Laurence Bianchini

Clinical and Biological Significance of CDK4 Amplification in Well-Differentiated and Dedifferentiated Liposarcomas

Purpose: The MDM2 and HMGA2 genes are consistently amplified in well-differentiated/dedifferentiated liposarcomas (WDLPS/DDLPS) whereas CDK4 is frequently but not always amplified in these tumors. Our goal was to determine whether the absence of CDK4 amplification was (a) correlated to a specific clinico-histopathologic profile; and (b) compensated by another genomic anomaly involving the CCND1/CDK4/P16INK4a/RB1/E2F pathway. Experimental Design: We compared the clinical characteristics of a series of 143 WDLPS/DDLPS with amplification of both MDM2 and CDK4 (MDM2+/CDK4+) to a series of 45 WDLPS/DDLPS with MDM2 amplification and no CDK4 amplification (MDM2+/CDK4-). We used fluorescence in situ hybridization, real time quantitative reverse transcription PCR, and immunohistochemistry to explore the status of CCND1, P16INK4a, P14ARF, and RB1. Results: We found that MDM2+/CDK4- WDLPS/DDLPS represent a distinct clinical subgroup with favorable prognostic features, including low-grade lipoma-like histology, peripheral location, and lower rate of recurrence. By using fluorescence in situ hybridization, we found that genomic aberrations expected to be alternative mechanisms for compensating the lack of CDK4 amplification, such as RB1 and CDKN2A deletions or CCND1 amplification, were very uncommon. In contrast, by using real time quantitative reverse transcription PCR and immunohistochemistry, we observed that overexpression of P16INK4a (and P14ARF) and CCND1 and reduced expression of RB1 were very frequent, independently of the CDK4 status. Conclusions: Our results underscore the complex coordinated regulation of the RB and p53 growth-control pathways in WDLPS/DDLPS. Because the absence of CDK4 amplification is not specifically counterbalanced by a genomic alteration of the CCND1/CDK4/P16INK4a/RB1/E2F pathway, CDK4 amplification may only represent a “MDM2-HMGA2-helper” in WDLPS/DDLPS tumorigenesis. (Clin Cancer Res 2009;15(18):5696–703)

Let-7 microRNA and HMGA2 levels of expression are not inversely linked in adipocytic tumors: analysis of 56 lipomas and liposarcomas with molecular cytogenetic data.

The aim of our study was first to assess the role of HMGA2 expression in the pathogenesis of adipocytic tumors (AT) and, second, to seek a potential correlation between overexpression of HMGA2 and let‐7 expression inhibition by analyzing a series of 56 benign and malignant AT with molecular cytogenetic data. We measured the levels of expression of HMGA2 mRNA and of eight members of the let‐7 microRNA family using quantitative RT‐PCR and expression of HMGA2 protein using immunohistochemistry. HMGA2 was highly overexpressed in 100% of well‐differentiated/dedifferentiated liposarcomas (WDLPS/DDLPS), all with HMGA2 amplification, and 100% of lipomas with HMGA2 rearrangement. Overexpression of HMGA2 mRNA was detected in 76% of lipomas without HMGA2 rearrangement. HMGA2 protein expression was detected in 100% of lipomas with HMGA2 rearrangement and 48% of lipomas without HMGA2 rearrangement. We detected decreased expression levels of some let‐7 members in a significant proportion of AT. Notably, let‐7b and let‐7g were inhibited in 61% of WDLPS/DDLPS. In lipomas, each type of let‐7 was inhibited in approximately one‐third of the cases. Although overexpression of both HMGA2 mRNA and protein in a majority of ordinary lipomas without HMGA2 structural rearrangement may have suggested a potential role for let‐7 microRNAs, we did not observe a significant link with let‐7 inhibition in such cases. Our results indicate that inhibition of let‐7 microRNA expression may participate in the deregulation of HMGA2 in AT but that this inhibition is neither a prominent stimulator for HMGA2 overexpression nor a surrogate to genomic HMGA2 rearrangements.

