Native and rearranged ALK copy number and rearranged ALK cell count in NSCLC: Implications for ALK inhibitor therapy.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. 7534-7534 ◽  
Author(s):  
D. Ross Camidge ◽  
Margaret Skokan ◽  
Porntip Kiatsimkul ◽  
Barbara Helfrich ◽  
Nathan Schulte ◽  
...  
Keyword(s):  
Cell Count ◽  
Cell Lines ◽  
Copy Number ◽  
Chromosomal Instability ◽  
Copy Number Gain ◽  
Diagnostic Techniques ◽  
Clinical Specimens ◽  
Alk Inhibitor ◽  
Positive Threshold

7534 Background: ALK rearranged NSCLC responds well to ALK inhibitors. Clinically, >15% cells showing rearrangements by break-apart FISH classifies tumors as ALK(+). Native ALK copy number gain has also been reported. We explored the significance of native and rearranged ALK copy number on crizotinib outcomes and whether >15% reflected a clear biological distinction in the frequency of ALK(+) cells. Methods: Copy number and genomic status of ALK assessed by FISH. A total of 1426 NSCLC clinical specimens, 174 ALK(+) and 1252 ALK(-) by standard criteria, and 26 NSCLC cell lines (2 ALK(+) and 24 ALK(-)) were investigated. Results: Native ALK gene mean copy number was significantly higher in ALK(-) than in ALK(+) cases (2.8, SD 0.93, range 1.2-11.4 vs. 1.8, SD 0.79; range 0.6 to 5.2; p<0.01). Frequency of native ALK copy number gain (≥3 copies/cell in ≥40% cells) was 19% in ALK(+) and 62% in ALK(-) tumors (p<0.001). In ALK(-) tumors, abundant focal amplification of native ALK was rare (0.8%); scanty amplification (<10% tumor cells) occurred in 1.1%, and duplication of the entire native ALK or of the ALK 3’ end occurred in 3.5%. Among ALK(-) cell lines, mean native ALK copy number ranged 2.1-6.9 and was not correlated with in vitro crizotinib sensitivity (IC50s 0.34-2.8 uM). In (+) patients, neither native or rearranged ALK copy number, nor percentage cell count correlated with maximal tumor shrinkage or PFS with crizotinib. Clinical specimens with 0-9%, 10-15%, 16-30%, 31-50% and >50% of ALK+ cells were found in 79.3%, 8.5%, 1.4%, 2.7% and 8.1% of cases, respectively. Conclusions: Lower native ALK copy number in ALK(+) NSCLC suggests ALK fusion occurs early, preceding chromosomal instability. Elevated native ALK copy number rarely reflects focal amplification and native ALK copy number increases alone are not associated with sensitivity to ALK inhibition in vitro. Neither native or rearranged copy number, nor positive cell count within ALK(+) tumors influences clinical benefit from ALK inhibition. As 8.5% of ALK(-) cases fall within 5% of the established >15% cell positive threshold, further investigation of ALK status by other diagnostic techniques in this subset may be warranted.

scholarly journals Centromere 17 copy number gain reflects chromosomal instability in breast cancer

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Kyoungyul Lee ◽  
Hyun Jeong Kim ◽  
Min Hye Jang ◽  
Sejoon Lee ◽  
Soomin Ahn ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Copy Number ◽  
Chromosomal Instability ◽  
Copy Number Gain ◽  
Next Generation Sequencing Data ◽  
Her2 Status ◽  
Sequencing Data ◽  
Her2 Positive ◽  
Ki 67 ◽  
Positive Linear Correlation

