EWS-Fli1 antisense oligodeoxynucleotide inhibits proliferation of human Ewing's sarcoma and primitive neuroectodermal tumor cells.

The translocation t(11;22) is a common chromosomal abnormality detected both in Ewing's sarcoma and in primitive neuroectodermal tumor cells. The translocation results in an EWS-Fli1 fusion gene, made up of the 5' half of the EWS gene on chromosome 22 fused to the 3' half of the Fli1 gene on chromosome 11. Recent studies have evaluated possible roles of the fusion gene products. However, the biological significance of EWS-Fli1 is still unknown. Using a competitive polymerase chain reaction technique, we show here that there might be a correlation between the expression levels of the EWS-Fli1 fusion gene and the proliferative activities of Ewing's sarcoma and primitive neuroectodermal tumor cells. When the EWS-Fli1 expression is inhibited by antisense oligodeoxynucleotides against the fusion RNA, the growth of the tumor cells is significantly reduced both in vitro and in vivo. The data further indicate the growth inhibition of the cells by the antisense sequence might be mediated by G0/G1 block in the cell cycle progression. These results suggest that EWS-Fli1 may play an important role in the proliferation of the tumor cells, and the EWS-Fli1 fusion RNA could be used as a target to inhibit the growth of Ewing's sarcoma and primitive neuroectodermal tumor with the specific antisense oligonucleotide.     

Interphase cytogenetics for the detection of the t(11;22)(q24;q12) in small round cell tumors.

Among the small round cell tumors differential diagnosis is particularly difficult for their undifferentiated or primitive character. In this mixed group of tumors, only the primitive neuroectodermal tumors, which include Ewing's sarcoma (ES), show the unique and consistent feature of the (11;22)(q24;q12) translocation, which can therefore be considered a hallmark of these neoplasias. We analyzed four primitive neuroectodermal tumor cell lines, one osteosarcoma cell line, and 11 patients by fluorescent in situ hybridization with cosmid clones 23.2 and 5.8, bracketing the t(11;22) at 11q24. Metaphase spreads from tumor cell lines, and from biopsy specimens of three patients with ES were analyzed. In the remaining eight patients comprising five ES, two small cell osteosarcomas and one chronic osteomyelitis, only nuclei preparations were available for analysis. We detected the t(11;22) in interphase nuclei of the four primitive neuroectodermal tumor cell lines, of three patients in which the karyotype demonstrated the translocation and in five cases of ES in which cytogenetic analysis had not been possible. Two cases of small cell osteosarcoma and one chronic osteomyelitis were also analyzed and were both normal with respect to the t(11;22). By analyzing cell lines and small round cell tumor samples by fluorescent in situ hybridization, we established that interphase cytogenetics is a rapid alternative to chromosomal analysis for the detection of the t(11;22) and represents an invaluable tool for the differential diagnosis of small round cell tumors.     

长骨造釉细胞瘤样尤文肉瘤临床病理观察

目的探讨长骨造釉细胞瘤样尤文肉瘤的诊断和鉴别诊断。方法应用常规组织病理和免疫组化观察1例造釉细胞瘤样尤文肉瘤并复习相关文献。结果造釉细胞瘤样尤文肉瘤与造釉细胞瘤有相似的组织学形态,keratin均（＋）,但造釉细胞瘤样尤文肉瘤vimentin、CD56、CD99和Syn也是（＋）。结论长骨造釉细胞瘤样尤文肉瘤是一种高度恶性的肿瘤,它与长骨造釉细胞瘤具有不同性质。     

