Osteosarcoma of the scapula arising in osteogenesis imperfecta

A case of osteosarcoma arising in the scapula of a 37 year old woman with severe congenital sporadic white sclera type osteogenesis imperfecta (OI) is presented. Osteosarcoma occurs as a sporadic rather than a related occurrence in patients with OI. A delay in the diagnosis of osteosarcoma may occur due to the similarity in symptomatology between the two conditions and because of the difficulty in differentiating between hyperplastic callus and tumor using conventional radiographic imaging techniques.     

Hyperplastic callus formation in osteogenesis imperfecta

A case of a child with the hyperplastic callus formation in association with osteogenesis imperfecta is presented. The soft tissue mass arising at the site of femoral fracture was clinically and radiologically suspected to be a hyperplastic callus, although a possibility of osteosarcoma could not be completely excluded. A drill biopsy of the soft tissue mass was performed which showed osteocartilaginous callus. The soft tissue mass disappeared following biopsy. This occurrence is also reported in previously described cases.     

Bcl-2,Cox-2,HSPs和p53在骨肉瘤患者中的表达及临床意义

目的:观察分析bcl-2,Cox-2,HSPs和p53蛋白等分子生物标志物在骨肉瘤患者中的表达差异性以及相关性,为临床病理诊断和治疗提供理论依据以及指导.方法:通过对所纳入的骨肉瘤患者典型组织进行切片以及SP染色,同期20例骨软骨瘤患者作为对照组.观察bcl-2,Cox-2,HSPs和p53等蛋白分子标志物的表达,使用X2检验分析上述蛋白表达在各组之间的差异性以及临床意义.结果:骨肉瘤患者bcl-2,Cox-2,Ki-67,HSP70,HSP90以及p53蛋白阳性表达率明显高于骨软骨瘤组,差异有统计学意义(P＜0.05);Vim蛋白的表达率在两组间差异无统计学意义(P＞0.05).结论:Bcl-2,Cox-2,HSP70,HSP90,Ki-67以及p53在骨肉瘤病灶形成以及进展过程中可能起重要的作用,并为临床病理学诊断以及精准医疗提供理论依据.     

Monoallelic deletion of the p53 gene through chromosomal translocation in a small cell osteosarcoma

Small cell osteosarcoma is a rare bone tumor of high-grade malignancy that most often arises in the metaphysis of long bones in the second decade of life. Cytogenetic and molecular genetic findings in small cell osteosarcoma are poorly defined. Conventional cytogenetic analysis of a small cell osteosarcoma arising in the proximal tibia of a 9-year-old male revealed a diploid chromosomal complement with complex structural rearrangements involving chromosomes 6, 16, and 17. Immunohistochemical assessment of p53 protein expression revealed nuclear p53 immunoreactivity in approximately 15% of the neoplastic cells. Subsequent fluorescence in situ hybridization (FISH) analyses confirmed loss of the p53 gene locus on the derivative chromosome 17 homolog and were negative for amplification of the MDM2, CDK4, c-MYC, HER-2/neu, CCND1, and COPS3 gene loci. To the best of our knowledge, this represents the first demonstration of monoallelic deletion of p53 in small cell osteosarcoma, suggesting that p53 alterations may play an important role in the development of small cell osteosarcoma as well as conventional osteosarcoma.     

Conditional mouse osteosarcoma, dependent on p53 loss and potentiated by loss of Rb, mimics the human disease

Osteosarcoma is the most common primary malignant tumor of bone. Analysis of familial cancer syndromes and sporadic cases has strongly implicated both p53 and pRb in its pathogenesis; however, the relative contribution of these mutations to the initiation of osteosarcoma is unclear. We describe here the generation and characterization of a genetically engineered mouse model in which all animals develop short latency malignant osteosarcoma. The genetically engineered mouse model is based on osteoblast-restricted deletion of p53 and pRb. Osteosarcoma development is dependent on loss of p53 and potentiated by loss of pRb, revealing a dominance of p53 mutation in the development of osteosarcoma. The model reproduces many of the defining features of human osteosarcoma including cytogenetic complexity and comparable gene expression signatures, histology, and metastatic behavior. Using a novel in silico methodology termed cytogenetic region enrichment analysis, we demonstrate high conservation of gene expression changes between murine osteosarcoma and known cytogentically rearranged loci from human osteosarcoma. Due to the strong similarity between murine osteosarcoma and human osteosarcoma in this model, this should provide a valuable platform for addressing the molecular genetics of osteosarcoma and for developing novel therapeutic strategies.     

