Frequent allelic deletion at the FHIT locus associated with p53 overexpression in squamous cell carcinoma subtype of Taiwanese non-small-cell lung cancers

The fragile histidine triad (FHIT) gene, encompassing the FRA3B fragile site at chromosome 3p14.2, is a tumour suppressor gene involved in different tumour types including non-small-cell lung cancers (NSCLCs). In the current study, we examined for allelic deletion at the FHIT locus in 58 primary and microdissected NSCLCs, for which a clinicopathologic profile was available. We found a loss of 87.7% in heterozygosity (LOH) frequency at one or more microsatellite markers (D3S1289, D3S2408, D3S1766, D3S1312, D3S1600). Allelic deletion of D3S1766 was related to tumour histology in 10 of 11 squamous cell carcinomas (90.9%) displaying LOH compared with nine of 17 adenocarcinomas (52.9%; P=0.049). Besides, in the subset of adenocarcinomas, a higher rate of LOH at D3S1289 was observed in male (six out of eight, 75%) than in female patients (four out of 17, 23.5%; P=0.028). However, FHIT LOH was not correlated overall with a variety of clinical parameters including sex, smoking status, staging, lymph node metastasis and survival. These results indicated that the high frequency of FHIT gene disruption was important in the development of both squamous cell carcinomas and adenocarcinomas. Furthermore, there was no association between LOH at FHIT and protein expression, suggesting the presence of complex mechanisms of Fhit inactivation. On the other hand, the association between FHIT LOH and p53 protein overexpression assessment reached statistical significance (P=0.026), implying that common alterations affect the two genes in tumour progression.     

Loss of heterozygosity at loci of candidate tumor suppressor genes in microdissected primary non-small cell lung cancer

To investigate the etiological association of allelic loss at chromosomal regions containing tumor suppressor genes (TSGs) in non-small cell lung cancer (NSCLC) in Taiwan, we examined 48 microdissected NSCLC samples for loss of heterozygosity (LOH) at nine loci where TSGs are localized nearby. The associations of LOH at each locus with clinicoparameters and prognosis were also examined. The frequent LOH was observed using markers, D3S1285 near the FHIT gene (58.3%), D17S938 near the p53 gene (56.7%), D9S925 near the p16 gene (54.5%), and D13S153 near the RB gene (47.6%). The occurrence of LOH at each TSG locus was compared with the patients' clinicoparameters. The incidence of LOH at D17S938 (p53 gene) and D3S4545 (VHL gene) was significantly higher in squamous carcinoma tumors than in adenocarcinoma tumors (P = 0.003 and 0.024, respectively). LOH of these two loci also occurred frequently in tumors from smoker patients compared to that from nonsmoker patients (P = 0.013 and 0.025, respectively). LOH at D13S153 (RB gene) was also associated with smoking (P = 0.008). In addition, the prognostic analyses indicated that the patients with LOH at D18S535 (18q21, near the SMAD2/4 gene) had significantly longer post-operative survival time compared to those without LOH (P = 0.03). Our results suggested that LOH at FHIT, p53, and p16 genes may occur frequently in NSCLC patients in Taiwan. In addition, LOH at p53, RB, and VHL may associate with smoking or squamous carcinoma patients and LOH at SMAD2/4 may be correlated with better prognosis.     

食管鳞癌FHIT基因等位缺失及其表达下调

背景与目的：食管癌3号染色体短臂(3p14)经常发生缺失或易位,脆性组氨酸三联体基因(fragile histidinetriad,FHIT)定位于3p14．2,在许多肿瘤中均发现此基因缺失或表达异常。然而,FHIT基因在食管癌中变化的形式以及FHIT改变在食管癌发生、发展中的作用尚未明确。本研究旨在探讨FHlT基因在食管鳞癌中的变化情况及其意义。方法：用微卫星分析方法观察80例食管鳞癌中FHIT基因的缺失,并用半定量逆转录聚合酶链反应法(reverse transeriptase-polymerase chain reaction,RT-PCR)分析其中20例食管鳞癌FHIT mRNA表达的变化。结果：FHIT基因内标志D3S3356、D3S3378、D3S3361在本系列的标本中为纯合子。FHIT基因旁侧微卫星位点D3S1234和D3S1540在癌旁组织具有较高的杂合度,信息个体中肿瘤组织的杂合性丢失(10ssofhelerozygosily,LOH)频率分别为57．69％(30／52)和67．86％(38／56)。在20例同时获得其RNA的标本中,15(75％)例出现FHIT基因的mRNA表达下调,且多发生于DNA水平伴有LOH的病例。但LOH发生与mRNA表达下调并不完全一致。结论：食管癌中FHIT基因频发异常,LOH是FHIT基因表达失调的主要机制,表观遗传学(epigenetic)变化可能与部分食管癌中FHIT基因的表达下调相关。     

