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CLINICAL OBSERVATIONS, INTERVENTIONS, AND THERAPEUTIC TRIALS
From the Division of Oncology, Department of Medicine
and the Department of Health Research and Policy, Stanford University
School of Medicine, CA.
We have cloned and characterized a novel human gene,
HGAL (human germinal center-associated lymphoma),
which predicts outcome in patients with diffuse large B-cell
lymphoma (DLBCL). The HGAL gene comprises 6 exons and
encodes a cytoplasmic protein of 178 amino acids that contains an
immunoreceptor tyrosine-based activation motif (ITAM). It is highly
expressed in germinal center (GC) lymphocytes and GC-derived lymphomas
and is homologous to the mouse GC-specific gene M17.
Expression of the HGAL gene is specifically induced in B
cells by interleukin-4 (IL-4). Patients with DLBCL expressing high
levels of HGAL mRNA demonstrate significantly longer overall survival
than do patients with low HGAL expression. This association was
independent of the clinical international prognostic index. High HGAL
mRNA expression should be used as a prognostic factor in DLBCL.
(Blood. 2003;101:433-440) Diffuse large B-cell lymphomas (DLBCLs) constitute
30% to 40% of adult non-Hodgkin lymphoma (NHL).1 There
is a consensus that DLBCL represents a diverse group of neoplasms with
heterogeneous genetic abnormalities, clinical features, treatment
responses, and prognoses.2 Previous attempts to
subclassify these neoplasms on morphologic grounds have been hampered
by irreproducibility, thus leading to their categorization as one
single group in the Revised European American Lymphoma (REAL)
classification.2,3 In view of this heterogeneity, only
50% of DLBCL patients are cured with standard chemotherapy. Therefore,
the establishment of prognostic models based on pretreatment
characteristics of patients or tumors is of paramount importance to
guide the choices of treatment intensities. The International
Prognostic Indicator (IPI), based on clinical characteristics at
diagnosis, has been constructed and successfully used to define
prognostic subgroups in DLBCL.4 However, the differences
in clinical features and in treatment responses of DLBCL are probably
caused by the marked genetic and molecular heterogeneity underlying
disease aggressiveness and tumor progression.
Examination of gene expression profiles in DLBCL tumors and application
of a pattern recognition algorithm termed hierarchical clustering
identified 2 molecularly distinct forms of the disease: germinal center
B-cell-like DLBCL, characterized by the expression of genes normally
expressed in germinal center B cells, and activated B-cell-like DLBCL,
characterized by the expression of genes normally induced during in
vitro activation of B cells.5 Patients with these 2 forms
of DLBCL were found to have very different prognoses: those with
germinal center B-cell-like DLBCL had a significantly better
overall survival than those with activated B-cell-like DLBCL. However,
the relative prognostic contribution of the individual genes defining
these 2 DLBCL subgroups could not be assessed by this method. This aim
can be achieved by using clinical data to supervise the discovery of
genes with expression patterns that correlate with outcome, as was
recently reported.6,7 This supervised approach may allow
identification of genes that play a role in determining prognosis and
pathophysiology, including the discovery of previously unknown genes of
major clinical relevance among the multiple expressed sequence tags
(ESTs) present on the arrays.
By conducting a search for genes predicting DLBCL outcome, we have
identified and cloned a novel gene, termed HGAL (human germinal center-associated lymphoma). This gene is mainly expressed in
germinal center (GC) B cells and is stimulated specifically by the
lymphokine interleukin-4 (IL-4).