Possible involvement of bcl-2 suppression in wild-type p53 gene-dependent cell growth repression in rat osteosarcoma cells

We recently obtained 3 cloned cell lines demonstrating the p53 mutation from a lung metastatic nodule of a rat transplantable osteosarcoma. In this study, we applied wild-type p53 gene transfer to the rat osteosarcoma cells by lipofection to investigate the effects on cell growth, expression of genes such as waf1/p21, bcl-2, and bax, and nucleosomal DNA fragmentation due to apoptosis. Reconstitution of the p53 gene inhibits cellular growth, and this growth-suppressive effect is partly due to apoptosis involving bcl-2 gene suppression in this tumor type. This rat osteosarcoma model is similar in biologic behavior to human cases and thus is very suitable for further investigation of tumorigenesis and gene therapy for osteosarcoma.     

Disruption of the Murine p53 Gene by Insertion of an Endogenous Retrovirus-like Element (ETn) in a Cell Line from Radiation-Induced Osteosarcoma

The mammalian genome harbors a large number of endogenous retroviruses and retrovirus-like elements. Increasing evidence is found that such elements can be activated and act as insertional mutagens. The activation of endogenous retroviral elements can be induced by a variety of environmental factors including irradiation. We have observed the insertion of a murine endogenous retrovirus-like ETn element into intron 4 of the p53 gene in an osteosarcoma cell line derived from a radiation-induced osteosarcoma. The insertion resulted in a p53-ETn-p53 fusion mRNA, a novel form of p53 mutation. This is the first report on insertion of an endogenous retroviral element into the p53 tumor suppressor gene. The data suggest that activated endogenous retroviruses and retrovirus-like elements might pose an enhanced risk for individuals exposed to noxae, which activate endogenous retroviral elements.     

Analysis of p53 expression in osteosarcoma of the jaw: Correlation with clinicopathologic and DNA ploidy findings

The objective of this study was to determine whether p53 expression correlated with clinicopathological and DNA ploidy in osteosarcoma of the jaw, a particular subtype of osteosarcoma associated with an older age at presentation, longer median survival, rare metastasis, and death due to uncontrolled local recurrence. Seventeen cases of osteosarcoma of the jaw were stained for p53 using CM1 antibody (Novocastra). Eight cases (47.5%) showed nuclear staining in more than 10% of the neoplastic nuclei. Ploidy analysis showed 13 aneuploid (eight p53+) and three diploid (none p53+) tumors. No significant relationship was found between p53 positivity and clinicomorphological features, DNA ploidy or survival. p53-positive cases were, however, more commonly associated with aneuploidy and chondroblastic differentiation. These results indicate that p53 immunostaining does not seem to provide prognostic information in osteosarcoma of the jaw.     

Colorectal carcinomas arising in the hyperplastic polyposis syndrome progress through the chromosomal instability pathway

