The postinduction level of minimal residual disease (MRD) was quantified with a competitive polymerase chain reaction (PCR) technique in 104 children with acute lymphoblastic leukemia (ALL) diagnosed between June 1993 and January 1998 and followed for a median of 4.2 years. A significant correlation was found between the MRD level on day 15 (D15) and day 29 (D29) after the start of induction therapy (r s ؍ 0.70, P < .0001). The 15 patients with T-cell disease had higher D29 MRD than those with B-lineage ALL (P ؍ .01). Age was positively related to D29 MRD (r s ؍ 0.32, P ؍ .001). The 16 patients who had a relapse had higher D15 and D29 MRD levels than the patients who stayed in remission (median levels D15, 1% versus 0.1%, P ؍ .03; D29, 0.4% versus 0.01%, P ؍ .0001). No patients with a MRD level less than 0.01% on D29 have so far had a relapse, whereas the 7-year probability of event-free survival for patients with higher MRD levels was 0.52 (P ؍ .0007). The group of patients with a D29 MRD less than 0.01% included patients with T-cell disease, white blood cell count more than 50 ؋ 10 9 /L at diagnosis, or age 10 years or older, and could not be identified by up-front criteria. The best-fit Cox model to predict the risk of relapse included D29 MRD (P ؍ .004) and age (P ؍ .009).
Summary
We hypothesized that Wilms tumour 1 gene (WT1) expression levels in acute myeloid leukaemia (AML) patients might have predictive value and reveal molecular relapse kinetics. WT1 level was determined at diagnosis, during therapy and post‐therapy follow‐up in 89 patients who reached first complete remission (CR1) (952 samples, median 8 samples/patient, range 2–38). CR1 bone marrow (BM) WT1 level above normal (based on 39 healthy donors) was an independent adverse prognostic factor regarding both disease‐free survival [hazard ratio (HR) 4·46, P = 0·001] and overall survival (HR 2·62, P = 0·019). By grouping 34 BM and 99 peripheral blood (PB) complete remission samples in monthly intervals prior to clinical relapse, disease development was delineated and a simple mathematical model constructed, that allowed for the prediction of relapse detection rates (RDRs) as well as median times [tms] from WT1 positivity to clinical relapse. BM sampling was required to obtain RDRs above 93% and tms above 67 d. Acceptable RDRs and tms (81% and 44 d, respectively) could be acquired by bimonthly PB sampling. In conclusion, CR1 WT1 expression is an independent prognostic factor in AML. According to our model, BM is superior for relapse prediction, but PB samples are useful when shorter sampling intervals are possible.
Individualized PCR strategies hamper comparability of molecular results between different laboratories in several fields of medicine. To harmonize BCR-ABL mRNA quantification an international multicenter trial involving 37 laboratories in 14 countries was initiated using 10 samples, each containing various dilutions (10, 2, 1 and 0.1%) of b3a2 or b2a2 BCR-ABL positive in normal leukocytes and negative controls. A novel control plasmid (pME-2) was designed for external calibration containing BCR-ABL and glucuronidase-b (GUS) sequences. Median BCR-ABL/ABL ratios were 9.1, 1.8, 0.85 and 0.11% in b3a2 samples and 9.5, 1.6, 0.84 and 0.11% in b2a2 samples. Median BCR-ABL/GUS ratios were 3.4, 0.77, 0.37 and 0.042% in b3a2 samples and 2.8, 0.48, 0.29 and 0.031% in b2a2 samples. The coefficients of variation were 0.62 for ratios BCR-ABL/ABL and 1.03 for ratios BCR-ABL/GUS. Five of 37 evaluable participating laboratories (13%) detected low BCR-ABL copy numbers in negative control samples; one laboratory failed to detect BCR-ABL in a low-level sample. We conclude that the use of a common control plasmid does indeed improve comparability of BCR-ABL mRNA quantification results. However, further standardizing efforts like introducing a calibrator and regular control rounds are needed.
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Recent studies have suggested that mutations in the mitochondrial genome (mtDNA) may play a role in the development and response to treatment for human cancer. The aim of this study was to investigate whether mtDNA variations have any prognostic relevance, to clarify the spectra of mtDNA variation and to determine whether there was any correlation to known prognostic factors in acute myeloid leukemia (AML). To elucidate this, we sequenced the entire mtDNA in 56 AML patients and 14 control subjects. When analyzing the biologic impact of the non-synonymous variations in the mtDNA coding genes, we found an inferior disease-free survival for patients exhibiting variations in the two most important catalytic genes of the complex IV of the oxidative phosphorylation complexes (OXPHOS), that is, the cytochrome c oxidase subunit I and the cytochrome c oxidase subunit II (hazard ratio 2.6, P = 0.03; multivariate analysis). In addition, the most frequent variation was the T16311C in the control region, which was found in 11 (20%) of the 56 patients. This observation was confirmed in another cohort of 173 diagnostic AML samples. In this expanded group, the T16311C variation tended to be associated with chromosomal abnormalities.
Amyloidosis is a shared name for several rare, complex and serious diseases caused by extra-cellular deposits of different misfolded proteins. Accurate characterization of the amyloid protein is essential for patient care. Immunoelectron microscopy (IEM) and laser microdissection followed by tandem mass spectrometry (LMD-MS) are new gold standards for molecular subtyping. Both methods perform superiorly to immunohistochemistry, but their complementarities, strengths and weaknesses across amyloid subtypes and organ biopsy origin remain undefined. Therefore, we performed a retrospective study of 106 Congo Red positive biopsies from different involved organs; heart, kidney, lung, gut mucosa, skin and bone marrow. IEM, performed with gold-labelled antibodies against kappa light chains, lambda light chains, transthyretin and amyloid A, identified specific staining of amyloid fibrils in 91.6%; in six biopsies amyloid fibrils were not identified, and in two, the fibril subtype could not be established. LMD-MS identified amyloid protein signature in 98.1%, but in nine the amyloid protein could not be clearly identified. MS identified protein subtype in 89.6%. Corresponding specificities ranged at organ level from 94-100%. Concordance was 89.6-100% for different amyloid subtypes. Importantly, combined use of both methods increased the diagnostic classification to 100%. Some variety in performances at organ level was observed.
SummaryReal-time quantitative polymerase chain reaction (qPCR) has been extensively validated for the detection of minimal residual disease (MRD) in acute myeloid leukaemia (AML). Meanwhile, multicolour flow cytometry (MFC) has received less attention because the so-called leukaemia-associated immunophenotypes (LAIPs) are generally of lower sensitivity and specificity, and prone to change during therapy. To improve MRD assessment by MFC, we here evaluate the combination of human Myeloid Inhibitory C-type Lectin (hMICL, also termed C-type lectin domain family 12, member A, CLEC12A) and CD 123 (also termed interleukin-3 receptor alpha, IL3RA) in combination with CD34 and CD117 (KIT), as an MRD assay in pre-clinical and clinical testing in 69 AML patients. Spiking experiments revealed that the assay could detect MRD down to 10 À4 in normal bone marrow with sensitivities equalling those of validated qPCR assays. Moreover, it provided at least one MFC MRD marker in 62/69 patients (90%). High levels of hMICL/ CD123 LAIPs at the post-induction time-point were a strong prognostic marker for relapse in patients in haematological complete remission (P < 0Á001). Finally, in post induction samples, hMICL/CD123 LAIPs were strongly correlated (r = 0Á676, P = 0Á0008) to applied qPCR targets. We conclude the hMICL/CD123-based MFC assay is a promising MRD tool in AML.
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