Deletions in the short arm of chromosome 17 (17p) involving the tumor suppressor TP53 occur in up to 20% of diffuse large B-cell lymphomas (DLBCLs). Although inactivation of both alleles of a tumor suppressor gene is usually required for tumor development, the overlap between TP53 deletions and mutations is poorly understood in DLBCLs, suggesting the possible existence of additional tumor suppressor genes in 17p. Using a bacterial artificial chromosome (BAC) and Phage 1 artificial chromosome (PAC) contig, we here define a minimally deleted region in DLBCLs encompassing approximately 0.8 MB telomeric to the TP53 locus. This genomic region harbors the tumor suppressor Hypermethylated in Cancer 1 (HIC1). Methylation-specific PCR demonstrated hypermethylation of HIC1 exon 1a in a substantial subset of DLBCLs, which is accompanied by simultaneous HIC1 deletion of the second allele in 90% of cases. In contrast, HIC1 inactivation by hypermethylation was rarely encountered in DLBCLs without concomitant loss of the second allele. DLBCL patients with complete inactivation of both HIC1 and TP53 may be characterized by an even inferior clinical course than patients with inactivation of TP53 alone, suggesting a functional cooperation between these two proteins. These findings strongly imply HIC1 as a novel tumor suppressor in a subset of DLBCLs.
Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin's lymphoma (NHL), comprising approximately 30–40% of lymphoid neoplasms (Jaffe et al., 2001). DLBCL represents a heterogeneous entity with respect to morphology, immunophenotype, cytogenetics, gene expression profiles and clinical outcome (Schlegelberger et al., 1999; Alizadeh et al., 2000; Jaffe et al., 2001; Rosenwald et al., 2002, 2003a; Savage et al., 2003). Therefore, the identification of molecular markers with prognostic significance is of great importance.
The pathogenesis of DLBCL reflects a multistep process involving distinctive recurring genetic lesions such as translocations affecting the BCL2 and BCL6 oncogenes. In addition, deletions in chromosome 17p occur in up to 20% of cases (Lossos, 2005). One critical regulator of cell survival targeted by this latter aberration is the tumor suppressor gene TP53. According to the ‘two-hit’ hypothesis, formulated by Knudson (1971), inactivation of both copies of a tumor suppressor gene is considered a pre-requisite for tumor development. Although the inactivaton of TP53 has been shown to be involved in the pathogenesis of DLBCL and correlates with clinical outcome (Ichikawa et al., 1997; Koduru et al., 1997; Stokke et al., 2000, 2001; Leroy et al., 2002), complete inactivation of TP53 by concurrent allelic deletion and mutation of the remaining allele has been demonstrated in few studies only.
Moreover, the deletion in chromosome 17p in DLBCL frequently affects the whole chromosomal short arm (Koduru et al., 1997; Stokke et al., 2001; Bea et al., 2005) raising the obvious question, whether inactivation of TP53 is the most important consequence of losses in 17p, or whether other tumor suppressor genes are also targeted within this deleted region. Frequent allelic losses at chromosome 17p13.3, independent of TP53 (17p13.1) deletions, have been reported in many types of human neoplasms, including carcinomas of the breast, lung, brain and ovary, as well as in hematopoietic malignancies, such as acute myeloid leukemias, acute lymphoblastic leukemias, chronic lymphocytic leukemias and malignant non-Hodgkin's lymphomas (B-NHL) (Saxena et al., 1992; Phillips et al., 1996; Sankar et al., 1998; Seitz et al., 2001; Konishi et al., 2003).
In this study, we performed a detailed analysis of the TP53 deletion and mutation status in DLBCL and frequently encountered monoallelic TP53 deletions, whereas the remaining allele was found to retain its functionality. Toward the isolation of a further, possibly disease- associated, gene in the 17p13.1-p13.3 deleted region, we performed a (detailed) deletion mapping of this critical region by fluorescence in situ hybridization (FISH) analysis with a contiguous set of bacterial artificial chromosome (BAC)/Phage 1 artificial chromosome (PAC) clones. We were able to identify a minimally deleted region in chromosome band 17p13.3 containing the tumor suppressor gene HIC1 (Hypermethylated in Cancer 1). Moreover, in DLBCLs carrying a monoallelic deletion of HIC1, the non-deleted allele was demonstrated to be frequently methylated, thus rendering HIC1 a potentially novel tumor suppressor in DLBCLs.
