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B-CLL is a lymphoproliferative disease characterized by a defect in spontaneous apoptosis that results in the accumulation of long-lived mature-looking lymphocytes.1 The disease has a prolonged natural history although the clinical course may be heterogeneous, with long-term survival of some individuals and aggressive progression in others. Patients with germline VH genes have a distinctly more malignant disease and a much shorter survival than those with SHMs.2, 3, 4, 5, 6
The aggressive clinical course in B-CLL is determined predominantly by a profound dysregulation of the immune system, with hypogammaglobulinemia and defects in cellular immunity.7 Indeed, it has been estimated that almost 60% of deaths linked to B-CLL are caused by bacterial or viral infections.8 Consequently to the overwhelming prevalence of the malignant B cell clone, B-CLL patients show much lower percentages of circulating T lymphocytes than healthy individuals.9, 10 In addition, Scrivener et al.11 have reported abnormalities of cell surface antigen expression on B-CLL patients' T cells that were not corrected by depletion of the malignant B-cell clone. This suggests the possibility that the T-cell perturbance in this neoplasm might be related, at least partially, to a depressed thymic function. In this regard, impaired thymopoietic capacity in B-CLL patients has been suggested by the low number of CD4+/CD45RA+ lymphocytes with a naive phenotype.12 Naive T cells are not necessarily an accurate marker of thymic function, however, whereas the frequency of sjTRECs in peripheral blood T cells is considered an accurate age-related marker of thymopoietic capacity.13, 14, 15, 16 sjTRECs are present in recent thymic emigrants as episomal DNA circles resulting from the rearrangement of T-cell receptor (TCR) α or β genes and are diluted progressively as a consequence of T cell peripheral division.17
We investigated the levels of sjTRECs generated from the rearrangement of the TCR α gene in peripheral blood mononuclear cells (PBMCs) of 30 untreated B-CLL patients at diagnosis and in age-matched healthy controls. We have also evaluated the hypothesis that sjTREC levels might be related to clinical course and to an accredited B-CLL prognostic marker represented by the somatic hypermutation status of the VH genes.
MATERIAL AND METHODS
Our study is based on a cohort of 30 B-CLL patients (informed consent was obtained from each participant). The mean age at diagnosis was 61.6 years (range = 49–70), 16 were males and 14 females. Twenty-four patients were diagnosed as early stage (Stage A) of the disease, with the remainders being in more advanced stages (Patients 2, 12, 17, 26, Stage B; Patients 7, 10, Stage C). A clinical follow-up of 5 years was available for B-CLL Patients 1–8, 10–12, 14–18 (Patients 1, 2, 6, 8, 10, 11, 15 showed progressive disease). Progression was assessed clinically on the basis of the following criteria recommended by the National Cancer Institute-sponsored workshop.18 Blood mononuclear cell counts (percentage of neoplastic CD5+/CD19+ cells), lymphocyte count doubling time; progression to a more advanced stage of disease; development of systemic symptoms; and a downward trend in hemoglobin concentration or platelet count. Disease in patients with at least one altered feature was defined “progressive,” whereas patients without altered features were considered to have “stable disease.” PBMCs from 18 age-matched healthy donors were obtained for sjTRECs comparison.
Genomic DNA extraction
Genomic DNA was purified from total PBMCs obtained by Ficoll/Hypaque (Pharmacia Upjohn, Gaithersburg, MD) density gradient centrifugation. PBMCs were lysed, digested with proteinase K (Sigma, St. Louis, MO) and nucleic acids were “salted out” and ethanol-precipitated.19
Measurement of signal joint (sj) TRECs in PBMCs
sjTRECs in PBMCs were quantitated by real-time PCR using the TaqMan assay with an ABI 7700 Prism detection system (Perkin-Elmer, Norwalk, CT). Amplification was carried out using 250 ng of genomic DNA for each sample. Specific primers were designed on the basis of a known sequence of the human TCR α locus (GenBank accession number AE000661): forward, 5′-CACATCCCTTTCAACCATGCT-3′ (nt 229214–229234); reverse, 3′-GCCAGCTGCAGGGTTTAGG-5′ (nt 140225–140244); probe, 5′-ACACCTCTGGTTTTTGTAAAGGTGCCCACT-3′ (nt 229236–229265). The PCR reaction contained Universal Master Mix 2X (Perkin Elmer Biosystem), 500 nM of each primer and 200 nM of probe. An external standard curve was plotted with a linear distribution (r = 0.99) between 10–105 copies of a plasmid (sj-pTREC) obtained by cloning a sjTREC fragment in a pZero Blunt vector (Invitrogen, La Jolla, CA). Inter-sample variation was monitored by measuring total cellular DNA using Sybr Green PCR (Perkin Elmer) with primers specific for human GAPDH (forward: 5′-ACCACAGTCCATGCCATCACT-3′; reverse: 5′-GGCCATCACGCCACAGITT-3′). Amplification conditions were 50°C for 2 min, 95°C for 15 min and 40 cycles at 95°C for 15 sec and 60°C for 1 min. sjTREC and GAPDH values were calculated using the ABI 7700 software; all samples were analyzed in duplicate and experiments were replicated to confirm the data. sjTREC levels were expressed as copies per 106 PBMCs, as described.14
