This was a retrospective study aiming to assess the clinical significance of BMP2 and BMP6 in patients with newly diagnosed multiple myeloma. Study endpoints were to evaluate the expression levels of BMP2 and BMP6 in NDMM and to determine whether BMP2 or BMP6 could serve as prognostic biomarkers for patients with NDMM. The study enrolled NDMM patients that were diagnosed and treated in the Department of Hematology of University Hospital of Larissa, Greece. Diagnosis of MM was based on the International Myeloma Working Group consensus criteria (14). The enrollment period was from March 2019 to December 2021. Nine healthy volunteers (male 5, median age 60, range 51–75) were additionally enrolled, at the same period of time. All participants provided written informed consent, prior to recruitment, to the use of their biological samples and clinical data in research and the study was reviewed and approved by the Institutional Review Board of the University Hospital of Larissa (approval code 21906) and adhered to the tenets of The Declaration of Helsinki. Clinical and laboratory data (collected prospectively) were retrieved from the electronic database and biological samples were retrieved from the department's biobank. All samples were acquired at the time of diagnosis and prior to treatment initiation.

Bone marrow aspirates were collected in EDTA tubes and immediately processed (within 2 h post sampling). Bone marrow mononuclear cells (BMMNCs) were isolated using density gradient separation with a Ficoll Paque Plus kit (MilliporeSigma) (Fig. S1). Bone marrow was diluted 1:1 with DPBS (Biowest) and was filtered through a 100 µm pore to remove cell clumps, clots and bone fragments. 4 ml of diluted cell suspension was carefully layered over 4 ml of Ficoll Paque Plus and an immediate centrifugation step (400 × g at 20°C for 30 min without brake) followed. The upper plasma layer was aspirated and the interphase was drawn for washing steps and further analyses. Cells were then washed with DPBS (Biowest), lysed with red blood cell lysis buffer (Cell Signaling Technology, Inc.) if required, aliquoted and stored as dry pellets at −80°C until further analysis. Total RNA was retrieved from BMMNCs using an E.Z.N.A. Total RNA kit I (Omega-Biotek Inc.) according to manufacturer's instructions. Briefly, 5×10⁶ pelleted cells were lysed using TRK lysis buffer and homogenized with a syringe. The lysate was then filtered through columns to remove debris and other contaminants. Washing steps were applied along with a middle DNA-digestion step and high-yield RNA was eventually eluted in 30 µl nuclease-free water. RNA concentration was quantified at 260 nm on a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Inc.). Prior to cDNA synthesis, purity and integrity of eluted RNA samples was verified by 1% agarose gel electrophoresis and by A260/280 (median ratio 2.08, range 1.8–2.14)-A260/230 (median ratio 1.93, range 1.4–2.2) absorbance ratios. As expected, agarose gel electrophoresis of RNA displayed the presence of 2 ribosomal bands (28S and 18S) with no smearing or any DNA band and the absorbance ratios were within accepted bounds (15–19). Total RNA was subsequently reverse transcribed into cDNA in a 20 µl reaction volume using a QuantiTect Reverse Transcription kit (Qiagen, GmbH). Approximately, 100 µg RNA template was incubated at 42°C with dNTPs, RNAse inhibitor, Quantiscript's Reverse Transcriptase, RT Buffer, RT Primer Mix along with RNase-free water. An initial incubation of 3 min at 95°C was applied to activate Quantiscript Reverse Transcriptase. cDNA was diluted and then deposited at −20°C. qPCR was carried out in a 36 well Rotor Gene Q (Qiagen) with CYBR green 1 as fluorescent dye. Β-2 microglobulin (B2M) was used as housekeeping gene. Intra-spanning primers with amplicon lengths of around 100 bp (acquired from Qiagen GmbH, GeneGlobe ID: SBH0312240 for B2M, GeneGlobe ID: SBH0638609 for BMP2 and GeneGlobe ID: SBH0513495 for BMP6) were used to ensure that traces of genomic DNA would not affect the PCR. Template cDNA was mixed with QuantiNova Probe PCR Master Mix and RNase-free water with volumes according to the manufacturer's protocol. The cycling conditions included an initial incubation step at 95°C for 2 min followed by 40 cycles of 95°C for 5 sec and 60°C for 10 sec. Specificity of each reaction was confirmed by resolving amplification products in a 2% agarose gel, stained with ethidium bromide (Fig. S2). Appropriate sample handling was verified by performing PCR reactions in duplicates. Cq values are presented as means. When for a specific sample the disagreement between the Cq measurements was greater than 1, the qPCR was performed for a third time and the final Cq value was calculated as the mean of the 3 measurements. Gene expression levels were analyzed using the Livak 2^−ΔΔCq method (20), after validating stable expression of B2M gene between patients and healthy individuals (median raw Cq value of patients 16.3, range 13.2–24.2 and median raw Cq value of healthy donors 18.5, range 14.4–19.8, P-value 0.1).

