Background: In acute myeloid leukemia (AML), the internal tandem duplication (ITD) in the juxtamembrane domain of the FLT3 (Fms-like tyrosine kinase 3) gene is one of the most frequent genetic alterations associated with poor prognosis. Methods: A complex evaluation of the analytical properties of the three most frequently used detection methods -PCR followed by agarose (AGE), polyacrylamide (PAGE) or capillary electrophoresis (CE) -was performed on 95 DNA samples obtained from 73 AML patients. Results: All the three methods verifi ed the presence of a mutant allele in 20 samples from 18 patients. AGE and PAGE could detect the presence of 1 % -2 % mutant allele, while the detection limit of CE was 0.28 % . However, acceptable reproducibility (inter-assay CV < 25 % ) of the mutant allele rate determination was only achievable above 1.5 % mutant/ total allele rate. The reproducibility of the ITD size determination by CE was much better, but the ITD size calculated by PeakScanner or GeneScan analysis was 7 % lower as compared to values obtained by DNA sequencing. The presence of multiple ITD was over-estimated by PAGE and AGE due to the formation of heteroduplexes.
Decreased absolute lymphocyte/monocyte ratio (LMR) in peripheral blood has been reported as an unfavorable prognostic marker in Hodgkin lymphoma. We aimed to investigate whether combining LMR and interim PET/CT scan result (PET2) confers stronger prognostic value than PET2 alone. 121 HL patients were investigated. LMR was calculated from a blood sample taken at the time of diagnosis. PET2 was carried out after the second chemotherapy cycle. Survival was calculated using the Kaplan-Meier method and significance was determined by log-rank test. Effect of variants on survival results was examined using univariate and multivariate analyses. Best LMR cut-off value was determined by receiver operating characteristic (ROC) curve. Best LMR cut-off value was 2.11 in the case of our patients (LMR > 2.11: favorable, LMR ≤ 2.11: unfavorable). Overall and progression-free survivals (OS/PFS) were significantly worse both in lower LMR (≤ 2.11) (OS: P = 0.041, PFS: P = 0.044) and PET2 positive groups (OS: P < 0.001, PFS: P < 0.001). In PET2 positive patient group (n = 32) the low LMR result meant a significantly worse OS (0.030) and PFS (0.001). Both LMR and PET2 proved to be independent prognostic factors on multivariate analysis, and strengthened each other's effect.
Leukemic cells often express markers, which are not characteristic of their particular cell lineage. In this study, we identified the ''A'' subunit of coagulation factor XIII (FXIII-A) in leukemic promyelocytes in de novo AML M3 cases. The cytoplasmic presence of factor XIII-A has previously been shown only in platelets/megakaryocytes and monocytes/macrophages. Furthermore, more recently we described the presence of FXIII-A in leukemic lymphoblasts. We studied 14 patients with this rare type of acute leukemia in a period of 4 years and investigated their bone marrow samples by 3-color flow cytometry upon diagnosis, mainly focusing on FXIII-A expression of leukemic cells. We detected FXIII-A also by ELISA, Western-blot, and confocal laser scanning microscopy. This was a homogenous group of AML M3 patients with translocation t(15;17)(q22;q21) detected by fluorescence in situ hybridization (FISH). In 10 out of 14 samples, FXIII-A was detectable by flow cytometry and was coexpressed with markers characteristic for leukemic promyleocytes (CD45dim/CD131/CD331/ CD1171/cyMPO1 and HLA-DR-/CD342/CD142/CD152). Staining for the markers GPIIb and GPIX were negative, and FXIII-A was identified in the cytoplasm of the cells by confocal microscopy in a relatively high quantity, as measured by ELISA. By Western blot analysis we could identify FXIII-A in the native 82 kDa form and in cleaved forms corresponding to cleavage products observed when purified FXIII-A was treated by human neutrophil elastase. This novel expression site of FXIII-A in AML M3 can be considered as a leukemia associated immunophenotype and may have pathophysiological significance. V C 2012 International Clinical Cytometry Society
We evaluated and validated a single-tube flow cytometric procedure for a simple six-parameter FCSS which has not only high diagnostic but also prognostic power.
The Bruton's tyrosine kinase (BTK) inhibitor ibrutinib has revolutionised the therapeutic landscape of chronic lymphocytic leukaemia (CLL). Acquired mutations emerging at position C481 in the BTK tyrosine kinase domain are the predominant genetic alterations associated with secondary ibrutinib resistance. To assess the correlation between disease progression, and the emergence and temporal dynamics of the most common resistance mutation BTK C481S , sensitive (10 À4 ) time-resolved screening was performed in 83 relapsed/refractory CLL patients during single-agent ibrutinib treatment. With a median follow-up time of 40 months, BTK C481S was detected in 48Á2% (40/83) of the patients, with 80Á0% (32/40) of them showing disease progression during the examined period. In these 32 cases, representing 72Á7% (32/44) of all patients experiencing relapse, emergence of the BTK C481S mutation preceded the symptoms of clinical relapse with a median of nine months. Subsequent Bcl-2 inhibition therapy applied in 28/32 patients harbouring BTK C481S and progressing on ibrutinib conferred clinical and molecular remission across the patients. Our study demonstrates the clinical value of sensitive BTK C481S monitoring with the largest longitudinally analysed real-world patient cohort reported to date and validates the feasibility of an early prediction of relapse in the majority of ibrutinib-treated relapsed/refractory CLL patients experiencing disease progression.
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