Microsatellite Instability in Chicken Lymphoma Induced by Gallid Herpesvirus 2

Microsatellite instability (MSI) has been found in a range of human tumors, and little is known of the links between MSI and herpesvirus. In order to investigate the relationship between MSI and Gallid herpesvirus 2 (GaHV-2)-induced lymphoma, fifteen Marek’s disease (MD) lymphomas were analyzed through using 46 microsatellite markers, which were amplified by PCR from DNA specimens of lymphoma and normal muscular tissues from the same chicken. PCR products were evaluated by denaturing polyacrylamide gel electrophoresis for MSI analysis. MSI was proved in all lymphomas, at least in one locus. Thirty of the 46 microsatellite markers had microsatellite alterations. These results suggested that GaHV-2-induced lymphoma in chickens is related to MSI, and this is the first report to demonstrate that MSI is associated with the GaHV-2 induced lymphoma in chicken.     

Identification of Marek''s disease virus nuclear antigen in latently infected lymphoblastoid cells.

A new Marek's disease virus (MDV) nuclear antigen (MDNA) was identified in two MDV-transformed T-lymphoblastoid cell lines, MKT-1 and MSB-1, derived from chickens bearing tumors induced by MDV. This MDNA was not detected in MSB-1 cells maintained in iododeoxyuridine, which activates the latent MDV genome. Moreover, it was not found in chicken embryo fibroblasts undergoing productive and cytolytic infection with MDV. Expression of MDNA is not related to strain pathogenicity in chickens, because chicken embryo fibroblasts productively infected with the pathogenic RBIB strain or the nonpathogenic CV-1 strain of MDV did not express this antigen. DNA-protein immunoprecipitation studies revealed that MDNA bound to two sites in the 190,00-base-pair (bp) MDV genome. One of these loci identified by MDNA obtained from MKT-1 and MSB-1 cells corresponded to a 476-bp segment within the short unique region of BamHI-A MDV DNA. A second locus located in a 280-bp segment within the short inverted repeat region of BamHI-A was also identified by MDNA from MSB-1 cells but not by MDNA obtained from MKT-1 cells. Analyses of the nucleotide sequence by DNase digestion showed that MDNA protected a 60-bp segment spanning a 22-bp palindromic sequence of the short unique region and a 103-bp sequence encompassing a 32-bp palindrome in the short inverted repeat region of BamHI-A MDV DNA.     

Deoxyribonucleic Acid of Marek''s Disease Virus in Virus-Induced Tumors

DNA was extracted from [(3)H]thymidine-labeled Marek's disease virus (MDV) and purified by two cycles of CsCl gradient centrifugation in a fixed-angle rotor. The DNA was transcribed in vitro into (32)P-labeled complementary RNA (cRNA). MDV cRNA did not hybridize with DNA from chicken embryo fibroblast cultures or from chicken spleen, but hybridized efficiently with DNA from MDV particles or MDV-infected cell cultures. Five Marek's disease tumors from different chickens and different organs (ovary, liver, testis) were all found to contain MDV DNA sequences. The relative amount of MDV DNA varied from tumor to tumor and was between 3 and 15 virus genome equivalents per cell. The content of virus DNA per cell in spleens from tumor-bearing chickens was much lower than in tumors from the same animals. MDV-infected cell cultures contained a large proportion (28-59%) of virus antigen-positive cells, as measured by immunofluorescence, but tumor cells were negative in this respect (<0.02% positive cells). These data indicate that MDV is present in a provirus form in tumor cells.     

Persistence of genomes of both herpesvirus of turkeys and Marek's disease virus in a chicken T-lymphoblastoid cell line

A cell line tentatively designated as MDCC-BO1(T), was established from spleen cells of an apparently healthy chicken inoculated with herpesvirus of turkey (HVT). BO1(T) cells were T lymphoblastoid cells and the more than 95% of them had Marek's disease (MD) tumor-associated surface antigen (MATSA). However, no viral internal antigens or membrane antigens could be demonstrated in them by immunofluorescence tests using chicken anti-HVT and -MD virus (MDV) sera. The virus could be rescued from BO1(T) cells by co-cultivation with chick embryo fibroblasts (CEF). The DNA of the rescued virus was characterized as HVT DNA by its sedimentation profile in a neutral glycerol gradient and its endonuclease Hind III cleavage-pattern. Ultrastructural studies on CEF infected with the rescued virus revealed the presence of HVT-like virions. However, DNA-DNA reassociation kinetics showed that the BO1(T) cells contained a few copies of NVT and also MDV genomes.     