Cell lines, normal tissues, and tumor specimens
Biopsy specimens from patients with primary untreated DLBCL (54 patients), follicle-center lymphoma (FCL) (21 patients), chronic lymphocytic leukemia (CLL) (16 patients), mantle cell lymphoma (MCL) (4 patients), nodal marginal zone lymphoma (MZL) (3 patients), and T-cell
lymphoblastic lymphoma (T-LL) (2 patients), classified according to the
Revised European-American Lymphoma Classification,2 were
used in this study. DLBCL tissues were chosen randomly from a
collection of specimens obtained during the course of diagnostic procedures between 1983 and 1993. All specimens were distinct from the
tumor samples used in our previous analysis of gene expression profiles
in DLBCL.5 Tumor tissues were stored either as
fresh-frozen biopsy specimens embedded in Tissue-Tek Optimal Cutting
Temperature (OCT) compound 4583 (Miles, Elkhart, IN) and preserved at
Germinal center (GC) B cells were purified from 3 human tonsils, as
previously described,5 and pooled. Peripheral blood mononuclear cells from healthy donors were isolated by
Ficoll-Hypaque density centrifugation (Amersham Pharmacia
Biotech, Piscataway, NJ). B cells were enriched to more than 90% by
human B-cell enrichment cocktail (StemCell Technologies, Vancouver, BC,
Canada), as determined by fluorescence-activated cell sorter (FACS)
analysis. Enriched B cells were plated at 5 × 106 cells
per well in 6-well plates (Corning Glassworks, Corning, NY) in IMDM
(Gibco BRL) supplemented with 2% fetal calf serum (FCS), 0.5% bovine
serum albumin (Sigma, St Louis, MO), 50 µg/mL human transferrin
(Sigma), 5 µg/mL bovine insulin (Sigma), and 15 µg/mL gentamicin
(Cellgro-Mediatech, Herndon, VA) at 37°C in 5%
CO2. The cells were stimulated with 100 U/mL IL-4 (R&D
Systems, Minneapolis, MN) and goat F(ab')2 antihuman µ antibodies (Biosource International, Camarillo, CA) for 6 and 24 hours.
For CD40 activation, purified B cells were stimulated by culture on
irradiated (55 Gy) CD40L-expressing mouse L cells (a gift of Yong-Jun
Liu, DNAX, Palo Alto, CA).
RNA isolation and reverse transcription reaction
cDNA cloning and sequencing Clone 814622 (GI: 2210537) was used for the design of 5' SMART rapid amplification of cDNA ends (RACE) polymerase chain reaction (PCR) primer, 5'-GCCAAAGAAGGGTAGTGGGATTACG-3'. We performed 5' SMART RACE cDNA amplification using Advantage 2 polymerase mix according to the manufacturer's protocol (Clontech). PCR amplicon was cloned into a TA-PCR cloning vector (Invitrogen, Carlsbad, CA). After the transformation of competent Escherichia coli (1 Shot INV F; Invitrogen) and plating on selective agar (50 µg/mL kanamycin, 40 µL of 40 µg/mL X-gal), 10 white colonies were picked for plasmid purification using QIAprep kit (Qiagen, Valencia, CA). DNA sequencing was performed on a 373 automatic DNA sequencer (Applied Biosystems) using ABI Prism Big Dye Terminator Kit (Perkin Elmer, Foster City, CA) as recommended by the manufacturer. HGAL full-length cloning was completed with 3' SMART RACE cDNA amplification. The cDNA sequence was confirmed by reverse transcription (RT)-PCR spanning the coding region of the gene.Northern blot analysis Total RNA from normal spleen (Ambion, Austin, TX) and DLBCL cell lines were size-fractionated in 1% agarose-glyoxal-dimethyl sulfoxide gels and transferred to Hybond N+ positively charged nylon membranes (Amersham Pharmacia Biotech, Piscataway, NJ) according to standard protocols. The membranes were hybridized in ULTRAhyb (Ambion) buffer with entire coding region HGAL cDNA probe, labeled with [ 32P]dATP (Amersham Pharmacia Biotech,
Piscataway, NJ) during asymmetric PCR.
