Prognostic significance of bcl-xL gene expression and apoptotic cell counts in follicular lymphoma

Follicular lymphoma is characterized primarily by defects in cell apoptosis rather than by cell proliferation.¹  Bcl-2, involved in follicular lymphoma, prevents cell apoptosis by blocking the mitochondrial pathway.^2,3  The long isoform of bcl-x, bcl-xL, acts as another important antiapoptotic factor of the bcl-2 family and is expressed in follicular lymphoma.⁴  Experimentally, functional differences exist between bcl-2 and bcl-xL. In bcl-2–deficient mice there is massive death of mature lymphocytes, whereas immature lymphocytes undergo apoptosis in bcl-x–deficient mice.⁵  In vitro, bcl-2 maintains the survival of resting T cells when bcl-xL prevents the apoptosis of activated T cells.⁶  Importantly, when bcl-xL is down-regulated, even if a constitutive level of bcl-2 is maintained, follicular lymphoma cells undergo apoptosis.⁷  Therefore, bcl-xL seems to play a key role in follicular lymphoma. However, the relation between bcl-xL gene expression and clinical features in patients with follicular lymphoma has not yet been reported.

In the present study, we assessed bcl-xL gene expression and apoptotic cell counts in 27 patients with follicular lymphoma using real-time quantitative reverse transcription–polymerase chain reaction (RT-PCR) and terminal deoxytransferase-catalyzed DNA nick-end labeling (TUNEL) assays. Overexpression of the bcl-xL gene in lymphoma cells was related to low numbers of apoptotic cells. Both factors reflect poor disease outcome in patients with follicular lymphoma.

Twenty-seven patients with follicular lymphoma—16 men and 11 women aged 21 to 82 years (median, 51 years)—with available frozen tumor specimen at diagnosis were included in this study. Histologic diagnoses were established according to the World Health Organization (WHO) classifications.⁸  Ten age- and sex-matched patients with reactive follicular hyperplasia were referred as controls. Approval for these studies was obtained from the Institut Universitaire D'Hématologie-Hôpital Saint-Louis institutional review board. Written informed consent was obtained from all patients for this study, in accordance with regulation of the Institut Universitaire D'Hématologie-Hôpital Saint-Louis.

Lymph nodes, surgically removed for diagnostic purposes, were immediately cut into 2 parts: one part was fixed in formaldehyde and further processed for paraffin embedding, and the other was snap frozen and stored at -80°C. A section was cut from each tissue block for systemic light microscopic control.

Frozen sections measuring 7 μm were prepared, immediately fixed in 70% ethanol, and stained with hematoxylin-eosin. The sections were dehydrated through a graded series of ethanol and xylene and air dried. For each patient, approximately 1500 lymphoma cells, corresponding to an average surface of 450 000 μm², were laser microdissected (PALM, Bernried, Germany) and catapulted into tubes for RNA extraction.

Total RNA was extracted from 10 frozen sections, each 10-μm thick, using the acid-guanidinium thiocyanate-phenol-chloroform method.⁹  First-strand cDNA was synthesized from 1 μg total RNA using Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA) and random hexamers according to the manufacturer's instructions. Total RNA from microdissected lymphoma cells was independently extracted and immediately reverse transcribed following the same protocol.

Quantitative RT-PCR was performed on an ABI PRISM 7700 system using the Pre-Developed TaqMan Assay Reagent specific to human bcl-xL and human transcription factor IID/TATA binding protein (TBP) gene expression quantification (PE Applied Biosystems, Warrington, United Kingdom), according to the manufacturer's instructions. The TBP gene was used as an endogenous control.

Quantification results were expressed in terms of the cycle threshold (CT) value according to the baseline adjusted to 0.05. The Jurkat cell, which expresses the bcl-xL gene,¹⁰  was used as the calibrator. The comparative CT method (PE Applied Biosystems) was used to quantify relative bcl-xL expression compared with the Jurkat cell. Briefly, the CT values were averaged for each triplicate. Differences between the mean CT values of bcl-xL and those of TBP were calculated as ΔCT_sample = CT bcl-xL - CT TBP and those of the ΔCT for the Jurkat cell as (ΔCT_calibrator = CT bcl-xL - CT TBP). Final results, the sample-calibrator ratio, expressed as N-fold differences of bcl-xL expression in the samples compared with Jurkat cell, were determined as 2 ^{-(ΔCTsample - ΔCTcalibrator)}.

