RESUMO
To investigate the clinical use of analyzing circulating tumor DNA in a clinical setting we present a pilot study comprising 93 patients from individuals with suspected lung cancer. The study aimed to evaluate the capability of analyzing circulating tumor DNA at the initial medical visit in order to detect genetic changes and mutations associated with lung cancer in plasma samples. Tumor DNA from plasma was extracted and analyzed with Next Generation Sequencing (NGS) and the result was compared with a matched tumor tissue collected in close connection from the same individual. Cancer-associated genetic mutations could be confirmed in about 60 percent of the plasma samples, and we observed a higher degree of conformance in patients with a more advanced disease. The results from the study provide valuable insights for an early clinical use of analyzing circulating tumor DNA in cases of suspected lung cancer, which could contribute to improving early diagnosis and treatment strategies for patients with lung cancer.
Assuntos
DNA Tumoral Circulante , Detecção Precoce de Câncer , Neoplasias Pulmonares , Mutação , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/diagnóstico , DNA Tumoral Circulante/sangue , DNA Tumoral Circulante/genética , Feminino , Pessoa de Meia-Idade , Detecção Precoce de Câncer/métodos , Projetos Piloto , Masculino , Idoso , Sequenciamento de Nucleotídeos em Larga Escala , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genética , Adulto , Idoso de 80 Anos ou maisRESUMO
There is limited knowledge of the biology of breast cancer (BC) brain metastasis (BM). We primarily aimed to determine the mutations in BCBM and to compare the mutational pattern with the matched primary breast cancer (BC). Secondary aims were to determine mutations in each subgroup (Luminal A-/B-like, HER2+ and TNBC) of BCBM, and to determine survival according to specific mutations. We investigated 57 BCBMs, including 46 cases with matched primary tumors (PT) by targeted Next Generation Sequencing (NGS) using the Cancer Hotspot Panel v2 (ThermoFisher Scientific) covering 207 targeted regions in 50 cancer related genes. Subtype according to immunohistochemistry was re-evaluated. NGS results fulfilling sequencing quality criteria were obtained from 52 BM and 41 PT, out of which 37 were matched pairs. Pathogenic mutations were detected in 66% of PTs (27/41), and 62% of BMs (32/52). TP53 mutations were most frequent; 49% (20/41) of PTs and 48% (25/52) in BMs, followed by PIK3CA mutations; 22% (9/42) in PTs and 25% (13/52) in BMs. Mutations in CDH1, EGFR, HRAS, RB1 CDKN2A and PTEN were detected in single pairs or single samples. Mutational pattern was discordant in 24% of matched pairs. We show a discordance of PIK3CA and TP53 mutations of roughly 25% indicating the need to develop methods to assess mutational status in brain metastasis where analysis of cell-free DNA from cerebrospinal fluid (CSF) has shown promising results.
Assuntos
Neoplasias Encefálicas/genética , Neoplasias Encefálicas/secundário , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Classe I de Fosfatidilinositol 3-Quinases/genética , Mutação , Proteína Supressora de Tumor p53/genética , Adulto , Idoso , Biomarcadores Tumorais/genética , Neoplasias Encefálicas/metabolismo , Neoplasias da Mama/metabolismo , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Análise de Sequência de DNA , Análise de SobrevidaRESUMO
BACKGROUND: Neoadjuvant tyrosine kinase inhibitor (TKI) therapy increases the chance of organ-preserving, radical resection in selected patients with gastrointestinal stromal tumors (GISTs). We aimed to evaluate systematic, immediate DNA sequencing of KIT and PDGFRA in pretreatment GIST tissue to guide neoadjuvant TKI therapy and optimize preoperative tumor response. METHODS: All patients who were candidates for neoadjuvant therapy of a suspected GIST [the study cohort (SC)] were prospectively included from January 2014 to March 2018. Patients were subjected to pretreatment endosonography-guided fine-needle biopsy (EUS-FNB) or transabdominal ultrasound-guided needle biopsy (TUS-NB), followed by immediate tumor DNA sequencing (< 2 weeks). A historic (2006-2013) reference cohort (RC) underwent work-up without sequencing before neoadjuvant imatinib (n = 42). The rate of optimal neoadjuvant therapy (TherapyOPTIMAL) was calculated, and the induced tumor size reduction (Tumor RegressionMAX, %) was evaluated by computed tomography (CT) scan. RESULTS: The success rate of pretreatment tumor DNA sequencing in the SC (n = 81) was 77/81 (95%) [EUS-FNB 71/74 (96%); TUS-NB 6/7 (86%)], with mutations localized in KIT (n = 58), PDGFRA (n = 18), or neither gene, wild type (n = 5). In patients with a final indication for neoadjuvant therapy, the TherapyOPTIMAL was higher in the SC compared with the RC [61/63 (97%) versus 33/42 (79%), p = 0.006], leading to a significantly higher Tumor RegressionMAX in patients treated with TKI (27% vs. 19%, p = 0.015). CONCLUSIONS: Pretreatment endosonography-guided biopsy sampling followed by immediate tumor DNA sequencing of KIT and PDGFRA is highly accurate and valuable in guiding neoadjuvant TKI therapy in GIST. This approach minimizes maltreatment with inappropriate regimens and leads to improved tumor size reduction before surgery.
