Daratumumab and isatuximab differentially affect CD38 detection on plasma cells in myeloma: Anti‐CD38 nanobody (clone JK36) and CD319 combination improve flow cytometric identification of plasma cells after targeted therapies

Summary

Inclusion of daratumumab and isatuximab in therapeutic regimens has improved outcomes in multiple myeloma patients. As these agents block CD38 detection, they may result in difficulties in flow cytometric (FC) identification of plasma cells (PCs). In addition, the heterogeneity of myeloma immunophenotype and the introduction of novel therapeutic agents necessitate FC panels with robust gating markers. To this end, we validated an 11‐colour PC panel that in addition to commonly used gating markers (CD45, CD38, CD138) includes CD319 and anti‐CD38 single variable heavy domain nanobody (clone JK36). A Boolean "or" function was applied to gate PCs using a combination of the above markers. Patterns and intensity of antigen expression were compared in samples from patients receiving daratumumab or isatuximab therapy, or neither. Daratumumab resulted in loss of detectable CD38 expression (clone HB‐7) on abnormal PCs, and JK36 overcame daratumumab blockage in almost all these cases. Interestingly, isatuximab showed an opposite pattern, with complete loss of CD38 detection by JK36, while retaining dim positivity by HB‐7. This combination of markers proved highly effective in identifying abnormal PCs after CD38‐directed therapies, supporting a need for redundancy of gating markers in FC panels.

Daratumumab and isatuximab differentially affect plasma cell identification by anti‐CD38 detection antibodies. HB‐7 and JK36 bind to CD38 epitopes E1 and E3, respectively, and co‐staining of plasma cells in the absence of CD38‐directed drugs results in a diagonal staining pattern (top left). Daratumumab (top middle) binds to epitope E1, masking CD38 detection by HB‐7. JK36 binds to epitope E3 and bypasses steric inhibition by daratumumab. On the other hand, isatuximab (top right) binds to epitope E3, preventing CD38 detection by JK36, while allowing its detection by HB‐7. When surface immunoglobulin light chain staining is performed (bottom panels), the presence of either daratumumab or isatuximab (both IgG‐kappa) on the surface of hematogones results in an artifactual kappa light chain restriction of hematogones.

INTRODUCTION

Despite substantial advances in the management of multiple myeloma (MM), this disease is still considered incurable, with frequent remissions and relapses. Introduction of small molecule anti‐myeloma agents such as immunomodulatory drugs and proteasome inhibitors to myeloma regimens has improved patient outcomes. In recent years, this has been complemented by the addition of CD38‐directed monoclonal antibodies (mAb), particularly daratumumab, that extended both progression‐free survival (PFS) and overall survival (OS) of treatment‐naïve or relapsed/refractory (R/R) MM patients. ¹

In addition to conventional myeloma response criteria that require the evaluation of serum protein electrophoresis, immunofixation and bone marrow (BM) PC count, in its last publication, the International Myeloma Working Group (IMWG) introduced new minimal/measurable residual disease (MRD)‐defined myeloma response categories that require negativity by standardized next‐generation sequencing (NGS) or next‐generation flow cytometry (NGF) assays with a minimum sensitivity of 10⁻⁵. ² Several studies have demonstrated that MRD negativity is associated with better outcomes as demonstrated by longer PFS and OS. ² , ³ , ⁴ In fact, only quantitative MRD level and myeloma cytogenetic profile are independent predictors of PFS and OS among MM patients. ⁴ Compared with molecular methods, FC has a shorter turn‐around time and wider availability, and can be applied to essentially all myeloma cases. In contrast, Food and Drug Administration (FDA)‐approved NGS‐based MRD assessment is only available in one platform, requires diagnostic sequence data and can be applied to approximately 90% of cases, ² , ⁵ , ⁶ limiting its universal application at the moment. As MRD information is being incorporated into clinical practice, there is an increasing need for laboratories to validate their own FC assays. Therefore, consensus guidelines have been developed to aid in optimal panel design and reduce variability. ⁷ , ⁸ , ⁹

