Heterogeneity of KRAS Mutation Status in Rectal Cancer

IntroductionAnti-EGFR targeted therapy is of increasing importance in advanced colorectal cancer and prior KRAS mutation testing is mandatory for therapy. However, at which occasions this should be performed is still under debate. We aimed to assess in patients with locally advanced rectal cancer whether there is intra-specimen KRAS heterogeneity prior to and upon preoperative chemoradiotherapy (CRT), and if there are any changes in KRAS mutation status due to this intervention.ResultsFor 20 (43%) out of the 47 patients, a KRAS mutation was detected. With 12 out of 20, the majority of these mutations affected codon 35. We did not obtained evidence that CRT results in changes of the KRAS mutation pattern. In addition, no intratumoral heterogeneity in the KRAS mutational status could be proven. This was true for both the biopsies prior to CRT and the resection specimens thereafter. The discrepancy observed in some samples when using the SNaPshot™ assay was due to insufficient sensitivity of this technique upon massive tumor regression by CRT as application of the therascreen® KRAS test revealed concordant results.ConclusionOur results indicate that the KRAS mutation status at the primary tumor site of rectal cancer is homogenous. Its assessment for therapeutic decisions is feasible in pre-therapeutic biopsies as well as in post-therapeutic resected specimens. The amount of viable tumor cells seems to be an important determinant for assay sensitivity and should thus be considered for selection of the analytical method.     

Recurrent Hyperparathyroidism and a Novel Nonsense Mutation in a Patient with Hyperparathyriodism-Jaw Tumor Syndrome

ObjectiveTo present the case of a hyperparathyroidism-jaw tumor (HPT-JT) patient with a novel nonsense mutation of the CDC73 gene.MethodsWe present the case of a patient with a history of three prior maxillectomies and two prior parathyroidectomies who presented with recurrent primary hyperparathyroidism (PHPT). We also briefly review the literature pertaining to HPT-JT.ResultsGenetic analysis revealed a novel nonsense mutation (c.85G>T; pGlu29) in exon 1 of CDC73. The patient’s son underwent genetic testing for a CDC73 mutation and was found to be negative.ConclusionHPT-JT is a rare condition characterized by PHPT and benign tumors of the mandible and maxilla. Up to 15% of HPT-JT patients with PHPT have parathyroid carcinoma. HPT-JT is associated with an inactivating mutation of CDC73, a gene that codes for the tumor suppressor protein parafibromin. This report expands our understanding of the genetics underlying this rare disorder and emphasizes the importance of early detection in order to prevent hypercalcemic complications such as parathyroid carcinoma. (Endocr. Pract. 2013;19:e134-e137)     

A Targeted Constitutive Mutation in the Apc Tumor Suppressor Gene Underlies Mammary But Not Intestinal Tumorigenesis

Germline mutations in the adenomatous polyposis coli (APC) gene are responsible for familial adenomatous polyposis (FAP), an autosomal dominant hereditary predisposition to the development of multiple colorectal adenomas and of a broad spectrum of extra-intestinal tumors. Moreover, somatic APC mutations play a rate-limiting and initiating role in the majority of sporadic colorectal cancers. Notwithstanding its multifunctional nature, the main tumor suppressing activity of the APC gene resides in its ability to regulate Wnt/β-catenin signaling. Notably, genotype–phenotype correlations have been established at the APC gene between the length and stability of the truncated proteins encoded by different mutant alleles, the corresponding levels of Wnt/β-catenin signaling activity they encode for, and the incidence and distribution of intestinal and extra-intestinal tumors. Here, we report a novel mouse model, Apc1572T, obtained by targeting a truncated mutation at codon 1572 in the endogenous Apc gene. This hypomorphic mutant allele results in intermediate levels of Wnt/β-catenin signaling activation when compared with other Apc mutations associated with multifocal intestinal tumors. Notwithstanding the constitutive nature of the mutation, Apc^+/1572T mice have no predisposition to intestinal cancer but develop multifocal mammary adenocarcinomas and subsequent pulmonary metastases in both genders. The histology of the Apc1572T primary mammary tumours is highly heterogeneous with luminal, myoepithelial, and squamous lineages and is reminiscent of metaplastic carcinoma of the breast in humans. The striking phenotype of Apc^+/1572T mice suggests that specific dosages of Wnt/β-catenin signaling activity differentially affect tissue homeostasis and initiate tumorigenesis in an organ-specific fashion.     

