Biomarker testing for advanced lung cancer by next-generation sequencing; a valid method to achieve a comprehensive glimpse at mutational landscape

Background: Next-generation sequencing (NGS) based assay for finding an actionable driver in non-small-cell lung cancer is a less used modality in clinical practice. With a long list of actionable targets, limited tissue, arduous single-gene assays, the alternative of NGS for broad testing in one experiment looks attractive. We report here our experience with NGS for biomarker testing in hundred advanced lung cancer patients. Methods: Predictive biomarker testing was performed using the Ion AmpliSeq™ Cancer Hotspot Panel V2 (30 tumors) and Oncomine™ Solid Tumor DNA and Oncomine™ Solid Tumor Fusion Transcript kit (70 tumors) on IonTorrent sequencing platform. Results: One-seventeen distinct aberrations were detected across 29 genes in eighty-six tumors. The most commonly mutated genes were TP53 (43% cases), EGFR (23% cases) and KRAS (17% cases). Thirty-four patients presented an actionable genetic variant for which targeted therapy is presently available, and fifty-two cases harbored non-actionable variants with the possibility of recruitment in clinical trials. NGS results were validated by individual tests for detecting EGFR mutation, ALK1 rearrangement, ROS1 fusion, and c-MET amplification. Compared to single test, NGS exhibited good agreement for detecting EGFR mutations and ALK1 fusion (sensitivity- 88.89%, specificity- 100%, Kappa-score 0.92 and sensitivity- 80%, specificity- 100%, Kappa-score 0.88; respectively). Further, the response of patients harboring tyrosine kinase inhibitor (TKI) sensitizing EGFR mutations was assessed. The progression-free-survival of EGFR positive patients on TKI therapy, harboring a concomitant mutation in PIK3CAmTOR and/or RAS-RAF-MAPK pathway gene and/or TP53 gene was inferior to those with sole-sensitizing EGFR mutation (2 months vs. 9.5 months, P = 0.015). Conclusions: This is the first study from South Asia looking into the analytical validity of NGS and describing the mutational landscape of lung cancer patients to study the impact of co-mutations on cancer biology and treatment outcome. Our study demonstrates the clinical utility of NGS testing for identifying actionable variants and making treatment decisions in advanced lung cancer

Triiodothyronine (T3) upregulates the expression of proto-oncogene TGFA independent of MAPK/ERK pathway activation in the human breast adenocarcinoma cell line, MCF7

ABSTRACT Objective: To verify the physiological action of triiodothyronine T3 on the expression of transforming growth factor α (TGFA) mRNA in MCF7 cells by inhibition of RNA Polymerase II and the MAPK/ERK pathway Materials and methods: The cell line was treated with T3 at a physiological dose (10−9M) for 10 minutes, 1 and 4 hour (h) in the presence or absence of the inhibitors, α-amanitin (RNA polymerase II inhibitor) and PD98059 (MAPK/ERK pathway inhibitor). TGFA mRNA expression was analyzed by RT-PCR. For data analysis, we used ANOVA, complemented with the Tukey test and Student t-test, with a minimum significance of 5%. Results: T3 increases the expression of TGFA mRNA in MCF7 cells in 4 h of treatment. Inhibition of RNA polymerase II modulates the effect of T3 treatment on the expression of TGFA in MCF7 cells. Activation of the MAPK/ERK pathway is not required for T3 to affect the expression of TGFA mRNA. Conclusion: Treatment with a physiological concentration of T3 after RNA polymerase II inhibition altered the expression of TGFA. Inhibition of the MAPK/ERK pathway after T3 treatment does not interfere with the TGFA gene expression in a breast adenocarcinoma cell line.

