Massively‐parallel sequencing assists the diagnosis and guided treatment of cancers of unknown primary

The clinical management of patients with cancer of unknown primary (CUP) is hampered by the absence of a definitive site of origin. We explored the utility of massively‐parallel (next‐generation) sequencing for the diagnosis of a primary site of origin and for the identification of novel treatment options. DNA enrichment by hybridization capture of 701 genes of clinical and/or biological importance, followed by massively‐parallel sequencing, was performed on 16 CUP patients who had defied attempts to identify a likely site of origin. We obtained high quality data from both fresh‐frozen and formalin‐fixed, paraffin‐embedded samples, demonstrating accessibility to routine diagnostic material. DNA copy‐number obtained by massively‐parallel sequencing was comparable to that obtained using oligonucleotide microarrays or quantitatively hybridized fluorescently tagged oligonucleotides. Sequencing to an average depth of 458‐fold enabled detection of somatically acquired single nucleotide mutations, insertions, deletions and copy‐number changes, and measurement of allelic frequency. Common cancer‐causing mutations were found in all cancers. Mutation profiling revealed therapeutic gene targets and pathways in 12/16 cases, providing novel treatment options. The presence of driver mutations that are enriched in certain known tumour types, together with mutational signatures indicative of exposure to sunlight or smoking, added to clinical, pathological, and molecular indicators of likely tissue of origin. Massively‐parallel DNA sequencing can therefore provide comprehensive mutation, DNA copy‐number, and mutational signature data that are of significant clinical value for a majority of CUP patients, providing both cumulative evidence for the diagnosis of primary site and options for future treatment. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Diagnosis and management of metastatic neoplasms with unknown primary

In cancer of unknown primary (CUP), metastases are clinically and histologically confirmed, but the primary tumor site remains elusive after extensive work-up. CUPs make up for 2–3% of all epithelial malignancies. The two prevailing histologies are adenocarcinomas and undifferentiated carcinomas, whereas squamous cell carcinomas, neuroendocrine carcinomas and rare histologies account for the remaining 10%.The diagnostic work-up in CUP relies strongly on a detailed immunohistological (IHC) analysis in order to characterize the tumor type, nowadays aided by molecular techniques. Diagnostics also include a thorough clinical examination, a basic lab draw with the most relevant tumor markers, and cross sectional imaging. Additional PET-CT is recommended in cervical lymph nodes suggestive of head and neck cancer and in limited metastases potentially treatable in curative intent.As for treatment, it is paramount to identify patients who fall into one of the six well defined “favorable” subset categories, namely extragonadal germ cell tumors, adenocarcinoma with isolated unilateral axillary lymph nodes in female patients, squamous cell carcinoma with neck lymph nodes, squamous cell carcinoma with inguinal lymph nodes, serous papillary peritoneal carcinomatosis in females and blastic bone metastasis in males with elevated PSA. These subsets are distinct both regarding the required treatment and the comparably favorable prognosis. Within the remaining “unfavorable” group, patients of colon and renal cancer type should be identified based on IHC and clinical picture, since the prognosis of these patients seems to improve with the use of therapy tailored to the presumed primary as well. For the few patients with limited metastases it should be assessed whether they are candidates for surgery, radiotherapy or surgery followed by irradiation in curative intent. The remaining majority of patients are treated with empiric palliative chemotherapy, typically a platinum – paclitaxel combination, though the level of evidence for this therapy recommendation is low. Gemcitabine alone or in combination can be used as an alternative.Decoding of the molecular profiles in CUP offers the prospect of targeted therapy with novel agents. However, there appears to be no uniform molecular pattern for CUP, and the observed molecular diversity thus poses a challenge to respective clinical trials.     

Expression profiling technology: its contribution to our understanding of breast cancer

Breast cancer is a complex genetic disease characterized by the accumulation of multiple molecular alterations. Routine clinical management of breast cancer relies on clinical and pathological factors, however. These seem insufficient to reflect the whole clinical heterogeneity of tumours and are less than perfectly adapted to each patient. Recent advances in human genome research and high-throughput molecular technologies have made it possible to tackle the molecular complexity of breast cancer and have contributed to the realization that the biological heterogeneity of breast cancer has implications for treatment. Gene expression profiling of breast cancer has been performed using several approaches. This review will describe the details of gene expression profiling of breast cancer, the different approaches and the impact on clinical management.     