The hyperplastic polyposis syndrome is characterized by the presence within the colon of multiple large hyperplastic polyps. We describe a case of hyperplastic polyposis syndrome associated with two synchronous carcinomas, one of which arises within a pre-existing hyperplastic lesion. Comparative genomic hybridization was used to determine genetic changes in both carcinomas and several associated hyperplastic lesions. Microsatellite analysis at five loci was performed on carcinomas and representative hyperplastic polyps, and p53 status was analyzed by immunohistochemistry. Both carcinomas showed multiple genetic aberrations, including high level gains of 8q and 13q, and loss of 5q. These changes were not seen in the hyperplastic polyps. Microsatellite instability was not seen in the carcinomas, four separate hyperplastic polyps, the hyperplastic polyp with mild adenomatous change associated with the carcinoma, or a separate serrated adenoma. Allelic imbalance in the cancers at D5S346 and D17S938 suggested allelic loss of both p53 and APC, as well as at the loci D13S263, D13S174, D13S159, and D18S49. An early invasive carcinoma in one hyperplastic polyp stained for p53 protein, but the associated hyperplastic polyp was negative. In this case, neoplastic progression followed the typical genetic pathway of common colorectal carcinoma and occurred synchronously with mutation of p53.     

Analysis of p53 tumor suppressor gene, H-ras protooncogene and proliferating cell nuclear antigen (PCNA) in squamous cell carcinomas of HRA/Skh mice following exposure to 8-methoxypsoralen (8-MOP) and UVA radiation (PUVA therapy)

Treatment with 8-methoxypsoralen (8-MOP) and ultraviolet radiation (primarily UVA), called PUVA therapy, has been used to treat different chronic skin diseases but led to a significant increased risk for skin cancer. The National Toxicology Program (NTP) performed a study in mice treated with PUVA that showed a significant increase in squamous cell carcinomas of the skin. In the present study, we evaluated the protein expression of p53 and PCNA and DNA mutations of p53 and H-ras genes in both hyperplastic and neoplastic squamous cell lesions from the NTP study. By immunohistochemical staining, protein expression of both p53 and PCNA was detected in 3/16 (19%) of hyperplastic lesions and 14/17 (82%) of SCCs in groups treated with both 8-MOP and UVA. The mutation frequency of p53 in SCCs from mice administered 8-MOP plus UVA was 15/17 (88%) with a predominant distribution of mutations in exon 6 (14/15 - 93%). No H-ras mutations were detected in the hyperplastic lesions/tumors. The mutagenic effect of PUVA on the p53 tumor suppressor gene may lead to a conformational modification and inactivation of the p53 protein, which are considered critical steps in PUVA-induced skin carcinogenesis. The p53 mutational frequency and patterns from our study were different from those reported in human PUVA-type tumors.     

Prognostic value of p53 alterations in human osteosarcoma: a meta analysis

Tumor suppressor gene p53 functions as the guardian of the human genome and mutations in p53 contribute to cancer development. However, studies that investigated the potential of p53 as a prognostic marker in osteosarcoma patients have yielded inconclusive results. Based on recommendation of the Cochrane Collaboration, this meta-analysis was conducted using data from the 17 published studies to evaluate the association of p53 alterations with clinical outcome of osteosarcoma patients. Different databases, including MEDLINE, PsycINFO, Scopus, EMBASE, and the Cochrane Central Register of Controlled Trials (CENTRAL) were searched. Prognostic value of p53 alterations was determined by risk ratio (RR). The data showed that p53-positive immunostaining tended to associate with decreased 2-year survival rates (RR, 1.94; 95% CI, 1.43 to 2.64; p < 0.0001, I² = 10%). However, the prediction value of RR was smaller with p53 expression than with p53 mutations. Moreover, patients who received neoadjuvant chemotherapy and surgery tended to have a stronger association between p53-positive staining and 2-year mortality compared to the patients treated with surgery only. However, p53-positive staining was not associated with 3-year (RR, 1.64; 95% CI, 0.84 to 3.20; P = 0.15; I² = 56%) and 5-year survival (RR, 1.25; 95% CI, 0.78 to 2.01; P = 0.36; I² = 70%). The data from the current study suggest that p53-positive osteosarcoma only predicted a decreased short-term survival rate, but not 3- or 5-year survival.     