AbstractChromosomal instability (CIN) is known to be associated with prognosis and treatment response in breast cancer. This study was conducted to determine whether copy number gain of centromere 17 (CEP17) reflects CIN, and to evaluate the prognostic and predictive value of CIN in breast cancer. CIN status was determined by summing copy number gains of four centromeric probes (CEP1, CEP8, CEP11, and CEP16) based on fluorescence in situ hybridization and CIN scores were calculated using next generation sequencing data. High CIN was associated with adverse clinicopatholgical parameters of breast cancer. Among them, positive HER2 status, high Ki-67 index and CEP17 copy number gain were found to be independent predictors of high CIN. High CIN was associated with poor clinical outcome of the patients in the whole group, as well as in luminal/HER2-negative and HER2-positive subtypes. CEP17 copy number was significantly higher in the high-CIN-score group than in the low-CIN-score group. A positive linear correlation between the mean CEP17 copy number and the CIN score was found. In conclusion, CEP17 copy number was confirmed as a useful predictor for CIN in breast cancer, and high CIN was revealed as an indicator of poor prognosis in breast cancer.

scholarly journals Biochemical Studies of (5-P-chlorophenyl -2-benzo 5, 6-coumarin-3-yelthylidene aminothiazole) as Antitumor Agent

2019 ◽  
pp. 1-16
Author(s):  
Faten Z. Mohamed ◽  
Mohamed S. Elghreeb ◽  
Moustafa S. Abdelhamid ◽  
Hazem A. Elbaz
Keyword(s):  
Antitumor Activity ◽  
Cell Count ◽  
Cell Lines ◽  
Cytotoxic Effect ◽  
Biological Activities ◽  
Control Cell ◽  
P53 Expression ◽  
Thiazole Derivative ◽  
Ehrlich Ascites

Heterocyclic compounds have a large spectrum of biological activities including antitumor activity. The present study describes the cytotoxic effect of newly synthesized thiazole derivative (TD2) that can prove effective antitumor activity on both in vivo and in vitro studies. Objective: the essential objective of this research is to prove the cytotoxic effect of newly synthesized thiazole derivative (TD2) up on EAC bearing mice and many kinds of human cell lines. Materials and Methods: Antitumor activity of TD2 was examined on EAC in Swiss albino Mice at dose of 2.5 mg/kg. TD2 was injected for 10 following days after transplantation of tumor. After one day of last dose and 18 hours of fasting, 7 Mice were sacrificed and the remaining was kept to evaluate ILS %. Antitumor activity of TD2 was assessed by inspecting tumor volume, tumor weight, viable cell count and nonviable cell count, hematological, biochemical and antioxidant parameters of mice. Results: TD2 demonstrated an inhibitory effect on both cancer cell lines in vitro and Ehrlich ascites cells in vivo.TD2 increased in life span of Ehrlich–bearing mice compared to control. Cell cycle and flow cytometric analysis revealed that TD2 directed Ehrlich cells toward apoptosis by increasing of P53 expression. Conclusion: It was concluded that TD2 have a potent antitumor activity against Ehrlich ascites carcinoma in mice beside a cytotoxic effect on MCF-7, PC3, HepG2 and HCT-116.

scholarly journals Exosomal transfer of miR-769-5p promotes osteosarcoma proliferation and metastasis by targeting DUSP16

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Wanshun Liu ◽  
Binyu Wang ◽  
Ao Duan ◽  
Kai Shen ◽  
Qi Zhang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Cell Lines ◽  
Mapk Signaling ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Clinical Specimens ◽  
Proliferation And Metastasis

Abstract Background Osteosarcoma (OS) is a malignant tumor originating from mesenchymal stem cells, and has an extremely high fatality rate and ability to metastasize. Although mounting evidence suggests that miR-769-5p is strongly associated with the malignant progression and poor prognosis of various tumors, the exact role of miR-769-5p in OS is still unclear. Therefore, this study aimed to explore the relationship between miR-769-5p and the malignant progression of OS, and its underlying mechanism of action. Methods miR-769-5p expression was analyzed in GSE28423 from the GEO database and measured in OS clinical specimens and cell lines. The effects of miR-769-5p on OS proliferation, migration and invasion were measured both in vivo and in vitro. In addition, bioinformatics analyses and luciferase reporter assays were used to explore the target genes of miR-769-5p. Rescue experiments were also conducted. Moreover, a co-culture model was used to test the cell interaction between bone mesenchymal stem cells (BMSC) and OS cells. Results We found that miR-769-5p is highly expressed in OS clinical specimens and cell lines. In vivo and in vitro experiments also showed that miR-769-5p significantly promoted the proliferation, migration and invasion of OS cells. Dual-specific phosphatase 16 (DUSP16) was negatively associated with miR-769-5p expression in OS cells and tissue samples and was validated as the downstream target by luciferase reporter assay and western blotting. Rescue experiments showed that DUSP16 reverses the effect of miR-769-5p on OS cells by negatively regulating the JNK/p38 MAPK signaling pathway. Additionally, the results of the co-culture of BMSCs and OS cells confirmed that miR-769-5p was transferred from BMSCs to OS cells through exosomes. Conclusions In summary, this study demonstrates for the first time that BMSC-derived exosomal miR-769-5p promotes OS proliferation and metastasis by targeting DUSP16 and activating the JNK/p38 MAPK signaling pathway, which could provide rationale for a new therapeutic strategy for OS.

