髓母细胞瘤10号染色体的杂合性丢失分析

目的探讨髓母细胞瘤的遗传学异常及其发病机制。方法通过微卫星分析（microsatellite analysis）方法,应用7个分别位于10号染色体长臂上PTEN（10q23）和DMBT1（10q25）基因位点的特异性标记物,分析18例髓母细胞瘤的杂合性丢失（loss of heterozygosity,LOH）。结果18例髓母细胞瘤中,位于10q23上的LOH比率为24％（9／37可分析标记）;位于10q25上的LOH比率为47％（9／19可分析标记）。结论在髓母细胞瘤中,染色体10q25上高比率的杂合性丢失提示,位于该位点上DMBT1基因的遗传学改变可能在髓母细胞瘤的发病机制中起着重要的作用。     

胶质母细胞瘤14号染色体杂合性丢失的初步研究

目的 寻找胶质母细胞瘤（glioblastoma,GBM)14号染色体上可能存在肿瘤抑制基因的杂合性丢失（loss of heterozygosity,LOH)aqfa,为发现和定位肿瘤抑制基因提供线索和依据。方法 应用聚合酶链反应方法,采用荧光标记引物和377型DNA序列自动分析仪,分析了20例GBM患者14号染色体上14个微卫星多态性标记的LOH。结果 在50％（10／20）GBM患者的14号染色体上观察到LOH,在38．2％（81／212）可提供信息位点存在LOH。14p和14q的LOH率分别为32％（6／19）、50％（10／20）。在位于14q31-32．3的D14S65位点、14q21-24.1的D14S63－D14S74位点间区域检测到了较高LOH率,分别为57．1％、46．7％－47．1％。在所测位点均未检测到微卫星不稳定（microsatellite instability,MI)。结论 染色体14q上等位基因的丢失可能在GBM分子水平发病机理中起着重要作用,14q31-32.3的D14S65位点、14q21-24.1的D14S63－D14S74位点间区域可能存在与GBM相关的肿瘤抑制基因。     

慢性粒细胞白血病8p22杂合性缺失的检测及意义

目的探讨8号染色体短臂（8p22）的杂合性缺失（LOH）在慢性粒细胞白血病（CML）发生和演变中的作用。方法采用多聚酶链反应（PCR）扩增、聚丙烯酰胺凝胶电泳和硝酸银染色技术,检测8p22上D8S511和D8S258位点的LOH。结果22例慢性期和8例加速期CML病人在8p22D8S511和D8S258位点均未发生LOH。4例急变期CML病人在D8S511位点上均未检出LOH,仅1例在D8S258位点发生LOH。5例正常对照组均未发生LOH。结论8p22上D8S511位点的LOH与CML各期的发生和演变无关;D8S258位点的LOH与CML慢性期和加速期的发生和演变无关．但可能与CML急变有关。     

人胶质母细胞瘤等位基因谱分析的研究

目的 探讨胶质母细胞瘤（glioblastoma,GBM)发病的分子遗传学机理，确定GBM的发生发展主要和哪些染色体或染色体区域有关，哪些染色体区域上可能存在与GBM相关的肿瘤抑制基因（tumor suppressor gene,TSG)。方法 应用聚合酶链反应技术，采用荧光标记的引物和377型DNA序列自动分析仪，对21例GBM的所有22对常染色体上共计382个微卫星位点进行了杂合性丢失（loss of heterozygosity,LOH)分析，相邻2个微卫星位点之间的平均遗传学距离为10cM。结果 在所有被检测的染色体臂上都观察到LOH，其中以染色体10q、10p、9p、17p和13q的LOH率最高（＞50％），这些染色体臂上已知的肿瘤抑制基因PTEN、DMBT1、p16、p53和Rb所在区域LOH率都较高；14q、3q、22q、11p、9q、19q上也存在较高的LOH率（＞40．5％）；首次发现多个共同微小丢失区域：9p22-23、10p12.2-14、10q21.3、13q12.1-14.1、13q14.3-31、17p11.2-12、17p13、3q24-27、11p12-13、14q31-32.3、14q21-24.1、22q13.2-13.3、4q35、4q31.1-31.2、6qtel、6q16.3。结论 GBM存在复杂的遗传学异常，涉及多条染色体臂。以10q、10p、9p、17p和13q的异常与GBM发生发展的关系最为密切。除了已知的肿瘤抑制基因PTEN、DMBT1、p16、p15、p53和Rb外，首次所发现的多个微小共同丢失区域上可能存在GBM相关的多个未知TSG。     

