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Development and Clinical Validation of a "Home-brew" Dual-Color FISH probe assay for HNRNPK

2015-06-15 13:56:12

The University of Texas M.D. Anderson Cancer Center; 1 September 2015; Annual Meeting and Exhibition Paper

Divya Narayanan, Kristina Regmi, Isaac Wun, Duy Truong, Ming Zhao, Sean Post, Carlos Bueso-Ramos, Peter Hu



Abstract


The heterogeneous nuclear ribonucleoprotein K(hnRNP K) has been linked to basic cellular functions. The HNRNP K gene, located at chromosome 9q21.32, has been observed as a deletion in certain subsets of AML patients. Recent RNA expression analyses have shown a reduction in HNRNP K expression from those AML patients with the gene deletion, but also an increased expression in those without the deletion. Therefore, an effective cytogenetic tool, i.e., home-brew FISH assays, has been used to evaluate the duplication and deletion of the HNRNP K gene, which can be representative of the overexpression and underexpression of hnRNP K that has significant impact on hematological malignancies. We developed dualcolor fluorescence in situ hybridization (FISH) assay using bacterial artificial chromosomes (BACs) RP11- 101L4 containing the entire HNRNP K gene (148kb) and a 198kb RP11-19G1 control probe to the 9p region. Three random bone marrow smears were used as controls. We used the statistical tool, BETAINV, to calculate the cut-off range for each signal pattern. The cut-off ranges were set as 6.8% for
duplication / addition of the red signal and 5.8% for deletion of the red signal. Based on metaphase analysis, the probes showed 100% specificity for the respective regions of HNRNP K and RP11-19G1. Nineteen bone marrow smears from various AML types were tested. Duplication pattern was found to be above the cut-off for 16 samples; while 3 samples with duplication pattern above the cut-off, also had deletion pattern above the cut-off. This suggests that multiple clones are present in the same patient sample, a phenomenon often observed in various haematological malignancies. Additional samples are currently being tested and their results will be compared against their cytogenetics G-banded analysis and other molecular analysis to further investigate these aberrations.



Introduction


Heterogeneous nuclear ribonucleoprotein K (hnRNP K) is a multifunctional signaling protein localized in the nucleus, cytoplasm, and mitochondria and is involved in the regulation of structural organization of the chromatin, transcription, pre-mRNA processing, splicing, mature mRNA transport to the cytoplasm,
translation, nuclear transport, signal transduction, and DNA repair. The cytoplasmic accumulation of hnRNP K was found to be crucial for metastasis, and further investigations have demonstrated that it is overexpressed in several human cancers and its abberrant cytoplasmic localization corresponds with worse
prognosis for patients. Various biochemical and cell line studies suggested hnRNP K negatively regulates the p53 pathways due to its reduced expression and also, positively regulates c-myc pathway due to its overexpression. This leads to the belief that its abnormal expression, in either direction, may have significant impact on tumor predisposition. However, in a subset of acute myeloid leukemia(AML), the HNRNP K gene region present in chromosome 9 (9p21.32) is found to be deleted, leading to reduction in its protein expression. Also, those AML patients who do not possess this deletion, exhibited an increase in hnRNP K expression. We have developed dual-color fluorescence in situ hybridization (FISH) assay, using bacterial artificial chromosomes (BACs). FISH, a simple yet powerful cytogenetic technique, has the ability to detect the genetic anomalities and also, to monitor the progression of aberration. Thus, it is a reliable technique for both research and diagnosis of hematological malignancies and solid tumors. Our aim is to validate the probe set specific to HNRNP K gene so that it can help to understand the expression spectrum of its protein in AML and also be a useful diagnostic tool to evaluate the prognosis due to hnRNP K protein in AML patients.



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