ABSTRACT Purpose: To determine molecular events involved in the tumorigenesis of phyllodes tumors (PT) and the role of each stromal (SC) and epithelial (EC) cell. Methods: Frozen breast samples enriched with epithelial and stromal cells from three fibroadenomas and 14 PT were retrieved and laser microdissected. Sanger and polymerase chain reaction-based sequencing of exon 2 MED12 and TERT promoter hotspot mutations were performed; 44K microarray platform was used to analyze gene expression. Results: All three fibroadenomas (FAs) presented mutations in MED12, but not in TERT, whose mutation was observed in five of the 14 PTs. EC and SC of each affected tumor displayed identical alterations. Of the total differentially expressed genes (DEG) (EC = 1,543 and SC = 850), 984 were EC-eDEGs and 291 were SC-eDEGs. We found a high similarity of diseases and functions enriched by both cell types, but dissimilarity in the number of enriched canonical pathways. Three signaling canonical pathways overlapping with EC and SC were predicted to be activated in one cell type and inactivated in the other, while no overlap in eDEGs was assigned to them. We also identified 13 EC-eDEGs and five SC-eDEGs enriched networks, in which the SC-eDEGs were able to segregate FA from PT samples. Conclusions: Identical TERT mutations from both SC and ES origins might affect the PTs tumorigenesis. Gene expression differences suggest coordinated molecular processes between these components with determinant differences acquired by SC, able to fully distinguish PTs from FAs lesions. Purpose (PT (SC Methods 1 microdissected reactionbased reaction based MED MED1 performed K Results (FAs alterations DEG (DEG 1543 543 1,54 850, 850 , 850) 98 ECeDEGs 29 SCeDEGs. SCeDEGs eDEGs. types other them networks Conclusions lesions 154 54 1,5 85 9 15 5 1, 8

OBJECTIVES: The present study aimed to contribute to the catalog of genetic mutations involved in the carcinogenic processes of uterine sarcomas (USs) and carcinosarcomas (UCSs), which may assist in the accurate diagnosis of, and selection of treatment regimens for, these conditions. METHODS: We performed gene-targeted next-generation sequencing (NGS) of 409 cancer-related genes in 15 US (7 uterine leiomyosarcoma [ULMS], 7 endometrial stromal sarcoma [ESS], 1 adenosarcoma [ADS]), 5 UCS, and 3 uterine leiomyoma (ULM) samples. Quality, frequency, and functional filters were applied to select putative somatic variants. RESULTS: Among the 23 samples evaluated in this study, 42 loss-of-function (LOF) mutations and 111 missense mutations were detected, with a total of 153 mutations. Among them, 66 mutations were observed in the Catalogue of Somatic Mutations in Cancer (COSMIC) database. TP53 (48%), ATM (22%), and PIK3CA (17%) were the most frequently mutated genes. With respect to specific tumor subtypes, ESS showed mutations in the PDE4DIP, IGTA10, and DST genes, UCS exhibited mutations in ERBB4, and ULMS showed exclusive alterations in NOTCH2 and HER2. Mutations in the KMT2A gene were observed exclusively in ULM and ULMS. In silico pathway analyses demonstrated that many genes mutated in ULMS and ESS have functions associated with the cellular response to hypoxia and cellular response to peptide hormone stimulus. In UCS and ADS, genes with most alterations have functions associated with phosphatidylinositol kinase activity and glycerophospholipid metabolic process. CONCLUSION: This preliminary study observed pathogenic mutations in US and UCS samples. Further studies with a larger cohort and functional analyses will foster the development of a precision medicine-based approach for the treatment of US and UCS.

Cancer-associated fibroblasts (CAF) influence tumor development at primary as well as in metastatic sites, but there have been no direct comparisons of the transcriptional profiles of stromal cells from different tumor sites. In this study, we used customized cDNA microarrays to compare the gene expression profile of stromal cells from primary tumor (CAF, n = 4), lymph node metastasis (N+, n = 3) and bone marrow (BM, n = 4) obtained from breast cancer patients. Biological validation was done in another 16 samples by RT-qPCR. Differences between CAF vs N+, CAF vs BM and N+ vs BM were represented by 20, 235 and 245 genes, respectively (SAM test, FDR < 0.01). Functional analysis revealed that genes related to development and morphogenesis were overrepresented. In a biological validation set, NOTCH2 was confirmed to be more expressed in N+ (vs CAF) and ADCY2, HECTD1, HNMT, LOX, MACF1, SLC1A3 and USP16 more expressed in BM (vs CAF). Only small differences were observed in the transcriptional profiles of fibroblasts from the primary tumor and lymph node of breast cancer patients, whereas greater differences were observed between bone marrow stromal cells and the other two sites. These differences may reflect the activities of distinct differentiation programs.