Daratumumab is a human immunoglobulin (Ig)G1‐kappa mAb that was approved in 2016 by the FDA for use in R/R‐MM in combination with conventional anti‐myeloma therapies, ¹ , ¹⁰ and later received approval as first‐line therapy in untreated patients. ¹¹ Widespread use of daratumumab in therapeutic regimens revolutionised the management of MM and led to remarkable outcome benefits. Isatuximab is a human/mouse chimeric IgG1‐kappa mAb that targets CD38 via an epitope distinct from that of daratumumab. ¹² Isatuximab in combination with pomalidomide and dexamethasone improved the outcome of adult patients with MM who have received at least two prior therapies ¹³ leading to its approval by the FDA for this group of patients in March 2020. Later, the FDA approved isatuximab in combination with carfilzomib and dexamethasone for patients with relapsed or refractory MM who have received one to three prior lines of therapy. ¹⁴ Recently, isatuximab, bortezomib, lenalidomide and dexamethasone combination was approved for newly diagnosed MM patients who are not eligible for autologous stem cell transplant. ¹⁵ While daratumumab's effect on PC FC results has been extensively studied, there is very limited such information about isatuximab, and the optimal panel to identify PCs in this setting is not clear.

Most reported FC panels use CD45, CD38 and CD138 combination to gate PCs. This combination, however, appears insufficient in light of widely used CD38‐directed antibodies. Daratumumab is known to block the CD38 epitope identified by most conventional anti‐CD38 antibodies. ¹⁶ , ¹⁷ This is further complicated by the relative instability of CD138 expression due to antigen shedding as samples age. ¹⁸ Therefore, it is conceivable that the detection sensitivity of FC panels is reduced after daratumumab, particularly with increased storage time, which can potentially result in false‐negative MRD results at low frequency of abnormal PCs. To overcome these challenges, several alternative markers have been evaluated, including anti‐CD38 multi‐epitope (ME) antibody from Cytognos, ³ , ¹⁶ , ¹⁹ CD38 variable heavy domain of heavy chain antibody (CD38 VHH, clone JK36), ¹⁶ , ²⁰ VS38c, ¹⁹ , ²¹ CD54, CD229 and CD319. ¹⁷ , ²² , ²³ Interestingly, there is limited agreement on the relative value of these markers, and preferred markers among different groups appear to range from VS38c ²¹ , ²⁴ to CD319 ¹⁷ and CD229. ²⁵

We developed an FC panel for diagnosis and monitoring of MM patients that in addition to CD38 and CD138 includes CD38 VHH (JK36) and CD319 and asked how this combination aids in identification of PCs after CD38‐directed therapies. We show that daratumumab and isatuximab differentially bind to CD38 leading to staining patterns unique to each drug.

MATERIALS AND METHODS

During the validation of our new PC FC panel on Becton Dickinson (BD) FACSLyric™ instruments, residual material from BM samples collected from patients with plasma cell neoplasms (PCNs) at diagnosis or follow‐up was evaluated by FC using a combination of antibodies listed in Table. A detailed staining procedure can be found in Supporting Information: Methods. Analysis was performed using Woodlist software (developed by Brent L. Wood). The lower limit of quantification (LLOQ) for the validated PC panel was established at 0.01%. In a small number of cases (depending on the clinical scenario), FC analysis of B cells (surface staining) was performed using a combination of antibodies listed in Table S2.

A total of 34 BM samples from patients with PCNs and no history of daratumumab or isatuximab therapy and 15 BM samples from patients with a history of daratumumab therapy were evaluated. Additional 10 BM samples from patients with a history of isatuximab therapy were added during and after the validation. Patients received daratumumab for an average duration of 180 days (range: 7–460 days) and received isatuximab for an average duration of 408 days (range: 123–750 days). The percentage of abnormal PCs ranged from 0.01% to 40.52% in patients not receiving CD38‐directed therapies (mean ± standard deviation [SD] of 7.55 ± 10.86), 0.05% to 59.96% in patients receiving daratumumab (mean ± SD of 7.79 ± 17.59) and less than 0.01% to 12.80% in patients receiving isatuximab (mean ± SD of 3.07 ± 4.93). Median fluorescent intensities (MFI) of CD38, CD38 VHH and CD319 on the abnormal PCs and hematogones were compared between samples from patients receiving daratumumab, isatuximab or neither. In addition, the relative value of CD138, CD38, CD38 VHH and CD319 in gating PCs after daratumumab or isatuximab was evaluated by comparing the abnormal PC percentage (among white blood cells) in the presence of various combinations of gating markers.

