荧光原位杂交与免疫组织化学方法对乳腺癌Her-2检测的比较

[目的]探讨荧光原位杂交(FISH)与免疫组织化学方法(IHC)检测乳腺癌组织中人类表皮生长因子受体2(Her-2)表达结果的差异.[方法]采用FISH和IHC法分别检测508例乳腺癌标本中Her-2基因扩增和Her-2蛋白表达状况.[结果]在IHC法检测Her-2蛋白表达0/+的104例(20.47％)标本中,FISH检出13例(2.56％)Her-2基因扩增,89例(17.52％)为非扩增,2例(0.39％)为可疑;Her-2蛋白表达4++的245例(48.23％)标本中,FISH检出121例(23.82％)Her-2基因扩增,120例(23.62％)为非扩增,4例(0.79％)为可疑;IHC在Her-2蛋白表达+++的159例(31.30％)标本中,FISH检出137例(26.97％) Her-2基因扩增,22例(4.33％)为非扩增.[结论]对于IHC法初筛Her-2蛋白为++～+++的患者必须进行FISH检测,以明确Her-2基因的状态,从而指导临床医师诊断治疗.     

应用FISH技术检测慢性淋巴细胞白血病ATM 和RB1基因缺失*

目的：探讨慢性淋巴细胞白血病（CLL）中11q22.3（ATM基因）和13q14（RB1 基因）的缺失情况以及荧光原位杂交（fluorescence in situ hybridization,FISH）技术检测慢性淋巴细胞白血病（CLL）染色体异常的价值,了解CLL 的分子遗传学特性。方法：分别用ATM和RB1 探针,运用荧光原位杂交（FISH）技术对10例CLL 确诊患者的11q22.3 和13q14染色体进行检测,并和常规细胞遗传学（Conventional cytogenetics,CC）检测方法,即G 显带法进行比较。结果：10例CLL 患者常规细胞遗传学检出2 例,其中t（5,9）,t（10,12）和+ 12各1 例;而FISH方法检出7 例至少有一种分子遗传学异常,del（11q22.3）4 例,纯合缺失2 例,del（13q14）7例,纯合缺失2 例。10例患者中4 例同时有del（11q22.3）和del（13q14）这2 种染色体异常。结论：FISH是一种在分析CLL 染色体异常方面较为快速、准确和敏感的有效技术,可提高染色体异常检出率,为CLL 提供较为准确的分子细胞遗传学信息,指导临床与预后分析。     

Increased Risk of Childhood Acute Lymphoblastic Leukemia (ALL) by Prenatal and Postnatal Exposure to High Voltage Power Lines : A Case Control Study in Isfahan,Iran

Childhood acute lymphoblastic leukemia (ALL) is one of the most common hematologic malignancies,accounting for one fourth of all childhood cancer cases. Exposure to environmental factors around the time ofconception or pregnancy can increase the risk of ALL in the offspring.This study aimed to evaluted the role ofprenatal and postnatal exposure to high voltage power lines on the incidence of childhood ALL.This cross-sectionalcase control study was carried out on 22 cases and 100 controls who were born and lived in low socioeconomicfamilies in Isfahan and hospitalized for therapeutic purposes in different hospitals from 2013-2014.With regardto the underlying risk factors, familial history and parental factors were noted but in this age, socioeonomic andzonal matched case control study, prenatal and childhood exposure to high voltage power lines was consideredas the most important environmental risk factors of ALL (p=0.006, OR=3.651, CI 95%, 1.692-7.878). As thepopulation was of low socioeconomic background, use of mobiles, computers and microwave was negligible.Moreover prenatal and postnatal exposure to indoor electrically charged objects was not determined to be asignificant environmental factor. Thus, pre and post natal exposure to high voltage power lines and living inpollutant regions as well as familial influence could be described as risk factors of ALL for the first time in alow socioeconomic status Iranian population     

卵巢上皮性癌 BRCA1 基因 mRNA 原位杂交检测的研究

目的研究在上皮性卵巢癌中BRCA1基因mRNA的表达情况及与病理参数的关系.方法采用原位杂交法检测 50 例卵巢上皮性癌组织中 BRCA1 基因 mRNA 的表达情况.结果BRCA1 mRNA 的阳性表达率在卵巢癌组织、良性肿瘤组织及正常组织中分别为 24%、81.8%和100%,癌组织中 BRCA1 基因的表达率显著低于良性肿瘤及正常组织,P＜0.01.随着卵巢癌分化程度的降低,BRCA1 基因表达缺失增多,P＜0.05;同时有淋巴结转移的癌组织,BRCA1 表达减少.结论BRCA1 基因 mRNA表达的减少可能与上皮性卵巢癌的发生发展有关,同时 BRCA1 mRNA 表达减少提示预后较差.     