Deletions in chromosome arm 17p are not strictly correlated with TP53 gene mutations
On the basis of the conventional cytogenetic analysis, a deletion in the short arm of chromosome 17 (17p) was detected in 34 of 172 DLBCLs analysed (20%) (Figure 1a). These 34 cases and 21 DLBCLs without 17p deletions were selected for a detailed deletion and mutation analysis of the TP53 gene at 17p13.1. By performing interphase FISH with a TP53 locus-specific BAC, a monoallelic TP53 deletion was observed in 28 of the 34 DLBCLs with 17p deletion (Figure 1b), while 6 cases displayed deletions in chromosome 17p by conventional karyotyping without affecting the TP53 locus.
Direct sequencing of PCR-amplified exons 2–11, which contain the entire coding region of TP53, revealed inactivating point mutations in the non-deleted allele in 8/28 cases with monoallelic TP53 deletions. Point mutations affecting one allele were detected in 3/27 DLBCLs with two gene copies (Table 1). In addition to the DNA sequencing strategy, which is conventionally used to search for TP53 gene alterations, we investigated a subset of 39 cases by use of the functional analysis of separated alleles in yeast (FASAY) assay. This approach detects deleterious/inactivating TP53 alterations by transforming tumor TP53 cDNA into yeast cells and subsequent discrimination of yeast colonies expressing functional p53 from those expressing non-functional p53 protein. By FASAY, inactivating TP53 gene alterations were found in 16 DLBCLs. Of these, seven mutations had also been identified by direct sequencing while nine mutations were exclusively observed applying the FASAY assay (Table 1). Therefore, FASAY increased the detection rate of TP53 mutations by more than 50%. Altogether, TP53 mutations were found in 13/28 DLBCLs (46%) with deletion of one gene copy, which resulted in a biallelic inactivation of TP53 (−/−) in those cases. In 22 of the 55 DLBCLs analysed, only one allele of the gene was found to be inactivated (+/−), either by deletion (in 15 tumors) or by mutation (in seven DLBCLs). A wild-type TP53 configuration (+/+) without deletion or mutation was observed in the remaining 20 DLBCLs (Table 1).
Because functional impairment of p53 is not necessarily caused by TP53 mutation, but can also be the result of deregulated protein degradation, we investigated the p53 protein expression in our series of patients by immunohistochemistry. Overexpression of the p53 protein (that is, expression of p53 in at least 20% of cells) was found in 22 cases, in 16 of them attributable to a TP53 mutation (Table 1, Figure 1c). In six cases, obviously, p53 accumulation was not caused by TP53 mutation, but rather due to impaired p53 degradation.
On the other hand, TP53 was found to be mutated in 4/33 DLBCL cases (12%) without detectable p53 immunostaining (Table 1). Taken together, aberrant expression of p53 protein was significantly correlated with a TP53 mutation status performing Fisher's exact test (P=0.0001).
In summary, inactivation of TP53 by deletion and/or mutation became evident in 35 of the analysed 55 DLBCLs (65%). Of note, two of the six cases with 17p deletions not affecting the TP53 gene locus did not present with any molecular genetic alteration of TP53 or aberrant accumulation of p53, indicating that TP53 was not altered in those cases (Table 1).
17p13.1-p13.3 deletion mapping points to HIC1 as a candidate gene in 17p13.3
Toward the isolation of a second disease-associated gene within the 17p deleted region, we investigated the chromosomal region telomeric to TP53 (17p13.1-p13.3) by detailed deletion mapping. On the basis of the cytogenetic analyses and FISH results with TP53 specific probes, 18 DLBCLs without 17p deletion (DLBCL nos. 1–18) and 5 DLBCLs with a 17p deletion not affecting the TP53 locus (DLBCL nos. 19, 20, 24, 25 and 27) were initially selected for deletion mapping. Dual-color interphase FISH was performed by hybridization of eight non-overlapping BAC clones that map to chromosomal bands 17p13.1-p13.3 in distances of approximately 0.5–1.5 MB. The analysed region was approximately 8 MB in size (Figure 2a). A total of 11 of the 23 DLBCLs analysed (DLBCL nos. 4, 5, 7, 8, 11, 17, 19, 20, 24, 25 and 27) showed a monoallelic deletion of the region recognized by RP11-433M14 in 17p13.3. A monoallelic deletion of RP11-74E22 was detected in one case (DLBCL no. 7), and loss of the chromosomal segment detected by RP11-64J4 in band 17p13.2 was observed in two DLBCLs (DLBCL nos. 17 and 24) (Figure 2b). In all cases, between 20 and 30% of cells were affected by the deletion, a number that was clearly above the threshold determined for these probes (10.8, 8.1 and 12.6%, respectively).