Statistical analysis was performed using Prism 8 (GraphPad software Inc., San Diego, CA). Numerical variables were compared using two‐tailed Mann–Whitney U‐test. Wilcoxon test was used for paired comparison of normal and abnormal PCs. Statistical significance was considered at p ≤ 0.05.

RESULTS

The tested FC panel includes commonly used PC gating markers (CD45, CD38, CD138) and two relatively newer markers: CD319 and CD38‐VHH (JK36). Single variable immunoglobulin domain nanobodies are derived from heavy chain antibodies that are naturally produced in camelids. Their small size and high solubility facilitate tissue penetration, and their extended complementary determining region 3 (CDR3) allows them to bind to epitopes hidden from conventional antibodies. ²⁶ , ²⁷ JK36 binds to an epitope of CD38 that differs from that of daratumumab and conventional anti‐CD38 antibodies, ²⁶ , ²⁷ , ²⁸ making it a valuable reagent for PC gating in this setting. ¹⁶ , ²⁰ Figure 1 illustrates the sequential steps in gating PCs. PCs are gated using a Boolean "or" function to include events in the PC regions of CD38 versus CD45 or CD138 versus CD45 or JK36 versus CD319. While not used in our gating, we found that JK36 versus CD45 and CD319 versus CD45 plots are as efficient as CD38 versus CD45 plot in gating abnormal PCs when combined with the CD138 versus CD45 plot (Figure S1). PCs are then evaluated for immunophenotypic aberrancy and light chain restriction. Aberrancies such as decreased to absent CD19, CD27, CD45 or CD81 or increased CD56 expression are used to gate the abnormal population using a ‘difference from normal’ approach (Figure 2). ⁷ , ⁸ In the absence of CD38‐directed therapies, JK36 intensity follows the intensity of the conventional anti‐CD38 antibody (HB‐7) on normal and abnormal PC populations resulting a diagonal expression pattern (Figures 1 and 2A). B‐cells, hematogones and natural killer (NK) cells are identified in the CD45 versus side scatter (SSC) defined lymphocyte gate using a combination of CD19, CD38, CD38 VHH, CD81 and CD319. Note that among haematopoietic cells, PCs have the highest expression of CD319 with intensities 0.5–1 log higher than brightest expression on lymphocytes.

FIGURE 1.

Plasma cell gating strategy. Initial gates (first row) are used to exclude events with fluidic problems, doublets and debris/low viability events. Briefly, a plot of all events versus APC‐H7 is used to confirm stable acquisition. A singlet gate is created on a plot of forward scatter height (FSC‐H) versus forward scatter area (FSC‐A). A plot of FSC‐A versus side scatter height (SSC‐H) is used as a viability gate to exclude low viability events with low FSC. Lymphocytes are gated as CD45‐high/low SSC events. Erythroid forms (orange) are defined as events lacking CD45, CD38, CD138, CD56 and CD319. White blood cells (WBC) are defined as all viable cells and not erythroids, and used as denominator to calculate plasma cell (PC) percentage. PCs are gated using a Boolean or function to include events in the PC regions of CD38 versus CD45 or CD138 versus CD45 or JK36 versus CD319 (second row). If one of these plots could not be used due to low expression of a PC‐specific marker, other plots from the second row could be replaced. PCs (kappa/blue, lambda/red) are then evaluated for immunophenotypic aberrancy using a ‘difference from normal’ approach, and light chain restriction. Within the lymphocyte gate (cyan), a combination of CD19, CD38, CD38 Variable Heavy domain of Heavy chain antibody (VHH), CD81 and CD319 is used to define mature B cells (kappa/light blue, lambda/violet; CD19‐positive CD38 or CD38‐VHH‐dim to negative, CD319‐negative), hematogones within the lymphocyte gate (yellow, CD19‐positive, CD38 or CD38 VHH‐positive, CD81‐bright, CD319‐negative, note, this gate excludes hematogones with lower CD45) and Natural Killer (NK) cells (brown, CD19‐negative, CD56‐positive, CD38 or CD38 VHH‐positive). Note that among lymphocytes, CD319 expression is highest on CD56‐positive cells reflecting NK cells and a subset of T cells.

