MYC amplification in breast cancer: a chromogenic in situ hybridisation study

AIMS: To analyse the correlation between MYC amplification and various clinicopathological features and outcome in a cohort of 245 patients with invasive breast carcinoma treated with surgery followed by anthracycline-based chemotherapy. Given the high prevalence of MYC amplification in tumours of BRCA1 mutation carriers and the similarities between these and sporadic "basal-like" carcinomas, the prevalence of MYC amplification in "basal-like" breast carcinomas was investigated. METHODS: MYC gene copy number was assessed on tissue microarrays containing duplicate cores of 245 invasive breast carcinomas by means of chromogenic in situ hybridisation using SpotLight C-MYC amplification probe and chromosome 8 centromeric probe (CEP8). Signals were evaluated at 400x magnification; 30 morphologically unequivocal neoplastic cells in each core were counted for the presence of the gene and CEP8 probes. RESULTS: Amplification was defined as a MYC:CEP8 ratio >2. Signals for both MYC and CEP8 were assessable in 196/245 (80%) tumours. MYC amplification was found in 19/196 cases (9.7%) and was not associated with tumour size, histological grade, positivity for oestrogen receptor, progesterone receptor, HER2, epidermal growth factor, cytokeratins 14, 5/6 and 17, MIB1 or p53. Only 4% of basal-like carcinomas showed MYC amplification, compared to 8.75% and 10.7% of luminal and HER2 tumours respectively. On univariate analysis, MYC amplification displayed a significant association with shorter metastasis-free and overall survival and proved to be an independent prognostic factor on multivariate survival analysis. CONCLUSION: MYC amplification is not associated with "basal-like" phenotype and proved to be an independent prognostic factor for breast cancer patients treated with anthracycline-based chemotherapy.     

脊髓性肌萎缩症SMN基因拷贝数定量分析

目的　探讨临床诊断为脊髓性肌萎缩症 (spinal muscular atrophy,SMA)而 PCR定性无运动神经元生存 (survival motor neuron,SMN )基因 T拷贝 (SMN - T)缺失患者的遗传基础 ;并探索 SMA表型与SMN基因 C(SMN- C)拷贝数的关系及 SMA患者及其直系亲属和正常人 SMN基因拷贝数的分布。方法对临床和病理诊断为 SMA ～ 型及少见型 4 5例患者、2 5名表型正常的 SMA直系亲属进行 SMN- T和SMN- C基因拷贝数定量分析 ,并与 33名正常人进行对比 ;所有对象均已经 PCR Dra 酶切法定性检测SMN基因 ,其中 ～ 型的 7例和 型的 2例为 SMN- T纯合缺失 ,余者无缺失。建立 SMN- T和囊性纤维化跨膜调节因子 (cystic fibrosis transmembrane conductance regulator,CFTR)的内标 ,所有标本进行非放射性、非荧光标记的多重竞争性 PCR,根据产物 SMN- T/ CFTR和 SMN- C/ CFTR比值 ,计算 SMN- T和SMN - C拷贝数。结果　 7例 ～ 型 SMN - T拷贝数均为 0 ; 型 2例拷贝数为 0 ,2例为 1个拷贝数 ,系杂合缺失 ,4例为 2个拷贝 ; 型及其他型患者均为 2个拷贝 ;直系亲属中 9例为 1个拷贝 ,系杂合缺失 ,其余及正常对照组均为 2个拷贝。SMN - C拷贝数在 SMA 型为≤ 2 , ～ 型为≤ 3, 型及其它型 SMA、直系亲属和正常对照组均为 0～ 3。结     

Parathyroid hormone-related protein in tissues of the emerging frog (Rana temporaria): immunohistochemistry and in situ hybridisation

Using antiserum to human parathyroid hormone-related protein (1–16) [PTHrP(1–16)] we have examined tissues of the common frog ( Rana temporaria ) for the presence of immunoreactive PTHrP (irPTHrP) at the stage of emergence from water to land. irPTHrP was detected in dorsal and ventral stratum granulosum of the skin, in the developing ovary, striated muscle and the choroid plexus epithelium of the brain as well as in the olfactory gland epithelium and olfactory lobe neurons of the brain. In the pituitary and hypothalamus irPTHrP protein could be demonstrated in the median eminence, infundibular stem and principally in the neural lobe and pars distalis of the pituitary with weak reaction in the pars intermedia. In situ hybridisation of the same tissues with an oligonucleotide probe to chicken PTHrP 55–65 clearly showed the presence of mRNA for PTHrP-like molecule in all the tissues containing irPTHrP. There was a major inconsistency in the pituitary in that the highest level of gene expression, assessed by in situ hybridisation, was found in the pars intermedia with only very low expression in the pars distalis and neural lobe and undetectable levels in the infundibular stem and median eminence. These observations suggest that tissues of the frog synthesise a PTHrP-like molecule but that in the pituitary the pars intermedia cells may export the protein to cells in other regions of the pituitary and hypothalamus.     

