The effect of azacitidine therapy on the M protein of MDS patients with concomitant MGUS.

Monoclonal gammopathy of undetermined significance (MGUS) is one of the most common pre‐malignant disorders and affects approximately 3.5% of the population who are over 50 years of age. The pathophysiological concept considers that multiple myeloma (MM) evolves from MGUS in a large proportion of patients. Despite the proposed risk factors, there are still no reliable biomarkers to predict which MGUS patients will develop MM. The prevalence of MGUS in patients with myelodysplastic syndrome (MDS) ranges between 2% and 10%1 and several agents are effective in the treatment of MDS and MGUS; however, the effect of azacitidine for MGUS has not yet been established in detail. The frequencies of TP53 mutations in MM/MGUS were 5%‐10%, and the presence of TP53 mutant clones has been associated with adverse outcomes.2 Another important distinction pertaining to the origin of plasma cell disorders is the involvement of interleukin (IL)‐6. We herein examined the effects of azacitidine on the M protein of MDS patients with concomitant MGUS, and the association of IL‐6 and TP53 mutations with MGUS.

Our retrospective study involved eight MDS patients with concomitant MGUS at the Japanese Red Cross Society Wakayama Medical Center between January 2010 and January 2018. Azacitidine was administered at 75 mg/m2/day for 7 days every 28 days. Responses were evaluated every 2 cycles using blood counts and marrow aspirates. Serum M‐protein was measured prior to the initiation and every 2 cycles of azacitidine treatment. The concentration of IL‐6 was determined prior to the initiation and every 2 cycles of azacitidine treatment using enzyme‐linked immunosorbent assay (ELISA) kits from Quantikine (R&D systems) based on the manufacturer's instructions. Bone marrow samples were obtained from all patients before the initiation and every 2 cycles of azacitidine treatment. Bone marrow sections (thickness of 2 μm) on SuperFrost microscope slides were de‐paraffinized and pre‐treated at 95°C for 7 min in citrate buffer (pH 6). In order to quantify and assess the distribution of hematopoietic cells and plasma cells, samples were stained for CD34 (Cell Marque Rocklin, CA) and CD138 (Dako Corporation, Carpinteria, CA). The DO‐7 antibody (Dako Cytomation, Denmark), which labels wild‐type and mutant‐type p53 proteins, was used to detect p53 protein expression. The entire trephine section was assessed for p53 protein nuclear staining in hematopoietic progenitor cells, as previously described.3 In order to minimize the possibility of false positive results, p53 protein expression was only considered to be positive if strong nuclear staining (score + 3) was present in at least 5% of hematopoietic cells in the entire BM section. G‐band karyotyping, immunophenotyping, serum protein electrophoresis, and immunoelectrophoresis were performed using standard procedures.

Seven patients had IgG MGUS except for 1 who had IgA MGUS. The serum M‐protein of all patients were ≥1.5 g/dL (median 2.6 g/dL, 1.8‐2.8 g/dL), and the FLC ratios of all patients were 7.47 (0.03‐17.2). Seven patients had high‐intermediate risk MGUS, and 1 patient had high risk MGUS using the Mayo Clinic risk stratification model to predict progression. The median IL‐6 level was 15.8 pg/mL (8.3‐37 pg/mL). A bone marrow examination showed hypercellular marrow with myelodysplasia‐related changes, numerous blasts, and plasma cells (Figure 1A). Strong nuclear p53 was detected in 5%‐30% (Figure 1B) and CD138 immunostaining was noted in 5%‐10% of plasma cells (Figure 1C) before the initiation of azacitidine treatment. A cytogenetic analysis revealed abnormal karyotypes in three patients, however, the specific chromosomal abnormalities, such as del(17p) and t(4;14), were not shown.

Figure 1

Bone marrow biopsies of the MGUS concomitant with MDS before (A, B, C) and after (D, E, F) azacitidine treatment (×40). A, Hypercellular marrow with myelodysplasia‐related changes, numerous blasts and plasma cells. B, Strong nuclear p53 immunostaining in numerous hematopoietic cells. C, CD138 immunostaining in 10% of plasma cells. D, Hypercellular marrow with myelodysplasia‐related changes and decreased blasts and plasma cells. E, Strong nuclear p53 immunostaining in a few hematopoietic cells. F, CD138 immunostaining in a few plasma cells

After two courses of azacitidine treatment, the median serum M protein levels (1.2 g/dL [0.7‐1.7 g/dL]) and IL‐6 levels (3.1 pg/mL [2.5‐5.4 pg/mL]) decreased, and the FLC ratios of all patients were normalized. A repeat marrow examination showed decreases in the number of myeloblasts and plasma cells (Figure 1D). Strong nuclear p53 (Figure 1E) was decreased in hematopoietic cells and CD138 immunostaining was detected in a few plasma cells (Figure 1F). The azacitidine treatment was continued (median: 25 cycles [8‐43 cycles]), and none of these patients showed progression of MGUS.

Azacitidine belongs to a class of cytosine analogues that was developed as an inhibitor of DNA methylation and has shown clinical efficacy toward MDS. In MM, DNA hypomethylation was reported as the predominant early change during myelomagenesis where it is gradually transformed to DNA hypomethylation in relapsed cases and during progression of the disease. Several reports showed that azacitidine was cytotoxic against MM cells and overcomes the growth and survival advantages provided by the BM microenvironment.4

The presence of TP53 mutations in MM/MGUS indicates a dismal prognosis: patients that exhibit a more aggressive disease course are more likely to have extramedullary disease and hypercalcemia and have shorter overall and progression‐free survival. Mikulasova et al. reported that TP53 mutations were not detected in samples from patients with MGUS and may indicate that the presence of TP53 mutations are drivers of MM progression.5

IL‐6 is a potent human myeloma‐cell growth factor, and its overproduction is known to play a critical role as an anti‐apoptosis‐inducing agent in MM. Khong et al. recently demonstrated that azacitidine exerts pleiotropic effects including the down‐regulation of anti‐apoptotic factors and JAK‐STAT signaling as well as the inhibition of NFκB in MM cell lines.6 In our study, the percentage of strongly p53‐positive bone marrow cells was greater than 5% at the time of the concomitant occurrence of MGUS and MDS with increase of serum IL‐6 levels; however, the levels of serum‐M protein, IL‐6 levels, the percentage of p53‐positive and CD138‐positive cells decreased after the treatment with azacitidine.

In this study, azacitidine was effective to delay the progression of MGUS. Although the mechanism underlying the progression of MGUS to MM is unknown, IL‐6 and TP53 mutations appear to contribute to the pathophysiology of MGUS. In conclusion, azacitidine have clear activity against MGUS and should be considered in the treatment strategy.

CONFLICT OF INTEREST

Nothing to report.