Development and evaluation of a secondary reference panel for BCR-ABL1 quantification on the International Scale.

Molecular monitoring of chronic myeloid leukemia patients using robust BCR-ABL1 tests standardized to the International Scale (IS) is key to proper disease management, especially when treatment cessation is considered. Most laboratories currently use a time-consuming sample exchange process with reference laboratories for IS calibration. A World Health Organization (WHO) BCR-ABL1 reference panel was developed (MR(1)-MR(4)), but access to the material is limited. In this study, we describe the development of the first cell-based secondary reference panel that is traceable to and faithfully replicates the WHO panel, with an additional MR(4.5) level. The secondary panel was calibrated to IS using digital PCR with ABL1, BCR and GUSB as reference genes and evaluated by 44 laboratories worldwide. Interestingly, we found that >40% of BCR-ABL1 assays showed signs of inadequate optimization such as poor linearity and suboptimal PCR efficiency. Nonetheless, when optimized sample inputs were used, >60% demonstrated satisfactory IS accuracy, precision and/or MR(4.5) sensitivity, and 58% obtained IS conversion factors from the secondary reference concordant with their current values. Correlation analysis indicated no significant alterations in %BCR-ABL1 results caused by different assay configurations. More assays achieved good precision and/or sensitivity than IS accuracy, indicating the need for better IS calibration mechanisms.

Light microscopic detection of BCR-ABL transcripts after in-cell RT-PCR: fusion gene expression might correlate with clinical evolution of chronic myeloid leukemia.

A procedure for in-cell amplification of the hybrid BCR-ABL mRNA by reverse transcription and polymerase chain reaction (RT-PCR) without extraction of the nucleic acids was performed directly in fixed and permeabilized cells of leukemia patients (22 patients with Ph'-positive chronic myeloid leukaemia-CML and 1 with Ph'-positive acute leukaemia-AL, as well as 7 Ph'-negative cases) and Ph'-positive human leukaemia cell lines (K562, LAMA-84, BV173). The labelling of the amplified sequences was done employing biotinylated primers and a second PCR in a semi-nested fashion with a low number of cycles. An enzymatic system based on biotin-streptavidin-chromogen reaction was used for the detection of labeled PCR product, thus producing a coloured product, visible to the eye under a standard light microscope. All samples from patients with cytogenetic and molecular evidence of BCR-ABL rearrangement showed specific cytoplasmic staining at the site of the amplified hybrid transcripts. It allowed definite distinction between positive and negative cells. K562, LAMA-84, BV173 cells were characterized with strong diffuse staining while an interesting finding of the present study was the presence of variable quantities of colour product in patients' samples which might be due to different mRNA expression. Early and intermediate stages of myeloid maturation showed more intense reactivity. Cases with an aggressive course of accelerated or blast phase CML and AL were found to have a considerable subset of cells with strongly expressed signal while cases in chronic phase were characterised with uniform weak to moderate reaction. Our observations support the hypothesis that the amount of BCR-ABL transcript expression within neoplastic cells may play a role in dictating the eventual behaviour of the leukaemic clone. Future studies at a single cell level of larger series of consecutive cases with a follow up might be able to identify those patients who are prone to transformation and provide certain indications for further therapeutic decisions.

Clonal heterogeneity in a patient with acute promyelocytic leukemia.

A 18-year-old man was diagnosed with acute promyelocytic leukemia (APL). The conventional cytogenetic analysis revealed normal karyotype 46, XY, t(15; 17). Reverse transcriptase polymerase chain reaction (RTPCR) identified PML-RARa chimeric transcripts. Complete remission (CR) was attained with 3 induction courses of Ara-C, daunorubicin and all-trans retinoic acid (ATRA). Three years later the patient relapsed. The blasts in bone marrow aspirate at relapse had AML-M3 morphology, and RT-PCR was positive for PML-RARa transcripts. The patient was treated with ATRA and daunorubicin without success. Two months later the blasts in bone marrow aspirate showed AML-M2 morphology, the karyotype was 47, XY, +8 and RT-PCR revealed the presence of AML1-ETO transcripts and absence of PML-RARa transcripts. The patient attained second CR with 3 induction courses -a course with Ara-C and daunorubicin and 2 courses with idarubicin, Ara-C and etoposide.

Philadelphia variant, t(5;9;22)(q13;q34;q11), in a case with chronic myeloid leukemia.

In this paper we report on a case of chronic myeloid leukemia (CML) with a Philadelphia variant involving chromosome 5 as a second change of the standard Philadelphia chromosome (Ph). Molecular analysis found a fusion gene BCR-ABL with participation of exons b3 and a2, respectively (b3a2). The molecular variant of the fusion gene BCR/ ABL or the rare involvement of chromosome 5 could possibly explain the mild course of the disease.