By FISH-mapping using two sets of contiguous clones, which recognize the genomic fragment around RP11-433M14 and the segment around RP11-74E22 and RP11-64J4, respectively (Figure 2a), we characterized further the observed deletions. Using the second set of clones, only two cases (DLBCL nos. 7 and 19) were shown to harbor small deletions in the respective region. In contrast, applying the second set, in all 9/11 cases with sufficient quality of the FISH experiments, we found a commonly deleted segment in 17p13.3 extending from BAC clones RP11-433M14 to RP11-667K14 (Figure 2b). The estimated size of this consensus deletion was 0.8 MB. Three cases exhibited a discontinuous loss of chromosomal material from 17p13.3 with retention of small segments within the consensus deletion region: Two hybridization signals were seen for CTD-2545H1 in DLBCL no. 4 and for RP11-4F24 in DLBCL nos. 5 and 24, while in all three cases chromosomal material detected by RP11-433M14 and RP11-667K14 was deleted. Of note, all of the five cases (DLBCL nos. 19, 20, 24, 25 and 27) harboring a cytogenetically detectable del(17p) without deletion of the TP53 locus were shown to contain a monoallelic deletion in 17p13.3 (Figure 2b, Table 1).
We subsequently investigated, whether the consensus deletion region in 17p13.3 was also affected by deletion in cases with TP53 deletion. To this end, we analysed 28 DLBCL tumors with loss of one TP53 gene copy (13 of them with a simultaneous mutation of the remaining allele) using our panel of eight BAC clones covering 17p13.1-p13.3 in combination with three clones recognizing the consensus deletion region in 17p13.3 (RP11-667K14, CTD-2545H1, RP11-433M14). In 19 of 28 DLBCLs, the whole arm of chromosome 17p was deleted. In four cases, the deletions extended to RP11-433M14. Thus, in those 23 cases the entire critical 17p13.3 region was lost. The remaining five DLBCLs (DLBCL nos. 30, 32, 35, 37 and 38) (Table 1) exhibited deletions extending to the centromeric part of this region recognized by RP11-667K14 and CTD-2545H1, which means that this part of the 17p13.3 deletion region was lost in all 28 DLBCL harboring a deletion of the TP53 locus.
According to the genome database (http://www.ncbi.nlm.nih.gov/mapview/), the consensus deletion region in 17p13.3 contains three candidate tumor suppressor genes, namely HIC1, Ovarian Cancer-Associated Gene 1 (OVCA1) and Ovarian Cancer-Associated Gene 2 (OVCA2). All three genes are localized in the centromeric part of the critical region recognized by RP11-667K14 and CTD-2545H1. Since an inactivation of HIC1 had previously been described in hematological neoplasms (Issa et al., 1997; Melki et al., 1999), we next examined whether 17p13.3 deletions detected by FISH affected the HIC1 gene locus. Applying a quantitative real-time PCR (qPCR) approach, we analysed the HIC1 gene locus in correlation to the TP53 locus in 22 DLBCLs. In all 15 DLBCLs tumors, which exhibited one FISH signal with the HIC1-specific BAC RP11-667K14, and DLBCL nos. 7, 19 and 23, that had not been analysed by HIC1-specific FISH, a monoallelic deletion of HIC1 was detected as indicated by HIC1/β-2-microglobulin (B2M) ratios between 0.06 and 0.61 (Figure 3, Table 1) and by HIC1/HBB ratios between 0.07 and 0.59. Importantly, all five cases with a partial loss of the 17p13.3 deletion segment analysed had a monoallelic deletion of HIC1. In 12 of the 18 DLBCLs with HIC1 deletion, the TP53/B2M ratio ranged between 0.78 and 1.59. For TP53/HBB a ratio between 0.88 and 1.44 was established, suggesting a concomitant wild-type TP53 status (Figure 3e). In four DLBCLs (nos. 2, 3, 21 and 22), the qPCR approach provided evidence for two HIC1 and two TP53 gene copies confirming the results of the FISH experiments.
HIC1 is inactivated by methylation in DLBCL
Hypermethylation of exon 1a, which codes for the 5′-untranslated region of the transcript, has been described as a mechanism for HIC1 gene inactivation. We, therefore, investigated the methylation status of HIC1 in our series of DLBCL cases by methylation-specific PCR (MS–PCR) amplification of exon 1a sequences. Of 49 DLBCLs that could be analysed, 30 cases (61%) showed evidence of hypermethylation (Table 1), while no methylation of HIC1 was detected in DNA samples derived from reactive lymph nodes or peripheral blood mononuclear cells from healthy volunteers. Representative MS–PCR results are illustrated in Figure 4. All positive tumor samples yielded PCR products when methylation-specific primers (M) and primers specific for the unmethylated allele (UM) were applied, indicating that each sample contained cell populations or alleles with a different HIC1 methylation status.
