Evaluating the HYDRASHIFT 2/4 Daratumumab assay: a powerful approach to assess treatment response in multiple myeloma

Introduction

Multiple myeloma (MM) is an incurable hematologic malignancy that requires long-term therapy and monitoring. Most MM patients undergo several cycles of remission and relapse, necessitating multiple lines of combination therapies, including proteasome inhibitors, immunomodulators, and therapeutic monoclonal antibodies (t-mAbs), among others [1]. Currently, the most widely used t-mAb is daratumumab, a human immunoglobulin IgG1κ monoclonal antibody (mAb) targeting the CD38 surface protein on myeloma cells [2]. Daratumumab has proven to be effective in treating both newly diagnosed [3], [4], [5], [6] and relapsed/refractory MM [7, 8]. However, as a human IgGκ immunoglobulin, daratumumab is detected on serum electrophoresis (EP) and immunofixation (IF), potentially causing false-positive interference [9, 10]. Misinterpretation of daratumumab as pathologic M-protein can lead to misidentification of a patient’s treatment response, based on the International Myeloma Working Group (IMWG) response criteria, and affect clinical decisions. Therefore, laboratory tests to distinguish daratumumab from endogenous M-protein are absolutely necessary.

Sebia’s HYDRASHIFT 2/4 Daratumumab assay has successfully discriminated between daratumumab and endogenous M-protein on gel IF [11]. This assay employs anti-daratumumab antiserum to bind to the daratumumab component and shift it outside the gamma globulin region, revealing the true presence of the patient’s M-protein. Although it is the only commercially available, FDA-approved, daratumumab-specific IF reflex assay, its performance has been evaluated in only a few studies with a limited number of samples [11], [12], [13]. In this study, we evaluated the performance of the HYDRASHIFT 2/4 Daratumumab assay in a relatively large number of MM patient samples and established an indication of the assay’s effectiveness based on our experience.

Materials and methods

Results

Interpretation of every gel IF conducted in this study reached a consensus by the three reviewers.

Precision and specificity

Before the implementation of the HYDRASHIFT assay, the daratumumab-induced Gκ band was observable in standard gel IF for both QC and patient samples, as shown in Figure 1A. The assay’s intra-day and inter-day variability were deemed satisfactory, as evidenced by the consistent appearance of the migration complex for both QC and patient samples following the application of the HYDRASHIFT assay, as illustrated in Figure 1B and C. This consistency indicates that the HYDRASHIFT assay effectively neutralizes the interference caused by daratumumab in the immunofixation process, thereby ensuring reliable and accurate detection of monoclonal proteins in the evaluated samples.

Figure 1:

Precision of HYDRASHIFT assay. (A) Standard gel IF of QC (Left) and the patient sample (Right). (B) Intra-day variability of HYDRASHIFT assay on QC (Left) and the patient sample (Right). Box in dotted line represents the migration complex. (C) Inter-day variability of HYDRASHIFT assay on QC (Left) and the patient sample (Right).

The specificity of the assay was confirmed to be acceptable, as the pre- and post-HYDRASHIFT IF results were identical across all four patient samples, with no migration complex observed (Figure 2). The HYDRASHIFT assay did not interfere with isatuximab or the patients’ endogenous M-protein, demonstrating excellent specificity.

Figure 2:

Specificity of HYDRASHIFT assay. (A) Pre- and post-HYDRASHIFT IF of patient with isatuximab infusion. (B) Pre- and post-HYDRASHIFT IF of patient with endogenous Gκ band. (C) Pre- and post-HYDRASHIFT IF of patient with endogenous Gλ band. (D) Pre- and post-HYDRASHIFT IF of patient with endogenous Aκ band.

After the application of the HYDRASHIFT assay, every daratumumab component migrated to the anodal end without leaving any trace of positive interference (Figure 1), while patients’ endogenous bands remained on their initial position (Figure 2). These results suggest the perfect separation capacity of the HYDRASHIFT assay, enabling full discrimination of daratumumab from patients’ endogenous M-protein.

Comparison analysis

A total of 113 serum samples from 68 multiple myeloma patients undergoing daratumumab therapy were analyzed. The pre- and post-HYDRASHIFT IF results were categorized into seven groups (Table 2). A migration complex was observed in categories 1–4 (n=91), indicating the successful elimination of the daratumumab-induced Gκ band by the HYDRASHIFT assay. Categories 1–3 showed persistence of endogenous M-protein, while category 4 indicated a complete response. No migration complex was observed in categories 5–7 (n=22), indicating the absence of a daratumumab-induced Gκ band. Categories 5 and 6 showed persistence of endogenous M-protein, whereas category 7 indicated a complete response. The time interval between the last daratumumab infusion and sample collection significantly differed between groups with (categories 1–4) and without (categories 5–7) a migration complex, with median intervals of 14 and 28 days, respectively (p<0.001) (Supplementary Figure 1).