Deletion of chromosome 3p14.2-p25 involving the VHL and FHIT genes in conventional renal cell carcinoma

Loss of heterozygosity (LOH) at chromosome 3p and inactivation of the VHL gene are associated with the development of conventional renal cell carcinomas (RCCs). Recently, it was suggested that LOH at the FHIT gene at 3p14.2 is an early event in the development of RCC and is characteristic for all types of RCC. We have analyzed 88 conventional, 30 papillary, and 22 chromophobe RCCs for LOH at the VHL and FHIT regions and at other loci on chromosome 3p. A continuous deletion of 3p14.2-p25 harboring the VHL and FHIT genes occurred in 96% of the conventional RCCs but only in 10% of the papillary RCCs and 18% of the chromophobe RCCs. Our data indicate that LOH at chromosome 3p14.2-p25 is specific for conventional RCC and that loss of one allele of both the VHL and FHIT genes occurs in early stage of tumorigenesis.     

Allelic loss at TP53 is not related to p53 protein overexpression in primary human endometrial carcinomas

We examined loss of heterozygosity (LOH) at the TP53 gene in primary human endometrial carcinomas (EC), and investigated the relationship between allelic loss, p53 protein overexpression, pRb-1 pathway alterations and MIB-1 proliferative activity. Applying the non-isotopic PCR-RFLP/VNTR-silver staining techniques, we investigated TP53 LOH in 46 tumors at four polymorphic loci. Out of 42 informative carcinomas, LOH was found in 19% of the cases studied. In general, there was no significant relationship between LOH and the clinical and pathological variables of cancer, including patient age, clinical stage, histological grade or depth of myometrial invasion. Interestingly, none of 7 tumors associated with hyperplasia revealed allelic imbalance, whereas 8 of 27 (30%) tumors without hyperplasia exhibited LOH (p=0.312; Fisher's exact test). Overexpression of nuclear p53 was not correlated with allelic loss at TP53 (p=0.336, Fisher's exact test). It is worth pointing out that p53 immunoreactivity was significantly related to proliferative activity of cancer (R=0.42, p=0.0037; Spearman's rank correlation test). A tendency towards a poorer outcome was reported in EC patients displaying TP53 LOH during short-time follow-up (p=0.093; log-rank test). None of the tumors simultaneously showed LOH at TP53 and RB1 genes (R=-0.211, p=0.16; Spearman's rank correlation test). p16INK4A alterations (LOH and gene deletion) occurred concomitantly, with 3 tumors showing the TP53 allelic loss, whereas the cyclin D1/cdk4 complex was overexpressed in a case with TP53 LOH. Altogether, losses at TP53 were not associated with p53 nuclear overexpression, but may affect a subset of EC patients characterized by an unfavorable prognosis at short-time follow-up. Allelic loss at TP53 seems to arise independently of LOH at the RB1 gene in carcinomas of the uterine corpus in humans. Disruptions at p16INK4A and/or cdk4/cyclin D1 concomitantly occurring with TP53 LOH may participate in the development of a subset of endometrioid-type ECs.     