复发前后多形性胶质母细胞瘤的等位基因谱分析

目的：研究多形性胶质母细胞瘤（glioblastoma multiforme,GBM)复发前后的分子遗传学变化，了解基因组范围内哪些染色体区域可能与GBM复发有关。方法：应用聚合酶链反应技术为基础的杂合性丢失（loss of heterozygosity,LOH)分析法，采用荧光标记的引物和377型DNA序列自动分析仪，检测了1例复发前后GBM所有22对常染色体上多达382个微卫星位点。相邻2个微卫星位点之间的平均距离为10cM。结果：对原发肿瘤标本的等位基因谱分析显示，染色体9p21上D9S157位点和10q21.3-26.3上D10S537、D10S185、D10S192、D10S597、D10S587、D10S217位点存在LOH。对复发肿瘤标本的研究显示，不但9p21和10q21.3-26.3上LOH的范围扩大，而且在其它多条染色体上也出现了LOH（包括1q,7p,7q,21q,20p,20q,10p,19p,19q)。结论：染色体9p和10q可能在该例GBM的发生中起着重要作用。尽管该病例复发前后的病理诊断相同，复发后GBM存在着更广泛的分子遗传学异常改变，可能伴随着更多肿瘤抑制基因的失活。     

应用基因组扫描对胃癌19号染色体进行杂合性缺失分析

目的 检测胃癌患者19号染色体微卫星位点的杂合性缺失（less of heterozygosity,LOH),以初步确定19号染色体上与胃癌相关基因连锁最密切的微卫星多态位点。方法 应用多重PCR对44例原发性胃癌患者中覆盖19号染色体上的22个微卫星位点（遗传距离在1．1－10．9cM之间）进行扩增。聚丙烯酰胺凝胶电泳分离PCR产物,Gene Scan^TM,Genotype^TM软件进行分析。结果 19个位点（19／22）检测出LOH阳性,总LOH频率为59％（26／44）,其中D19S571位点的LOH频率最高（21．43％）。结论 高频的LOH位点附近,可能存在未知的胃癌相关基因。     

脆性组氨酸三联体(FHIT)基因

人类第3号染色体上的基因座在不同的肿瘤组织中出现高频率的杂合性丢失(LOH),提示该部位可能是抑癌基因潜伏的位点。最近克隆的脆性组氨酸三联体基因(fragile histidine triad,FHIT)可能是定位于染色体3p14.2的一个抑癌基因,该基因在肿瘤组织中广泛地缺失为研究肿瘤发生机制提供了新线索。     

头颈部鳞癌的微卫星不稳定性和杂合性丢失的初步研究

目的:探讨头颈部鳞癌的微卫星不稳定性(MSI)及杂合性丢失(LOH)。方法:选择来自3、5、6、8、9、13、17和18号染色体的15个微卫星标志对36例头颈部鳞癌标本和相应的外周血进行微卫星分析。结果:36例头颈部鳞癌中,27.8%(10/36)分别有1-8个位点存在MSI,MSI发生率较高的位点为:D17S520(22.9%)、D6S105(16.7%)和D8S264(13.9%)。在9p21-p22和3p14等处存在一定的LOH。微卫星异常的检出率与肿瘤分期、分级无相关性。结论:提示MSI是头颈部鳞癌中较为常见的遗传学变化,染色体9p21-p22和3p14区域可能存在与头颈部鳞癌有关的抑癌基因。     

