肺癌组织中白介素-8基因表达的实时定量PCR研究

目的建立白介素-8 (IL-8)基因表达的绝对定量实时PCR检测技术.方法应用实时定量PCR和ELISA方法检测2株人肺癌细胞H460及A549中IL-8 mRNA表达及蛋白分泌.同时对9例正常肺及44例非小细胞肺癌(NSCLC)组织中IL-8 mRNA进行分析.结果应用实时定量PCR方法能够对肺癌细胞中IL-8 mRNA拷贝数进行定量分析,IL-8表达值与蛋白分泌量呈正相关.正常肺组织中IL-8基因表达值为4.87±1.69 (单位:拷贝数/104 GAPDH拷贝),肺癌组织为17.04±23.96,两组间差异有统计学显著意义(P＝0.002).若以正常肺组织均数的2倍(9.74)和4倍(19.48)分别作为IL-8阳性表达及过表达的界限,52.3%(23/44例)的肺癌组织IL-8表达增高,其中IL-8阳性表达率为29.5 %(13/44例),过表达率为22.7 %(10/44例).统计分析显示,在女性、鳞癌和晚期肿瘤(III期及T2～T4)中IL-8过表达率明显增高,具有统计学显著差异(P<0.05).结论应用实时定量PCR技术能够对IL-8表达水平进行定量分析,IL-8过表达与患者性别、组织类型和病期密切相关.     

Quantification of micrometastases in lymph nodes of colorectal cancer using real-time fluorescence polymerase chain reaction

The expression of carcinoembryonic antigen (CEA) mRNA was assessed in 102 lymph nodes (LNs) obtained from seven colorectal cancer patients by both the conventional non-quantitative RT-PCR and quantitative RT-PCR. The number of CEA-expressing cells was calculated compared with CEA-expressing MKN-45 cell line as a standard control. Using the quantitative RT-PCR, the relative number of CEA-expressing cells ranged between 1.3x103 and 5.7x106 in 16 histologically positive LNs and between 2.3x101 and 8.1x105 in 10 histologically negative and RT-PCR positive LNs. In both histologically and RT-PCR negative LNs, the relative cell number was <4.0x102. Our results demonstrated that quantifying the amount of metastasis might enhance the reliability of RT-PCR detection assay as a diagnostic tool for the detection of cancer micrometastases.     

The immunocytochemical detection of axillary micrometastases in breast cancer.

The histological detection of tumour metastases in axillary lymph nodes from cases of breast carcinoma is of major prognostic significance, but may be difficult when metastases are of microscopic size. We have therefore investigated whether immunohistological techniques can increase the accuracy of metastasis detection in axillary lymph nodes. Forty-five cases of breast carcinoma were studied, in all of whom the axillary lymph nodes had been reported as free of metastases. Paraffin sections from these cases were stained by immunoenzymatic techniques, using monoclonal antibodies directed against human milk fat globule membrane antigen ("anti-EMA") and against epithelial intermediate filaments ("anti-keratin"). In 4/12 cases of lobular carcinoma and in 3/33 cases of ductal carcinoma, previously unsuspected micrometastases were revealed by immunohistological staining, representing an overall increase in detection rate of 15% (and of 33% for the lobular carcinoma cases). In addition to this group of 45 histologically "negative" biopsies, 12 samples were studied in which only a proportion of the nodes had been reported as containing tumour. In 5 of these cases immunostaining revealed previously undetected metastases. These findings suggest that immunohistological analysis may have a routine role to play in the staging of breast carcinoma. It is noted that the 15% increase in diagnostic accuracy achieved in the present study is comparable to the proportion of breast carcinoma patients in whom disseminated disease develops despite their axillary lymph nodes being reported as tumour-free at the time of surgery.     

Clonal analysis of human breast cancer by means of the polymerase chain reaction.

