhães, Pedro D. Oliveira, Achiléa L. Bittencourt, Lourdes Farre, Fatah Kashanchi.

Abstract: BackgroundAdult T-cell leukemia/lymphoma (ATL) is a mature T-cell neoplasia etiologically linked to HTLV-1. Manifestations of ATL are diverse and different clinical types with different tissue involvement and aggressiveness have been described. The mechanisms that lead to the development of ATL clinical types have not yet been clarified. Considering that in ATL patients HTLV-1 infection generally occurs in childhood, a multistep carcinogenesis model has been proposed. Microsatellite alterations are important genetic events in cancer development and these alterations have been reported in the aggressive types of ATL. Little is known about oncogenesis of the less aggressive types.Methodology/Principal FindingsIn this study we investigated the role of the microsatellite alterations in the pathogenesis mediated by HTLV-1 in the different types of ATL. We examined the presence of microsatellite instability (MSI) and loss of heterozigosity (LOH) in matched pair samples (tumoral and normal) of 24 patients with less aggressive types (smoldering and chronic) and in aggressive types (acute and lymphoma) of ATL. Four microsatellite markers D10S190, D10S191, D1391 and DCC were analyzed. MSI was found in four patients, three smoldering and one chronic, and LOH in four patients, three smoldering and one acute. None of the smoldering patients with microsatellite alterations progressed to aggressive ATL.Conclusions/SignificanceTo our knowledge, this is the first report describing the presence of MSI and LOH in the less aggressive types of ATL. These results indicate that microsatellite alterations may participate in the development of the less aggressive types of ATL.

Partial Text: Adult T-cell leukemia/lymphoma (ATL) is a severe mature T-cell neoplasia caused by human T-cell lymphotropic virus type-1 (HTLV-1) [1]. It is estimated that there are approximately 5–10 million HLV-1 carriers worldwide, distributed in endemic areas which include Japan, Caribbean islands, Iran, Central and West Africa and South America [2]. The lifetime risk of developing ATL is estimated to be 2–5% [3],[4]. In Japan, there are about 700 new ATL patients every year [5]. HTLV-1 infection is endemic in the state of Bahia in Northeastern Brazil, where several cases of ATL have already been reported [6]. In Bahia, 14% of the ATL patients also present HTLV-1-associated myelopathy/tropicalspastic paraparesis (HAM/TSP) [6], a chronic inflammatory disease of the central nervous system [7]. ATL is originally classified into four clinical types: acute, chronic, lymphoma and smoldering [8]. The acute and lymphoma types have a poor prognosis, whereas the chronic and smoldering types have a less aggressive clinical course [6]. Because there is a long latency period between infection and the development of ATL, a multistep carcinogenesis model has been suggested for this neoplasia [9]. However, the oncogenic mechanisms implicated in the development of the different clinical types of ATL have yet to be established [10]. Microsatellites are short tandem repeats of DNA with one to six base pairs, which are very polymorphic and interspersed throughout the human genome. Alterations such as microsatellite instability (MSI) and loss of heterozygosity (LOH) have been documented in various tumor types, including hematological malignancies. MSI is associated with the failure of the DNA mismatch repair system [11]. LOH represents somatic allelic deletion in tumor DNA and may be related to inactivation of a tumor suppressor gene. MSI alterations have been reported in the aggressive ATL in Japan [12]–[15]. Hatta et al. [12] investigated 22 cases of acute and lymphoma types of ATL and found MSI in 40% of them. These authors suggested that MSI may be involved in the progression of ATL. Hayami et al. [13] studied 18 ATL patients, 14 of whom had the acute, three the chronic and one the smoldering type. These authors found MSI in 22% of the cases, all with the acute form of ATL. They suggested that genomic instability may be associated with tumor progression rather than to the development of ATL itself. LOH was reported in two studies including the acute and lymphoma types [14],[15]. The aim of the present study was to investigate the role of MSI and LOH in the pathogenesis mediated by HTLV-1 in patients with different clinical types of ATL.