前列腺癌及前列腺上皮内瘤6号染色体等位基因杂合子丢失分析

目的：检测原发性前列腺癌及高级别前列腺上皮内瘤（PIN）6号染色体等位基因杂合子丢失（LOH）及其意义。方法：经显微切割技术切获取前列腺癌及PIN各10例患者DNA,采用聚合酶链反应（PCR）及微卫星多态性技术,对6号染色体上的20个微卫星标志位点LOH进行检测。结果：10例原发性前列腺中有8例在6号染色上至少有1个位点检测到LOH,6p21-6q23及6q25-6q27为2个高频LOH区,10例高级别PIN检测6号染色体20个位点,有5例各有1个位点检测到LOH,结论：前列腺癌中存在6号染色体的高频LOH区,分别位于6q21-6q23,6q25-6q27区,编码细胞周期素C及胰岛素样生长因子Ⅱ受体的基因为此2区侯选的抑癌基因,它们可能与前列腺癌的发生发展有关。     

髓母细胞瘤比较基因组杂交分析及ERBB-2异常表达的意义

目的研究髓母细胞瘤全基因组的遗传学异常,探讨癌基因的异常表达在髓母细胞瘤发病机制中的作用以及与预后的关系。方法应用比较基因组杂交（comparative genomic hybridization,CGH）技术检测14例髓母细胞瘤全基因组的遗传学改变;同时,在扩大系列的29例髓母细胞瘤中,应用荧光原位杂交（fluorescence in situ hybridization,FISH）和免疫组化染色分别检测ERBB-2在基因水平和蛋白水平的表达。结果（1）CGH结果显示,在所有14例髓母细胞瘤标本中,每一条染色体臂上都检测到了染色体的失衡（获得或丢失）,最常见的染色体异常为17q（85．7％）和7q（35．7％）的获得,以及8p（50％）、16q（28．6％）和17p（35．7％）的丢失;（2）FISH检测中,44．5％（13／29例）的肿瘤细胞有ERBB-2基因的异常表达;（3）免疫组化结果显示,37．9％（11／29例）的病例有抗体c-erbB-2的阳性表达;（4）在预后较差的16例患者中,56％（9／16例）的病例有ERBB-2的过度表达。结论CGH研究发现了髓母细胞瘤全基因组的染色体失衡。在染色体17q特异性位点上ERBB-2基因的异常改变很可能在髓母细胞瘤的发病机制中起着重要的作用,其过度表达与患者的预后密切相关。     

Allelotyping analysis at chromosome arm 8p of high-grade prostatic intraepithelial neoplasia and incidental, latent, and clinical prostate cancers

In this study, we used 7 informative microsatellite markers at 8p22, 23.1, and 23.2 in Japanese patients to compare frequency of loss of heterozygosity (LOH) in 53 lesions of high-grade prostatic intraepithelial neoplasia (HGPIN), 38 cases (38 lesions) of incidental prostate cancer (IPC), 31 cases (41 lesions) of latent prostate cancer (LPC), and 102 cases (168 lesions) of clinical prostate cancer (CPC). The frequency of LOH at 8p22-23.2 with at least 1 marker was 0%, 33%, 57%, and 51% in the HGPIN, IPC, LPC, and CPC cases, respectively. No statistically significant difference was found at 8p22-23.2 between the types of prostate cancer. However, the frequency of 8p22 deletion was significantly higher in CPC and LPC cases than in IPC cases (P = 0.0003) or lesions (P = 0.0017). The frequency of LOH at 8p22 and 8p23.1 loci in high-grade tumors was significantly higher than in low-grade tumors in both the LPCs/IPCs and CPCs (P < 0.05). Allelic loss at 8p22 was significantly more frequent in CPC than in IPC (P = 0.002) and in pT4 CPC than in earlier-stage CPC (P = 0.038). These findings suggest that deletion of 8p is an important event in both the initiation and metastasis of prostate cancer. The extremely high frequency of LOH at 8p22-23.1 in high-grade tumors suggests the existence of a novel putative tumor-suppressor gene associated with the progression of prostate cancer. These results should be useful in identifying the target gene of deletion at 8p.     