Assuntos
Neoplasias Gastrointestinais/diagnóstico por imagem , Tumores do Estroma Gastrointestinal/diagnóstico por imagem , Mutação , Proteínas Proto-Oncogênicas c-kit/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Análise de Sequência de DNA/métodos , Idoso , Biópsia por Agulha Fina , Endossonografia , Feminino , Neoplasias Gastrointestinais/genética , Neoplasias Gastrointestinais/terapia , Tumores do Estroma Gastrointestinal/genética , Tumores do Estroma Gastrointestinal/terapia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Terapia Neoadjuvante , Medicina de Precisão , Período Pré-Operatório , Estudos ProspectivosRESUMO
BACKGROUND: Brain metastases (BM) are a feared progression of breast cancer (BC) with impact on quality of life and survival. Despite improved treatments, it is believed patients suffering from BM are increasing. AIMS: To study potential changes in the number of BM, the possible links between BC subgroup and extent of BM with prognosis. To investigate the interval between primary BC/extra cranial recurrence, and diagnosis of BM in the years 1994-2014. PATIENTS AND METHODS: Clinical data from 191 patients with BM diagnosed 1994-2014, was retrieved from charts. Primary tumours where re-evaluated histologically. RESULTS: There was an increase of BM in 5 years cohorts (1994-99 (n = 9); 2000-04 (n = 36); 2005-09 (n = 60); 2010-14 (n = 86)). We found no difference in the time interval from primary BC to BM but an insignificant increase in time from extra cranial relapse to development of BM in the time periods 1994-2004 and 2005-2014 of 15.5 and 25.0 months (p = 0.0612). Survival after BM was 7 months (95% CI 6-10) with a statistically significant difference between HER2 positive and TNBC with an inferior outcome for the latter (p = 0.018) whilst no differences were present when Luminal BC were compared with HER2 positive BC (p = 0.073). CONCLUSIONS: We show an increase of BM over time whilst the time span from primary BC to BM is unchanged supports earlier findings that adjuvant treatments have little preventive function. Time from extra cranial recurrence to BM was prolonged with one year. Patients with TNBC or more advance extent of BM had the shortest survival with BM.
Assuntos
Neoplasias Encefálicas/secundário , Neoplasias da Mama/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/classificação , Progressão da Doença , Feminino , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos Retrospectivos , Análise de Sobrevida , Suécia/epidemiologiaRESUMO
AIM: To evaluate endoscopic ultrasound (EUS)-guided biopsies for the pretreatment characterization of gastrointestinal stromal tumors (GIST) to personalize the management of patients. METHODS: All patients with lesions suspected to be GIST who were referred for EUS-sampling at a tertiary Swedish center were eligible for inclusion 2006-2015. During the observational study phase (2006-2011), routine fine-needle-aspiration (EUS-FNA) was performed. In 2012-2015, we converted to an interventional, randomized protocol with dual sampling EUS-FNA and fine-needle-biopsy-sampling (EUS-FNB) for all lesions. c-KIT- and DOG-1-immunostaining was attempted in all samples and a manual count of the Ki-67-index was performed. FNB-sampled tissue and the resected specimens were subjected to Sanger sequencing of the KIT and platelet-derived growth factor alpha (PDGFRA) genes. RESULTS: In all, 64 unique patients with GIST were included, and of these, 38 were subjected to pretreatment dual sampling. EUS-FNB had a higher diagnostic sensitivity when compared head-to-head with EUS-FNA (98% vs 58%, P < 0.001) and was more adequate for Ki-67-indexing (Ki-67EUS) (92% vs 40%, P < 0.001). Sequencing of EUS-biopsies was successful in 43/44 (98%) patients, and the mutation profiles (KIT-mutation 73%, PDGFRA-mutation 18%, wild-type 7%) were fully congruent with those detected in the corresponding resected specimens. In imatinib-naïve patients, the Ki-67EUS was comparable with the Ki-67-index in the corresponding surgical specimens (Ki-67SURG) (2.7% vs 2.9%, P = 0.68). In patients treated with neoadjuvant imatinib who also carried mutations indicating sensitivity, the Ki-67EUS was higher than the Ki-67SURG (2.5% vs 0.2%, P = 0.005), with a significant reduction in the Ki-67-index of -91.5% (95%CI: -82.4 to -96.0, P = 0.005). CONCLUSION: EUS-guided biopsy sampling is accurate for the pretreatment diagnosis and characterization of GISTs and allows the prediction and evaluation of tumor response to neoadjuvant imatinib therapy.