Table 2:

Comparison and categorization of the pre- and post-HYDRASHIFT IF results.

Category no.	Presence of migration complex	Pre-HYDRASHIFT IF result	Post-HYDRASHIFT IF result	Disease status	No. of samples	BM study result	Interpretation of the monoclonal components
1a	Yes (n=91)	Gκ + Non-Gκ	Non-Gκa	P	21a	ND	Daratumumab + endogenous M-protein (non-Gκ)
2		Gκ + Gκ	Gκ	P	12	ND	Daratumumab + endogenous M-protein (Gκ)
3		Gκ	Gκ	P	16	ND	Daratumumab + endogenous M-protein (Gκ)
4b		Gκ	No band	CR	42	CR (3b)	Daratumumab
5c	No (n=22)	Non-Gκ	Non-Gκc	P	3c	ND	Endogenous M-protein (non-Gκ)
6d		Gκ	Gκ	P	17	P (1d)	Endogenous M-protein (Gκ)
7		No band	No band	CR	2	ND	Absent

1. Non-Gκ refers to the isotype of the monoclonal band other than Gκ. ^aIsotypes of category 1 consist of 14 Gλ, four λ, two Aλ, and one Aκ + λ. ^bThree samples in category 4 revealed complete remission on BM study, consistent with the post-HYDRASHIFT IF result. ^cIsotypes of category 5 consist of one Aκ + κ, one κ, and one λ. ^dOne sample in category 6 revealed disease persistence on BM study, consistent with the post-HYDRASHIFT IF result. CR, complete response; IF, immunofixation; ND, not done; P, persistence.

The IF patterns of patients treated with daratumumab and exhibiting a migration complex were classified into four categories (1–4), based on the isotype of endogenous M-protein and disease progression. Representative cases for each category are illustrated in Figure 3. An oligoclonal band pattern on pre-HYDRASHIFT IF (Figure 3A and B) suggested the coexistence of daratumumab and endogenous M-protein. A single Gκ band pattern on pre-HYDRASHIFT IF indicated two possibilities: formation by co-migration of daratumumab and endogenous M-protein (Figure 3C) or solely due to daratumumab (Figure 3D).

Figure 3:

Representative cases with migration complex on the comparison analysis. Daratumumab and patients’ endogenous M-protein were fully discriminated. (A) Pre- and post-HYDRASHIFT IF of category 1 (Gκ + Non-Gκ → Non-Gκ). (B) Pre- and post-HYDRASHIFT IF of category 2 (Gκ + Gκ → Gκ). (C) Pre- and post-HYDRASHIFT IF of category 3 (Gκ → Gκ). (D) Pre- and post-HYDRASHIFT IF of category 4 (Gκ → No band).

Among 36 patients tested two or more times with the HYDRASHIFT assay, three experienced a change in their disease status, as detailed in Table 3. Patients A and C exhibited relapse of multiple myeloma; the 2nd follow-up (f/u) for patient A showed positive findings on post-HYDRASHIFT IF and urine IF, indicating relapse. Similarly, the 2nd f/u for patient C showed positive results on post-HYDRASHIFT IF and serum EP, suggesting a relapse. Patient B demonstrated complete remission at the 2nd f/u, as evidenced by negative results on post-HYDRASHIFT IF and other tests.

Table 3:

Serial follow-up of pre- and post-HYDRASHIFT IF results on three patients with change on disease status.

Patient	Follow-up	Sample collection date	Chemotherapy cycle	Pre-HYDRASHIFT IF result	Post-HYDRASHIFT IF result	Serum EP	Urine EP	Urine IF	BM study	Disease status
A	#1	2022.07.21	C8D1	No band	No band	N	ND	N	ND	CR
	#2	2023.01.31	C15D1	Gκ	Gκ	N	ND	Gκ	ND	P
B	#1	2022.08.30	C8D1	Gκ + Gκ	Gκ	N	N	N	ND	P
	#2	2022.11.22	C11D1	Gκ	No band	N	N	N	ND	CR
	#3	2023.02.14	C14D1	Gκ	No band	N	N	N	ND	CR
C	#1	2022.11.29	C20D1	Gκ	No band	N	N	N	ND	CR
	#2	2023.03.21	C24D1	Gκ + Gκ	Gκ	M (<0.1)	N	N	ND	P
	#3	2023.04.18	C25D1	Gκ + Gκ	Gκ	M (0.45)	N	N	ND	P
	#4	2023.05.04	C25D1	Gκ + Gκ	Gκ	M (1.55)	N	Gκ	ND	P
	#5	2023.06.02	C25D1	Gκ	Gκ	M (0.46)	N	Gκ	ND	P

1. The number in the parenthesis indicates the amount of M-protein in g/dL unit. CR, complete response; M, monoclonal peak; N, no peak or band; ND, not done; P, persistence.