scholarly journals Dysregulated Aid/Apobec Family Proteins Promote Genomic Instability in Multiple Myeloma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 803-803
Author(s):  
Srikanth Talluri ◽  
Mehmet Kemal Samur ◽  
Leutz Buon ◽  
Stekla A Megan ◽  
Purushothama Nanjappa ◽  
...  
Keyword(s):  
Genomic Instability ◽  
Cell Lines ◽  
Copy Number ◽  
Cytidine Deaminase ◽  
Myeloma Cell ◽  
Mrna Editing ◽  
Genomic Changes ◽  
Elevated Expression ◽  
Apobec Family

Abstract The AID/APOBEC family of cytidine deaminase proteins includes AID (activity induced deaminase), and 10 related APOBEC enzymes (A1, A2, A3A, A3B, A3C, A3D, A3F, A3G, A3H and A4). AID has been well-studied for its role in somatic hyper mutation and class switch recombination of immunoglobulin genes whereas APOBECs (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like) have been shown to have roles in mRNA editing and in antiviral immunity. Dysregulated activity of APOBECs causes C >T transitions or C>G, C>A transversions in DNA. We have recently shown APOBEC signature mutation pattern in multiple myeloma (MM) genomes (Bolli et al Nat. Comm. 2014), and interestingly, the APOBEC mutation signature correlates with sub clonal diversity in myeloma. A role for the AID/APOBECs in generation of somatic mutations has also been proposed in a variety of other cancers based on identification of APOBEC signature mutations In order to understand which APOBECs are dysregulated in myeloma, we performed RNA sequencing analysis of primary myeloma cells from 409 newly-diagnosed MM patients and myeloma cell lines. Our analysis showed elevated expression of several APOBEC family members; mainly A3A, A3B, A3C, and A3G. We then optimized a plasmid-based functional assay and found high cytidine deaminase activity in extracts from a number of myeloma cell lines and patient derived CD138+ cells compared to CD138+ cells from healthy donors, suggesting that APOBECs are dysregulated in myeloma. We then investigated the impact of elevated APOBEC expression/function on overall genome maintenance and acquisition of genomic changes (such as amplifications, deletions) overtime. We used shRNA-mediated knockdown of specific APOBEC proteins in myeloma cell lines and investigated the acquisition of genomic changes in control and knockdown cells during their growth in culture, using SNP (Single Nucleotide Polymorphism) arrays and WGS (whole genome sequencing) platforms. Our results with both approaches showed significant reduction in the accumulation of copy number changes (both amplifications and deletions) and overall mutation load after APOBEC knockdown. Evaluation with both the SNP and WGS showed that when control and APOBEC knockdown cells were cultured for three weeks, the acquisition of new copy number and mutational changes throughout genome were reduced by ~50%. We next investigated the relationship between APOBEC expression/activity in MM and other DNA repair pathways. Using an in vitro HR activity assay, we measured HR activity in extracts from control and APOBEC knockdown cells. Depletion of APOBEC proteins resulted in 50-80% reduction in in vitro HR activity of the extracts. We also evaluated correlation between HR activity and gene expression using RNA-seq data from myeloma cells derived from 100 patients at diagnosis and identified the genes whose expression correlated with HR activity. Elevated expression of APOBECs 3D, 3G and 3F significantly correlated with high HR activity (R=0.3; P≤0.02), suggesting their relevance to HR. Analyzing genomic copy number information for each patient we have also observed significant correlation between higher expression of A3G and increased genomic instability in this dataset (P=0.0045). In summary, our study shows that dysregulated APOBECs induce mutations and genomic instability, and inhibiting APOBEC activity could reduce the rate of accumulation of ongoing genomic changes. This data sheds light on biology of the disease as well as clonal evolution. Disclosures Munshi: Amgen: Consultancy; Oncopep: Patents & Royalties; Celgene: Consultancy; Janssen: Consultancy; Takeda: Consultancy; Merck: Consultancy; Pfizer: Consultancy.