基于锁核酸及等位位点特异性扩增技术的KRAS基因突变检测荧光定量PCR方法研究

基于等位位点特异性扩增的原理,设计锁核酸修饰KRAS基因突变特异性扩增引物,结合封阻探针技术,建立检测KRAS基因突变的荧光定量PCR方法。结果发现,锁核酸修饰的引物及探针可显著提高等位位点特异性扩增技术用于复杂样本中的微量基因突变检测的敏感度,该技术检测KRAS基因突变的敏感性可达0.01%~0.1%。进一步用建立的荧光定量PCR方法检测52例结直肠癌患者血浆标本,并用DNA测序法作为对照,同时用健康人血浆标本建立阴性检测结果判读标准,以初步评价该方法应用于循环DNA中KRAS基因突变检测的可行性。结果发现结直肠癌患者KRAS基因突变主要是G12C、G12A和G12R,而且q PCR法的阳性检出率为46.15%,高于DNA测序法(13.46%),阴性结果与DNA测序法的符合率为100%。此外,结直肠癌患者外周血KRAS基因的突变检出率与文献报道组织标本中的突变检出率及常见突变类型基本相符。上述结果说明该方法检测循环肿瘤DNA(circulating tumor DNA,ct DNA)具有较高的可靠性,可以用于肿瘤患者循环血液中KRAS基因突变的检测。     

High BRAF Mutation Frequency and Marked Survival Differences in Subgroups According to KRAS/BRAF Mutation Status and Tumor Tissue Availability in a Prospective Population-Based Metastatic Colorectal Cancer Cohort

RAS and BRAF mutations impact treatment and prognosis of metastatic colorectal cancer patients (mCRC), but the knowledge is based on trial patients usually not representative for the general cancer population. Patient characteristics, treatment and efficacy according to KRAS, BRAF and MSI status were analyzed in a prospectively collected unselected population-based cohort of 798 non-resectable mCRC patients. The cohort contained many patients with poor performance status (39% PS 2-4) and elderly (37% age>75), groups usually not included in clinical trials. Patients without available tissue micro array (TMA) (42%) had worse prognostic factors and inferior survival (all patients; 7m vs 11m, chemotherapy-treated;12m vs 17m). The 92 patients (21%) with BRAF mutation had a poor prognosis regardless of microsatellite instability, but receipt of 1-2^nd chemotherapy was similar to wildtype BRAF patients. Median survival in this cohort varied from 1 month in BRAF mutated patients not given chemotherapy to 26 months in wildtype KRAS/BRAF patients <75 years in good PS. TMA availability, BRAF mutation and KRAS mutation were all independent prognostic factors for survival. The observed 21% BRAF mutation incidence is higher than the previously and repeatedly reported incidence of 5-12% in mCRC. Screening for BRAF mutations before selection of treatment is relevant for many patients, especially outside clinical trials. A BRAF mutation only partly explained the very poor prognosis of many mCRC patients. Survival in unselected metastatic colorectal cancer patients is extremely variable and subgroups have an extremely short survival compared to trial patients. Patients without available TMA had worse prognostic factors and shorter survival, which questions the total generalizability of present TMA studies and implies that we lack information on the biologically worst mCRC cases. Lack of available tissue is an important underexposed issue which introduces sample bias, and this should be recognized more clearly when conclusions are made from translational mCRC studies.     

