WCRJ 2016; 3 (4): e799 A COST-EFFECTIVENESS METHOD FOR DETECTION OF ABL MUTATIONS IN PATIENTS WHO DEVELOPED IMATINIB RESISTANCE C. FIERRO1, T. MUTO1, M. TREMATERRA1, F. MORANO1, S. PUGLIESE2, M. DI PAOLO2, A. TROISI2, G. CRESCENTE3 1Hematology and Cellular Immunology Clinical Biochemystry A.O. dei Colli Monaldi, Naples Italy 2CETAC Research Centre, Caserta, Italy 3Italian Association of Pharmacogenomics and Molecular Diagnostics, Caserta, Italy Abstract – Background: Despite high response rate to specific tyrosine kinase inhibitors (TKI), resistances have been observed in patients in treatment with Imatinib because acquired mutations into BCR-ABL1 kinase domain (KD). This can prove a challenge and underlines the importance of utilizing a detection method that is easy, sensitive, reproducible and cost-effective. Materials and Methods: We developed a high specific and sensitive detection assay in order to quickly and easily identify T315I mutation in gene ABL patients by Peptide Nucleic Acid (PNA) directed PCR clamping. Results: The experimental design forecasts that both PNA and PCR primer mutant target sites overlap, thus leading to a direct competition towards complementary DNA (cDNA). 25 μM of ol- igo-PNA was enough to perfect matching PNA/cDNA duplex template in wild-type ABL sequence and PCR amplification is suppressed. Contemporary, in the mutant DNA (I315I) the PNA/cDNA du- plex hybridization fail and PCR can be performed. Conclusions: This detection method is easy, sensitive, reproducible and very cheap. Sequencing method could be restricted as confirmation method only in doubtful mutant samples. Finally, this platform should be ideal for small laboratory that processing few samples. KEYWORDS: PNA Clamping PCR, BCR-Abl, T315I, Imatinib, Sequencing, Chronic myeloid leukemia, Philadelphia chromosome. INTRODUCTION tyrosine kinase inhibitors2. Among different mu- tations identified the frequently observed T315I Chronic myeloid leukemia (CML) is character- is of particular concern since it is not effectively ized by the presence of the Philadelphia chro- targeted by the majority of TKIs so far available3. mosome (Ph+) resulting from a translocation be- The only drug showing activity against I315I tween chromosomes 9 and 221. Ph chromosome positive CML is ponatinib4. creates the BCR-ABL fusion gene coding for a Currently, the recommended method for BCR- constitutive active tyrosine kinase ABL protein. ABL mutation detection is the sequencing of the Despite high response rate to specific tyrosine KD of ABL gene from exon 4 to 10. This is time kinase inhibitor (TKI), primary and secondary consuming and it allows reaching a maximum resistance has been observed: upfront resistance sensitivity of 10-15% of mutant DNA in a large is defined as lack of initial response and acquired excess amount of wild-type (wt) DNA5. resistance is defined as loss of an established re- The latter point represents a limit, as fre- sponse. BCR-ABL kinase domain (KD) mutations quently mutated clones may be present at a lower represent a well-established cause of resistance to percentage6. Corresponding Author: Carla Fierro, BS, Ph.D; e-mail: [email protected] 1 A relative new technique such the “ultra deep TABLE 1. Most important methodics applied for screening sequencing” allows to reach a very high level of e identification of mutation in ABL. sensitivity but it is far from been routinely appli- Method Autor Analitic cable in world-wide laboratories7. sensibility The availability of a simple, sensitive and DNA sequencing Gorre et al12 10-20% quick assay, allowing a rapid detection of the Shah et al13 T315I mutation is therefore crucial, as the de- Nested PCR+ Brandford et al14 10% tection of this mutation represents an important Sequencing Hochhaus et al15 element in clinical decision for CML patients. D-HPLC Soverini et al16 17 Since far time are used different techniques Irving et al Deiniger et al18 capable of identifying individual polymorphisms. Ernst et al19 1-5 % Among these, some have received validation for Pyrosequencing Khorashad et al20 1-5% clinical/diagnostic purposes [Single-Strand Con- HRM Polàkovà et al21 1-5% formational Polymorphism (SSCP) and Restric- Double Gradient Sorel et al22 tion Fragment Length Polymorphism (RFLP)] Denaturing Electrophoresis and others are still in an experimental phase of MALDI-TOF Vivante et al23 validation [Denaturing High Performance Liq- Allele specific Roche-lestienne et al24 1% uid Chromatography (D-HPLC), High Resolution PCR Gruber et al25 Melt (HRM) GENE-chip, MALDI-TOF, PNA Pelzackermann et al26 27 Clamping PCR]. A good example of the compara- Scorpion Probe Khorashad et al 0.1 % PNA fluorescent- Kreuzer et al28 0.1% tive sensitivity and specificity of the method listed clamping-PCR is summarized on (Table 1), which reporting the screening of point mutations in the ABL gene in patients with CML. Among all the techniques for the discrimination of point mutations, the PNA plate