Importância da identificação das mutações do protooncogene RET e sua atuação no desenvolvimento dos diversos fenótipos das neoplasias endócrinas múltiplas tipo 2 / Importance of RET proto-oncogene mutations identification and its performance in thedevelopment of the multiples endocrines neoplasias type 2 several phenotypes

A hereditariedade autossômica dominante da neoplasia endócrina múltipla tipo 2 (NEM 2) relaciona-se à ativação do proto-oncogene RET, através de mutações missense. As mutações do RET são encontradas em 95% dos casos índices de NEM 2 e apresentam relação direta entre sua localização codon específica e os diversos fenótipos desenvolvidos, dentre eles, carcinoma medular datireóide, feocromocitoma e/ou hiperparatireoidismo. Baseando-se em análises bioquímicas e genéticas, é possível efetuar um diagnósticoprematuro, viabilizando a intervenção cirúrgica em tempo hábil. A periodicidade da monitorização bioquímica é ditada pelo fenótipopresente, pelas manifestações clínicas familiares e pelo genótipo RET. A recomendação da análise genética deve ser feita a todos indivíduos afetados e também a seus ascendentes e descendentes diretos, caso alguma mutação esteja presente; permitindo identificar os portadores de mutações RET, previamente ao início da sintomatologia. Neste trabalho, serão discutidos os aspectos molecularesdos diversos fenótipos da NEM 2, bem como a importância da identificação genotípica do proto-oncogene RET e sua interação com os testes bioquímicos visando o diagnóstico precoce, prevenção, monitorização, screening familiar e, portanto, maior sobrevidado paciente.

The dominant autossomic hereditarity of the multiple endocrine neoplasia type 2 (MEN 2) is related to RET proto-oncogene activation, through mutations missense. RET mutations are found in 95% of MEN 2 index cases and present direct relation between its specific localization codon and the diverse developed phenotypes, among them, medullary thyroid carcinoma, pheochromocytoma and/or hyperparathyroidism. Being based on biochemists and genetics analyses, it is possible to perform a premature diagnosis, making possible a surgical intervention in the right time. The biochemist monitoring regularity is determined by present phenotype, the familiar clinical manifestations and RET genotype. The recommendation of the genetic analysis must be made to all affected individuals and also their ascendants and descendants, in case some mutation is present, allowing to identify the RET mutations carriers previously to the beginning of the symptomatology. In this work, the molecular aspects of MEN 2 diverse phenotypes will be discussed, as well as the importance of the RET proto-oncogene genotypic identification and its interaction with the biochemists tests aiming the precocious diagnosis, prevention, monitoring, familiar screening e, therefore, the patient’s longer survival.

Detecção e rastreamento de mutações no proto-oncogene rET em pacientes com neoplasia endócrina múltipla tipo 2 por meio de eletroforese em gel sensível à conformação / RET prot-oncogene mutations screening and detection in patients with multiple endocrine neoplasia type 2 using conformation sensitive gel electrophoresis

A neoplasia endócrina múltipla tipo 2 é uma síndrome tumoral hereditária, onde o RET apresenta mutações germinativas causadoras da patologia. Com o diagnóstico genético, a tireoidectomia total tornou-se curativa para indivíduos assintomáticos portadores de mutações. Nosso objetivo é validar a metodologia de eletroforese em gel sensível à conformação (CSGE) no rastreamento de mutações do RET, comparativamente ao polimorfismo conformacional de cadeia simples (SSCP) e seqüenciamento genético em famílias portadoras das mutações: Cys620Arg (NEM-2A + HSCR e CMT-F); Cys634Arg (NEM-2A); Cys634Tyr (CMT-F); Val648Ile (NEM-2A); Val804Met (CMT-F) e Met918Thr (NEM-2B). Os polimorfismos rastreados foram: Gly691Ser, Leu769Leu e Ser904Ser. Ambas metodologias de rastreamento demonstraram-se sensíveis para alterações genéticas do RET exceto para a mutação Val804Met e polimorfismo Ser904Ser.

Multiple endocrine neoplasia type 2 (MEN-2) is inherited tumor syndrome, presenting RET germline mutations as causing-disease. With genetic diagnosis, prophylactic total thyroidectomy became curative for asymptomatic individuals carrying RET mutations. Our objective is to validate conformation sensitive gel electrophoresis (CSGE) methodology for RET genetic screening mutation, comparatively to single-strand conformation polymorphism (SSCP) and automatic sequencing in mutations carrying families: Cys620Arg (MEN-2A + HSCR and FMTC); Cys634Arg (MEN-2A); Cys634Tyr (FMTC); Val648Ile (MEN-2A); Val804Met (FMTC) and Met918Thr (MEN-2B). Polymorphisms were also screened: Gly691Ser, Leu769Leu and Ser904Ser. Both screening methodologies were sensible for RET genetic alterations except for Val804Met mutation and Ser904Ser polymorphism.