Integrating CRISPR screening with tumor genomic analyses to identify therapeutic targets in hepatocellular carcinomas (HCC) independent of P53.

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. e14659-e14659
Author(s):  
Ankur Sheel ◽  
SuetYan Kwan ◽  
Wen Xue
Keyword(s):  
Cell Proliferation ◽  
Cell Growth ◽  
Cell Lines ◽  
Chromosomal Instability ◽  
Cancer Genomics ◽  
Essential Gene ◽  
Patient Data ◽  
Underlying Mechanisms

e14659 Background: Hepatocellular carcinoma (HCC) is an aggressive subtype of liver cancer with few effective treatments. Moreover, the underlying mechanisms that drive HCC pathogenesis remain poorly characterized. Identifying genes and pathways essential for HCC cell growth will aid the development of new targeted therapies for HCC. Furthermore, the P53 pathway is frequently mutated in HCC therefore identifying targets with therapeutic efficacy irrespective of P53 status is warranted. Methods: To identify kinases essential for HCC proliferation, we performed a kinome wide CRISPR screen in human HCC cell lines with varying P53 mutations and validated our findings using CRISPR-Cas9 mediated genetic manipulations in human HCC cell lines in-vitro and in-vivo. Furthermore, we performed an integrated cancer genomics analyses using patient data from TCGA and the NCI to validate the relevancy of our findings. Results: We identified transformation/transcription domain-associated protein (TRRAP) as an essential gene for HCC cell proliferation. we show that depletion of TRRAP or its co-factor, histone acetyltransferase KAT5, inhibits HCC cell growth via induction of P53, P21 and RB-independent senescence in-vitro and in-vivo. Furthermore, we find that TRRAP is upregulated in HCC patient samples independent of TP53 mutations. Integrated cancer genomics analyses using both HCC patient data derived from TCGA and from RNA-sequencing of our in-vitro model identified a chromosomal instability signature that was regulated by TRRAP/KAT5 in-vitro. Furthermore this chromosomal instability signature was also upregulated in HCC patients. Finally, we identify TOP2A as a target in this pathway as genetic depletion of TOP2A inhibited cell growth via induction of senescence. Conclusions: Our results uncover a role for TRRAP/KAT5 in promoting HCC cell proliferation via activation of mitotic genes in order to potentiate a chromosomal instability signature. Our findings suggest that targeting the TRRAP/KAT5 complex and TOP2A is a therapeutic strategy for HCC, even in tumors that have escaped P53 and RB tumor suppressive programs.

Good and Poor Response Gene Expression Signatures to Proteasome Inhibitors Using a Mouse Model of Multiple Myeloma

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1843-1843
Author(s):  
Holly Stessman ◽  
Linda B. Baughn ◽  
Aaron G. Sarver ◽  
Aatif Mansoor ◽  
Tzu G. Wu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Cell Lines ◽  
Copy Number ◽  
Research Funding ◽  
Proteasome Inhibitors ◽  
Gene Signature ◽  
Mouse Tumor ◽  
Tumor Cell Lines

