Detection of BCR-ABL T315I mutation by peptide nucleic acid directed PCR clamping and by peptide nucleic acid FISH
- Valentina Rosso†1,
- Enrico Bracco†2,
- Roberto Pedrola1,
- Sonia Carturan1,
- Elisabetta Signorino1,
- Jessica Petiti1,
- Chiara Calabrese1,
- Paolo Nicoli1,
- Marco De Gobbi1,
- Valentina Gaidano1,
- Daniela Gallo1,
- Stefano Ulisciani1,
- Carmen Fava1,
- Giovanna Rege-Cambrin1,
- Francesco Frassoni3,
- Giuseppe Saglio1 and
- Daniela Cilloni1Email author
© Rosso et al. 2015
Received: 13 June 2015
Accepted: 15 June 2015
Published: 3 July 2015
Mutations of the BCR-ABL1 fusion gene represent a well established cause of resistance to tyrosine kinase inhibitors. Among the different mutations identified T315I is of particular concern since it is not effectively targeted by the majority of Tyrosine Kinase Inhibitors so far available. We developed a novel assay based on peptide nucleic acid (PNA) technology coupled to immunofluorescence microscopy (PNA-FISH) for the specific detection at a single cell level of BCR-ABL T315I mutation thus improving both, diagnostic resolution and the study of clonal prevalence. Furthermore we developed an additional method based on PNA directed PCR-clamping for the fast and easy detection of the mutation.
The PNA directed PCR clamping allows to detect an amount of mutated template as low as 0.5 %. This method is highly sensitive, specific and cheap and could be applied even in laboratory not equipped for more sophisticated analysis. Furthermore, the PNA FISH method allows to identify a small amount of progenitor cells still present after therapy with specific inhibitors.
We present here two different methods based on PNA for the detection of T315I useful for different purposes. PNA-FISH can be used to study clonal evolution. In addition, this method could help in the study of compound mutations being able to identify two different mutations in a single cell. PNA directed PCR clamping although not superior to sequencing can be applied worldwide even in laboratory not equipped to search for mutations.
KeywordsBCR-ABL1 T315I mutation Chronic Myeloid Leukemia PNA
Chronic myeloid leukemia (CML) is characterized by the presence of the Philadelphia chromosome (Ph+) resulting from a translocation between chromosomes 9 and 22 . Ph chromosome gives origin to the BCR-ABL1 fusion gene coding for a constitutive active tyrosine kinase protein. Despite high response rate to specific tyrosine kinase inhibitors (TKI), primary and secondary resistance have been observed: upfront resistance is defined as lack of initial response and acquired resistance is defined as loss of an established response. BCR-ABL1 kinase domain (KD) mutations represent a well established cause of resistance to tyrosine kinase inhibitors . Among different mutations identified the frequently observed T315I is of particular concern since it is not effectively targeted by the majority of TKIs so far available . The only drug showing activity against T315I positive CML is ponatinib. 
Currently, the recommended method for BCR-ABL1 mutation detection is the sequencing of the KD . This is time consuming and it allows to reach a maximum sensitivity of 10–15 %.
The latter point represents a limit, as frequently mutated clones may be present at a lower percentage .
A relative new technique such the “ultra deep sequencing” allows to reach a very high level of sensitivity but it is far from been routinely applicable in world-wide laboratories .
The availability of a simple, sensitive and quick assay, allowing a rapid detection of the T315I mutation is therefore crucial, as the detection of this mutation represents an important element in clinical decision for CML patients.
Peptide Nucleic Acid (PNA) is a potent DNA mimic in terms of sequence specific hybridization. PNA/DNA is thermally more stable than DNA/DNA or DNA/RNA duplexes,  but PNA sequences cannot be extended by DNA polymerase . As consequence, PNA/DNA duplex suppresses DNA amplification. Furthermore, PNA/DNA hybridization shows a greater single-base-pair mismatch discrimination than the corresponding DNA/DNA duplex.
In addition, in order to identify the presence of BCR-ABL1 T315I mutation at the single-cell level, we set up a fluorescently-labelled PNA probe, coupled to FISH technology.
This method allows to distinguish single mutated cells from wild type cells in the context of Ph positive hematopoiesis. Here we report the two strategies (PNA clamping and PNA FISH) for a highly sensitive and very specific detection of BCR-ABL1 T315I, applied in the clinical setting of BCR-ABL T315I monitoring.