Molecular Cytogenetic Analysis of Gene Rearrangements in Childhood Acute Lymphoblastic Leukemia

The aims of this study were to estimate the incidences of BCR/ABL, MLL, TEL/AML1 rearrangements, and p16 deletions in childhood acute lymphoblastic leukemia (ALL), to identify new abnormalities, and to demonstrate the usefulness of interphase fluorescence in situ hybridization (FISH). We performed G-banding analysis and FISH using probes for BCR/ABL, MLL, TEL/AML1 rearrangements, and p16 deletions on 65 childhood ALL patients diagnosed and uniformly treated at a single hospital. Gene rearrangements were identified in 73.8% of the patients using the combination of G-banding and FISH, while the chromosomal abnormalities were identified in 49.2% using G-banding alone. Gene rearrangements were disclosed by FISH in 24 (72.7%) of 33 patients with normal karyotype or no mitotic cell in G-banding. Among the gene rearrangements detected by FISH, the most common gene rearrangement was p16 deletion (20.3%) and the incidences of others were 14.1% for TEL/AML1, 11.3% for MLL, and 1.8% for BCR/ABL translocations. Infrequent or new aberrations such as AML1 amplification, MLL deletion, ABL deletion, and TEL/AML1 fusion with AML1 deletion were also observed. We established the rough incidences of gene rearrangements in childhood ALL, found new abnormalities and demonstrated the diagnostic capability of interphase FISH to identify cryptic chromosome aberrations.

A primary study on the gene expression profiling of human brain contusion by cDNA microarray / 法医学杂志

OBJECTIVE@#To screen the differential expression of oncogenes and tumors suppressed genes(OTS genes) after human brain contusion by cDNA microrarray.@*METHODS@#The total RNAs isolated from normal and contusion human brain tissues were purified by Oligotex to obtain mRNAs. Both sources of mRNAs were reversely transcribed to cDNAs with the incorporation of fluorescent dUTP to prepare the hybridization probes. The probe from normal tissue and the contusion brain tissue were labeled with Cy3-dUTP and Cy5-dUTP respectively. The mixed probes were hybridized to the BioDoor Chip OTS-2.2S, a cDNA microarray which contains 227 oncogenes and tumors suppressed genes. After high-stringent washing, the cDNA microarray was scanned for the fluorescent signals and showed differences between two tissues.@*RESULTS@#Among the 227 target genes, 3 genes including Human carcinoma associated HOJ-1 (HoJ-1), Human KIAAOO65 gene,Human retinoblastoma related protein (p107) gene, showed distinct deference in expression level between the human brain contusion tissue and normal tissue.@*CONCLUSION@#The 3 genes in the brain contusion was significantly the differential expression by OTS 2.2S cDNA microarray. Further analysis of these genes will be helpful to understand the molecular mechanism of brain injury and utilization in forensic medicine.

Virus y cáncer humano: genética molecular del cáncer cervicouterino / Virus and human carcinoma: molecular genetic and cervical carcinoma

El cáncer es un proceso multifactorial y con múltiples etapas. Los protooncogenes, antioncogenes y virus oncogénicos están involucrados en el desarrollo de diversas neoplasias. La expresión alterada de los protooncogenes (por mutaciones, rearreglos o amplificaciones), así como la cooperación entre ellos puede llevar a la célula a una estado transformado. Los genes p53 y RB codifican para dos proteínas antioncogénicas que regulan las decisiones celulares de proliferación o diferenciación. La ausencia de RB (pérdida de ambos alelos) lleva a la liberación de factores de transcripción. p53 en su forma mutada, favorece el crecimiento celular. El cáncer cervicouterino ejemplifica claramente la intervención de este tipo de factores en su desarrollo. Los papilomavirus humanos genitales (PVH) estan implicados en su etiología como iniciadores de la proliferación celular. La inactivación de las proteínas antioncogénicas p53 y p105 RB porparte de los oncogenes virales E6 y E7, respectivamente, mantiene el estado de divición celular continua. Adicionalmente, la participación de ooncogenes expresados en forma alterada (c-myc) y otros cofactores contribuyen a modificar los periodos de latencia y la gravedad de la enfermedad

