Detection of a monoclonal antibody therapy (ofatumumab) by serum protein and immunofixation electrophoresis

We would like to bring to the attention of your readers the increasing possibility of ‘false-positive’ serum protein electrophoresis (SPE) and serum immunofixation electrophoresis (IFE) results in patients receiving monoclonal antibody therapeutics (MATs). In a recent clinical trial investigating ofatumumab for the treatment of Waldenstrom macroglobulinemia, we observed seven patients who [along with their previously described monoclonal immunoglobulin M (IgMs)] had faint monoclonal IgG-kappa appear during therapy. In these patients, the faint monoclonal proteins were always identified at the same location on the SPE and IFE gels near the cathode. Where follow-up testing was available, the IgG-kappa was shown to disappear after completion of therapy. Communication with the ordering clinician and review of the electronic medical record revealed ongoing therapy with ofatumumab (Arzerra®; GlaxoSmithKline, Research Triangle Park, NC, USA). Ofatumumab is a human monoclonal IgG1-kappa antibody that targets CD20. Figure 1 shows example serum IFEs from a 72-year-old female before and during treatment with ofatumumab given in five doses (one dose, 300 mg; four doses, 2000 mg over 4 weeks). The patient’s previously identified monoclonal IgM-kappa is visible on both IFEs, although a faint monoclonal IgG-kappa is evident during ofatumumab therapy. A trace band corresponding to the monoclonal IgG-kappa was also detected by SPE in this patient (<0·5 g/l; not shown). Detection of ofatumumab by serum immunofixation electrophoresis (IFE). Top: the baseline IFE of a patient with Waldenstrom macroglobulinemia. A monoclonal IgM-kappa is evident. Bottom: the same patient during a course of ofatumumab therapy. A faint monoclonal IgG-kappa (←) is now also evident. This monoclonal protein is also visible on the acid-fixed electrophoresis (ELP) lane. The anode and cathode are indicated on the right hand side. The top right portion of each scanned image was deleted to remove patient identifiers. HYDRASYS 9 IF kits (Sebia Inc., Norcross, GA, USA) were used for immunofixation. Similar findings with other MATs – siltuximab, rituximab (Rituxan®), trastuzumab (Herceptin®), bevacizumab (Avastin®), infliximab (Remicade®), cetuximab (Erbitux®) and adalimumab (Humira®) – were recently described in an excellent report by McCudden et al (2010). Another study has demonstrated transient identification of monoclonal proteins in patients treated with efalizumab (Raptiva®, a humanized IgG1-kappa antibody) (Prignano, 2010). While probable MATs were detected by SPE and IFE in the efalizumab study, some newly identified antibodies were biclonal or consisted of unrelated subtypes (for example, IgM). A transient monoclonal and/or oligoclonal response to certain therapies (even to MATs) can exist. These ‘atypical’ IFE patterns, for example, are frequently observed after high-dose chemotherapy and/or transplantation. More recently, atypical IFE patterns have been described with non-myeloablative treatment of multiple myeloma, where they were even shown to correlate with favourable clinical response (Mark et al, 2008). As MATs are commonly included in many chemotherapeutic regimens, it may become difficult to differentiate atypical IFE patterns from MATs in many patients. On a technical note, detection of MATs by serum IFE depends on their species of origin, as IFE kits typically use anti-human reagent antibodies for clonal characterization. These reagent antibodies may have no (or at least minimal) ability to recognize non-human immunoglobulins. Detection of MATs by SPE (using either agarose or capillary methods), however, would not depend on their species of origin, as there are no reagent antibodies in these methods. A mouse-derived MAT (the ‘-omabs’) might be therefore be detectable by SPE but not IFE, while the human (‘-mumab’), humanized (‘-zumab’), and many if not all chimeric mouse-human (‘-ximab’) MATs are far more likely to be detectable by both SPE and IFE. Decreased sensitivity of agarose SPE to MAT interference when compared with capillary or immunofixation methods, however, has also been observed (McCudden et al, 2010). Such differences (along with the variation of methods used between laboratories and the differences in MAT dosing between patients) may limit our ability to reliably detect MATs in a given patient. Determining that a new monoclonal component observed on SPE and IFE may be due to a MAT remains a challenging task. This process is assisted by a detailed clinical history, the presence of expected heavy and light chain subtypes based on the MAT suspected, and the gradual disappearance of this component after therapy is completed. As clinical laboratories are rarely provided with extensive patient history, it is likely that faint monoclonal components unknowingly due to MATs are being reported. This may lead clinicians to falsely suspect conditions such as monoclonal gammopathy of undetermined significance (MGUS). Furthermore, MATs are increasingly being used in numerous medical disciplines, including allergy/immunology, dermatology, gastroenterology, haematology, oncology, rheumatology, and organ transplantation. Greater awareness of potential MAT detection by SPE and IFE assays by clinicians and laboratorians will help to prevent unnecessary diagnostic work-ups in these cases. The authors have no conflict of interest to disclose.