Amplification Of Other Genes Research Articles

Detection of MDM2 amplification by shallow whole genome sequencing of cell-free DNA of patients with dedifferentiated liposarcoma.

High-level amplification of MDM2 and other genes in the 12q13–15 locus is a hallmark genetic feature of well-differentiated and dedifferentiated liposarcomas (WDLPS and DDLPS, respectively). Detection of this genomic aberration in plasma cell-free DNA may be a clinically useful assay for non-invasive distinction between these liposarcomas and other retroperitoneal tumors in differential diagnosis, and might be useful for the early detection of disease recurrence. In this study, we performed shallow whole genome sequencing of cell-free DNA extracted from 10 plasma samples from 3 patients with DDLPS and 1 patient with WDLPS. In addition, we studied 31 plasma samples from 11 patients with other types of soft tissue tumors. We detected MDM2 amplification in cell-free DNA of 2 of 3 patients with DDLPS. By applying a genome-wide approach to the analysis of cell-free DNA, we also detected amplification of other genes that are known to be recurrently affected in DDLPS. Based on the analysis of one patient with DDLPS with longitudinal plasma samples available, we show that tracking MDM2 amplification in cell-free DNA may be potentially useful for evaluation of response to treatment. The patient with WDLPS and patients with other soft tissue tumors in differential diagnosis were negative for the MDM2 amplification in cell-free DNA. In summary, we demonstrate the feasibility of detecting amplification of MDM2 and other DDLPS-associated genes in plasma cell-free DNA using technology that is already routinely applied for other clinical indications. Our results may have clinical implications for improved diagnosis and surveillance of patients with retroperitoneal tumors.

RAC1 Alterations Induce Acquired Dabrafenib Resistance in Association with Anaplastic Transformation in a Papillary Thyroid Cancer Patient.

Simple SummaryWe identified an acquired RAC1 (P34R) mutation in the metastatic tumor of a BRAF-mutated papillary thyroid cancer patient treated with the BRAF inhibitor dabrafenib. Further investigations uncovered a high RAC1 copy number in the metastatic sample and its derived cell line. We demonstrated that an increase in RAC1 copy numbers could lead to increased tumor cell growth independently of the RAC1 (P34R) mutation. Further, we identified polyploidy of chromosome 7 in the metastatic sample and derived cell line, which accounted for amplification of other genes, their increased expression and therefore their ability to also induce resistance to therapy.BRAF-activating mutations are the most frequent driver mutations in papillary thyroid cancer (PTC). Targeted inhibitors such as dabrafenib have been used in advanced BRAF-mutated PTC; however, acquired resistance to the drug is common and little is known about other effectors that may play integral roles in this resistance. In addition, the induction of PTC dedifferentiation into highly aggressive KRAS-driven anaplastic thyroid cancer (ATC) has been reported. We detected a novel RAC1 (P34R) mutation acquired during dabrafenib treatment in a progressive metastatic lesion with ATC phenotype. To identify a potential functional link between this novel mutation and tumor dedifferentiation, we developed a cell line derived from the metastatic lesion and compared its behavior to isogenic cell lines and primary tumor samples. Our data demonstrated that RAC1 mutations induce changes in cell morphology, reorganization of F-actin almost exclusively at the cell cortex, and changes in cell adhesion properties. We also established that RAC1 amplification, with or without mutation, is sufficient to drive cell proliferation and resistance to BRAF inhibition. Further, we identified polyploidy of chromosome 7, which harbors RAC1, in both the metastatic lesion and its derived cell line. Copy number amplification and overexpression of other genes located on this chromosome, such as TWIST1, EGFR, and MET were also detected, which might also lead to dabrafenib resistance. Our study suggests that polyploidy leading to increased expression of specific genes, particularly those located on chromosome 7, should be considered when analyzing aggressive thyroid tumor samples and in further treatments.

Acquired resistance mechanisms of EGFR-TKI in advanced non-small cell lung cancer

Since the development of molecular biology, the treatment of advanced non-small cell cancer is shifting from traditional chemotherapy into molecular targeted therapy with genotyping as a guide′s help. The most widely used is epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs). With the application of EGFR-TKIs, the resistances to EGFR inhibitors are paid more and more attention, in recent years. The main mechanisms of acquired resistances are as follows: secondary mutation of the EGFR gene, amplification of c-MET, Her2 and other target genes, histological transformation, activation of the bypass mechanisms, loss of p53, the relief of negative feedback loops, overlap of mechanisms, etc. Key words: Carcinoma, non-small-cell lung; Drug resistance, neoplasm; Molecular targeted therapy

The CRKL gene encoding an adaptor protein is amplified, overexpressed, and a possible therapeutic target in gastric cancer