Role of GSTM1 (Present/Null) and GSTP1 (Ile105Val) Polymorphisms in Susceptibility to Acute Lymphoblastic Leukemia among the South Indian Population

Acute lymphoblastic leukaemia (ALL) is the most common pediatric malignancy worldwide. The origin ofthis disease may be explained by a combination of genetic and environmental factors. Glutathione-s-transferasesare a multi-gene family of enzymes involved in the detoxification of a wide variety of environmental carcinogens.A total of 92 immunophenotyped cases (below 25 years of age) and 150 cord blood controls were here analysedby PCR for GSTM1(Present/Null) and RQ-PCR allelic discrimination assay for GSTP1(Ile105Val). We found asignificant increased risk for ALL with the GSTM1 null genotype (OR: 1.96, 95%CI=1.08-3.57), but no significantrisk was found with the GSTP1 (Ile/Val) genotype (OR: 1.32, 95%CI = 0.74-2.37) and the GSTP1 Val/Val genotype(OR: 1.41, 95%CI=0.5-3.96) alone. Combined analysis of GSTM1 and GSTP1 showed significant higher riskassociated with the GSTM1 (null/null) and GSTP1 [(Ile/Val)/ (Val/Val)] genotype (OR=2.78: 95%CI=1.16-6.69).     

Mutation Screening and Association Study of the Folylpolyglutamate Synthetase (FPGS) Gene with Susceptibility to Childhood Acute Lymphoblastic Leukemia

Background: Folylpolyglutamate synthetase (FPGS), an important enzyme in the folate metabolic pathway,plays a central role in intracellular accumulation of folate and antifolate in several mammalian cell types. Loss ofFPGS activity results in decreased cellular levels of antifolates and consequently to polyglutamatable antifolatesin acute lymphoblastic leukemia (ALL). Materials and Methods: During May 1997 and December 2003, 134children diagnosed with ALL were recruited from one hospital in Thailand. We performed a mutation analysis inthe coding regions of the FPGS gene and the association between single nucleotide polymorphisms (SNPs) withinFPGS in a case-control sample of childhood ALL patients. Mutation screening was conducted by polymerasechain reaction-single strand conformation polymorphism (PCR-SSCP) and subsequently with direct sequencing(n=72). Association analysis between common FPGS variants and ALL risk was done in 98 childhood ALL casesand 95 healthy volunteers recruited as controls. Results: Seven SNPs in the FPGS coding region were identifiedby mutation analysis, 3 of which (IVS13+55C>T, g.1297T>G, and g.1508C>T) were recognized as novel SNPs.Association analysis revealed 3 of 6 SNPs to confer significant increase in ALL risk these being rs7039798 (p=0.014, OR=2.14), rs1544105 (p=0.010, OR= 2.24), and rs10106 (p=0.026, OR= 1.99). Conclusions: These findingssuggested that common genetic polymorphisms in the FPGS coding region including rs7039789, rs1544105, andrs10106 are significantly associated with increased ALL risk in Thai children.     