FIGURE 2.

Representative FC plots of BM samples from patients with plasma cell neoplasms in the absence (A) or presence (B and C) of daratumumab therapy. Abnormal PCs (green, in contrast to normal polytypic PCs in kappa/blue or lambda/red) were identified by aberrant expression of CD19, CD27, CD45, CD56 or CD81 in combination with light chain restriction. Note the diagonal pattern of CD38 (HB‐7) versus CD38 VHH (JK36) in (A) due to binding to different epitopes of the same antigen. Also, note the absence of detectable CD38 expression using HB‐7 on all haematopoietic cells including PCs after daratumumab therapy (B and C) that is overcome by JK36. The population with apparent CD38‐positivity is eosinophils with high autofluorescence. BM, bone marrow; FC, flow cytometry; PCs, plasma cells.

As shown previously, ¹⁶ , ²⁹ daratumumab resulted in loss of detectable CD38 by conventional anti‐CD38 antibody HB‐7 on normal and abnormal PCs (Figure 2B,C). Figure 3A demonstrates complete loss of CD38 detection using HB‐7. JK36 restored CD38 detection and PCs could be gated using a combination of JK36 and either CD45 or CD319 (Figure 2B,C). Of note, the intensity of CD38 on PCs as detected by JK36 was significantly lower in daratumumab‐treated patients as compared to untreated patients with an average reduction in MFI of sevenfold after daratumumab (Figure 3B, also see Figure 2A against 2B and 2C), However ‘CD38 rescue’ (Figure 3C) was sufficient to gate the majority of PCs in most cases (see Figure 2B,C as examples). CD319 was equally effective in identifying PCs after daratumumab (Figure 2B,C). While CD319 MFI was slightly lower in abnormal PCs compared to their normal counterparts (Figure 3D), no significant difference in CD319 intensity on abnormal PCs was noted after daratumumab (Figure 3E). Finally, we evaluated the relative value of CD138, JK36 and CD319 in gating abnormal PCs after daratumumab (Figure 3F). Among two marker combinations, CD319 + JK36 combination gated higher number of PCs than CD138 + JK36, while no differences were seen when CD138 + CD319 was compared with CD138 + JK36 or CD319 + JK36. When a single marker was used for gating, JK36 and CD319 were similarly effective, and both were superior to CD138 in gating abnormal PCs.

FIGURE 3.

Daratumumab effect on CD38 detection using conventional anti‐CD38 antibody (HB‐7) and CD38 VHH (JK36). (A) Complete loss of detectable CD38 using HB‐7 on abnormal PCs after daratumumab (n = 15) as compared to plasma cell neoplasms without daratumumab (n = 34). (B) While JK36 staining shows decreased CD38 intensity after daratumumab therapy, there is sufficient ‘CD38 rescue’ (C) to gate PCs (also see Figure 2B,C for examples). (D) CD319 intensity on paired normal and abnormal PCs in samples untreated with CD38‐directed therapies (n = 24). (E) CD319 intensity on abnormal PCs with (n = 15) or without (n = 34) daratumumab therapy. (F) Frequency of abnormal PCs in after daratumumab therapy (n = 15) using full panel (including CD138, JK36 and CD319), or in the absence of one gating marker (CD138 + JK36, CD138 + CD319 or CD319 + JK36 combinations) or two gating markers (CD319, JK36, CD138 or HB‐7 alone). ns, not significant; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001. PCs, plasma cells.