Alterations in the INK4a/ARF locus and their effects on the growth of human osteosarcoma cell lines

Two different proteins, p16(INK4a) and p14(ARF), encoded by the INK4a/ARF locus play important roles in the RB and p53 pathways, respectively. This study was performed to determine genetic and epigenetic alterations in the INK4a/ARF locus and their effects on the growth of osteosarcoma. Among six cell lines examined, both p16(INK4a) and p14(ARF) exons were homozygously deleted in two cell lines, MG63 and HOS, and both p16(INK4a) and p14(ARF) promoters were methylated in one cell line, U2OS. Wild-type mRNA and proteins for p16(INK4a) and p14(ARF) were expressed in three other cell lines, SaOS2, HuO9, and G292. Transfection studies were performed using two cell lines, U2OS and MG63. Both the RB and p53 genes were wild types in U2OS, whereas p53 but not RB was mutated in MG63. Both p16(INK4a) and p14(ARF) suppressed the growth of U2OS, whereas p16(INK4a) but not p14(ARF) suppressed the growth of MG63. p53 only did not suppress the growth of MG63 either; however, coexpression of p14(ARF) with p53 increased the fraction of the G0/G1 phase in MG63 cells. The data presented here demonstrate the importance of genetic and epigenetic alterations in the INK4a/ARF locus for the growth of osteosarcoma and thus will be useful to further understand the biologic behavior of osteosarcoma in association with the defects in the p53 and RB pathways.     

Adenovirus-mediated p53 tumor suppressor gene therapy of osteosarcoma

The clinical outcome for osteosarcoma (OS) remains discouraging despite efforts to optimize treatment using conventional modalities including surgery, radiotherapy and chemotherapy. Novel therapeutic approaches based on our expanding understanding of the mechanisms of tumor cell killing have the potential to alter this situation. Tumor suppressor gene therapy aims to restore the function of a tumor suppressor gene lost or functionally inactivated in cancer cells. One such molecule, the p53 tumor suppressor gene plays a critical role in safeguarding the integrity of the genome and preventing tumorigenesis. Introduction of wild-type (wt) p53 into transformed cells has been shown to be lethal for most cancer cells in vitro, but clinical trials of p53 gene replacement have had limited success. Analysis of these clinical trials highlighted the insufficient efficacy of current vectors and low proapoptotic activity of wt p53 as a single agent in vivo. In this review, a contemporary summarization of the current status of adenovirus-mediated p53 gene therapy of OS is presented. Advancement in our understanding of p53 tumor suppressor activity, the molecular biology of chemoresistant OS, and recent advances in tumor targeting with adenoviral vectors are also addressed. Based on these parameters, prospects for future investigations are proposed.     

MicroRNA-34a Inhibits Human Osteosarcoma Proliferation by Downregulating Ether à go-go 1 Expression

Aberrant expression of MicroRNAs (miRNAs) has been implicated in several types of cancer. As a direct target gene of p53, miR-34a has been suggested to mediate the tumor suppressor function of p53. Ether à go-go 1 (Eag1) channel is overexpressed in a variety of cancers and plays important roles in cancer progression. However, the link between miR-34a and Eag1 in cancer is unclear. In this study, we used human osteosarcoma as the model to demonstrate that miR-34a was significantly downregulated in osteosarcoma tissues and cell lines compared with normal brain tissues and osteoblastic cell line. Next we evaluated the role of miR-34a in the regulation of osteosarcoma cell proliferation by CCK-8 and colony formation assays. The results showed that overexpression of miR-34a inhibited the proliferation of MG-63 and Saos-2 cells. Furthermore, xenograft nude mice model showed that miR-34a inhibited osteosarcoma growth in vivo. Mechanistically, we found that overexpression of miR-34a led to decreased Eag1 expression in osteosarcoma cells while inhibition of miR-34a increased Eag1 expression. Taken together, our results suggest that miR-34a could inhibit osteosarcoma growth via the down regulation of Eag1 expression.     

