Characterization of the common fragile site FRA9E and its potential role in ovarian cancer

Common fragile sites (CFSs) are regions of profound genomic instability that have been hypothesized to play a role in cancer. The major aim of this study was to locate a fragile region associated with ovarian cancer. Differential display (DD)-PCR analysis comparing normal ovarian epithelial cultures and ovarian cancer cell lines identified pregnancy-associated plasma protein-A (PAPPA) because of its frequent loss of expression (LOE) in ovarian cancer cell lines. PAPPA is localized to human chromosome 9q32-33.1, a region associated with significant loss of heterozygosity (LOH) in ovarian tumors (>50%) and in close proximity to the FRA9E CFS. FISH analysis determined that PAPPA was contained within the distal end of FRA9E. Characterization of FRA9E determined that aphidicolin-induced instability extended over 9 Mb, identifying FRA9E as the largest CFS characterized to date. Comprehensive LOH analysis revealed several distinct peaks of LOH within FRA9E. Semiquantitative RT-PCR analysis of 16 genes contained within FRA9E indicated that genes showing LOE in ovarian tumors coincided with regions of high LOH. PAPPA displayed the most significant loss (72%). This study provides evidence to suggest that instability within FRA9E may play an important role in the development of ovarian cancer and lends further support for the hypothesis that CFSs may be causally related to cancer.     

A 700-kb physical map of a region of 16q23.2 homozygously deleted in multiple cancers and spanning the common fragile site FRA16D

We have identified a >600-kb region at 16q23.2 that is homozygously deleted from malignant ovarian ascites using representational difference analysis. Overlapping homozygous deletions were also observed in the colon carcinoma cell line HCT116 and a xenograft established from the small cell lung cancer cell line WX330. This region coincides with that described previously by others as showing loss of heterozygosity in prostate and breast cancers (C. Li et al., Genes Chromosomes Cancer, 24: 175-182, 1999; A. Latil et al., Cancer Res., 57: 1058-1062, 1997; K. Driouch et al., Genes Chromosomes Cancer, 19: 185-191, 1997; A. Iida et al., Br. J. Cancer, 75: 264-267, 1997). In addition, the minimally deleted region spans the common fragile site FRA16D. We have constructed a 700-kb physical map encompassing the deleted region. By fluorescence in situ hybridization of aphidicolin-induced metaphase chromosomes, we have preliminary data to suggest that P1-derived bacterial artificial chromosome clones from the contig lie on both sides of FRA16D. This is confirmed by extensive fluorescence in situ hybridization analysis of the region reported in the accompanying article (M. Mangelsdorf et al., Cancer Res., 60: 1683-1689, 2000) and is consistent with an involvement of this common fragile site in the loss of 16q23.2 material in various cancer types. The minimally deleted region of approximately 210 kb has been characterized using our own markers and public domain markers. Eleven distinct expressed sequences mapped to the region, providing a basis for identifying the predicted tumor suppressor gene in this region.     

Lack of large intragenic rearrangements in dihydropyrimidine dehydrogenase (DPYD) gene in fluoropyrimidine-treated patients with high-grade toxicity

Purpose  Deficiency of dihydropyrimidine dehydrogenase (DPD) has been associated with severe fluoropyrimidines (FP) toxicity. Mutations in DPD-coding gene (DPYD) were shown to increase the risk of severe toxicity in FP-treated cancer patients. However, the majority of DPYD alterations characterized in these patients has been considered as polymorphisms and known deleterious mutations are rare and present in only limited subgroup of patients with high toxicity. Recently, the common fragile site FRA1E was mapped within DPYD locus but intragenic rearrangements in DPYD gene were not studied so far. Methods  We performed the analysis of intragenic rearrangements of DPYD using multiplex ligation-dependent probe amplification in 68 patients with high-grade gastrointestinal and/or hematological toxicity developed at the beginning of FP treatment. Results  We did not detect any deletion/duplication of one or more DPYD exons in analyzed patients. Conclusions  We assume that rearrangements in DPYD gene play insignificant role in the development of serious FP-related toxicity. I. Ticha, P. Kleiblova contributed equally to this work.     

