Thalidomide and its derivatives, lenalidomide and pomalidomide (also known as IMiDs), have significantly changed the treatment landscape of multiple myeloma, and the recent discovery of cereblon (CRBN) as their direct biological target has led to a deeper understanding of their complex mechanism of action. In an effort to comprehend the precise mechanisms behind the development of IMiD resistance and examine whether it is potentially reversible, we established lenalidomide‐resistant (‐LR) and pomalidomide‐resistant (‐PR) human myeloma cell lines from two IMiD‐sensitive cell lines, OPM2 and NCI‐H929, by continuous culture in the presence of lenalidomide or pomalidomide for 4–6 months, until acquirement of stable resistance. By assessing genome‐wide DNA methylation and chromatin accessibility in these cell lines, we found that acquired IMiD resistance is associated with an increase in genome‐wide DNA methylation and an even greater reduction in chromatin accessibility. Transcriptome analysis confirmed that resistant cell lines are mainly characterized by a reduction in gene expression, identifying SMAD3 as a commonly downregulated gene in IMiD‐resistant cell lines. Moreover, we show that these changes are potentially reversible, as combination of 5‐azacytidine and EPZ‐6438 not only restored the observed accessibility changes and the expression of SMAD3, but also resensitized the resistant cells to both lenalidomide and pomalidomide. Interestingly, the resensitization process was independent of CRBN. Our data suggest that simultaneous inhibition of DNA methyl transferases and EZH2 leads to an extensive epigenetic reprogramming which allows myeloma cells to (re)gain sensitivity to IMiDs.

Immunomodulatory drugs (IMiDs), such as lenalidomide and pomalidomide, may induce significant remissions in multiple myeloma (MM) patients, but relapses are frequently observed and the underlying molecular mechanisms for this are not completely understood. Circular RNAs (circRNAs) constitute an emerging class of non-coding RNAs with important roles in cancer. Here, we profiled genome-wide expression patterns of circRNAs in IMiD-sensitive MM cells and their resistant counterparts as well as in IMiD-resistant cells treated with specific epigenetic drugs alone or in combination. We found that genome-wide circRNA expression patterns reflect IMiD sensitivity and ciRS-7 (also known as CDR1as) was the most downregulated circRNA upon acquired resistance. The depletion of ciRS-7 correlated with increased methylation levels of the promoter CpG island of its host gene, LINC00632. Expression of LINC00632 and ciRS-7 was partly restored by treatment with a combination of an EZH2 inhibitor (EPZ-6438) and a DNA methyl transferase inhibitor (5-azacytidine), which also restores the IMiD sensitivity of the cells. However, knockdown of ciRS-7 did not affect IMiD sensitivity and we found that ciRS-7 also becomes epigenetically silenced after prolonged cell culture without drug-exposure. In conclusion, we found that genome-wide circRNA expression patterns reflect IMiD sensitivity in an in vitro model of acquired resistance.

The immunomodulatory drug thalidomide, and its analogs, lenalidomide, and pomalidomide (IMiDs), have become essential components of the standard treatment for multiple myeloma (MM), and have led to significant improvement of survival in patients with this devastating disease. Cereblon (CRBN), the direct target of IMiDs, has been proposed as a predictive biomarker of response to IMiDs. Using standard immunohistochemistry in formalin-fixed paraffin embedded (FFPE) bone marrow samples of 23 patients treated with a lenalidomide-containing regimen, we found that the malignant plasma cells of all the patients stained positive for CRBN, IKZF1, and IKZF3, regardless of sensitivity to IMiDs. Moreover, we detected no mutations in CRBN, IKZF1, IKZF3, CUL4A, or IRF4, but found expanded TP53-mutated clones in two out of seven sequential samples. Thus, our data argue against the use of CRBN and its downstream targets as predictive biomarkers of IMiD response in MM and confirm clonal evolution patterns during lenalidomide resistance.

Myelodysplastic syndrome (MDS) is a heterogeneous group of clonal hematopoietic disorders. MDS is frequently associated with deletions on chromosome 5q as well as aberrant DNA methylation patterns including hypermethylation of key tumor suppressors. We have previously shown that hypermethylation and silencing of the non-coding RNA VTRNA2-1 are correlated with poor outcomes in acute myeloid leukemia patients. In this study, we find that VTRNA1-2 and VTRNA1-3, both located on chromosome 5q, can be regulated and silenced by promoter DNA methylation, and that the hypomethylating agent 5-aza-2-deoxycytidine causes reactivation these genes. In normal hematopoiesis, we find that vault RNAs (vtRNAs) show differential methylation between various hematopoietic cell populations, indicating that allele-specific methylation events may occur during hematopoiesis. In addition, we show that VTRNA1-3 promoter hypermethylation is frequent in lower risk MDS patients and is associated with a decreased overall survival.

Objective: The aim of this study was to evaluate the diagnostic and prognostic role of multiparameter flow cytometry (FC) in patients with idiopathic cytopenia of undetermined significance (ICUS) and clonal cytopenia of undetermined significance (CCUS).Methods: We performed FC using a standardized panel and two different diagnostic algorithms (Ogata, Wells) in a well‐characterized cohort of 79 patients with ICUS/CCUS and compared it with a retrospective blinded morphological evaluation and data from targeted next‐generation DNA sequencing of 20 myelodysplastic syndrome (MDS)‐related genes.Results: Our data show that FC has low sensitivity in distinguishing CCUS from ICUS patients (40.5% for Ogata score and 59.5% for Wells score). The Wells score was suggestive of dysplasia in ICUS/CCUS patients with concurrent morphological signs of dysplasia in the bone marrow (following re‐evaluation by two hematopathologists) and in CCUS patients with a higher mutational burden. Eight patients with ICUS/CCUS from our cohort progressed to another myeloid malignancy (MDS, acute myeloid leukemia, or chronic myelomonocytic leukemia), all showing flow cytometric signs of dysplasia.Conclusion: FC performs poorly in diagnosing CCUS versus ICUS. However, it can potentially provide prognostic information in cytopenic patients by identifying a subgroup of patients with a higher grade of dysplasia, higher mutational burden, and higher risk of progression and, together with mutational screening, also identify a group of patients who might require morphological reassessment of dysplastic changes in their bone marrow.

Thrombotic thrombocytopenic purpura (TTP) is a rare and potentially fatal disease for which rapid diagnosis is crucial for patient outcomes. Deficient activity (< 10%) of the liver enzyme, ADAMTS13, is the pathophysiological hallmark of TTP, and measurement of the enzyme activity can establish the diagnosis of TTP with high accuracy. Thus, along with the clinical history, appropriate laboratory assessment of a suspected case of TTP is essential for diagnosis and treatment. Here, we present a review of the available laboratory tests that can assist clinicians in establishing the diagnosis of TTP, with special focus on ADAMTS13 assays, including the measurement of the antigen and activity, and detection of autoantibodies to ADAMTS13.