The Q-LAMP Method Represents a Valid and Rapid Alternative for the Detection of the BCR-ABL1 Rearrangement in Philadelphia-Positive Leukemias.

Stefania Stella,Silvia Rita Vitale,Claudia Sargas Simarro,Luigia Luciano,Enrico Marco Gottardi,Francesco Grimaldi,Fabio Stagno,Lucrezia Pironi,Eva Barragan Gonzalez,Santa Errichiello,Paolo Vigneri,Carmen Fava,Barbara Izzo,Valeria Favout

doi:10.3390/ijms20246106

Abstract

Molecular detection of the BCR-ABL1 fusion transcripts is necessary for the genetic confirmation of a chronic myeloid leukemia diagnosis and for the risk classification of acute lymphoblastic leukemia. BCR-ABL1 mRNAs are usually identified using a conventional RT-PCR technique according to the BIOMED-1 method. In this study, we evaluated 122 BCR-ABL1-positive samples with the Q-LAMP assay to establish if this technology may represent a valid alternative to the qualitative BIOMED-1 PCR technique usually employed for the detection and the discrimination of the common BCR-ABL1 transcripts (p190 and p210 isoforms). We found a 100% concordance rate between the two methods. Specifically, the p190- and p210-positive samples were amplified by Q-LAMP with a median threshold time (Tt) of 26.70 min (range: 24.45–31.80 min) and 20.26 min (range: 15.25-34.57 min), respectively. A median time of 19.63 was observed in samples displaying both (e13a2/e14a2) p210 isoforms. Moreover, the Q-LAMP assay allowed recognition of the BCR-ABL1 e13a2 and e14a2 isoforms (median Tts 18.48 for e13a2 vs. 26.08 min for e14a2; p < 0.001). Finally, 20 samples harboring rare BCR-ABL1 isoforms (e1a3, e13a3, e14a3, and e19a2) were correctly identified by the Q-LAMP assay. We conclude that the Q-LAMP assay may represent a faster and valid alternative to the qualitative BIOMED-1 RT-PCR for the diagnosis at BCR-ABL1-positive leukemias, especially when samples are analyzed in centers with restricted resources and/or limited technical expertise.

Highlights

The Philadelphia (Ph) chromosome is the first cytogenetic marker associated with a malignant neoplasia as it is present in 95% of chronic myeloid leukemia (CML) cases, in 3-5% of pediatric acute lymphoblastic leukemia (ALL), and in 15%–20% of adult ALL [1,2,3].The Ph chromosome is generated by a reciprocal translocation between the long arms of chromosomes 9 and 22 t(9;22) (q34;q11) with the ABL1 oncogene juxtaposed to the breakpoint cluster region (BCR) gene on chromosome 22 [4]
In about 75% of BCR-ABL1-positive ALL, but
We evaluate if the Q-loop-mediated isothermal amplification (LAMP) technology may represent a valid alternative to the BIOMED-1 PCR method for the detection and the discrimination of the common BCR-ABL1 transcripts in Ph-positive leukemia patients

Summary

Introduction

The Ph chromosome is generated by a reciprocal translocation between the long arms of chromosomes 9 and 22 t(9;22) (q34;q11) with the ABL1 oncogene juxtaposed to the breakpoint cluster region (BCR) gene on chromosome 22 [4] This cytogenetic alteration gives rise to the BCR-ABL1 chimeric gene encoding for an oncoprotein with constitutive tyrosine kinase (TK) activity that alters the proliferation rates, survival signaling, immunological interactions, and cytoskeleton dynamics of hematopoietic stem cells [5,6,7,8,9]. The breakpoint in the BCR gene is most frequently located downstream of exon 13 or exon 14 (e13 and e14, previously referred to as exons b2 and b3), in the major cluster region (M-BCR), while the most common breakpoint in the ABL1 gene is upstream of exon 2 (a2) These breakpoints lead to the e13a2 or e14a2 BCR-ABL1 rearrangements, which encode for a 210 kDa protein called p210. Other infrequent breakpoints involve BCR exons 6 or 8 (e6a2 or e8a2) or ABL1 exon 3 (e13a3 or e14a3) [1,10,11,12,13,14]

Methods

Results

Conclusion