PNA-PCR clamping for BCR-ABL1 T315I mutation
The study was approved by the local ethic committee of San Luigi Hospital, Orbassano, Turin. After written informed consent BM aspirates were obtained from 17 imatinib resistant CML patient and 1 Ph + Acute Lymphoblastic Leukemia (ALL), all displaying T315I mutation detected by Sanger Sequencing. In addition, as negative control, 25 CML patients without mutations and 15 healthy subjects were examined. Detection of BCR-ABL1 was performed by capillary Sanger Sequencing method and analyzed by sequencing with BigDye terminator v3.1 (Applied Biosystem, Foster City, California CA) and capillary electrophoresis on ABI PRISM 3130XL (Applied Biosystem, Foster City, California CA). The sensitivity of this method was previously estimated by serial dilutions experiments to be approximately10 %.
Primer Sequences were as follow:
PNA competitor: OO-atatcatcactgagttcat-Lys
Competitor PNA sequence was designed to perfectly match WT template sequence.
PNA-PCR clamping conditions for BCR-ABL1 T315I mutation detection were as follow:
1st step PCR: 94 °C 3 min, (94 °C 30 s, 55 °C 30 s, 72 °C 1 min 40 s) for 40 cycles, 72 °C 5 min; 2nd step PCR: 94 °C 3 min, (94 °C 30 s, 65 °C 20 s, 55 °C 50 s, 72 °C 15 s) for 30 cycles. Sensitivity was assessed mixing, at different ratio, mutated (T315I) and WT template. Dilutions were as follow: 100, 20, 10, 5, 1, 0.5 and 0 % T315I mutated versus WT template. PCR amplification was carried-out in absence (−) or in presence (+) of competitor PNA, at a concentration 3× greater than primer FWD. The amplification performed without (−) PNA represents an internal positive control displaying the efficiency of template amplification.
PNA FISH for the detection of BCR-ABL1 T315I mutation
In addition, for the detection of T315I mutation in progenitor cells we set up a method based on fluorescently-labelled PNA probe (PNA FISH). CD34+ cells were enriched by magnetic cell sorting (MACS; Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s protocol. PNA probe was designed on the human BCR-ABL1 T315I cDNA. The single nucleotide mismatch falls just in the middle of the sequence. The probe has been further tagged by fluorescinated dye at its amino-terminus. The sequence is as follow: Alexa488-OO-TATCATTGAGT-Lys. Detection of BCR-ABL T315 by PNA FISH was performed as previously described . At least 500 cells have been evaluated for each sample. A positive control is added in each reaction to distinguish between negative results and lack of hybridization.
Results and discussion
PNA FISH and PNA direct PCR clamping allow to detect T315I mutation in CML patients
This technique allowed us to identify T315I mutation at a single cell level. In particular we applied this technique to CD34+ cells to investigate the mutation in the progenitor cell compartment.
We suggest that this approach could be extended to other relevant and frequent BCR-ABL1 mutations thus allowing to drive clinical decisions after TKI failure. Importantly, it is now known that the emergence of compound mutations in a BCR-ABL1 allele usually confer ponatinib resistance. More in general, it was shown that BCR-ABL1 compound mutants confer different levels of TKI resistance , thus requiring a rational and patient adapted selection of drugs to optimize the clinical outcome. PNA-FISH allows to identify compound mutation in single cells and to predict response to therapy. Furthermore, PNA-FISH technology allows to provide an answer to many questions including the possibility of the persistence of mutated stem/progenitor cells in patients in MMR and the significance of the presence of small mutated clones at diagnosis. Finally, it allows to follow up clonal evolution during TKI therapy.
Acute lymphoblastic leukemia
Chronic myeloid leukemia
Fluorescence in situ hybridization
Magnetic activated cell sorting
Major molecular remission
Polymerase chain reaction
Peptide nucleic acid
Tyrosine kinase inhibitors
This work was supported by a special grant from “AIRC 5 per mille” to the AGIMM group (AIRC-Gruppo Italiano Malattie Mieloproliferative); for a complete list of AGIMM investigators see at http://www.progettoagimm.it. The study was partially funded by grants from: PRIN, AIL (Associazione Italiana contro le Leucemie), and by Regione Piemonte.
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