Assuntos
Antineoplásicos/uso terapêutico , Aspiração por Agulha Fina Guiada por Ultrassom Endoscópico/métodos , Tumores do Estroma Gastrointestinal/diagnóstico , Tumores do Estroma Gastrointestinal/tratamento farmacológico , Mesilato de Imatinib/uso terapêutico , Adulto , Idoso , Idoso de 80 Anos ou mais , Anoctamina-1/genética , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Tumores do Estroma Gastrointestinal/genética , Tumores do Estroma Gastrointestinal/patologia , Humanos , Antígeno Ki-67/genética , Masculino , Pessoa de Meia-Idade , Mutação , Terapia Neoadjuvante/métodos , Proteínas de Neoplasias/genética , Estudos Prospectivos , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-kit/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Sensibilidade e Especificidade , Análise de Sequência de DNA , Resultado do Tratamento , Adulto JovemRESUMO
Comprehensive genetic profiling by massively parallel sequencing, commonly known as next generation sequencing (NGS), is becoming the foundation of personalized oncology. For sarcomas very few targeted treatments are currently in routine use. In clinical practice the preoperative diagnostic workup of soft tissue tumours largely relies on core needle biopsies. Although mostly sufficient for histopathological diagnosis, only very limited amounts of formalin fixated paraffin embedded tissue are often available for predictive mutation analysis. Targeted NGS may thus open up new possibilities for comprehensive characterization of scarce biopsies. We therefore set out to search for driver mutations by NGS in a cohort of 55 clinically and morphologically well characterized sarcomas using low input of DNA from formalin fixated paraffin embedded tissues. The aim was to investigate if there are any recurrent or targetable aberrations in cancer driver genes in addition to known chromosome translocations in different types of sarcomas. We employed a panel covering 207 mutation hotspots in 50 cancer-associated genes to analyse DNA from nine gastrointestinal stromal tumours, 14 synovial sarcomas, seven myxoid liposarcomas, 22 Ewing sarcomas and three Ewing-like small round cell tumours at a large sequencing depth to detect also mutations that are subclonal or occur at low allele frequencies. We found nine mutations in eight different potential driver genes, some of which are potentially actionable by currently existing targeted therapies. Even though no recurrent mutations in driver genes were found in the different sarcoma groups, we show that targeted NGS-based sequencing is clearly feasible in a diagnostic setting with very limited amounts of paraffin embedded tissue and may provide novel insights into mesenchymal cell signalling and potentially druggable targets. Interestingly, we also identify five non-synonymous sequence variants in 4 established cancer driver genes in DNA from normal tissue from sarcoma patients that may possibly predispose or contribute to neoplastic development.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Mutação , Sarcoma/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias Ósseas/genética , Criança , Pré-Escolar , Feminino , Neoplasias Gastrointestinais/genética , Tumores do Estroma Gastrointestinal/genética , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Sarcoma/patologia , Sarcoma de Ewing/genética , Adulto JovemRESUMO
Mesenchymal chondrosarcoma is an extremely rare malignant tumour that most commonly originates in the bone, but is also present in extraskeletal sites. The tumour is morphologically characterized by a biphasic pattern of small round cells and islands of cartilage. Spinal mesenchymal chondrosarcomas are even rarer and, therefore, few investigations exist regarding the biological behaviour of the tumours. In the present study, we report a case of a 10-year-old female presenting with 9 months of back pain and radiographic findings of an intradural lesion measuring 1.5 cm at the level of Th4. The tumour was completely excised and subjected to pathological analyses. Following detection of the HEY1-NCOA2 fusion gene, the tumour was morphologically and immunohistochemically defined as an intradural mesenchymal chondrosarcoma attached to the dura mater. In this study, we validate the recent identification of the fusion gene HEY1-NCOA2 in paediatric extraskeletal mesenchymal chondrosarcomas. The relevant literature is reviewed and further discussed in relation to our findings.