Upregulation of miR‑95-3p inhibits growth of osteosarcoma by targeting HDGF

Osteosarcoma is the most common bone malignancy and miR-95-3p plays an important role in multiple cancers. The purpose of this study was to explore the effect and potential mechanism of miR-95-3p on the growth of osteosarcoma. In vitro, the osteosarcoma cell lines, SAOS-2 and U2OS cells, were transfected with miR-95-agomir to assess the role of miR-95-3p in proliferation and apoptosis of osteosarcoma cells. We determined that overexpression of miR-95-3p significantly attenuated cell proliferation but enhanced apoptosis in SAOS-2 and U2OS cells. We also found that overexpression of miR-95-3p in osteosarcoma cells downregulated the expression of hepatoma-derived growth factor (HDGF). Next, knockdown of HDGF by siRNA targeting HDGF clearly inhibited cell proliferation and induced apoptosis in U2OS cells. In vivo, a tumor formation assay in BALB/c nude mice was conducted by injecting the pre-miR-95 or control vector lentivirus-infected U2OS cells to determine the effect of miR-95-3p on the growth of osteosarcoma. Results showed miR-95-3p overexpression inhibited the osteosarcoma growth and downregulated the HDGF expression in xenografted tumor. For mechanism study, we co-transfected HDGF/pcDNA3.1 plasmid and miR-95-agomir to U2OS cells, and we demonstrated that overexpression of HDGF could attenuate the effects of miR-95-3p on U2OS cell proliferation, apoptosis and migration. These findings indicated that miR-95-3p might act as a potential tumor suppressor in osteosarcoma by targeting HDGF. Thus, miR-95-3p may become a potential therapeutic in treatment of osteosarcoma.     

骨肿瘤MDM2和p53基因的改变

目的从基因水平研究ＭＤＭ２和ｐ５３基因在骨肿瘤中的表达，探讨其在骨肿瘤发生发展中的作用。方法用地高辛标记原位杂交技术研究了３８例骨肿瘤（骨肉瘤１２例，软骨肉瘤１０例，骨巨细胞瘤１４例，软骨母细胞瘤２例）ＭＤＭ２和ｐ５３的表达情况，并分析两种基因表达之间的相互关系。结果ＭＤＭ２在骨肉瘤、软骨肉瘤和骨巨细胞瘤中的阳性率分别为４１．７％、５０．０％和３５．７％。ｐ５３的阳性率分别为５８．３％、４０．０％和２１．４％。２例软骨母细胞瘤ＭＤＭ２和ｐ５３均为阳性。ＭＤＭ２与ｐ５３基因表达呈显著正相关（Ｐ＜０．００５）。结论ＭＤＭ２和ｐ５３基因改变是骨肿瘤的一种常见现象，可能参与骨肿瘤的发生与发展。     

c-myc、p53和p16的表达及GNAS1基因突变在骨的纤维结构不良中的意义

目的 检测c-myc、p53和p16蛋白在骨的纤维结构不良(FD)中的表达及其意义,检测FD中GNAS1基因第8外显子突变,探讨FD的病变性质.方法 采用免疫组织化学SP法检测35例FD(包括1例FD恶变,1例Mazabraud综合征)及20例对照组(10例骨痂、10例骨肉瘤)中c-myc、p53和p16蛋白表达.采用基因组DNA抽提、PCR扩增及基因测序的方法检测35例FD中GNAS1基因第8外显子突变情况.结果 91%(32/35)FD中检测到c-myc蛋白表达,与阴性对照组相比差异具有统计学意义(P=0.001).p53阳性表达仅出现于1例FD合并骨肉瘤变病例中.p16蛋白在34例FD中阳性,与阳性对照组相比差异具有统计学意义(P=0.001).35例FD中,12例GNAS1基因第8外显子DNA扩增成功,其中2例(1例Mazabraud综合征的FD;1例单骨性FD)检测到GNAS1基因突变.结论 c-myc可能是除c-fos外的又一FD相关的原癌基因,p16基因的异常表达在FD的形成过程中可能起重要作用,p53蛋白过表达有助于FD恶变的预测及预后的判断.FD中存在有GNAS1的基因突变.FD是多种因素共同作用导致的骨成熟障碍的肿瘤性病变.     