Discussion

This study represents the largest collection of patient samples among the performance evaluation studies for the HYDRASHIFT assay [11], [12], [13], comprising a total of 113 serum samples from 68 daratumumab-treated MM patients for the comparison analysis of pre- and post-HYDRASHIFT IF.

Out of the 113 samples, 22 exhibited no visible migration complex. This absence could be attributed to low serum daratumumab concentrations, falling below the limit of visualization. Caillon et al. demonstrated that migration complexes become difficult to visualize when daratumumab concentration drops below 0.2 g/L [12]. Daratumumab pharmacokinetic data indicate that serum concentrations may dip below 0.2 g/L after reaching steady state [15]. However, the high variability in serum daratumumab concentrations among patients made it impossible to precisely estimate the concentration. Therefore, our analysis concentrated on factors influencing serum daratumumab concentration: the daratumumab infusion cycle, dosage, and sampling timing. Firstly, the number of daratumumab infusion cycles was sufficient to assume a steady state in all samples. Secondly, the daratumumab dosage was consistently set at 16 mg/kg for all patients. Thirdly, the interval between serum sampling and daratumumab infusion was significantly longer in the group without the migration complex (n=22) compared to the group with the migration complex (n=91), as shown in Supplementary Figure 1. We hypothesize that this extended interval led to the relatively lower serum daratumumab concentrations, resulting in the absence of a visible migration complex.

Before the adoption of the HYDRASHIFT assay, interpreting serum EP and IF results in daratumumab-treated patients involved correlating these results with other tests. Historical data, including serum EP/IF, urine EP/IF, free light chain ratio, and even BM studies, were thoroughly reviewed to detect pathologic M-protein presence. Despite these efforts, uncertainty persisted when a single Gκ band was identified in initial serum IF tests, making it difficult to ascertain whether the band represented the patient’s M-protein (category 6), daratumumab (category 4), or the coexistence of both monoclonal antibodies (category 3). Consequently, the HYDRASHIFT assay is required for samples displaying a single Gκ band on initial serum IF, where the patient’s M-protein and daratumumab may have co-migrated due to their similar electrophoretic mobility. In our study, categories 3, 4, and 6 necessitated the use of the HYDRASHIFT assay for accurate treatment response evaluation. Additionally, Kirchhoff et al. reported that the HYDRASHIFT assay is unnecessary for the patients with M-spike of over 0.2 g/dL, the maximum serum concentration of daratumumab, since high M-spike quantitation itself implies the presence of pathologic M-protein and persistent myeloma [16]. A total of 53 samples revealed M-protein of less than 0.2 g/dL on serum EP, consisting of seven samples in category 3, 42 samples in category 4 and four samples in category 6. Hence, 47 % (53/113) of the samples in our study would truly require the HYDRASHIFT assay. On the other hand, when two distinct bands are seen on initial serum IF (categories 1 and 2), distinguishing the patient’s M-protein from daratumumab becomes feasible. Nevertheless, the HYDRASHIFT assay remains beneficial for identifying rare, treatment-related, endogenous oligoclonal bands [11, 17, 18].

The HYDRASHIFT assay proved invaluable in detecting changes in disease status during routine f/u tests. Without it, the disease status determined by initial serum immunofixation (IF) would have led to the misclassification of the 2nd and 3rd f/u of patient B and the 1st f/u of patient C (referenced in Table 3) as persistent disease. However, the application of post-HYDRASHIFT IF revealed the disappearance of the monoclonal Gκ band, indicating disease remission. For patient B, complete remission (CR) was achieved by the 2nd f/u, and as planned, daratumumab monotherapy concluded on cycle 24 without further treatment. For patient C, CR was reached by the 1st f/u, and daratumumab monotherapy concluded on cycle 25 as scheduled. Unfortunately, the 2nd f/u for patient C showed disease relapse, prompting a change in the treatment regimen to a combination of pomalidomide, cyclophosphamide, and dexamethasone. The utility of the HYDRASHIFT assay was particularly evident in these three instances (2nd and 3rd f/u for patient B, and 1st f/u for patient C), where each case exhibited a single Gκ band on initial serum IF. Cases presenting a single Gκ band are susceptible to misinterpretation, and the HYDRASHIFT assay stands as the only commercially available, FDA-approved method capable of eliminating false-positive IF results, underscoring its importance in accurate disease status evaluation.

