Somatic mutations in the promoter region of telomerase reverse transcriptase (TERT) gene, mainly at positions c.-124 and c.-146 bp, are frequent in several human cancers; yet its presence in ...gastrointestinal stromal tumor (GIST) has not been reported to date. Herein, we searched for the presence and clinicopathological association of TERT promoter mutations in genomic DNA from 130 bona fide GISTs. We found TERT promoter mutations in 3.8% (5/130) of GISTs. The c.-124C>T mutation was the most common event, present in 2.3% (3/130), and the c.-146C>T mutation in 1.5% (2/130) of GISTs. No significant association was observed between TERT promoter mutation and patient's clinicopathological features. The present study establishes the low frequency (4%) of TERT promoter mutations in GISTs. Further studies are required to confirm our findings and to elucidate the hypothetical biological and clinical impact of TERT promoter mutation in GIST pathogenesis.
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DOBA, EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, IZUM, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, SIK, UILJ, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Microsatellite instability (MSI) testing has been advocated for all newly diagnosed colorectal cancer patients. One of the most common tests is composed by a pentaplex panel of mononucleotides ...markers (NR-27, NR-21, NR-24, BAT-25, and BAT-26), which allows the analysis of MSI in tumors without the need of reference DNA. For that, it is fundamental to establish a quasi-monomorphic variation range (QMVR) for each marker. Herein, we aimed to establish the QMVR in a Brazilian healthy population, to evaluate the feasibility of MSI determination of tumors, without the matching normal DNA. Furthermore, we intend to assess their ancestry using specific ancestry-informative markers (AIMs) and correlate with QMVR. The QMVR was assessed in 214 individuals, through a pentaplex PCR followed by fragment analysis. The ancestry analysis was done by 46 AIMs in a single multiplex PCR followed by capillary electrophoresis. Following QMVR establishment, we observed 23 individuals with alleles outside the QMVR. Importantly, none of them exhibited more than one marker outside the range. Therefore, individuals with instability at ≥2 markers would be accurately classified as MSI. The European ancestry proportion was the most frequent (67.5%), followed by the African (19.6%). The comparison of the individuals with alleles within (n=191) and outside (n=23) the QMVR showed statistical difference in the proportions of European and African alleles, confirming the higher polymorphic nature of African ancestry. In conclusion, the present study reports an accurate methodology to assess MSI status without matched-normal DNA and independently of the ethnicity, even in the highly admixed population of Brazil.
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DOBA, EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, IZUM, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, SIK, UILJ, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Malignant pleural mesothelioma (MPM) is a highly lethal disease comprising a heterogeneous group of tumors with challenging to predict biological behavior. The diagnosis is complex, and the ...histologic classification includes 2 major subtypes of MPM: epithelioid (∼60% of cases) and sarcomatous (∼20%). Its identification depends upon pathological investigation supported by clinical and radiological evidence and more recently ancillary molecular testing. Treatment options are currently limited, with no known targeted therapies available.
To elucidate the mutation profile of driver tumor suppressor and oncogenic genes in a cohort of Brazilian patients.
We sequenced 16 driver genes in a series of 43 Brazilian malignant mesothelioma (MM) patients from 3 distinct Brazilian centers. Genomic DNA was extracted from formalin-fixed paraffin-embedded tumor tissue blocks, and the TERT promoter region was amplified by PCR followed by direct capillary sequencing. The Illumina TruSight Tumor 15 was used to evaluate 250 amplicons from 15 genes associated with solid tumors (AKT1, GNA11, NRAS, BRAF, GNAQ, PDGFRA, EGFR, KIT, PIK3CA, ERBB2, KRAS, RET, FOXL2, MET,and TP53). Library preparation with the TruSight Tumor 15 was performed before sequencing at the MiSeq platform. Data analysis was performed using Sophia DDM software.
Out of 43 MPM patients, 38 (88.4%) were epithelioid subtype and 5 (11.6%) were sarcomatoid histotype. Asbestos exposure was present in 15 (39.5%) patients with epithelioid MPM and 3 (60%) patients with sarcomatoid MPM. We found a TERT promoter mutation in 11.6% of MM, and the c.-146C>T mutation was the most common event. The next-generation sequencing was successful in 33 cases. A total of 18 samples showed at least 1 pathogenic, with a median of 1.8 variants, ranging from 1 to 6. The most mutated genes were TP53 and ERBB2 with 7 variants each, followed by NRAS BRAF, PI3KCA, EGFR and PDGFRA with 2 variants each. KIT, AKT1, and FOXL2 genes exhibited 1 variant each. Interestingly, 2 variants observed in the PDGFRA gene are classic imatinib-sensitive therapy.