Abstract Abstract 1843 The proteasome inhibitor bortezomib (Bz) has been used extensively and with much success in the treatment of multiple myeloma (MM) patients; however, patients eventually relapse, many as non-responders to subsequent treatments with Bz making drug resistance a significant problem. Here we utilized cell lines created using a iMycCa/Bcl-xL transgenic mouse model of MM (Cheung, et al. J Clin Invest (2004) 113: 1763) to identify 1) gene expression signatures of Bz response, 2) differences in gene expression between sensitive and resistant cell lines, and 3) cytogenetic abnormalities associated with Bz sensitive and resistant phenotypes. The iMycCa/Bcl-xL transgenic mice develop plasma cell tumors with 100% penetrance and have shown strikingly strong similarities to human MM by extensive gene expression profiling (GEP), spectral karyotyping and histology (Boylan, et al. Cancer Res (2007) 67: 4069). Six cell lines created from these mice were dose escalated with Bz over approximately six months to create Bz resistant (BzR) cell lines with approximately 5–8 fold increase in IC50 to Bz compared to their sensitive counterparts. The BzR characteristics were stable, as lines grown in the absence of drug for as long as 6 months maintained drug resistance upon subsequent challenge. Notably, BzR lines showed cross resistance to other investigational proteasome inhibitors (MLN9708 and carfilzomib) while maintaining sensitivity to other chemotherapeutic agents (dexamethasone and melphalan), suggesting a common mechanism of emerging resistance to proteasome inhibitors. The results of GEP of these mouse tumor cell lines treated with Bz were compared with a recently published human drug trial where GEP was completed prior to and 48 hours after a “test dose” of Bz was administered to patients (Shaughnessy, et al. Blood (2011), ahead of print). In the mouse tumor cell lines, 116 genes were differentially expressed upon in vitro Bz treatment (p=0.001, ≥1.5 fold change). Between the mouse and human drug response data sets was an overlapping common 27-gene signature (p=1×10−25, Fishers exact test) of Bz-induced expression changes that has not previously been described. Time points were collected in these mouse cell line GEP experiments at 0, 2, 8, 16, and 24 hours after Bz treatment. A comparison of the Bz sensitive and derived BzR lines prior to drug treatment revealed a 50 gene signature (p=0.05, ≥2 fold change) that distinguishes three pairs of sensitive and resistant lines. Gene-set enrichment analyses have revealed significant pathways that are differentially regulated in the sensitive and resistant responses. Additional GEP differences were seen when time course expression patterns were examined from Bz sensitive compared to resistant tumor lines. Thus, GEP signatures that distinguish tumor lethality from resistance were identified both prior to Bz treatment, as well as in the early response to Bz. In addition, array comparative genomic hybridization on 4 pairs of mouse Bz sensitive and established BzR lines revealed not only gross differences in copy number between the differentially responding groups of cells but copy number abnormalities that may be unique to the emerging resistance. Taken together, these data indicate that this model is useful for the identification of good and poor Bz response signatures in MM. These signatures are currently being evaluated in human tumor cells from single agent bortezomib phase II and phase III clinical trials. Because the in vitro adapted tumor mouse lines can be genetically manipulated using lentiviral vectors, this model can be used as a preclinical platform to validate existing gene models with respect to Bz response, something that cannot be done using human patients. Subsequent transfer of manipulated lines into syngeneic, immunocompetent recipients can further test Bz response in vivo presenting a significant advantage of this robust mouse MM model system over other in vitro systems. Disclosures: Stessman: Millennium: The Takeda Oncology Company: Research Funding. Mansoor:Millennium: The Takeda Oncology Company: Research Funding. Janz:Millennium: The Takeda Oncology Company: Research Funding. Van Ness:Millennium: The Takeda Oncology Company: Research Funding.