Targeting Lyn inhibits tumor growth and metastasis in Ewing's sarcoma

Src family tyrosine kinases (SFK) play an important role in growth and metastasis of many types of human malignancies. However, their significance in Ewing's sarcoma remains to be elucidated. The purpose of this study was to evaluate the role of Lyn, one member of the SFK, in Ewing's sarcoma growth and metastasis and to determine whether a SFK inhibitor can induce Ewing's tumor regression. Lyn was expressed and activated in TC71, A4573, and SK-ES human Ewing's sarcoma cells. Lyn expression was seen in 13 of 15 patient tumor samples, 6 of which showed Lyn activation. Specific inhibition of Lyn using small interfering RNA significantly decreased primary tumor growth and lytic activity, and also reduced lung metastases in vivo. Down-regulation of Lyn resulted in decreased invasive capacity of tumor cells in vitro. AP23994, a small-molecule SFK inhibitor, decreased Lyn kinase activity and suppressed TC71 cell growth in vitro in a dose-dependent manner. Furthermore, treatment of mice bearing s.c. TC71 tumors with AP23994 or with polyethylenimine/Lyn-small interfering RNA gene therapy resulted in reduced Lyn kinase activity and significant tumor growth suppression. EWS/FLI-1, which is translocation fusion protein associated with Ewing's sarcoma, regulated Lyn gene expression and kinase activity. These data suggest that targeting Lyn may be a new therapeutic approach in treatment of Ewing's sarcoma.     

Usefulness of long‐distance inverse polymerase chain reaction for molecular detection of 14q32 translocation in a clinical setting

All mature B‐cell leukaemias and lymphomas have a clonal Ig gene recombination, and half of them have a reciprocal chromosomal translocation involving the 14q32 locus. The 14q32 translocation partners are variable, such as BCL‐2, BCL‐1 and BCL‐6, thus accounting for the difficulty in molecular detection by the current genomic polymerase chain reaction (PCR) method. To identify B‐cell clones efficiently with an Ig gene rearrangement and reciprocal inter‐chromosomal translocation, we verified the usefulness, in a practical laboratory setting, of our modified long‐distance inverse (LDI) PCR method for detecting IgH gene rearrangements involving inter‐ and intra‐chromosomal segments. The total run time of this LDI PCR method was 5.5?h. Using 24 samples of mature B‐cell leukaemias and lymphomas, the modified LDI PCR gave clonally rearranged amplicons in 83?% (20/24) of cases. Direct sequencing results of the amplicons revealed inter‐chromosomal translocations in 5 cases (25?%) and intra‐chromosomal rearrangements in the remaining 15 cases (75?%). The partners of the inter‐chromosomal translocation consisted of the 11q13.3 segment containing a partial BCL1 sequence in 3 cases; 18q21.3 segment containing a partial BCL2 sequence in one case; and a segment of 7q11.2 in one case. We present an LDI PCR‐based methodology for the efficient identification of 14q32 translocations, with modifications to reduce the total run time to within one day.     

Insulin-like growth factor I expression by tumors of neuroectodermal origin with the t(11;22) chromosomal translocation. A potential autocrine growth factor.

Expression of insulin-like growth factor I (IGF-I) mRNA by some tumor cell lines of neuroectodermal origin has been described. To further explore the significance of IGF-I mRNA expression in these tumors, a more extensive analysis was performed. Most (9 of 10) neuroectodermal tumor cell lines with a t(11;22) translocation (primitive neuroectodermal tumor [PNET], Ewing's sarcoma, esthesioneuroblastoma) expressed IGF-I mRNA, whereas 0 of 15 cell lines without the translocation (PNET, neuroblastoma) expressed IGF-I. Furthermore, inasmuch as all neuroblastoma (12 of 12) cell lines examined expressed IGF-II RNA, the pattern of IGF expression could distinguish between these closely related tumors. CHP-100, a PNET cell line with the t(11;22) translocation, was shown to secrete both IGF-I protein and an IGF binding protein, IGFBP-2. This cell line also expressed the type I IGF receptor mRNA, and blockade of this receptor by a monoclonal antibody (alpha IR3) inhibited serum-free growth. These data demonstrate that IGF-I expression is a property of neuroectodermal tumors with a t(11;22) translocation and that interruption of an IGF-I autocrine loop inhibits the growth of these tumor cells.     