RESUMO
DDIT3, also known as GADD153 or CHOP, encodes a basic leucine zipper transcription factor of the dimer forming C/EBP family. DDIT3 is known as a key regulator of cellular stress response, but its target genes and functions are not well characterized. Here, we applied a genome wide microarray based expression analysis to identify DDIT3 target genes and functions. By analyzing cells carrying tamoxifen inducible DDIT3 expression constructs we show distinct gene expression profiles for cells with cytoplasmic and nuclear localized DDIT3. Of 175 target genes identified only 3 were regulated by DDIT3 in both cellular localizations. More than two thirds of the genes were downregulated, supporting a role for DDIT3 as a dominant negative factor that could act by either cytoplasmic or nuclear sequestration of dimer forming transcription factor partners. Functional annotation of target genes showed cell migration, proliferation and apoptosis/survival as the most affected categories. Cytoplasmic DDIT3 affected more migration associated genes, while nuclear DDIT3 regulated more cell cycle controlling genes. Cell culture experiments confirmed that cytoplasmic DDIT3 inhibited migration, while nuclear DDIT3 caused a G1 cell cycle arrest. Promoters of target genes showed no common sequence motifs, reflecting that DDIT3 forms heterodimers with several alternative transcription factors that bind to different motifs. We conclude that expression of cytoplasmic DDIT3 regulated 94 genes. Nuclear translocation of DDIT3 regulated 81 additional genes linked to functions already affected by cytoplasmic DDIT3. Characterization of DDIT3 regulated functions helps understanding its role in stress response and involvement in cancer and degenerative disorders.
Assuntos
Núcleo Celular/genética , Citoplasma/metabolismo , Fibroblastos/metabolismo , Fibrossarcoma/metabolismo , Lipossarcoma/metabolismo , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismo , Antineoplásicos Hormonais/farmacologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Western Blotting , Adesão Celular , Ciclo Celular , Movimento Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Proliferação de Células , Células Cultivadas , Citoplasma/efeitos dos fármacos , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibrossarcoma/tratamento farmacológico , Fibrossarcoma/genética , Citometria de Fluxo , Perfilação da Expressão Gênica , Proteínas de Fluorescência Verde/genética , Humanos , Lipossarcoma/tratamento farmacológico , Lipossarcoma/genética , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tamoxifeno/farmacologiaRESUMO
BACKGROUND: The FUS-DDIT3 fusion oncogene encodes an abnormal transcription factor that has a causative role in the development of myxoid/round-cell liposarcomas (MLS/RCLS). We have previously identified FLT1 (VEGFR1) as a candidate downstream target gene of FUS-DDIT3. The aim of this study was to investigate expression of FLT1 and its ligands in MLS cells. METHODS: HT1080 human fibrosarcoma cells were transiently transfected with FUS-DDIT3-GFP variant constructs and FLT1 expression was measured by quantitative real-time PCR. In addition, FLT1, PGF, VEGFA and VEGFB expression was measured in MLS/RCLS cell lines, MLS/RCLS tumors and in normal adiopocytes. We analyzed nine cases of MLS/RCLS and one cell line xenografted in mice for FLT1 protein expression using immunohistochemistry. MLS/RCLS cell lines were also analyzed for FLT1 by immunofluorescence and western blot. MLS/RCLS cell lines were additionally treated with FLT1 tyrosine kinase inhibitors and assayed for alterations in proliferation rate. RESULTS: FLT1 expression was dramatically increased in transfected cells stably expressing FUS-DDIT3 and present at high levels in cell lines derived from MLS. The FLT1 protein showed a strong nuclear expression in cells of MLS tissue as well as in cultured MLS cells, which was confirmed by cellular fractionation. Tissue array analysis showed a nuclear expression of the FLT1 protein also in several other tumor and normal cell types including normal adipocytes. The FLT1 ligand coding gene PGF was highly expressed in cultured MLS cells compared to normal adipocytes while the other ligand genes VEGFA and VEGFB were expressed to lower levels. A more heterogeneous expression pattern of these genes were observed in tumor samples. No changes in proliferation rate of MLS cells were detected at concentrations for which the kinase inhibitors have shown specific inhibition of FLT1. CONCLUSIONS: Our results imply that FLT1 is induced as an indirect downstream effect of FUS-DDIT3 expression in MLS. This could be a consequence of the ability of FUS-DDIT3 to hijack parts of normal adipose tissue development and reprogram primary cells to a liposarcoma-like phenotype. The findings of nuclear FLT1 protein and expression of corresponding ligands in MLS and normal tissues may have implications for tissue homeostasis and tumor development through auto- or intracrine signaling.