应用荧光标记多重PCR对原发性胃癌进行微卫星不稳定性检测

为探讨 19号染色体微卫星不稳定性 (microsatelliteinstability ,MSI)在原发性胃癌发生中的作用 ,选择覆盖 19号染色体的 2 6个高分辨 (<5cM)微卫星标记 ,采用FAM、TET和HEX3种不同颜色的荧光染料标记微卫星引物 ,应用荧光标记多重PCR对 44例原发性胃癌及其正常对照组织进行扩增 ,产物在ABI3 77测序仪上经变性聚丙烯酰胺凝胶电泳 ,通过GeneScanTM及GenotyperTM 软件进行图像收集和MSI分析。同时采用PCR SSLP 银染技术检测了其中4个位点的MSI状态。 2 6个微卫星位点中 ,2 2个 (84 62 % )存在MSI,以D19S40 2MSI发生频率最高 (2 2 73 % ) ,平均MSI频率为 8 3 %。 44例胃癌中 ,3 4例 (77 2 7% )在一个或一个以上位点出现MSI。MSI作为胃粘膜癌变过程中一种可能的分子病理学机制 ,可作为胃癌分子检测的指标之一     

Latent Marek's disease virus can be activated from its chromosomally integrated state in herpesvirus-transformed lymphoma cells.

Marek's disease virus (MDV), a lymphotropic herpesvirus, induces T-cell lymphomas in chicken, its natural host. The lymphoma cells are latently infected with MDV but the viral contribution to the transformed phenotype is not understood. To investigate the virus-cell interaction, we focused on the status of MDV in the transformed cells. By the use of highly sensitive fluorescent in situ hybridization with metaphase chromosomes, we found (i) MDV DNA to be randomly integrated at multiple sites in the chromosomes of primary lymphoma cells from chicken tissues; (ii) extrachromosomal, circular MDV genomes were absent and linear virion DNA was usually not detectable in the latently infected lymphoma cells; (iii) the pattern of integration sites revealed the clonal origin of the tumour cells; which (iv) was retained in in vitro established cell lines derived from primary lymphomas; (v) activation of the lytic phase of MDV's life cycle occurred in vitro suggesting that MDV can escape from its integrated status by an unknown mechanism.     

High frequency of microsatellite instability in sporadic colorectal cancer patients in Iran

Microsatellite instability in sporadic colorectal cancer patients was assessed, and the clinicopathological associations were evaluated in northeastern Iran, which is a high-risk region for gastrointestinal malignancies. Microsatellite instability (MSI) status of tumoral tissue, compared to normal tissue, was assessed with a standard panel of MSI markers on paraffin-embedded surgically resected tissues from 67 consecutive sporadic colorectal cancer patients. Eleven of the patients were under 40 years old. Female patients were significantly younger than male patients (mean age 54.2 vs 62.1 years, P = 0.020). MSI analysis revealed 18 cases of MSI-H (26.9%), 11 MSI-L (16.4%) and 38 MSS (microsatellite stable tumors; 56.7%). While a greater proportion of patients consisted of males, 56.7 vs 43.3% females, MSI-H was more frequent in females (34.5 vs 21.5%). MSI was associated with proximal location of tumor (P = 0.003) and lower stages of tumor (P = 0.002), while MSS tumors were associated with node metastasis. MSI has a higher frequency in sporadic colorectal cancer patients, suggesting that molecular epidemiology of the genetic alterations involved in colorectal cancer carcinogenesis has a different pattern in the Iranian population, which deserves further epidemiological attention. The high frequency of MSI-H in this population suggests that we should look at microsatellite instability prior to chemotherapy to determine the most appropriate chemotherapeutic strategy in our population.     