Clonality of human breast cancer was analyzed in small DNA samples prepared from cryostat sections, by means of the polymerase chain reaction (PCR). The method used for clonal analysis was based on restriction fragment length polymorphism of X-chromosome-linked phosphoglycerokinase (PGK) gene and on the differential methylation of the PGK gene due to random inactivation of one of two X-chromosomes by methylation in females. All the 20 breast cancer samples analyzed by the PCR-based method were monoclonal in origin and adjacent normal breast tissues were polyclonal. When DNA samples were prepared from widely separated sites of cancers, every sample was found to be monoclonal, always exhibiting inactivation of the same X-chromosome in each tumor. The study on sensitivity showed that the PCR-based method for clonal analysis can detect the presence of monoclonal cells against a polyclonal background when the monoclonal cell population is 50% or more. These results demonstrate that clonal analysis by means of PCR offers a good method for studying the clonality in small DNA samples prepared from cryostat sections of tumors. This method could be applied to distinguish between benign (polyclonal) and malignant (monoclonal) breast lesions.     

Selection of mRNA markers for detection of lymph node micrometastases in breast cancer patients

The aim of this study was to search for specific and sensitive mRNA markers or a combination of markers for RT-PCR detection of micrometastases in axillary lymph nodes (LNs) from patients with breast cancer. LNs (n=177) from 17 patients were examined with Cytokeratin20 (CK20), melanoma-associated genes (MAGE1, MAGE3), carcinoembryonic antigen (CEA), prostate-specific antigen (PSA), mammaglobin (MGB1) and mammaglobin B (MGB2) as molecular markers. CK20, MAGE1 and MAGE3 were slightly positive in primary tumors and CEA, PSA, MGB1 and MGB2 were highly positive. MGB1 and MGB2 were 100% positive in HE-positive LNs while CEA and PSA were only 35.7% and 57.1% positive. MGB1 and MGB2 were also 30.1% and 17.8% positive in HE-negative nodes. Thus, MGB1 and MGB2 are specific and a combination of the two should be useful for detection of micrometastases in axillary LNs of breast cancer patients.     

Quantitative Detection of Interleukin 8 Gene Expression in Lung Cancer by Real-time Polymerase Chain Reaction

Objective To develop a method for absolute quantification of interleukin 8 (IL—8) mRNA by using real-time polymerase chain reaction (PCR). Methods The IL —8 mRNA and protein expression in 2 human lung cancer cell lines, H460 and A549, were evaluated by real-time PCR and ELISA. The IL-8 mRNA expression in 9 cases of normal lung tissue and 44 cases of non-small cell lung cancer (NSCLC) were examined. Results The IL—8 mRNA copy number in a given sample can be measured by real-time PCR. The gene expression of IL—8 is correlated with its protein secretion. The normalized value of IL—8 expression was 4.87±1.69 (copies/ 10⁴ GAPDH copies) in normal lung tissue and 17.04 ±23.96 in NSCLC, respectively. The difference between these two groups is statistically significant (P=0.002). Using 9.74 and 19.48 as cut-off points for positive expression and overexpression of IL—8, 52.3%(23/44cases) of NSCLC were found to express an increased level of IL-8, among which 29.5% (13/ 44cases) were defined as positive expression and 22.7%(10/44cases) as overexpression. Statistical analysis indicated that IL—8 overexpression was significantly increased in female cancers, squamous carcinoma, and in late stages of disease (P<0.05). Conclusion The IL—8 gene expression can be determined by a real-time PCR technique. IL -8 overexpression is correlated with gender, histopathology and stages of the disease.     

Detection of breast cancer micrometastases in bone marrow using reverse-transcriptase chain reaction and southern hybridization

Thirty-flveto40%ofallpatientswithbreastcarcinoma,includingupt024%ofpatientswithn0evidence0fmetastasesatthetimeofdiagnosis,willrelapseafterprimarytherapy.l"']Themostreliableprognosticindicesofaxillarylymphnodestatusandsizeofprimarytumorcannotpredictinwh0mthediseasewillrecur.Bonemarrowisafrequentandreadilyaccessiblesiteofmetastases.Inup8O%ofpatients,therelaPsedevelopsstariingfrombonemarrowmetastasesats0mepointinthepr0cessoftheirillness.l3]Currentmethodstodetectboneinvolvement,suchasX-rayandbon…     

Quantitative methylation-specific polymerase chain reaction gene patterns in urine sediment distinguish prostate cancer patients from control subjects.