We concluded that Brazilian MPM harbor mutation in classic tumor suppressor and oncogenic genes, which might help in the guidance of personalized treatment of MPM.
TERT Promoter Mutations in Soft Tissue Sarcomas Campanella, Nathália C.; Penna, Valter; Abrahão-Machado, Lucas Faria ...
The International journal of biological markers,
01/2016, Volume:
31, Issue:
1
Journal Article
Peer reviewed
Open access
Introduction
Oncogenic hotspot mutations in the promoter region of the TERT gene have been identified in several cancer types as being associated with a worse outcome. Additionally, a polymorphism ...(rs2853669) in the TERTpromoter region was reported to modify the survival of TERT-mutated patients. Our aim is to determine the frequency of c.-124 C>T and c.-146 C>T TERT mutations and to genotype the rs2853669 polymorphism in a series of 68 soft tissue sarcomas (STS) comprising 22 histological subtypes.
Methods
PCR was performed, followed by direct sequencing of a fragment of TERT containing the hotspots and the rs2853669.
Results
We found TERTmutations in 4/68 (5.9%) STSs including 1 pleomorphic liposarcoma (1/1), 1 dedifferentiated liposarcoma (1/1) and 2 myxoid liposarcomas (2/9). The variant C allele of rs2853669 was found in 54.8% (34/62) of all STSs and in 75% (3/4) of TERT-mutated cases. TERT mutations were associated with younger age, and the C allele of the rs2853669 was associated with high histological grade (2 and 3). No association was found between TERT mutation status or rs2853669 genotype and patient prognosis.
Conclusions
We showed that TERT promoter mutation is not a recurrent event in STS and is present in particular histological subtypes.
The microsatellite instability (MSI) phenotype may constitute an important biomarker for patient response to immunotherapy, particularly to anti-programmed death-1 inhibitors. MSI is a type of ...genomic instability caused by a defect in DNA mismatch repair (MMR) proteins, which is present mainly in colorectal cancer and its hereditary form, hereditary nonpolyposis colorectal cancer. Gastrointestinal stromal tumor (GIST) development is associated with activating mutations of KIT proto-oncogene receptor tyrosine kinase (KIT) or platelet-derived growth factor receptor α (PDGFRA), which are oncogenes that predict the response to imatinib mesylate. In addition to KIT/PDGFRA mutations, other molecular alterations are important in GIST development. In GISTs, the characterization of the MSI phenotype is scarce and the results are not consensual. The present study aimed to assess MSI in a series of 79 GISTs. The evaluation of MSI was performed by pentaplex polymerase chain reaction comprising five markers, followed by capillary electrophoresis. The expression of MMR proteins was evaluated by immunohistochemistry. Regarding the KIT/PDGFRA/B-Raf proto-oncogene, serine/threonine kinase molecular profile of the 79 GISTs, 83.6% of the tumors possessed KIT mutations, 10.1% had PDGFRA mutations and 6.3% were triple wild-type. The mutated-PDGFRA cases were associated with gastric location and a lower mitotic index compared with KIT-mutated and wild-types, and these patients were more likely to be alive and without cancer. MSI analysis identified 4 cases with instability in one marker, however, additional evaluation of normal tissue and immunohistochemical staining of MMR proteins confirmed their microsatellite-stable nature. The results of the present study indicated that MSI is not involved in GIST tumorigenesis and, therefore, cannot serve as a biomarker to immunotherapy response in GIST.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Gastrointestinal stromal tumors (GISTs) owe their development to the activating mutations in mast/stem cell growth factor receptor (KIT) or platelet-derived growth factor receptor A (PDGFRA) ...oncogenes. Both these KIT and PDGFRA oncogenes are members of the type III transmembrane receptor tyrosine kinase family that stimulates intracellular signaling pathways controlling cell proliferation, adhesion, apoptosis, survival, and differentiation. The presence and type of KIT/PDGFRA mutations help to predict the imatinib mesylate therapy, a selective tyrosine kinase inhibitor. Moreover, there is reported a small proportion of wild-type GISTs for both KIT and PDGFRA genes, and tumors more often acquire secondary mutations on KIT, that results into imatinib resistance. New patents to the GISTs imatinib resistant have recently been introduced. At present, sunitinib, is prescribed as second line therapy for patients with imatinibresistant or imatinib-intolerant GIST, and a number of other drugs, such as masitinib and valatinib, are in the pipeline. The present research focuses on GISTs diagnostic, prognostic and therapeutic biomarkers and addresses the development of novel patents for the treatment of these patients.
