Resolution of dysglycaemia after treatment of monoclonal gammopathy of endocrine significance

Abstract In very rare cases of monoclonal gammopathy, insulin-binding paraprotein can cause disabling hypoglycaemia. We report a 67-year-old man re-evaluated for hyperinsulinaemic hypoglycaemia that persisted despite distal pancreatectomy. He had no medical history of diabetes mellitus or autoimmune disease but was being monitored for an IgG kappa monoclonal gammopathy of undetermined significance. On glucose tolerance testing, hyperglycaemia occurred at 60 min (glucose 216 mg/dL) and hypoglycaemia at 300 min (52 mg/dL) concurrent with an apparent plasma insulin concentration of 52 850 pmol/L on immunoassay. Laboratory investigation revealed an IgG2 kappa with very high binding capacity but low affinity (Kd 1.43 × 10−6 mol/L) for insulin. The monoclonal gammopathy was restaged as smouldering myeloma not warranting plasma cell–directed therapy from a haematological standpoint. Plasma exchange reduced paraprotein levels and improved fasting capillary glucose concentrations. Lenalidomide was used to treat disabling hypoglycaemia, successfully depleting paraprotein and leading to resolution of symptoms.


Introduction
Insulin autoimmune syndrome (IAS, Hirata disease) is a rare cause of hyperinsulinaemic hypoglycaemia characterised by insulin-binding autoantibodies in exogenous insulin-naïve individuals. 1,2In affected patients, antibody binding reduces the action of acutely secreted insulin and delays clearance of plasma insulin resulting in increased insulin concentration with raised insulin/C-peptide molar ratio. 1,3,4Insulin immunocomplexes act as reservoirs, with insulin-glucose mismatch leading to post-prandial and/or fasting hypoglycaemia.][7][8][9][10][11][12][13][14][15][16][17] Both entities are characterised by very high plasma insulin concentrations but can vary in severity of hypoglycaemia at presentation.Laboratory investigation is not standardised, yet obtaining evidence that insulinbinding globulin is due to PCD rather than co-existence of IAS with coincidental asymptomatic PCD can have clinical utility.While IAS may be mild, and spontaneously resolve, disabling hypoglycaemia may persist in some patients and immunosuppressive regimens employed. 1In contrast, cytotoxic chemotherapy has been used in monoclonal insulin-binding antibody-associated multiple myeloma (MM). 8,12,15,16 significance (MGUS). 10We now describe the clinical and laboratory investigation of a man with severe recurrent hypoglycaemia after pancreatectomy, who was ultimately confirmed as having an insulin-binding paraprotein.He experienced symptom resolution following treatment with lenalidomide and dexamethasone.

Insulin and C-peptide immunoassay
Plasma insulin and C-peptide were measured using the DiaSorin LIAISON XL chemiluminescence analyser.Plasma dilution for insulin analysis was undertaken using assay diluent.

Insulin immunocomplex detection
Polyethylene glycol (PEG) precipitation studies of plasma, and gel filtration chromatography (GFC) of plasma post addition of recombinant human insulin, were undertaken as previously published. 18

Radioligand binding assays
Insulin-binding affinity by antibody was assessed in a 10-fold dilution with anti-insulin antibody negative serum, with immune complexes precipitated using a 50:50 mixture of protein A Sepharose and protein G Sepharose as previously outlined. 1,19,20Kd (mol/L) was calculated by non-linear regression analysis using a 1-site model (R 2 value .98),assuming equal antibody binding by labelled ([125-I]-A14) and unlabelled insulin.

Electrophoresis and light chain capture
Capillary zone electrophoresis was undertaken using the Helena Biosciences V8 system.Insulin-binding light chain was determined using the PureProteome™ Kappa Ig Binder and Lambda Ig Binder Magnetic Beads as directed by manufacturer's instructions.Patient triplicate mean results were compared with results from an IAS sample used as positive control and an anti-insulin antibody-negative control sample.

Case presentation
A 67-year-old man was referred with a 10-year history of episodic loss of consciousness preceded by sweating, palpitation, fatigue, and intense hunger, resolving with refined carbohydrates.
Previously, evaluation at another centre confirmed fasting hyperinsulinaemic hypoglycaemia and a possible Ga-68 DOTATATEavid pancreatic lesion.He underwent distal pancreatectomy for suspected insulinoma; histology showed an 8 mm nodule without evidence of an endocrine tumour.Four years later, he was diagnosed with MGUS and monitored expectantly. 25Hypoglycaemia symptoms worsened during fasting and postprandially with hypoglycaemic unawareness ultimately developing.He had never been prescribed insulin or oral hypoglycaemic agents and had no family history of diabetes mellitus or autoimmune conditions.His body mass index was 32.2 kg/m 2 ; examination was otherwise unremarkable.
Given the SM, determining whether the anti-insulin antibodies were due to IAS or PCD would direct management.Radioligand binding studies and light chain capture assays confirmed the principal insulin-binding globulin was IgG (Figure 3A) of IgG2 subclass (Figure 3B) and kappa light chain (Figure 3C) consistent with the M-protein.
Due to the COVID-19 pandemic, he was unable to attend follow-up appointments so was speculatively prescribed prednisolone (20-40 mg daily).A weight increase of 6.5 kg ensued, and recurrent hypoglycaemia with loss of consciousness persisted.
Smouldering myeloma reassessment 12 months later revealed no indication to treat from a haematological perspective.Paraprotein increased to 25 g/L and anti-insulin IgG to 242 mg/L with follow-up GFC results confirming a further rise in insulin-binding capacity (Figure 2B).Given ongoing life-threatening hypoglycaemia and inefficacy of prednisolone, the patient agreed to a trial of plasma cell-directed therapy (PCDT), namely lenalidomide 25 mg daily for 21/28-day cycle with dexamethasone 40 mg once weekly.Non-neutropenic sepsis developed within a week, and attempted dose reduction to lenalidomide 20 mg once daily resulted in a second similar hospital presentation.
To assess the potential benefit of insulin-binding antibody depletion, he underwent a 3-day course of plasma exchange. 1rior to this, fasting capillary blood glucose (FCBG) ranged from 54 to 65 mg/dL, paraprotein level 16 g/L with anti-insulin IgG 159 mg/L, and some reduction in insulin-binding capacity (Figure 2B).Following plasma exchange, paraprotein fell to 2 g/L with equivocal residual insulin binding by day 2 (Figure 2B) and increased FCBG ranging from 92 to 153 mg/dL, providing proof of concept and support for ongoing PCDT.
Reduced dose lenalidomide 5 mg once daily for 21/28-day cycle was commenced, and improvement in glycaemic control followed within 3 months with FCBGs ranging from 67 to 110 mg/dL.This regimen has continued for over 18 months with no side effects and no hypoglycaemic symptoms.