Three discrete areas within the chromosomal 8p21.3-23 region are associated with the development of breast carcinoma of Indian patients

Deletion in the 22.9 -Mb chromosomal (chr.) 8p21.3-23 region has been shown to be necessary for the development of breast carcinoma (CaBr). In this study, we have attempted to detect the minimal deleted region(s) in the chr.8p21.3-23 region in 62 primary breast lesions having 56 CaBr tumors and six other breast lesions of Indian patients using 15 microsatellite markers. The loss of heterozygosity (LOH) was observed for at least one marker in 96.4% (54/56) of the CaBr samples. Three discrete minimal deleted regions with high frequencies of LOH (39-65%) were identified in the chromosomal 8p23.1-23.2 (D1), 8p23.1 (D2) and 8p 21.3-22 (D3) regions within 2.03, 0.41, 2.47 Mb, respectively. No significant correlation was observed with the high deleted regions and the different clinicopathological parameters. Interestingly, 51.8% (29/56) CaBr samples showed either loss of chr.8p or interstitial deletions in this arm, indicating the importance of chr.8p in the development of CaBr. The pattern of allelic loss in the bilateral lesions had indicated that the lesions were clonal in origin and probably the deletion in the D3 region was the early event among the D1-D3 regions. Thus, our data have indicated that the D1-D3 regions could harbor candidate tumor suppressor gene(s) (TSGs) associated with the development of CaBr.     

Definition of a region of loss of heterozygosity at chromosome 11q in cervical carcinoma.

Chromosomal deletions at segment 11q23-q24 have been identified in a variety of human epithelial tumors, including cervical carcinoma (CC), indicating the presence in this region of at least a tumor suppressor gene (TSG) involved in the development of these neoplasms. To localize the 11q deletion target more precisely, 54 primary cervical carcinomas were examined for loss of heterozygosity (LOH) using a panel of microsatellite DNA markers mapping to 11p.15 and spanning region 11q23-qter. Nineteen tumors were found to have LOH at chromosome 11q. The highest frequency of LOH was observed at locus APOC-3, located in 11q23.1-q23.2, which was deleted in 42% of the informative cases. In contrast, LOH was infrequent at distal 11q in current series of CC. The smallest common region of loss included APOC-3 and was defined distally by marker D11S925 in region 11q23. The present data strongly suggest that the 11q suppressor gene(s) involved in cervical tumorigenesis is likely to be located at chromosome region 11q22-q23.     

Allelic loss of chromosomal arm 8p in breast cancer progression.

Loss of heterozygosity (LOH) of chromosomal arm 8p has been reported to occur at high frequency for a number of common forms of human cancer, including breast cancer. The objectives of this study were to define the regions on this chromosomal arm that are likely to contain breast cancer tumor suppressor genes and to determine when loss of chromosomal arm 8p occurs during breast cancer progression. For mapping the tumor suppressor gene loci, we evaluated 60 cases of infiltrating ductal cancer for allelic loss using 14 microsatellite markers mapped to this chromosomal arm and found LOH of 8p in 36 (60%) of the tumors. Whereas most of these tumors had allelic loss at all informative markers, five tumors had partial loss of 8p affecting two nonoverlapping regions. LOH for all but one of the tumors with 8p loss involved the region between markers D8S560 and D8S518 at 8p21.3-p23.3, suggesting that this is the locus of a breast cancer tumor suppressor gene. We then studied LOH of 8p in 38 cases of ductal carcinoma in situ (DCIS) with multiple individually microdissected tumor foci evaluated for each case. LOH of 8p was found in 14 of the DCIS cases (36%), including 6 of 16 cases of low histological grade and 8 of 22 cases of intermediate or high histological grade. In four of these DCIS cases, 8p LOH was seen in some but not all of the multiple tumor foci examined. These data suggest that during the evolution of these tumors, LOH of 8p occurred after loss of other chromosomal arms that were lost in all tumor foci. Thus, LOH of 8p, particularly 8p21.3-p23, is a common genetic alteration in infiltrating and in situ breast cancer. Although 8p LOH is common even in low histological grade DCIS, this allelic loss often appears to be preceded by loss of other alleles in the evolution of breast cancer.     