Angiotensin II stimulates proliferation of adventitial fibroblasts cultured from rat aortic explants

It has been proposed that the local renin-angiotensin system is activated in the adventitia after vascular injury. However, the physiological role of Angiotensin II (Ang II) in the adventitia has not been studied at a cellular level. This study was designed to assess the role of Ang II in the growth response of cultured adventitial fibroblasts (AFs). Adventitial explants of the rat thoracic aorta showed outgrowth of AFs within 5-7 days. Ang II caused hyperplastic response of AF cultures. The Ang II-induced mitogenic response of AFs was mediated primarily by the AT1 receptor. Ang II caused a rapid induction of immediate early genes (c-fos, c-myc and jun B). Induction of c-fos expression was fully blocked by an AT1 receptor antagonist but not by an AT2 receptor antagonist. Epidermal growth factor (EGF), platelet-derived growth factor-BB (PDGF-BB) and basic fibroblast growth factor (bFGF) induced DNA synthesis in AFs. Co-stimulation of AFs with the growth factors and Ang II potentiated the incorporation of 3H-thymidine into DNA. Results from this study indicate that Ang II causes mitogenesis of AFs via AT1 receptor stimulation and potentiates the responses to other mitogens. These data suggest that the Ang II may play an important role in regulating AF function during vascular remodeling following arterial injury.

Oxidants, anti-oxidants, essential fatty acids, eicosanoids, cytokines, gene/oncogene expression and apoptosis in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an auto-immune disease in which free radicals, eicosanoids, cytokines and nitric oxide seem to play a major role. An increase in the generation of superoxide anion and excess of interleukin-2 (IL-2), tumour necrosis factor (TNF) and pro-inflammatory eicosanoids and a fall in the production of nitric oxide and anti-oxidants such as superoxide dismutase and glutathione peroxidase seems to occur during the active phase of the disease. Thus, an imbalance in the pro-and anti-inflammatory molecules and a change in the delicate balance between the oxidants and anti-oxidants seems to have a vital role in the pathophysiology of SLE. In addition, a defect in the apoptosis of pro-inflammatory T cells may perpetuate the chronic inflammatory process in SLE. Thus, methods designed to suppress the generation of free radicals. IL-1, IL-2 and TNF and of eicosanoids and augment the concentrations of nitric oxide and anti-oxidants and enhance the apoptotic death of the pro-inflammatory T cells may be benefit in the management of SLE. Recent studies suggest that essential fatty acids and their metabolites, whose levels were found to be low in SLE, may restore the balance between pro- and anti-inflammatory molecules, oxidants and anti-oxidants and induce apoptosis of T cell.

Diagnostico precoz de neoplasia endocrina multiple tipo 2 (MEN 2) por la deteccion de portadores de mutaciones en el proto-oncogen RET / Early diagnosis of multiple endocrine neoplasia type 2 by detection of matated RET proto-oncogene carriers