p53 and mdm2 expression in colorectal carcinoma: a correlative analysis with clinical staging and histological parameters

Colorectal carcinoma (CRC) is rare in the Indian subcontinent. The two-hit carcinogenic theory has been well established from the turn of this century, in which p53 is described as a late marker in colorectal carcinogenesis, and murine double minute 2 (mdm2) has not yet been correlated with colorectal carcinoma. With the recent introduction of anti-mdm2 oligonucleotides, it is apparent that if its role can be established in colorectal carcinogenesis, the DNA conservation of the wild p53 strain would be achieved therapeutically because mdm2 conserves the wild p53 strain. We studied 32 cases with adequate number of controls and found a positive correlation (P = .001) between these proteins. These proteins were expressed in tumor adjacent to the hyperplastic mucosa, dysplastic mucosa, and aberrant crypt foci, unlike in studies in the West. The expression of these proteins was correlated with aggressive tumor behavior. All these indicate significant diagnostic, therapeutic, and prognostic roles for these proteins.     

Osteosarcoma arising in fibrous dysplasia,confirmed by mutational analysis of GNAS gene

Malignancy arising in fibrous dysplasia (FD) is rare. Approximately 100 cases have been reported so far, and osteosarcoma is the most common malignancy. We report a case of osteosarcoma in a 33-year-old Japanese man with monostotic FD of the right proximal femur from the age of 16 years. Histologically, relatively well-differentiated osteosarcoma was found in the FD lesion. Immunohistochemically, the FD was negative for p53 or MDM2, and the MIB-1 index was less than 1%, whereas the osteosarcoma was positive for both p53 and MDM2, and the MIB-1 index was up to 15%. The FD and osteosarcoma were negative for CDK4. Fluorescent in situ hybridization assay showed no amplification of the MDM2 gene, indicating that the osteosarcoma was a conventional osteosarcoma, not an intraosseous well-differentiated type. The original cell of malignancy in FD is unclear. Malignancy can be potentially derived from dysplastic cells in the area of the FD or cells in the adjacent normal tissues. GNAS gene mutation has recently been reported for fibrous dysplasia and the mutation is highly specific to fibrous dysplasia among fibro-osseous lesions including osteosarcoma.In this case, point mutations of GNAS were found in the FD and osteosarcoma but not in the adjacent normal tissues, suggesting that osteosarcoma was derived from the spindle cells of FD. This is the first report to clearly show that osteosarcoma is derived from the spindle cells in fibrous dysplasia (FD).     

A role of histone H4 hypoacetylation in vascular endothelial growth factor expression in colon mucosa adjacent to implanted cancer in athymic mice cecum.

Expression of angiogenic factors is upregulated in hyperplastic mucosa adjacent to colon cancer, and this upregulation is closely associated with cancer growth and metastasis. We investigated the role of histone acetylation in vascular endothelial growth factor (VEGF) expression in hyperplastic mucosa adjacent to orthotopic colon cancer in mice. In the hyperplastic mucosa adjacent to KM12SM tumor in the cecum of athymic mice, VEGF upregulation was associated with hypoxia-inducible factor (HIF)-1alpha induction. The hyperplastic mucosa also showed hypoacetylation of histone H4 and reduction of both p53 and von Hippel-Lindau (VHL) proteins. To examine the effects of growth factors and cytokines on histone acetylation and levels of p53, VHL and HIF-1alpha, the rat intestinal epithelial cell line IEC6 was treated with epidermal growth factor (EGF) and interleukin (IL)-15 for 35 days. Acetylated histone H4, p53 protein and ubiquitinated protein levels were reduced, whereas HIF-1alpha production was upregulated in EGF- and IL-15-treated IEC6 cells. These findings suggest that EGF- or IL-15-induced histone H4 hypoacetylation is associated with repression of p53 and VHL genes in intestinal epithelial cells. The subsequent suppression of protein ubiquitination leads to upregulation of VEGF production by HIF-1alpha retention.