PURPOSE: Aberrant promoter hypermethylation of several known or putative tumor suppressor genes occurs frequently during the pathogenesis of prostate cancers and is a promising marker for cancer detection. We sought to develop a test for prostate cancer based on a quantitative methylation-specific polymerase chain reaction (QMSP) of multiple genes in urine sediment DNA. PATIENTS AND METHODS: We tested urine sediment DNA for aberrant methylation of nine gene promoters (p16INK4a, p14(ARF), MGMT, GSTP1, RARbeta2, CDH1 [E-cadherin], TIMP3, Rassf1A, and APC) from 52 patients with prostate cancer and 21 matched primary tumors by quantitative fluorogenic real-time polymerase chain reaction. We also analyzed urine sediments from 91 age-matched individuals without any history of genitourinary malignancy as controls. RESULTS: Promoter hypermethylation of at least one of the genes studied was detected in urine samples from all 52 prostate cancer patients. Urine samples from the 91 controls without evidence of genitourinary cancer revealed no methylation of the p16, ARF, MGMT, and GSTP1 gene promoters, whereas methylation of RARbeta2, TIMP3, CDH1, Rassf1A, and APC was detected at low levels. CONCLUSION: Overall, methylation found in urine samples matched the methylation status in the primary tumor. A combination of only four genes (p16, ARF, MGMT, and GSTP1) would theoretically allow us to detect 87% of prostate cancers with 100% specificity. Our data support further development of the noninvasive QMSP assay in urine DNA for early detection and surveillance of prostate cancer.     

实时荧光定量RT-PCR检测胃癌腹腔冲洗液CK20、CEA mRNA

目的研究实时荧光定量RT-PCR方法定量检测胃癌患者腹腔冲洗液中CK20、CEA mRNA的表达,并探讨其临床意义.方法通过实时定量荧光RT-PCR方法,对56例胃癌患者术中腹腔冲洗液中定量检测CK20、CEA mRNA的表达.结果浆膜受侵患者CEA mRNA的表达率61.5%(16/26),明显高于未及浆膜者30.0%(9/30)(P＜0.05).浆膜受侵患者CK20 mRNA的表达率46.2%(12/26)与未及浆膜者56.7%(17/30)(P>0.05)差异无显著性.结论实时定量荧光RT-PCR检测CEA mRNA可作为胃癌腹腔脱落癌细胞的诊断或腹膜转移的预测,指导术中干预治疗.     

Bone marrow micrometastases in breast cancer patients

We designed three new four–drug cisplatin–containing combinations and evaluated their activity in a randomized phase II study including patients with locally advanced (stage III) and locally recurrent breast carcinoma.All combinations included methotrexate (M) on day 1 and cisplatin (P) on day 2 (MVAC–like combinations) and differed from one another by the addition of Epirubicin (Epi), Vincristine (V), Etoposide (E), Mitomycin (Mi). Based on the administered agents, they were named MPEMi, MPEpiE, MPEpiV. The combinations were randomly assigned to 101 patients, 57 with locally advanced and 44 with locally recurrent breast carcinoma. Response was evaluated after 4 cycles.The complete response (CR) rates were 7 and 43 and the CR plus partial response (PR) rates were 84 and 89 in locally advanced and in locally recurrent disease, respectively. In locally advanced disease, a pathologic CR (pCR) was assessed in seven of 57 patients (12). There were no significant differences among the three combinations. The toxicities were at times severe, but generally tolerable, as demonstrated by the high cumulative doses of the drugs received by the patients.In conclusion, these three innovative chemotherapy regimens induced high CR plus PR rates in the neoadjuvant treatment of stage III and of locally recurrent breast carcinoma, and a high rate of pCR in stage III disease. These regimens warrant testing in phase III trials.     