Assuntos
Núcleo Celular/metabolismo , Fibrossarcoma/metabolismo , Lipossarcoma Mixoide/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Proteínas da Gravidez/metabolismo , Proteína FUS de Ligação a RNA/metabolismo , Transdução de Sinais , Fator de Transcrição CHOP/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Western Blotting , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/patologia , Proliferação de Células , Relação Dose-Resposta a Droga , Fibrossarcoma/genética , Fibrossarcoma/patologia , Imunofluorescência , Humanos , Imuno-Histoquímica , Ligantes , Lipossarcoma Mixoide/genética , Lipossarcoma Mixoide/patologia , Camundongos , Camundongos SCID , Transplante de Neoplasias , Proteínas de Fusão Oncogênica/genética , Fator de Crescimento Placentário , Proteínas da Gravidez/genética , Inibidores de Proteínas Quinases/farmacologia , Interferência de RNA , Proteína FUS de Ligação a RNA/genética , Proteínas Recombinantes de Fusão/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição CHOP/genética , Transfecção , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator B de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
Myxoid/round cell liposarcoma (MLS/RCLS) is the most common subtype of liposarcoma. Most MLS/RCLS carry a t(12;16) translocation, resulting in a FUS-DDIT3 fusion gene. We investigated the role of the FUS-DDIT3 fusion in the development of MLS/RCLS in FUS-DDIT3- and DDIT3-transfected human HT1080 sarcoma cells. Cells expressing FUS-DDIT3 and DDIT3 grew as liposarcomas in severe combined immunodeficient mice and exhibited a capillary network morphology that was similar to networks of MLS/RCLS. Microarray-based comparison of HT1080, the transfected cells, and an MLS/RCLS-derived cell line showed that the FUS-DDIT3- and DDIT3-transfected variants shifted toward an MLS/RCLS-like expression pattern. DDIT3-transfected cells responded in vitro to adipogenic factors by accumulation of fat and transformation to a lipoblast-like morphology. In conclusion, because the fusion oncogene FUS-DDIT3 and the normal DDIT3 induce a liposarcoma phenotype when expressed in a primitive sarcoma cell line, MLS/RCLS may develop from cell types other than preadipocytes. This may explain the preferential occurrence of MLS/RCLS in nonadipose tissues. In addition, development of lipoblasts and the typical MLS/RCLS capillary network could be an effect of the DDIT3 transcription factor partner of the fusion oncogene.
Assuntos
Fibrossarcoma/irrigação sanguínea , Regulação Neoplásica da Expressão Gênica , Lipossarcoma Mixoide/irrigação sanguínea , Proteína FUS de Ligação a RNA/fisiologia , Fator de Transcrição CHOP/fisiologia , Adipogenia , Animais , Análise por Conglomerados , Regulação para Baixo , Feminino , Fibrossarcoma/metabolismo , Humanos , Lipossarcoma Mixoide/metabolismo , Camundongos , Camundongos SCID , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas de Fusão Oncogênica/fisiologia , Proteína FUS de Ligação a RNA/genética , Proteína FUS de Ligação a RNA/metabolismo , Fator de Transcrição CHOP/metabolismo , Transfecção , Regulação para CimaRESUMO
The myxoid/round cell liposarcoma oncogene FUS-DDIT3 is the result of a translocation derived gene fusion between the splicing factor FUS and DDIT3. In order to investigate the downstream targets of DDIT3, and the transforming effects of the FUS-DDIT3 fusion protein, we have introduced DDIT3-GFP and FUS-DDIT3-GFP constructs into a human fibrosarcoma cell line. The gene expression profiles of stable transfectants were compared to the original fibrosarcoma cell line by microarray analysis. We here report that the NFkappaB and C/EBP beta controlled gene IL6 is upregulated in DDIT3- and FUS-DDIT3-expressing fibrosarcoma cell lines and in myxoid liposarcoma cell lines. Strong expression of the tumor associated multifunctional cytokine interleukin 6 was confirmed both at mRNA and protein level. Knockdown experiments using siRNA against CEBPB transcripts showed that the effect of FUS-DDIT3 on IL6 expression is C/EBP beta dependent. Chromatin immunoprecipitation revealed direct interaction between the IL6 promoter and the C/EBP beta protein. In addition, the effect of DDIT3 and FUS-DDIT3 on the expression of other acute phase genes was examined using real-time PCR. We demonstrate for the first time that DDIT3 and FUS-DDIT3 show opposite transcriptional regulation of IL8 and suggest that FUS-DDIT3 may affect the synergistic activation of promoters regulated by C/EBP beta and NFkappaB.