In 27/30 (90%) DLBCLs showing hypermethylation of HIC1, a simultaneous HIC1 deletion was present, indicating biallelic inactivation of HIC1 (−/−). Notably, 5/5 cases with a 17p deletion not affecting the TP53 gene locus, which could be analysed by MS–PCR, presented with an inactivation of both HIC1 alleles (including two cases with neither a deletion nor mutation of TP53). In three cases, HIC1 methylation was not associated with an allelic loss of HIC1 suggesting a heterozygous HIC1 status (+/−) or, alternatively, an inactivation of both HIC1 alleles by methylation (−/−). No hypermethylation of HIC1 was observed in 19 DLBCLs, 11 of which carried a monoallelic deletion of HIC1 (+/−). No evidence of either HIC1 deletion or hypermethylation was detected in eight DLBCLs (+/+) (Table 1).
Altogether, in 49 DLBCL tumors from this study, both TP53 and HIC1 genes were analysed. In the majority of cases (31 DLBCLs, 63%) both genes were concomitantly inactivated by deletion and/or point mutation, overexpression or methylation. In seven DLBCLs with wild-type TP53, HIC1 was completely inactivated by both hypermethylation and allelic loss and in three cases with wild-type TP53 a partial HIC1 inactivation by methylation or deletion was detected. In contrast, only one DLBCL with an inactivated TP53 but wild-type HIC1 gene was observed and no evidence of either TP53 or HIC1 inactivation was found in seven DLBCLs (14%) (Table 1). Considering the recently reported gender specificity among HIC1 heterozygous mice (Chen et al., 2003), we investigated if there was any gender predominance among our HIC1 heterozygous (HIC1+/−) DLBCL cases. However, no gender specificity was observed (five male and six female patients among 11 HIC1+/− DLBCLs, see Table 1). Likewise, no gender predominance was present within HIC1+/+ and HIC1−/− DLBCL (Table 1).
HIC1 status does not influence SIRT1 gene expression in DLBCL
Since the inactivation of HIC1 has been reported to result in an overexpression of SIRT1, leading to deacetylation and inactivation of p53 (Chen et al., 2005), the SIRT1 gene expression level was determined in 10 DLBCLs with complete inactivation of HIC1 (−/−) and in 8 cases with wild-type HIC1 (+/+). SIRT1 gene expression was found to be highly variable showing a 15-fold range between the cases with highest and lowest expression level (data not shown). No significant differences in SIRT1 expression level was detected when comparing DLBCL cases with HIC1+/+ and HIC1−/− status (P=0.143), or comparing HIC1+/+ and HIC1−/− tumor samples with cDNA from reactive lymph node specimens (P=0.129 and P=0.424, respectively).
TP53 and HIC1 inactivations are negative prognostic indicators in DLBCL
In recent studies, a functional cooperation of TP53 and HIC1 in tumor formation has been elucidated, providing evidence that the simultaneous inactivation of TP53 and HIC1 influences tumor aggressiveness in mice (Chen et al., 2004). We, therefore, compared the clinical course of DLBCL patients with respect to the TP53 and HIC1 status. Clinical data were available from 40/55 DLBCL patients from this series. Although showing a trend, neither TP53 mutations (Figure 5a) nor deletions (Figure 5b) alone were significantly associated with impaired survival in our cohort. Biallelic inactivation of TP53 (−/−), however, clearly predicted for inferior outcome compared to DLBCL patients with wild-type TP53 (+/+) (P=0.0079, Figure 5c). We next tested, whether concurrent and complete inactivation of both TP53 and HIC1 genes was associated with shorter survival. Wild-type TP53 (+/+) and wild-type HIC1 (+/+) were identified in seven DLBCLs and a simultaneous heterozygous inactivation status of both TP53 (+/−) and HIC1 (+/−) was detected in seven DLBCLs. All of these cases had clinical data available. In eight DLBCLs, concurrent biallelic inactivation of both TP53 (−/−) and HIC1 (−/−) was observed and clinical data were available in four cases. Even though the numbers are small, a significant reduction of length of survival in DLBCL patients harboring concurrent and complete inactivation of TP53 and HIC1 was evident (P=0.0022; Figure 5d).
To assess a potential role of HIC1 inactivation in the progression of the disease we compared the survival times of patients with wild type and inactivated HIC1. We observed that inactivation of HIC1 by deletion and/or methylation led to a reduction of the survival time in patients characterized by wild-type TP53 (+/+): Patients with wild-type HIC1 (+/+) lived 71.6 months (n=7; range=5–162 months) on average, while those with inactivated HIC1 (+/− and −/−) already died after 38.5 months (n=6; range=1–126 months) on average. In addition, complete inactivation of HIC1 (−/−) seems to increase the pathogenic effect of TP53 inactivation in TP53 (−/−) patients: While none of the 4 patients with complete TP53 (−/−) and HIC1 (−/−) inactivation lived longer than 12 months, 2/3 patients with completely inactivated TP53 (−/−) but retention of one active HIC1 allele (+/−) lived 13 and 144 months (Table 1).