El MEN 2 es un síndrome hereditario autosómico dominante, en el cual mutaciones del RET dan origen a tres fenotipos diferentes: carcinoma medular de tiroides familiar (CMTF), MEN 2A y MEN 2B. La identificación de mutaciones en el proto-oncogen RET predice el desarrollo de la enfermedad antes de las evidencias clínicas y bioquímicas. En este trabajo se identificaron portadores del RET por caracterización de mutaciones en pacientes y sus familiares. Se estudiaron 21 familias con CMTF (5 y 6 miembros), 4 con MEN 2A (dos de 5, una de 4 y otra de 3 miembros) y 2 con MEN 2B (5 y 1 miembros). Se obtuvieron muestras de ADN de sangre, en todos los casos y de tejido de feocromocitoma y/o tejido tiroideo en los operados. Se utilizó PCR para amplificar los exones 10, 11 y 16 con oligonucleótidos específicos, realizándose secuenciación directa de los fragmentos. En las familias con CMTF y con MEN 2A se encontraron mutaciones en el codón 634 del exón 11 en 16 sujetos, dectándose 9 casos con la mutación TGC r CGC (cisteína a arginina), 3 con TGC r TAC (cisteína a tirosina) y 4 con TGC r TTC (cisteína a fenilalanina). En los pacientes con MEN 2 B se encontró una mutación en el codón 918 del exón 16 ATG r ACG (meitonina a treonina). En tejido tumoral se detectó la misma mutación que en sangre periférica. El diagnóstico de MEN 2 fue confirmado en los 8 pacientes y detectado en 10 familiares. En los 5 portadores tiroidectomizados se encontró hiperplasia de células C o microcarcinoma in situ en 2 niños (9 y 12 años) y CMT en 3 adultos. La detección temprana de mutaciones del RET, especialmente en familiares seguida por tiroidectomía total, podría prevenir el desarrollo de CMT, modificado el desenlace fatal que ocurre cuando es diagnosticado tardíamente.

Perda do sítio de restriçäo fok-I no codon 918 do proto-oncogenese RET na neoplasia endócrina múltipla tipo 2B / Fok-1 restriction site loss in codon 918 of RET proto-oncogene in type 2b multiple endocrine neoplasm

Descreveu-se o uso de método simplificado de detecção de perda do sítio de restrição que envolve o codon 918 do protooncogene RET, usando-se digestão direta com a enzima de restrição fok-I. Este método foi aplicado na análise do DNA genômico do sangue periférico de uma paciente com neoplasia endócrina múltipla tipo 2B (NEM-2B), seus pais e irmão. Descartou-se a existência da mutação nos parentes e confirmou-se padrão eletroforético de DNA compatível com mutação no codon 918 na paciente estudada. Estes achados sugerem que esta mutação tenha ocorrido durante a gametogênese de um dos pais da afetada. Com este método elimina-se a necessidade do seqüenciamento gênico direto, economizando tempo e gastos com o exame. Propõe-se que este procedimento possa ser usado na detecção desta mutação em indivíduos pertencentes ao grupo de risco para NEM-2B, desde que cerca de 93 por cento dos pacientes com esta doença apresentam mutação no codon 918.

Carcinogênese colorretal e gástrica / Colorectal and stomach carcinogenis

A grande maioria dos autores, hoje, concorda que o câncer é o resultado do acúmulo de alteraçoes adquiridas e herdadas na seqüência de DNA de oncogenes e genes supressores tumorais. A ativaçao dos oncogenes, geralmente, resulta em proliferaçao celular descontrolada, devido à exacerbaçao da funçao dos seus produtos na célula. As progressoes tumorais no cólon e estômago parecem ser reguladas por duas vias genéticas, envolvendo a ativaçao de oncogenes, c-myc e c-met, respectivamente, e de diversos genes supressores tumorais. A grande maioria dos genes supressores tumorais inativados no câncer de cólon, p53 e APC/MCC, também o sao no câncer gástrico.

Proto-oncogene mutations in human breast cancer [HBC]

This is a preliminary study of gene mutations in HBC. The genetic organizations of C-myc, L-myc and new oncogenes were examined in tumors from 10 patients with poorly differentiated infiltrating ductal cell carcinoma with epsilateral axillary lymph node metastasis. Genetic abnormalities in 6 cases was observed. 2 tumors has only new sequences amplified, one had only L-myc and 3 had both. The amplification of new and L-myc was found in 2 pregnant patients, 1 presented with bone metastases. New oncogene was amplified in a patient who developed contralateral breast cancer and bone metastasis within 1 year from diagnosis. This study suggested that L-myc and new mutations may be useful prognostic indicators. The alteration of new and L-myc genes in tumors from pregnant patients warrants further investigation. Additional studies are in progress