The HYDRASHIFT assay offers notable user convenience, as it integrates seamlessly with the existing analyzers (Hydrasys 2 scan focusing) by requiring only the simple addition of an anti-daratumumab reagent, in contrast to the original protocol. We explored the possibility of the HYDRASHIFT assay interfering with other monoclonal antibodies involving the IgG heavy chain or kappa light chain, given that the anti-daratumumab antiserum is applied to the IgG and kappa lanes of the gel. A specificity analysis was carried out, confirming that the HYDRASHIFT assay does not affect isatuximab and patients’ pathologic M-proteins with isotypes of Gκ, Gλ, and Aκ. However, we were unable to test specificity for other isotypes containing IgG or kappa, such as Mκ, due to the absence of specimens. Isotypes Dκ and Eκ were also not tested because of their rare occurrence.

Another limitation of our study is the self-determined study design and acceptance criteria, given the lack of existing guidelines for evaluating the performance of non-binary examinations with three or more categorical results [19]. Only one article provided a comprehensive method for verifying precision, accuracy, and conducting comparison studies for non-binary qualitative examinations [20]. However, applying this verification protocol was impractical due to the complexity of the experiments, the unavailability of QC samples, and the lack of alternative assays capable of mitigating the effect of daratumumab. Additionally, the determination of disease status may have been limited due to the lack of BM studies, considering their pivotal role in diagnosing MM. Only four BM studies were conducted on the exact day of sample submission (Table 2), owing to practical difficulties such as the lengthy interval between follow-up BM studies, whereas other tests – serum and urine EP, urine IF and post-HYDRASHIFT IF – were thoroughly performed on every patient sample. Moreover, the use of other t-mAbs – erlantamab, talquetamab, teclistamab (Table 1) – was neglected in this study, presuming that they would not induce false-positive interference in serum EP or IF tests. The dosage of these other t-mAbs, in moles per patient’s body weight, was less than 15 % of the daratumumab dosage, indicating their negligible impact on interference compared to that of daratumumab. Furthermore, as these t-mAbs have recently gained clinical approval, their effects on immunofixation analysis are unknown, necessitating further evaluation.

The HYDRASHIFT assay stands out as a distinct method that differentiates between patients’ M-protein and daratumumab, yet it faces certain limitations. Specifically, the HYDRASHIFT assay is tailored for daratumumab and does not address interferences caused by other t-mAbs, as illustrated in Figure 2A. This limitation becomes increasingly relevant as new combinations of t-mAbs are introduced in clinical practice. To fully eliminate interferences from t-mAbs, antisera specific to each t-mAb need to be developed. For example, the HYDRASHIFT 2/4 isatuximab assay by Sebia successfully removes isatuximab interference using anti-isatuximab antiserum [21]. Moreover, assays that counteract the interference from both daratumumab and nivolumab have been recently formulated [13]. To bypass the limitations inherent to the IF method, quantitative assays employing mass spectrometry (MS) have been adopted [22], [23], [24], [25], [26]. MS-based assays are capable of accurately quantifying M-protein without interference from multiple t-mAbs, owing to the unique mass-to-charge ratio of M-protein compared to that of t-mAbs. From the perspective of detecting minimal residual disease, MS-based assays offer sensitivity and minimal invasiveness, making them a compelling alternative to BM studies [23, 26]. Despite their advantages, MS-based methods have not been broadly adopted for routine monitoring due to challenges such as cost, maintenance, and the need for standardization. Therefore, during the transition from electrophoretic to the forthcoming mass-spectrometric era in M-protein diagnostics, the HYDRASHIFT assay remains an essential tool. Its specificity for daratumumab, while a limitation in the face of emerging t-mAbs, underscores the need for continued development in diagnostic assays to accommodate evolving therapeutic strategies.

Conclusions

As daratumumab continues to gain prominence as a primary therapy for multiple myeloma [27, 28], the demand for the HYDRASHIFT assay is expected to rise. The clinical adoption of this highly precise and specific assay is critical for providing accurate monitoring of treatment responses. Particularly, samples from patients treated with daratumumab that display a single Gκ band on standard gel IF are prime candidates for the HYDRASHIFT assay. This tool addresses a significant challenge in the interpretation of IF results, ensuring that clinicians can differentiate between the therapeutic monoclonal antibody and the patient’s own monoclonal proteins. Thus, the HYDRASHIFT assay plays a vital role in optimizing patient care by enabling more precise assessments of disease status and treatment efficacy.