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  • Priyadarshini John, MD

Silent Saboteurs: Detecting Monoclonal Immunoglobulins and Determining Clinical Outcomes in Monoclonal Gammopathy of Renal Significance (MGRS) with Undetectable Clones

Illustrated by: Corina Teodosiu, MD

AcademicCME (www.academiccme.com) is accrediting this educational activity for CE and CME for clinician learners. Please go to https://academiccme.com/kicr_blogposts/ to claim credit for participation.


The term monoclonal gammopathy of renal significance (MGRS) was introduced in 2012 for the first time by the International Kidney and Monoclonal Gammopathy Research Group (IKMG) to outline the individuals who had renal injury secondary to monoclonal protein deposition while not meeting other pathologic criteria. In 2017, the definition of MGRS was modified to define it as a clonal proliferative disorder where there is production of nephrotoxic monoclonal protein. Hence, the presence of monoclonal immunoglobulins (M proteins) suggests the presence of an underlying clonal proliferative disorder, and a renal biopsy is necessary to diagnose, classify and treat patients with MGRS. These monoclonal immunoglobulins are heterogeneous in nature. The structure of M proteins can vary from having pentameric IgM to just having free light chains. The quantity of M proteins also ranges from gm/L of serum to almost nil. Clonal plasma cell proliferative disorders have numerous presentations, treatment algorithms and varied outcomes depending on the type of monoclonal immunoglobulin secreted, their quantity and pathogenicity.


The main mechanisms of MGRS related kidney injury are classified into direct and indirect pathways. The direct mode of renal injury is through deposition of monoclonal protein, or its light chain and heavy chain components, in various parts of the kidney. The indirect mode of renal injury is through monoclonal immunoglobulin activation of the alternative complement pathway, by producing antibodies against the complement proteins. So the culprit monoclonal immunoglobulin diseases potentially include all B cell and plasma cell proliferative disorders, which otherwise do not satisfy the criteria for hematological disease.


The diagnostic criteria for MGRS includes the following;

(1)Clonal Immunoglobulin related kidney involvement.

(2)Clone production is insufficient to cause overt hematological malignancy.


Evaluation of suspected MGRS can be challenging. Diagnostic modalities of the MGRS spectrum include renal biopsy and a thorough hematological assessment. Renal biopsy includes light microscopy, immunofluorescence (IF) with immunoperoxidase/immunohistochemistry and transmission electron microscopy (TEM). Typing of IgG subclass with restriction assists in diagnosis. TEM comprises an important part of the evaluation. Comprehensive description of findings of EM are summarized here. Hematological assessment consists of identification of monoclonal immunoglobulin in serum or urine and characterization of specific clones. The various tests available to detect nephropathic clones include: bone marrow sampline, blood or lymph node sampline, flow cytometry with immunotyping, bone marrow biopsy immunohistochemistry for kappa and lambda, and molecular biology techniques incorporating high-throughput sequencing assay from bone marrow mRNA encoding light chain variable segment immunoglobulins, RACE-RepSeq, which is sensitive to detectigt minimal clonal disease.


Methods for Identification of monoclonal immunoglobulin

Often MGRS is associated with low levels of monoclonal protein secondary to a B cell or plasma cell clone. Low sensitivity of protein electrophoresis makes it inefficient to detect the low levels of circulating monoclonal proteins. Light chains, with their shorter half-lives, make protein electrophoresis even more challenging. Immunofixation remains the gold standard for identification and typing of monoclonal immunoglobulins. Immunofixation is more sensitive than electrophoresis. Immunoblotting, though not widely available, is of great value in diagnosing heavy chain amyloidosis,where it identifies the deletion of constant domain of the heavy chain  which is pathognomonic of heavy chain amyloidosis.Immunoblotting also aids in characterization of IgG heavy chain. Unfortunately, due to the diverse presentations of MGRS, a battery of tests must be performed to identify, quantify and prognosticate the underlying monoclonal protein. Electrophoresis and immunofixation should be performed on both  serum and urine samples. Katzmann et al found that combination of tests including SPEP, free light chain assay and immunofixation electrophoresis increase the sensitivity to around 97.4% in detection of monoclonal protein. However immunofixation electrophoresis is costly and requires a great number of limited reagents and resources. 


Serum Protein Electrophoresis: Agarose gel electrophoresis, or capillary zone electrophoresis, are commonly used methods for the SPEP. In monoclonal gammopathies the M protein appears as a restricted zone of migration. Kyle et al, in a series of approximately 1000 myeloma patients, observed the majority of monoclonal spike distribution was in the gamma region followed by the junction between beta-gamma, beta region and minority in alpha 2 fraction.


the serum protein electrophoresis curve in monoclonal gammopathies

The limitations of SPEP are as follows;


  1. In Monoclonal light chain myeloma, M spike might not be detected as these are excreted through the kidney and have a low molecular weight.

  2. Monoclonal IgAs might be hidden behind a beta peak.

  3. Monoclonal IgD spikes might be too small to detect. IgD is primarily found on the surface of B lymphocytes and usually accounts for only about 0.25% of total immunoglobulins.


