Characterization of in vitro and in vivo hypomethylating effects of decitabine in acute myeloid leukemia by a rapid, specific and sensitive LC-MS/MS method

DNA hypermethylation is a common finding in malignant cells and has been explored as a therapeutic target for hypomethylating agents (e.g., decitabine). Detection of changes in DNA methylation might serve as a pharmacodynamic endpoint to establish the biological activity of these agents and predict clinical response. We developed and validated a rapid, sensitive and specific LC-MS/MS method for determination of global DNA methylation (GDM) in vitro and in vivo. Ratios of 5-methyl-2′-deoxycytidine (5mdC) to the internal standard 2-deoxyguanosine (2dG) in mass signal were used to quantify GDM levels. The assay was validated in a linear range from 40 fmol to 200 pmol 5mdC. The intra-day precision values ranged from 2.8 to 9.9% and the inter-day values from 1.1 to 15.0%. The accuracy of the assay varied between 96.7 and 109.5%. This method was initially applied for characterization of decitabine-induced GDM changes in in-vitro-treated leukemia cells. Following exposure to 2.5 μM decitabine, GDM decreased to ∼50% of the baseline value. The clinical applicability of this method was then demonstrated in bone marrow samples from patients with acute myeloid leukemia treated with decitabine. Our data support the use of our LC-MS/MS method for clinical pharmacodynamic determination of changes in GDM in vivo

An international study to standardize the detection and quantitation of BCR-ABL transcripts from stabilized peripheral blood preparations by quantitative RT-PCR

Due to the lack of comparability of BCR-ABL mRNA quantification results generated by various methodologies in different laboratories, an international multicenter trial was started with the participation of six laboratories (platforms: LightCycler™, LC, n=3; TaqMan™, TM, n=3). One hundred and eighty-six PB samples derived from healthy donors were spiked with serial dilutions (1:20 to 1:2×106) of b2a2, b3a2 or e1a2 BCR-ABL positive white blood cells (WBC) from leukemic patients. After PAXgene™ stabilization, blinding, freezing and distribution, standardized RNA extraction, cDNA synthesis, PCR protocols and data evaluation were carried out. There was no significant difference in the results achieved using LC and TM technologies, but a considerable overall variation (CV=0.74 for ratios BCR-ABL/ABL). Up to a dilution of 1:1,000, 27/30 of the 2.5 mL samples tested positive. For higher dilutions, a PB volume of 5 or 10 ml was required to improve sensitivity. The study showed the feasibility of RQ-PCR standardization independent of the PCR machine used

Single Center Experience Using Monoclonal COVID-19 Antibodies in the Management of Immunocompromised Patients with COVID-19

The medical care of immunocompromised patients with COVID-19 infection causes major hurdles in the management of these patients in clinical practice. However, poor responses to vaccinations in patients with oncological or autoimmune diseases require rapid action and effective care in this fragile patient population. Monoclonal antibodies (mAb) offer an effective therapeutic option with a favorable toxicity profile. We have retrospectively reviewed the first 100 patients treated with mAb in our clinic and assessed the individual vaccine response, side effects of mAb, hospitalization rate and mortality. None of the outpatients treated with mAb had to be hospitalized. In particular, the third SARS-CoV-2 vaccination had a significant effect on the seroconversion (37.5% vs. 77.8% positive patients) in the entire group of patients studied. No side effects of 3°/4° were observed following mAb administration; the mortality in the entire cohort was 7%. Our data and experience show good effectiveness and a favorable tolerability profile of mAb, supporting the feasibility of this therapy in everyday clinical practice. Of note, in immunocompromised patients, both the vaccination status and success need to be recorded in a systematic manner and taken into account in terms of therapeutic intervention using mAb in case of a SARS-CoV-2 infection

The Urine Light Chain/eGFR Quotient as a Tool to Rule out Cast Nephropathy in Myeloma-Associated Kidney Failure

Kidney involvement with resulting kidney failure leads to increased mortality in patients with multiple myeloma (MM). Cast nephropathy (CN), in particular, if left untreated, quickly leads to kidney failure requiring dialysis and has a very poor prognosis for the affected patient. The gold standard for diagnosing kidney involvement is a kidney biopsy. However, due to bleeding risk, this cannot be done in every patient. We recently reported that a quotient of urine light chain (LCurine) to glomerular filtration rate (eGFR) is a non-invasive diagnostic tool for patients with kidney involvement in MM. But this quotient has not yet been tested in everyday clinical practice. In this study, our LCurine/eGFR ratio was tested on 67 patients in two centers. Enrollment took place between January 2019 and September 2023. A total of 18 of the 67 patients had CN. With the threshold defined in our initial paper, we were able to show a sensitivity of 100% with a specificity of 85.7% for CN in patients with MM. As a result, the LCurine/eGFR quotient recognizes 100% of all CN and can therefore detect this group, which has a very poor prognosis, without the need for a kidney biopsy

Clinical relevance of mutations and gene-expression changes in adult acute myeloid leukemia with normal cytogenetics: are we ready for a prognostically prioritized molecular classification?

Recent molecular analyses of leukemic blasts from pretreatment marrow or blood of patients with acute myeloid leukemia (AML) and a normal karyotype, the largest cytogenetic subset (ie, 40%-49%) of AML, have revealed a striking heterogeneity with regard to the presence of acquired gene mutations and changes in gene expression. Multiple submicroscopic genetic alterations with prognostic significance have been discovered, including internal tandem duplication of the FLT3 gene, mutations in the NPM1 gene, partial tandem duplication of the MLL gene, high expression of the BAALC gene, and mutations in the CEBPA gene. Application of gene-expression profiling has also identified a gene-expression signature that appears to separate cytogenetically normal AML patients into prognostic subgroups, although gene-expression signature-based classifiers predicting outcome for individual patients with greater accuracy are needed. These and similar future findings are likely to have a major impact on the clinical management of cytogenetically normal AML not only in prognostication but also in selection of appropriate treatment, since many of the identified genetic alterations already constitute or will potentially become targets for specific therapeutic intervention. In this report, we review prognostic genetic findings in karyotypically normal AML and discuss their clinical implications