The evolution of malignant and reactive γδ + T cell clones in a relapse T-ALL case after allogeneic stem cell transplantation
© Chen et al.; licensee BioMed Central Ltd. 2013
Received: 20 April 2013
Accepted: 10 July 2013
Published: 12 July 2013
To improve the outcome of patients with T-cell acute lymphoblastic leukemia (T-ALL), characterization of the biological features of T-ALL blast cells and the immune status of patients with T-ALL is needed to identify specific therapeutic strategies.
Using a novel approach based on the combination of fine-tiling comparative genomic hybridization (FT-CGH) and ligation-mediated PCR (LM-PCR), we molecularly identified a malignant γδ + T cell clone with a Vδ5Dδ2Jδ1 rearrangement that was paired with a T cell receptor (TCR) VγI and comprised a Vγ1Vδ5 T cell clone in a relapse T-ALL patient. This malignant Vδ5 T cell clone disappeared after chemotherapy, but the clone was detected again when disease relapsed post allogeneic hematopoietic stem cell transplantation (allo-HSCT) at 100 weeks. Using PCR and GeneScan analyses, the distribution and clonality of the TCR Vγ and Vδ subfamilies were examined before and after allo-HSCT in the patient. A reactive T cell clone with a Vδ4Dδ3Jδ1 rearrangement was identified in all samples taken at different time points (i.e., 4, 8, 68, 100 and 108 weeks after allo-HSCT). The expression of this Vδ4+ T cell clone was higher in the patient during complete remission (CR) post allo-HSCT and at disease relapse.
This study established a sensitive methodology to detect T cell subclones, which may be used to monitor minimal residual disease and immune reconstitution.
T-cell acute lymphoblastic leukemia (T-ALL) comprises 25% of adult ALL cases, and its outcome is poorly understood. Among patients with T-ALL, approximately 40% achieve long-term remission [1–3]. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains one of the best options for curing T-ALL. However, many patients cannot find an HLA-matched donor; therefore, haploidentical/mismatched HSCTs may be an alternative treatment for T-ALL [4, 5]. The high T-ALL failure rate is mainly the result of an insufficient understanding of T-ALL biology, which hampers the identification of reliable prognostic factors that enable appropriate therapy adjustment . To improve T-ALL outcome, characterization of the biological features of T-ALL blast cells and the immune status of patients is needed to design specific therapeutic strategies [6–10]. T-ALL is generally considered to be a clonal disorder that arises from the expansion of committed lymphoid precursors, and leukemic clones in different patients vary due to the T cell receptor (TCR) gene rearrangements that occur during T-cell differentiation [1, 11]. Moreover, TCR rearrangements also provide different recombination breakpoints that lead to the creation of fusion genes . TCR rearrangement analysis may be used to determine T-ALL immunogenetic characteristics, and TCR rearrangements may be characterized by leukemia antigen-reactive T cell clones, which are thought to be specific to anti-leukemic cytotoxic T cells [13, 14].
In this study, using a novel approach based on the combination of fine-tiling comparative genomic hybridization (FT-CGH) and ligation-mediated PCR (LM-PCR) , which combines PCR and the GeneScan techniques [16, 17], we molecularly characterized the malignant and reactive γδ + T cell clones in a patient with T-ALL before and after relapse 100 weeks post allo-HSCT.
Clinical therapy details for the patient with relapse T-ALL
Blast cells (%) post-treatment
BM / PB
CTX, VCR, ADM, DXM
11 / 3
MTX + DXM
MTX, Ara-c , DXM
37 / 3
CTX, Ara-c, TPT
47 / 1
Ara-c + DXM
Allo-HSCT (conditioning regimen: Flu, BU/CY)
1 / 0
MTX + DXM
GVHD (Grade II)
0.5 / 0
GVHD under control
1.5 / 0
MTX + DXM
0 / 0
(Ara-c + MTX + DXM) x 8 times
VDS, NVT, L-ASPDXM
3 / 0
MTX + DXM (03.05 2012) Ara-c + DXM (25.05.2012)
VCR, NVT, L-ASP, DXM, CTX
1 / 0
Clinical details of the collected samples
4 W post allo-HSCT
8 W post allo-HSCT
68 W post allo-HSCT
Malignant T-ALL clone
To characterize the cellular T-ALL features and the T cell clonality at different time points before and after allo-HSCT and at relapse post allo-HSCT, which may identify a factor associated with outcome, we analyzed the TCR breakpoint loci to identify chromosomal translocations and malignant T cell clones by the FT-CGH, LM-PCR, RT-PCT and GeneScan techniques [15–17].
FT-CGH using overlapping oligonucleotides designed to cover an entire genomic region of interest is a valuable tool for high-resolution chromosomal breakpoint characterization . To achieve high resolution CGH (<1 kb), which is necessary for subsequent in vitro DNA amplification, a custom designed high-density, fine-tiling long oligonucleotide array consisting of 385,000 oligonucleotides 40–60 bp in length was prepared using Maskless Array Synthesizer (MAS) technology (NimbleGen Systems; Reykjavik, Iceland). This array, covering 24 Mb of genome, was selected using the human genome browser hg18 assembly (University of California, Santa Cruz). The array included TCR αδ and IgH loci, which are located on chromosome 14q11 (Chr14: 21,130 -22,130 kb) and 14q32 (Chr14: 105,080 -106,360 kb), respectively, and known to be frequently involved in chromosomal alterations in lymphoid malignancies. The neighboring oligonucleotides with an average distance of 63 bp were grouped in 200, 400, and 1,000 bp clusters. After normalization with reference DNA from the HEK293 T cell line, the mean fluorescence was analyzed using the SignalMap software (NimbleGen) .
The identification of malignant T-ALL cell clones was performed at different times using Southern blot, PCR, RT-PCR, GeneScan, FT-CGH, and next generation sequencing spectratyping . The distribution profiles and clonality of the TCR repertoire in T cells could be characterized using RT-PCR and GeneScan . While an advantage of the FT-CGH and LM-PCR techniques is that they can identify chromosomal breakpoints and unique, high percentage T cell clones in a sample, they cannot characterize the polyclonal TCR subfamily distribution or a small fraction of T cell clones . The novel, exhaustive T cell repertoire sequencing technique can directly measure the TCR repertoire size of at least 1 million clonotypes . Therefore, the combination of the FT-CGH, LM-PCR, PCR, and GeneScan techniques for characterizing T cell malignancies is an ideal serial method not only for identifying abnormal chromosome rearrangements in malignant T cell clones but also for detecting the evolution of malignant T cell clones for the diagnosis, prognosis, and evaluation of reactive T cell clones to characterize the immune status of patients and develop specific immunotherapies.
Clonally expanded reactive T cell clone
In summary, the evolution of malignant TCR γδ + and reactive T cell clones was identified in a patient with relapse T-ALL before and after allo-HSCT and at relapse post allo-HSCT. The techniques used in this study establish the sensitive detection of malignant and reactive T cell clones, and the identified T cell clones may serve not only as biomarkers for minimal residual disease detection but also as anti-leukemia immune status indicators in patients who achieved CR.
This work was supported by grants from the National Natural Science Foundation of China (No. 30871091, 91129720 and 81270604), the Collaborated grant for HK-Macao-TW of the Ministry of Science and Technology (2012DFH30060), the Fundamental Research Funds for the Central Universities (No. 21610603, 21612116) and the Guangdong Science & Technology Project (No. 2012B050600023).
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