Cell apoptosis was confirmed by in situ detection of fragmented DNA, using TUNEL assay,¹¹  on deparaffinized 5-μm–thick sections, treated with proteinase K (20 μg/mL) for 15 minutes at room temperature.

The number of apoptotic lymphoma cells was assessed blindly by 2 pathologists, who did not know the bcl-xL levels, on an Olympus Provis AX 70 microscope (Olympus Optical, Tokyo, Japan), with wide-field eyepiece number 26.5. At 400× magnification, this wide-field eyepiece provided a field size of 0.344 mm². Results are expressed as the mean number of cells per field at 400× magnification.

Patient characteristics were compared using χ² analysis and the Fisher exact test for categorical variables and the Wilcoxon test for continuous variables. Overall survival was measured from the date of diagnosis to either death from any cause or the end date of December 31, 2002. When the end date was not reached, the data were censored at the date of the last follow-up evaluation. Survival functions were estimated using the Kaplan-Meier method and compared using the log-rank test. Multivariate survival analysis was performed using a Cox regression model. Differences were considered significant when the 2-sided P < .05. All statistical analyses were performed using SAS 8.2 (SAS Institute, Cary, NC) and Splus 2000 (MathSoft Inc, Berkeley, CA) software.

Compared with the patients with reactive follicular hyperplasia, the bcl-xL gene was overexpressed in whole lymph node sections (median, 5.5 [range, 1.2-28.1] vs 2.3 [range, 1.6-3.2]; P = .0115).

Because lymph node sections contain not only lymphoma cells but also stromal cells, lymphoma cells were further isolated by laser microdissection. The higher level of bcl-xL in microdissected lymphoma cells compared with whole lymph node sections (median, 11.0 [range, 1.9-59.6] vs 5.5 [range, 1.2-28.1]; P = .0379) demonstrated that bcl-xL was expressed by lymphoma cells.

The number of the apoptotic lymphoma cells on TUNEL assays (Figure 1) ranged from 5 to 108 (median, 38) and were inversely correlated with the bcl-xL gene expression level on lymph node sections (r = -0.7552) and microdissected lymphoma cells (r = -0.7736).

This is in accordance with experimental data showing that the bcl-xL gene prevents apoptosis.²  Bcl-xL–overexpressing mice showed enhanced survival of B cells,¹²  whereas bcl-x–deficient mice had extensive lymphocyte apoptosis.⁵  In vitro, B lymphocytes expressing high levels of bcl-xL were resistant to Fas-induced cell apoptosis.¹³  In contrast, the down-regulation of bcl-xL enhanced transforming growth factor-β (TGF-β)–induced apoptosis of cultured B lymphoma cells.¹⁴ 

Overexpression of bcl-xL and low numbers of apoptotic lymphoma cells were associated with multiple extranodal sites, elevated lactate dehydrogenase (LDH) levels, and an International Prognostic Index (IPI) indicating high risk in the 27 patients with follicular lymphoma (Table 1). Moreover, both factors were significantly related to short overall survival times (Figure 1).

To our knowledge, this is the only study of bcl-xL gene expression on disease outcome in patients with follicular lymphoma. Experimental data on B-lymphoma cells demonstrated that bcl-xL gene overexpression caused resistance to apoptosis induced by anti-immunoglobulin M (anti-IgM) or by chemotherapeutic agents.^15,16  This mechanism might favor lymphoma development and result in poor prognoses for follicular lymphoma patients. Further studies are needed to assess the value of bcl-xL as a therapeutic target in patients with follicular lymphoma who are at high risk.

We thank Christian Bastard for critical review of the manuscript, Janet Jacobson for English editing, Luc Legrès and Allison Desveaux for engineering assistance, Sylvie Corre for data management, and Laboratoire Photo d'Hématologie for illustrations.