Impact of gene expression profiling in lymphoma diagnosis and prognosis

Lymphoma classification has changed several times over time as our understanding of normal and malignant lymphocyte biology has advanced. This has improved prognostication, but there remain large diagnostic groups with diverse outcomes. In an attempt to refine diagnosis and prognostic power in these, global gene expression profiling (GEP) has been used to further improve our understanding of lymphoma. This review will cover the impact of GEP on the diagnosis, prognosis, biological understanding and identification of novel treatments for the main types of lymphoma, as well its translation to clinical practice. Specifically, it will cover the use of GEP to identify prognostic subgroups within existing diagnostic categories, in an attempt to improve prognostication in those subgroups with wide variation in outcome. Many of these studies have given additional novel insights into the biology of lymphoma, including the role of the immune system and the stromal environment. The improved understanding that these studies have given have suggested possible new treatments, linking diagnosis, prognosis, biological understanding and improved treatment.     

Gene expression profiling of colorectal cancer and metastases divides tumours according to their clinicopathological stage

Gene expression profiling of matched colorectal carcinomas and metastases could reveal key molecular events involved in tumour progression and metastasis. Expression profiles have been created from 25 colorectal carcinomas (CRCs, pT1-4), corresponding normal colonic mucosa, and 14 liver metastases using cDNA arrays containing 1176 cancer-related genes (Clontech). Hierarchical clustering clearly distinguished carcinomas from non-cancerous tissues, separated tumours into high-stage (pT4 and extensive lymph node or distant metastases) and low-stage (< or =pT3) groups, and correlated with the histopathological classification in 87% (33/38 cases). Most primary tumours and matched liver metastases clustered on terminal branches of the dendrogram. Statistical analysis (Mann-Whitney U-test) revealed 40 tumour-specific genes (29 up-regulated, 11 down-regulated) which allowed identification of malignant tissue samples by cluster analysis. A specific expression signature in matching metastases was not found, but a set of 23 classifier genes with statistically significant expression patterns in high- and low-stage tumours was identified. These genes may represent important targets in colorectal carcinogenesis and might provide useful clinicopathological tools in the management of colorectal cancer.     

基于DNA芯片的细菌基因表达谱技术的建立与评价

目的建立和优化基于全基因组DNA芯片的细菌基因表达谱技术，并用实时定量RT-PCR对此技术平台进行评价。方法提取并纯化鼠疫耶尔森菌总RNA，以六核苷酸随机引物（N6）和／或基因组特异引物（GDP）逆转录合成cDNA，用CY染料直接或间接标记后，与包括鼠疫耶尔森菌4005个ORF的全基因组芯片杂交，通过芯片扫描仪获得表达谱分析结果，归一化后进行分析。结果用实时定量PCR技术验证。结果采用N6＋GDP混合引物以间接法标记获得了较为理想的结果，表达谱技术与实时定量PER技术所得结果高度相关，证明了此技术的可靠性和实验设计与数据分析的合理性。结论合理的设计和归一化的分析方法是基于DNA芯片的细菌基因表达谱分析成功的关键，该技术的建立为细菌功能基因组研究奠定了基础。     

Asthma-predictive genetic markers in gene expression profiling of peripheral blood mononuclear cells

Purpose

We sought to identify asthma-related genes and to examine the potential of these genes to predict asthma, based on expression levels.

Methods

The subjects were 42 asthmatics and 10 normal healthy controls. PBMC RNA was subjected to microarray analysis using a 35K array; t-tests were used to identify genes that were expressed differentially between the two groups. A multiple logistic regression analysis was applied to the differentially expressed genes, and area under the curve (AUC) values from receiver operating characteristic (ROC) curves were obtained.

Results

In total, 170 genes were selected using the following criteria: P≤0.001 and ≥2-fold change. Among these genes, 57 were up-regulated and 113 were down-regulated in asthmatics versus normal controls. A multiple logistic regression analysis was done using more stringent criteria (P≤0.001 and ≥5-fold change), and eight genes were selected as candidate asthma biomarkers. Using these genes, 255 models (2⁸-1) were generated. Among them, only 85 showed P≤0.05 by multiple logistic regression analysis. Based on the AUCs from ROC curves for the 85 models, we found that the best model consisted of the genes MEPE, MLSTD1, and TRIM37. The model showed 0.9928 of the AUC with 98% sensitivity and 80% specificity.

