Multiplex Ligation-Dependent Probe Amplification Analysis on Capillary Electrophoresis Instruments for a Rapid Gene Copy Number Study

Annotated DNA samples that had been previously analyzed were tested using multiplex ligation-dependent probe amplification (MLPA) assays containing probes targeting BRCA1, BRCA2, and MMR (MLH1/MSH2 genes) and the 9p21 chromosomal region. MLPA polymerase chain reaction products were separated on a capillary electrophoresis platform, and the data were analyzed using GeneMapper v4.0 software (Applied Biosystems, Foster City, CA). After signal normalization, loci regions that had undergone deletions or duplications were identified using the GeneMapper Report Manager and verified using the DyeScale functionality. The results highlight an easy-to-use, optimal sample preparation and analysis workflow that can be used for both small- and large-scale studies.     

Improved Detection of Deletions and Duplications in the DMD Gene Using the Multiplex Ligation-Dependent Probe Amplification (MLPA) Method

The multiplex ligation-dependent probe amplification (MLPA) assay is the most powerful tool in screening for deletions and duplications in the dystrophin gene in patients with Duchenne and Becker muscular dystrophy (DMD/BMD). The efficacy of the assay was validated by testing 20 unrelated male patients with DMD/BMD who had already been screened by multiplex PCR (mPCR). We detected two duplications that had been missed by mPCR. In one DMD patient showing an ambiguous MLPA result, a novel mutation (c.3808_3809insG) was identified. MLPA improved the mutation detection rate of mPCR by 15 %. The results of our study (1) confirmed MLPA to be the method of choice for detecting DMD gene rearrangements in DMD/BMD patients, (2) showed that ambiguous MLPA amplification products should be verified by other methods, and (3) indicated that the MLPA method could be used in screening even for small mutations located in the probe-binding regions.     

Development of a Flow-Trough Microarray based Reverse Transcriptase Multiplex Ligation-Dependent Probe Amplification Assay for the Detection of European Bunyaviruses

It is suspected that apart from tick-borne encephalitis virus several additional European Arboviruses such as the sandfly borne Toscana virus, sandfly fever Sicilian virus and sandfly fever Naples virus, mosquito-borne Tahyna virus, Inkoo virus, Batai virus and tick-borne Uukuniemi virus cause aseptic meningo-encephalitis or febrile disease in Europe. Currently, the microarray technology is developing rapidly and there are many efforts to apply it to infectious diseases diagnostics. In order to arrive at an assay system useful for high throughput analysis of samples from aseptic meningo-encephalitis cases the authors developed a combined multiplex ligation-dependent probe amplification and flow-through microarray assay for the detection of European Bunyaviruses. These results show that this combined assay indeed is highly sensitive, and specific for the accurate detection of multiple viruses.     

多重连接依赖性探针扩增技术及其应用进展

多重连接依赖性探针扩增(MLPA)是一种高通量、针对待测核酸中靶序列进行定性和相对定量分析的新技术。该技术具有分辨率高、操作简便、设备要求低等诸多优势而广泛用于检测人类基因组内拷贝数变异(CNV)。近年来,MLPA在技术与应用上又有许多新的发展,如运用化学合成法制备3'、5'探针,MLPA在基因甲基化检测、基因表达水平分析、基因部分片段重复区域拷贝数分析及转基因基因分型中的应用,并且MLPA与基因芯片微阵列技术的结合,使得多重连接探针扩增真正具备了高通量检测能力。本文就MLPA技术及其应用进展作一综述。     

Spontaneous Epithelial-Mesenchymal Transition and Resistance to HER-2-Targeted Therapies in HER-2-Positive Luminal Breast Cancer

Resistance to trastuzumab, a rationally designed HER-2-targeting antibody, remains a major hurdle in the management of HER-2-positive breast cancer. Preclinical studies suggest the mechanisms of trastuzumab resistance are numerous. Unfortunately, the majority of these studies are based around HER-2-positive (HER-2+) luminal cell lines. The role of epithelial to mesenchymal transition (EMT), a genetic program that confers a basal phenotype, may represent a novel mechanism of escape for HER-2+ luminal cells from trastuzumab treatment. Here we investigated this possibility using a model of clonal selection in HER-2+ luminal breast cancer cells. Following a random isolation and expansion of “colony clusters” from SKBR-3 cell lines, several colony clusters underwent a spontaneous EMT in-vitro. In addition to expression of conventional EMT markers, all mesenchymal colony clusters displayed a predominant CD44+/CD24- phenotype with decreased HER-2 expression and elevated levels of a β1-integrin isoform with a high degree of N-glycosylation. Treatment with a β1-integrin function-blocking antibody, AIIB2, preferentially decreased the N-glycosylated form of β1-integrin, impaired mammosphere formation and restored epithelial phenotype in mesenchymal colony clusters. Using this model we provide the first clear evidence that resistance to trastuzumab (and lapatinib) can occur spontaneously as HER-2+ cells shift from a luminal to a basal/mesenchymal phenotype following EMT. While the major determinant of trastuzumab resistance in mesenchymal colony clusters is likely the down regulation of the HER-2 protein, our evidence suggests that multiple factors may contribute, including expression of N-glycosylated β1-integrin.     