TEL/AML1、BCR/ABL、E2A/PBX1、MLL/AF4阳性儿童急性淋巴细胞白血病的临床特点及预后

目的探讨儿童急性淋巴细胞白血病（ALL）TEL/AML1、BCR/ABL、E2A/PBX1、MLL/AF4四种融合基因的表达及其临床诊治意义。方法用实时荧光定量PCR(RQ-PCR)方法检测TEL/AML1、BCR/ABL（p190和p210两种亚型）、E2A/PBX1、MLL/AF4四种融合基因在312例ALL患儿中的表达情况,并总结融合基因阳性患儿的临床和生物学特点、治疗反应及预后情况。结果（1）融合基因阳性共120例,TEL/AML1阳性组72例(23.1%),BCR/ABL阳性组22例(7.1%),p190 16例（5.1%）、p210 6例（1.9%）,E2A/PBX1阳性组18例(5.8%),MLL/AF4阳性组8例(2.6%)。（2）TEL/AML1阳性组诊断时WBC平均值为(18.02±6.45)×109/L,诱导化疗结束完全缓解率(CR)100%,随访期间无复发;BCR/ABL阳性组发病时年龄(9.40±3.55)岁,诱导化疗结束CR 81.8%,强化治疗末仍有6例融合基因未转阴,随访中有8例（36.3%）复发,8例死亡;E2A/PBX1阳性组全部按高危标准给予化疗,诱导化疗结束CR 88.9%,强化治疗期末仍有3例基因微量表达,随访中2例复发;MLL/AF4阳性组发病时WBC(38.41±9.30)×109/L,强化治疗期末仍有2例基因呈阳性,随访中均复发死亡。结论融合基因可作为ALL危险度分层、监测微小残留病、判断预后的重要指标之一。     

Simultaneous Detection of BCL-2 Protein, Trisomy 12, Retinoblastoma and P53 Monoallelic Gene Deletions in B-Cell Chronic Lymphocytic Leukemia by Fluorescence in Situ Hybridization (FISH): Relation to Disease Status

Various genetic abnormalities are often found in B-CLL, but their relative importance in the pathogenesis and evolution of the disease has not been adequately clarified. We studied the expression of bcl-2 protein and the possible simultaneous occurrence of bcl-2 overexpression, trisomy 12 and the Rbl and p53 gene deletions in 38 patients with B-CLL by combining immunophenotyping and dual color interphase FISH. We also looked for correlation between the genetic abnormalities and clinical parameters such as stage, disease duration from diagnosis to the time of study and overall survival. High expression of the bcl-2 protein was found in 76.3% of the patients (29/38). Trisomy 12 was found in 37% of cases (14/38) and Rbl monoallelic gene deletion in 42% (16/38). The percentage of cells with hemizygous Rbl deletion ranged from 13 to 18%. Monoallelic deletion of p53 was found in 29% of cases (11/38). The number of cells with only one signal ranged from 28 to 98%. Patients in stage A had on average, less than one abnormality, while patients in stage C had 2.6 abnormalities. Patients appeared to accumulate genetic abnormalities with time. Bcl-2 overexpression was found early in the course of the disease. Trisomy 12 appeared later, at about the same time as Rbl deletion, but was not associated with adverse prognosis. Monoallelic deletion of p53 gene appeared rather late in the course of the disease and was associated with advanced stage. Despite the fact that more deaths occurred in the group of patients with three or four abnormalities and the presence of p53 gene deletion, differences in survival were not statistically significant, probably due to the limited number of patients in each group. A larger group of patients studied in a prospective manner will better clarify these issues in the future.     

PD-L1 Expression by Two Complementary Diagnostic Assays and mRNA In Situ Hybridization in Small Cell Lung Cancer

IntroductionTherapeutic antibodies to immune checkpoints show promising results. Programmed death-ligand 1 (PD-L1), an immune checkpoint ligand, blocks the cancer immunity cycle by binding the PD-L1 receptor (programmed death 1). We investigated PD-L1 protein expression and messenger RNA (mRNA) levels in SCLC.MethodsPD-L1 protein expression and mRNA levels were determined by immunohistochemistry (IHC) with SP142 and Dako 28-8 PD-L1 antibodies and in situ hybridization in primary tumor tissue microarrays in both tumor cells and tumor-infiltrating immune cells (TIICs) obtained from a limited-disease SCLC cohort of 98 patients. An additional cohort of 96 tumor specimens from patients with extensive-disease SCLC was assessed for PD-L1 protein expression in tumor cells with Dako 28-8 antibody only.ResultsThe overall prevalence of PD-L1 protein expression in tumor cells was 16.5%. In the limited-disease cohort, the prevalences of PD-L1 protein expression in tumor cells with SP142 and Dako 28-8 were 14.7% and 19.4% (tumor proportion score cutoff ≥1%) and PD-L1 mRNA ISH expression was positive in 15.5% of tumor samples. Increased PD-L1 protein/mRNA expression was associated with the presence of more TIICs (p < 0.05). The extensive-disease cohort demonstrated a 14.9% positivity of PD-L1 protein expression in tumor cells with Dako 28-8 antibody.ConclusionsA subset of SCLCs is characterized by positive PD-L1 and/or mRNA expression in tumor cells. Higher PD-L1 and mRNA expression correlate with more infiltration of TIICs. The prevalence of PD-L1 in SCLC is lower than that published for NSCLC. The predictive role of PD-L1 expression in SCLC treatment remains to be established.     