Microsatellite instability typing in serum and tissue of patients with colorectal cancer: comparing real time PCR with hybridization probe and high-performance liquid chromatography

Allelic variation of BAT-25 (a 25-repeat quasimonomorphic poly T) and BAT-26 (a 26-repeat quasimonomorphic polyA) loci as two mononucleotide microsatellite markers, were analyzed with high-performance liquid chromatography (HPLC) compared with Real-Time PCR using hybridization probes. BAT-26 and BAT-25 markers were used to determine an appropriate screening technique with high sensitivity and specificity to diagnose microsatellite instability (MSI) status in patients with colorectal cancer (CRC). One of the pathways in colorectal tumor genesis is microsatellite instability (MSI⁺). MSI is detected in about 15 % of all CRCs; 3 % are of these are associated with Lynch syndrome and the other 12 % are caused by sporadic. Colorectal tumors with MSI have distinctive features compared with microsatellite stable tumors. Due to the high percentage of MSI⁺ CRC in Iran, screening of this type of CRC is imperative. Two markers were analyzed in tissues and sera of 44 normal volunteers and tumor and matched normal mucosal tissues as well as sera of 44 patients with sporadic CRC. The sensitivity and specificity of BAT-26 with real time PCR method (Hybridization probe) were 100 % in comparison with sequencing method as the gold standard, while HPLC had a lower sensitivity and specificity. According to HPLC data, BAT-26 was more sensitive than BAT-25 in identifying MSI tumors. Therefore, MSI typing using the BAT-26 hybridization probe method compared to HPLC could be considered as an accurate method for diagnosing MSI in CRC tumors but not in serum circulating DNAs.     

Combined p53/Bax mutation results in extremely poor prognosis in gastric carcinoma with low microsatellite instability

Gastric cancer is highly refractory to DNA-damaging therapies. We therefore studied both gene mutation and protein expression of p53 and Bax in a cohort of 116 patients with gastric cancer who underwent R0-resection with a curative intent. Bax mutation was independent from severe microsatellite instability (MSI), that is, global mismatch repair deficiency as determined by analysis of BAT-25/BAT-26 microsatellite markers. Thus, Bax-frameshift mutation is a feature of tumors with low MSI. In contrast and as expected, no p53 mutations were observed in the microsatellite instable tumors. p53 Mutation or p53 overexpression did not have an impact on disease prognosis. p53-Inactivation was, however, associated with an extremely poor prognosis in the subgroup of patients with Bax-mutated tumors. Thus, we show for the first time that the combined mutation of p53 and Bax, two key regulators of the mitochondrial apoptosis pathway, results in an extremely aggressive tumor biology and poor clinical prognosis.     

结直肠腺瘤中的微卫星不稳定性类型

应用微切割 聚合酶链反应 单链长度多态性 (PCR SSLP)的方法 ,检测 1 6个微卫星位点在 5 9例 6 2个结直肠腺瘤标本的微卫星不稳定性状态 .结果表明 :腺瘤 1 6个位点的总微卫星不稳定性(microsatelliteinstability ,MSI)发生率为 1 4 4 % ,MSI H所占的比率为 9 7% ;在 1 0例可以同时微切割得到腺瘤和癌变成分的病例中 ,腺瘤和癌变成分在每个微卫星位点的改变情况不完全相同 ,并且当在某一位点同时表现为阳性时 ,部分凝胶电泳的图像相同 ,而部分不同 ;在某些位点表现为癌变成分的异常条带泳动速度更快 ,说明序列比腺瘤中更短 ;MSI H与病人的年龄、性别、腺瘤发生部位和病理学亚型之间未见统计学差异 ,但MSI H组的平均年龄 (5 6 5 0± 1 1 38)低于MSI L组 (6 0 36±1 1 34) ,女性所占比率 (5 6 )明显高于男性 ,6例MSI H中无 1例组织学类型为管状腺瘤 ;各位点在MSI H组的MSI改变率明显高于MSI L组 ,在TGFβRⅡ (A) 1 0 、hMSH6、TCF4、BAT2 6等位点有明显差异 (P <0 0 5 ,其中BAT2 6的P <0 0 1 ) .可以推断 :在结直肠癌发生发展的早期即腺瘤阶段即可表现微卫星不稳定性 ;微卫星不稳定性可以随结直肠肿瘤的发展过程而发展 ,并且特定的微卫星位点的改变可能仅发生于肿瘤进程的特定阶段 ;在结直肠癌     

食管鳞癌MLH1基因改变及其与微卫星不稳定的关系

MLH1是位于3p21．3上的一个DNA错配修复基因,其异常与多种肿瘤相关。为探索食管鳞癌中MLH1基因改变及其与微卫星不稳定(MSI)的关联情况,采用微卫星分析和RT—PCR方法检测了14个微卫星标志在食管癌中的状况及MLH1转录水平的表达,发现35％的食管癌出现至少一个微卫星的不稳定,66．7％的肿瘤在MLH1基因内标志D3S1611位点表现为杂合性丢失,但是MLH1没有明显的mRNA表达下调。MSI与食管癌分期、分级、淋巴结转移、患者年龄和性别等参数及MLH1基因杂合性丢失(LOH)之间无统计学意义的相关性。这些结果表明：食管癌中MLH1存在较高频率的等位基因丢失,但其mRNA表达水平并无明显异常;所测微卫星标志的不稳定是食管癌的频发事件,与MLH1基因LOH不存在必然联系。     

Retention of oncogenicity by a Marek's disease virus mutant lacking six unique short region genes.