Loss of heterozygosity in 8p is associated with microinvasion in colorectal carcinoma

Loss of heterozygosity (LOH) from the short arm of chromosome 8 is frequent in a variety of malignancies, suggesting the presence of a tumor suppressor gene in this region. Previous studies suggested that this deletion may correlate with higher clinicopathologic stages in colorectal cancer, but others did not support this finding; in part, this difficulty is due to the low heterozygosity of the RFLP markers that were used. Here we report on a preliminary investigation in which we used highly informative microsatellite markers to determine whether deletions of 8p are correlated with poor prognostic features. Paraffin-embedded tumor tissue from 15 patients was analyzed with a panel of three microsatellite markers that are known to be sites of frequent LOH. Fourteen of the 15 cases were informative with at least one marker, and 7 showed LOH. Analysis of clinical features showed that there was no relation of 8p LOH with patient age or tumor stage, grade, location, or pattern of growth. However, a statistically significant correlation was seen between LOH and lymphatic, vascular, or perineural microinvasion (Fisher exact test, P = 0.01). This histologic feature is known to be a stage-independent indicator of prognosis. Our data suggest that 8p LOH may be associated with poor outcome and demonstrate the utility of these microsatellite markers for its detection.     

BCL10 is not a major target for frequent loss of 1p in testicular germ cell tumors

The deletion of chromosome 1p is one of the frequent genetic alterations found in testicular germ cell tumors (GCTs), suggesting the presence of a tumor suppressor gene. BCL10, which was identified as a gene altered in mucosa-associated lymphoid tissue lymphoma, has been mapped at 1p22. The gene has been reported to be mutated in a variety of human cancers. In this study, we investigated the allelic deletions on 1p and the mutation of BCL10 in 51 GCTs comprising 30 seminomas and 21 non-seminomatous germ cell tumors. Loss of heterozygosity (LOH) on 1p was tested using three microsatellite markers. The search for BCL10 mutations in each of the three exons was screened by a single-stranded conformation polymorphism (SSCP) analysis and samples with abnormal bandshifts were directly sequenced. LOH at at least one locus tested was found in 42% (21/49) of the tumors (43% of seminomas and 38% of NSGCTs). SSCP and direct sequence analyses revealed that there were single nucleotide polymorphisms at codon 5, 8, 162, and intron 1. However, there were no somatic mutations of BCL10 in the 51 tumors. In support of the previous studies, our results demonstrated that LOH on 1p is frequent in both seminomas and NSGCTs, indicating that there is an important tumor suppressor on 1p in GCT. However, the results indicate that BCL10 is not a candidate target gene of the 1p deletion.     

Loss of heterozygosity reveals non-VHL allelic loss in hemangioblastomas at 22q13

Hemangioblastomas (HBs) are low-grade (World Health Organization grade I/IV) central nervous system (CNS) tumors that frequently contain VHL (3p26) mutations. They occur sporadically and in von Hippel Lindau (VHL) disease. Encoded pVHL aids degradation of hypoxia-inducible factors (HIFs) in the presence of normal oxygen levels. HBs provide an in vivo view of HIF effects within a CNS tumor. Typically, HBs are cystic tumors containing a mural nodule formed by noninvasive, vacuolated stromal cells that are embedded in a network of capillaries. Nine HBs, consecutively resected from 8 patients at our institution during a recent 2-year time span, were evaluated for additional losses of tumor suppressor genes. Non-VHL microsatellites studied for loss of heterozygosity (LOH) are near tumor suppressor genes lost in gliomas, pituitary adenomas, several CNS tumors on 22q, neurofibromatosis 1, and colon carcinomas (13, 2, 2, 1, and 2 markers for each, respectively). LOH in the region of 3p21.3-3p26.3 occurred in 3 of 8 HBs informative for at least 1 marker (D3S1539, D3S2303, or D3S2373). By using 2 markers (D22S417 and D22S532) for 22q13.2, LOH was found in 5 of 8 informative HBs. All 3 HBs with allelic losses near VHL also showed LOH at 22q13.2. No consistent losses were found with markers for 1p34, LMYC, 5q21, 5q32, 9p21, 10q23, 17p13, and 19q13. LOH for the 22q13.2 region in HBs suggests that the loss of another tumor suppressor gene is involved in the pathogenesis of HBs in addition to VHL. Absence of LOH for glioma markers is consistent with the low-grade behavior of HBs.     

