Combination of baseline FDG PET/CT total metabolic tumour volume and gene expression profile have a robust predictive value in patients with diffuse large B-cell lymphoma.

PURPOSE: This study evaluated the predictive significance of total metabolic tumour volume (TMTV) measured on baseline FDG PET/CT and its value in addition to gene expression profiling using a new method of gene analysis (rapid reverse transcriptase multiplex ligation-dependent probe amplification assay, RT-MLPA) in patients with diffuse large B-cell lymphoma treated with R-CHOP or R-CHOP-like chemotherapies. METHODS: The analysis included 114 patients. TMTV was measured using a 41% SUVmax threshold and tumours were classified into GCB or ABC subtypes according to the RT-MLPA assay. RESULTS: The median follow-up was 40 months. the 5-year progression-free survival (PFS) was 54% and the 5-year overall survival (OS) was 62%. The optimal TMTV cut-off value was 261 cm3. In 59 patients with a high TMTV the 5-year PFS and OS were 37% and 39%, respectively, in comparison with 72% and 83%, respectively, in 55 patients with a low TMTV (p = 0.0002 for PFS, p < 0.0001 for OS). ABC status was significantly associated with a worse prognosis. TMTV combined with molecular data identified three groups with very different outcomes: (1) patients with a low TMTV whatever their phenotype (n = 55), (2) patients with a high TMTV and GCB phenotype (n = 33), and (3) patients with a high TMTV and ABC phenotype (n = 26). In the three groups, 5-year PFS rates were 72%, 51% and 17% (p < 0.0001), and 5-year OS rates were 83%, 55% and 17% (p < 0.0001), respectively. In multivariate analysis, TMTV, ABC/GCB phenotype and International Prognostic Index were independent predictive factors for both PFS and OS (p < 0.05 for both). CONCLUSIONS: This integrated risk model could lead to more accurate selection of patients that would allow better individualization of therapy.

BCL2 expression but not MYC and BCL2 coexpression predicts survival in elderly patients with diffuse large B-cell lymphoma independently of cell of origin in the phase 3 LNH03-6B trial.

BACKGROUND: Our aim was to evaluate whether the cell of origin (COO) as defined by the Hans algorithm and MYC/BCL2 coexpression, which are the two main biological risk factors in elderly patients treated with rituximab, cyclophosphamide, doxorubicin hydrochloride, vincristine, and prednisolone (R-CHOP), maintain their prognostic value in a large prospective clinical trial. PATIENTS AND METHODS: We evaluated 285 paraffin-embedded samples from patients (60-80 years of age) enrolled in the Lymphoma Study Association trial LNH03-6B who were treated with R-CHOP. We correlated the COO defined by the transcriptome according to the Wright algorithm with that defined by the Hans algorithm in a subset of 62 tumors with available frozen tissue samples. RESULTS: The non-germinal center B-cell-like phenotype according to the Hans algorithm and BCL2 expression (but not MYC and BCL2 coexpression) predicted worse progression-free survival [hazard ratio (HR)=1.78, P = 0.003 and HR = 1.79, P = 0.003, respectively] and overall survival (HR = 1.85, P = 0.005 and HR = 1.67, P = 0.02, respectively) independently of the International Prognostic Index. The correlation between the Hans algorithm and the Wright algorithm was 91%, with an almost perfect concordance according to a kappa test (0.81). CONCLUSIONS: Our results suggest that immunohistochemically defined COO remains a useful tool for predicting prognosis in diffuse large B-cell lymphoma when performed under optimized standardized conditions and that BCL2 expression may help to identify elderly patients at risk for relapse and who could potentially respond to anti-BCL2 targeted agents. In this prospective phase III trial, the coexpression of MYC and BCL2 does not appear to predict worse survival. CLINICAL TRIAL NUMBER: NCT00144755.

The costimulatory molecule CD70 is regulated by distinct molecular mechanisms and is associated with overall survival in diffuse large B-cell lymphoma.

