Identification of rare atypical BCR-ABL1 transcript: A case report

CML is characterized by the presence of a BCR-ABL1 fusion transcript. Several guidelines have been published for its detection and molecular monitoring. Here, a case is described of chronic myeloid leukemia presenting in the blast phase with a rare variant transcript, with a discussion on possible red flags in its detection and genetic testing and description of the patient's clinical characteristics. This case highlights the pitfalls of using real-time quantitative reverse-transcription polymerase chain reaction (RQ-PCR) for diagnosis of CML, especially when the clinical picture and the test results are discordant.

Outcome and prognostic factors of children with Philadelphia chromosome-positive acute lymphoblastic leukemia treated with imatinib followed by allogeneic hematopoietic cell transplantation in first remission

BACKGROUND: Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) is a subset of ALL with poor prognosis. Here, we analyzed the outcomes and prognostic factors of children with Ph+ ALL who received imatinib and chemotherapy followed by allogeneic hematopoietic cell transplantation (HCT) in first complete remission (CR). METHODS: Thirty-one Ph+ ALL patients (female 10) diagnosed from January 2005 to December 2016 were included in the study. All patients were treated with imatinib and chemotherapy before HCT. Bone marrow (BM) evaluations included real-time quantitative polymerase chain reaction (RQ-PCR) study of the BCR-ABL1 fusion transcript. All patients received HCT with total body irradiation (TBI)-based conditioning at a median of 6.4 (range, 4.2–47.1) months from diagnosis. RESULTS: Compared to values at diagnosis, the median decrement of RQ-PCR value post-consolidation, and prior to HCT was −3.7 Log and −4.8 Log, respectively. The 5-year event-free survival (EFS) and overall survival of the patients were 64.5±9.4% (20/31) and 75.0±8.3% (23/31) respectively. Events included relapse (N=5) and death in CR post-HCT (N=6). The 5-year incidence of molecular relapse was 30.9±9.1% (9/31). An RQ-PCR decrement of at least −4 Log post-consolidation significantly predicted lower incidence of molecular relapse: 7.7±7.7% for ≥−4 Log decrement, 50.0±13.8% for <−4 Log decrement (P=0.027). CONCLUSION: Decrement in RQ-PCR for the BCR-ABL1 transcript that was determined after consolidation was the only significant prognostic factor for incidence of molecular relapse. In the post-induction TKI initiation setting, steadfast imatinib treatment during consolidation may allow for optimum post-HCT outcomes.

Minimal residual disease detection in Tunisian B-acute lymphoblastic leukemia based on immunoglobulin gene rearrangements

IGH gene rearrangement and IGK-Kde gene deletion can be used as molecular markers for the assessment of B lineage acute lymphoblastic leukemia (B-ALL). Minimal residual disease detected based on those markers is currently the most reliable prognosis factor in B-ALL. The aim of this study was to use clonal IGH/IGK-Kde gene rearrangements to confirm B-ALL diagnosis and to evaluate the treatment outcome of Tunisian leukemic patients by monitoring the minimal residual disease (MRD) after induction chemotherapy. Seventeen consecutive newly diagnosed B-ALL patients were investigated by multiplex PCR assay and real time quantitative PCR according to BIOMED 2 conditions. The vast majority of clonal VH-JH rearrangements included VH3 gene. For IGK deletion, clonal VK1f/6-Kde recombinations were mainly identified. These rearrangements were quantified to follow-up seven B-ALL after induction using patient-specific ASO. Four patients had an undetectable level of MRD with a sensitivity of up to 10-5. This molecular approach allowed identification of prognosis risk group and adequate therapeutic decision. The IGK-Kde and IGH gene rearrangements might be used for diagnosis and MRD monitoring of B-ALL, introduced for the first time in Tunisian laboratories.

FAMLF is a target of miR-181b in Burkitt lymphoma

Burkitt lymphoma (BL) is a highly malignant non-Hodgkin's lymphoma that is closely related to the abnormal expression of genes. Familial acute myelogenous leukemia related factor (FAMLF; GenBank accession No. EF413001.1) is a novel gene that was cloned by our research group, and miR-181b is located in the intron of the FAMLF gene. To verify the role of miR-181b and FAMLF in BL, RNAhybrid software was used to predict target site of miR-181b on FAMLF and real-time quantitative PCR (RQ-PCR) was used to detect expression of miR-181b and FAMLF in BL patients, Raji cells and unaffected individuals. miR-181b was then transfected into Raji and CA46 cell lines and FAMLF expression was examined by RQ-PCR and western blotting. Further, Raji cells viability and proliferation were detected by MTT and clone formation, and Raji cell cycle and apoptosis were detected by flow cytometry. The results showed that miR-181b can bind to bases 21–42 of the FAMLF 5′ untranslated region (UTR), FAMLF was highly expressed and miR-181b was lowly expressed in BL patients compared with unaffected individuals. FAMLF expression was significantly and inversely correlated to miR-181b expression, and miR-181b negatively regulated FAMLF at posttranscriptional and translational levels. A dual-luciferase reporter gene assay identified that the 5′ UTR of FAMLF mRNA contained putative binding sites for miR-181b. Down-regulation of FAMLF by miR-181b arrested cell cycle, inhibited cell viability and proliferation in a BL cell line model. Our findings explain a new mechanism of BL pathogenesis and may also have implications in the therapy of FAMLF-overexpressing BL.