Ewing's sarcoma: transplantation in nude rat

Eight Ewing's sarcoma, primary tumor or metastasis, have been transplanted in Nude Rats. These tumors grow slowly and only in female rats. One of them has been maintained for 13 months with 5 passages. It has conserved all the characteristics of the primary tumor, histologic and ultramicroscopic morphology, glycogen secretion and cytogenetic modification (11.22 translocation). The graft of Ewing's sarcoma to Nu/Nu rats is a valuable system to get more material in good condition to study the nature and the origin of Ewing's cells, to test the new chemotherapy trials and to prepare and test the monoclonal antibodies.     

E1A gene therapy inhibits angiogenesis in a Ewing's sarcoma animal model

We assessed vascular endothelial growth factor (VEGF) expression in four different human Ewing's sarcoma cell lines (TC71, SK-ES, RD, and A4573) and in tumors in nude mice induced following s.c. injection of TC71 cells. Three of the four cell lines (TC71, SK-ES, and A4573) expressed significantly higher levels of VEGF than did normal human osteoblasts. Transfection of the adenovirus type 5 early region 1A (E1A) gene into TC71 cells down-regulated VEGF expression in vitro. In the mice bearing TC71 cell tumors, intratumoral injections of an adenoviral vector containing the E1A gene (Ad-E1A) decreased VEGF expression, inhibited tumor growth, and increased the survival rates in comparison with the mice given injections of PBS or an adenoviral vector containing beta-galactosidase (Ad-beta-gal). E1A gene therapy also significantly reduced blood vessel density and induced cell apoptosis in the tumors. These results demonstrate that E1A gene therapy inhibits angiogenesis, most likely by suppression of VEGF expression. Thus, E1A gene therapy may be a new therapeutic approach for Ewing's sarcoma.     

Burkitt's lymphoma cell line bearing surface IgA and negative for nuclear antigen of Epstein-Barr virus (EBNA)

A new continuous cell line, TL-1, was established in tissue culture from Burkitt's lymphoma arising at the ileocecal region. It has been maintained for the past two years. The cells were small and round, and grew in a suspension culture. Population doubling time was approximately 36 hours, The cells revealed the chromosomal abnormality i.e. translocation between 8 and 14 chromosomes, which was reported on some Burkitt's lymphomas. This line was negative for nuclear antigen of EB virus (EBNA) and bore surface IgA and not any receptors. Inoculums of 10(7) cells produced tumors in the athymic nude mice. The histological appearance of the tumor was consistent with the Burkitt's lymphoma. Ultrastructural findings showed nuclear blebs and clefts. As far as we know, this was the first cell line of Burkitt's lymphoma with t(8q-; 14q+) in Japan and perhaps the first lymphoma cell line with surface IgA.     

Basic fibroblast growth factor as a growth inhibitor for cultured human tumor cells.

Basic fibroblast growth factor (bFGF) stimulates the proliferation of many cells and it is found in a wide variety of normal or transformed tissues. As demonstrated here, bFGF is also present in cultured human Ewing's sarcoma cells. Unexpectedly, however, bFGF isolated from these cells inhibits their own proliferation, indicating that bFGF can act as an endogenous (autocrine) growth inhibitor for cultured Ewing's sarcoma cells. Since bFGF also inhibits the proliferation of some further tumor cells, but stimulates that of others, it can be considered a bifunctional regulator of tumor cell proliferation. The autocrine growth-inhibitory effect of bFGF in Ewing's sarcoma cells may explain the low mitotic activity of Ewing's sarcomas.     

一个上皮样肉瘤细胞株的分子细胞遗传学研究

对建成的一个上皮样肉瘤(ES)细胞株进行了染色体分析和DNA印迹实验研究。结果表明,该瘤株染色体众数45,丢失1个22号染色体;结构异常为del(3)(q21.1)以及5、6、9和12号衍生染色体。用定位于人染色体3q13.1-q22的pPCC41A2克隆作为DNA标识探针,在该瘤株DNA中检出PCCB基因的部分缺失。本研究结果提示del(3)(q21.1)及PCCB基因的改变可能与ES癌变有关。     

Establishment of a sarcoma cell line, MS-K, expressing Ki-ras proto-oncogene product from mouse bone marrow stromal cells