Abnormal transcripts of FHIT gene in Chinese brain tumors

FHIT located at chromosome 3p14.2 was discovered and proposed as a candidate tumor suppressor gene in several cancers. To determine whether the FHIT gene at 3p14.2 is altered in Chinese brain tumors, we examined 13 brain tumors for deletions within FHIT locus. Evaluation of the FHIT gene in the panel of brain tumors led to a comprehensive mutation analysis. The complete sequence of the FHIT gene was determined and deletions between exon 5-8 were found in all 13 cases. In addition, single point mutation of amino acid from two glioblastoma and one atypical meningioma cases and multiple amino acid mutations from one pituitary tumor were observed. Our results support the hypothesis that FHIT gene alteration is involved in tumorigenic development of human neoplasms.     

Comparative analysis of loss of heterozygosity of specific chromosome 3, 13, 17, and X loci and TP53 mutations in human epithelial ovarian cancer

We previously reported the identification of three minimal regions of deletion on the short arm of chromosome 3 (3p) in epithelial ovarian tumor specimens, suggesting that the inactivation of tumor-suppressor genes in these regions may be important in terms of ovarian tumorigenesis. Another previous study of ovarian cancer observed that allele loss of chromosome 179 was frequently found in ovarian tumors that also showed loss of heterozygosity (LOH) of chromosomes 3p, 13q, 17p, and Xp. In an independent study, we also reported a high frequency of LOH for selected chromosome 17 loci in high-grade and late-stage ovarian tumors. We have extended our LOH analysis of chromosome 3p to include 102 ovarian tumor specimens (29 and 73 samples were previously examined for LOH of chromosome 3p and 17 markers, respectively), using additional polymorphic markers, to assess the coordinate LOH of loci representing the three chromosome 3p minimal regions of deletions [von Hippel-Lindau syndrome (VHL), thyroid hormone receptor beta, and fragile histidine triad (FHIT)] and LOH of other important loci [tumor protein 53 (TP53), breast cancer 1 early onset (BRCA1), breast cancer 2 early onset, retinoblastoma 1, ornithine carbamoyltransferase, and androgen receptor] or somatic mutations in TP53. There was a significant association between LOH of any chromosome 3p marker and LOH of any chromosome 17 marker (P = 0.026). The frequency of LOH at the TP53 locus was higher in the group of samples that displayed LOH of a 3p marker (P = 0.019), as was the frequency of LOH at the BRCA1 locus (P = 0.014). LOH of chromosome 3p was noted in four specimens that did not display LOH of either the BRCA1 or the TP53 locus, indicating that LOH of these loci need not precede LOH of the chromosome 3p loci. We found a significant association between LOH of the VHL (3p25) locus and LOH of any chromosome 17 marker (P = 0.005), suggesting that there may be an important relationship, in the tumorigenesis of epithelial ovarian cancer, between a gene at 3p25 and a gene located on chromosome 17. Our results indicate that inactivation of p53 by somatic mutation is unlikely to be a prerequisite to chromosome 3p LOH, because we found no significant association between mutations in TP53 and LOH of the three chromosome 3p loci. The frequency of LOH at the FHIT locus at 3p14 increased significantly with advancing age at diagnosis (P = 0.018), as did the frequency of somatic TP53 mutations (P = 0.008).     

Multiple regions with allelic loss at chromosome 3 in superficial multifocal bladder tumors

We have examined six patients with multiple low grade, low stage superficial multifocal bladder tumors with surrounding tissues for loss of heterozygosity (LOH) and microsatellite instability at chromosome 3, totaling 76 samples. The majority (4/5) of the patients had LOH at or close to the fragile histidine triad (FHIT) gene (3p14.2; D3S1300), which is a candidate tumor suppressor gene for many cancer types. One patient showed a consistent LOH with four adjacent markers around FHIT region in all the tumors whereas in the corresponding surrounding tissues the heterozygosity was retained. In addition to the region near FHIT, two other regions had frequent allelic losses - one near the p telomere (3p25-26; D3S3050) and another near the q telomere (3q27; D3S2418). The largest numbers of LOH in the surrounding tissues were found at these regions (3/5 at D3S3050 and 2/5 at D3S2418). The D3S3050 marker is located at 3p26-3p25, near the Von Hippel-Lindau (VHL) tumor suppressor gene locus. LOH that were more random were found at 3q21.3-25.2 (D3S1744) and at 3p12-3p11 (D3S2465). Taken together, at least three regions at chromosome 3p25-26, 3p14.2 and 3q27 seem to have frequent loss of heterozygosity in multifocal superficial bladder tumors. We also performed a phylogenetic-type analysis to find out common changes and the degree of heterogeneity. The overall heterogeneity was low within a given patient: in all cases the majority of the tumor samples arranged in a single branch with a common origin. This point of origin varied from patient to patient, which is compatible with the earlier studies demonstrating the heterogeneity of the single primary bladder tumors. However, the phylogenetic-type analysis suggests that the FHIT region contains often the very first alterations at chromosome 3.     