BackgroundGenomic DNA amplification is a genetic factor involved in cancer, and some oncogenes, such as ERBB2, are highly amplified in gastric cancer. We searched for the possible amplification of other genes in gastric cancer.Methods and ResultsA genome-wide single nucleotide polymorphism microarray analysis was performed using three cell lines of differentiated gastric cancers, and 22 genes (including ERBB2) in five highly amplified chromosome regions (with a copy number of more than 6) were identified. Particular attention was paid to the CRKL gene, the product of which is an adaptor protein containing Src homology 2 and 3 (SH2/SH3) domains. An extremely high CRKL copy number was confirmed in the MKN74 gastric cancer cell line using fluorescence in situ hybridization (FISH), and a high level of CRKL expression was also observed in the cells. The RNA-interference-mediated knockdown of CRKL in MKN74 disclosed the ability of CRKL to upregulate gastric cell proliferation. An immunohistochemical analysis revealed that CRKL protein was overexpressed in 24.4% (88/360) of the primary gastric cancers that were analyzed. The CRKL copy number was also examined in 360 primary gastric cancers using a FISH analysis, and CRKL amplification was found to be associated with CRKL overexpression. Finally, we showed that MKN74 cells with CRKL amplification were responsive to the dual Src/BCR-ABL kinase inhibitor BMS354825, likely via the inhibition of CRKL phosphorylation, and that the proliferation of MKN74 cells was suppressed by treatment with a CRKL-targeting peptide.ConclusionThese results suggested that CRKL protein is overexpressed in a subset of gastric cancers and is associated with CRKL amplification in gastric cancer. Furthermore, our results suggested that CRKL protein has the ability to regulate gastric cell proliferation and has the potential to serve as a molecular therapy target for gastric cancer.

Biologie moléculaire des sarcomes

Sarcomas represent a heterogeneous group of tumors with a complex and poorly reproducible classification. However, in the last ten years, several specific genetic alterations have been described allowing a molecular classification with: 1) sarcomas with a specific translocation which can be used as a diagnostic marker. These translocations can be demonstrated by RT-PCR or by FISH with commercially available break apart probes; 2) sarcomas with simple genomic profile showing amplification of a few genes. Well differentiated liposarcomas, dedifferentiated liposarcomas and intimal sarcomas show a simple genomic profile characterised by MDM2 and CDK4 amplifications associated with amplification of other genes in dedifferentiated liposarcomas; 3) sarcomas with activating mutations: about 90% of GIST show activating mutation of a receptor tyrosine kinase gene, either KIT or PDGFRA. The most frequent mutation involves exon 11 of KIT followed by exon 9 of KIT and exon 18 of PDGFRA. Demonstration of these mutations is useful for the diagnosis of CD117 negative GIST, for predicting response to imatinib and to explain secondary resistance to imatinib; 4) sarcomas with inactivating mutations: malignant rhabdoid tumors show biallelic inactivation of INI1 gene with a lost of INI1 expression which can be demonstrated by immunohistochemistry; 5) other sarcomas usually show a complex genomic profile characterised by numerous gains and losses of genes with a frequent loss of RB1 and alterations of P53. Leiomyosarcomas, pleomorphic rhabdomyosarcomas, pleomorphic liposarcomas, myxofibrosarcomas, poorly differentiated sarcomas (so-called MFH and fibrosarcomas) belong to this category and show no specific molecular abnormality.

Disorders in cell circuitry during multistage carcinogenesis: the role of homeostasis.

The multistage process of carcinogenesis involves the progressive acquisition of mutations, and epigenetic abnormalities in the expression, of multiple genes that have highly diverse functions. An important group of these genes are involved in cell cycle control. Thus, cyclin D1 is frequently overexpressed in a varety of human cancers. Cylin D1 plays a critical role in carcinogenesis because (i) overexpression enhances cell transformation and tumorigenesis, and enhances the amplification of other genes, and (ii) an antisense cyclin D1 cDNA reverts the malignant phenotype of carcinoma cells. Therefore, cyclin D1 may be a useful biomarker in molecular epidemiology studies, and inhibitors of its function may be useful in both cancer chemoprevention and therapy. We discovered a paradoxical increase in the cell cycle inhibitors protein p27(Kip1) in a subset of human cancers, and obtained evidence for homeostatic feedback loops between cyclins D1 or E and p27(Kip1). Furthermore, derivatives of HT29 colon cancer cells with increased levels of p27(Kip1) showed increased sensitivity to induction of differentiation. This may explain why decreased p27(Kip1) in a subset of human cancers is associated with a high grade (poorly differentiated) histology and poor prognosis. Agents that increase cellular levels of p27(Kip1) may, therefore, also be useful in cancer therapy. Using an antisense Rb oligonucleotide we obtained evidence that the paradoxical increase in pRb often seen in human colon cancers protects these cells from growth inhibition and apopotosis. On the basis of these, and other findings, we hypothesize that homeostatic feedback mechanisms play a critical role in multistage carcinogenesis. Furthermore, because of their bizarre circuitry, cancer cells suffer from 'gene addiction' and 'gene hypersensitivity' disorders that might be exploited in both cancer prevention and chemotherapy.