A síndrome de Lynch representa de 1-7% de todos os casos de câncer colorretal. É uma síndrome de herança autossômica dominante que predispõe ao câncer e é causada por mutações nos genes de reparo de ácido desoxirribonucléico (DNA). Desde a descoberta dos principais genes com função de reparo de DNA, mutações nos genes MSH2, MLH1, MSH6, PMS2 e PMS1 estão relacionadas com a susceptibilidade à síndrome de Lynch. Outro gene, MLH3, tem sido proposto como tendo papel na predisposição à síndrome de Lynch, porém mutações de significância clínica nesse gene não são claras. De acordo com o banco de dados InSiGHT (International Society for Gastrointestinal Hereditary Tumors), aproximadamente 500 diferentes mutações associadas à síndrome de Lynch são conhecidas, envolvendo primeiramente MLH1 (50%), MSH2 (40%) e outros (10%). Grandes progressos têm ocorrido para nosso entendimento das bases moleculares da síndrome de Lynch. A caracterização molecular será a forma mais precisa para definirmos a síndrome de Lynch e irá fornecer informações preditivas mais precisas sobre o risco de câncer colorretal e extra-colônico, além de permitir regimes otimizados de manejo.

Lynch syndrome represents 1-7% of all cases of colorectal cancer and is an autosomal-dominant inherited cancer predisposition syndrome caused by germline mutations in deoxyribonucleic acid (DNA) mismatch repair genes. Since the discovery of the major human genes with DNA mismatch repair function, mutations in five of them have been correlated with susceptibility to Lynch syndrome: mutS homolog 2 (MSH2); mutL homolog 1 (MLH1); mutS homolog 6 (MSH6); postmeiotic segregation increased 2 (PMS2); and postmeiotic segregation increased 1 (PMS1). It has been proposed that one additional mismatch repair gene, mutL homolog 3 (MLH3), also plays a role in Lynch syndrome predisposition, but the clinical significance of mutations in this gene is less clear. According to the InSiGHT database (International Society for Gastrointestinal Hereditary Tumors), approximately 500 different LS-associated mismatch repair gene mutations are known, primarily involving MLH1 (50%) and MSH2 (40%), while others account for 10%. Much progress has been made in understanding the molecular basis of Lynch Syndrome. Molecular characterization will be the most accurate way of defining Lynch syndrome and will provide predictive information of greater accuracy regarding the risks of colon and extracolonic cancer and enable optimal cancer surveillance regimens.

Visando o desenvolvimento de vetores homólogos de transformação de cloroplastos para tomate (Lycopersicon esculentum L.), fragmentos cloroplastidiais de tomate cv. IAC-Santa Clara foram clonados e analisados. Isolamento e clonagem de fragmentos PstI/SalI de cloroplastos de tomate deram origem aos plasmídios pIJB1 e pIJB2, contendo fragmentos de 8,6 pb e 6,4 pb respectivamente. Seqüenciamento e análise dos fragmentos revelaram que as seqüências apresentam de 93 a 100 % de identidade com os respectivos fragmentos em tabaco nas regiões codificadoras. Espaços intergênicos mostraram-se menos conservados, sugerindo que divergências evolutivas ocorreram principalmente em regiões não diretamente envolvidas na tradução. A diferença mais marcante encontrada está em um fragmento de 437 pb presente em tabaco na região intergênica entre os genes trnE e trnT, mas ausente no DNA cloroplastidial de tomate. Análise do padrão de restrição das seqüências cloroplastidiais apontou sítios únicos de restrição, sugerindo potencial uso dessas seqüências para a construção de vetores de transformação de cloroplastos. O recente sucesso na expressão de genes exógenos de interesse agronômico em cloroplastos de plantas sugere que essa tecnologia promete revolucionar o conceito e a aplicação da engenharia genética no melhoramento de plantas.