非小细胞肺癌EGFR荧光原位杂交与免疫组化检测的比较

目的探讨荧光原位杂交（FISH）技术和免疫组化（IHC）法检测石蜡标本非小细胞肺癌（NSCLC）EGFR基因扩增及蛋白表达水平的差异性。方法采用FISH和IHC分别检测27例NSCLC患者石蜡标本EGFR基因和蛋白表达,对2种方法的检测结果进行对比分析。结果 14例IHC法EGFR表达（3＋）的标本中有9例FISH显示阳性（64.29%）,其中5例为EGFR基因高多体性扩增（55.56%）,4例为EGFR基因扩增（44.44%）;6例IHC（2＋）的标本中仅1例为高多体性扩增（16.67%）;2例IHC（1＋）及5例IHC（-）标本均无EGFR基因扩增。结论 IHC法初筛（3＋）、（2＋）的标本与FISH检测的符合率较低,提示对IHC检测EGFR表达为（3＋）及（2＋）并选择靶向药物治疗的病例,应采用FISH法对EGFR基因表达作进一步检测。     

多色荧光原位杂交方法同时检测人精子染色体数目畸变和结构畸变

背景与目的:建立可以同时检测人精子染色体数目畸变和结构畸变的多色荧光原位杂交技术.材料与方法:使用2条1号染色体探针(着丝粒和末端探针),2条18号染色体着丝粒探针,分别用地高辛或生物素标记,与人精子核DNA进行荧光原位杂交,用CY3-链亲和素检测生物素探针杂交信号;用与FITC结合的抗地高辛抗体检测地高辛信号,结果:在Nikon荧光显微镜下,可以清楚看到精子头部呈现兰色的荧光信号背景下,有红色荧光点信号(1号染色体着丝粒),绿色荧光信号(1号染色体末端),和黄色荧光信号(18号染色体着丝粒部位红、绿两种荧光信号的混合色).本方法能够同时检测到精子1号,18号染色体非整倍体率(双体率,缺体率),1号染色体短臂和着丝粒结构畸变(重复率,缺失率),以及二倍体精子率3种染色体异常.用建立的方法检测14名正常人135 937条精子,测得1号染色体双体率、缺体率分别为0.045%和0.048%;18号染色双体率、缺体率分别为0.053%和0.045%;二倍体精子率为0.061%;1号染色体末端重复率、缺失率分别为0.082%、0.069%,1号着丝粒重复率、缺失率分别为0.075%,0.060%;均在文献报道范围内.结论:本研究建立的多色FISH可用于测定正常人和环境化学物接触人群精子染色体数目畸变和结构畸变.     

遗传学和免疫表型分析在急性粒单核细胞白血病临床诊断中的意义

 目的　探讨染色体G显带、荧光原位杂交和流式细胞术在急性粒单核细胞白血病临床诊断、鉴别诊断和预后中的意义。方法　运用染色体G显带和流式细胞术 ,对 15例骨髓细胞形态学拟诊为急性髓系白血病M4型 (AML M4 )的患者进行核型分析和免疫分型 ,并用间期荧光原位杂交技术 (inter phasalfluorescenceinsituhybridization ,I FISH)对其中 6例患者进行检测。结果　染色体G显带核型分析显示正常核型 6例 ,伴有inv(16 ) (p13;q2 2 )异常 2例 ,伴有del(16 ) (q2 2 )、 X ,5 q ,t(8;2 1) (q2 2 ;q2 2 )、t(9 ;2 2 ) (q34;q11)、t(11;17)和t(11;?)异常各 1例 ,另有 2例无分裂相可供分析。I FISH对 3例患者进行CBFβ基因重排检测 ,2例阳性 ,1例阴性 ;对这例阴性患者进一步检测AML1/ETO融合基因 ,结果阳性 ;3例患者检测了MLL基因重排 ,2例阳性 ,1例阴性但伴有t(9;2 2 )异常 ,BCR/ABL融合基因阳性。免疫分型提示 14例患者有髓系和单核系分化抗...     

