How to assess clinical response in multiple myeloma

In this interview, Cerba Research explains how its teams were able to assess the clinical response in patients with multiple myeloma (MM) receiving monoclonal antibody treatment.

Can you give our readers a brief overview of multiple myeloma (MM)?

In multiple myeloma (MM), a type of bone marrow cancer, malignant plasma cells secrete large amounts of monoclonal immunoglobulin protein (M protein), which can be detected by serum protein electrophoresis (SPEP) or immunofixation electrophoresis (IFE).

International Myeloma Working Group (IMWG) criteria dictate that patients’ serum samples must be negative for M protein by SPEP/IFE to determine complete response (CR) or stringent CR (sCR).

What is the role of monoclonal antibodies and why are they important?

Monoclonal antibodies have shown therapeutic efficacy in a range of malignancies, but they can complicate the interpretation of IFE data. For example, daratumumab is a CD38 IgG1κ monoclonal antibody (mAb) in clinical development for the treatment of MM⁵ which has demonstrated clinical responses that increase over time. This requires the evaluation of CR/sCR by SPEP/IFE.

About half of patients with MM produce an IgG1κ M protein. In a subset of patients, either daratumumab or the daratumumab anti-idiotype complex may co-migrate with endogenous M protein. Steady-state concentrations of daratumumab (dosed at 16 mg/kg weekly, bimonthly, and then monthly) are readily detectable in most SPEP and IFE assays.

What are the main goals in assessing clinical response in MM?

The primary objective is to validate and implement a daratumumab interferential reflex assay (DIRA) that distinguishes M protein from daratumumab, as assessed by IFE, to determine whether additional testing to assess CR/sCR is warranted (ie, bone marrow examination). To do this, we obtain human serum samples from patients with MM (n=51) from a commercial source or from patients treated with daratumumab in clinical studies (n=33).

How are the clinical samples evaluated?

Cerba performs a battery of serum IFE assays using Maxikit Hydragel 9IF kits (Sebia Electrophoresis, Norcross, GA) according to manufacturer’s specifications.

Antisera against immunoglobulins gamma (IgG), alpha and mu heavy chains and free and bound kappa (κ) and lambda light chains are used to determine the monoclonal protein present in each sample.

We then incubate serum samples from baseline-treated and daratumumab-treated patients with or without anti-idiotype mAb (mouse anti-huMaxCD38; clone 5-3-9-4) for 15 minutes at room temperature before using them with IFE of IgG and Igκ analyze antisera.

To demonstrate that the anti-idiotype antibody binds and translocates daratumumab without affecting the detection and migration of endogenous M protein, commercially available serum samples from MM patients (n=51) were spiked with daratumumab, anti-idiotype or Daratumumab spiked + anti-idiotype (500 and 1000 µg/ml; 1:1 ratio IgG and Igκ) and were then analyzed by IFE to assess changes in M ​​protein migration.

What are the other important parts of the analysis that need to be determined in order to assess whether the assessment is effective?

There are several factors that we must consider. The lower limit of detection (LOD) is determined by evaluating daratumumab ± anti-idiotype over a clinically relevant dynamic range to identify the lowest concentration detected by ≥ 1 parameter (daratumumab-Igg, daratumumab + anti-idiotype complex -IgG, daratumumab-Igκ, daratumumab + anti-idiotype Igκ for IFE; daratumumab or daratumumab + anti-idiotype by SPEP).

We then ensure reproducibility. We performed three independent runs on 10 samples from daratumumab-treated patients who had achieved PR or better and M protein ≤ 5 g/dL. These runs were performed with DIRA and the results (DIRA positive or DIRA negative) then checked for reproducibility. Finally, two independent reviewers review and interpret all results to verify agreement.

After completing the DIRA assessment, what do you expect to see in the results?

In a specific case, the DIRA template used daratumumab ± anti-idiotype as controls for the therapeutic antibody migration and the daratumumab anti-idiotype shifted pairs.

Baseline and post-treatment serum ± anti-idiotype were then compared to determine if M protein remained after switching to daratumumab. DIRA-positive results showed M protein in this case, while DIRA-negative results showed only a shift of daratumumab but no residual M protein (lanes 8, 12; Figure 4).

We also determined the lower limit of detection (LOD) in MM serum samples using IFE at 100 µg/mL in 9 out of 10 samples by ≥1 parameter and at 200 µg/mL in 10 out of 10 samples. In the same samples analyzed by SPEP, either daratumumab and daratumumab plus anti-idiotype complex could be identified at 100 µg/mL in 3 out of 10 samples and at 200 µg/mL in 10 out of 10 samples.

In 10 out of 10 (100%) patient samples treated with daratumumab, results were consistent across all three independent runs, demonstrating a reasonable level of DIRA reproducibility and concordance.

What Makes DIRA a Good Method for Assessing Clinical Response in Multiple Myeloma?

DIRA enables the identification of clinical responses. In one study, a total of 33 samples from daratumumab-treated patients from multiple different studies were evaluated for clinical response with DIRA; Thirteen patients (39%) were DiRA negative, 10 of whom had confirmed CR based on bone marrow and FLC, while 20 (61%) were DIRA positive, meaning they continue to be monitored.

As previously mentioned, DIRA is a specific, reproducible method that can confirm the interference of daratumumab on serum IFE at 100 to 200 µg/mL. In addition, DIRA-negative status signals that additional testing is needed to verify CR/sCR and IMWG response criteria, and changes may be needed if mAbs are approved for the treatment of MM.

Illustration 1. MM cells secrete large amounts of M protein, which is detectable by SPEP. Photo credit: Cerba Research

figure 2 Therapeutic antibodies may impair the ability to confirm clinical results more than excellent partial responses. Photo credit: Cerba Research

figure 3 Schematic representation of DIRA-positive and DIRA-negative patient samples treated with daratumumab. Photo credit: Cerba Research

figure 4 Example of DIRA positive and DIRA negative patient samples treated with daratumumab. Photo credit: Cerba Research

Figure 5. Specificity of anti-idiotype MAb. Photo credit: Cerba Research

Figure 6. Reproducibility of DIRA results between independent experiments. Photo credit: Cerba Research

Table 1. Agreement of expert ratings between experiments. Source: Cerba Research

dara, daratumumab; anti-id, anti-idiotype; Yes / Yes; n, no; VgPR, very good partial response.

About Cerba Research

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