The varying clinical course in DLBCL is thought to be in part reflected by the heterogeneity of underlying genetic alterations. Besides recurrent chromosomal translocations affecting BCL2, BCL6 and CMYC oncogenes, nonrandom gains and losses of chromosomal material, such as amplifications of the c-REL locus in the short arm of chromosome 2, are also frequently encountered in DLBCL (Lossos, 2005). Recurring deletions of genetic material in 17p have been observed in up to 20% of DLBCLs (Jaffe et al., 2001; Lossos, 2005), and deletions as well as mutations of TP53, which is mapped to 17p13.1, have been shown to confer an inferior clinical prognosis (Ichikawa et al., 1997; Stokke et al., 2000, 2001). According to Knudson's (1971) ‘two-hit’ hypothesis, inactivation of both copies of a tumor suppressor gene is required for tumor formation, but 17p deletions including TP53, TP53 mutations and nuclear p53 overexpression, a commonly used surrogate marker for TP53 mutations in diagnostic pathology, do not necessarily overlap in DLBCL. This was also evident in our series, in which 15/55 (27%) DLBCLs carrying a monoallelic TP53 deletion did not show evidence of concurring TP53 mutations in the second allele. Vice versa, a substantial subset of cases with TP53 mutations (7/55, 13%) did not show a simultaneous deletion of the TP53 locus in the other allele. Since only few studies in DLBCL investigated both TP53 alleles for possible alterations to discriminate between monoallelic and biallelic inactivations (Koduru et al., 1997; Stokke et al., 2000), we performed a combined cytogenetic, FISH and mutational analysis of the gene in 55 DLBCLs. To increase the sensitivity of the analysis, we sequenced the entire TP53 coding region (exons 2–11) on the genomic level, in contrast to several other studies that preferentially focused on exons 5 through 9, the hot spot of TP53 mutations. Importantly, we also performed FASAY followed by cDNA sequencing of single TP53-transformed yeast clones. FASAY distinguishes yeast colonies expressing functional p53 protein encoded by a normal p53 cDNA sequence from colonies producing a mutant, non-functional p53 protein encoded by mutant cDNA simply on the basis of color (Ishioka et al., 1993; Flaman et al., 1995) and has been proven to be highly sensitive and reliable (Duddy et al., 2000; Meinhold-Heerlein et al., 2001). Indeed, the detection rate of TP53 mutations in our cohort was increased by approximately 50% upon FASAY, which could be explained by the fact that direct sequencing of genomic DNA was hampered by a relatively low tumor cell fraction carrying TP53 mutations. Taken together, a substantial subset of DLBCL (15 cases out of 55 DLBCLs in the present study, 27%) with monoallelic TP53 deletions did not harbor TP53 mutations in the second allele and it appears unlikely that we missed a significant number of TP53 mutations given our combined direct sequencing and FASAY approach.
This finding provides support for the hypothesis that genomic deletions involving the TP53 gene locus may not always affect TP53 itself and raises the obvious question whether other tumor suppressor genes might be targeted by deletions in this genomic region. In hematological malignancies, there is already some evidence of allelic losses in 17p occurring independently from, or outside of, TP53, as demonstrated in few cases of acute myeloid leukemias, acute lymphoblastic leukemias, chronic lymphocytic leukemias and malignant lymphomas (Sankar et al., 1998).
Detailed deletion mapping of the region telomeric to TP53 using a BAC-/PAC-contig covering the chromosomal region from 17p13.1 to 17p13.3 resulted in the delineation of a 0.8 MB consensus deletion region in band 17p13.3 in 11 DLBCL cases. This critical region contains the three putative tumor suppressor genes HIC1, OVCA1 and OVCA2. OVCA1 and OVCA2 were identified by allelic deletion mapping and positional cloning. These two genes are expressed from the same genetic locus using different promoters. Forced overexpression of OVCA1 reduced cell growth of ovarian cancer cell lines providing evidence for its tumor-suppressive role (Schultz et al., 1996). In contrast to OVCA1, overexpression of OVCA2 did not alter cell proliferation of ovarian cancer cell lines (Chen and Behringer, 2004). The homology to α-β-hydrolases, however, suggests a putative enzymatic activity in retinoid-induced growth arrest, differentiation and apoptosis (Prowse et al., 2002).