A sarcoma cell line, MS-K, was established from a long-term culture of mouse bone marrow stromal cells. When inoculated into syngeneic 3HC/HeNS1c mice, the cells formed large necrosis-free tumors, but there were no apparent changes in hematological features or in general conditions of tumor-bearing mice. The tumor had a fibroblastic appearance, was well vasculated and differentiated into adipocytes at the peripheral region. Immunohistochemical studies revealed that the cells were positive for vimentin and S-100 protein, indicating that the cells were of lipoblast origin. A significant amount of fat-deposition was induced in the cytoplasm of the cells when MS-K cells were cultured in the presence of hydrocortisone and insulin. Antibody-staining for oncogene products showed that the cells were negative for c-fos but strongly positive for Ki-ras. MS-K cells did not adhere hemopoietic stem cells or support their proliferation, which contrasts with previously established MS-1, which is a hemopoietic-supportive and stem cell-adherent lipoblast cell line. These properties of MS-K and MS-1 should be useful in the identification of the surface structures for stem cell anchorage.     

Clonal expansion in follicular lymphoma occurs subsequent to antigenic selection

The genesis of human follicular lymphoma (FL) is a multistep process. The initial event is thought to be the chromosomal translocation t(14;18)(q32;q21) juxtaposing the bcl-2 proto-oncogene with the immunoglobulin (Ig) H chain locus joining segment (JH) as an error of D-J or V-D joining in the pre-B cell. However, FL is recognized clinically as a tumor of surface Ig (sIg)-positive B cells with morphologic and phenotypic similarities to the centrocyte of the secondary immune response. Thus, additional steps must be involved in the clonal expansion of the FL tumor cell beyond the activation of bcl-2 as a consequence of the t(14;18) translocation. Like the normal centrocyte, somatic mutations accumulate in the variable (V) genes of FL tumor B cells. To determine if clonal expansion of FL occurs before or after the development of the malignant follicle, we sought to examine the evolution of the FL V gene from its unmutated germline (GL) counterpart. To obtain the GL gene we first cloned the productively rearranged V gene of patient MT FL and obtained the clone rMTF. A hybridization probe derived from the 2.1-kb region upstream of the V gene in clone rMTF identified a single band in Southern blot hybridization of GL DNA. This probe was used to screen a size-selected library, and candidate GL V genes were isolated. Two identical clones, MTGL1 and 2, proved to have upstream regions (USRs) that were colinear with the USR of the rMTF. Thus, the MTGL clones represent the unmutated GL V genes, which were productively rearranged in the MT FL. Comparison of the GL V gene sequence to a consensus of MT FL V gene sequences revealed 42 mutations, demonstrating that malignant clonal expansion occurred subsequent to the activation of somatic mutation, presumably in the malignant follicle. Furthermore, the individual FL V gene sequences segregated into two distinct patterns of mutation. The major population represented 71% of the clones, and the minor population 29%. To investigate possible mechanisms for the parallel selection of distinct tumor cell populations, we analyzed the pattern of silent and replacement mutations within the V gene sequences. We found that in the framework regions (FRs) of both populations there were significantly fewer replacement changes than expected, suggesting that negative selective pressure was maintaining the structural integrity of the sIg. In contrast, the complementarity determining regions (CDRs), which make up the antigen binding domain of Ig, had an excess of replacement changes, suggesting positive selection for altered ligand binding.     

低度恶性纤维黏液样肉瘤2例报告及文献复习

目的 探讨低度恶性纤维黏液样肉瘤（LGFS）的临床病理特点、诊断和预后。方法 对2例LGFS进行光镜、电镜、组化和免疫组化观察并结合文献进行分析。结果 肿瘤由纤维性区和黏液样区混合构成,二者呈交错相间排列;纤维性区瘤细胞呈旋涡状排列;黏液样区瘤细胞散在;细胞核轻度异型;PAS染色黏液样区和肿瘤细胞浆内均为阴性,免疫组化标记vimentin、NSE阳性。电镜观察瘤细胞呈平细胞与肌纤维母细胞的特点。结论 LGFS是一种来源于纤维母细胞独立存在进展缓慢的低度恶性软组织肉瘤,易误诊为良性。病理诊断依赖于组织学、免疫组化和电镜观察,电镜在该瘤的诊断和鉴别诊断中具有重要价值。     