amplification. Competitor PNA sequence Clamping PCR is of great interest as it is capable was designed to perfectly match wild-type (wt) of identifying minimal amounts of variant gene template sequence. Therefore, when a single base containing the mutation within a sample almost pair mismatch occurs (like in the case of T315I) exclusively “wild-type” (for example, identifica- PNA-template stability is strongly impaired and tion of small quantities of cancer cells bearing a DNA amplification favored. mutation within a biopsy where there is an excess In this report, we describe a new assay method of normal tissue)8. based on 3′-Peptide nucleic acid (PNA) and modi- Oligo-PNA is a potent DNA mimic in terms fied reverse primer able to clump the amplification of sequence specific with high hybridization of wild-type (wt) DNA. This detection method is affinity. The duplex PNA/DNA thermodynamic easy, sensitive, reproducible and cost-effective. is more stable than DNA/DNA or DNA/RNA This method should be perfect in terms of costs duplexes9, but PNA sequences cannot be ex- for small laboratory that processing few samples. tended by DNA polymerase10. As consequence, PNA/DNA duplex suppresses DNA PCR ampli- fication. Furthermore, PNA/DNA hybridization shows a greater single-base-pair mismatch dis- MATERIALS AND METHODS crimination than the corresponding DNA/DNA duplex. Positive controls Based on this premise and previous data, we developed a novel and sensitive detection assay Positive controls of the conventional PCR and in order to quickly and easily identify T315I sequencing are represented from the cellular mutation in CML patients by PNA directed PCR line K562 in which the gene of BCR is present clamping11. The experimental design forecasts in fusion BCR/ABL p210 (exon 14, exon 2 of that both PNA and PCR primer target sites over- Abl) with b3a2. The rearrangement controls for lap, thus leading to a direct competition towards the mutational screening of PNA Clamping PCR complementary DNA. When perfect matching are represent by a plasmid of 2710 bases (bp) occurs PNA/DNA template hybridization is fa- synthesized from the Gen company Script (The vored more than primer-DNA template duplex Biology Crow USA) in which it has been intro- and DNA amplification is suppressed. Converse- duced the sequence of 643 bp of the ABL gene ly, a single mismatch destabilizes the PNA/DNA comprised between exon 4and exon the 7, that it template duplex, favoring the hybridization be- contains the mutations that will come taken in tween template and primer thus allowing tem- consideration. 2 A COST-EFFECTIVENESS METHOD FOR DETECTION OF ABL MUTATIONS IN PATIENTS WHO DEVELOPED IMATINIB RESISTANCE Sampling 40U RNAasi inibitor (Life Technologies, Milan, Italy) and 200U of Reverse Transcriptase “Super White blood cells (WBC) from bone marrow Script III” (Life Technologies, Milan, Italy). The and peripheral blood are obtained for erithrolisys reaction was incubated for 60 minutes to 42°C using ipotonic solutions made up of ammonium and subsequently for 15 minutes to 75°C. chloride and potassium bicarbonate. The pellet cellular it comes washed in PBS (NaCl 137 mM, KCl 2.7 mM, Na2HPO4.7H 2O, 8.1 mM, KH2PO4 Selective amplification of transcribed 1.9 mM) and quoted in 2 tubes for the extraction fusion gene BCR/ABL of nucleic acids. For the extraction of the DNA the pellet it comes frozen to -20°C, while for the For the mutational screening uses a amplicon extraction of the RNA, the share it comes mixture obtained from a conventional PCR that uses a in phenol/guanidinic acid solution (TRIZOL Invi- primer that it recognizes the gene in the region of trogen, Milan, Italy) and frozen to -20°C. the Major-Breakpoint Cluster Region (M-BCR), post on exon the 12 of BCR and a designed other on exon 7 of ABL. The Forward BCR exon 12 Isolation of RNA primer sequence is 5’-TCC GCT GAC CAT CAA TAA GG-3’ and ABL Reverse 5’-CCA GAC GTC For the extraction of the RNA total, approximate- GGA CTT GAT GG-3. ly 10 million of WBC was combined to 1 mL of This PCR product is used as template for the TRIZOL (Invitrogen, Milan, Italy), added 0.2 mL next PCR-screening for one requirement: of Clorophorme. After centrifugation to 12.000 g 1. To increase the sensibility of amplification for 15 minute to 4°C was evident two phases: one 2. To select the allele BCR-ABL rearrangement organic (down) and one watery (upper). The water in the neoplastic cells excluding the wild-type phase overhanging comes captured and added to ABL (that it major increases the specificity of one equal amount of isopropanol. The nucleic the analysis). acid falls after centrifuge under shape of knows The amplification was performed with 1 μl them. Eventual polluting agents are efficiently of cDNA in a mixture of 25 mL of 10 x Buf- washing by successive 75%ethanol step. The pel- fer (Roche, Milan, Italy) in which is contained let of RNA is resolved in 30 mL of RNAasi-free 1.5mm MgCl2, 200 nM of both primers, 200 nM water.