IHC、FISH和RT-PCR检测对EML4-ALK重排的一致性

棘皮动物微管结合蛋白-间变性淋巴瘤激酶(echinoderm microtubule-associated protein-like4-anaplastic lymphoma kinase,EML4-ALK)在肺癌中已成为第二个最重的驱动致癌基因,在4%~6%的肺腺癌中EML4-ALK已经成为第一个可以靶向治疗的融合基因位点.伴随着ALK分离探针荧光原位杂交(fluorescent in situ hybridization,FISH)试剂盒的上市,克唑替尼已经被批准治疗ALK阳性的进展期非小细胞肺癌(non-smallcelllungcancer,NSCLC).然而,一种靶向药物的成功主取决于一种敏感且特异的筛选实验方法来检测分子药物作用的靶点.以作者的经验看,用RT-PCR来检测EML4-ALK,比用FISH和免疫组化(immunohistochemistry,IHC)方法更敏感,结果更可靠.尽管通过FISH检测ALK已经经过大量的临床实验验证,然而该方法在技术层面仍存在许多具有挑战性的问题,而通过IHC和RT-PCR方法检测ALK仍需临床进一步的探索.     

非小细胞肺癌中ALK蛋白表达与临床病理相关

目的探讨间变性淋巴瘤激酶(ALK)与非小细胞肺癌(NSCLC)的病理特征之间的关系。方法用VENTANA ALK(D5F3)免疫组织化学法(IHC)检测125例NSCLC患者手术切除的石蜡标本中ALK蛋白的表达情况,用荧光原位杂交(FISH)检测ALK蛋白表达阳性病例EML4-ALK融合基因表达情况,分析ALK蛋白的表达与NSCLC临床病理特征及预后之间的关系。结果 125例患者中,ALK蛋白阳性表达15例(12.0%),主要定位于细胞质内,少数位于细胞膜上;ALK蛋白在有吸烟史患者中的阳性表达率显著低于无吸烟史者,在腺癌中阳性表达率显著高于非腺癌,在高-中分化癌中阳性表达率显著低于低分化癌(P0.05);Kaplan-Meier分析显示ALK阳性的患者预后较阴性好(P0.05)。15例ALK蛋白阳性病例经FISH验证,10例为FISH阳性。结论 ALK蛋白的表达与NSCLC患者是否有吸烟史、临床病理类型以及肿瘤分化程度有紧密联系。IHC可以作为可靠的ALK筛查手段,提高ALK的检出率。FISH检测对确诊ALK蛋白阳性肺癌具有重要意义。     

非小细胞肺癌中Ventana ALK免疫组化异质性分析

目的探讨非小细胞肺癌(non-small cell lung cancer, NSCLC)中Ventana间变性淋巴瘤激酶(anaplastic lymphoma kinase, ALK)免疫组化异质性的分子变异特点和临床病理特征。方法收集2 228例NSCLC进行Ventana ALK(D5F3)免疫组化染色,并采用双盲法进行评价。对其中的Ventana ALK异质性病例行FISH和下一代测序(next generation sequencing, NGS)检测。结果 Ventana ALK阳性201例(9.0%),异质性10例(0.4%),阴性2 017例(90.5%)。Ventana ALK异质性病例包括2例大细胞神经内分泌癌、1例淋巴上皮瘤样癌和7例鳞状细胞癌。Ventana ALK异质性病例中强阳性肿瘤细胞占1%～30%。FISH检测ALK分离信号为0～12%,均为FISH阴性。9例ALK异质性病例成功经NGS检测,均未见ALK基因变异(包括基因融合、拷贝数变异、插入/缺失或单核苷酸变异)。结论 Ventana ALK异质性NSCLC应行其他分子病理学检测以进一步确认其ALK基因状态。     