We previously reported the construction of Marek's disease virus (MDV) strains having mutations in various genes that map to the unique short (US) region of the viral genome (J.L. Cantello, A.S. Anderson, A. Francesconi, and R.W. Morgan, J. Virol. 65:1584-1588, 1991; M.S. Parcells, A.S. Anderson, and R.W. Morgan, Virus Genes 9:5-13, 1994; M.S. Parcells, A.S. Anderson, and R.W. Morgan, J. Virol. 68:8239-8253, 1994). These strains were constructed by using a high-passage-level serotype 1 MDV strain which grew well in chicken embryo fibroblasts. Despite the growth of the parent and mutant viruses in cell culture, in vivo studies were limited by poor growth of these strains in chickens. One of the mutants studied lacked 4.5 kbp of US region DNA and contained the lacZ gene of Escherichia coli inserted at the site of the deletion. The deletion removed MDV homologs to the US1, US2, and US10 genes of herpes simplex virus type 1 as well as three MDV-specific open reading frames. We now report the construction of a mutant MDV containing a similar deletion in the US region of the highly oncogenic RB1B strain. This mutant, RB1B delta 4.5lac, had a growth impairment in established chicken embryo fibroblasts similar to that described previously for MDVs lacking a functional US1 gene. In chickens, RB1B delta 4.5lac showed decreased early cytolytic infection, mortality, tumor incidence, and horizontal transmission. Several lymphoblastoid cell lines were established from RB1B delta 4.5lac-induced tumors, and virus reactivated from these cell lines was LacZ+. These results indicate that the deleted genes are nonessential for the transformation of chicken T cells or for the establishment and maintenance of latency. On the basis of the growth impairment observed for RB1B delta 4.5lac in cell culture and in vivo, we conclude that deletion of these genes affects the lytic replication of MDV. This is the first MDV mutant constructed in the RB1B oncogenic strain, and the methodology described herein provides for the direct examination of MDV-encoded determinants of oncogenicity.     

Analysis of genome instability in breast cancer

Breast cancer is a heterogeneous disease, previously associated with genomic instability. Our aim was to analyze microsatellite markers in order to determine patterns and levels of instability, as well as possible correlations with histopathological parameters. Polymerase chain reaction was used to characterize microsatellite instability (MSI) and loss of heterozygosity (LOH) in 107 breast carcinomas at twelve microsatellite loci. Some of the markers were selected because of their relation to steroid hormone metabolism, which seems to be related to sporadic breast cancer risk. D5S346 and D17S250 markers showed a statistically significant frequency of MSI. LOH in D3S1611, D17S250, AR and ER-β were associated with some parameters of worse prognosis. Marker group analysis showed that CYP19, AR and ER-β were related to histological grade III, ER-negative and PR-negative cases. Our results suggest that marker group analysis may be preferred to the single marker strategy, being predictive of worst prognosis when single markers are unable to provide such information. A further evaluation of steroid metabolism genes and their association with low penetrance genes in breast cancer may be useful.     

Macrophage restriction of Marek's disease virus replication and lymphoma cell proliferation.

Macrophages are shown to restrict the replication of Marek's disease virus (MDV) and isotope uptake by spleen cells from chickens bearing Marek's disease (MD) tumors. The titer of virus from duck embryo fibroblasts (DEF) co-cultivation with MDV-spleen cells pretreated to deplete marcophages was 4- to 18-fold higher than with untreated cells. Treated MDV-spleen cells increased isotope uptake by 2-fold. These restrictive activities are attributable to macrophage regulation of cell proliferation.     

EMAST Is Associated with a Poor Prognosis in Microsatellite Instable Metastatic Colorectal Cancer

Purpose

To determine the frequency and prognostic value of elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) in metastatic colorectal cancer (mCRC) patients in relation to microsatellite instability (MSI) status and MSH3 protein expression.