Genotypic analysis of pulmonary Langerhans cell histiocytosis

Reported studies show that the systemic form of Langerhans cell histiocytosis (LCH) is a clonal expansion of Langerhans cells (LC) associated with aberrant expression of several oncogenes or tumor-suppressor genes. LCH of the lung is a heterogenous group of lesions thought to be a reactive rather than neoplastic process. The histogenesis of the LCH of the lung is uncertain, and to date there are no studies investigating its underlying molecular abnormalities. We performed comparative genotypic analysis by using allelic loss (LOH) of polymorphic microsatellite markers associated with tumor suppressor genes. Fourteen cases of formalin-fixed, paraffin-embedded LCH of the lung were studied. Microdissection of a total of 26 nodules from 14 patients and paired reference lung tissue was performed under stereomicroscopic visualization. To evaluate allelic loss, we used a panel of 11 polymorphic microsatellite markers that were situated at or near tumor suppressor genes on chromosomes 1p, 1q, 3p, 5p, 9p, 17p, and 22q. The PCR products were analyzed by using capillary electrophoresis to identify germline heterozygous alleles and LOH. Allelic loss at 1 or more tumor suppressor gene loci was identified in 19 of 24 nodules. The total fractional allelic loss (FAL) ranged from 6% (1q) to 41% (22q), with a mean of 22%. The FAL in individual cases ranged from 0 (7 nodules) to 57% (1 nodule). Fifteen discordant allelic losses at 1 to 3 chromosomal loci were identified in 8 patients with multiple synchronous nodules. Our results show that LOH of tumor suppressor genes is present in the LCH of the lung, and they indicate that the putative tumor suppressor genes situated on chromosomes 9p and 22q may play a role in the development of a subset of the LCH of the lung.     

A 3-Mb physical map of the chromosome region 8p21.3-p22, including a 600-kb region commonly deleted in human hepatocellular carcinoma,colorectal cancer,and non-small cell lung cancer

To isolate a putative tumor suppressor gene(s), we have constructed a physical map and a detailed deletion map of chromosome region 8p21.3-p22, where loss of heterozygosity (LOH) has been frequently seen in human hepatocellular carcinomas (HCC), colorectal cancers (CRC), and non-small cell lung cancers (NSCLC). The smallest commonly deleted region at 8p21.3-p22 in HCC and CRC was between the loci defined by CI8-245 and CI8-2644; in NSCLC, a region between CI8-1051 and CI8-2644 was commonly deleted. A contiguous physical map of 12 cosmid markers in the 8p21.3-p22 region was constructed by means of multi-color fluorescence in situ hybridization (FISH) and pulsed-field gel electrophoresis (PFGE). On the basis of this physical map, which spans roughly 3.1 Mb, the estimated sizes of the commonly deleted regions were at most 1.2 Mb in HCC and CRC and 0.6 Mb in NSCLC. As four of the 12 physically ordered markers are located within the 0.6 Mb region commonly deleted in all three tumor types, nearly one fourth to one fifth of the target region has already been covered with cosmid inserts. Genes Chrom Cancer 10:7–14 (1994). © 1994 Wiley-Liss, Inc.