Novel approach to quantitative polymerase chain reaction using real-time detection: Application to the detection of gene amplification in breast cancer

Gene amplification is a common event in the progression of human cancers, and amplified oncogenes have been shown to have diagnostic, prognostic and therapeutic relevance. A kinetic quantitative polymerase-chain-reaction (PCR) method, based on fluorescent TaqMan methodology and a new instrument (ABI Prism 7700 Sequence Detection System) capable of measuring fluorescence in real-time, was used to quantify gene amplification in tumor DNA. Reactions are characterized by the point during cycling when PCR amplification is still in the exponential phase, rather than the amount of PCR product accumulated after a fixed number of cycles. None of the reaction components is limited during the exponential phase, meaning that values are highly reproducible in reactions starting with the same copy number. This greatly improves the precision of DNA quantification. Moreover, real-time PCR does not require post-PCR sample handling, thereby preventing potential PCR-product carry-over contamination; it possesses a wide dynamic range of quantification and results in much faster and higher sample throughput. The real-time PCR method, was used to develop and validate a simple and rapid assay for the detection and quantification of the 3 most frequently amplified genes (myc, ccnd1 and erbB2) in breast tumors. Extra copies of myc, ccnd1 and erbB2 were observed in 10, 23 and 15%, respectively, of 108 breast-tumor DNA; the largest observed numbers of gene copies were 4.6, 18.6 and 15.1, respectively. These results correlated well with those of Southern blotting. The use of this new semi-automated technique will make molecular analysis of human cancers simpler and more reliable, and should find broad applications in clinical and research settings. Int. J. Cancer 78:661–666, 1998. © 1998 Wiley-Liss, Inc.     

Comparison between immunocytochemical and polymerase chain reaction techniques for detection of oestrogen receptor and transforming growth factor beta in breast cancer.

The utility of the polymerase chain reaction (PCR) as a technique for determining the expression of transforming growth factor beta (TGF-beta) and of the oestrogen receptor (ER) in clinical breast cancer tissue was examined. PCR analysis was compared with immunocytochemical assays for TGF-beta and for ER. Seventy confirmed breast carcinoma samples were analysed for ER using both techniques with a statistically highly significant concordance (P < 0.001) between the two methods. Nineteen samples were observed to be ER positive and 46 samples were found to be ER negative by both techniques. Forty-eight samples were analysed for TGF-beta using both PCR and immunocytochemistry. Of the 24 samples observed to be positive for TGF-beta by immunocytochemistry, all were found to be positive for TGF-beta mRNA (PCR). Similarly, the 24 samples observed to be TGF-beta negative by immunocytochemistry were also negative for TGF-beta mRNA, indicating 100% specificity and 100% sensitivity of the PCR technique. PCR is therefore considered a viable technique for analysis of both ER and TGF-beta in small samples such as fine-needle aspirates.     

Fluorescent methylation-specific polymerase chain reaction for DNA-based detection of prostate cancer in bodily fluids

Promoter hypermethylation of the glutathione S-transferase P1 gene (GSTP1) is the most frequent DNA alteration in prostatic carcinoma. Because this epigenetic DNA alteration can be reliably detected by methylation-specific PCR (MSP), we applied this new technique for molecular detection of prostate cancer in various human bodily fluids. We investigated GSTP1 promoter hypermethylation in DNA isolated from plasma, serum, ejaculate, and urine after prostate massage and from prostate carcinoma tissues from 33 patients with prostate cancer and 26 control patients with benign prostatic hyperplasia (BPH). Fluorescently labeled MSP products were analyzed on an automated gene sequencer. Whereas GSTP1 promoter hypermethylation was not detectable by MSP in prostate tissue and bodily fluids from patients with BPH, we found it in 94% of tumors (16 of 17), 72% of plasma or serum samples (23 of 32), 50% of ejaculate (4 of 8) and 36% of urine (4 of 11) from patients with prostate cancer. Additionally, MSP identified circulating tumor cells in 30% (10 of 33) of prostate cancer patients. Analysis of GSTP1 promoter hypermethylation by MSP thus provides a specific tool for molecular diagnosis of prostate cancer in bodily fluids.