Material and Methods

The frequency of EMAST was evaluated in mCRC patients with MSI tumors and microsatellite stable (MSS) tumors. A literature overview was performed to compare the frequency of EMAST in our study with existing data. Immunohistochemistry for MSH3 was compared with EMAST status. Outcome was studied in terms of overall survival (OS) of mCRC patients with MSI and MSS tumors.

Results

EMAST was evaluated in 89 patients with MSI tumors (including 39 patients with Lynch syndrome) and 94 patients with MSS tumors. EMAST was observed in 45.9% (84 out of 183) of patients, with an increased frequency in MSI tumors (79.8% versus 13.8%, p < 0.001). We found no correlation between EMAST and MSH3 protein expression. There was no effect of EMAST on prognosis in patients with MSS tumors, but patients with MSI / non-EMAST tumors had a significantly better prognosis than patients with MSI / EMAST tumors (OS: HR 3.22, 95% CI 1.25-8.30).

Conclusion

Frequency of EMAST was increased in mCRC patients with MSI tumors, compared to MSS tumors. Our data suggest that the presence of EMAST correlates with worse OS in these patients. There was no effect of EMAST on the prognosis of patients with MSS tumors. A limitation of our study is the small number of patients in our subgroup analysis.     

Transformation of chicken embryo fibroblasts and tumor induction by the middle T antigen of polyomavirus carried in an avian retroviral vector.

The middle T antigen of polyomavirus transformed primary chicken embryo fibroblasts when expressed from a replication-competent avian retrovirus. This in vitro-constructed retrovirus, SRMT1, is a variant of Rous sarcoma virus that encodes the middle T antigen in place of v-src. Inoculation of SRMT1 into 1-week-old chickens rapidly induced hemangiomas and hemangiosarcomas. As shown with mammalian cells infected with polyomavirus, polyomavirus middle T antigen appears to be associated with p60c-src in chicken cells infected with SRMT1. When lysates of SRMT1-infected cells immunoprecipitated with either a monoclonal antibody against p60src or anti-T serum were assayed in an in vitro kinase reaction, the middle T antigen was heavily phosphorylated. To see whether an excess of p60c-src could alter the extent of phosphorylation of the middle T protein or the process of cell transformation by middle T, cells were doubly infected with SRMT1 and NY501, a virus which overexpresses p60c-src. Doubly infected chicken embryo fibroblasts transformed with the same kinetics and were morphologically indistinguishable from chicken embryo fibroblasts infected with SRMT1 alone. Phosphorylation of the middle T antigen was elevated two- to fivefold relative to cells infected only with SRMT1.     

Induction of host gene expression following infection of chicken embryo fibroblasts with oncogenic Marek's disease virus

Microarrays containing 1,126 nonredundant cDNAs selected from a chicken activated T-cell expressed sequence tag database (http://chickest.udel.edu) were used to examine changes in host cell gene expression that accompany infection of chicken embryo fibroblasts (CEF) with Marek's disease virus (MDV). Host genes that were reproducibly induced by infection of CEF with the oncogenic RB1B strain of MDV included macrophage inflammatory protein, interferon response factor 1, interferon-inducible protein, quiescence-specific protein, thymic shared antigen 1, major histocompatibility complex (MHC) class I, MHC class II, beta(2)-microglobulin, clusterin, interleukin-13 receptor alpha chain, ovotransferrin, a serine/threonine kinase, and avian leukosis virus subgroup J glycoprotein.     

Clinicopathologic features in colorectal cancer patients with microsatellite instability

The microsatellite instability (MSI) mutational pathway is critical to carcinogenesis in a small but significant proportion of colorectal cancers. While MSI is identified in most cancers in individuals with hereditary non-polyposis colorectal cancer, the majority of MSI tumors are found in individuals with sporadic disease. Colorectal cancers arising as a result of MSI have distinct clinicopathologic features distinguishing them from those with microsatellite stability. MSI colorectal cancers affect a larger percentage of women, are usually localized proximal to the splenic flexure, and have a higher incidence of synchronous and metachronous tumors. They are associated with a mucinous histology, tumor-infiltrating lymphocytes, a Crohn's-like inflammatory response, and a higher grade but lower stage. Overall survival is better in individuals with MSI. The benefit of chemotherapy in MSI colorectal cancers, with and without lymph node metastases, remains unclear.