Detection of BCR-ABL T315I mutation by peptide nucleic acid directed PCR clamping and by peptide nucleic acid FISH

Biomarker Research

Valentina Rosso³  Enrico Bracco¹  Roberto Pedrola³  Sonia Carturan³  Elisabetta Signorino³  Jessica Petiti³  Chiara Calabrese³  Paolo Nicoli³  Marco De Gobbi³  Valentina Gaidano³  Daniela Gallo³  Stefano Ulisciani³  Carmen Fava³  Giovanna Rege-Cambrin³  Francesco Frassoni²  Giuseppe Saglio³  Daniela Cilloni³ 

[1] Department of Oncology, University of Turin, Turin, Italy

[2] Department of Pediatric Hemato-Oncology and Stem Cell and Cellular Therapy Laboratory, Institute G. Gaslini, Largo G Gaslini, Genoa 16147, Italy

[3] Department of Clinical and Biological Sciences, University of Turin, Turin, Italy

关键词: PNA;    Chronic Myeloid Leukemia;    T315I mutation;    BCR-ABL1;   

Others  :  1219056
DOI  :  10.1186/s40364-015-0039-y

 received in 2015-06-13, accepted in 2015-06-15,  发布年份 2015

【 摘 要 】

Background

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.

Results

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.

Conclusions

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.

【 授权许可】

   
2015 Rosso et al.

【 图 表 】

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【 参考文献 】

• [1]Groffen J, Stephenson JR, Heisterkamp N, de Klein A, Bartram CR, Grosveld G. Philadelphia chromosomal breakpoints are clustered within a limited region, bcr, on chromosome 22. Cell. 1984; 36:93-9.
• [2]Soverini S, Hochhaus A, Nicolini FE, Gruber F, Lange T, Saglio G et al.. BCR-ABL kinase domain mutation analysis in chronic myeloid leukemia patients treated with tyrosine kinase inhibitors: recommendations from an expert panel on behalf of European LeukemiaNet. Blood. 2011; 118:1208-15.
• [3]Hughes T, Saglio G, Branford S, Soverini S, Kim DW, Müller MC et al.. Impact of baseline BCR-ABL mutations on response to nilotinib in patients with chronic myeloid leukemia in chronic phase. J Clin Oncol. 2009; 27:4204-10.
• [4]Cortes JE, Kim DW, Pinilla-Ibarz J, le Coutre P, Paquette R, Chuah C et al.. Kantarjian H A phase 2 trial of ponatinib in Philadelphia chromosome-positive leukemias. N Engl J Med. 2013; 369:1783-96.
• [5]Quintás-Cardama A, Gibbons DL, Kantarjian H, Talpaz M, Donato N, Cortes J. Sequencing of subcloned PCR products facilitates earlier detection of BCR-ABL1 (T315I) and other mutants compared to direct sequencing of the ABL1 kinase domain. Leukemia. 2008; 22:885-8.
• [6]Preuner S, Mitterbauer G, Mannhalter C, Herndlhofer S, Sperr WR, Valent P et al.. Quantitative monitoring of BCR/ABL1 mutants for surveillance of subclone-evolution, expansion, and depletion in chronic myeloid leukaemia. Eur J Cancer. 2012; 48:233-6.
• [7]Soverini S, De Benedittis C, Machova Polakova K, Brouckova A, Horner D, Iacono M et al.. Unraveling the complexity of tyrosine kinase inhibitor-resistant populations by ultra-deep sequencing of the BCR-ABL kinase domain. Blood. 2013; 122:1634-48.
• [8]Sugimoto N, Yamamoto K, Satoh N. Positional effect of single bulge nucleotide on PNA (peptide nucleic acid)/DNA hybrid stability. Nucleic Acids Symp Ser. 1999; 42:95-6.
• [9]Sugimoto N, Satoh N, Yamamoto K. Comparison of thermodynamic stabilities between PNA (peptide nucleic acid)/DNA hybrid duplexes and DNA/DNA duplexes. Nucleic Acids Symp Ser. 1999; 42:93-4.
• [10]Bracco E, Rosso V, Serra A, Carnuccio F, Gaidano V, Nicoli P et al.. Design and application of a novel PNA probe for the detection at single cell level of JAK2V617F mutation in Myeloproliferative Neoplasms. BMC Cancer. 2013; 13:348. BioMed Central Full Text
• [11]Zabriskie MS, Eide CA, Tantravahi SK, Vellore NA, Estrada J, Nicolini FE et al.. BCR-ABL1 compound mutations combining key kinase domain positions confer clinical resistance to ponatinib in Ph chromosome-positive leukemia. Cancer Cell. 2014; 26:428-42.