Book reviews

ABSTRACT The Editor tells us that he chose the title Molecular Biology of Cancer Genes rather than Molecular Biology of Oncogenes “because the book deals not only with genes that cause cancer but also those that are involved in other ways in the neoplastic process’. That is an immense field to cover in less than three hundred pages, so it is of interest to see what criteria might have been used to permit a rational selection of subjects to be made. Unfortunately, neither the Editor’s introduction nor the list of contents offers any clue. It is difficult, for example, to see why there is a chapter on ras oncogenes in human cancer, but no chapter devoted to any other single oncogene; why there is no discussion of the general characteristics of oncogenes as a special class of gene; why there is a chapter on the amplification of the myc oncogene in prostatic carcinoma cells, but no chapters on the amplification of other genes in other tumours and none on the biological significance of gene amplification as a whole; why there is a chapter devoted to oncogenes in lung cancer (a good chapter, by the way), but no chapter on the more deeply studied aberrations in colorectal carcinoma. Why is the behaviour of NCAM in small lung cell cancer more interesting than that of other adhesion molecules in other cancers? Why are tumour-suppressor genes, now the main centre of attention in the genetic analysis of tumour cells, dealt with (inadequately) in less than ten pages? Why is there no mention of the profound analysis of tumorigenic mutations in Drosophila! (Drosophila isn’t even in the index.) The answer in each case, must, I fear, be that the choice of articles was determined by the Editor’s personal range of interest and by the catchment area from which he was able to collect contributions. The end result is a heterogeneous collection of articles varying, as usual, both in their quality and in the depth of their analysis. Two or three of the fourteen chapters are good reviews of their subjects (although, of course, already dated); but as a balanced overview of current research on genes determining cancer, to say nothing of genes associated in other ways with cancer, the book fails. To succeed within so small a compass, a book of this kind must select the most important general features of contemporary research in the area and expound them in an intellectually coherent way. A collection of disparate essays by specialists writing in detail about their own particular experimental interests won’t do.

Amplification of butyrylcholinesterase and acetylcholinesterase genes in normal and tumor tissues: putative relationship to organophosphorous poisoning.

Cholinesterases are ubiquitous carboxylesterase type B enzymes capable of hydrolyzing the neurotransmitter acetylcholine which are transiently expressed in multiple germline, embryonic, and tumor cells. The acute poisoning effects of various organophosphorous compounds are generally attributed to their irreversible covalent interaction with cholinesterases and block of their catalytic activities. We have recently found a de novo inheritable amplification of a CHE gene encoding defective butyrylcholinesterase (acylcholine acyl hydrolase; EC 3.1.1.8) in a family under prolonged exposure to the agricultural organophosphorous insecticide methyl parathion. Further analysis revealed that both the CHE and the ACHE genes, encoding acetylcholinesterase (acetylcholine acetyl hydrolase; EC 3.1.1.7), are amplified in leukemias and platelet disorders and that the tumorigenic expression of these genes in ovarian carcinomas is associated with their frequent coamplification in these tumors. The amplification of CHE and ACHE genes in normal and tumor tissues might be analogous to the well-known amplification of other genes encoding target proteins to toxic compounds. As such, it could provide cells a selection advantage when exposed to organophosphorous poisons. Further, since cholinesterases appear to play developmentally important roles in multiple cell types, the amplification and overexpression of their corresponding genes might affect fertility, be related to the progression of various tumor types, and bear upon the ecological and clinical risks involved with the common use of organophosphorous poisons.

Increased expression of the epidermal growth factor receptor gene in malignant gliomas is invariably associated with gene amplification.

Primary malignant gliomas from 63 patients were analyzed to determine the relationship between amplification of the gene encoding the epidermal growth factor receptor (EGFR) and expression of the corresponding mRNA. Twenty-four tumors were found to have amplified the EGFR gene and amplification of other genes occurred in three additional tumors. Hybridization with synthetic RNA probes was used to quantitate mRNA levels in situ. All 24 tumors with amplification of the EGFR gene had high levels of expression of this gene, while none of the 39 tumors without amplification had increased levels. This shows that, in human gliomas, large increases in the expression of the EGFR gene are invariably associated with alterations in gene structure.

Selective Gene Amplification in Mammalian Cells after Exposure to 60 Co g Rays, 241 Am a Particles, or uv Light

Simian Virus 40 wild type (SV40)-transformed Chinese hamster embryo cells (Co631) contain about five viral copies integrated per cell genome. These SV40 sequences were used as endogenous indicator genes to study the response of mammalian cells to radiation at the gene level. An increase in copy number was detected by dispersed cell blotting and Southern analysis in combination with specific DNA hybridization. All types of radiation tested induce a 15- to 25-fold amplification of SV40 sequences without producing intact virus. The amplification is dose dependent and increases with time after irradiation: a maximum effect is observed at Day 3 after alpha particle or uv exposure and at Day 6 after gamma-ray exposure. A RBE of 6 can be calculated for alpha particles if amplification rates at Day 3 are compared. However, when the maximum effect is considered independent of time, no difference between different types of radiation is observed. Southern blots of genomic DNA show that not all integrated SV40 sequences are amplified upon radiation. Amplified sequences are found either in restriction fragments of relatively high molecular weight or in unit size fragments. SV40 amplification is selective in that the amplification of other genes, e.g., of alpha-actin, dhfr (dihydrofolate reductase), and of two oncogenes of the ras family (Kirsten ras and Harvey ras), was below detection level.