Real-time polymerase chain reaction measurement of HGAL mRNA expression HGAL mRNA expression was measured using the TaqMan technology on an ABI Prism 7900HT Sequence Detector System (Applied Biosystems). Probe and primers were designed using Primer Express software (Applied Biosystems) and were chosen to hybridize to sequences at the junction between 2 exons to avoid amplification of genomic DNA, as follows: forward primer, 5'-CCCAAAACGAAAATGAAAGAATGT-3' (900 nM); reverse primer, 5'-GGGTATAGCACAGCTCCTCTGAGTA-3' (900 nM); probe, 5'-CCATCCAGGACAATGT-3' (250 nM), labeled with 6-carboxy-fluorescein phosphoramidite (FAM) at the 5' end and with nonfluorescent quencher (NFQ) at the 3' end. PCR reactions were prepared in a final volume of 20 µL, with final concentrations of 1× TaqMan Universal PCR Master Mix (Applied Biosystems) and cDNA equivalent to 20 ng input RNA. Reaction mixtures were assembled at 4°C, followed by PCR consisting of 50°C UNG initiation for 2 minutes, AmpliTaq Gold activation at 95°C for 10 minutes, followed by 95°C for 15 seconds and 60°C for 1 minute, for 40 cycles. Each PCR run included the 5 points of the calibration curve for HGAL and GAPDH (5-fold diluted human RNA), a no-template control, the calibrator Raji cDNA, and the patient's samples, all in triplicate. Threshold cycle (Ct) was chosen at 10 times the standard deviation of the baseline fluorescence signal of the first few PCR cycles.Because the quality of the RNA (ie, extent of RNA degradation) and consequently the amount of cDNA added to each reaction were difficult to assess, we also quantified the level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the endogenous RNA control using a commercially available kit (PE Applied Biosystems), and each sample was normalized on the basis of its GAPDH content, as was previously reported.8 For each experimental sample, the amount of the target HGAL and the endogenous reference (GAPDH) were determined from the calibration curves obtained by serial dilution of control human RNA (Applied Biosystems). HGAL amount was then divided by the endogenous reference (GAPDH) amount to obtain a normalized value. To allow the relative expression of HGAL gene to be compared across all the tested samples, HGAL/GAPDH expression ratios were also normalized to the HGAL/GAPDH values concomitantly measured in Raji cells (calibrator), as suggested in ABI 7700 User Bulletin 2 (Applied Biosystems). Search for HGAL gene mutations To determine whether the HGAL gene is somatically mutated, high-molecular-weight DNA was extracted from 5.0 × 106 cells by a commercially available kit (QIAamp Tissue Kit; Qiagen, Valencia, CA) from 19 NHL (8 DLBCL and 11 FCL) specimens, 2 DLBCL cell lines and normal T lymphocytes from 2 tumor specimens, enriched by CD3 microbeads (Miltenyi Biotec, Auburn, CA). Approximately 1000 bp from the transcription initiation site, including the first exon and the 5' portion of the first intron, were amplified and directly sequenced using 2 pairs of primers: Mut-1 forward-5'-AGCACAAGGCAAGAAGGAAGTG-3'; Mut-1 reverse- 5'-CTGAAAGAGGGTGGTGATTTTGAC-3'; Mut-2 forward- 5'-GTCAAAATCACCACCCTCTTTCAG-3'; and Mut-2 reverse- 5'-TCCTGTGTTCCACTCTCCAGTAGC.PCR was performed in a final volume of 50 µL containing 0.5 µM of each primer, 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl2, 200 µM each dNTP, and 2.5 U Taq DNA polymerase (Gibco BRL). The PCR conditions were 1 cycle at 94°C for 5 minutes, 56°C (Mut-1 primers) or 54.5°C (Mut-2 primers) for 1 minute, and 72°C for 3 minutes, 30 cycles at 94°C for 30 seconds, 56°C (Mut-1 primers) or 54.5°C (Mut-2 primers) for 30 seconds, and 72°C for 30 seconds, and 1 cycle at 72°C for 7 minutes. PCR products were analyzed by 2% agarose gel electrophoresis and stained with ethidium bromide. Bands