Angiofibroma of soft tissue with fibrohistiocytic features and intratumor genetic heterogeneity of NCOA2 gene rearrangement revealed by chromogenic in situ hybridization: A case report

Angiofibroma of soft tissue is a recently described soft tissue tumor that is characterized by fibroblastic spindle tumor cells with arborizing capillary proliferation. Cytogenetically, it harbors a specific fusion gene involving the nuclear receptor coactivator 2 (NCOA2) gene. We report here additional new pathological and cytogenetic features. A soft tissue tumor in the left thigh of 73‐year‐old female was investigated. Microscopically, histiocytoid tumor cells were scattered in an edematous background with branching capillary proliferation. Immunohistochemically, we identified that the tumor cells were positive for histiocytic markers such as CD68 and CD163. Rearrangement of the NCOA2 gene was detected successfully by chromogenic in situ hybridization; however, abnormal signal patterns were observed in only a small subset of tumor cells. Unlike typical tumors with bland spindle cells, the present tumor needs to be distinguished from myxoid, dendritic and clear cell tumors. This case may suggest that angiofibroma of soft tissue is not in the center of the fibroblastic/myofibroblastic tumor group, but rather shows a fibrohistiocytic nature. We also found intratumor genetic heterogeneity, which is uncommon for a translocation‐associated tumor. Therefore, careful evaluation is required to detect the gene rearrangement in this tumor entity.     

Evaluation of HER2 gene amplification in invasive breast cancer using a dual‐color chromogenic in situ hybridization (dual CISH)

Fluorescence in situ hybridization (FISH) assay is considered the ‘gold standard’ for evaluation of HER2/neu (HER2) gene status, however, it is difficult to recognize morphologic features of tumors using fluorescence microscopy. Thus, chromogenic in situ hybridization (CISH) has been proposed as an alternative method to evaluate HER2 gene amplification. Here, we examined the dual color CISH (dual CISH) method which provides information regarding the copy number of the HER2 gene and chromosome 17 centromere from a single slide. We examined 40 cases of invasive ductal carcinomas of the breast that were resected surgically. HER2 gene status was assessed with FISH (Abbott) and dual CISH (Dako). HER2 gene amplification status was classified according to the guidelines of the American Society of Clinical Oncology and College of American Pathologists (ASCO/CAP). Comparison of the cut‐off values for HER2/chromosome 17 centromere copy number ratio obtained by dual CISH and FISH showed that there was almost perfect agreement between two methods (Kappa coefficient 0.96). The results of the two commercial products were almost consistent for evaluation of HER2 gene counts on the sections. The current study proved that dual CISH is comparable with FISH for evaluating HER2 gene status.     

一种进行基因表达研究的有效技术--小鼠全胚胎RNA原位杂交

目的　建立小鼠整体胚胎水平研究基因表达的方法。　方法　采用地高辛配基标记的造血相关基因 Runx1和神经发生相关基因 Runx3RNA探针 ,对 10 .5～ 14天小鼠全胚胎进行 RNA原位杂交 ,通过检测胚胎组织中 m RNA的存在状况来观察基因的表达。结果　在小鼠胚胎观察到 Runx1和 Runx3基因在不同组织中的清晰的杂交信号 ;不同探针和不同大小的胚胎需要不同的最适蛋白酶 K处理条件。　结论　小鼠全胚胎 RNA原位杂交技术是一种有效的研究基因表达的方法 ,可以从整体水平反映基因表达的全貌 ,在功能基因组学时代将具有很大的应用潜力 ,为基因表达研究提供了一种可与 L ac Z染色和免疫组织化学媲美的选择。蛋白酶 K处理条件是否适当是小鼠全胚胎 RNA原位杂交成功的关键因素。     

Laser capture microdissection reveals dose-response of gene expression in situ consequent to asbestos exposure

The genes that mediate fibroproliferative lung disease remain to be defined. Prior studies from our laboratory showed by in situ hybridization and immunohistochemistry that the genes coding for tumour necrosis factor alpha, transforming growth factor beta, the platelet-derived growth factor A and B isoforms, and alpha-1 pro-collagen are expressed in fibroproliferative lesions that develop quickly after asbestos inhalation. These five genes, along with matrix metalloproteinase 9, a collagenase found to be increased in several lung diseases, are known to control matrix production and cell proliferation in humans and animals. Here we show by laser capture microdissection that (i) The six genes are expressed at significantly higher levels in the asbestos-exposed mice when comparing the same anatomic regions 'captured' in unexposed mice. (ii) The bronchiolar-alveolar duct (BAD) junctions, where the greatest number of fibres initially deposit, were always significantly higher than the other anatomic regions for each gene. The first alveolar duct bifurcation (ADB) generally was higher than the second ADB, the ADBs were always significantly higher than the airway walls and pleura, and the airway walls and pleura were generally higher than the unexposed tissues. (iii) Animals exposed for 3 days always exhibited significantly higher levels of gene expression at the BAD junctions and ADBs than animals exposed for 2 days. To our knowledge, this is the first demonstration of a dose-response to a toxic particle in situ, and this response appears to be dependent on the number of fibres that deposits at the individual anatomic site.     