放射性药物在肿瘤靶向治疗中的应用

放射性药物指供临床诊断或治疗用的放射性核素制剂或其标记化合物。放射性核素靶向治疗是利用对肿瘤细胞具有特异高亲和力的分子载体将核素定向导入特定的肿瘤组织,对肿瘤进行治疗。与传统的放疗和化疗相比,其具有选择性杀伤肿瘤细胞的特点。随着核医学的发展,SPECT/CT、PET/CT的普及,新靶点的发现和新型放射性药物的研发,利用放射性药物进行靶向治疗在肿瘤临床治疗中占据的地位越来越重要。本文简述了放射性药物的分类、组成及特点;综述了针对肿瘤相关抗原的放射免疫药物在非霍奇金淋巴瘤、结直肠癌和前列腺癌中的应用;受体介导的放射性核素药物在治疗神经内分泌肿瘤、前列腺癌和乳腺癌中的临床应用以及基于基因修饰的放射性药物在肿瘤靶向治疗中的实验研究进展。最后总结了放射性药物在肿瘤靶向治疗中的应用前景与面临的挑战,以期为靶向治疗肿瘤的放射性药物的开发和临床应用提供一些参考。     

Direct Detection and Differentiation of Pathogenic Leptospira Species Using a Multi-Gene Targeted Real Time PCR Approach

Leptospirosis is a growing public and veterinary health concern caused by pathogenic species of Leptospira. Rapid and reliable laboratory tests for the direct detection of leptospiral infections in animals are in high demand not only to improve diagnosis but also for understanding the epidemiology of the disease. In this work we describe a novel and simple TaqMan-based multi-gene targeted real-time PCR approach able to detect and differentiate Leptospira interrogans, L. kirschneri, L. borgpeteresenii and L. noguchii, which constitute the veterinary most relevant pathogenic species of Leptospira. The method uses sets of species-specific probes, and respective flanking primers, designed from ompL1 and secY gene sequences. To monitor the presence of inhibitors, a duplex amplification assay targeting both the mammal β-actin and the leptospiral lipL32 genes was implemented. The analytical sensitivity of all primer and probe sets was estimated to be <10 genome equivalents (GE) in the reaction mixture. Application of the amplification reactions on genomic DNA from a variety of pathogenic and non-pathogenic Leptospira strains and other non-related bacteria revealed a 100% analytical specificity. Additionally, pathogenic leptospires were successfully detected in five out of 29 tissue samples from animals (Mus spp., Rattus spp., Dolichotis patagonum and Sus domesticus). Two samples were infected with L. borgpetersenii, two with L. interrogans and one with L. kirschneri. The possibility to detect and identify these pathogenic agents to the species level in domestic and wildlife animals reinforces the diagnostic information and will enhance our understanding of the epidemiology of leptopirosis.     

Assessment of Ovarian Cancer Spheroid Attachment and Invasion of Mesothelial Cells in Real Time

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not accurately model the behaviors of cancer cells within a nonadherent environment, as cancer cells inherently aggregate into multicellular structures which contribute to the metastatic process by attaching to and invading the peritoneal lining to form secondary tumors. To model this important stage of ovarian cancer metastasis, multicellular aggregates, or spheroids, can be generated from established ovarian cancer cell lines maintained under nonadherent conditions. To mimic the peritoneal microenvironment encountered by tumor cells in vivo, a spheroid-mesothelial co-culture model was established in which preformed spheroids are plated on top of a human mesothelial cell monolayer, formed over an extracellular matrix barrier. Methods were then developed using a real-time cell analyzer to conduct quantitative real time measurements of the invasive capacity of different ovarian cancer cell lines grown as spheroids. This approach allows for the continuous measurement of invasion over long periods of time, which has several advantages over traditional endpoint assays and more laborious real time microscopy image analyses. In short, this method enables a rapid, determination of factors which regulate the interactions between ovarian cancer spheroid cells invading through mesothelial and matrix barriers over time.     

