Investigating the utility of saliva immunoglobulins for the detection of myeloma and using myeloma proteins to clarify partition between oral and systemic immunity.

OBJECTIVES: Myeloma is characterised by the presence of monoclonal immunoglobulin (M-protein) and the free light chain (FLC) in blood. We investigated whether these M-proteins and FLC are detectable in myeloma patients' saliva to evaluate its utility for non-invasive screening and monitoring of haematological malignancies. METHODS: A total of 57 patients with monoclonal gammopathy and 26 age-matched healthy participants provided paired serum and saliva samples for immunoglobulin characterisation and quantification. RESULTS: Myeloma patients had IgG or IgA M-protein levels ranging up to five times and FLC levels up to a thousand times normal levels of polyclonal immunoglobulins. Despite these highly elevated levels, only two IgG and no IgA M-proteins or FLC could be detected in paired saliva samples. Most patients had reduced levels of serum polyclonal immunoglobulins, but all had normal levels of salivary IgA. CONCLUSIONS: Immunoglobulin transfer from blood is not determined by levels in the systemic circulation and more likely dictated by periodontal inflammation and the integrity of the oral epithelium. Immunoglobulins secreted by bone marrow plasma cells do not substantially enter saliva, which represents a poor medium for myeloma diagnosis. These findings, along with normal salivary IgA levels despite systemic immunoparesis, support a strong partitioning of oral from systemic humoral immunity.

Response comparison of multiple myeloma and monoclonal gammopathy of undetermined significance to the same anti-myeloma therapy: a retrospective cohort study.

BACKGROUND: Multiple myeloma is consistently preceded by monoclonal gammopathy of undetermined significance (MGUS), which is usually only treated by a form of anti-multiple myeloma therapy if it is causing substantial disease through deposition of secreted M proteins. However, studies comparing how MGUS and multiple myeloma plasma cell clones respond to these therapies are scarce. Biclonal gammopathy multiple myeloma is characterised by the coexistence of an active multiple myeloma clone and a benign MGUS clone, and thus provides a unique model to assess the responses of separate clones to the same anti-multiple myeloma therapy, in the same patient, at the same time. We aimed to identify how MGUS and multiple myeloma plasma cell clones responded to anti-multiple myeloma therapy in patients newly diagnosed with biclonal gammopathy multiple myeloma. METHODS: In this retrospective cohort study, we identified patients with biclonal gammopathy multiple myeloma by central laboratory analysis of 6399 newly diagnosed patients with multiple myeloma enrolled in three UK clinical trials (Myeloma IX, Myeloma XI, and TEAMM) between July 7, 2004, and June 2, 2015. In addition to the inclusion criteria of these trials, our study necessitated at trial entry the presence of two distinct M proteins in immunofixation electrophoresis. The primary endpoint was difference in response achieved with anti-multiple myeloma therapy on MGUS (which we defined as M2) and multiple myeloma (M1) clones-overall, within patients, and between therapy types-with international therapy response criteria assessed with χ2 analyses. We analysed by intention to treat. FINDINGS: 44 patients with biclonal gammopathy multiple myeloma with IgG or IgA MGUS clones were subsequently identified from the three trials and then longitudinally monitored. 41 (93%) of M1 clones had a response to therapy (either complete response, very good partial response, partial response, or minor response) compared with only 28 (64%) of M2 clones (p=0·0010). For the 20 patients who received intensive therapy, there was no difference between the proportion of responding clones in M1 (19 [95%]) and M2 (15 [75%], p=0·13). However, for the 17 patients who received non-intensive therapy, 16 (94%) of M1 clones had a response compared with ten [59%] of M2 clones (p=0·031). When examining clones within the same patient, 30 (68%) of 44 individual patients had different levels of responses within the M1 and M2 clones. One patient exhibited M2 progression to myeloma and subsequently died. INTERPRETATION: These results show that, in patients with biclonal gammopathy multiple myeloma, anti-multiple myeloma therapies exert a greater depth of response against multiple myeloma plasma cell clones than MGUS plasma cell clones. Although some MGUS clones exhibited a complete response, many did not respond, which suggests that the underlying features that render multiple myeloma plasma cells susceptible to therapy are present in only some MGUS plasma cell clones. To determine MGUS clone susceptibly to therapy, future studies might seek to identify, with biclonal gammopathy multiple myeloma as an investigative model, the genetic and epigenetic alterations that affect whether MGUS plasma cell clones are responsive to anti-multiple myeloma therapy. FUNDING: National Institute of Health Research, Medical Research Council, and Cancer Research UK.

Active multiple myeloma suppresses and typically eliminates coexisting MGUS.