Identification of the human/mouse syntenic common fragile site FRA7K/Fra12C1--relation of FRA7K and other human common fragile sites on chromosome 7 to evolutionary breakpoints

Common fragile sites (CFSs) are expressed as chromosome gaps in cells of different species including human and mouse as a result of the inhibition of DNA replication. They may serve as hot spots for DNA breakage in processes such as tumorigenesis and chromosome evolution. Using multicolor fluorescence in situ hybridization mapping, the authors describe here human CFS FRA7K on chromosome band 7q31.1 and its murine homolog Fra12C1. Within the syntenic FRA7K/Fra12C1 region lies the IMMP2L/Immp2l gene with a size of 899/983 kb. The authors further mapped 2 amplification breakpoints of the breast cancer cell line SKBR3 to the CFSs FRA7G and FRA7H. The 5 molecularly defined CFSs on chromosome 7 do not preferentially colocalize with synteny breaks between the human and mouse genomes and with intragenomic duplications that have occurred during chromosome evolution. In addition, in contrast to all currently reported data, CFSs in chromosome band 7q31 do not show increased DNA helix flexibility in comparison with control regions without CFS expression.     

Identification of unstable sequences within the common fragile site at 3p14.2: implications for the mechanism of deletions within fragile histidine triad gene/common fragile site at 3p14.2 in tumors

The FRA3B, at 3p14.2, lies within the fragile histidine triad (FHIT) gene and is the most highly expressed of the common fragile sites observed when DNA replication is perturbed by aphidicolin. Common fragile sites are highly unstable regions of the genome. Large intragenic deletions within FHIT, localized within the FRA3B sequences, have been identified in a variety of tumor cells. To characterize the FRA3B deletions in tumor cells and identify FRA3B sequences that are required for fragile site induction, we used microcell-mediated chromosome transfer to isolate hybrid cell clones that retain chromosome 3 homologues with various deletions within FRA3B. Detailed molecular mapping of the FHIT/FRA3B locus in the resultant hybrid cells revealed a complex pattern of instability within FRA3B. Each tumor cell line contained multiple chromosome 3 homologues with variable deletion patterns, often with discontinuous deletions, suggesting that the process of breakage and repair within FRA3B is an ongoing one. By comparing the approximate location of the breakpoints in the hybrid clones, we identified 11 recurring breakpoint/repair regions within the FRA3B. A comparison of the frequency of breaks/gaps within FRA3B in the hybrid clones with various deletions of FRA3B sequences revealed that the loss of FRA3B sequences does not reduce the overall rate of breakage and instability within the remaining FRA3B sequences. The majority of breaks occurred in the proximal portion of the FRA3B, in a 300-kb interval between exon 4 and the proximal 50 kb of intron 5. Our observations suggest that there is no single sequence within the FRA3B that influences breakage or recombination within this region; however, we cannot rule out the presence of multiple "hot spots" within the FHIT/FRA3B locus. Together, the results suggest that factors other than the DNA sequence per se are responsible for the formation of DNA breaks/gaps.     

High-throughput genotyping by DHPLC of the dihydropyrimidine dehydrogenase gene implicated in (fluoro)pyrimidine catabolism

Dihydropyrimidine dehydrogenase (DPD) is the first and rate-limiting enzyme in the degradation of pyrimidines and pyrimidine base analogs including the anticancer drugs 5-fluorouracil (5-FU) and Xeloda. A decreased DPD enzyme activity has been described in cancer patients experiencing severe and life-threatening toxicity after 5-FU treatment and distinct sequence variants in the DPD gene (DPYD) have been associated with impaired enzyme function. The most prominent mutation in the DPD deficient patient group, a mutation in the splicing donor consensus sequence of intron 14, IVS14+1g>a, resulting in a truncated protein, has been observed in the Caucasian population at frequencies as high as 0.91%-0.94%. This underlines the need for a test system for DPYD mutations in patients undergoing chemotherapy with 5-FU or with Xeloda. To set up a fast and sensitive method to identify variant DPYD alleles, we analyzed 50 healthy individuals by denaturing high performance liquid chromatography (DHPLC). A primer set spanning the whole coding region and the exon-intron boundaries of DPYD was used. In addition, a cDNA-based assay was developed to rapidly identify the 165 base pair deletion in the corresponding RNA of IVS14+1g>a mutation carriers. The optimal mutation detection was elaborated for each of the PCR fragments. DHPLC analysis detected 5 different genetic alterations occurring in the coding region of the gene, as well as 10 intronic sequence variants, respectively. In conclusion, high-throughput screening for DPYD variants by DHPLC may be a reliable tool in the investigation of the molecular basis of DPD deficiency. Furthermore, it will help to identify patients at risk for toxic side effects upon chemotherapy using 5-FU and will facilitate individual treatment of patients.     