Complex t(5;8) involving the CSPG2 and PTK2B genes in a case of dermatofibrosarcoma protuberans without the COL1A1-PDGFB fusion

Dermatofibrosarcoma protuberans (DFSP) is a rare, dermal neoplasm of intermediate malignancy. It is made of spindle-shaped tumor cells in a storiform pattern positive for CD34. Cytogenetically, DFSP cells are characterized by either supernumerary ring chromosomes composed of sequences derived from chromosomes 17 and 22 or more rarely of translocations t(17;22). These chromosomal rearrangements lead to the formation of a specific chimeric gene fusing COL1A1 to PDGFB. So far, the COL1A1-PDGFB fusion gene remains the sole fusion gene identified in DFSP. However, some observations suggest that genes, other than COL1A1 and PDGFB, might be involved in some DFSP cases. We report in this paper a DFSP case presenting as a unique chromosomal abnormality a complex translocation between chromosomes 5 and 8. This is the first report of a DFSP case where the lack of chromosomes 17 and 22 rearrangement and the absence of COL1A1-PDGFB fusion gene have been demonstrated. Using fluorescence in situ hybridization analysis, we showed that the CSPG2 gene at 5q14.3 and the PTK2B gene at 8p21.2 were disrupted by this rearrangement. Although rare, the existence of cases of DFSP negative for the COL1A1-PDGFB fusion has to be taken in consideration when performing molecular diagnosis for a tumor suspected to be a DFSP.

SREBP-1c/MicroRNA 33b Genomic Loci Control Adipocyte Differentiation.

ABSTRACT White adipose tissue (WAT) is essential for maintaining metabolic function, especially during obesity. The intronic microRNAs miR-33a and miR-33b, located within the genes encoding sterol regulatory element-binding protein 2 (SREBP-2) and SREBP-1, respectively, are transcribed in concert with their host genes and function alongside them to regulate cholesterol, fatty acid, and glucose metabolism. SREBP-1 is highly expressed in mature WAT and plays a critical role in promoting in vitro adipocyte differentiation. It is unknown whether miR-33b is induced during or involved in adipogenesis. This is in part due to loss of miR-33b in rodents, precluding in vivo assessment of the impact of miR-33b using standard mouse models. This work demonstrates that miR-33b is highly induced upon differentiation of human preadipocytes, along with SREBP-1. We further report that miR-33b is an important regulator of adipogenesis, as inhibition of miR-33b enhanced lipid droplet accumulation. Conversely, overexpression of miR-33b impaired preadipocyte proliferation and reduced lipid droplet formation and the induction of peroxisome proliferator-activated receptor γ (PPARγ) target genes during differentiation. These effects may be mediated by targeting of HMGA2, cyclin-dependent kinase 6 (CDK6), and other predicted miR-33b targets. Together, these findings demonstrate a novel role of miR-33b in the regulation of adipocyte differentiation, with important implications for the development of obesity and metabolic disease.

Peroxisome proliferator-activated receptor β/δ (PPARβ/δ) is highly expressed in liposarcoma and promotes migration and proliferation.

Aberrations of specialized metabolic pathways might be implicated in the development of neoplasias. Peroxisome proliferator‐activated receptors (PPARs) are ligand‐activated transcription factors with important functions in metabolism. PPARβ/δ and PPARγ act in the proliferation and differentiation of adipose tissue progenitor cells. Thus, a potential use of PPARγ agonists for the treatment of liposarcoma had been suggested, but clinical trials failed to detect beneficial effects. We show here that PPARδ is highly expressed in liposarcoma compared to lipoma and correlates with proliferation. Stimulation of liposarcoma cell lines with a specific PPARδ agonist increases proliferation, which is abolished by a PPARδ–siRNA or a specific PPARδ antagonist. Expression of the adipose tissue secretory factor leptin is lower in liposarcoma compared to lipoma and leptin reduces proliferation of liposarcoma cell lines. PPARδ activation stimulates cell migration whereas leptin diminishes it. We demonstrate that PPARδ directly represses leptin as: (a) leptin becomes down‐regulated upon PPARδ activation; (b) PPARδ represses leptin promoter activity in different sarcoma cell lines; (c) deletion of a PPAR/RxR binding element in the leptin promoter abolishes repression by PPARδ; and (d) in chromatin immunoprecipitation we confirm in vivo binding of PPARδ to the leptin promoter. Our data suggest inhibition of PPARδ as a potential novel strategy to reduce liposarcoma cell proliferation. Copyright