Characterization of a novel myeloma cell line, MM.1

A myeloma cell line, MM.1, has been established from the peripheral blood cells of a patient with immunoglobulin A myeloma. MM.1 grows in suspension either singly or in small clusters and secretes lambda-light chain. Phenotypically, MM.1 cells lack most B cell antigens, but they do express human leukocyte antigen DR, PCA-1, and T9 and T10 antigens. Molecular analysis of MM.1 demonstrates that it is negative for the presence of the Epstein-Barr virus genome. Southern analysis of MM.1 detected a rearrangement of the lambda-light chain gene, and Northern analysis revealed high levels of lambda gene expression. Cytogenetic analysis of the MM.1 cell line revealed the presence of seven related chromosomally abnormal cell lines characterized by numerical and structural aberrations, and it revealed five nonclonal abnormal cells. The most notable abnormality is a reciprocal translocation involving band q24.3 of chromosome 12 and band q32.3 of chromosome 14; translocations involving 14q32 are frequently observed in neoplasms of B cell origin.     

滑膜肉瘤32例临床病理分析

目的 探讨滑膜肉瘤的形态特点和鉴别诊断。方法 收集原诊断为滑膜肉瘤的病例66例、恶性周围神经鞘膜瘤10例、纤维肉瘤6例、平滑肌肉瘤13例，进行形态学观察，并采用10种抗体做免疫组化染色。结果 66例原诊滑膜肉瘤的病例中，最终确诊为滑膜肉瘤者27例，诊断准确率40．9％。最易误诊为滑膜肉瘤的肿瘤是恶性周围神经鞘膜瘤，占31．8％。5例滑膜肉瘤最初误诊为其他梭形细胞肉瘤。组织学双向分化是其主要特点，分单相纤维型，双相型和低分化型，未见单相上皮型病例。免疫组化瘤细胞表达CK和／或EMA，同时vimentin阳性，S－100和PGP9.5也有一定阳性率。bcl-2蛋白阳性率高，多为弥漫性。其他几种梭形细胞肉瘤不表达CK和EMA，bcl-2蛋白阳性率低或局灶阳性。结论 滑膜肉瘤是一种容易误诊的软组织肉瘤，免疫组化同时表达上皮性和间叶性标记。最易误诊的肿瘤是恶性周围神经鞘膜瘤。bcl-2蛋白检测对滑膜肉瘤鉴别诊断有一定帮助。     

脊柱尤文肉瘤的临床及病理特征和MRI表现

目的探讨脊柱尤文肉瘤的临床、病理及MRI特点。方法回顾性分析9例经手术病理或穿刺活检证实的脊柱尤文肉瘤的临床、病理特征及MRI表现。结果9例脊柱尤文肉瘤中,发生于颈椎、胸椎和腰椎各2例,骶椎3例。6例进行了MRI检查,均显示椎体骨骼信号异常,在T1WI上4例表现为中等信号,2例表现为稍低信号;在T2WI上表现为稍高信号,钆喷替酸葡甲胺（Gd-DTPA）增强后不均匀强化。3例表现为溶骨性骨质破坏、椎体压缩变扁;3例表现为椎体轻度膨胀性溶骨性破坏。相邻的椎间隙无狭窄、椎间盘信号无异常;均见周围明显的软组织肿块,增强后明显强化。HE染色并镜下观察肿瘤细胞均呈小圆形,不伴有Homer-Wright（H-W）菊形团排列;PAS染色阳性6例,NSE弱阳性2例。结论脊柱原发性尤文肉瘤的MRI表现有一定的特点,但缺乏特异性。当患者为儿童或青少年,且影像学表现为溶骨性骨质破坏伴明显的软组织肿块时需考虑尤文肉瘤的可能。