Conclusions

MEPE, MLSTD1, and TRIM37 may be useful biomarkers for asthma.     

Gene expression profiling of endocrine tumors using DNA microarrays: progress and promise

Genomewide profiling of gene expression, made possible by the development of DNA microarray technology and more powerful by the sequencing of the human genome, has led to advances in tumor classification and biomarker discovery for the common types of human neoplasia. Application of this approach to the field of endocrine neoplasia is in its infancy, although some progress has been recently reported. In this review, the progress to date is summarized and the promise of DNA microarray analysis in conjunction with tissue array immunohistochemistry to significantly impact endocrine tumor diagnosis and prognosis is discussed.     

Best practices for the extraction of genomic DNA from formalin-fixed paraffin-embedded tumor tissue for cancer genomic profiling tests

Recently, two cancer genomic profiling tests have been approved in Japan and implemented in routine clinical practice: the FDA-approved FoundationOne CDx test, and the OncoGuide NCC Oncopanel test. The quality and quantity of DNA significantly affects the sequencing results; therefore, preparing a sufficient amount of high-quality DNA for clinical cancer genomic profiling tests is important. We examined the best practices for the extraction of cancer genomic DNA from formalin-fixed paraffin-embedded (FFPE) tumor tissues of pancreatic, lung and colon cancer specimens. We found that the quality of cancer genomic DNA extracted from 10-μm-thick FFPE samples improved significantly, compared with that from 4-μm-thick FFPE samples, suggesting that 10-μm-thick FFPE samples are preferable for clinical cancer genomic profiling tests. For convenience, we created a quick reference table for calculating the required number of FFPE slides.     

云南省病毒性脑炎和不明原因发热患者的相关病毒IgM抗体检测及分析

目的 调查云南省病毒性脑炎和不明原因发热病例的发病病因.方法 在云南省部分地区采集病毒性脑炎和不明原因发热患者血清和脑脊液标本,用ELISA法检测患者标本中包括虫媒病毒、肠道病毒和呼吸道病毒中15种病毒的IgM抗体.结果 在云南省共采集病毒性脑炎患者血清和脑脊液标本526份,不明原因发热患者血清标本221份.经ELISA法检测,病毒性脑炎患者标本中乙型脑炎病毒、柯萨奇病毒、埃可病毒、版纳病毒、单纯疱疹病毒、腮腺炎病毒、登革热病毒和罗斯河病毒的IgM抗体阳性率依次为50.95％(阳性数=268)、21.48％ (113)、19.58％(103)、7.41％ (39)、3.04％(16)、1.71％(9)、1.00％(5)和0.38％(2);不明原因发热患者血清中乙型脑炎病毒、登革热病毒、单纯疱疹病毒、版纳病毒、腮腺炎病毒、柯萨奇病毒、埃可病毒、罗斯河病毒和巴马森林病毒的IgM抗体阳性率依次为21.27％ (47)、20.36％ (45)、13.12％(29)、12.67％ (28)、11.76％ (26)、4.52％(10)、1.81％(4)、1.81％(4)和0.45％(1).乙型脑炎病毒、登革热病毒、版纳病毒、罗斯河病毒和巴马森林病毒感染以7-10月为主,柯萨奇病毒、埃可病毒、单纯疱疹病毒和腮腺炎病毒感染以7-8月居多.结论 乙型脑炎病毒是云南省夏秋季病毒性脑炎流行的主要病原体,柯萨奇、埃可和单纯疱疹病毒是散发性病毒性脑炎的重要病原;登革、版纳、单纯疱疹和腮腺炎病毒是不明原因发热的重要病毒病原并可能存在罗斯河和巴马森林等蚊媒病毒的流行.     