During and after the validation of PC panel, we encountered some BM samples from patients receiving isatuximab (Table S3). We found that isatuximab results in a different pattern of CD38 detection by HB‐7 and JK36. In contrast to daratumumab, CD38 could be detected using HB‐7 antibody (Figures 4 and 5A), although generally at a lower intensity compared to samples without CD38‐directed therapy (data not shown). In contrast, isatuximab completely blocks CD38 detection using JK36 (Figures 4 and 5B), likely secondary to epitope blockage. This pattern was observed in both normal and abnormal PCs (data not shown). No significant difference in the intensity of CD319 on PCs after daratumumab and isatuximab therapy was noted (Figure 5C) suggestive of similar effectiveness of CD319 in gating PCs after these therapies. We compared the relative value of CD138, HB‐7 and CD319 in gating abnormal PCs after isatuximab therapy (Figure 5D). Among two marker combinations, no difference in effectiveness of CD138 + HB‐7, CD138 + CD319 and CD319 + HB‐7 was noted. Moreover, HB‐7, CD319 and CD138, when used as single markers, were similarly effective in gating abnormal PCs. Of note, 7 of 10 isatuximab‐treated patients had documented a history of daratumumab therapy. However, daratumumab was discontinued 10–59 months before the initiation of isatuximab (average of 33 months). No significant difference in the intensity of HB‐7 on abnormal PCs between patients with or without a history of daratumumab therapy was present in this limited number of cases (data not shown). Similar differential effect of daratumumab and isatuximab was observed on CD38 detection in hematogones (Figure 5E–G).

FIGURE 4.

Representative FC plots of a BM sample from a MM patient after isatuximab therapy. Abnormal PCs (green) show dim CD38 positivity using HB‐7 antibody, but demonstrate complete loss of detectable CD38 using JK36. BM, bone marrow; FC, flow cytometry; MM, multiple myeloma; PCs, plasma cells.

FIGURE 5.

Comparison of CD38 (HB‐7) and CD38‐VHH (JK36) and CD319 staining intensity (A–C, respectively) on abnormal PCs after daratumumab (n = 15) and isatuximab (n = 7) therapy. (D) Frequency of abnormal PCs after isatuximab therapy (n = 7) using full panel (including CD138, HB‐7 and CD319), or in the absence of one gating marker (CD138 + HB‐7, CD138 + CD319 or CD319 + HB‐7 combinations) or two gating markers (HB‐7, CD319, CD138 or JK36 alone). (E–G) Staining intensity and pattern on hematogones after daratumumab (n = 10) or isatuximab (n = 10) therapy, or in the absence of both targeted therapies (n = 26) (yellow, hematogones; light blue/kappa or violet/lambda positive polytypic B cells, overlap on dot plots). ns, not significant; ****p ≤ 0.0001; PCs, plasma cells.

A pitfall in the flow cytometric work‐up of bone marrow samples after daratumumab therapy is artifactual kappa light chain restriction of hematogones when surface light chain staining is performed during evaluation of B cells. ²⁹ This is due to daratumumab (IgG‐kappa) binding to CD38 on hematogones that are detected by fluorochrome‐labelled anti‐kappa antibody. We asked if a similar pattern could be observed after isatuximab (also IgG‐kappa) therapy. As expected, all (6 out of 6) tested isatuximab‐treated samples showed artifactual surface kappa light chain restriction of hematogones (Figure 6 and Table S3).

FIGURE 6.

Daratumumab and isatuximab effect on surface immunoglobulin light chain staining of hematogones and B cells (cyan: Stage‐1 hematogones, orange: Stage 2 hematogones, blue/kappa or red/lambda positive mature polytypic B cells). Note the artifactual kappa light chain restriction of stage 1 and stage 2 hematogones after daratumumab and isatuximab therapies.