Discussion
Reports of hypoglycaemia caused by insulin-binding antibodies secreted in PCD are very rare.Hypoglycaemia may be postprandial, postabsorptive, or fasting, and, as in our patient, symptoms can be disabling.The very high plasma insulin concentration, increased insulin/C-peptide molar ratio, and positive anti-insulin IgG result were indistinguishable from that of IAS. 1 Detectable C-peptide immunoreactivity concurrent in a hypoglycaemic sample was of high molecular weight and could represent antibody-bound proinsulin crossreactivity in immunoassay. 1,26he investigative approach relied upon anti-insulin antibody assays not typically available outside a specialist laboratory, including GFC, RBS, and light chain capture assays.Results of capillary zone electrophoresis of serum (A), following addition of anti-kappa (B), and following addition of anti-lambda (C).Results were consistent with a monoclonal band, which was reduced with anti-kappa but not anti-lambda.

B.Grant et al. K27
1][12][13][14][15][16] Binding affinity was lower than previously demonstrated in an IAS cohort using the same methodology. 18][29][30][31] They are most commonly of the IgG class without predominance of a particular subclass or light chain. 32Further examination in this case confirmed the insulin-binding antibody to be IgG2 kappa, consistent with the paraprotein and increased serum concentration of IgG2.
In the context of severe dysglycaemia requiring clinical intervention, discriminating the 2 entities had important management implications: although aberrant insulin kinetics are induced by a similar mechanism in both polyclonal IAS and monoclonal insulin binding, autoantibody production in IAS may be self-limiting, while in PCD, targeted PCDT is needed for antibody depletion.Following initial side effects that followed initiation of PCDT, plasma exchange provided proof of principle that antibody depletion would improve glucose control and lead to symptomatic improvement.Lenalidomide was recommenced at a lower dose that resulted in resolution of symptoms and the absence of side effects.Although lenalidomide use with dexamethasone has been reported in MM and insulin-binding antibodies, 16 we believe this is the first reported successful use of combination therapy in SM with insulin-binding paraprotein, whereby disabling hypoglycaemia directed therapy.Results expressed as percentage recovery of insulin immunoreactivity (%).Depletion of paraprotein with anti-kappa but not anti-lambda was confirmed with capillary zone electrophoresis (Figure 1B and C).InsAb +, positive control; InsAb -, negative control.Patient results are consistent with anti-insulin kappa.

Figure 2 .
Figure 2. Prolonged oral glucose tolerance test (A).Fasting capillary blood glucose measured pre, and venous blood glucose measured post, 75 g oral glucose load.(Venous sample collected at time 0 was unsuitable for analysis due to haemolysis.)Gel filtration chromatography of plasma post addition of synthetic human insulin (B).Results from presentation, follow-up, pre plasma exchange, and post plasma exchange are shown.Elution volumes of immunoglobulin G (IgG), albumin (Alb), and monomeric insulin (mIns) are shown (molecular weights 150, 66.5, and 5.8 kDa, respectively).High-molecular-weight insulin (HMWIns) is antibody-bound insulin that elutes with immunoglobulin, and the slur between peaks is produced by dissociation of immunocomplexes during filtration.Radioligand binding assay (C).Data were consistent with an antibody of high capacity and low affinity (Kd 1.43 × 10 −6 mol/L) to bind insulin.

Figure 1 .
Figure 1.Capillary zone electrophoresis pre/post light chain capture.Results of capillary zone electrophoresis of serum (A), following addition of anti-kappa (B), and following addition of anti-lambda (C).Results were consistent with a monoclonal band, which was reduced with anti-kappa but not anti-lambda.

Figure 3 .
Figure 3. Insulin binding by antibody class (A).Results are expressed as arbitrary units (AU) derived from a logarithmic standard curve.Data demonstrated most insulin binding using protein A Sepharose (GB-PAS) and protein G Sepharose (PGS), consistent with anti-insulin IgG.Insulin binding by IgG subclass (B).Results are expressed as nano unit of insulin bound per millilitre of serum (nU/mL).Data are consistent with anti-insulin IgG2.Insulin recovery following light chain capture (C).Results expressed as percentage recovery of insulin immunoreactivity (%).Depletion of paraprotein with anti-kappa but not anti-lambda was confirmed with capillary zone electrophoresis (Figure1B and C).InsAb +, positive control; InsAb -, negative control.Patient results are consistent with anti-insulin kappa.