胃癌高表达cDNA片段W41的克隆及组织表达谱分析

目的：从人胃癌组织中克隆新的相关易感基因。方法：利用cDNA末端快速扩增PCR技术得到了扩增片段，将其克隆、核苷酸序列分析，并将序列在GenBank进行同源性比较。利用Northern杂交、多组织Northern及基因表达系列分析了所得基因片段的组织表达谱。结果：获得1条533bp带有poly(A)尾的cDNA片段，可见加尾信号AATAAA，与GenBank基因数据库同源比较，该序列未见与任何已知基因同源，登录GenBank(登录号为AF325202）。该序列在胃癌组织的表达强度高于对应正常组织，多组织Northern及基因表达系列分析表明该序列在多种肿瘤组织中高表达，在正常组织中表达减弱。结论：得到了1条与胃癌可能相关的新的cDNA序列。     

Benzodiazepines use and breast cancer risk: A population-based study and gene expression profiling evidence

The aim of this study was to investigate whether long-term use of Benzodiazepines (BZDs) is associated with breast cancer risk through the combination of population-based observational and gene expression profiling evidence. We conducted a population-based case-control study by using 1998 to 2009 year Taiwan National Health Insurance Research Database and investigated the association between BZDs use and breast cancer risk. We selected subjects age of >20 years old and six eligible controls matched for age, sex and the index date (i.e., free of any cancer at the case diagnosis date) by using propensity scores. A bioinformatics analysis approach was also performed for the identification of oncogenesis effects of BZDs on breast cancer. We used breast cancer gene expression data from the Cancer Genome Atlas and perturbagen signatures of BZDs from the Library of Integrated Cellular Signatures database in order to identify the oncogenesis effects of BZDs on breast cancer. We found evidence of increased breast cancer risk for diazepam (OR, 1.16; 95%CI, 0.95–1.42; connectivity score [CS], 0.3016), zolpidem (OR, 1.11; 95%CI, 0.95–1.30; CS, 0.2738), but not for lorazepam (OR, 1.04; 95%CI, 0.89–1.23; CS, -0.2952) consistently in both methods. The finding for alparazolam was contradictory from the two methods. Diazepam and zolpidem trends showed association, although not statistically significant, with breast cancer risk in both epidemiological and bioinformatics analyses outcomes. The methodological value of our study is in introducing the way of combining epidemiological and bioinformatics approaches in order to answer a common scientific question. Combining the two approaches would be a substantial step towards uncovering, validation and further application of previously unknown scientific knowledge to the emerging field of precision medicine informatics.     

Expression profiling and interferon-beta regulation of liver metastases in colorectal cancer cells

Liver metastasis is the major cause of death among colorectal cancer patients. Many gene products have been associated with the colon cancer cells' ability to metastasize to the liver, including carcinoembryonic antigen (CEA) and mucins. In this study we examined changes in expression of 384 genes in a model of human colorectal cancer metastasis in nude mice. Using DNA microarrays, we compared expression between MIP-101 cells, a poorly metastatic human colon cancer cell line, with an interferon-beta (IFN-β) resistant subline of MIP-101 (β-MIP) that is metastatic to the liver. Treatment of β-MIP cells with increasing concentrations of IFN-β caused a reversion to the non-metastatic phenotype. The array data showed down-regulation of genes involved in apoptosis in β-MIP cells and their return to the MIP-101 pattern upon IFN-β treatment. Cluster analysis also showed involvement of genes belonging to cell cycle, angiogenesis and invasion pathways. Selected genes were chosen to validate the microarray data by semi-quantitative RT-PCR. Association between gene expression pattern and metastatic phenotype was verified by intra-splenic injection in nude mice. The number of genes examined in this study was small, but carefully selected. Significant changes associated with cell growth and survival were observed, which gave the metastatic cells an advantage to grow in the liver. This information may help identifying new markers for colorectal cancer prognosis as well as aid the development of new therapeutic approaches. This revised version was published online in July 2006 with corrections to the Cover Date.     

