case_images: PMC6280987_01
case_images: Map of the most recurrent amino acid substitutions in the BCR-ABL1 kinase domain in Ph+ clinical samples.. Notes: Highlighted mutations are the most frequent, usually associated with IM resistance. Numbering of residues is according to normal ABL1 protein. Key structural motifs within the kinase domain are indicated as follows: P-loop, phosphate binding loop; IM binding, ATP/IM binding region; C-loop, kinase catalytic domain; A-loop, activation loop; C-lobe, C-terminal lobe.. Abbreviations: IM, imatinib; Ph+, Philadelphia chromosome positive.
case_images: ott-11-8589Fig1.jpg
case_images: PMC6280987_01_ott-11-8589Fig1.jpg
case_images: f1-ott-11-8589
case_images: Molecular analysis of the patient's white blood cells revealed the expression of P190BCR-ABL1 ("Methods" section in Supplementary materials and Figure S1), the most common isoform in Ph+ ALL.
case_images: Cytogenetic (A) and molecular (B) analyses of Ph+ ALL patient's bone marrow sample.. Notes: (A) Interphase FISH image depicting a Ph+ nucleus, 46,XY,t(9;22)(q34.1;q11.2), using LSI BCR/ABL1 Dual Color, Dual Fusion translocation probe. The image was provided by the Hematology Department of Hospital dos Capuchos. (B) ABL1 and BCR-ABL1 molecular analyses: (B1) control PCR amplification of ABL1 (expected amplicon size=296 bp) and (B2) nested-PCR amplification of e1a2 (P190BCR-ABL1) isoform (expected amplicon size=381 bp). PCR products (1 microL) were resolved via 2% agarose gel electrophoresis. Ladder, GeneRuler DNA Ladder Mix SM0331.. Abbreviations: ALL, acute lymphoblastic leukemia; FISH, fluorescence in situ hybridization; NTC, non-template control; Ph+, Philadelphia chromosome positive.
case_images: ott-11-8589Fig2.jpg
case_images: PMC6280987_01_ott-11-8589Fig2.jpg
case_images: f2-ott-11-8589
case_images: A 31-year-old male was diagnosed with B-ALL (92% blasts), characterized as Ph+ by cytogenetics (70% fluorescence in situ hybridization [FISH]-positive; Figure 2A; "Methods" section in Supplementary materials). However, minimal residual disease (MRD) was detected via reverse transcription nested-PCR according to the standardized procedure established by van Dongen et al ("Methods" section in Supplementary materials and Table S1), which shows a sensitivity level of 10-4 for BCR-ABL1 transcripts (Figure 2B).
case_images: Ph+ ALL patient's novel BCR-ABL1 point mutation and disease molecular monitoring.. Notes: (A) P190BCR-ABL1 protein structure indicating the position of the detected mutation. Sequencing chromatograms displaying: (A1) BCR-ABL1 nucleotide sequence from Ph+ ALL patient's white blood cells at the time of diagnosis; (A2) BCR-ABL1 nucleotide sequence from Ph+ ALL patient's white blood cells at day 29 after diagnosis and treatment initiation. Chromatogram peak analysis indicates that the variation is present in 20% of the total Ph+ clones; (A3) ABL1 nucleotide sequence from Ph+ ALL patient's epithelial cells; and (A4) ABL1 from control individual (WT). P-loop: phosphate binding loop; IM binding: ATP/IM binding region; C-loop: kinase catalytic domain; A-loop: activation loop; and C-lobe: C-terminal lobe. (B) BCR-ABL1 mRNA relative quantification in bone marrow samples at the time of diagnosis and follow-up. qPCR data were normalized to ABL1 and compared to transcript levels at the time of diagnosis. Patient started induction chemotherapy and dasatinib after diagnosis. Treatment was switched to bosutinib 5 months after diagnosis and maintained for 7 weeks (until the 7-month time point).. Abbreviations: ALL, acute lymphoblastic leukemia; IM, imatinib; Ph+, Philadelphia chromosome positive; WT, wild-type.
case_images: ott-11-8589Fig3.jpg
case_images: PMC6280987_01_ott-11-8589Fig3.jpg
case_images: f3-ott-11-8589
case_images: Mutational analysis via Sanger sequencing ("Methods" section in Supplementary materials) showed a point mutation in the ABL1 domain of the fusion transcript, c.1319A.G, which lies in the region that translates for the C-terminal lobe of the kinase domain, p.Tyr440Cys (Figure 3). Despite this variation being present in 20% of the BCR-ABL1-expressing cells at follow-up (day 29; Figure 3A2), it had not been identified at diagnosis (day 1; Figure 3A1). This was confirmed by the analysis of the genomic DNA of the patient's epithelial cells (Figure 3A3). After 7 weeks on bosutinib, BCR-ABL1 levels did decrease when compared to those on dasatinib (Figure 3B).