In diffuse large B-cell lymphomas (DLBCL), a recurrent deletion of the 19p13 region has recently been described. CD70 and TNFSF9 genes are suspected tumor suppressor genes, but previous studies suggest an oncogenic role for CD70. Therefore, we studied the consequences of variation in CD70 copy number and epigenetic modifications on CD70 expression. Copy-number variation was investigated in 144 de novo DLBCL tissues by comparative genomic hybridization array and quantitative multiplex PCR. Gene expression was assessed by quantitative RT-PCR, and CD70 promoter methylation was determined by pyrosequencing. The 19p13.3.2 region was deleted in 21 (14.6%) cases, which allowed the minimal commonly deleted region of 57 Kb that exclusively includes the CD70 gene to be defined. Homozygous deletions were observed in four (2.7%) cases, and acquired single-nucleotide variations of CD70 were detected in nine (6.3%) cases. CD70 was highly expressed in both germinal centre B-cell-like (GCB) and activated B-cell-like (ABC) DLBCL compared to normal tissue, with distinct molecular mechanisms of mRNA expression regulation. A gene dosage effect was observed in the GCB subtype, whereas promoter methylation was the predominant mechanism of down regulation in the ABC subtype. However, high CD70 expression levels correlated to shorter overall survival in both the GCB (P = 0.0021) and the ABC (P =0.0158) subtypes. In conclusion, CD70 is targeted by recurrent deletions, somatic mutations and promoter hypermethylation, but its high level of expression is related to an unfavorable outcome, indicating that this molecule may constitute a potential therapeutic target in selected DLBCL.

The isotype of the BCR as a surrogate for the GCB and ABC molecular subtypes in diffuse large B-cell lymphoma.

Gene expression profiling has identified two major molecular subtypes of diffuse large B-cell lymphoma (DLBCL) that are histologically indistinguishable but differ in cure rates. Here, we investigated whether the isotype of the B-cell receptor (BCR) expressed by the tumoral cells correlated with the molecular subtype and survival. Gene expression analysis clustered the 53 patients included in this study into three subgroups, 17 germinal center B-cell-like (GCB) cases, 26 activated B-cell-like (ABC) cases and 10 intermediate cases. The molecular subtype was correlated with the isotype, as 15/17 GCB cases expressed a secondary isotype (immunoglobulin (Ig)G or IgA), whereas 24/26 ABC cases expressed a primary isotype (IgM or IgD) (P<0.0001). There was a trend toward a worse outcome for patients with an ABC DLBCL and a shorter overall survival for patients with IgM+ tumor (P=0.21 and 0.014, respectively). Finally, fluorescence in situ hybridization (FISH) analysis revealed a striking asymmetric pattern, as the IGHM gene is conserved only on the productive IGH allele in most IgM+ tumors. Taken together, these data indicate that the isotype of the BCR is a reliable indicator for the GCB and ABC subtypes in DLBCL, and suggest that the conservation of an IgM is required for ABC DLBCL lymphomagenesis to occur.

Follicular lymphoma cell niche: identification of a preeminent IL-4-dependent T(FH)-B cell axis.

Follicular lymphoma (FL) B cells contract tight connections with their microenvironment, which governs the pathogenesis and progression of the disease. Indeed, specific immune response gene signatures, obtained from whole biopsy samples, have been associated with patient survival. In this study, we performed gene expression profiling of purified B cell and non-B cell compartments obtained from FL and reactive lymph nodes. We identified 677 non-redundant genes defining the FL interface and involving 26 FL-specific functional networks. This approach highlighted an interleukin-4 (IL-4)-centered pathway associated with an activation of signal transducer and activator of transcription 6 (STAT6), which favors overexpression of IL-4-target genes. In addition, FL microenvironment was characterized by a strong enrichment in follicular helper T cells (T(FH)), as demonstrated through transcriptomic and flow cytometry analyses. The majority of phospho-STAT6(pos) B cells were located at the vicinity of cells expressing the programmed death 1 (PD-1) T(FH) marker. Moreover, purified FL-derived T(FH), expressed IL4 at very high levels compared with purified tonsil-derived T(FH) or non-T(FH) microenvironment. Altogether, our study demonstrated that tumor-infiltrating T(FH) specifically express functional IL-4 in FL, creating an IL-4-dependent T(FH)-B cell axis. This cross talk could sustain FL pathogenesis and represent a new potential therapeutic target.

Recurrent genomic aberrations combined with deletions of various tumour suppressor genes may deregulate the G1/S transition in CD4+CD56+ haematodermic neoplasms and contribute to the aggressiveness of the disease.