SS18阳性滑膜肉瘤中ALK及C-met的表达及其临床意义

目的观察ALK及C-met在滑膜肉瘤中的表达,探讨两者表达与临床病理特征的关系及意义。方法对82例经形态学及免疫表型诊断为滑膜肉瘤的组织微阵列行SS18 FISH检测,并采用免疫组化法检测ALK(D5F3)、ALK(5A4)及C-met的表达,对ALK/C-met蛋白高表达者行相应ALK/C-met FISH及EML4-ALK RT-PCR检测。结果SS18阳性滑膜肉瘤有38例,其中ALK(D5F3)阳性率为18.4%(7/38),高表达率为2.6%(1/38),但ALK(5A4)均阴性。唯一ALK(D5F3)蛋白高表达者ALK FISH阳性,但EML4-ALK RT-PCR阴性,并发生肺转移。C-met阳性率为18.4%(7/38),高表达率为10.5%(4/38),阳性组和阴性组间在组织学分型及组织学分级上差异有统计学意义(P<0.05),且高表达者的病死率或转移率达75%(3/4);但4例C-met FISH均阴性。结论对滑膜肉瘤ALK蛋白初筛,选用克隆号D5F3更为合适。ALK/C-met高表达者预后欠佳,滑膜肉瘤中ALK及C-met有一定阳性率,提示ALK/C-met可能是该亚型的潜在治疗靶点。     

肺鳞状细胞癌EGFR/ALK的基因状态

目的:探讨肺鳞状细胞癌各亚型中EGFR和ALK的基因状态.方法:应用ARMS方法检测肺鳞状细胞癌石蜡组织中EGFR基因突变和ALK融合基因情况.结果:218例肺鳞状细胞癌样本中,E GFR基因突变率为4.59％(10/218),19del和L858R各为2.29％(5/218).ALK融合基因阳性率为6.14％(7/114).结论:肺鳞状细胞癌存在一定比例EGFR基因突变和ALK融合基因阳性,肺鳞状细胞癌EGFR基因和ALK融合基因常规检测不可忽视.     

伴有EML4-ALK基因融合的肺ALK重排软组织肿瘤1例

患者女, 66岁, 反复发热伴胸闷15 d入院。胸部CT检查:左肺上叶见一截面积大小11.4 cm×7.2 cm的软组织密度影, 边界尚清, 周围有分叶。随后患者行左肺上叶切除及肺门淋巴结清扫手术。镜下观察:肿瘤细胞呈上皮样, 细胞核染色质粗颗粒状, 缺乏明显的核仁, 细胞质少, 偏嗜酸性, 细胞边界不清楚。肿瘤细胞呈实性片状排列或形成模糊的结节, 可见细胞稀疏区域和密集区域混杂存在, 稀疏区的肿瘤细胞可形成孤立的旋涡状结构, 伴有富于黏液样和透明玻璃样变的间质。免疫组织化学染色结果:CD34、波形蛋白、INI1阳性, S-100蛋白部分阳性, 间变性淋巴瘤激酶(ALK)弱阳性, Ki-67阳性指数30%。肿瘤融合基因RNA检测:ALK基因重排, 与EML4融合, 产生EML4-ALK融合基因。熟悉掌握ALK重排软组织肿瘤的临床病理学特征, 对于软组织肿瘤精准分类以及对高度侵袭性肿瘤采取ALK抑制剂靶向治疗至关重要。     

结直肠腺癌享有ALK融合基因：12例临床病理和分子遗传学特征并文献复习

正结直肠腺癌(CRC)是全球癌症相关死亡的主要原因之一,其发生主要由癌基因或抑癌基因突变引起。研究发现一小部分CRC涉及受体酪氨激酶(RTK)基因融合,通常以3区与5区伴侣基因融合形成酪氨酸激酶嵌合基因/癌基因而致癌,其与间变性淋巴瘤激酶(ALK)具有高度同源性。该实验以确定致癌基因ALK融合所致CRC的发生率及其临床病理和遗传学特征为目的,选取8 150例CRC中12例(0.15%)免疫组化ALK(D5F3)阳性的CRC,经RNA二代测序(NGS)证实该组肿瘤含CAD-ALK(1例)、DIAPH2-ALK(2例)、EML4-ALK(2例)、LOC101929227-ALK(1例)、     