FHIT基因与子宫颈癌的相关性研究

脆性组氨酸三联体（FHIT）基因是近年来发现的定位于人3号染色体短臂（3p14．2）上的候选抑癌基因。许多研究表明其表达改变与人类多种肿瘤关系密切。FHIT基因可能通过调控细胞周期，诱导细胞凋亡发挥抑制肿瘤的作用，其失活机制主要表现为启动子区域CPG岛甲基化、缺失及异常转录。本文重点综述FHIT基因的生物学特性及在宫颈癌发生发展中的作用。     

FHIT基因与肿瘤

脆性组氨酸三联体(FHIT)基因是一种定位于人类染色体3p14.2上的抑癌基因,在多种器官中都有表达,其包含的脆性位点FRA3B,是基因组中最脆弱的部分.FHIT可促进细胞凋亡,抑制细胞增殖和细胞癌变.FHIT基因高度甲基化、基因缺失、FRA3B脆性位点的改变以及蛋白合成过程中的异常,可导致FHIT基因功能缺失,促进多种肿瘤的发生与发展.将野生型FHIT导入FHIT表达降低或表达缺失的肿瘤细胞中可促进肿瘤细胞凋亡.FHIT基因与其他基因联合治疗可能为肿瘤的治疗提供新的方向.     

Loss of heterozygosity at the short arm of chromosome 3 in microdissected cervical intraepithelial neoplasia

Loss of heterozygosity (LOH) is a common genetic finding in many human neoplasms, including cervical cancer. The detection of LOH at specific loci in the precursor of cervical cancer, cervical intraepithelial neoplasia (CIN) may help in elucidating the evolution of this cancer, which has a clearly defined histological premalignant phase. However, molecular genetic investigation of CIN is difficult because many of the lesions are very small and sometimes ill defined topographically. In this study we analyzed eighteen polymorphic microsatellite repeats on chromosome 3p in CINs using a method of primer extension pre-amplification (PEP) for whole genome amplification combined with microdissection. These markers encompass chromosome region 3pter-3p12. LOH at one or more loci was detected in five (33%) out of the 15 informative cases with low grade CIN (CIN 1), while 22 (92%) out of 24 cases with high grade CIN (CIN 2 and 3) (P<0.01). The highest incidence (41%) of LOH was detected at locus D3S1038 (3p26.1-3p25.2). Frequent LOH (more than 20%) was also found at other loci including D3S1110 (3p25.3-3p25.1) (31%), D3S656 (3p25.1) (24%), D3S1076 (3p21.2-3p21.1) (29%), D3S1300 (3p21.1-3p14.2) (24%), D3S1600 (3p14.2-3p14.1) (24%), and D3S1079 (3p13) (25%). The results from this study taken together with others indicate that the genetic alterations on chromosome 3p are common in high grade of CIN and are probably early events in cervical carcinogenesis. Tumor suppressor gene(s) that play a role in cervical neoplasm may be located on the short arm of chromosome 3, likely at or near 3p26.1-25.1, 3p21.2-21.1, and 3p14.2-13.     