电穿孔法介导醛脱氢酶基因与多药耐药基因的转移和表达

背景与目的：将不同类型的耐药基因同时导入造血细胞,以扩大耐药范围和进行体内选择是基因治疗的重要策略,这类载体基因片段较长,进行基因转移有一定的难度。本研究旨在寻找安全有效的长片段基因转移G1Na-AIM以NdeⅠ酶切线性化,电穿孔法转导PA317细胞,经长春新碱（VCR）和4－氢过氧化环磷酰胺（4－HC）选择后,应用Southern blot法确定原病毒的整合,逆转录聚合酶链反应（RT－PCR）和流式细胞术（FCM）分析转移基因的表达,筑巢式聚合酶链反应（nested PCR)检验转移系统的安全性。结果：电穿孔法有效介导了ALDH1与MDR1基因的同时转移,Southern blot法证实ALDH1与MDR1基因稳定整合至宿主细胞基因组,RT－PCR检测到转移基因的转录,FCM测定下游基因MDR1蛋白表达增高4倍,转导率达98％。nested PCR未检测到辅助病毒（env)。结论：电穿孔法安全有效地介导了ALDH1与MDR1基因的共移和高表达。     

Lethal outcome of a patient with a complete dihydropyrimidine dehydrogenase (DPD) deficiency after administration of 5-fluorouracil: frequency of the common IVS14+1G>A mutation causing DPD deficiency.

Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme in the catabolism of 5-fluorouracil (5FU), and it is suggested that patients with a partial deficiency of this enzyme are at risk from developing a severe 5FU-associated toxicity. In this study, we demonstrated that a lethal toxicity after a treatment with 5FU was attributable to a complete deficiency of DPD. Analysis of the DPD gene for the presence of mutations showed that the patient was homozygous for a G-->A mutation in the invariant GT splice donor site flanking exon 14 (IVS14+1G>A). As a consequence, no significant residual activity of DPD was detected in peripheral blood mononuclear cells. To determine the frequency of the IVS14+1G>A mutation in the Dutch population, we developed a novel PCR-based method allowing the rapid analysis of the IVS14+1G>A mutation by RFLP. Screening for the presence of this mutation in 1357 Caucasians showed an allele frequency of 0.91%. In our view, the apparently high prevalence of the IVS14+1G>A mutation in the normal population, with 1.8% heterozygotes, warrants genetic screening for the presence of this mutation in cancer patients before the administration of 5FU.     

Clinical relevance of the concentrations of both pyrimidine nucleoside phosphorylase (PyNPase) and dihydropyrimidine dehydrogenase (DPD) in colorectal cancer

BACKGROUND: Pyrimidine nucleoside phosphorylase (PyNPase) converts 5'-deoxy-5-fluorouridine (5'-DFUR) to 5'-fluorouracil (5-FU), which exerts an anti-cancer effect before being catabolized by dihydropyrimidine dehydrogenase (DPD). We examined the possible correlation of the tissue concentrations of both PyNPase and DPD with the clinicopathological features of colorectal cancer. METHODS: In 36 cases of colorectal cancer, the concentrations of both PyNPase and DPD in fresh-frozen samples from either tumor or normal tissue were quantified using ELISA. RESULTS: The concentration of PyNPase was found to be significantly higher in the tumor than in the normal tissue (p = 0.001), whereas DPD showed no difference. The tumor/normal tissue ratio of PyNPase was higher in advanced stage cases, and also in the presence of liver metastasis, lymph node metastasis and vessel invasion (each p < 0.05). On the other hand, the tumor/normal tissue ratio of DPD was also higher in advanced stage cases and also in the presence of vessel invasion (each p < 0.05), thus indicating a poor response to 5-FU. The PyNPase/DPD ratio, which is known to be correlated with the tissue concentration of 5'-DFUR, was higher in the tumor than in the normal tissue (p = 0.001). CONCLUSIONS: The tumor/normal tissue ratios of both PyNPase and DPD might be useful candidates for predicting the prognosis of colorectal cancer. The PyNPase/DPD ratio was higher in the tumor tissue than in the normal tissue; however, further investigations are needed to clarify the effectiveness of fluoropyrimidine therapy.     