DISCUSSION

We developed an 11‐colour FC panel that includes CD38 VHH (JK36) and CD319 in addition to commonly used PC markers CD38 and CD138 for identification of PCs. Redundancy in gating markers and plots was created to overcome challenges caused by the heterogeneity of myeloma immunophenotype, CD38‐directed therapies and relative CD138 instability. In line with previous studies, ¹⁷ , ²² we found that, among haematopoietic cells, CD319 expression is highest on PCs. In addition, CD319 is expressed on abnormal PCs in the vast majority of PC neoplasms with a slightly reduced intensity compared to normal PCs. In combination with CD45, CD319 is a reliable marker to gate PCs. In contrast to a previous study, ¹⁷ we did not observe any significant reduction in the intensity of CD319 on abnormal PCs after daratumumab or isatuximab therapy, a feature that supports its use in the MRD setting after such therapies. Of note, we did not have any patient in our cohort that received elotuzumab, a CD319‐directed mAb that is FDA‐approved for treatment of R/R‐MM. ³⁰ A previous study showed that incubation of PCs with elotuzumab does not significantly block CD319 detection, ¹⁷ making it a suitable marker for PC gating in that setting as well.

There is limited information about the utility of JK36 after CD38‐directed therapies. Oberle et al. ¹⁶ evaluated CD38 expression on PCs from a daratumumab‐treated patient 6 weeks after discontinuation of daratumumab using conventional anti‐CD38 antibodies (clones T16 and LS198‐4‐3), CD38 ME antibody and JK36. Staining with T16 was completely blocked by daratumumab and LS198‐4‐3 and CD38 ME showed dim staining, whereas JK36 showed complete restoration of CD38 staining. ElMaraashly et al. ²⁰ evaluated CD38 expression in 23 daratumumab‐treated patients using conventional anti‐CD38 antibody (clone T16) and JK36. While T16 staining failed to stain CD38 in all 23 cases, JK36 showed restoration of CD38 staining in 21 of 23 patients. Interestingly, the absence of JK36 staining was observed in two cases, suggestive of CD38 depletion as a mechanism of resistance to daratumumab.

Several lines of evidence indicate that CD38 intensity on PCs is correlated with response to daratumumab. CD38 expression on primary myeloma cells before initiation of therapy was higher in patients with at least partial response versus non‐responders after therapy. ³¹ In addition, CD38 expression level on myeloma cells was associated with daratumumab‐induced antibody‐dependent cell‐mediated cytotoxicity (ADCC) and complement‐dependent cytotoxicity (CDC), ³² and all‐trans retinoic acid (ATRA) induced CD38 upregulation leading to enhanced daratumumab‐mediated ADCC and CDC. ³¹ , ³² Daratumumab therapy results in rapid reduction of CD38 antigen expression on PCs through different mechanisms including selection of cells with lower CD38 expression, trogocytosis of CD38‐daratumumab complexes by FCγ‐receptor expressing cells and internalization of CD38 on myeloma cell. ³¹ This reduction is transient and CD38 expression is restored approximately 6 months after discontinuation of daratumumab. ³¹ In line with the above studies, we found approximately sevenfold reduction in CD38 intensity in daratumumab‐treated samples using JK36 nanobody (Figure 3B). Despite this, it was possible to gate all or part of PC population using JK36 in combination with CD45 or CD319. In daratumumab‐treated samples, the presence of either CD319 or JK36 in addition to CD138, or a combination of CD319 + JK36 allowed for inclusion of more than 90% of total abnormal PCs (Figure 3F), whereas use of CD138 in isolation could recover only two‐thirds of PCs. This may result in false‐negative MRD results when disease is present at low levels.

While uncommon, complete loss of CD38 due to gene deletion can happen as an escape mechanism in a subset of myeloma patients after CD38‐directed therapy. ³³ Since conventional CD38 antibodies cannot differentiate between daratumumab steric hindrance and CD38 loss due to gene deletion, inclusion of JK36 would be helpful in such distinction. Moreover, as evidence is suggestive of a relationship between CD38 intensity and daratumumab induced cytotoxicity, inclusion of JK36 may be of value in guiding CD38‐directed therapies.