EGFR突变的非小细胞肺癌患者EML4-ALK融合基因的检测及其临床特征分析

目的: 检测EML4-ALK融合基因在表皮生长因子受体(epidermal growth factor receptor, EGFR)突变的非小细胞肺癌(non-small-cell lung cancer, NSCLC)人群中的突变率,并分析其与临床特征的关系。方法: 入选的102例NSCLC患者均为中国人,且至少满足以下1个入选条件:女性、不吸烟/少吸烟和肺腺癌。将102例患者的组织标本采用多重逆转录聚合酶链反应 (multiplex RT-PCR)的方法检测其EML4-ALK融合基因的突变率;对EML4-ALK阳性患者的组织标本采用DNA扩增后直接测序的方法来检测其EGFR(18~21号外显子)及Kirsten鼠肉瘤基因(Kirsten rat sarcoma,KRAS)(1、2号外显子)的突变情况。结果: 102例非小细胞肺癌患者的组织标本,有8例(7.8%)存在EML4-ALK融合基因突变,其中7例为突变体1(variant 1,V1),1例为突变体2(variant 2,V2);这8例EML4-ALK阳性患者组织标本的EGFR(18~21号外显子)及KRAS(1、2号外显子)均为野生型。8例阳性患者中,5例患者的年龄小于总体患者的平均年龄(59±10)岁,占62.5%(5/8);女性患者6例,占75%(6/8);不吸烟患者7例,占87.5%(7/8);腺癌患者5例,占62.5%(5/8)。结论: EML4-ALK融合基因突变代表了NSCLC的一个新的分子亚型,EML4-ALK突变与EGFR及KRAS突变是不共存的。     

伴ALK融合的甲状腺乳头状癌3例临床病理分析

目的 探讨伴ALK融合的甲状腺乳头状癌(ALK-papillary thyroid carcinoma, ALK-PTC)的临床病理学特征。方法 收集3例ALK-PTC的临床病理资料，行HE、免疫组化、FISH及二代测序，并复习相关文献。结果 3例ALK-PTC中，女性2例，男性1例，年龄38～53岁，平均年龄44岁。肿块最大径0.6～2.5 cm。镜下见肿瘤以滤泡状排列为主，局灶区呈乳头状排列。免疫表型：肿瘤细胞CK19、Galectin-3、HBME-1、TTF-1和ALK(D5F3)均阳性，TPO、CD56、BRAF、NTRK和ROS1均阴性。FISH检测示ALK基因融合均阳性。例1 RNA测序结果示HIF1A-ALK融合，例2和例3 RNA测序结果示EML4-ALK融合。结论 ALK-PTC相对少见，组织学形态及免疫表型具有特征性，其准确诊断对治疗和判断预后具有重要意义。     

非小细胞肺癌p63基因的表达及其与3号染色体位点变化的关系

目的　探讨非小细胞肺癌 p6 3基因的蛋白表达水平及其与定位在 3号染色体 2 7～ 2 9区域改变的关系。 方法　应用比较基因组杂交 (CGH)技术对 70例原发性肺鳞状细胞癌和肺腺癌标本进行染色体不平衡性分析。采用组织芯片技术构建12 2例原发性非小细胞肺癌石蜡包埋标本组织芯片 ,并采用免疫组织化学方法检测p6 3蛋白表达情况。比较 p6 3蛋白表达及其与 3号染色体末端改变的关系。结果　CGH分析结果发现 ,30例鳞状细胞癌 2 4例出现 3q2 7～ 2 9区域DNA拷贝数目的增加 ,4 0例腺癌仅 8例发现 3q2 7～ 2 9区域DNA获得。p6 3免疫组化染色结果显示 :5 0例 (84 75 % )鳞状细胞癌免疫组化染色为阳性 ;3例大细胞肺癌中 2例 (6 6 6 6 % )为阳性反应 ;腺癌中仅有 1例 (1 6 7% )为阳性。p6 3蛋白的阳性表达率与患者的年龄、性别、肿瘤的分级、肿瘤的转移以及生存率无关 (P >0 0 5 )。p6 3免疫组化阳性率与 3q2 7～ 2 9区域的改变比较结果显示 :p6 3免疫组化阳性反应与 3号染色体长臂 2 7～ 2 9区域的DNA扩增呈正相关 (P <0 0 1)。结论　p6 3基因的扩增与肺鳞状细胞癌发生和发展有密切的关系     

Schizophrenia in a North Swedish geographical isolate, 1900–1977. Epidemiology, genetics and biochemistry