of appropriate size were excised from the gels and purified by adsorption to a silica matrix (QIAquick columns; Qiagen). Amplicons were directly sequenced in both directions using the same primers. Statistical analysis To identify genes potentially predicting DLBCL outcome, we have applied the significance analysis of microarrays (SAM) method9 to previously published DLBCL gene expression profiling data.5 The SAM method ranks genes according to their Cox proportional hazards regression score. A significant gene list is formed by cutting off the ranked list at a given threshold. The threshold is chosen by assessment of the false discovery rate (FDR), estimated by random permutation of the survival times. We chose an FDR of 15%.Comparison of clinical characteristics between DLBCL patients with high and low HGAL expression was performed by Student t test for age and by Fisher exact test for all the other variables using GraphPad Prism version 2.0 software (San Diego, CA). Overall survival (OS) time of patients with DLBCL was calculated from the date of diagnosis until the date of death or last follow-up examination. Disease-free survival (DFS) was measured as the interval between the date of complete remission after induction treatment and the date of relapse, death, or last follow-up evaluation. Survival curves were estimated using the product-limit method of Kaplan-Meier and were compared using the log-rank test. Multivariate regression analysis according to the Cox proportional hazards regression model10 with OS as the dependent variable was used to adjust the effect of HGAL expression for IPI and BCL6 expression. Two-tailed P < .05 was considered significant.
Identification and cloning of the HGAL gene We performed an analysis of previously reported data on the gene expression in DLBCL5 supervised by survival information. For this purpose, we used the SAM method.9 This analysis identified 234 genes whose expression correlated strongly with clinical outcome of DLBCL patients (data not shown). The gene whose expression best predicted DLBCL OS was an EST UniGene cluster 49614 (clone 814622; GI: 2210537). It had nucleotide sequence homology to the previously reported mouse GC-specific gene
M17.11 In this data set, DLBCL patients with
mRNA expression of clone 814622 greater than the median expression of
all analyzed patients exhibited significantly longer OS
(P = .008) than patients with low expression (Figure
1). By hierarchical clustering, this gene
was contained within the GC gene cluster. On the microarray, its
expression was high in GC lymphocytes, intermediate in memory B cells,
and relatively low in peripheral blood B cells.5 In
tumors, its expression was high in FCL tumors, low in CLL, and
heterogeneous in DLBCL specimens.5 These observations
suggested that this gene is up-regulated at specific stages of B-cell
differentiation, especially in GC lymphocytes. A full-length cDNA was
cloned from sorted GC lymphocytes and from the Ramos cell line and was
termed HGAL (GenBank accession number AF521911) (Figure
2A). The full-length sequence of HGAL
cDNA perfectly matched another EST on the array (clone 1339726; GI:
2883522), which also was among the top genes that predicted DLBCL
outcome and had an expression pattern similar to that of clone
814622.5 PCR amplification of the HGAL mRNA by 3' RACE
revealed the presence of 2 ampliconsa major amplicon of 1659 bp and a
minor amplicon of approximately 3500 bp, the latter containing the
sequence of the shorter amplicon and differing in the length of the 3'
untranslated sequence. Northern blot analysis of normal spleen and
several NHL cell lines confirmed the PCR findings and demonstrated the
presence of 2 RNA transcripts of approximately 1.7 kb (major
transcript) and 3.5 kb (Figure 3).