PCR amplification of Ig VH genes and analysis
The VHDJH region for each patient was amplified by PCR from 1 μg PBMCs genomic DNA using consensus primers in the framework 1 [FR1c: AGGTGCAGCTG(GC)(AT)G(GC)(AGT)GG] and in the joining [JHc: ACCTGAGGAGACGGTGACC(AG)(GT)(GT)GT] regions. PCR was carried out in a final volume of 50 μl, with 50 pmol of each primer, 200 μM dNTPs, 1 mM MgCl2, 10% of DMSO and 1 U Taq DNA polymerase (GIBCO). Amplification consisted of an initial denaturation step at 94°C for 3 min followed by 34 cycles at 94°C for 1 min, at 62°C 1 min and 72°C for 2 min, with a final extension step for 10 min at 72°C. The number of SHMs was determined by comparing the VHDJH sequence of each patient to the germline VH genes with the highest homology using the Ig-BLAST or the V base database. Based on the number of somatic mutations detected in these genes, the cases were classified as “unmutated” or “mutated.” Consistent with current convention, “unmutated” genes were defined as those with <2% differences from the most similar VH germline, whereas “mutated” genes were those with >2% difference from the closest germline.
sjTREC analysis in B-CLL patients and in age-matched healthy controls
Due to the difficulties in purifying normal (non-neoplastic) PBMCs from peripheral blood of B-CLL patients, the number of sjTRECs was evaluated directly in total PBMCs. This approach has been validated previously in healthy donors14, 20 and used recently to determine sjTREC levels in children with T and B cell acute lymphoblastic leukemia.21
To determine the sensitivity of the assay and to exclude a potential confounding effect of tumor DNA deriving from monoclonal B cells, 3 healthy donors with different levels of sjTRECs were selected. Genomic DNA from their PBMCs was serially diluted with genomic DNA of 3 tumor cell lines not containing sjTRECs. By this approach, variable concentrations of DNA from normal lymphocytes ranging from 3–96% were prepared. No interference of tumor DNA was observed, because the amount of sjTRECs showed linearity and reproducibility even at concentrations as low as 6% of normal DNA, indicating that sjTRECs could be reliably quantified even in B-CLL samples containing up to 94% of tumor DNA (data not shown). When the concentrations of normal DNA were lower than 6% sjTREC levels did not show linearity and were not reproducible.
sjTREC levels were then evaluated in 30 untreated B-CLL patients at diagnosis selected to have normal PBMCs ≥6% and an age at diagnosis ≤70 years and, for comparison, in 18 age-matched healthy donors.
Genomic DNA of each healthy donor was diluted with genomic DNA from tumor cell lines lacking sjTRECs to obtain samples containing 96% to 6% normal DNA. Results were plotted to verify linearity. The mean regression coefficient of the 18 curves was r = 0.98+0.02. The number of sjTRECs in PBMCs of normal donors is represented in Figure 1 in terms of mean values with a 95% confidence interval (CI); sjTREC levels in PBMCs of the 30 B-CLL patients are reported for each individual according to the percentage of normal PBMCs. Normal or non-neoplastic PBMCs were defined as the proportion of PBMCs that scored negative for CD19 and CD5 coexpression and varied from 6–43% of total PBMC population. Nine B-CLL patients showed sjTREC levels in the range of normal donors (Patients 4, 6, 8, 14, 15, 18, 24, 25, 27) whereas 2 individuals (Patients 7, 26) showed sjTREC concentrations exceeding the range of normal donors. The remaining 19 patients had either decreased or non-detectable sjTRECs in their peripheral blood compartment.
In the small set of 16 patients, for whom a 5-year observation period was available, no correlation between the levels of sjTRECs at diagnosis and disease evolution was observed. Three of 7 patients with normal or higher sjTREC levels, and 4 of 9 patients with low sjTREC levels showed disease progression.
VH genes mutations in B-CLL patients
The VH region for each patient was amplified from PBMCs with consensus primers in the framework 1 and in the joining regions. The number of SHMs was then determined by comparing the VH sequences to the germline VH genes with the highest homology. Sixteen patients expressed VH3 family genes, 10 expressed VH1, 3 patients VH4, and 1 patient VH2 (Table I). SHMs were observed in 17 cases, whereas 13 patients had leukemic VH genes with ≥98% sequence homology with the nearest germline gene.