Over 200 schizophrenic patients belonging to three major and interrelated pedigree complexes have been investigated over the past 30 years in a North Swedish geographically isolated population, presently numbering about 6,000. An intensive investigation of a number of biochemical correlates and genetic markers in a few selected families belonging to one of the major pedigrees has indicated new strategies for the current research program.
Schizophrenia, as defined operationally, is significantly associated with decreased activities of two enzymes (1) blood platelet monoamine oxidase, (2) plasma dopamine-β-hydroxylase, and (3) with the genetic marker Gc² (group specific antigen). Both enzymes are subject to genetic variation. A positive score for linkage between schizophrenia and low plasma DBH activity has been calculated, but, so far, available data are insufficient for discrimination between linkage and partial contribution of genetically controlled low plasma DBH to the pathogenesis of the disease. Alternatively, both mechanisms could be involved.
As a model for continued research, schizophrenia is explained as based on a double dominant-recessive genotype (Aabb), representing a vulnerability which in about 50 % of cases develops into clinical schizophrenia. It is suggested that the dominant mutation (A) operates on or affects MAO activity, and that the recessive genotype (bb) is instrumental in low variates of DBH activity and very likely such variates within the normal range of physiological variation. Moreover, it is suggested that the combined effects of MAO- and DBH-reduced efficiency on the metabolism of e.g. dopamine could be an essential pathogenic mechanism for the schizophrenic illness which is segregating in this population.     

The universal muscular chain reaction,muscle spasm,Torsions, ruptures and extravasations. Chameleons of pathology and some manifestations of simple muscular disorders

Most bodily functions require the coordinated actions of complementary and supplementary paired muscle groups. Where this essential muscular cooperation is lacking, hollow organs may burst and others become literally screwed up, giving rise to many similar spastic diseases such as Torticollis, Twisted ovarian cyst, Torsion of the Testis, Volvulus of the intestines, Varicose Veins, Megacolon, Aortamegaly, Scoliosis, Erb's Palsy, Peyronie's Disease, Main-en-Griffe, Undescended Foot (Pes Cavus), Talipes, Strabismus. Spasm is “panenepidemic” and unclassified examples of Torsion Dystonia and Dyskinesia really are as common as debt and taxes.     

The dominant diseases of modernized societies as omega-3 essential fatty acid deficiency syndrome: Substrate beriberi

About 1900, modern food selection and processing caused widespread $\text{epidemics}$ of the B vitamin deficiency diseases of beriberi and pellagra which, for genetic reasons, often expressed as different diseases ranging from bowel and heart disease to dermatoses and psychoses. But the B vitamins merely help convert essential fatty acids (EFA) into the prostaglandin (PG) tissue regulators and it now turns out that, through hydrogenation, milling and selection of w3-poor southern foods, we have also been systematically depleting, by as much as 90%, a newly discovered trace Nordic EFA (w3) of special importance to primates and sole precursor of the PG3(4) series, even as a concurrent fiber deficiency increases body demand for EFA. Since substrate EFA is processed by many B vitamin catalysts, an EFA deficiency will mimic a $\text{panh}$ypovitaminosis B, i.e., a mixture of $\text{substrate}$ beriberi and $\text{substrate}$ pellagra resembling vitamin beriberi and pellagra but exhibiting as even more diverse $\text{endemic}$ disease. This would consitute a second stage of the Modern Malnutrition and explain why some workers now hold the dominant diseases of modermized societies to be new, nutritionally based, pellagraform yet lipid-related and to range, once again, from heart disease to psychosis. It is an assumption that our dominant diseases are unrelated to each other or are merely revealed by our diagnostic acumen and therapeutic success; and that hydrogenating millions of tons of food oils annually, to destroy the rancidity producing w3-EFA, is safe for $\text{primates}$. Extensive beriberiform disease is reported here in 32 typical cases taken from medical practice which responds strikingly to linseed oil supplements (60% w3-EFA) in confirmation of identical results in Capuchins.     