CD4+CD56+ haematodermic neoplasms (HDN) constitute a rare disease characterized by aggressive clinical behaviour and a poor prognosis. Tumour cells from HDN are leukaemic counterparts of plasmacytoid dendritic cells (pDCs). Despite increased knowledge of the ontogenetic origin of these tumours, the genetic causes and oncogenic signalling events involved in malignant transformation are still unknown. To delineate novel candidate regions and disease-related genes, we studied nine typical CD4+CD56+ HDN cases using genome-wide high-resolution array comparative genomic hybridization (CGH). Genomic imbalances, which were predominantly losses, were frequently detected. Gross genomic losses or gains involving an entire chromosome were observed in eight cases. The most frequent imbalances were deletions of chromosome 9, chromosome 13 and partial losses affecting 17p or 12p. Combinations of deletions of tumour suppressor genes (TSG), namely RB1, CDKN1B (p27), CDKN2A, (p16(ink4a), p14(arf)) or TP53 (p53), were observed in all cases. These results indicate that deletion events altering G1/S regulation are crucial for HDN oncogenesis. Furthermore, in addition to frequent sporadic gene losses, in one case we observed a 8q24 interstitial deletion that brought MYC closer to miR-30b/miR-30d, which may be related to their deregulation. Taken together, these results indicate that in addition to frequent G1/S checkpoint alterations, various genetic events could contribute to the chemoresistance of the tumour.

Mapping of MYC breakpoints in 8q24 rearrangements involving non-immunoglobulin partners in B-cell lymphomas.

Chromosomal translocations joining the immunoglobulin (IG) and MYC genes have been extensively reported in Burkitt's and non-Burkitt's lymphomas but data concerning MYC rearrangements with non-IG partners are scarce. In this study, 8q24 breakpoints from 17 B-cell lymphomas involving non-IG loci were mapped by fluorescence in situ hybridization (FISH). In seven cases the breakpoint was inside a small region encompassing MYC: in one t(7;8)(p12;q24) and two t(3;8)(q27;q24), it was telomeric to MYC whereas in four cases, one t(2;8)(p15;q24) and three t(8;9)(q24;p13) it was located in a 85 kb region encompassing MYC. In these seven cases, partner regions identified by FISH contained genes known to be involved in lymphomagenesis, namely BCL6, BCL11A, PAX5 and IKAROS. Breakpoints were cloned in two t(8;9)(q24;p13), 2.5 and 7 kb downstream from MYC and several hundred kb 5' to PAX5 on chromosome 9, joining MYC to ZCCHC7 and to ZBTB5 exon 2, two genes encoding zinc-finger proteins. In these seven cases, MYC expression measured by quantitative reverse transcription-polymerase chain reaction (RT-PCR) was significantly higher when compared to that of patients without 8q24 rearrangement (P=0.006). These results suggest that these rearrangements are the consequence of a non-random process targeting MYC together with non-IG genes involved in lymphocyte differentiation and lymphoma progression.

Two patterns of chromosomal breakpoint locations on the immunoglobulin heavy-chain locus in B-cell lymphomas with t(3;14)(q27;q32): relevance to histology.

The t(3;14)(q27;q32) is the most common translocation involving BCL6 in B-cell lymphoma. Although this translocation was predominantly associated with diffuse large B-cell lymphoma (DLBCL), recent studies have shown that it can also be found in follicular lymphomas (FL), often associated with a large cell component. To further investigate the relationship that might exist between this translocation and the phenotype of the tumors, we studied 34 lymphomas with a t(3;14)(q27;q32). Twenty cases were DLBCL, 14 FL and most cases, regardless of histology, were negative for the expression of CD10 (26/32, 81%). We identified the IGH switch region involved in the translocation for 32 cases. Our data indicate that in DLBCL most breakpoints involve the switch mu (17/19; 89%), whereas in FL most involve a switch gamma (9/13; 70%) (P=0.0016, Fisher's exact test). This correlation between the histology and the structure of the translocated allele suggests that the lymphomas with Smu and Sgamma translocations may originate from different cells, or that the substituted regulatory regions that come to deregulate BCL6 may affect the presentation of the disease.

Clinical and biological relevance of single-nucleotide polymorphisms and acquired somatic mutations of the BCL6 first intron in follicular lymphoma.