Alignment of the sequenced cDNA to the GenBank database and BLAST search of the human genome identified a perfectly aligning genomic DNA sequence (AC024964 and NT_022484). The gene was located on chromosome 3q13. Comparison of the genomic sequence to the cDNA sequence revealed that HGAL spans 11 kb. Recognition of a Kozak sequence and search for the longest open reading frame (ORF) led to the identification of a putative ORF extending from exon 1 to exon 6 and encoding a 178-amino acid (aa) protein, with 51% identity and 62% similarity to the mouse GC-expressed M17 protein.11 The HGAL gene product had a hydrophilic profile with no predicted transmembrane domain, and it lacked a nuclear localization sequence. The C-terminal portion of HGAL protein is proline rich. Similar to mouse M17 protein,11 HGAL contains a modified immunoreceptor tyrosine-based activation motif (D/EX7D/EX2YX2LX7YX2L, termed ITAM)12 (Figure 2B). ITAM fragments are usually found within the cytoplasmic domains of transmembrane receptors, and they play a role in signal transduction in B and T lymphocytes. HGAL and M17 are the only 2 known nonreceptor proteins that contain such a motif. However, the spacing between the 2 YXXL regions is greater than 7 aa. A potential contact site to SH2 domains was identified at aa positions 102 to 105 (YENV).13 The 3' untranslated sequence of HGAL contains several cryptic polyadenylation sites and ATTTA motifs capable of mediating rapid degradation of mRNAs.14 Analysis of HGAL expression in normal tissues, NHL cell lines, and tumor specimens Expression of HGAL was further evaluated by real-time RT-PCR among normal tissues. The highest HGAL mRNA expression was observed in GC cells, thus confirming our previous array observations, followed by thymus and spleen (Figure 4A). All nonhematopoietic and nonlymphopoietic organs, except lung, expressed only trace amounts of HGAL. Examination of HGAL mRNA expression in malignant cell lines reveled variable expression in B-cell NHL cell lines, minimal expression in Jurkat T cells, and no expression in nonlymphoid HL60 and K562 cell lines (Figure 4B). We subsequently analyzed HGAL expression in a spectrum of NHL tumor specimens (Figure 4C) that were distinct from specimens analyzed by DNA microarrays. HGAL expression was high in all FCL and low in all CLL, all T-LL, and most MCL specimens. A single MCL specimen that demonstrated high HGAL expression contained multiple normal GC. HGAL expression in DLBCL was highly variable. In some cases it was not expressed at all, whereas in other cases its expression levels were similar to those of FCL specimens. These observed patterns of expression in normal and malignant tissues confirmed our previous array observations (Figure 1B) demonstrating that HGAL is highly expressed in GC lymphocytes and GC-derived tumors. High expression in thymus and relatively low expression in peripheral blood lymphocytes (PBLs) suggested that some immature T lymphocytes might also express high HGAL mRNA levels. However, the limited number of analyzed immature T cell tumors did not exhibit high HGAL expression.
Analysis of HGAL mutations Given that in normal lymphocyte subsets and in NHL HGAL exhibited the highest expression in GC lymphocytes and GC-derived tumors, both of which exhibit somatic mutational activity affecting Ig, BCL6, and other genes,15-22 we evaluated whether the HGAL gene might also be the target of somatic mutations. In the Ig and BCL6 genes, mutations are mainly observed in 1000 to 2000 bp 3' to the transcription initiation site. Consequently, we sequenced a DNA region of approximately 1000 bp starting at and downstream from the transcription initiation site of HGAL in 19 NHL specimens and 2 cell lines. No mutations from the germline sequence were found in any of these specimens. Four polymorphic variants, also detected in T cells isolated from the 2 lymphoma specimens, were identified at the following positions, numbered according to sequence AC024964: in the untranslated portion of exon 1 at position 160869 C/T (allele prevalence 0.90 and 0.10), in intron 1 at positions 160976 T/C (allele prevalence 0.81 and 0.19), 161049 G/A (allele prevalence 0.90 and 0.10), and 161640 T/C (allele prevalence 0.95 and 0.05).HGAL expression is induced by IL-4 To determine how HGAL expression might be involved in physiologic responses, peripheral blood B lymphocytes were stimulated for 6 and 24 hours with anti-µ antibodies, IL-4, or cells expressing human CD40 ligand (t-CD40L). Following stimulation, total RNA was extracted and HGAL mRNA expression was assessed by real-time RT-PCR. As shown in Figure 5, anti-µ antibodies (0.5 µg/mL) or stimulation with CD40L did not affect HGAL mRNA expression, even though these stimuli induced the expression of activation markers (CD69 and CD71), as expected (data not shown). Remarkably, IL-4 stimulated HGAL mRNA expression by 4- to 5-fold, an effect that persisted for 24 hours (Figure 5). No synergistic effect in the induction of HGAL mRNA expression was observed on costimulation with IL-4 and anti-µ antibodies. These results suggest that HGAL is specifically involved in the response to IL-4.