喉癌中13号染色体杂合性丢失研究

目的　为初步限定喉癌中 13号染色体抑癌基因的缺失区域和为发现及定位抑癌基因提供线索和依据。方法　应用聚合酶链反应 ( PCR) ,筛查了 58例喉癌 (包括 3例原位癌 )组织中染色体13q上 D13S765( 13q13) ,RB1.2 0 ( 13q14.2 ) ,D13S133( 13q14.3) ,D13S318( 13q2 1) 4个座位微卫星多态标记的杂合性丢失。结果　 3例原位癌中无一例发生 13q的杂合性丢失 ,55例浸润癌中在 13q一个以上座位出现杂合性丢失的频率为 4 5% ( 2 4 / 53) ,D13S765座位的杂合性丢失的频率最高 ,为 52 %( 2 2 / 4 2 )。结论　喉癌组织中染色体 13q缺失区域在 D13S765( 13q13)座位附近 ,RB1的近端 ,即在D13S765座位附近存在着与喉癌发生发展密切相关的抑癌基因 ,可能包括 RB1基因 ,它的失活与浸润期喉癌发生发展密切相关。     

Role of FHIT in human cancer.

Through investigation of hemizygous and homozygous deletions in common human cancers, including lung cancer, we have cloned and characterized a gene at chromosome region 3p14.2, FHIT, that is inactivated in epithelial tumors, particularly in tumors resulting from exposure to environmental carcinogens. In some tumors, particularly those associated with environmental carcinogens, alterations in the FHIT gene occur quite early in the development of cancer. In other cancers, Fhit inactivation seems to be a later event, possibly associated with progression to more aggressive neoplasias. Thus, detection of Fhit expression by immunohistochemistry in premalignant and malignant tissues may provide important diagnostic and prognostic information.     

Two-hit inactivation of FHIT by loss of heterozygosity and hypermethylation in breast cancer.

PURPOSE: The FHIT gene, which spans the FRA3B fragile site at chromosome 3p14.2, is a candidate tumor suppressor gene in breast carcinomas. In this study, we would like to delineate more precisely its role in breast tumorigenesis. EXPERIMENTAL DESIGN: To confirm the tumorigenic role of FHIT, 46 sporadic invasive ductal carcinomas of the breast were tested for the "two hits" required to inactivate this gene. Microsatellite loss of heterozygosity (LOH) was considered as the first hit. To examine the possibility that hypermethylation serves as the second hit for FHIT inactivation, methylation of 5'-CpG islands of FHIT was analyzed by methylation-specific PCR. RESULTS: LOH was detected in 8 of 40 informative tumors, and hypermethylation was observed in 22 of 46 (48%) cases. Aberrant FHIT protein expression was found in 31 of 46 (67%) cases examined. All seven tumors showing both LOH and hypermethylation showed complete loss of Fhit protein expression. In addition, a significant positive association was found between the existence of LOH and 5'-CpG island hypermethylation (P = 0.04), which was consistent with the two-hit model. CONCLUSIONS: To our knowledge, this study provides the first evidence that biallelic inactivation of FHIT by LOH and hypermethylation leads to the complete inactivation of FHIT gene in patients with breast cancer. Silencing of the FHIT gene by promoter hypermethylation occurs in primary breast carcinomas, especially those with LOH. These findings support a role for this tumor suppressor gene in sporadic breast tumorigenesis.     

Allelic imbalance at chromosome region 11q23 in cervical carcinomas

The long arm of chromosome 11 has received much scrutiny as a high frequency of deletions of various sites has been observed in different tumour types, indicating the presence of putative tumour suppressor genes. In the present study, 81 primary cervical carcinomas were examined for allelic imbalance (AI) using nine microsatellite markers, mapping to the chromosomal region 11q23.1 where the ATM gene is located. AI at any locus in the region was found in 34 of 81 (42%) tumours. AI frequencies varied from 12 to 31% for the different markers used, with the highest frequency at marker D11S1294. Based on the findings of 17 cases with restricted areas of deletions, four chromosomal regions of possible importance in cervical carcinomas could be distinguished. The first region is located between the markers D11S1325 and D11S1819, the second region between D11S2179 and D11S1294, the third region between D11S1778 and D11S1818 and the fourth region between D11S1818 and D11S1347. The second region may thus contain part of the ATM gene. No association between AI of any marker and histopathological or clinical parameters was seen. When comparing the AI findings of the different loci with TP53 protein overexpression, the only significant association found was with D11S2179 located within the ATM gene. The results indicate that a tumour suppressor gene (or genes) on chromosome 11q.23.1 may be involved in carcinogenesis of the cervix and the involvement of the ATM gene remains a possibility.