Differential Expression of CD269 and CD317 Genes in Bone Marrow of Patients with Multiple Myeloma / 现代检验医学杂志

Objective To explore the differential expression of CD269 and CD317 in patients with multiple myeloma(MM). Methods Newly diagnosed samles from patients of MM(20 cases)and iron deficiency anemia(20 cases),40 cases in total (from 06/2015 to 08/2013,the Department of Hematology,Central Hospital of Zhuzhou City)were collected.Real-time quantitative PCR(RQ-PCR)tests were used to detect the relative expression of CD269 and CD317 in bone marrow sam-ples,and the results were statistically analyzed with clinical features.Results The relative expression levels of CD269 and CD317 in patients with multiple myeloma(4.418±4.568,4.327±2.876)were significantly higher than those in the control group(0.600±0.838,1.033±1.335),the difference was statistically significant(t=3.676,4.646,all P<0.05)respective-ly,while not related with the gender,age(P>0.05).There was no correlation between the expression of CD269 and CD317 (r=0.041,P=0.864),but positively correlated with the ratio of myeloma cells(r=0.495,P=0.026;r=0.533,P=0.016).Conclusion CD269 and CD317 were highly expressed in patients with multiple myeloma and may be involved in the pathogenesis of multiple myeloma.

Real-time quantifi cation for BCR-ABL transcripts in chronic myeloid leukaemia patients in UKMMC, Malaysia

Molecular pathogenesis of chronic myeloid leukemia (CML) is well established and molecular monitoring for patients with CML has become an important practice in the management of patients on imatinib therapy. In the present study, we report the use of RQ-PCR method for detection of BCR-ABL fusion gene for our CML cases. We performed a two-step RQ-PCR on bone marrow aspirates or peripheral blood of 37 CML patients. Quantitative expression of BCR-ABL fusion gene was carried out relative to the expression of a housekeeping gene as endogenous control to compensate for uneven cell numbers, RNA quality, or variations in reverse transcription effi ciencies. Twenty-four of these patients were pre-treated with hydroxyurea or alpha interferon prior to the imatinib therapy. Their BCR-ABL fusion gene levels were monitored for 18 months. All samples processed were evaluable. The PCR amplifi cation effi ciency of the ABL gene is 90.5% (0.2158) and the BCR-ABL gene, 93.4% (0.1573).

Evaluation of BiosewoomTM Real-Q Cytomegalovirus Quantification kit for Cytomegalovirus Viral Load Measure / 대한진단검사의학회지

BACKGROUND: Rapid and accurate laboratory tests are essential to detect cytomegalovirus (CMV) infections in solid organs and haematopoietic stem cell transplant recipients. We assessed the realtime quantitative PCR (RQ-PCR) technology for its usefulness in detecting CMV DNA. METHODS: We evaluated the analytical performance of CMV RQ-PCR using Real-Q Cytomegalovirus Quantification kit (BioSewoom Inc., Korea). To evaluate its clinical utility, we also compared it to pp65 antigenemia test, an immunostaining method, on 343 samples of total 84 patients, including 63 transplant recipients. RESULTS: The detection limit of RQ-PCR was 63 copies/mL and none of hepatitis B virus, hepatitis C virus, or human immunodeficiency virus showed a cross-reactivity with CMV. Total coefficient of variation (CV) was 10.4-19.5%. It detected CMV DNA in a linear range from 1 x 10(2) to 5 x 10(11) copies/mL (P<10(-13), R2=0.9994). The qualitative positive rates of pp65 antigenemia test and RQ-PCR were 4.7%, 16.3%, respectively and concordance rate between the two tests was 84.8% (K=0.221, P<10(-6)). In comparison of quantitative results, the correlation between two tests was significant (r=0.45, P<10(-17)). In comparison among three groups by pp65 antigen level, CMV DNA level obtained with RQ-PCR increased significantly (P<10(-3) and P<10(-7), respectively). CONCLUSIONS: The RQ-PCR is easier to perform than the immunostaining method, has good analytical performance and reflects the blood level of viral DNA well. It may be a new method substituting the pp65 antigenemia test. Further studies determining RQ-PCR value starting pre-emptive therapy will be required.