BACKGROUND: Myeloma is consistently preceded by premalignant monoclonal gammopathy of undetermined significance (MGUS). In >5% of MGUS patients there is a second MGUS clone (biclonal gammopathy of undetermined significance; BGUS), yet, at myeloma diagnosis, presentation of biclonal gammopathy myeloma (BGMy) is considered less frequent, implying that myeloma eradicates coexisting MGUS. METHODS: In the largest study of its kind, we assessed BGMy frequency amongst 6399 newly diagnosed myeloma patients enrolled in recent UK clinical trials. RESULTS: Compared to expected prevalence (i.e., >5% of MGUS have BGUS), only 58 of 6399 (0.91%) newly diagnosed myeloma patients had BGMy, indicating myeloma typically eliminates coexistent MGUS. In these 58 BGMy cases, the MGUS plasma cell clone was greatly suppressed in size compared to typical levels observed in conventional MGUS; contrarily, the MGUS clone did not inhibit the myeloma plasma cell clone in BGMy. CONCLUSION: Myeloma eliminates the majority of competing MGUS, and when it does not, the MGUS clone is substantially reduced in size.

Development of a rapid and quantitative lateral flow assay for the simultaneous measurement of serum κ and λ immunoglobulin free light chains (FLC): inception of a new near-patient FLC screening tool.

BACKGROUND: Serum free light chains (FLC) are sensitive biomarkers used for the diagnosis and management of plasma cell dyscrasias, such as multiple myeloma (MM), and are central to clinical screening algorithms and therapy response criteria. We have developed a portable, near-patient, lateral-flow test (Seralite®) that quantitates serum FLC in 10 min, and is designed to eliminate sample processing delays and accelerate decision-making in the clinic. METHODS: Assay interference, imprecision, lot-to-lot variability, linearity, and the utility of a competitive-inhibition design for the elimination of antigen-excess ('hook effect') were assessed. Reference ranges were calculated from 91 healthy donor sera. Preliminary clinical validation was conducted by retrospective analysis of sera from 329 patients. Quantitative and diagnostic results were compared to Freelite®. RESULTS: Seralite® gave a broad competitive-inhibition calibration curve from below 2.5 mg/L to above 200 mg/L, provided good assay linearity (between 1.6 and 208.7 mg/L for κ FLC and between 3.5 and 249.7 mg/L for λ FLC) and sensitivity (1.4 mg/L for κ FLC and 1.7 mg/L for λ FLC), and eliminated anomalous results from antigen-excess. Seralite® gave good diagnostic concordance with Freelite® (Roche Hitachi Cobas C501) identifying an abnormal FLC ratio and FLC difference in 209 patients with newly diagnosed MM and differentiating these patients from normal healthy donors with polyclonal FLC. CONCLUSIONS: Seralite® sensitively quantitates FLC and rapidly identifies clinical conditions where FLC are abnormal, including MM.

Development of a highly-sensitive multi-plex assay using monoclonal antibodies for the simultaneous measurement of kappa and lambda immunoglobulin free light chains in serum and urine.

Monoclonal κ and λ immunoglobulin free light chain (FLC) paraproteins in serum and urine are important markers in the diagnosis and monitoring of B cell dyscrasias. Current nephelometric and turbidimetric methods that use sheep polyclonal antisera to quantify serum FLC have a number of well-observed limitations. In this report, we describe an improved method using specific mouse anti-human FLC monoclonal antibodies (mAbs). Anti-κ and anti-λ FLC mAbs were, separately, covalently coupled to polystyrene Xmap® beads and assayed, simultaneously, in a multi-plex format by Luminex® (mAb assay). The mAbs displayed no cross-reactivity to bound LC, the alternate LC type, or other human proteins and had improved sensitivity and specificity over immunofixation electrophoresis (IFE) and Freelite™. The assay gives good linearity and sensitivity (<1 mg/L), and the competitive inhibition format gave a broad calibration curve up to 437.5 mg/L and prevented anomalous results for samples in antigen excess i.e. high FLC levels. The mAbs displayed good concordance with Freelite™ for the quantitation of normal polyclonal FLC in plasma from healthy donors (n=249). The mAb assay identified all monoclonal FLC in serum from consecutive patient samples (n=1000; 50.1% with monoclonal paraprotein by serum IFE), and all FLC in a large cohort of urine samples tested for Bence Jones proteins (n=13090; 22.8% with monoclonal κ, 9.0% with monoclonal λ, and 0.8% with poly LC detected by urine IFE). Importantly this shows that the mAbs are at least close to the ideal of detecting FLC from all patients and neoplastic plasma cell clones. Given the overall effectiveness of the anti-FLC mAbs, further clinical validation is now warranted on serial samples from a range of patients with B cell disorders. Use of these mAbs on other assay platforms should also be investigated.