Molecular cytogenetics of pediatric adipocytic tumors

Both epidemiologic and cytogenetic data on pediatric adipose tissue tumors are scarce. Pediatric adipose tumors are mainly represented by lipomas, though only 28 cytogenetic descriptions of pediatric lipoma have been reported to date. Similar to adult cases, most of these pediatric lipomas harbored rearrangements of the chromosomal regions 12q14-q15 and 6p21, involving the HMGA2 and HMGA1 genes. Further cytogenetic studies of pediatric lipoma would be useful to determinate whether some partner genes of HMGA2, such as NFIB, may have a specific role in the early onset of these tumors. Cytogenetically, the best documented pediatric adipose tumor is lipoblastoma, which is the second most frequent adipose tumor in children. Chromosomal alterations in lipoblastoma, observed in 61% of cases studied by conventional cytogenetics, typically involve the 8q11-q12 region. The target gene of this rearrangement is PLAG1. Anomalies of PLAG1 have been observed in 70% of cases of pediatric adipose tumors studied by molecular cytogenetics methods, such as fluorescence in situ hybridization (FISH) or comparative genomic hybridization on array (array-CGH). The rare described cases of malignant pediatric adipose tumors in children are mostly myxoid liposarcomas. In the 27 cases explored at the genetic level, all pediatric myxoid liposarcomas showed the classical rearrangement of the DDIT3 gene at 12q13. In conclusion, the epidemiology and the prevalence of histological types of adipose tissue tumors differ in the pediatric population compared with adults, whereas chromosomal and genic rearrangements are similar to those of adult cases in each histological type.

Syndecan-1 regulates adipogenesis: new insights in dedifferentiated liposarcoma tumorigenesis

Syndecan-1 (SDC1/CD138) is one of the main cell surface proteoglycans and is involved in crucial biological processes. Only a few studies have analyzed the role of SDC1 in mesenchymal tumor pathogenesis. In particular, its involvement in adipose tissue tumors has never been investigated. Dedifferentiated liposarcoma, one of the most frequent types of malignant adipose tumors, has a high potential of recurrence and metastastic evolution. Classical chemotherapy is inefficient in metastatic dedifferentiated liposarcoma and novel biological markers are needed for improving its treatment. In this study, we have analyzed the expression of SDC1 in well-differentiated/dedifferentiated liposarcomas and showed that SDC1 is highly overexpressed in dedifferentiated liposarcoma compared with normal adipose tissue and lipomas. Silencing of SDC1 in liposarcoma cells impaired cell viability and proliferation. Using the human multipotent adipose-derived stem cell model of human adipogenesis, we showed that SDC1 promotes proliferation of undifferentiated adipocyte progenitors and inhibits their adipogenic differentiation. Altogether, our results support the hypothesis that SDC1 might be involved in liposarcomagenesis. It might play a prominent role in the dedifferentiation process occurring when well-differentiated liposarcoma progress to dedifferentiated liposarcoma. Targeting SDC1 in these tumors might provide a novel therapeutic strategy.

Prognostic value of HMGA2, CDK4, and JUN amplification in well-differentiated and dedifferentiated liposarcomas

HMGA2, CDK4, and JUN genes have been described as frequently coamplified with MDM2 in atypical lipomatous tumor, well-differentiated liposarcoma, and dedifferentiated liposarcoma. We studied the frequency of amplification of these genes in a series of 48 dedifferentiated liposarcomas and 68 atypical lipomatous tumors/well-differentiated liposarcomas. We correlated their amplification status with clinicopathological features and outcomes. Histologically, both CDK4 (P=0.007) and JUN (P=0.005) amplifications were associated with dedifferentiated liposarcoma, whereas amplification of the proximal parts of HMGA2 (5′-untranslated region (UTR) and exons 1–3) was associated with atypical lipomatous tumor/well-differentiated liposarcoma (P=0.01). CDK4 amplification was associated with axial tumors. Amplification of 5′-UTR and exons 1–3 of HMGA2 was associated with primary status and grade 1. Shorter overall survival was correlated with: age >64 years (P=0.03), chemotherapy used in first intent (P<0.001), no surgery (P=0.003), grade 3 (P<0.001), distant metastasis (P<0.001), node involvement (P=0.006), and CDK4 amplification (P=0.07). In multivariate analysis, distant metastasis (HR=8.8) and grade 3 (HR=18.2) were associated with shorter overall survival. A shorter recurrence-free survival was associated with dedifferentiated liposarcoma (P<0.001), grade 3 (P<0.001), node involvement (P<0.001), distant metastasis (P=0.02), recurrent status (P=0.009), axial location (P=0.001), and with molecular features such as CDK4 (P=0.05) and JUN amplification (P=0.07). Amplification of 5′-UTR and exons 1–3 (P=0.08) and 3′-UTR (P=0.01) of HMGA2 were associated with longer recurrence-free survival. Distant metastasis was associated with shorter recurrence-free survival (HR=5.8) in multivariate analysis. Dedifferentiated liposarcoma type was associated with axial location, grade 3 and recurrent status. In conclusion, we showed that the amplification of HMGA2 was associated with the atypical lipomatous tumor/well-differentiated liposarcoma histological type and a good prognosis, whereas CDK4 and JUN amplifications were associated with dedifferentiated liposarcoma histology and a bad prognosis. In addition, we also provided the first description of the molecular evolution of a well-differentiated liposarcoma into four successive dedifferentiated liposarcoma relapses, which was consistent with our general observations.