Genetic modifications of the BCL6 gene in lymphoma include translocations, deletions, and somatic mutations (SM) of the 5' noncoding region. Three single-nucleotide polymorphisms (SNPs) of the major mutation cluster region (MMC) have been reported, including two substitutions (397G/C, 502G/A) and one deletion (520DeltaT). Clinical and biological relevance of these SNPs are unknown. Based on a case-control study, BCL6 SNPs frequencies were assessed in 97 t(14;18) follicular lymphomas (FL) and in 54 lymphomas with 3q27 rearrangement. Allele frequencies were similar in the FL and controls groups. The 397 G/C genotype was correlated to a higher-grade transformation risk (P=0.02). SM were observed in 39.1% of FL and were characterized by a clustering distribution (hot spots spanning position 420-435, 106-127, and 590-600). No correlation between genotypes or acquired mutational status and BCL6 expression was demonstrated. However, gel mobility-shift assays, using SNPs containing probes show results representative for protein/DNA complexes. This study demonstrates that the first BCL6 intron is a highly variable region as a consequence of both SNP and SM, which may contribute to biology and outcome of FL.

Gene transcription in hepatocytes during the acute phase of a systemic inflammation: from transcription factors to target genes.

During an acute, systemic inflammation, the liver is triggered by blood-borne pro-inflammatory cytokines such as Tumor Necrosis Factor alpha, Interleukin-1beta and Interleukin-6. The end result is an up- or down-regulated synthesis and/or activation of liver-enriched transcription factors that in turn regulate many target genes coding for resident or secreted acute phase proteins. In this review, various classifications of these acute phase proteins are presented. Major inflammation-driven changes in the synthesis and/or activity of the hepatic transcription factors are illustrated. Some of their up- or down-regulated target genes are used as paradigms of the various transcriptional mechanisms that take place on gene promoters during an acute, systemic inflammation. Finally, further specific features of inflammation-associated gene transcription in liver from acute phase onset to resolution are provided.

Long-range identification of hepatocyte nuclear factor-3 (FoxA) high and low-affinity binding sites with a chimeric nuclease.

Identifying the complete set of transcription factors that bind the promoter and other regulatory regions of a gene of interest is an essential step in functional genomics. We have developed an original assay for the systematic detection of hepatocyte nuclear factor-3 (HNF-3) binding sites within cloned promoters. This assay is based on expression of a recombinant enzyme, HNF-3beta/FN, that is comprised of the rat HNF-3beta DNA-binding domain and the non-specific nuclease domain of the FokI restriction enzyme. Southern analysis of target plasmids with proven HNF-3 binding sites showed that HNF-3beta/FN was able to specifically cut both DNA strands in the vicinity of these binding sites, whereas mutagenized binding sites were no longer cleaved. Likewise, as yet undescribed HNF-3 binding sites were detected easily over a distance spanning several thousand bases. The functionality of such binding sites was confirmed by electromobility shift assay. Furthermore, the extent of cleavage by HNF-3beta/FN at a given binding site was tightly correlated with the affinity of a natural HNF-3beta molecule for this site. This novel approach can be extended to other transcription factors for long-range identification of functional transcription factor binding sites in genes.

Chromosomal assignments of mammalian genes with an acute inflammation-regulated expression in liver.

A set of acute inflammation-regulated genes expressed in liver has been assigned to rat, mouse, and human chromosomes by detecting species-specific PCR amplicons in rat(x)mouse or mouse(x)hamster somatic cell hybrids or radiation hybrids or by in silico matches of corresponding rat cDNAs to various libraries of previously assigned rat, mouse, or human genes or expressed-sequence tags. This allowed us to assign 24, 22, and 21 inflammation-regulated genes to rat, mouse, and human chromosomes, respectively. From these assignments as well as those previously determined for a larger set of genes with an acute inflammation-regulated transcription in liver, we further investigated whether such genes are clustered onto given chromosomes. A cluster was found on rat Chromosome (Chr) 6q with a conserved synteny on mouse Chr 12 and human Chr 14q13-q32, and another cluster previously reported on human Chr 1q has been extended with five further genes. Our data suggest that during an acute inflammation, a higher-order regulation may control some liver-expressed genes that share a given chromosome area.

Characterisation of BCL2-JH rearrangements in follicular lymphoma: PCR detection of 3' BCL2 breakpoints and evidence of a new cluster.

