Identification Of Gene Amplification Research Articles

Abstract 5123: Characterization of genetic mutation spectra and identification of gene amplification and fusion variants in cell-free nucleic acid from cultured cancer cell media and liquid biopsy specimens using Oncomine™ Pan-Cancer Cell-Free Assay

Abstract Currently the standard practice in tumor biomarker research still relies on invasive tumor biopsy from local sites following by molecular testing or NGS assay. However, this only provides a very limited characterization of tumor composition. Recent studies in non-invasive biomarker research have demonstrated the potential advantages of using cell-free nucleic acids isolated from blood plasma to study genetic heterogeneity of tumor population and dissect the complex cancer clonal architecture. However, it has been challenging to apply to practical research due to low sample yield and sensitivity of detection approaches. Moreover, presence of both cfDNA and cfRNA requires methods capable of interrogating both types of analytes to maximize the utility of each plasma sample and characterize the comprehensive spectrum of mutations including single nucleotide variants, gene amplifications, and structural variants. Recently developed Oncomine™ Pan-Cancer Cell-free Assay employs an amplification-based approach from Ion Torrent NGS technology and achieves exceptional sensitivity and specificity with input amount as little as 20 ng. It includes the most comprehensive genetic content to simultaneously interrogate both cfDNA and cfRNA. Multiple libraries were pooled together for templating on Ion Chef™ and sequenced on Ion GeneStudio S5 systems. In this study, a panel of cancer cell lines harboring multiple variant types were selected and cultured for extended time after apoptosis. We were able to extract cell-free nucleic acids that were released into the cell media and mimic the circulating DNA profile of liquid biopsy samples using in vitro model. Using Oncomine™ Pan-Cancer Cell-free Assay, we successfully detected all the expected variants in these cancer cell lines including gene amplification (MET, ERBB2, CDK4) and fusion variants (ALK fusion and MET exon skipping). Subsequently, we applied this assay to a set of longitudinal liquid biopsy samples collected from a human subject with NSCLC during the course of 15 months. The results showed that a well-known TP53 mutation R248Q was consistently detected in the longitudinal samples with varying allelic frequencies. Interestingly, additional gene amplifications including MET, CDK4 and FGFR3 were identified at late time points. Furthermore, these observations were confirmed by digital PCR and concordant with FISH analyses in solid tumor. Overall, this study demonstrates that Pan-Cancer assay provides a unique and complete NGS solution for comprehensive genetic mutation assessment using in vitro and in vivo liquid biopsy models. Citation Format: Ru Cao, Kris Lea, Madhu Jasti, Jeff Schageman, Khalid Hanif, Yanchun Li, Jian Gu, Varun Bagai, Priyanka Kshatriya, Harriet Wikman, Sonja Loges, Kelli Bramlett. Characterization of genetic mutation spectra and identification of gene amplification and fusion variants in cell-free nucleic acid from cultured cancer cell media and liquid biopsy specimens using Oncomine™ Pan-Cancer Cell-Free Assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5123.

Abstract A099: Safety, tolerability, pharmacokinetics, and efficacy of pan-fibroblast growth factor receptor inhibitor rogaratinib in Japanese patients with refractory, locally advanced metastatic solid tumors selected with a novel, mRNA-based patient selection strategy

Abstract Background: Fibroblast growth factor receptor (FGFR)-mediated pathways are considered to be promising anticancer therapy targets. There are several types of genetic evidence that support an oncogenic function of FGFRs: identification of gene amplifications, activating mutations, chromosomal translocations, and aberrant splicing at the post-transcriptional level. Since FGFR expression is regulated not only by genetic but also by epigenetic mechanisms, examination of tumor FGFR mRNA levels may identify cancer patients likely to benefit from rogaratinib (BAY 1163877), an oral, potent small-molecule pan-FGFR inhibitor. We conducted a phase I study of rogaratinib in Japanese subjects with refractory, locally advanced or metastatic solid tumors selected by elevated tumor FGFR1-3 mRNA expression to evaluate the safety, tolerability, pharmacokinetics, and preliminary efficacy (NCT02592785). Methods: Japanese subjects were enrolled at 600 mg BID (Cohort1) or 800 mg BID as the recommended phase 2 dose (Cohort 2) on a continuous 21-day cycle based on high FGFR1-3 mRNA expression. Archival or newly obtained formalin-fixed, paraffin-embedded (FFPE) tumor tissue samples were analyzed for elevated FGFR1-3 mRNA levels by RNA in situ hybridization (RNAscope®; Advanced Cell Diagnostics, Inc., Newark, CA) and Nanostring® assay (NanoString Technologies, Inc., Seattle, WA). Tumor response was assessed according to Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1. Results: In the prescreening phase of this study, 62 FFPE samples from cancer patients with several tumor types were assessed for FGFR1-3 tumor mRNA expression levels and the valid prescreening results were obtained from 48 samples. Among them, 16 samples (16/48, 33%) were found to overexpress FGFR mRNA. Overall, 9 FGFR mRNA-positive patients were enrolled and treated with rogaratinib, 3 in Cohort 1 and 6 in Cohort 2. All subjects had experienced more than one adverse event (AE). Most of them were Grade 1 or 2. No DLT, no SAE, no Grade 4 or 5 AEs were reported. Two G3 drug-related AEs were hyponatremia and increased ALT. The most common treatment related AE was hyperphosphatemia with Grade 1 or 2 and all cases were controllable with anti-hyperphosphatemia treatment. PK exposure in Japanese subjects was comparable to Western subjects. Eight patients could be advanced to at least one tumor response assessment by CT imaging. As a result, 1 patient with epipharynx cancer achieved a partial response (PR) while 5 patients had stable disease (SD) as best response in target lesions, including three SD patients with tumor shrinkage compared to baseline CT imaging. Conclusions: Rogaratinib showed a good tolerability with a confirmed PR and a disease control rate (DCR) of 56% (5/9). We concluded that 800 mg BID is safe and well tolerable to Japanese patients confirming the global recommended dose. The high prevalence of FGFR mRNA overexpression in tumors as well as the clinical response of FGFR-positive tumors support an mRNA-based patient selection for FGFR inhibitor treatment of cancer. Citation Format: Makoto Tahara, Shunji Takahashi, Matthias Ocker, Stuart Ince, Hendrik Nogai, Changliang Zhang, Fumiyoshi Sei, Peter Ellinghaus. Safety, tolerability, pharmacokinetics, and efficacy of pan-fibroblast growth factor receptor inhibitor rogaratinib in Japanese patients with refractory, locally advanced metastatic solid tumors selected with a novel, mRNA-based patient selection strategy [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A099.