Identification of PPAP2B as a novel recurrent translocation partner gene of HMGA2 in lipomas

Most lipomas are characterized by translocations involving the HMGA2 gene in 12q14.3. These rearrangements lead to the fusion of HMGA2 with an ectopic sequence from the translocation chromosome partner. Only five fusion partners of HMGA2 have been identified in lipomas so far. The identification of novel fusion partners of HMGA2 is important not only for diagnosis in soft tissue tumors but also because these genes might have an oncogenic role in other tumors. We observed that t(1;12)(p32;q14) was the second most frequent translocation in our series of lipomas after t(3;12)(q28;q14.3). We detected overexpression of HMGA2 mRNA and protein in all t(1;12)(p32;q14) lipomas. We used a fluorescence in situ hybridization‐based positional cloning strategy to characterize the 1p32 breakpoint. In 11 cases, we identified PPAP2B, a member of the lipid phosphate phosphatases family as the 1p32 target gene. Reverse transcription‐polymerase chain reaction analysis followed by nucleotide sequencing of the fusion transcript indicated that HMGA2 3′ untranslated region (3′UTR) fused with exon 6 of PPAP2B in one case. In other t(1;12) cases, the breakpoint was extragenic, located in the 3′region flanking PPAP2B 3′UTR. Moreover, in one case showing a t(1;6)(p32;p21) we observed a rearrangement of PPAP2B and HMGA1, which suggests that HMGA1 might also be a fusion partner for PPAP2B. Our results also revealed that adipocytic differentiation of human mesenchymal stem cells derived from adipose tissue was associated with a significant decrease in PPAP2B mRNA expression suggesting that PPAP2B might play a role in adipogenesis.

A newly characterized human well-differentiated liposarcoma cell line contains amplifications of the 12q12-21 and 10p11-14 regions

While surgery is the usual treatment for localized well-differentiated and dedifferentiated liposarcomas (WDLPS/DDLPS), the therapeutic options for patients with advanced disease are limited. The classical antimitotic treatments are most often inefficient. The establishment of genetically characterized cell lines is therefore crucial for providing in vitro models for novel targeted therapies. We have used spectral karyotyping, fluorescence in situ hybridization with whole chromosome painting and locus-specific probes, and array-comparative genomic hybridization to identify the chromosomal and molecular alterations of a novel cell line established from a recurring sclerosing WDLPS. The karyotype was hypertriploid and showed multiple structural anomalies. All cells retained the presence of a giant marker chromosome that had been previously identified in the primary cell cultures. This giant chromosome contained high-level amplification of chromosomal regions 12q13-21 and lacked the alpha-satellite centromeric sequences associated with WDLPS/DDLPS. The 12q amplicon was large, containing 370 amplified genes. The DNA copy number ranged from 3 to 57. The highest levels of amplification were observed at 12q14.3 for GNS, WIF1, and HMGA2. We analyzed the mRNA expression status by real-time reverse transcription polymerase chain reaction for six genes from this amplicon: MDM2, HMGA2, CDK4, TSPAN31, WIF1, and YEATS4. mRNA overexpression was correlated with genomic amplification. A second amplicon originating from 10p11-14 was also present in the giant marker chromosome. The 10p amplicon contained 62 genes, including oncogenes such as MLLT10, previously described in chimeric fusion with MLL in leukemias, NEBL, and BMI1.