HIC1 encodes a zinc-finger transcription factor that represses transcription (Deltour et al., 1999, 2002; Pinte et al., 2004) and functions as a tumor suppressor in vivo (Wales et al., 1995). Homozygous inactivation of HIC1 in knockout mice results in embryonic and perinatal lethality, while a gender-dependent tumor spectrum was observed in case of heterozygous HIC1 inactivation, with a predominance of epithelial cancers in males and lymphoid and mesenchymal tumors in females. In addition, it was proposed that loss of one HIC1 allele enhances the aggressiveness of tumors in mice (Chen et al., 2003). However, in contrast to a recent report by Chen et al. (2003), we did not observe a female predominance among HIC1 heterozygous DLBCL patients. Since, HIC1 has been reported to be frequently inactivated by hypermethylation in hematological malignancies (Issa et al., 1997; Melki et al., 1999) as well as in solid tumors (Kanai et al., 1999; Dong et al., 2001; Rathi et al., 2003; Waha et al., 2004), we focused our investigation on this gene, in particular its inactivation by methylation. According to the results of our study, several lines of evidence suggest a putative tumor suppressor role for HIC1 in DLBCL. First, 61% of the cases in our study cohort presented with methylated HIC1 (exon 1a) DNA, whereas, in line with previous reports (Issa et al., 1997; Melki et al., 1999), no hypermethylation was observed in reactive lymph nodes or other control specimens. In tumor specimens yielding methylated products, unmethylated products were invariably present in addition. This finding was expected given the regular presence of non-malignant bystander cells in the lymphoma specimens and in view of the fact that only a tumor cell subclone may be affected by HIC1 alterations. Second, 90% of DLBCL with HIC1 hypermethylation showed a simultaneous HIC1 deletion of the second allele indicating biallelic inactivation of HIC1. Third, complete inactivation of HIC1 by both hypermethylation and allelic loss was identified in seven DLBCLs with wild-type TP53 status. Finally, survival data suggest that DLBCL patients with complete inactivation of both TP53 and HIC1 may have an even inferior clinical course than patients with complete inactivation of TP53 alone. In addition, patients with a wild-type TP53 background presented with shorter survival times when HIC1 was inactivated than those with an unaffected HIC1 gene. As a note of caution, it should be mentioned that all of the DLBCL patients included in the present study received an anthrancyclin-based (CHOP-like) therapy without additional anti-CD20 immunotherapy. Clearly, any prognostic significance of HIC1 inactivation will have to be studied in the context of prospective clinical trials using current treatment approaches.
The possible synergetic effect of simultaneous TP53 and HIC1 inactivation in the clinical course of DLBCL patients is paralleled in a TP53/HIC1 double heterozygous knockout mouse model that suggests a functional cooperation between these proteins, as the loss of HIC1 function leads to earlier tumor formation and increased aggressiveness in TP53 altered mice (Chen et al., 2004).
Interestingly, there is a functional link between HIC1 and TP53, and both genes may cooperate in their activity to suppress tumor formation. HIC1 is a direct target of TP53 (Wales et al., 1995; Guerardel et al., 2001; Britschgi et al., 2006) and represses the SIRT1 deacetylase as part of a regulatory loop. Therefore, inactivation of HIC1 has been reported to result in an overexpression of SIRT1, leading to deacetylation and inactivation of p53 by negative modulation of its DNA-binding capacity (Chen et al., 2005).
Nevertheless, it remains to be elucidated whether HIC1 status is involved in the regulation of SIRT1 expression in DLBCL. Although a direct regulation of SIRT1 gene expression by functional HIC1 has been demonstrated (Chen et al., 2005), we failed to observe significant changes in SIRT1 mRNA levels comparing DLBCL patients with wild-type HIC1 (+/+) and patients with complete inactivation of HIC1 (−/−). Intriguingly, the repression of SIRT1 gene expression does not appear to be affected on the basis by functional HIC1 solely, but also by the availability of the redox-sensitive HIC1-corepressor CtBP and the concentration of NADH in the nucleus. The reduction of NADH results in impaired formation of HIC1–CtBP complexes, therefore leading to decreased HIC1-mediated repression, which is associated with increased SIRT1 gene expression independent of HIC1-status (Zhang et al., 2007). Moreover, previous studies reported an increase in SIRT1 protein in the liver of fasted mice while mRNA levels remained constant, suggesting that SIRT1 is regulated post-transcriptionally (Rodgers et al., 2005). Since enhanced protein expression of SIRT1 has been demonstrated in human cancers (Hida et al., 2007; Huffman et al., 2007) as well as in tumors from genetically altered mice (Chen et al., 2005), it will be interesting to determine whether increased SIRT1 protein level is observed in DLBCL.