Impacts of excision repair cross-complementing gene 1 (ERCC1), dihydropyrimidine dehydrogenase, and epidermal growth factor receptor on the outcomes of patients with advanced gastric cancer

Using laser-captured microdissection and a real-time RT-PCR assay, we quantitatively evaluated mRNA levels of the following biomarkers in paraffin-embedded gastric cancer (GC) specimens obtained by surgical resection or biopsy: excision repair cross-complementing gene 1 (ERCC1), dihydropyrimidine dehydrogenase (DPD), methylenetetrahydrofolate reductase (MTHFR), epidermal growth factor receptor (EGFR), and five other biomarkers related to anticancer drug sensitivity. The study group comprised 140 patients who received first-line chemotherapy for advanced GC. All cancer specimens were obtained before chemotherapy. In patients who received first-line S-1 monotherapy (69 patients), low MTHFR expression correlated with a higher response rate (low: 44.9% vs high: 6.3%; P=0.006). In patients given first-line cisplatin-based regimens (combined with S-1 or irinotecan) (43 patients), low ERCC1 correlated with a higher response rate (low: 55.6% vs high: 18.8%; P=0.008). Multivariate survival analysis of all patients demonstrated that high ERCC1 (hazard ratio (HR): 2.38 (95% CI: 1.55-3.67)), high DPD (HR: 2.04 (1.37-3.02)), low EGFR (HR: 0.34 (0.20-0.56)), and an elevated serum alkaline phosphatase level (HR: 1.00 (1.001-1.002)) were significant predictors of poor survival. Our results suggest that these biomarkers are useful predictors of clinical outcomes in patients with advanced GC.     

Clinical application of immunoreactivity of dihydropyrimidine dehydrogenase (DPD) in gastric scirrhous carcinoma treated with S-1, a new DPD inhibitory fluoropyrimidine

BACKGROUND: A highly specific antibody against recombinant human dihydropyrimidine dehydrogenase (DPD) has been developed to immunohistochemically assess DPD expression in tumors. A new oral DPD inhibitory fluoropyrimidine (DIF), S-1, is reportedly effective against gastric scirrhous carcinoma. PATIENTS AND METHODS: In this study, the relationship between immunoreactivity to DPD in biopsy specimens and the effects of chemotherapy were investigated in 61 patients treated with first-line fluoropyrimidine-based chemotherapy (S-1:DIF, 5-FU:non-DIF) for gastric scirrhous carcinoma. RESULTS: The response rate was significantly higher in patients with DPD-positive tumors than in those with DPD-negative tumors in the S-1 group (45.5%, 10.0%: p < 0.05), as compared to the 5-FU group (0%, 5.6%: p = 0.398). According to the median survival time, there was no significant difference between patients with DPD-positive tumors (364 days) and those with DPD-negative tumors (406 days; p = 0.626) in either the S-1 group or the 5-FU group (181 days and 256 days, respectively; p = 0.543). CONCLUSION: This study indicates that S-1 may be effective even in gastric scirrhous carcinoma with a high level of DPD activity.     

Clinical implications of dihydropyrimidine dehydrogenase (DPD) deficiency in patients with severe 5-fluorouracil-associated toxicity: identification of new mutations in the DPD gene.

Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme in the catabolism of 5-fluorouracil (5FU), and it is suggested that patients with a partial deficiency of this enzyme are at risk for developing a severe 5FU-associated toxicity. To evaluate the importance of this specific type of inborn error of pyrimidine metabolism in the etiology of 5FU toxicity, an analysis of the DPD activity, the DPD gene, and the clinical presentation of patients suffering from severe toxicity after the administration of 5FU was performed. Our study demonstrated that in 59% of the cases, a decreased DPD activity could be detected in peripheral blood mononuclear cells. It was observed that 55% of patients with a decreased DPD activity suffered from grade IV neutropenia compared with 13% of patients with a normal DPD activity (P = 0.01). Furthermore, the onset of toxicity occurred, on average, twice as fast in patients with low DPD activity as compared with patients with a normal DPD activity (10.0 +/- 7.6 versus 19.1 +/- 15.3 days; P < 0.05). Analysis of the DPD gene of 14 patients with a reduced DPD activity revealed the presence of mutations in 11 of 14 patients, with the splice site mutation IVS14+1G-->A being the most abundant one (6 of 14 patients; 43%). Two novel missense mutations 496A-->G (M166V) and 2846A-->T (D949V) were detected in exon 6 and exon 22, respectively. Our results demonstrated that at least 57% (8 of 14) of the patients with a reduced DPD activity have a molecular basis for their deficient phenotype.