基于磁性纳米材料的肿瘤靶向治疗研究进展

磁性纳米材料具有独特的磁学性质，可响应外磁场，产生力、热等效应。如在静磁场下将药物磁靶向递送至肿瘤部位；低频交变磁场下可将纳米药物主动渗透至病灶部位，实现瘤内均一分布；中频交变磁场作用下磁滞损耗产生热和增强的活性氧，用于肿瘤治疗。磁性纳米材料同时具有尺寸依赖的磁学性质以及表面多功能化等特点，可将磁靶向、分子靶向以及磁热疗联合。此外，磁性纳米材料具有磁共振成像性能以及纳米酶催化特性，使其在肿瘤诊疗一体化治疗方面获得了广泛应用。近年来，纳米给药系统不断被优化，基于磁性纳米材料的肿瘤靶向治疗也得到了长足的发展。鉴于此，本文围绕提高靶向肿瘤治疗效果，从磁靶向药物治疗、被动靶向磁热疗和主动分子靶向磁热疗、纳米酶特性以及诊疗一体化应用等几方面出发，综述了基于磁性纳米材料的肿瘤靶向治疗研究进展。     

Market Assessment of Tuberculosis Diagnostics in Brazil in 2012

Background

Improved diagnostics for the diagnosis of tuberculosis (TB) are urgently needed. However, test developers and investors require market size data to support new product development. This study assessed the served available market for TB diagnostics in Brazil in 2012 and the market segmentation in the public and private sectors.

Methods

Data were collected on test volumes done in the public and private sectors for the diagnosis of latent and active TB, drug susceptibility testing and treatment follow-up. Tests included were tuberculin skin tests, interferon-gamma releases assays, smear microscopy, solid and liquid cultures, nucleic acid amplification tests and phenotypic drug susceptibility tests. The data were collected by means of an electronic survey via the Brazilian State laboratories and from sales information provided by manufacturers. Test costs for the public sector were calculated using a components approach, while costs for the private sector were based on prices paid by patients. The overall market value (expenditure) for the entire country was calculated using the public sector test costs.

Results

During 2012, an estimated total of 2.4 million TB diagnostic tests were done in Brazil, resulting in an estimated overall market value of USD 17.2 million. The public sector accounted for 91% of the test volumes and 88% of the market value. Smear microscopy was the most commonly test (n = 1.3 million; 55% of total) at an estimated value of USD 3.7 million. Culture overall (n = 302,761) represented 13% of test volumes and 40% (USD 6.9 million) of the market value. On average, USD 208 was spent on TB diagnostics for every notified TB patient in Brazil, in 2012.

Conclusion

The TB diagnostics market value in Brazil in 2012 was over USD 17 million. These study results will help test developers to understand the current and potential market for replacement or add-on diagnostic technologies.     

Considering the Role of Time Budgets on Copy-Error Rates in Material Culture Traditions: An Experimental Assessment

Ethnographic research highlights that there are constraints placed on the time available to produce cultural artefacts in differing circumstances. Given that copying error, or cultural ‘mutation’, can have important implications for the evolutionary processes involved in material culture change, it is essential to explore empirically how such ‘time constraints’ affect patterns of artefactual variation. Here, we report an experiment that systematically tests whether, and how, varying time constraints affect shape copying error rates. A total of 90 participants copied the shape of a 3D ‘target handaxe form’ using a standardized foam block and a plastic knife. Three distinct ‘time conditions’ were examined, whereupon participants had either 20, 15, or 10 minutes to complete the task. One aim of this study was to determine whether reducing production time produced a proportional increase in copy error rates across all conditions, or whether the concept of a task specific ‘threshold’ might be a more appropriate manner to model the effect of time budgets on copy-error rates. We found that mean levels of shape copying error increased when production time was reduced. However, there were no statistically significant differences between the 20 minute and 15 minute conditions. Significant differences were only obtained between conditions when production time was reduced to 10 minutes. Hence, our results more strongly support the hypothesis that the effects of time constraints on copying error are best modelled according to a ‘threshold’ effect, below which mutation rates increase more markedly. Our results also suggest that ‘time budgets’ available in the past will have generated varying patterns of shape variation, potentially affecting spatial and temporal trends seen in the archaeological record. Hence, ‘time-budgeting’ factors need to be given greater consideration in evolutionary models of material culture change.     