Evaluation of BiosewoomTM Real-Q Cytomegalovirus Quantification kit for Cytomegalovirus Viral Load Measure / 대한진단검사의학회지

BACKGROUND: Rapid and accurate laboratory tests are essential to detect cytomegalovirus (CMV) infections in solid organs and haematopoietic stem cell transplant recipients. We assessed the realtime quantitative PCR (RQ-PCR) technology for its usefulness in detecting CMV DNA. METHODS: We evaluated the analytical performance of CMV RQ-PCR using Real-Q Cytomegalovirus Quantification kit (BioSewoom Inc., Korea). To evaluate its clinical utility, we also compared it to pp65 antigenemia test, an immunostaining method, on 343 samples of total 84 patients, including 63 transplant recipients. RESULTS: The detection limit of RQ-PCR was 63 copies/mL and none of hepatitis B virus, hepatitis C virus, or human immunodeficiency virus showed a cross-reactivity with CMV. Total coefficient of variation (CV) was 10.4-19.5%. It detected CMV DNA in a linear range from 1 x 10(2) to 5 x 10(11) copies/mL (P<10(-13), R2=0.9994). The qualitative positive rates of pp65 antigenemia test and RQ-PCR were 4.7%, 16.3%, respectively and concordance rate between the two tests was 84.8% (K=0.221, P<10(-6)). In comparison of quantitative results, the correlation between two tests was significant (r=0.45, P<10(-17)). In comparison among three groups by pp65 antigen level, CMV DNA level obtained with RQ-PCR increased significantly (P<10(-3) and P<10(-7), respectively). CONCLUSIONS: The RQ-PCR is easier to perform than the immunostaining method, has good analytical performance and reflects the blood level of viral DNA well. It may be a new method substituting the pp65 antigenemia test. Further studies determining RQ-PCR value starting pre-emptive therapy will be required.

Differential molecular response of the transcripts B2A2 and B3A2 to imatinib mesylate in chronic myeloid leukemia

Chronic myeloid leukemia (CML) originates from the hematopoietic stem cell and is characterized by the reciprocal translocation t(9;22)(q34;q11), which results in the BCR-ABL fusion gene on chromosome 22q-, also known as the Philadelphia chromosome. This chimeric gene codes for a cytoplasmic protein with constitutive tyrosine-kinase activity, responsible for cellular transformation and leukemogenesis in CML. The aim of this observational cohort study was to discriminate and quantify BCR-ABL transcripts in the peripheral blood of patients with CML who were treated with imatinib mesylate (Glivec, Novartis). Twenty-two patients were followed for six months during treatment. Quantitative real time polymerase chain reaction was performed before treatment and after 3 and 6 months from treatment initiation. As compared with the third month, there was a significant decrease in BCR-ABL expression in the sixth month of treatment (P = 0.0002). At the sixth month, there was a significant difference in the levels of the two major transcripts of BCR-ABL, B2A2 and B3A2 (P = 0.0347), indicating that B2A2 may be more sensitive to imatinib. The results of our study indicate that imatinib is able to modify the natural history of CML, and raise the hypothesis that patients who express the B2A2 transcript may have a better prognosis.

Assessment of bcr/abl expression by real-time quantitative PCR in chronic myeloid leukemia patients after imatinib mesylate treatment / 解放军医学杂志

Objective To set up a new real-time quantitative PCR method for the detection of minimal residual disease in chronic myeloid leukemia patients, and to assess the bcr/abl fusion gene expression in chronic myeloid leukemia patients before and after treatment with imatinib mesylate by real-time quantitative PCR method. Methods The bcr/abl fusion gene expression in 30 patients with bcr/abl-positive chronic myeloid leukemia was analyzed by using real-time quantitative reverse transcription PCR (RQ-PCR) method. The patients treated with imatinib in a dose of 400mg/d for 1 year and 2 years were also examined (8 cases for each). In 19 new patients the same study was also conducted. Results The real time quantitative PCR method could detect 10 copies in the test. The average bcr/abl expression levels in new patients or patients who had been treated with imatinib for 1 year and 2 years were 68.18%?26.67%, 0.16%?0.15% and 0.04%?0.02%, respectively. The average logarithm reduction values after treatment were 2.82 in the first year and 3.36 in the second year. In 25% of patients (4/16) negative FISH results could not be obtained, but it was much lower than that of before imatinib-treatment. When FISH became negative, RQ-PCR showed positive results. Conclusions RQ-PCR is a more sensitive technique in the detection of bcr/abl fusion gene than the FISH. It is an important way to monitor the tumor cell during the treatment with imatinib mesylate in chronic myeloid leukemia patients.