荧光原位杂交技术检测BCR/ABL融合基因的研究

目的建立荧光原位杂交技术平台,应用FISH技术检测在CML检测BCR/ABL融合基因,探讨FISH技术在在CML中应用的价值。方法应用FISH对BCR/ABL探针进行前期的验证,建立正常阈值,再应用该探针检测CML中BCR/ABL融合基因,进行临床检测评估。结果主要假阳性信号模式的正常阈值为1G1R1F 11%、1G1R2F 2%。75例样本FISH检测出48例阳性,15例经细胞遗传学检测,12例检测结果与FISH结果一致,3例CG为阴性,FISH检测为阳性。结论荧光原位杂交技术应用于临床检测之前应进行探针的前期验证,制定一套规范实验流程,且FISH技术在CML诊断、分型、临床治疗方案的制定、预后的判断以及微小残留病变检测上均有重要的价值。     

Determination of HER2 amplification in primary breast cancer using dual‐colour chromogenic in situ hybridization is comparable to fluorescence in situ hybridization: a European multicentre study involving 168 specimens

García‐Caballero T, Grabau D, Green A R, Gregory J, Schad A, Kohlwes E, Ellis I O, Watts S & Mollerup J
(2010) Histopathology 56, 472–480
Determination of HER2 amplification in primary breast cancer using dual‐colour chromogenic in situ hybridization is comparable to fluorescence in situ hybridization: a European multicentre study involving 168 specimens Aims: Fluorescence in situ hybridization (FISH) can be used to reveal several genomic imbalances relevant to proper cancer diagnosis and to the correct treatment regime. However, FISH requires expensive and advanced fluorescence microscopes in addition to expertise in fluorescence microscopy. To determine whether a newly developed dual‐colour chromogenic in situ hybridization (CISH) method is a suitable alternative to FISH, we analysed the human epidermal growth factor receptor 2 gene (HER2) amplification level of 168 breast cancer specimens using dual‐colour CISH and FISH and compared the results. Methods and results: We found 100% agreement between HER2 status determined by FISH and dual‐colour CISH. Furthermore, we observed that the time used to score slides was significantly reduced by 28% in dual‐colour CISH compared with the FISH protocol. Concordance between HER2 protein status and dual‐colour CISH or FISH was equally good with an overall agreement of 96.8%. Correlation between the HER2/centromere 17 gene ratios obtained with dual‐colour CISH and FISH was highly significant with an overall correlation coefficient (ρ) of 0.96. Conclusions: We conclude that dual‐colour CISH and bright field microscopy are excellent alternatives to FISH when analysing the HER2 status of primary breast cancer.     

Dengue virus inoculation to human skin explants: an effective approach to assess in situ the early infection and the effects on cutaneous dendritic cells

Although dengue virus (DV) enters through skin while mosquitoes feed, early contacts remain unexplored regarding the cutaneous viral fate and in situ immune responses. We addressed this by exposing healthy, non-cadaveric, freshly obtained human skin explants to a human DV2 isolate. We demonstrated negative-strand DV-RNA and non-structural protein-1, both suggestive of viral replication in skin. Although control, mock-infected and DV-infected explants showed less (MHC-CII(+)/CD1a(+)/Langerin+) Langerhans cells, deranged morphology and decreased frequency were more apparent in DV-infected explants. Whereas DV+ cells were infrequent in epidermis and completely absent in dermis, some areas of basal epidermis were clearly DV+, presumably keratinocytes, cells where TUNEL positivity revealed apoptosis. Unlike fresh, control and mock-infected skin, DV-infected explants expressed CD80 and CD83, indicative of dendritic cell (DC) activation and maturation, respectively. However, sequential sections indicated that these cells were not DV+, suggesting that activated/mature DCs capable of priming T cells, probably, were not infected. Alternatively, the occasionally infected epidermal DC might not have reached maturation. Interestingly, skin DV infection apparently uncouples the DC activation/maturation process from another crucial DC function, the subsequent migration into dermis. This was suggested, because upon cutaneous DV infection, the few emerging CD83+ (mature) DCs remained within the outer epidermis, while no dermal CD83+ DCs were observed. These paradoxical effects might represent unknown DV subversion strategies. This approach is relatively easy, quick (results in 48 h), economical for developing countries where dengue is re-emerging and advantageous to evaluate in situ viral biology, immunity and immunopathology and potential antiviral strategies.     