Application of reverse transcription polymerase chain reaction for analysis of multidrug resistance in patients with acute myeloblastic leukemia.

PURPOSE: To test the multidrug resistance 1 (MDR1) gene expression in acute myeloid leukemia (AML) patients using the reverse transcription polymerase chain reaction (RT-PCR) in order to assess the application of the method for routine screening. MATERIALS AND METHODS: 39 AML patients (mean age 43.9-/+18.2 years) and 10 healthy volunteers were studied by simultaneous amplification of the MDR1 and beta2-microglobulin (beta2-M) mRNA, as an internal control. Semi-quantitative characterization of MDR1 expression was done using a 4-grade scoring system: negative (-/+), weakly positive(-/+), moderately positive (+), strongly positive(++), according to the intensity of the MDR1 and beta2-M bands. RESULTS: The amplification of the MDR1 in healthy individuals yielded no products in 9 samples and in 1 case a (-/+) reaction was observed. In AML patients, RT-PCR revealed no MDR1 product (normal level) in 19 (48.7%) and (+)/(++) reaction (overexpression) in 15 (38.5%) patients. In the remaining 5 (12.8%) patients a (-/+) reaction was found, comparable to that of the (-/+) healthy individual (the level of MDR1 expression could not be defined). MDR1 overexpression was seen in 73.3% of the M1/M2 patients, and only in 14.3% of the M3 and M4/M5 patients. Besides, the MDR1(- )/(-/+)patients were significantly younger than MDR1(+)/ (++) patients (mean age 38.4-/+19.5/43.0-/+8.7 years, versus 51.1-/+16.7/65.0-/+4.3 years). CONCLUSION: RT-PCR allows for the identification of patients with moderate and higher levels of MDR1 overexpression, while the weak positive reactions require additional testing. Besides, our data support the observation that the level of expression might be associated with the French-American-British (FAB) subtype of AML and with the age of the patients.

Wilms' tumor protein and FLT3-internal tandem duplication expression in patients with de novo acute myeloid leukemia.

Bone marrow samples of 30 patients with de novo adult acute myeloid leukemia (AML) were analyzed for Wt1 and FLT3-internal tandem duplication (FLT3-ITD) expression measured by western blot and reverse transcription-polymerase chain reaction analysis, respectively. Wt1 was detected in 53·3% of AML patients (16/30), while FLT3-ITD in 23·3% (7/30). The high Wt1 expression correlated with the presence of FLT3-ITD (P = 0·014) and lower rate of complete remission (P = 0·023). The cumulative survival in AML patients was affected significantly by the presence of FLT3-ITD, being lower in the FLT3-ITD (+) group (6·0±2·4 months) compared to the FLT3-ITD (-) patients (17·9±3·3; P = 0·04). The expression of FLT3-ITD could probably activate Wt1 expression in AML blast cells and thus might contribute to its oncogenic function to provide cells with survival advantages in vivo. The detection of both molecular markers (Wt1 and/or FLT3-ITD) may be helpful in defining high risk AML patients that need special therapeutic strategies.

Very early onset of an acute myeloid leukemia in an adult patient with B-cell lymphoblastic leukemia.

We report on a case of a 30-year-old male with acute B-lymphoblastic leukemia (B-ALL) with immunophenotype CD19(+), CD22(+), CD20(+), CD10(+), with aberrant expression of CD13 and CD117, and IgH gene rearrangements. Three months after treatment with GMALL-2003 and Ida/FLAG protocols bone marrow showed predominance of blasts with myeloid morphology and phenotype MPO(+), CD13(+), CD33(+), CD64(+), CD15(+), CD56(+), EVI-1 gene overexpression and lack of IgH rearrangements. The case is the first report of a very early emergence of myeloid leukemia during the induction treatment for B-ALL in an adult patient. Different pathogenetic mechanisms are discussed - clonal evolution or selection, lineage switch or development of a de novo or therapy-induced leukemia.

Acute megakaryoblastic leukaemia with extreme thrombocytosis and p190(bcr/abl)rearrangement.

Acute megakaryoblastic leukaemia (AML-M7) is an uncommon disease, composing 0.5-1.2% of newly diagnosed adult acute myeloid leukaemias (AML). It is characterised by higher incidence and complexity of cytogenetic abnormalities. We report a rare case of Philadelphia (Ph) chromosome-positive AML-M7, presenting with extreme thrombocytosis and having a poor outcome. The diagnosis was established on the basis of morphological and flow cytometry data for megakaryoblastic proliferation in the bone marrow. Cytogenetics revealed 47,XX,+8,t(9;22)(q34;q11), and p190(BCR-ABL)-rearrangement was detected. MDR1-gene overexpression was not demonstrated; however, the patient was resistant to therapy and died in 6 months. The reported case contributes to the overt heterogeneity of Ph-positive AMLs, which warrants further investigation and understanding.