Oncogene-independent resistance in Philadelphia chromosome - positive (Ph+) acute lymphoblastic leukemia (ALL) is mediated by activation of AKT/mTOR pathway

Tyrosine kinase inhibitors (TKIs) such as imatinib, nilotinib, dasatinib, and ponatinib have significantly improved the life expectancy of Philadelphia chromosome-positive (Ph⁺) acute lymphocytic leukemia (ALL) patients; however, resistance to TKIs remains a major clinical challenge. Point mutations in the tyrosine kinase domain (TKD) of BCR-ABL1 have emerged as the predominant cause of acquired resistance. In approximately 30% of patients, the mechanism of resistance to TKIs remains elusive. This study aimed to investigate mechanisms of nonmutational resistance in Ph⁺ ALL. Here we report the development of a nonmutational resistance cell line SupB15-RT; conferring resistance to approved ABL kinase inhibitors (AKIs) and allosteric inhibitors GNF-2, ABL001, and crizotinib, except for dasatinib (IC90 50nM), a multitarget kinase inhibitor. We found that the AKT/mTOR pathway is activated in these cells and their proliferation inhibited by Torin-1 with an IC50 of 24.7 nM. These observations were confirmed using 3 different ALL patient-derived long term cultures (PDLTCs): (1) HP (BCR-ABL1 negative), (2) PH (BCR-ABL1 positive and responsive to TKIs) and (3) BV (BCR-ABL1 positive and nonmutational resistant to TKIs). Furthermore, Torin-1 and NVP-BEZ235 induced apoptosis in PH and BV cells but not in HP cells.Our experiments provide evidence of the involvement of AKT/mTOR pathway in the evolution of nonmutational resistance in Ph⁺ ALL which will assist in developing novel targeted therapy for Ph⁺ ALL patients with BCR-ABL1 independent nonmutational resistance.     

Regulatory Effect of Resveratrol and Prednisolone on MDR1 Protein Expression in Acute Lymphoblastic Leukemia Cell Line (CCRF-CEM)

Objective: Chemotherapy is the most widely recognized technique to regard leukemia and also different sorts ofhuman tumors. In any case, tranquilize protection has stayed as the primary test against the adequacy of medications.Besides, having different unfriendly impacts, chemotherapy drugs are getting to be traded by characteristic modalities forgrowth treatment. In such manner, natural segments, for example, resveratrol and prednisolone have been recognized tosharpen the leukemic cells to modified cell demise through an arrangement of complex procedures. In this investigation,we have analyzed effect of 15, 50 and 100μM of resveratrol and 700μM of prednisolone on the human multidrugprotection quality 1 (MDR1) as a notable marker for cell sedate protection. We assessed the impact of resveratrol andprednisolone on MDR1 protein expression in the CCRF-CEM cell line as an agent for intense lymphoblastic leukemia.The investigation was planned to clear up whether. Materials and methods: CCRF-CEM cells linage get underdrug treatment with use of resveratrol and prednisolone. Western blot use at 24 and 48 hours with different doses ofresveratrol and prednisolone to analysis of MDR1 expression changes. Results: Effect of 15, 50, and 100 micro molarof resveratrol and 700 micro molars of prednisolone on CCRF-CEM cells led to the MDR1 decrease. Western blot usefor evaluation of MDR1 protein expression changes. Conclusion: In the present study, we observed that resveratroland prednisolone, with a dose-dependent effect, can reduce the expression of the MDR1 protein. This reduction ofexpression demonstrates that resveratrol and prednisolone can overcome to drug resistance created by MDR1.     

Chromosomal Analysis of Purified B-Chronic Lymphocytic Leukemia Lymphocyte Cultures: Comparison with Whole Blood Cultures and in Situ Hybridization

Chromosomal analysis of stimulated whole blood cells and purified B lymphocytes was performed in 13 stage A(0) and 1 stage C(1V) chronic lymphocytic leukemia (B-CLL) patients. Abnormal clones were found in 6 cases in purified B lymphocytes cultures and in a single one in whole blood cultures. In situ hybridization with a chromosome 12 probe was in accordance with the chromosomal analysis of purified B-CLL lymphocytes and not with the results obtained using whole blood culture. Cytogenetic analysis of isolated B cells is simple and sensitive. It enhances the detection of abnormal clones in B-CLL and applied to larger series of patients, it should allow a precise evaluation of the incidence of chromosomal abnormalities in CLL and of their clinical (prognostic) significance.