Real-time polymerase chain reaction analysis of MDM2 and CDK4 expression using total RNA from core-needle biopsies is useful for diagnosing adipocytic tumors

Taro Sasaki 0 Akira Ogose 0 Hiroyuki Kawashima 0 Tetsuo Hotta 0 Hiroshi Hatano Takashi Ariizumi Hajime Umezu Riuko Ohashi Tsuyoshi Tohyama Naohito Tanabe Naoto Endo 0 0 Division of Orthopedic Surgery, Niigata University Graduate School of Medical and Dental Sciences , 757-1, Asahimachi-dori, Niigata City, Niigata 951-8510 , Japan Background: Diagnosing adipocytic tumors can be challenging because it is often difficult to morphologically distinguish between benign, intermediate and malignant adipocytic tumors, and other sarcomas that are histologically similar. Recently, a number of tumor-specific chromosome translocations and associated fusion genes have been identified in adipocytic tumors and atypical lipomatous tumors/well-differentiated liposarcomas (ALT/WDL), which have a supernumerary ring and/or giant chromosome marker with amplified sequences of the MDM2 and CDK4 genes. The purpose of this study was to investigate whether quantitative real-time polymerase chain reaction (PCR) could be used to amplify MDM2 and CDK4 from total RNA samples obtained from core-needle biopsy sections for the diagnosis of ALT/WDL. Methods: A series of lipoma (n = 124) and ALT/WDL (n = 44) cases were analyzed for cytogenetic analysis and lipoma fusion genes, as well as for MDM2 and CDK4 expression by real-time PCR. Moreover, the expression of MDM2 and CDK4 in whole tissue sections was compared with that in core-needle biopsy sections of the same tumor in order to determine whether real-time PCR could be used to distinguish ALT/WDL from lipoma at the preoperative stage. Results: In whole tissue sections, the medians for MDM2 and CDK4 expression in ALT/WDL were higher than those in the lipomas (P < 0.05). Moreover, karyotype subdivisions with rings and/or giant chromosomes had higher MDM2 and CDK4 expression levels compared to karyotypes with 12q13-15 rearrangements, other abnormal karyotypes, and normal karyotypes (P < 0.05). On the other hand, MDM2 and CDK4 expression levels in core-needle biopsy sections were similar to those in whole-tissue sections (MDM2: P = 0.6, CDK4: P = 0.8, Wilcoxon signed-rank test). Conclusion: Quantitative real-time PCR of total RNA can be used to evaluate the MDM2 and CDK4 expression levels in core-needle biopsies and may be useful for distinguishing ALT/WDL from adipocytic tumors. Thus, total RNA from core-needle biopsy sections may have potential as a routine diagnostic tool for other tumors where gene overexpression is a feature of the tumor. - Background Adipocytic tumors represent the largest group of soft tissue tumors [1]. The diagnosis of adipocytic tumors is primarily based on clinical features and histologic patterns [2]. However, the distinction between lipomas and atypical lipomatous tumors/well-differentiated liposarcomas (ALT/ WDL) may be difficult to distinguish morphologically. Cytogenetic studies of adipocytic tumors have revealed a clear association between chromosomal findings and clinicohistopathological features [3,4]. Clonal chromosome aberrations have been found in nearly 60% of all lipomas [4], of which two-thirds are rearrangements involving the 12q13-15 chromosomal region. A variety of rearrangements, mainly involving the 6p and 13q regions, are observed in the remaining lipoma cases [5-7]. In tumors with aberrations involving 12q13-15 region, the high mobility group protein gene (HMGA2, also known as HMGIC) on chromosome 12 is rearranged. These aberrations may also result in the creation of chimeric genes, in which the HMGA2 gene is fused to multiple genes. The most frequent gene aberration in lipomas is HMGA2/LPP [8]. ALT/WDL and dedifferentiated liposarcomas (DDL) most often have a supernumerary ring and giant marker chromosomes composed of amplified sequences from the 12q13-15 region [9,10], including the murine doubleminute type 2 gene (MDM2) and the cyclin-dependent kinase 4 gene (CDK4) [11-13]. Amplification of the 12q1315 region has not been observed in lipoma, and the MDM2 and CDK4 proteins are known to be overexpressed in ALT/WDL but not in lipoma [14]. Immunohistochemistry for MDM2 and CDK4 plays a helpful role in the differential diagnosis of adipocytic tumors. Aleixo et al. [15] reported that MDM2 has very high sensitivity (100%) in the identification of ALT/WDL among lipomas, but has low specificity (58.8%), whereas CDK4 has low sensitivity (68.4%), but high specificity (88.2%). Immunohistochemistry may be used to demonstrate MDM2 and CDK4 amplification, but the sections sometimes show several staining patterns such as diffuse, moderate, and focal positivity. Categorization of these staining patterns has been developed differently by different researchers, making it difficult to compare studies effectively. The use of minimally invasive biopsies to diagnose soft tissue tumors has become increasingly common. On the other hand, ALT/WDL can be difficult to distinguish morphologically from benign lipomatous lesions, especially with limited material in which the diagnostic features of scattered atypical cells are not present because of heterogeneity of the neoplasm. However, distinguishing benign lipomatous tumors from ALT/WDL is important at primary biopsy. In this study, we used whole tissue sections from surgically resected specimens to retrospectively analyze cytogenetic findings by quantifying MDM2 and CDK4 expression levels in lipomas and ALT/WDL with realtime polymerase chain reaction (PCR) from total RNA. We evaluated the clinical utility of measuring MDM2 and CDK4 expression levels to establish a diagnosis of adipocytic tumors, with the aim of making a distinction between lipoma and ALT/WDL. Moreover, we compared the results of MDM2 and CDK4 expression in whole tissue sections with those in core-needle biopsy sections in order to investigate whether real-time PCR for MDM2 and CDK4 could be used to distinguish between ALT/WDL and lipoma prior to surgery. Methods Specimens Tumor samples were obtained from patients that underwent surgical resection at Niigata University Hospital between August 2001 and December 2012. In total, 124 cases of lipoma and 44 cases of ALT/WDL were studied (Additional file 1: Table S1). In all cases, the diagnosis of lipoma or ALT/WDL was established according to the World Health Organization (WHO) Classification of Tumors [2] by using hematoxylin and eosin-stained tissue sections from the surgical resection specimens. Two experienced pathologists independently reviewed the cases in which it was difficult to distinguish between lipoma and ALT/WDL. There were 159 primary and 9 recurrent tumors. The patient cohort consisted of 96 men and 72 women between 24 and 86 years of age (mean 59.0 years; range 2486 years). The samples were taken from both core-needle biopsy sections and whole tissue sections of the adipose tissue tumors. Some of the samples represent paired whole tissue sections and core-needle biopsy sections from the same tumor. Core-needle biopsy sections were sampled p (...truncated)