In conclusion, our study provides evidence that deletions in chromosome 17p are not strictly associated with inactivation of TP53 and suggests HIC1 as a novel, clinically relevant, tumor suppressor gene in DLBCL. Therapeutic approaches aiming at the restoration of HIC1 function, for example, by demethylating agents and chromatin-remodeling drugs, may therefore be beneficial in a subset of DLBCL patients with altered HIC1 expression.
Materials and methods
Tumor specimens and clinical data
A total of 172 DLBCL specimens, referred to the Department of Pathology at the University of Würzburg between 1990 and 2004, were classified according to the criteria of the World Health Organization (WHO) classification system (Jaffe et al., 2001). Tumors were selected for the study on the basis of the availability of cytogenetics. The TP53 and HIC1 status was analysed in detail in 55 DLBCLs including 34 cases with a deletion in 17p as well as in 21 cases with two copies of 17p as proven by cytogenetics. Clinical data were available from 40 of these 55 DLBCLs. All patients had received anthracycline-based chemotherapy without rituximab. Median follow-up was 2.3 years and 60% of patients died during this period. The median age of patients was 64 years and 44% were men. The study was approved by the Local Ethics Committee of the University of Würzburg, Germany.
Chromosome spreads were prepared from lymphocyte short-term cultures of all 172 DLBCL cases according to standard protocols (Lichter et al., 1995). After classical Giemsa-trypsin banding, the karyotypes were constructed according to the International System for Human Cytogenetic Nomenclature (ISCN) (Shaffer and Tommerup, 2005). Identical structural aberrations, or genetic gains, in two or more metaphases and identical genetic losses in at least three metaphases were defined as clonal.
Immunohistochemical staining for the p53 protein was performed on freshly cut slides from formalin-fixed paraffin-embedded tissues using the DO-7 antibody (1:50; DAKO, Glostrup, Denmark), as reported previously (Rudiger et al., 1998). The staining for p53 was considered positive when ⩾20% of tumor cell nuclei were stained distinctively from the background.
Isolation of genomic DNA and direct sequencing of the TP53 gene
DNA was extracted using the phenol–chloroform extraction method according to previously published protocols (Zettl et al., 2000). Genomic DNA corresponding to exons 2–11 of TP53 was amplified by PCR as described previously (Pinyol et al., 2000) using the oligonucleotide primers 5′-IndexTermCACAGGAAGCCGAGCTGTC-3′ and 5′-IndexTermGGGGACTGTAGATGGGTGAA-3′ (exons 2 and 3), 5′-IndexTermTTGGAAGTGTCTCATGCTG-3′ and 5′-IndexTermCAGGCATTGAAGTCTCATGG-3′ (exon 4), 5′-IndexTermGGAGGTGCTTACGCATGTTT-3′ and 5′-IndexTermTGGGGTTATAGGGAGGTCAA-3′ (exons 5 and 6), 5′-IndexTermGCACTGGCCTCATCTTGG-35′ and 5′-IndexTermTGAAAGCTGGTCTGGTCCTT-3′ (exons 7–9), 5′-IndexTermCCATCTTTTACTCAGGTAC -3′ and 5′-IndexTermGAAGGCAGGATGAGAATG-3′ (exon 10), 5′-IndexTermTTCCCGTTGTCCCAGCCTTAG-3′ and 5′-IndexTermCAAGCAAGGGTTCAAAGA-3′ (exon 11). PCR fragments were analysed on 1% agarose gels, purified with Gel Extraction Systems (Marligen Biosciences Inc., Ijamsville, MD, USA) and sequenced using BigDye Terminator v3.1 cycle sequencing on an ABI PRISM3100-Avant Genetic Analyzer (Applied Biosystems, Foster City, CA, USA).
Functional analysis of separated alleles in yeast and Split Assay
FASAY and Split Assays were performed on 39 DLBCLS as described previously (Waridel et al., 1997; Smardova et al., 2001). Briefly, RNA was extracted from fresh-frozen material using TrizolReagent (Invitrogen, GmbH, Karlsruhe, Germany). cDNA synthesis was carried out using the iScript cDNA Synthesis Kit according to the manufacturer's instructions (BioRad, München, Germany). After transformation of yeast cells with TP53 cDNA, single clones were used to isolate cDNA. cDNA sequencing of at least five clones per DLBCL sample was performed as reported previously (Nenutil et al., 2005).