Molecular Characterization Of The Anti-Leukemic Effects Of Single Agent Eltrombopag

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4923-4923
Author(s):  
Uwe Platzbecker ◽  
Katja Sockel ◽  
Claudia Schönefeldt ◽  
Daniel Nowak ◽  
Susann Helas ◽  
...  
Keyword(s):  
Complete Remission ◽  
Disease Progression ◽  
Cell Line ◽  
Cell Lines ◽  
Copy Number ◽  
Single Agent ◽  
Leukemia Cell ◽  
Leukemia Cell Line ◽  
Dose Dependent

Abstract Introduction Eltrombopag (EP) is a small-molecule, nonpeptide thrombopoietin receptor (TPO-R) agonist which has been shown in-vitro to inhibit leukemia cell growth. The underlying mechanism is still under investigation. Methods We report a patient with NPM1 mutated/FLT3 negative refractory AML who achieved a complete remission during treatment with single agent EP within the PMA112509 trial. In this patient we conducted sequential molecular analyses out of the bone marrow to study the underlying molecular mechanisms. Therefore, samples prior to EP, at remission and relapse were subjected to genome-wide copy number analysis using Affymetrix SNP 6.0 array in search for acquired copy number alterations (CNA). To screen for alterations in commonly mutated genes in AML, samples were further subjected to a next generation deep sequencing assay (NGS) of mutational hotspots in the genes ASXL1, CBL, DNMT3A, ETV6, EZH2, IDH1/2, KRAS, NPM1, NRAS, RUNX1, SF3B1, SRSF2, TET2, TP53, U2AF1 and ZRSR2. Sequencing was performed on the 454 GS Junior platform (Roche applied science). Moreover we investigated the expression of TPO-R (CD110) by different assays in cell lines and primary AML samples. To study the TPO-R dependency of potential antineoplastic EP effects we studied the effects of lentiviral TPO-R knockdown and single agent EP on the vitality and cell cycling of TPO-expressing and non-expression leukemia cell lines. Results By using NGS we followed the NPM1+ mutation (NPM1 c.864incTCTG) load in this patient and found a concomitant decline (prior EP: 12.6%, at CR: 1.1%) but not disappearance of NPM1+ cells and a reemergence (15.2%) together with a clonal evolution and development of a NRAS c.37G>C mutation during disease progression (Figure 1) while a SNP-array demonstrated no additional CNA at disease progression. Real time PCR analysis demonstrated TPO-R expression at all time points analyzed which declined during complete remission(TPO-R/GAPDH: prior EP: 56.7%, at CR: 32.3%). These results prompted us to study TPO-R expression of blasts by flow cytometry in de novo AML samples (n=72) at diagnosis. In fact, TPO-R was expressed only in 33 of 72 AML patients but across all FAB and cytogenetic subgroups. The median MFI (mean fluorescence intensity) of CD110 was 2-fold higher on blasts than on CD110 positive lymphocytes. Interestingly, there were some differences with regards to the mutational status, since the NPM mutation was documented more frequently in CD110 negative than in CD110 positive AML cases (26% vs. 10%). These data were confirmed by Taqman-PCR in an independent cohort (n=57) with a nearly three fold lower expression of TPO-R on NPM1+/FLT3- compared to NPM1-/FLT3- (p=0.0163) cases. Next, we sought to clarify if TPO-R expressing AML cell lines are dependent on TPO-R expression. Knockdown of TPO-R by lentivirally transferred shRNA resulted in down-regulation and rapid cell death in the TPO-R expressing megakaryoblastic cell line (CMK). However, treatment with EP in-vitro at doses ranging from 1 to 10 µg/ml lead to a dose-dependent decrease in the cell division rate and vitality not only in CMK but also in cell-lines with weak or absent surface TPO-R expression (e.g. KG1a, a human acute myeloid leukemia cell line or OCI-AML3, a NPM1+ myeloid cell line). In parallel, a significant counterregulatory upregulation of TPO-R mRNA was observed which was dose-dependent (KG1a, p=0.0014). Conclusion These data demonstrate that TPO-R is heterogeneously expressed across all AML subtypes but absent in the majority of NPM1+/FLT3- cases. The clinical response seen in our patient with a refractory NPM1+ AML further provides evidence to the fact that single agent EP can exert potent anti-leukemic effects in-vivo. These effects seem to be mediated rather independently of TPO-R expression. Disclosures: Platzbecker: GlaxoSmithKline: Honoraria, Research Funding.

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