Hypo- und Hypermagnesämie aus kardiologischer Sicht

Zusammenfassung Eine Reihe pathologischer Zustände bedingen Magnesiummangel. Zustände mit Hypermagnesämie sind ebenfalls bekannt, doch wesentlich seltener. Für den Kardiologen beachtenswert ist, daß unter Therapie mit bestimmten Diuretica bei Herzinsuffizienz, bei Herzinfarkt, Kardiomyopathie, Digitalisintoxikation und bestimmten Herzrhythmusstörungen Hypomagnesämie beobachtet wurde. Leider kann in der klinischen Routine nur ein extracelluläres Magnesiumdefizit durch Serumbestimmungen gemessen werden; über Magnesiummangel einzelner Organe kann nichts ausgesagt werden. Hinweise für Magnesiummangel geben aber neben der Messung des Serumspiegels Anamnese, klinischer Befund, bestimmte EKG-Veränderungen wie auch evtl. Hypokalämie, ein Zustand, bei dem sich oft — besonders bei Aldosteronismus — parallele Veränderungen zeigten.Tierexperimente deuten darauf hin, daß infarktähnliche Läsionen unter Magnesiummangel entstehen, doch ob Herzinfarkt beim Menschen durch Magnesiummangel ausgelöst werden kann, ist noch ungeklärt. In Leichenherzen zeigte sich im Infarktgebiet neben Calciumakkumulation signifikanter Magnesiumverlust, wobei unklar blieb, ob sich Ursache oder Folge des Infarktes widerspiegelten. Falls ein ursächlicher Zusammenhang besteht, ist er im Myokardstoffwechsel selbst zu suchen, wie bei der Alkoholkardiomyopathie, wo myokardialer Magnesiummangel zumindest als pathogenetischer Teilfaktor anerkannt wird. Andererseits versucht man aber auch Beziehungen zwischen Atherosklerose, Blutgerinnung und Hypomagnesämie herzustellen, in der Meinung, daß Magnesiummangel auch über den coronaren Pathomechanismus des Herzinfarktes wirken könnte. Sicher scheint, daß gewisse EKG-Veränderungen und Herzrhythmusstörungen durch einen irritierten Magnesiumhaushalt bedingt sein können, da sie bei Gabe bzw. Entzug von Magnesium verschwinden. Daß Magnesiummangel die Glykosidtoleranz verringert, wird tierexperimentell bestätigt. Unter Hypomagnesämie bewirkt Acetylstrophanthidin eher und länger Rhythmusstörungen als ohne, außerdem lassen diese sich durch Magnesiumgaben eliminieren. Da in gewissen Fällen spontane und digitalisinduzierte Herzrythmusstörungen durch Magnesiuminjektionen beseitigt wurden, scheint Magnesium als Therapeuticum angebracht. Einsatz verschiedener Magnesiumsalze bei Angina pectoris, degenerativen Herzerkrankungen und Herzinsuffizienz ohne geprüften und offensichtlich gestörten Magnesiumhaushalt ist fragwürdig, weil keine eindeutigen klinischen Erfolgsbeweise vorliegen. Immerhin mag es aber larvierte, durch Serumbestimmungen nicht erfaßbare Mangelzustände geben. Allgemein erscheint es aus kardiologischer Sicht ratsam, den Magnesiumhaushalt zu überwachen und in entsprechenden Fällen auszugleichen, um möglichen Myokardläsionen oder fatalen Herzrhythmusstörungen entgegenzuwirken.     

Environmental risk factors and their role in the management of atopic dermatitis

Introduction: The etiology of atopic dermatitis (AD) is multifactorial with interaction between genetics, immune and environmental factors.

Areas covered: We review the role of prenatal exposures, irritants and pruritogens, pathogens, climate factors, including temperature, humidity, ultraviolet radiation, outdoor and indoor air pollutants, tobacco smoke exposure, water hardness, urban vs. rural living, diet, breastfeeding, probiotics and prebiotics on AD.

Expert commentary: The increased global prevalence of AD cannot be attributed to genetics alone, suggesting that evolving environmental exposures may trigger and/or flare disease in predisposed individuals. There is a complex interplay between different environmental factors, including individual use of personal care products and exposure to climate, pollution, food and other exogenous factors. Understanding these complex risk factors is crucial to developing targeted interventions to prevent the disease in millions. Moreover, patients require counseling on optimal regimens for minimization of exposure to irritants and pruritogens and other harmful exposures.     