荧光原位杂交检测乳腺癌HER-2基因的应用价值

目的:探讨荧光原位杂交(FISH)在检测乳腺癌组织HER-2基因扩增的应用价值.方法:收集50例手术切除的乳腺癌标本.经过处理和切片,FISH法检测细胞HER-2基因扩增情况,IHC法检测细胞膜上HER-2蛋白表达情况.kappa检验法对两种检测结果的一致性进行统计分析.结果:FISH法与IHC法检测结果总符合率为90%(K=0.75,P<0.05).在本组胶东半岛女性乳腺癌人群中HER-2基因扩增率为28%.结论:FISH法检测乳腺癌HER-2基因敏感性及稳定性较高,可作为最终确诊HER-2基因状态的方法.     

Genetic diagnosis of neurofibromatosis type 1: targeted next- generation sequencing with Multiple Ligation-Dependent Probe Amplification analysis

Background

Neurofibromatosis type 1 (NF1) is a dominantly inherited tumor predisposition syndrome that targets the peripheral nervous system. It is caused by mutations of the NF1 gene which serve as a negative regulator of the cellular Ras/MAPK (mitogen-activated protein kinases) signaling pathway. Owing to the complexity in some parts of clinical diagnoses and the need for better understanding of its molecular relationships, a genetic characterization of this disorder will be helpful in the clinical setting.

Methods

In this study, we present a customized targeted gene panel of NF1/KRAS/BRAF/p53 and SPRED1 genes combined with Multiple Ligation-Dependent Probe Amplification analysis for the NF1 mutation screening in a cohort of patients clinically suspected as NF1.

Results

In this study, we identified 73 NF1 mutations and two BRAF novel variants from 100 NF1 patients who were suspected as having NF1. These genetic alterations are heterogeneous and distribute in a complicated way without clustering in either cysteine–serine-rich domain or within the GAP-related domain. We also detected fifteen multi-exon deletions within the NF1 gene by MLPA Analysis.

Conclusions

Our results suggested that a genetic screening using a NGS panel with high coverage of Ras–signaling components combined with Multiple Ligation-Dependent Probe Amplification analysis will enable differential diagnosis of patients with overlapping clinical features.

     

曲妥珠单抗联合多西紫杉醇在HER-2 阳性晚期乳腺癌中的应用

目的:探讨曲妥珠单抗联合多西紫杉醇在HER-2阳性晚期乳腺癌中的应用。方法:收取2010年2月至2016年1月我院收治的82例HER-2阳性晚期乳腺癌患者作为研究对象,根据不同治疗方案分为观察组及对照组。观察组43例患者给予曲妥珠单抗联合多西紫杉醇治疗,多西紫杉醇以3周为1个周期连续用药4个周期,曲妥珠单抗连续使用8周~52周。对照组39例患者只给予曲妥珠单抗单药治疗。对两组患者临床疗效、临床受益反应指数以及不良反应发生情况进行观察与比较。结果:51P1察组总有效率(60.47%)高于对照组(43.59%),但差异无统计学意义(P0.05)。观察组疾病控制率(90.70%)显著高于对照组(71.79%),差异有统计学意义(P0.05)。观察组临床受益反应有效率(76.74%)显著高于对照组(41.03%)差异有统计学意义(P0.05)。两组患者不良反应无显著差异(P0.05)。结论:曲妥珠单抗联合多西紫杉醇对于HER-2阳性晚期乳腺癌有较好的临床疗效及安全性,患者临床受益高。     

The Added Value of Medical Testing in Underwriting Life Insurance

Background

In present-day life-insurance medical underwriting practice the risk assessment starts with a standard health declaration (SHD). Indication for additional medical screening depends predominantly on age and amount of insured capital. From a medical perspective it is questionable whether there is an association between the level of insured capital and medical risk in terms of mortality. The aim of the study is to examine the prognostic value of parameters from the health declaration and application form on extra mortality based on results from additional medical testing.