With the view of developing chloroplast transformation vectors based on homologous targeting regions for tomato (Lycopersicon esculentum L.), plastid DNA fragments of tomato cv. IAC-Santa Clara were cloned and analyzed. Isolation and cloning of PstI/SalI chloroplast fragments into the pBlueScript vectors yielded the plasmids pIJB1 and pIJB2 with cpDNA fragments of 8.6 kb and 6.4 kb, respectively. DNA sequencing and computer analyses revealed that the tomato sequences cloned display from 93 to 100 % of identity to the respective fragments in tobacco, which is more pronounced in coding regions. The intergenic spacers are somewhat less conserved suggesting that evolutionary divergences occurred mainly in these putative non-coding regions. The most remarkable difference found is a 437 bp sequence present in tobacco chloroplast genome in the intergenic region of the genes trnE and trnT but completely absent in the tomato chloroplast DNA. Analysis of restriction enzyme digestion patterns revealed several unique restriction sites in the intergenic spacer regions suggesting potential utility of these sequences in species-specific vector construction for tomato chloroplast transformation.

Quinases de proteínas relacionadas a SNF1 (SnRK) podem desempenhar um papel importante na regulação da expressão gênica em células vegetais. Essa família de proteínas regulatórias é representada pela quinase de proteínas SNF1 (sucrose non-fermenting -1) em Saccharomyces cerevisiae, AMPKs (quinase de proteínas ativadas por AMP) em células de mamíferos e SnRKs (quinase de proteínas relacionadas a SNF1) em células vegetais. A família de SnRKs foi reorganizada em três subfamílias de acordo com suas relações filogenéticas com base nas seqüências de aminoácidos das proteínas. Membros das subfamílias de SnRKs foram identificados em diversas plantas. Existem evidências mostrando que essa família de proteínas está envolvida em resposta a estresses (nutricional e ambiental), apesar de seu papel não ser totalmente compreendido. Nesse trabalho nós identificamos 22 contíguos de ESTs (expressed sequence tags) de cana-de-açúcar codificando SnRKs putativas. O alinhamento das seqüências de aminoácidos das SnRKs putativas de cana-de-açúcar com seqüências de aminoácidos de SnRKs identificadas em outras plantas revelaram um domínio catalítico N-terminal altamente conservado. Além disso, nossos resultados indicaram que em cana-de-açúcar há pelo menos um membro de cada subfamília de SnRKs. Análise do padrão de expressão dos contíguos de EST codificando para SnRK putativas nas 26 bibliotecas selecionadas do banco de dados do Sucest, indicou que membros dessa família de quinases são expressos por toda planta. Membros de cada subfamília não apresentaram padrões de expressão específicos, sugerindo que suas funções não estão correlacionadas com sua relação filogenética, com base nas seqüências de aminoácidos da região N-terminal.

Sucrose non-fermenting-1-related protein kinases (SnRKs) may play a major role in regulating gene expression in plant cells. This family of regulatory proteins is represented by sucrose non-fermenting-1 (SNF1) protein kinase in Saccharomyces cerevisiae, AMP-activated protein kinases (AMPKs) in mammals and SnRKs in higher plants. The SnRK family has been reorganized into three subfamilies according to the evolutionary relationships of their amino acid sequences. Members of the SnRK subfamily have been identified in several plants. There is evidence that they are involved in the nutritional and/or environmental stress response, although their roles are not yet well understood. We have identified at least 22 sugarcane expressed sequence tag (EST) contigs encoding putative SnRKs. The amino acid sequence alignment of both putative sugarcane SnRKs and known SnRKs revealed a highly conserved N-terminal catalytic domain. Our results indicated that sugarcane has at least one member of each SnRK subfamily. Expression pattern analysis of sugarcane EST-contigs encoding putative SnRKs in 26 selected cDNA libraries from the sugarcane expressed sequence tag SUCEST database has indicated that members of this family are expressed throughout the plant. Members of the same subfamily showed no specific expression patterns, suggesting that their functions are not related to their phylogenic relationships based on N-terminal amino acid sequence phylogenetic relationships.