Follicular lymphomas (FL) are closely associated with a t(14;18)(q32;q21) translocation, leading to a bcl2 protein over-production. This translocation probably constitutes a very early step in the development of the disease. Besides the cytogenetic assay, t(14;18) detection can be achieved using either Southern blot or polymerase chain reaction (PCR). Since 1990, several publications have reported discrepancies between the results of cytogenetic and molecular analysis of t(14;18). Using methods able to explore long DNA fragments, several authors reported breakpoints located outside the usual breakpoint regions. However, these techniques cannot be easily used in routine. The aim of this study was to develop a simple PCR assay to amplify rearrangements usually not detected in FL. We selected a group of 83 patients with a t(14;18) on cytogenetic analysis: using usual probes and primers, 54/83 (65.1%) showed a MBR rearrangement, 7/83 (8.4%) were mcr positive and 22/83 (26.5%) remained negative. Among these 22 rearrangements, nine could be detected using this new PCR assay. Four breakpoints were located in a 20 bp area suggesting a recurrent breakpoint cluster close to an Alu repetitive sequence. Finally, remaining negative cases (13/83, 15.6%) suggest that other breakpoints are located between the MBR and mcr regions.

Fetuin-B, a second member of the fetuin family in mammals.

A set of orthologous plasma proteins found in human, sheep, pig, cow and rodents, now collectively designated fetuin-A, constitutes the fetuin family. Fetuin-A has been identified as a major protein during fetal life and is also involved in important functions such as inhibition of the insulin receptor tyrosine kinase activity, protease inhibitory activities and development-associated regulation of calcium metabolism and osteogenesis. Furthermore, fetuin-A is a key partner in the recovery phase of an acute inflammatory response. We now describe a second protein of the fetuin family, called fetuin-B, which is found at least in human and rodents. On grounds of domain homology, overall conservation of cysteine residues and chromosomal assignments of the corresponding genes in these species, fetuin-B is unambiguously a paralogue of fetuin-A. Yet, fetuin-A and fetuin-B exhibit significant differences at the amino acid sequence level, notably including variations with respect to the archetypal fetuin-specific signature. Differences and similarities in terms of gene regulation were also observed. Indeed, studies performed during development in rat and mouse showed for the first time high expression of a member of the fetuin family in adulthood, as shown with the fetuin-B mRNA in rat. However, like its fetuin-A counterpart, the fetuin-B mRNA level is down-regulated during the acute phase of experimentally induced inflammation in rat.

An array of binding sites for hepatocyte nuclear factor 4 of high and low affinities modulates the liver-specific enhancer for the human alpha1-microglobulin/bikunin precursor.

Alpha1-Microglobulin and bikunin are two plasma glycoproteins encoded by a gene for alpha1-microglobulin/bikunin precursor (AMBP). The strict liver-specific transcription of the AMBP gene is controlled by an elaborate and remote enhancer made of six clustered boxes numbered 1 to 6 (core enhancer) that are binding sites for the hepatocyte-enriched nuclear factors HNF-1, HNF-4, HNF-3, HNF-1, HNF-3 and HNF-4 respectively. Three further boxes, 7 to 9, have now been found in the enhancer area in a position 5' of box 2, 5' of box 1 and 3' of box 6, respectively. Electrophoretic mobility-shift assays with nuclear extracts from the HepG2 hepatoma cell line demonstrated that boxes 7 and 8 are both functional HNF-4-binding sites of high and low affinity respectively, whereas no binding capacity of box 9 was detected by this method. Transfection of HepG2 and Chinese hamster ovary cells with chloramphenicol acetyltransferase constructs harbouring the core or extended AMBP enhancer with wild-type or mutated boxes and co-transfection with expression plasmids for a wild-type or defective HNF-4 identified box 7 as an essential element for the basal activity of this enhancer. The response of boxes 7 and 8 varies with the level of HNF-4 in cells. Box 9 exhibits a repressor activity that can be detected when box 8 is ablated. In vivo this corresponds to conditions of low box 8 occupancy when the intracellular level of HNF-4 is limited. These results reinforce the view that the AMBP enhancer is a quite elaborate and unusual example of a modular enhancer whose activity is fine-tuned by the level of cognate nuclear factors in the cell.