Methods

A history register-based cohort study was conducted including about 15.000 application files accepted between 2007 and 2010. Blood pressure, lipids, cotinine and glucose levels were used as dependent variables in logistic regression models. Resampling validation was applied using 250 bootstrap samples to calculate area under the curves (AUC’s). The AUC was used to discriminate between persons with and without at least 25% extra mortality.

Results

BMI and the overall assessment of the health declaration by an insurance physician or medical underwriter showed the strongest discrimination in multivariable analysis. Including all variables at minimum cut-off levels resulted in an AUC of 0.710 while by using a model with BMI, the assessment of the health declaration and gender, the AUC was 0.708. Including all variables at maximum cut-off levels lead to an AUC of 0.743 while a model with BMI, the assessment of the health declaration and age resulted in an AUC of 0.741.

Conclusions

The outcome of this study shows that BMI and the overall assessment of the health declaration were the dominant variables to discriminate between applicants for life-insurance with and without at least 25 percent extra mortality. The variable insured capital set by insurers as factor for additional medical testing could not be established in this study population. The indication for additional medical testing at underwriting life-insurance can possibly be done on limited variables instead of the obligatory medical testing based on age and the amount of insured capital.     

基于四引物扩增受阻突变体系PCR 的多重乳腺癌相关SNP 位点检测方法的建立

为提高单核苷酸多态性检测的通量, 引入多重嵌合引物PCR 和毛细管电泳对四引物扩增受阻突变体系PCR 进行改进. 针对乳腺癌位点rs4784227(C>T), rs1219648(G>A)和rs3803662(T>C)设计特异性嵌合引物, 经一次PCR扩增后, 通过毛细管电泳分析产物长度, 同时确定3 个位点的基因型. 70 份全血和口腔拭子样本, 电泳结果均与测序一致, 实现成功分型. 本方法仅需一次PCR 和一次毛细管电泳即可获得3 个位点的分型结果, 操作简单、快速准确.     

乳腺癌细胞表皮生长因子受体、HER-2对雌激素受体的调控及三苯氧胺耐药机制

雌激素受体(ER)在乳腺癌的发生和发展中起重要作用,抗雌激素治疗的内分泌治疗为重要的治疗方案,但易产生三苯氧胺(TAM)的耐药性而使治疗失效,原因之一可能是由于表皮生长因子受体(EGFR)和HER-2高表达引起ER磷酸化所致。本文概述了其中的分子机制和可能涉及的传导通路等。     

Prognostic Value of Molecular Breast Cancer Subtypes based on Her2, ESR1, PGR and Ki67 mRNA-Expression in Muscle Invasive Bladder Cancer

INTRODUCTION: Gene expression analyses have identified similarities between bladder and breast cancer, where clinical risk stratification is based on Her2, ESR1, PGR and Ki67 expression. The aim of the study was to assess the respective marker gene expression in patients treated with radical cystectomy for muscle-invasive bladder cancer (MIBC) and to evaluate the applicability of breast cancer subtypes for MIBC risk stratification. MATERIALS & METHODS: 102 patients treated with radical cystectomy for MIBC were assessed. Using routine FFPE tissue and an IVD validated kit, mRNA expression was measured by single step RT-qPCR. Partition test were employed to define cut-off values for high or low marker gene expression. Association of expression with outcome was assessed using Kaplan-Meier analysis and multivariate cox regression analysis. Finally, we performed validation of our results in the MD-Anderson cohort (n = 57). RESULTS: Cancer specific survival (CSS) was impaired in patients with high gene expression of Her2 (P = 0.0009) and ESR1 (P = 0.04). In the multivariate regression model Her2 expression remained significant for the prediction of CSS (HR = 2.11, CI 1.11-4.21, P = 0.024). Furthermore, molecular stratification by breast cancer subgroups was significant (P = 0.023) for CSS prediction. Especially the differentiation between Her2-positive and Luminal A (HR = 4.41, CI 1.53-18.71, P = 0.004) and Luminal B (HR = 1.96, CI 0.99-4.08, P = 0.053) respectively was an independent prognostic parameter for CSS. External validation resulted in comparable risk stratification with differences in fractional subgroups distribution. CONCLUSION: Gene expression of Her2, ESR1, PGR, Ki67 and corresponding breast cancer subtypes allow a risk-stratification in MIBC, whereby Her2 overexpressing tumors reveal a particularly poor prognosis.     

Expression of Autophagy-Related Proteins According to Androgen Receptor and HER-2 Status in Estrogen Receptor-Negative Breast Cancer

Purpose

The purpose of this study was to investigate the expression of autophagy-related proteins in relation to androgen receptor (AR) status in estrogen receptor (ER)-negative breast cancers.

Methods

We extracted 334 ER-negative breast cancer samples to construct tissue microarrays (TMAs), which were immunohistochemically stained for autophagy-related proteins (beclin-1, LC3A, LC3B, p62) and for AR and HER-2.

