The term personalized medicine has been employed in widely different contexts and has acquired status as one of the most often used keywords recently. In this review we take it to understand the ...application of modern diagnostic medicine and therapeutics to patient with the purpose of eradicating disease or alleviating symptoms in a manner, where all actions are based on detailed knowledge of the condition of the individual patient. Applying these concepts should lead to optimization of clinical decision-making and, in its utmost consequence, a substantial decrease in costs incurred for hospitalization and follow-up. The latter is based on the evidence that for many disorders “less but more targeted” will mean improved outcome. Acute myeloid leukemia (AML) is the most common acute leukemia in adults and is a major challenge in terms of diagnosis, care, follow-up and therapy. Thus, in population-based analyses, overall survival is only just exceeding 40% with major reasons for treatment failure. For these reasons, AML has been intensely studied during the recent decades. With the development of multiparametric flow cytometry, it allows us to get an accurate diagnosis and immunophenotypic profiles of AML. In addition, there is now an abundance of knowledge regarding its cytogenetic and molecular background. These enable us to follow the amount of disease down to the minutest quantity with a high resolution of molecular details. Finally, based on knowledge of these variables in the single patient cytoreduction is now being refined to therapies targeted to the molecular changes in the patient.
An increasing body of data has demonstrated that the traditional concept of morphologic complete remission in acute myeloid leukemia, in which less than 5% myeloblasts is regarded as a sufficient ...response criterion, is not biologically sound. Fortunately, the quantitative reverse-transcribed polymerase chain reaction (RT-PCR) method seems to be a promising alternative because of its high degree of preclinical standardization and extreme sensitivity on the background of an accurate day-to-day estimate of sample quality. Widespread implementation of this has, however, to some extent been hampered by the lack of knowledge of how and when to measure minimal residual disease levels and, even more importantly, how to react preemptively on a molecular relapse defined by a PCR reversal. Thus, only few prospective studies have been published to date to clinically validate this assay. Here, we discuss outstanding issues in the clinical implementation of RT-PCR for fusion transcripts, mutated and overexpressed genes in acute myeloid leukemia patients in complete remission, and propose a set of guidelines, which can be used when designing prospective trials aimed at validating the use of RT-PCR as well as for following these patients based on mathematical models for disease recurrence recently developed in our laboratory.
The article by Morgenstern et al., cited in this Editorial Comment, is published in issue 174:6 (http://onlinelibrary.wiley.com/doi/10.1111/bjh.14160/full).
Methylation-sensitive high resolution melting (MS-HRM) methodology is able to recognise heterogeneously methylated sequences by their characteristic melting profiles. To further analyse ...heterogeneously methylated sequences, we adopted a digital approach to MS-HRM (dMS-HRM) that involves the amplification of single templates after limiting dilution to quantify and to determine the degree of methylation. We used this approach to study methylation of the CDKN2B (p15) cell cycle progression inhibitor gene which is inactivated by DNA methylation in haematological malignancies of the myeloid lineage. Its promoter region usually shows heterogeneous methylation and is only rarely fully methylated. The methylation status of CDKN2B can be used as a biomarker of response to treatment. Therefore the accurate characterisation of its methylation is desirable.
MS-HRM was used to assess CDKN2B methylation in acute myeloid leukaemia (AML) samples. All the AML samples that were methylated at the CDKN2B promoter (40/93) showed varying degrees of heterogeneous methylation. Six representative samples were selected for further study. dMS-HRM was used to simultaneously count the methylated alleles and assess the degree of methylation. Direct sequencing of selected dMS-HRM products was used to determine the exact DNA methylation pattern and confirmed the degree of methylation estimated by dMS-HRM.
dMS-HRM is a powerful technique for the analysis of methylation in CDKN2B and other heterogeneously methylated genes. It eliminates both PCR and cloning bias towards either methylated or unmethylated DNA. Potentially complex information is simplified into a digital output, allowing counting of methylated and unmethylated alleles and providing an overall picture of methylation at the given locus. Downstream sequencing is minimised as dMS-HRM acts as a screen to select only methylated clones for further analysis.
Purpose: Promoter hypermethylation of, for example, tumor-suppressor genes, is considered to be an important step in cancerogenesis
and a negative risk factor for survival in patients with ...myelodysplastic syndromes (MDS); however, its role for response to
therapy has not been determined. This study was designed to assess the effect of methylation status on the outcome of conventional
induction chemotherapy.
Experimental Design: Sixty patients with high-risk MDS or acute myeloid leukemia following MDS were treated with standard doses of daunorubicin
and 1-β- d -arabinofuranosylcytosine. Standard prognostic variables and methylation status of the P15 ink4b ( P15 ), E-cadherin ( CDH ), and hypermethylated in cancer 1 ( HIC ) genes were analyzed before treatment.
Results: Forty percent of the patients achieved complete remission (CR). CR rate was lower in patients with high WBC counts ( P = 0.03) and high CD34 expression on bone marrow cells ( P = 0.02). Whereas P15 status alone was not significantly associated with CR rate ( P = 0.25), no patient with hypermethylation of all three genes achieved CR ( P = 0.03). Moreover, patients with CDH methylation showed a significantly lower CR rate ( P = 0.008), and CDH methylation retained its prognostic value also in the multivariate analysis. Hypermethylation was associated with increased
CD34 expression, but not with other known predictive factors for response, such as cytogenetic profile.
Conclusions: We show for the first time a significant effect of methylation status on the outcome of conventional chemotherapy in high-risk
MDS and acute myelogenous leukemia following MDS. Provided confirmed in an independent study, our results should be used as
a basis for therapeutic decision-making in this patient group.
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