HGAL mRNA expression is a predictor of DLBCL survival Array data (Figure 1A) suggested that high HGAL mRNA expression predicts improved OS of DLBCL. To confirm this observation, we analyzed the correlation between HGAL mRNA expression and DLBCL survival in an independent group of DLBCL patients by real-time RT-PCR. For this analysis we chose a value of 0.73 for HGAL expression based on HGAL mRNA expression in GC lymphocytes from 3 normal tonsil specimens. With a median follow-up of 49 months (range, 6-171 months), the OS rate was significantly higher in the DLBCL patients with high HGAL gene expression than in the DLBCL patients with low HGAL gene expression (median OS, 67 and 33 months, respectively; P = .01) (Figure 6A). The patient with the longest OS of 171 months in the group with high HGAL expression died of a cause unrelated to lymphoma (Figure 6A). To further assess the strength of HGAL in predicting survival, we analyzed its effect on survival when HGAL expression was considered as a continuous variable; exact expression values were converted to ranks to avoid giving undue influence to outlying points. Again, higher HGAL expression correlated with longer OS (P = .01). Clinical characteristics at presentation of patients with high (0.73 or higher) and low (lower than 0.73) HGAL gene expression are presented in Table 1. Patients with low HGAL gene expression tended to have higher levels of lactate dehydrogenase (LDH), but no difference in the distribution of other components of IPI was observed. In addition, no differences in complete remission rates were observed in DLBCL groups with high (0.73 or higher) and low (lower than 0.73) HGAL gene expression. Median DFS in DLBCL with low HGAL expression was 20 months, but it was not reached in DLBCL with high HGAL expression. Differences in DLBCL DFS curves approached but did not reach statistical significance (P = .08; data not shown), probably because of the relatively small number of analyzed patients. Patients with DLBCL tumors expressing high HGAL had significantly better failure-free survival than patients with low HGAL expression (P = .01). We next analyzed whether HGAL expression could add to the OS prognostic value of the IPI. Because of the small number of patients in each IPI score subgroup, we combined patients with low or low-intermediate (low clinical risk) and high-intermediate or high (high clinical risk) scores. Considering patients with low clinical risk separately, as judged by the IPI, patients in the high (0.73 or higher) HGAL gene expression group had a distinctly better OS than patients with low (lower than 0.73) HGAL gene expression (P = .04) (Figure 6B). Interestingly, only a few (3 of 13) patients with high clinical risk, as judged by IPI, had high HGAL gene expression, thus preventing meaningful statistical analysis of the possible predictive effect of HGAL gene expression on OS in this group of patients. We next assessed the strength of HGAL in predicting survival in comparison to the IPI. IPI alone did not reach statistical significance in predicting OS in our group of patients, probably because of the small sample size in each IPI category. In multivariate Cox regression analyses that included IPI scores or IPI individual components and HGAL, with OS as the dependent variable, only HGAL mRNA expression was an independent predictor of OS in DLBCL patients (Table 2, P = .02). HGAL expression still predicted DLBCL OS as assessed by a log-likelihood ratio test. This analysis confirmed that HGAL gene expression might accurately stratify patients into good and bad prognostic groups, even in a small group of patients in which IPI does not predict the outcome.