Results

There were 127 AR-positive cases and 207 AR-negative cases, and 140 HER-2-positive cases and 194 HER-2 negative cases. The AR-negative group was associated with tumoral LC3A expression (P<0.001), while the AR-positive group was associated with tumoral BNIP3 expression (P<0.001). Tumoral LC3A was most highly expressed in the AR-negative and HER-2 negative group, while stromal LC3A showed the highest expression in the AR-negative and HER-2-positive group. Tumoral BNIP3 and stromal BNIP3 were highest in the AR-positive and HER-2-negative group. In the AR-positive and HER-2-negative group, stromal p62 positivity was an independent factor that was statistically significant in its association with shorter disease-free survival (DFS) (Hazard ratio: 10.21, 95% CI: 1.130–92.31, P = 0.039). Shorter DFS was associated with tumoral LC3A positivity (Hazard ratio: 10.28, 95% CI: 2.068–51.19, P = 0.004) in the AR-negative and HER-2-positive group.

Conclusion

In ER-negative breast cancers, AR status was associated with expression of different types of autophagy-related proteins. Tumoral LC3A was most highly expressed in AR-negative breast cancers, while tumor BNIP3 was highest in AR-positive breast cancers.     

超声检查在乳腺癌诊断中价值的探讨

目的:探讨二维及彩色多普勒超声在乳腺癌诊断方面的临床应用价值。方法:对60例经手术病理证实的乳腺肿块病例的二维及彩色多普勒超声检查与病理结果进行回顾性分析。结果:乳腺癌诊断中,大多数恶性肿块表现为形态不规则、边缘粗糙、边界不清、内部回声不均匀、后方回声衰减。恶性肿块的血流显示率明显高,血流分布以Ⅱ、Ⅲ级为主。结论:二维声像图及彩色多普勒在乳腺癌诊断方面有较高的临床应用价值。由于良恶性肿物的影像互有交叉,其诊断符合率并非是100%。须两者联合应用,综合分析,方可提高超声对乳腺良恶性肿块的诊断符合率。     

超声检查在乳腺癌诊断中价值的探讨

目的：探讨二维及彩色多普勒超声在乳腺癌诊断方面的临床应用价值。方法：对60例经手术病理证实的乳腺肿块病例的二维及彩色多普勒超声检查与病理结果进行回顾性分析。结果：乳腺癌诊断中,大多数恶性肿块表现为形态不规则、边缘粗糙、边界不清、内部回声不均匀、后方回声衰减。恶性肿块的血流显示率明显高,血流分布以Ⅱ、Ⅲ级为主。结论：二维声像图及彩色多普勒在乳腺癌诊断方面有较高的临床应用价值。由于良恶性肿物的影像互有交叉,其诊断符合率并非是100%。须两者联合应用,综合分析,方可提高超声对乳腺良恶性肿块的诊断符合率。     

Modulation of Invasive Phenotype by Interstitial Pressure-Driven Convection in Aggregates of Human Breast Cancer Cells

This paper reports the effect of elevated pressure on the invasive phenotype of patterned three-dimensional (3D) aggregates of MDA-MB-231 human breast cancer cells. We found that the directionality of the interstitial pressure profile altered the frequency of invasion by cells located at the surface of an aggregate. In particular, application of pressure at one end of an aggregate suppressed invasion at the opposite end. Experimental alteration of the configuration of cell aggregates and computational modeling of the resulting flow and solute concentration profiles revealed that elevated pressure inhibited invasion by altering the chemical composition of the interstitial fluid near the surface of the aggregate. Our data reveal a link between hydrostatic pressure, interstitial convection, and invasion.     

GSTs同工酶基因在乳腺癌中的扩增与基因的表达

采用ＤＮＡ和ＲＮＡ的斑点杂交分析方法,对３２例乳腺癌和相应的癌旁正常组织中ＧＳＴ－π、ＧＳＴ－α和ＧＳＴ－μ基因的ＤＮＡ扩增和ＲＮＡ转录表达情况进行研究,发现ＧＳＴ－π在乳腺癌中存在基因扩增和明显的ｍＲＮＡ表达升高,ＧＳＴ－π基因表达调控主要在转录水平进行的;ＧＳＴ－α和ＧＳＴ－μ在乳腺癌中表达水平较低,但仍可见α和μ类ＧＳＴ同工酶ｍＲＮＡ转录在肿瘤和正常组织中发生了较大的变化。结合乳腺癌中雌激素受体（ＥＲ）表达情况还发现ＧＳＴ－π表达水平与ＥＲ的表达存在负相关性。