BAC/PAC map construction, clone preparation and labeling
To obtain appropriate FISH probes for the detection of deletions on chromosome 17p, clones were selected using the Clone Registry database (http://www.ncbi.nlm.nih.gov/genome/clone) and comparing sequence data by BLAST-search (http://www.ncbi.nlm.nih.gov/BLAST/) in the genome sequence data. The BAC- and PAC-clones RP11-199F11, RP11-333E1, RP11-314A20, RP11-167N20, RP11-545O6, RP11-208J12, RP11-147K16, RP11-64J4, CTD-2309O5, CTD-2513A7, CTD-3060P21, CTB-11O23, RP11-74E22, RP11-667K14, CTD-2545H1, RP11-4F24, RP11-433M14, CTD-2231E3, RP11-818O24 and RP5-1029F21 were obtained from the libraries RPCIB753 and RPCIP704 of the RZPD German Resource Center for Genome Research (Berlin, Germany).
After DNA isolation according to standard protocols (Macherey-Nagel, Düren, Germany), probes were generated by nick translation with biotin-16-dUTP or digoxigenin-11-dUTP (Roche Diagnostics, Mannheim, Germany).
Interphase-FISH was performed on cytogenetic preparations or tumor cells isolated from frozen tumor tissues according to a standard protocol (Lichter et al., 1995). Hybridization and immunodetection were performed as described previously (von Bergh et al., 2000; Haralambieva et al., 2002). Cytogenetic preparations of five reactive lymph node specimens were used to determine the cutoff level for each probe. At least 100 intact nuclei per case were evaluated on a Leica fluorescence microscope (Leica Microsystems, Bensheim, Germany). Illustrations were documented using the ISIS imaging system (MetaSystems, Altlussheim, Germany).
To detect genomic losses of HIC1 and TP53 in the tumor specimens, quantitative real-time PCR (qPCR) using genomic DNA was performed as described previously (Rosenwald et al., 2003b). The B2M locus in chromosome band 15q21 and the hemoglobin-β (HBB) locus in 11q15 were selected as reference genes. To determine the cutoff level for genomic deletions affecting the HIC1 and TP53 gene loci, control DNA samples were prepared from peripheral blood mononuclear cells obtained from five healthy volunteers. The cutoff ratios for HIC1/B2M and TP53/B2M were 0.67 and 0.65, respectively. For HIC1/HBB and TP53/HBB cutoff ratios of 0.65 and 0.71 were calculated, respectively. Previously described primers for B2M and HIC1 were applied (Goff et al., 2000; Guerardel et al., 2001). Primers for TP53 amplification were 5′-IndexTermTTTGGGTCTTTGAACCCTTG-3′ (sense) and 5′-IndexTermCCACAACAAAACACCAGTG-3′ (antisense). To amplify HBB following oligonucleotides were used: 5′-IndexTermACCCTTAGGCTGCTGGTGG-3′ (sense) and 5′-IndexTermGGAGTGGACAGATCCCCAAA-3′ (antisense).
SIRT1 gene expression levels were determined by Taqman real-time quantitative RT–PCR (qRT–PCR) with pre-developed assays (Applied Biosystems, Darmstadt, Germany; SIRT1: Hs_01009006_m1, B2M: HS_00187842_m1) and calculated with the standard curve method using cDNA of reactive lymph node specimen and B2M as endogenous control.
Methylation analysis of HIC1
DNA methylation analysis was performed by using the EZ DNA Methylation-Gold Kit (Zymo Research, Orange, CA, USA) according to the manufacturer's instructions. MS–PCR was carried out as described previously (Herman et al., 1996). Primers used to amplify the 5′-untranslated region (exon 1a) of the HIC1 gene were reported previously (Dong et al., 2001). All samples were analysed with both methylation-specific primers (M) and primers specific for the unmethylated allele (UM). The bisulfite-treated DNA of five healthy volunteers and five reactive lymph node tissue sections served as negative controls. As a positive control, bisulfite-modified DNA of the Raji cell line was used. MS–PCR products were separated on 3% agarose gels and visualized by ethidium bromide staining.
Survival analyses were performed by using the Kaplan–Meier method. The statistical significance of associations between the TP53 or HIC1 status and survival was determined by using the log-rank test. P<0.05 was considered as statistically significant. For all analyses the SPSS software V12.0 (SPSS Inc., Chicago, IL, USA) was used.
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We thank Heike Brueckner, Irina Eichelbroenner, Doris Hetzer and Petra Stempfle for their excellent technical assistance. H S is a member of the Graduiertenkolleg 639 (Tumorinstabilität) which is supported by the Deutsche Forschungsgesellschaft (DFG). AR is supported by the Interdisciplinary Center for Clinical Research (IZKF), University of Würzburg.
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Stöcklein, H., Smardova, J., Macak, J. et al. Detailed mapping of chromosome 17p deletions reveals HIC1 as a novel tumor suppressor gene candidate telomeric to TP53 in diffuse large B-cell lymphoma. Oncogene 27, 2613–2625 (2008). https://doi.org/10.1038/sj.onc.1210901
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