We have recently reported that BCL6 expression is a good
predictor of OS in DLBCL patients.8 Consequently, we
wanted to compare the prognostic value of HGAL, BCL6, or
their combination on DLBCL outcome (Figure
7). High expression of each gene
predicted better DLBCL survival with a similar statistical power
(derivation DLBCL group, P = .007 and
P = .008; validation DLBCL group, P = .01 and
P = .01, for BCL68 and
HGAL, respectively). DLBCL patients with high expression of
both genes had a better OS than DLBCL patients with low expression of
both genes (P = .0006; Figure 7). Patients whose tumors
exhibited high expression of at least one of these genes also had
better OS than patients in whom expression of both genes was low
(P = .015; Figure 7). There was a trend for better OS in
patients whose tumors exhibited high expression of both genes than
those whose tumors had high expression of only 1 of these 2 genes, but
the trend did not reach statistical significance. Multivariate Cox
regression analysis that included the individual components of the IPI
and HGAL and BCL6 with OS as the dependent variable demonstrated that BCL6 expression was the best
independent OS predictor (P = .02), followed by
HGAL expression (P = .08).
Recent application of cDNA microarray gene expression profiling to DLBCL specimens has significantly advanced our understanding of this heterogeneous disease. Definition of gene expression signatures characteristic of GC and of activated normal lymphocytes resulted in recognition of 2 DLBCL subtypes, based on their cell of origin: GC B-cell-like DLBCL and activated B-cell-like DLBCL.5 These 2 subtypes of DLBCL exhibited different survival outcomes, though the study was not designed to find gene expression patterns that predict survival. Further evidence for meaningful biologic differences between these DLBCL subgroups was provided by analysis of immunoglobulin gene mutations18 and BCL-2 gene translocations.23 Moreover, array analysis identified signaling pathways that are abnormally active in activated B-cell-like DLBCL but not in GC B-cell-like DLBCL. Members of these pathways have been suggested as attractive targets for therapeutic interventions.24 The subdivision of DLBCL into GC B-cell-like and activated B-cell-like types has recently been confirmed in a large and independent collection of DLBCL patients,7 and a third group has also been identified. In the present work we demonstrate yet another potential of gene
expression profiling Our study also shows that HGAL has prognostic significance in DLBCL tumors. Remarkably, in an independent group of DLBCL patients, gene expression profiling recently demonstrated that EST corresponding to HGAL mRNA was among the 16 genes predicting DLBCL outcome.7 The survival curves of our 2 DLBCL patient groups, assessed by cDNA microarrays in the first group and by real-time PCR in the second group, were similar, with 60% and 20% of DLBCL patients with high and low HGAL expression, respectively, surviving for more than 50 months after diagnosis. Interestingly, during the initial 24 months following diagnosis, the survival of DLBCL patients with high and low HGAL mRNA expression was similar. Conversely, after this initial period, patients with low HGAL mRNA expression died more often than did patients with high HGAL expression. Whether the better survival in the high HGAL expression group could be attributed to a specific function of the gene or simply to the fact that it identifies tumors that originate from GC lymphocytes is unknown. Our previous observations indicate that not every GC marker has prognostic significance. Improved survival of DLBCL patients with tumors expressing high BCL6 mRNA, but not necessarily of those expressing high levels of CD10,8 suggests a gene-specific effect or an unrecognized stage of GC lymphocyte ontology, from which the tumors originate. Regarding the first possibility of a gene-specific effect on tumor survival, it is notable that HGAL (as demonstrated in this study) and BCL627 are IL-4-inducible genes. IL-4 is a growth and differentiation factor for normal B cells. Distinct effects were reported in malignant B cells. It has been demonstrated that IL-4 induced in vitro inhibition of growth in 60% of lymphoma specimens.28 Other studies indicate that IL-4 provides growth-inhibitory signals to NHL cells, activated through their surface immunoglobulin receptors.29 Recently, an inverse association between tumor levels of IL-4 and lymphoma proliferation have been noted.30 Consequently, it is possible that high mRNA expressions of HGAL and BCL6 are markers of tumors in which IL-4 exhibits antilymphoma effects. This