Transglutaminase‐1 gene mutations in autosomal recessive congenital ichthyosis: Summary of mutations (including 23 novel) and modeling of TGase‐1

Autosomal recessive congenital ichthyosis (ARCI) is a heterogeneous group of rare cornification diseases. Germline mutations in TGM1 are the most common cause of ARCI in the United States. TGM1 encodes for the TGase‐1 enzyme that functions in the formation of the cornified cell envelope. Structurally defective or attenuated cornified cell envelop have been shown in epidermal scales and appendages of ARCI patients with TGM1 mutations. We review the clinical manifestations as well as the molecular genetics of ARCI. In addition, we characterized 115 TGM1 mutations reported in 234 patients from diverse racial and ethnic backgrounds (Caucasion Americans, Norwegians, Swedish, Finnish, German, Swiss, French, Italian, Dutch, Portuguese, Hispanics, Iranian, Tunisian, Moroccan, Egyptian, Afghani, Hungarian, African Americans, Korean, Japanese and South African). We report 23 novel mutations: 71 (62%) missense; 20 (17%) nonsense; 9 (8%) deletion; 8 (7%) splice‐site, and 7 (6%) insertion. The c.877‐2A>G was the most commonly reported TGM1 mutation accounting for 34% (147 of 435) of all TGM1 mutant alleles reported to date. It had been shown that this mutation is common among North American and Norwegian patients due to a founder effect. Thirty‐one percent (36 of 115) of all mutations and 41% (29 of 71) of missense mutations occurred in arginine residues in TGase‐1. Forty‐nine percent (35 of 71) of missense mutations were within CpG dinucleotides, and 74% (26/35) of these mutations were C>T or G>A transitions. We constructed a model of human TGase‐1 and showed that all mutated arginines that reside in the two beta‐barrel domains and two (R142 and R143) in the beta‐sandwich are located at domain interfaces. In conclusion, this study expands the TGM1 mutation spectrum and summarizes the current knowledge of TGM1 mutations. The high frequency of mutated arginine codons in TGM1 may be due to the deamination of 5′ methylated CpG dinucleotides. Hum Mutat 0, 1–12, 2009. © 2009 Wiley‐Liss, Inc.     

中国人消化道肿瘤发病的遗传背景因素——错配修复基因hMLH1错义突变Va1384Asp

目的 探讨中国人中存在的错配修复基因ｈＭＬＨ１ Ｖａ１３８４Ａｓｐ在大肠癌,胃癌,食道癌发病中的作用。方法 中国汉族人１０１例大肠癌患者,７９例胃癌患者,７６例食道癌患者,７９例和７６例胃癌和食道癌患者的亲属,１００名正常对照,各取正常体细胞,提取基因组ＤＮＡ。ＰＣＲ－ＳＳＣＰ和ＤＮＡ序列分析技术检测ｈＭＬＨ１基因的第１２外显子,比较分析Ｖａｌ３８４Ａｓｐ的检出率。     

In vitro marker gene expression analyses in human peripheral blood mononuclear cells: A tool to assess safety of influenza vaccines in humans

Vaccines are inoculated in healthy individuals from children to the elderly, and thus high levels of safety and consistency of vaccine quality in each lot must meet the required specifications by using preclinical and lot release testing. Because vaccines are inoculated into humans, recapitulation of biological reactions in humans should be considered for test methods. We have developed a new method to evaluate the safety of influenza vaccines using biomarker gene expression in mouse and rat models. Some biomarker genes are already known to be expressed in human lymphocytes, macrophages and dendritic cells; therefore, we considered some of these genes might be common biomarkers for human and mice to evaluate influenza vaccine safety. In this study, we used human peripheral blood mononuclear cells (PBMC) as a primary assessment tool to confirm the usefulness of potential marker genes in humans. Analysis of marker gene expression in PBMC revealed biomarker gene expressions were dose-relatedly increased in toxic reference influenza vaccine (RE)-stimulated PBMC. Although some marker genes showed increased expression in hemagglutinin split vaccine-stimulated PBMC, their expression levels were lower than that of RE in PBMC from two different donors. Many marker gene expressions correlated with chemokine production. Marker genes such as IRF7 were associated with other Type 1 interferon (IFN)-associated signals and were highly expressed in the CD304⁺ plasmacytoid dendritic cell (pDC) population. These results suggest PBMC and their marker genes may be useful for vaccine safety evaluation in humans.