Table I. VH family, SHMS and sjTRECs in B-CLL patients
The VH sequences have the following accession numbers: AY291299, AY291297, AJ389186, AY291310, AY291298, AJ389176, AY291296, AY291311, AY291301, AY291300, AY291308, AY291303, AY291307, AY291305, AY291306, AY291302, AY291309, AY291304, AJ389171, AY300822, AJ389178, AY300822, AY300821, AY311491, AY311492, AY311493, AY311494, AY311495, AY311496, AY311497.
Normal, high or low compared to healthy donors.
The relationship between disease course and the presence or absence of VH SHMs was analyzed in the 16 patients for whom a 5-year observation period was available. The absence of VH mutations was related to disease progression because 5 of 6 patients without VH mutations progressed in their disease, whereas only 2 of 10 patients with VH mutations showed progression (p = 0.02, Fisher's χ2 test). Frequencies of SHMs and relation to disease progression confirm data reported by other investigators.3, 4, 5, 6
Of the 17 cases with SHMs in the VH region, 8 had sjTREC levels in the range of or higher than normal donors, whereas only 3 of 13 cases without SHMs had normal sjTREC levels (Table I). Although not statistically significant (p = 0.17, Fisher's χ2 test) because of the limited number of cases, these differences could suggest an association between the presence of SHMs and a normal sjTREC count.
Our study demonstrates that a marked decrease in sjTREC levels occurs in about 60% of B-CLL patients at the time of diagnosis in comparison to age-matched healthy subjects. sjTREC levels are reported to be influenced by thymic output on one hand and by naive T cell proliferation and differentiation into a mature phenotype on the other.16, 22 A potential pitfall in the measurement of TRECs as markers of thymic function may appear in cases of increased peripheral T cell division, as shown in HIV infection23 and rheumatoid arthritis.24 Because low sjTREC levels were found even in patients at the initial stage of disease (15 of 24 patients at Stage A), it is unlikely that the sjTREC decrease was a consequence of increased T cell proliferation or differentiation to a “memory” phenotype, reported in more advanced stage B-CLL patients.12
In terms of pathogenesis the causes of thymic failure in B-CLL remain unclear. Leukemic B cells may produce cytokines capable of interfering with thymic cell proliferation. Alternatively, B-CLL membrane surface molecules might interact directly with thymic cells and cause their dysfunction. This latter mechanism has been invoked in the pathogenesis of B-CLL associated hypogammaglobulinemia. Indeed, T cells activated in the presence of leukemic B cells, have been shown to up-regulate CD30 expression and to acquire a CD30-dependent Ig class-switch inhibitory activity.25 Thus, by altering the microenvironment of secondary lymphoid organs, leukemic B cells might generate a milieu that fosters CD30-dependent immune suppression rather than CD40-dependent immune help. Upregulation of CD30 by leukemic B cells could be implicated in thymic failure in B-CLL because CD30, belonging to the tumor necrosis factor (TNF) receptor family, may function like CD95/Fas and the TNF receptor 1 (TNFR1) in mediating cell death signals. In this regard, Fas and TNFR1 are expressed mainly by mature T cells, whereas CD30 appears to be the primary death-signaling molecule in thymocytes.26
It can not be excluded that the decreased sjTREC levels observed in B-CLL patients depend on a redistribution of sjTREC-expressing naïve T cells from the peripheral blood into secondary lymphoid organs in response to chemokine released by the neoplastic cells.
Low levels of sjTRECs in 4 of 6 patients with disease at Stage B or C is consistent with a previous study demonstrating that the percentage of CD4+CD45RA+ lymphocytes in B-CLL patients is negatively associated with an advanced clinical stage.12 There is no obvious explanation for the exceedingly high sjTREC content observed in Patients 7 and 26 (Stage C and B, respectively), although high levels of sjTRECs have been reported in individuals with thymomas.27
In conclusion, our study demonstrates that sjTREC levels can be quantified in total PBMCs of B-CLL patients even when the percentage of normal lymphocytes is as low as 6% and suggests that thymopoiesis, being likely decreased in about 60% of B-CLL patients, possibly contributes to the cellular immune deficiency that is a hallmark of this leukemia. These findings will need to be further confirmed in a larger cohort of B-CLL patients whereas longitudinal studies will be crucial for establishing a definitive role of thymic function in the immunological defects associated with this leukemia.
The authors thank Dr. D. Douek (Vaccine Research Center, NIH, Bethesda) for his help in setting-up and optimizing sjTRECs quantification, Dr. D. Morelli for providing samples of healthy donors and Mrs. L. Mameli for contributing to manuscript preparation.