In situ hybridisation: background and applications

<i>In situ</i> hybridisation (ISH) offers molecular diagnosis in a morphological context. Over the last 20 years the technique has moved from research tool to mainstream pathology test with the advent of chromogenic assays, an increasing variety of commercially available probes, and automated hybridisation platforms. The technique can be used to detect DNA or RNA depending on the hybridisation protocol, and can be applied at both light microscopy (LM) and electron microscopy (EM) levels. Troubleshooting problems requires a basic understanding of the technique including probe design and stringency. ISH offers the possibility of simultaneous detection of multiple molecular targets, and/or co-detection of gene and protein targets (via IHC), allowing phenotyping of a cell containing a molecular target, or co-analysis of a target gene and its expression. Current clinical applications include detection of virus (EBV, HPV), analysis of chromosomal translocations or losses (haematological malignancies, sarcomas, small blue cell tumours, gliomas), identification of gene amplification (HER2, EGFR), and tracking of host vs donor cells in sex-mismatched transplant patients (Y chromosome).

Detection of the NUP214-ABL Rearrangement in T-ALL by High-Density Oligonucleotide Arrays: Evidence of the Value of This Approach in the Identification of Gene Amplifications.

The translocation t(9;22), hallmark of CML, is also detected in B-lineage acute lymphoblastic leukemia (ALL). This aberration leads to the BCR/ABL rearrangement that induces a constitutive activation of the tyrosine kinase ABL1. Although BCR/ABL rearrangements were previously considered a rare event in T-ALL, recent data showed the involvement of ABL1 also in this malignancy. Specifically, a new fusion gene involving ABL1 and NUP214, both located at the 9q34 region, has been reported. This abnormality was detected by FISH analysis and reconfirmed by array CGH and PCR analysis. We previously evaluated by microarrays a set of 128 adults with ALL: leukemic cells from 33 patients were of T-cell origin. In the present study, we analyzed the expression levels of ABL1, with the aim of identifying T-ALL cases who may have an involvement of ABL1. Overall, mean expression levels of ABL1 were: pre-B BCR/ABL+ ALL: 1202.71 ± 67.15; pre-B BCR/ABL- ALL: 629.06 ± 23.91; T-ALL: 758.56 ± 42.26, indicating that ABL1 is more highly expressed in T-lineage ALL than in BCR/ABL-B-lineage ALL. Detailed analysis of the T-ALL subset identified 3 cases who had very high levels of ABL1, comparable to those expressed in the BCR/ABL+ cases, and 1 further case with ABL1 expression levels higher than the mean values of BCR/ABL+ cases. Notably, 3/4 patients were refractory to induction chemotherapy. To understand the mechanisms underlying overexpression of ABL1, reverse transcriptase PCR (RT-PCR) was performed and showed the presence of the NUP214-ABL1 in the case having the highest levels of ABL1, whereas the remaining 3 cases were negative. FISH analysis confirmed the presence of NUP214-ABL1 in this case. Quantitative PCR (Q-PCR) of the ABL1 gene was also performed on the same set of T-ALL cases and showed a high degree of correlation between microarray analysis and Q-PCR. HOX11 expression was increased in 2/3 cases, but was not exclusive of these samples; HOX11L2 expression was not evaluated because this transcript was not represented in the platform used. However, FISH analysis excluded the presence of the t(5;14) in all 3 cases. Conventional cytogenetics revealed a normal karyotype in 1 patient, a 4p deletion and an additional marker chromosome in 1, a del(6q) in the case with the highest ABL expression, and was not evaluable in 1. Similarly, CGH analysis showed unbalances in all cases. None of these cases carried a deletion of CDKN2A. Following these findings, we evaluated by both Q-PCR and RT-PCR 15 newly diagnosed T-ALL patients: none of them showed high levels of ABL1. In summary, these results show the value of microarray analysis for the identification of cases carrying specific chromosomal amplifications, i.e. NUP214-ABL1. To our knowledge, this is the first report that identifies this DNA amplification by investigating RNA expression levels of the corresponding transcript. Moreover, our results indicate that this aberration is less frequent than previously reported, being detected in our overall series in 2% of cases. Further investigations are ongoing on the remaining 3 samples to explain the mechanisms underlying ABL overexpression.