腺病毒载体在肿瘤靶向性基因治疗中的应用

近年来,腺病毒载体广泛应用于恶性肿瘤的靶向性基因治疗。对传统腺病毒载体的改造主要有以下策略：(1)通过改变腺病毒的嗜性(tropism),使之具有感染宿主细胞的靶向性;(2)在转录水平控制外源性基因的表达;(3)可选择性裂解肿瘤细胞的有复制能力的腺病毒(replication-competent adenovirus,RCA)。更多安全、高效的靶向性腺病毒载体将应用于肿瘤基因治疗中。     

Mutation detection in KRAS Exon 1 by constant denaturant capillary electrophoresis in 96 parallel capillaries

Mutations in KRAS exon 1 oncogene are frequently found in colon carcinomas. A correlation between the mutated KRAS and the prognosis and outcome of treatment of colon cancer patients was reported in the literature. The object of our work was to establish a high-throughput method with high sensitivity to enable screening of tumor mutation status of KRAS exon 1 in large groups of colon cancer patients. KRAS exon 1 sequences from DNA isolated from 191 sporadic colon cancers were PCR amplified using one primer labeled with fluorescein and a second primer extended by a GC-clamp. After PCR amplification samples were subjected to automated 96-array constant denaturant capillary electrophoresis using a modified MegaBACE 1000 sequencing instrument. Mutant samples were identified by characteristic peak patterns. The sensitivity of detection of a mutant allele in a background of the wild-type alleles was 0.3%. Using the 96-array instrument a typical screening of 191 samples for KRAS mutation status could be performed within 2 h. A KRAS exon 1 mutation was found in 66 of 191 (34.6%) of the samples. The 96-array constant denaturant capillary electrophoresis provides an opportunity for the high-sensitivity screening of large cancer populations for KRAS exon 1 mutations.     

Mutation Detection in Patients with Retinal Dystrophies Using Targeted Next Generation Sequencing

Retinal dystrophies (RD) constitute a group of blinding diseases that are characterized by clinical variability and pronounced genetic heterogeneity. The different nonsyndromic and syndromic forms of RD can be attributed to mutations in more than 200 genes. Consequently, next generation sequencing (NGS) technologies are among the most promising approaches to identify mutations in RD. We screened a large cohort of patients comprising 89 independent cases and families with various subforms of RD applying different NGS platforms. While mutation screening in 50 cases was performed using a RD gene capture panel, 47 cases were analyzed using whole exome sequencing. One family was analyzed using whole genome sequencing. A detection rate of 61% was achieved including mutations in 34 known and two novel RD genes. A total of 69 distinct mutations were identified, including 39 novel mutations. Notably, genetic findings in several families were not consistent with the initial clinical diagnosis. Clinical reassessment resulted in refinement of the clinical diagnosis in some of these families and confirmed the broad clinical spectrum associated with mutations in RD genes.     

Inactivation and Inducible Oncogenic Mutation of p53 in Gene Targeted Pigs

Mutation of the tumor suppressor p53 plays a major role in human carcinogenesis. Here we describe gene-targeted porcine mesenchymal stem cells (MSCs) and live pigs carrying a latent TP53^R167H mutant allele, orthologous to oncogenic human mutant TP53^R175H and mouse Trp53^R172H, that can be activated by Cre recombination. MSCs carrying the latent TP53^R167H mutant allele were analyzed in vitro. Homozygous cells were p53 deficient, and on continued culture exhibited more rapid proliferation, anchorage independent growth, and resistance to the apoptosis-inducing chemotherapeutic drug doxorubicin, all characteristic of cellular transformation. Cre mediated recombination activated the latent TP53^R167H allele as predicted, and in homozygous cells expressed mutant p53-R167H protein at a level ten-fold greater than wild-type MSCs, consistent with the elevated levels found in human cancer cells. Gene targeted MSCs were used for nuclear transfer and fifteen viable piglets were produced carrying the latent TP53^R167H mutant allele in heterozygous form. These animals will allow study of p53 deficiency and expression of mutant p53-R167H to model human germline, or spontaneous somatic p53 mutation. This work represents the first inactivation and mutation of the gatekeeper tumor suppressor gene TP53 in a non-rodent mammal.