Statistical analysis was performed with the GraphPad Prism version 10 (Dotmatics). Results were considered significant when P-values were <0.05. The normality of distribution was assessed using a Shapiro-Wilk test. For non-parametric data, a Mann Whitney U test was used to compare differences between two groups. For parametric data, an unpaired Student's t-test was applied. The Fisher's exact test was used to identify differences between the proportions of categories in two independent groups. Univariate and multivariate analyses were carried out by Cox regression analysis. Pearson correlation was performed to assess the linear relationship between two continuous variables and Spearman correlation was used for categorical data. Kaplan-Meier (KM) curves were applied to plot survival and progression-free survival. The log-rank test was used to compare the distribution of time to event between 2 groups.

A total of 18 newly diagnosed patients with MM were included in the present study along with 9 additional healthy volunteers who served as controls (male 5, median age 60 years, range 51–75 years). Patient baseline characteristics are shown in Table I. Of the patients with MM, the median age was 62 years, 50% were male and most had IgG MM (50%). The median percentage of bone marrow infiltration was 55% (range 10–90%) and the median beta-2 microglobulin levels were 4.9 mg/l (range 2.3–14.1). A total of 33% of patients presented with elevated LDH levels. High risk cytogenetics were observed in 5 patients (27%). Regarding the ISS staging, 16% were ISS-1, 38% were ISS-2 and 44% were ISS-3. The majority of patients presented with bone disease (72%). The median follow-up period was 50 months (range 4–67). Patients were primarily treated either with VCD or VRD as induction therapy and response was evaluated after five cycles of upfront therapy.

To assess whether BMP2 and BMP6 are differentially expressed in multiple myeloma, the expression levels of BMP2 and BMP6 genes between NDMM patients and healthy controls were assessed. BMP6 mRNA abundance was 5-fold higher in the NDMM group compared to the HD group (unpaired Student's t-test, P=0.0004) (Fig. 1A). In contrast, BMP2 expression was not significantly different between the two groups (Mann Whitney U test, P=0.5) (Fig. 1B). Based on these results, BMP6 was further studied regarding the clinical impact of its expression. Patients were categorized into two groups based on the expression levels of BMP6. The median ΔCq value of the NDMM group was used as the threshold to divide the patients into high and low BMP6 expression groups, each with 9 patients. In the high expression group, the median ΔCq value was 10.1 (range 9.2–11) while in the low expression group the median ΔCq value was 11.8 (range 11.2–13.7). The characteristics of each group are summarized in Table II.

The overall response rate (≥PR) was 77% (n=7/9) for the high expression group and 88% (n=8/9) for the low expression group, whereas the ≥ VGPR rate was 55% (n=5/9) and 44% (n=4/9), respectively. Response patterns are shown in Fig. 1C. The median progression free survival (calculated using the Kaplan Meier curves) was 30 months for the high expression group compared to 46 months for the low expression group (log-rank HR=1.6, 95% CI: 0.4–5.3, P=0.3) (Fig. 1D). The median estimated overall survival was 42 months for the high expression group and was not reached for the low expression group (log-rank HR=1.9, 95% CI: 0.4–7.8, P=0.4) (Fig. 1E). In the univariate analysis, age (P=0.06, HR=1.06, 95% CI: 0.99–1.13), LDH levels (P=0.2, HR=1.006, 95% CI: 0.99–1.01), percentage of bone marrow infiltration (P=0.3, HR=3.9, 95% CI: 0.1–80), b2-microglobulin levels (P=0.5, HR=1.07, 95% CI: 0.8–1.3) and ΔCq values of BMP6 (P=0.1, HR=0.6, 95% CI: 0.3–1.1) were factors affecting survival. Whereas, for most factors statistical significance was not reached, they are described as affecting survival based on the hazard ratio values. In the multivariate analysis, age (P=0.05, HR=1.07, 95% CI: 0.99–1.16) was the only factor affecting survival. Pearson and Spearman correlation analyses (Fig. S3) did not show any correlation between BMP6 ΔCq values and age (r=0.02, P=0.9), LDH levels (r=−0.08, P=0.7), b2-microglobulin levels (r-0.2, P=0.4), percentage of bone marrow infiltration (r=−0.1, P=0.4) and ISS stage (r_s=0.1, P=0.6) thus limiting the confounding factors for the subgroup analysis.

Next, whether BMP6 expression affected patient's bone status at diagnosis was assessed, given that the BMP family of proteins is a crucial signaling cascade for bone homeostasis and that BMP6 specifically is an osteogenic mediator (21,22). The presence of more than 3 spinal osteolytic lesions was used as the cut-off point to divide the cohort into patients with highly active bone disease and patients with less active bone disease. A total of 33% (n=3/9) of patients from the high BMP6 expression group exhibited highly active bone disease compared to 55% (n=5/9) of patients from the low BMP6 expression group (Fisher's exact test, P=0.6). Similarly, the rate of skeletal-related-events (based on the presence of at least one fracture, the need for bone-related radiotherapy and the need for bone-related surgery) was 22% (n=2/9) for the high BMP6 expression group and 55% (n=5/9) for the low BMP6 expression group (Fisher's exact test, P=0.3).