HLA genes in Amerindians from Mexico San Vicente Tancuayalab Teenek/Huastecos

Huastecos or Teenek Amerindians are presently living at North East Mexico (San Luis Potosi State). They have probably one of the most ancient culture of Mexico and Central America together with Mayas and Olmec groups with which also show close relationships. Proximity to Atlantic Ocean/Mexican Gulf originated that Spaniards had very early contact with them at about 1519 CE or before. In the present paper we have aimed to study HLA gene profile which may be useful for HLA and disease epidemiology and transplant programs in Teeneks. HLA-DRB1*04:07, -DRB1*14:06 and -DRB1*04:11 have been found in high frequency like in other Amerindian groups. High frequency typical Amerindians HLA extended haplotypes have been found, such as A*02-B*35-DRB1*04:07-DQB1*03:02; A*68-B*39-DRB1*04:07-DQB1*03:02 and A*02-B*39-DRB1*04:07-DQB1*03:02; also new haplotypes have been described, like A*02-B*52-DRB1*04:11-DQB1*03:02, A*68-B*35-DRB1*14:02-DQB1*03:01 and A*68-B*40-DRB1*16:02-DQB1*03:01. Genetic proximity is observed not only to linguistically close Mayans, but also to Mazatecans, Mixtecans and Zapotecans, who speak an altogether different languages; it shows once more that genes and languages do not correlate. This population was greatly diminished after European contact between 1500 and 1600 years CE; in fact, North and South America First Inhabitants population was brought from 80 down to 8 million people because of diseases (i.e.: measles, smallpox or influenza), slavery and war.     

'Very sore nights and days': the child's experience of illness in early modern England, c.1580-1720

Sick children were ubiquitous in early modern England, and yet they have received very little attention from historians. Taking the elusive perspective of the child, this article explores the physical, emotional, and spiritual experience of illness in England between approximately 1580 and 1720. What was it like being ill and suffering pain? How did the young respond emotionally to the anticipation of death? It is argued that children’s experiences were characterised by profound ambivalence: illness could be terrifying and distressing, but also a source of emotional and spiritual fulfilment and joy. This interpretation challenges the common assumption amongst medical historians that the experiences of early modern patients were utterly miserable. It also sheds light on children’s emotional feelings for their parents, a subject often overlooked in the historiography of childhood. The primary sources used in this article include diaries, autobiographies, letters, the biographies of pious children, printed possession cases, doctors’ casebooks, and theological treatises concerning the afterlife.     

Guidelines for the Validation and Use of Immunoassays for Determination of Introduced Proteins in Biotechnology Enhanced Crops and Derived Food Ingredients

Recent advancements in agricultural biotechnology have created a need for analytical techniques to determine introduced proteins in crops enhanced through modern biotechnology techniques. These proteins are expressed in plant tissues and may be present in food ingredients. Immunoassays are ideally suited for protein detection and may be used as both quantitative and threshold methods. Microplate ELISA and lateral flow devices are two of the most commonly used immunoassay formats for agricultural biotechnology applications. This paper provides general background information and a discussion of criteria for the validation and application of immunochemical methods to the analysis of proteins introduced into plants and food ingredients using biotechnology methods. It is the result of a collaborative effort of members of the Analytical Environmental Immunochemical Consortium. This collaborative effort represents the combined expertise of several organizations to reach consensus on establishing guidelines for the validation and use of immunoassays. Further, the paper offers developers and users a consistent approach to adopting the technology as well as aid in producing accurate and meaningful results.     

Ultracryotomy: development and application (with examples from the yeast cytology) (author's transl)

The preparation steps usually necessary for obtaining ultrathin frozen sections of biological material (chemical prefixation, enclosing, cryoprotective treatment, freezing, sectioning, and post-staining the sections for transmission electron microscopy) are submitted to a critical analysis. The application of cryo-ultramicrotomy, in particularly for cytochemical purposes, is reviewed. Fundamental considerations of chemical prefixation and poststaining are supported by examples from yeast cytology. Furthermore, the efficiency of the cryo-ultramicrotomy (electron optical resolution of ultrastructural details) is demonstrated on yeast cells and protoplasts.