Global gene expression profiling of formalin-fixed paraffin-embedded tumor samples: a comparison to snap-frozen material using oligonucleotide microarrays

Oligonucleotide microarrays are widely used to investigate gene expression in a large-scale approach. A major limitation is the dependency on frozen material to obtain high-quality ribonucleic acid because most clinical specimens are formalin-fixed and paraffin-embedded (FFPE). The ability to analyze these samples using microarrays would enlarge the investigable sample stocks manifold. We conducted a comparison of snap-frozen and FFPE tissues investigating two malignomas. Gene expression profiles were obtained from both materials of the tumors. Independently processed triplicates of snap-frozen and FFPE specimen, respectively, were two-round-amplified and hybridized on Affymetrix GeneChips (Palo Alto, CA, USA). Differentially expressed genes were identified in both FFPE and frozen material. All replicates had a correlation coefficient (R) of greater than 0.95 after normalization. Only direct comparison of FFPE to frozen replicates resulted in a mean R of 0.86, rendering a "mixed" investigation unfeasible. More than 50% (419 genes) of the more than fivefold differentially expressed genes (800 in FFPE, 685 in frozen material) were detected concomitantly regardless of the material used, which is similar to other comparisons of different gene expression analysis platforms. Thus, global gene expression analyses using solely FFPE material seem to be feasible with nearly comparable results to frozen tissue studies.     

人类survivin基因的克隆及在胃粘膜中表达的初步分析

目的：克隆人类Survivin(SVV)基因并分析其在胃粘膜中的表达情况。方法：采用逆转录聚合酶链反应从人胃癌组织中克隆SVV基因，地高辛标记cRNA探针，原位杂交检测其在胃粘膜中的表达。结果：得到两个SVV基因cDNA克隆、即SVV－S4A与SVV－S1B，前者与已知SVV基因cDNA序列相同，后者发生了SVV第3外显子丢失。原位杂交显示SVV－S4A主要表达于胃癌细胞的胞质，SVV－S1B主要表达于正常胃组织。结论：SVV－S4A和SVV－S1B在胃癌组织与正常胃粘膜组织中的表达存在不同特征。     

Co-infection of scrub typhus and leptospirosis in patients with pyrexia of unknown origin in Longding district of Arunachal Pradesh in 2013

Background: Scrub typhus and leptospirosis are bacterial zoonotic disease causing high morbidity and mortality. The seasonal outbreak of pyrexia is common in Arunachal Pradesh (AP); many times the disease remains undiagnosed. Objective: An outbreak of pyrexia of unknown origin (PUO) occurred in Longding district of Arunachal Pradesh in 2013, with 108 deaths, which was investigated to elucidate the cause of illness. Methodology: Blood samples from the affected region with acute pyrexia were collected, and screened for the malaria parasite, scrub typhus IgM and leptospira IgM. Results: Scrub typhus IgM was reactive in 97% (30/31), and 25% (8/31) cases were co-infected with leptospira. Incidentally, scrub typhus reactive (67%) and leptospira co-infection (62.7%) were higher in females. Record of previous 3 years (2011–2013) from Longding, Community Health Centre showed an increase in indoor pyrexia cases by 2-fold or more during October and November. Conclusion: The present study is the first report of co-infection of scrub typhus with leptospirosis from Northeast India. Medical officers in this region should take scrub typhus and leptospirosis in their differential diagnosis of patients with PUO for early diagnosis and effective treatment.     

结直肠癌组织中K-ras基因突变的分子病理学检测分析

目的:应用实时荧光定量PCR方法探讨K-ras基因突变情况及其临床病理意义。方法:收集71例结直肠癌石蜡组织,使用实时荧光定量PCR法检测K-ras基因状态。结果:结直肠癌中K-ras基因突变率为35.22%(25/71),发现7种突变(Gly12Asp,Gly12Val,Gly12Cys,Gly12Ser,Gly12Ala,Gly12Arg和Gly13Asp),其中1例为Gly12Val和Gly12Arg双突变,其中56.00%(14/25)的突变发生在第十二密码子的第二位碱基,且最常见类型为Gly12Asp。K-ras基因突变率在男性组中低于女性组(χ2=7.904,P=0.005),在无淋巴结转移组中低于有淋巴结转移组(χ2=5.851,P=0.016),差异有统计学意义,但K-ras基因突变与其他临床病理参数(年龄、肿瘤位置、浸润深度、组织学类型及Dukes’分期)差异均无统计学意义(P>0.05)。结论:女性或有淋巴结转移结直肠癌患者K-ras基因突变多见,可作为筛查是否进行分子靶向治疗的重点人群。