As a relatively new agent in the treatment of myeloma, the effect of isatuximab on CD38 expression patterns is not well studied. Isatuximab and daratumumab bind to non‐overlapping epitopes of CD38, ³⁴ , ³⁵ , ³⁶ leading to distinct staining patterns with our FC panel. Daratumumab binds to epitope E1 on CD38 ²⁷ , ²⁸ that overlaps with the epitope recognized by the HB‐7 antibody ³⁴ and is distinct from epitope E3 recognized by JK36. ²⁷ , ²⁸ , ³⁵ In contrast, isatuximab binds to epitope E3 ³⁵ blocking CD38 detection by JK36 and not by HB‐7. Our data suggest that CD138, HB‐7 and CD319 are similarly effective in gating abnormal PCs after isatuximab therapy, although small differences may not be detected given the small size of our isatuximab‐treated cohort. Decreased CD38 intensity on PCs and hematogones after isatuximab therapy, as detected by HB‐7 (Figure 5E and data not shown) may be due to similar mechanisms involved in reducing CD38 intensity after daratumumab, although the possibility of partial steric collision between isatuximab and HB‐7 leading to reduced binding of the latter cannot be excluded. Importantly, our data emphasize the need for the inclusion of both HB‐7 and JK36 in FC panels to overcome steric hinderance caused by either anti‐CD38 drugs.

Finally, we demonstrated that isatuximab therapy results in artifactual surface kappa light chain restriction of marrow hematogones. This is similar to the previously described daratumumab effect on hematogones. ²⁹ It is important to note that in post‐therapy myeloma samples, high level of suspicion for CD38‐directed therapy should be applied when unusual patterns of surface light chain expression by B cell is noted to avoid over‐interpretation of a monoclonal B‐cell population.

In summary, we developed an FC panel with redundancy in PC gating markers and showed that both JK36 and CD319 are excellent markers for PC identification after CD38‐directed therapies. We believe that the added cost associated with a higher number of PC gating markers (estimated increased cost of 2% of total assay cost per each added antibody) is well justified by improving the quality of the assay that translates into improved patient care, reducing the number of reflex NGS assays and the ability to tackle the challenges associated with a growing list of targeted therapies. Finally, we demonstrated that treatment with daratumumab and isatuximab leads to distinct CD38 staining patterns that result from epitope overlap with CD38 detection reagents (Table 1). Understanding these patterns is essential for flow cytometry laboratories and pathologists to guide decisions about panel design and help interpret results in the context of the patient's treatment history.

TABLE 1.

Daratumumab and isatuximab effect on CD38 detection on plasma cells and surface light chain staining of hematogones.

Therapy	Isotype	CD38 (HB‐7)	CD38 VHH (JK36)	Surface light chain on hematogones
Daratumumab	IgG‐kappa	Negative	Often positive (decreased)	Artifactual kappa restriction
Isatuximab	IgG‐kappa	Often positive (decreased)	Negative	Artifactual kappa restriction

AUTHOR CONTRIBUTIONS

AS designed the study, analysed data, interpreted results and wrote the manuscript. TC designed the analysis template, performed assays and analysed data. VW performed assays, analysed data and wrote a portion of the manuscript. OK performed assays and analysed data. JRF analysed data and interpreted results. SC designed the study and analysis template, analysed data and interpreted results. All authors reviewed the manuscript and approved the final version.

CONFLICT OF INTEREST STATEMENT

The authors have no conflict of interest to declare.

ETHICS STATEMENT

This study was approved by the Institutional Review Board at the University of Washington (IRB #50111).

Supporting information

Data S1.

Figure S1.

Table S1.

Table S2.

Table S3.

Shameli A, Catey T, Woodings V, Koepke O, Fromm JR, Cherian S. Daratumumab and isatuximab differentially affect CD38 detection on plasma cells in myeloma: Anti‐CD38 nanobody (clone JK36) and CD319 combination improve flow cytometric identification of plasma cells after targeted therapies. Br J Haematol. 2025;207(3):757–766. 10.1111/bjh.20218

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available on request from the corresponding author.