Abstract
Introduction: Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumor on the gastrointestinal tract, and are characterized by hotspot mutations in KIT and PDGFRA genes, ...which are predictive of imatinib-based therapy response. We showed previously that RKIP, a metastasis suppressor gene, is lost in about 17% of GIST, and it was associated with an adverse outcome. RKIP is plays a pivotal role in several signaling pathways, namely MAPK, NF-κβ, and GSK3β however a multidimensional integrative analysis to uncover its biological impact was never done in cancer and in particular in GIST.
Experimental procedures: We established a RKIP knockout GIST cell line (GIST-T1 KO) by CRISPR/Cas9 system. The differential gene and microRNA expression profile between GIST-T1 RKIP-KO and GIST-T1 negative control cells were assessed using the NanoString nCounter® technology and the nCounter® PanCancer Pathways and nCounter® miRNA Expression assays (NanoString Technologies). All procedures including sample preparation (100 ng of RNA), hybridization, detection and scanning were performed according to manufacture's instructions (NanoString Technologies). In a second step, we performed a global proteomic analysis using the Acquity UPLC M-Class chromatography system (Waters Co) linked to the mass spectrometer Synapt G2-Si (Waters Co). The protein
identification and quantification were performed in Progenesis QI for Proteomics (Waters Co) software.
Results: The nCounter gene expression analysis showed 56 genes differentially expressed: 18 downregulated and 38 upregulated genes in RKIP KO cells. The altered genes are especially involved in PI3k-AKT, MAPK and Ras signaling pathways. The global proteomic characterization showed 50 proteins differentially expressed: 5 were downregulated and 44 upregulated proteins in RKIP KO cells. In microRNA expression profile analysis we found 133 downregulated microRNAs in RKIP KO cells. Taking together, these findings uncover new molecules that are going to screen in a cohort of GIST patients.
Conclusion: For the first time, using a multidimensional integrative analysis we identified driver candidates and pathways, which will contribute the uncovered the biological role of RKIP in cancer.
Citation Format: Nathália C. Campanella, Matias Melendez, Leticia Ferro Leal, Adriane Feijo Evangelista, Vitor Marcel Faça, Rui Manuel Reis. Integrative molecular analysis uncovers key molecules and signaling pathways regulated by RKIP in gastrointestinal stromal tumors (GISTs) abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3998.
Background
The T-box transcription factor Brachyury was recently reported to be upregulated and associated with prognosis in solid tumors. Here, we proposed to evaluate the potential use of Brachyury ...protein expression as a new prognostic biomarker in gastrointestinal stromal tumors (GIST).
Methods
Brachyury protein expression was analyzed by immunohistochemistry in a cohort of 63
bona fide
GIST patients. Brachyury expression profiles were correlated with patients’ clinicopathological features and prognostic impact. Additionally, an
in silico
analysis was performed using the Oncomine database to assess Brachyury alterations at DNA and mRNA levels in GISTs.
Results
We found that Brachyury was overexpressed in the majority (81.0 %) of primary GISTs. We observed Brachyury staining in the nucleus alone in 4.8 % of cases, 23.8 % depicted only cytoplasm staining, and 52.4 % of cases exhibited both nucleus and cytoplasm immunostaining. The presence of Brachyury was associated with aggressive GIST clinicopathological features. Particularly, Brachyury nuclear (with or without cytoplasm) staining was associated with the presence of metastasis, while cytoplasm sublocalization alone was correlated with poor patient survival.
Conclusions
Herein, we demonstrate that Brachyury is overexpressed in GISTs and is associated with worse outcome, constituting a novel prognostic biomarker and a putative target for GIST treatment.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
The biological importance of human papillomavirus (HPV) in the field of medicine – related to cervical carcinogenesis – has been extensively reported in the last decades. For the first time, a direct ...correlation between cause and effect to explain a cancer development was completely achieved in medical research. Consequently, the Nobel Prize was awarded to HZ Hausen in 2008 for his efforts to understand the effects of persistent infection of oncogenic types of HPV and malignancy transformation. The aim of the present review was to summarize the principal elements of HPV characteristics and their importance in oncology.
It is established that HPV is the main etiologic agent for the development of cervical cancer. With the evolution of diagnosis and molecular biology, many tools have become essential for an early diagnosis and thereby, considerably reducing mortality. Molecular biology continues to advance and provide new perspectives with the use of reverse-transcription PCR in automation and genotyping through next-generation sequencing. This article aims to provide an overview of what is currently used in HPV diagnostic and research and future perspectives with the help of technologies such as next-generation sequencing for screening and vaccination.
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