group of DLBCL tumors has better clinical outcomes. It is also possible that HGAL and BCL6 are markers of distinct lymphocyte differentiation stages from which a subset of DLBCL tumors arise. Simultaneous evaluation of the prognostic significance of expression of HGAL and BCL6 genes allowed patients to be subclassified into 3 groups (Figure 7): one group with high expression of both genes, one group with low expression of both genes, and one group highly expressing only one of these genes. These groups appear to differ in OS. Interestingly, all of these groups have similar initial decreases in survival, indicating that other factors are more important in determining early death. After this initial period, there was a plateau in OS of patients whose tumors expressed high levels of both genes in contrast to continuous deaths observed in patients highly expressing only one of these genes. Because the number of patients in each group was small, further studies are needed to establish whether patients with high expression of both genes have a better or similar OS than patients whose tumors exhibit high expression of only one of these genes. Interestingly, in contrast to heterogeneous patterns of DLBCL, all FCL tumors express high levels of HGAL and BCL6, thus suggesting their uniform origin from a single B-cell counterpart. With the advance of microarray technology, better understanding of DLBCL pathophysiology, and identification of expression signatures that predict tumor outcome,5-7 we will be able to improve the management of, and perhaps find new targets for the therapy for, lymphoma. Simple assays, such as RT-PCR or immunoperoxidase staining, will eventually be developed based on the most useful predictive markers. BCL68 and HGAL must now be added to BCL2,31-34 survivin,35 and short lists of other genes identified by microarray studies6,7 as candidates for routine clinical application. In summary, we have cloned and characterized a new gene, HGAL, which is highly expressed in GC lymphocytes and GC-derived tumors. Moreover, we demonstrate that HGAL mRNA expression is an important predictor of OS in patients with DLBCL. HGAL expression defined DLBCL subgroups with distinct outcomes even in patients at low risk, defined by the currently used clinical index (IPI). Further studies will elucidate the biologic function of this gene and will confirm its prognostic value alone or in combination with other genes.
R.L. is an American Cancer Society Clinical Research Professor.
Submitted July 1, 2002; accepted August 15, 2002.
Supported by grants NIH CA33399 and CA34233.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Ronald Levy, Division of Oncology, Stanford University School of Medicine, 269 Campus Dr, CCSR Bldg, Rm 1105, Stanford, CA 94305-5306; e-mail: levy{at}leland.stanford.edu.
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Top
Abstract
Introduction
mercaptoethanol.
80°C or as frozen viable cell suspensions in 10% dimethyl
sulfoxide in liquid nitrogen. DLBCL specimens were chosen based on
diagnosis of primary DLBCL; availability of the tissue specimen
obtained at diagnosis, before the initiation of therapy; treatment with an anthracycline-containing chemotherapy; and follow-up at Stanford University Hospital and availability of the outcome data. In all DLBCL
patients chosen for the study, disease dissemination was evaluated
before treatment by physical examination, bone marrow biopsy, and
computed tomography of the chest, abdomen, and pelvis. All patients
were staged according to the Ann Arbor system. IPI scores could be
determined from the records of 49 of these patients. All DLBCL tumors
had the histologic appearance of centroblastic large-cell lymphomas
demonstrating diffuse patterns of involvement without evidence of
residual reactive follicles.
) and
low (lower than the median expression of all analyzed patients, 
)
HGAL mRNA expression as determined in previous microarray analysis of
DLBCL.
), MZL (n = 3; 
), MCL
(n = 4; 
), T-LL (n = 2; 
), and sorted and pooled GC cells
from 3 normal tonsils (
). HGAL mRNA expression was measured by
real-time quantitative PCR. HGAL mRNA expression is a relative
expression of HGAL normalized to GAPDH in relation to the HGAL/GAPDH
ratio of the Raji cell line (see "Patients, materials, and
methods").
) HGAL mRNA
expression. HGAL-high cases, n = 24; HGAL-low
cases, n = 30. (B) Kaplan-Meier curves of OS in patients with DLBCL
of low clinical risk (IPI score, 0-2) grouped on the basis of
HGAL gene expression. HGAL 0.73 or higher, 
B activity is required for survival of activated B-cell-like diffuse large B cell lymphoma cells.
J Exp Med.
2001;194:1861-1874