The study included 24 patients with ATL diagnosed at the Professor Edgard Santos Teaching Hospital at the Federal University of Bahia (UFBA), Brazil. Five patients had the acute, nine the chronic, nine the smoldering and one the lymphoma-type [8] (Table 1). Additional samples from patients #10 and #19 were collected after ATL progression from the smoldering to the chronic and from the chronic to the acute phase, respectively. All cases of smoldering ATL were non-leukemic [8], presented only skin lesions and none or less than 4% of atypical cells in peripheral blood smears. Serology for HTLV-1 was performed by enzyme-linked immunosorbent assay (ELISA, Ortho Clinical Diagnostics Inc., USA) and confirmed by Western blot (HTLV Blot 2.4, Genelabs Technologies). All patients were HIV-negative. Eight patients had association with HAM/TSP (Table 1). The diagnosis and classification of ATL and HAM/TSP were based on established criteria [7],[8]. Matched pair samples (tumoral and normal samples from the same patient) were evaluated. DNA was extracted from peripheral blood mononuclear cells (PBMC) using the Cell Culture DNA Mini Kit (Qiagen, Valencia, CA). ) The PBMC were collected prior to the treatment with an association of zidovudine and interferon-α (AZT/INF-α) or chemotherapy, except for patient #11 who has been treated with AZT/INF-α for the last five years. DNA control from each patient was extracted from normal samples (oral mucosa, hair root, nail specimens and/or normal skin or lung tissues included in paraffin blocks), using the QIAamp DNA investigator Kit (Qiagen, Valencia, CA) or QIAamp DNA FFPE tissue kit (Qiagen, Valencia, CA). Microsatellite alterations were investigated using four markers: D10S190 and D10S191 (located at chromosome 10), D11S1391 (located at chromosome 11) and DCC 18S21 (located at chromosome 18). These loci were selected because they are known to present a higher frequency of MSI in aggressive ATL [12]. The primer sequences for PCR amplification of each marker were obtained from the UniSTS database ( For each marker, the forward primer was fluorescently labeled with FAM, VIC, NED or PET (Applied Biosystems, Foster City, CA). PCR products were combined with formamide and LIZ-500 size standard and analyzed by capillary electrophoresis using an ABI 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA). Data analysis was performed using the Peak Scanner Software (Applied Biosystems, Foster City, CA). MSI was determined by PCR comparing the amplification pattern in the tumoral and normal samples from the same patient. A method based on the intensity of the amplification signal was used to determine LOH [16]. The ratio between the amplification intensity of PBMC samples and that of the normal tissue samples was calculated as previously described [16]. In this analysis, ratio values ≤ 0.6 or > 1.67 were considered representative of LOH. Only patients who were heterozygous for a given locus were considered informative for LOH analysis. The HTLV-1 proviral load was quantified by real-time PCR using 30 ng of DNA and the TaqMan Fast Universal PCR Master Mix kit (Applied Biosystems) in the 7500 Fast Real-Time PCR System (Applied Biosystems). The HTLV-1 tax gene was selected for quantification of viral copies and normalization was carried out using the beta-globin gene.

MSI was found in four of the 24 patients evaluated (16.6%) (Table 1; Fig. 1A), three of which consisted of smoldering and one of chronic ATL. Two patients were males and two females. In the smoldering ATL, MSI was found in only one locus, in chromosome 10 in two patients (in marker D10S190 in patient #6 and in D10S191 in patient #8) and in chromosome 18 in the other.

The frequency of MSI in ATL patients in the current study was lower compared to previous reports [12],[13]. This may be partially due to the few acute and lymphoma ATL patients who were included. However, the absence of MSI in six acute patients was unexpected. Interestingly, we also found MSI in the loci previously reported in ATL patients from Japan [12], another endemic area for ATL.



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