Immunofixation electrophoresis: Immunofixation has a higher sensitivity by 10 fold in comparison to protein electrophoresis, with a threshold of limit detection of approximately 10 mg/dL. Usually IFE is performed with antisera to IgG, IgM, IgA,  kappa and lambda chains. Delta and epsilon antisera are used only if a light chain is detected without a heavy chain.


Nephelometry immunoglobulin quantification: Nephelometry estimates both monoclonal and polyclonal fractions of immunoglobulins. Hence the utility of nephelometry is limited in regular settings and is used in special situations like:


  1. When there is minimal polyclonal synthesis.

  2. When IgG M spike is more than 30 g/L when dye saturation stands as a limitation

  3. When IgA M spike migrates to beta fraction.


Serum free light chain assay: sFLC assay estimates quantitative measurement of kappa and lambda chains by using antibodies against the epitopes of light chains with a sensitivity of less than 5 mg/L. The various assays which are  used include  immunonephelometric assays based on polyclonal antibodies (FreeLite assay), and assays based on monoclonal antibodies (N latex serum free light chains). Reference range for normal kappa , lambda and k/𝛌 ratio are  3.3-19.4 mg/l, 5.7-26.3 mg/l, 0.26-1.65 respectively. In the setting of renal dysfunction, the main channel of excretion of free light chains is by means of  reticuloendothelial pinocytosis at the level of proximal tubules, but at a rate much lesser than that of kidney and hence there is a slight increase in the normal ratio of k/λ to 0.37-3.17. The ratio remains unaltered in the N latex free light chain assay. The main limitations of the assay include

 

  1. Low levels of serum free light chains remain undetectable with this assay. 

  2. 24 hour collection can be cumbersome.


Cryoglobulins: Testing for cryoglobulins is also necessary in the work up of patients with MGRS. Cryoglobulins are special proteins with a property of precipitation upon cooling and dissolving upon heating. The presence of serum precipitate after 24 hours in an ice  bath implies cryoglobulinemia, and the concentration of cryocrit is estimated by centrifugation of the tube. Immunofixation of the precipitate can be performed on washed, resuspended and warmed cryoprecipitate by the antibodies with specific heavy/light chains. Type 1 cryoglobulins are usually monoclonal IgG, IgGA, IgGM and sometimes monoclonal light chains. Type2 cryoglobulins are usually a mixture of monoclonal and polyclonal. Type 3 cryoglobulins are frequently polyclonal. The clinical manifestations of cryoglobulins depend on the temperature at which cryoglobulin precipitates, rather than the amount of monoclonal protein.


During analysis of  the samples for cryoglobulin it is crucial to maintain the temperature during collection and processing as cryoglobulins would precipitate below 370C and are prone to false negative errors. Majority of the cryoglobulins coexist with rheumatoid factor as this is an immune mechanism which causes clearance of infection and activation of complement. Absence or low levels of rheumatoid factor narrows diagnosis to monoclonal gammopathy. Hence testing for rheumatoid factor in the evaluation of cryoglobulinemia is of paramount importance.

The following table summarizes different methods of identification of monoclonal immunoglobulin protein.

Methods for identifying monoclonal proteins

Kidney Biopsy

After the renal biopsy confirms the monoclonal protein related injury, IF staining for IgG subclass helps in confirmation of deposition of an intact monoclonal IgG. IgG3 is highly kidney avid because of its properties including its large molecular weight, positive charge and tendency for self aggregation. IgG3 kappa is seen in proliferative glomerulonephritis associated with monoclonal immune deposits (PGNIMD). Light chain restriction also helps in narrowing down the diagnosis. Lambda light chains are commonly associated with amyloidosis, while Kappa light chains are mostly associated with MIDD. See our KI Reports Community educational blog for more on MGRS.


Clonal identification: Bone Marrow aspiration with biopsy is the initial investigation of choice. Few malignancies like chronic lymphoid leukemia are diagnosed enough with flow cytometry from peripheral blood. Adjunct analysis on bone marrow include cytogenetics, fluorescence in situ hybridisation (FISH) and next generation DNA sequencing. Nevertheless despite exhaustive evaluation, clonal detection rate can be as low as just 30% as is observed in PGNMID, making it difficult to treat. The chance of non-detectability is directly proportional to identification of the monoclonal immunoglobulin, which is as low as 17%. CT with PET and whole body MR is of immense use in identifying the suspicious lesions like that of plasmacytoma or biopsy worthy lymph nodes, which can aid in diagnosis. Congo red stain on bone marrow can identify AL amyloidosis in 60% of the cases. A MYD88 mutation is often seen in 80% of the cases of lymphoplasmacytic clones.


The most common clones identified are B cell clones, plasma cell clones and lymphoplasmacytic clones. Plasma cell clones are more commonly seen in light chain amyloidosis, MIDD, and Fanconi syndrome. B cell clones are more often associated with immunotactoid GN. Lymphoplasmacytic clones are most often linked to cryoglobulinemic glomerulonephritis.


Clonal identification is one of the most important strategies to guides treatment. B cell clones typically secrete IgG or IgM, plasma cell clones secrete all 5 types of immunoglobulins and light chains, whereas lymphoplasmacytic clones secrete IgM monoclonal immunoglobulins.


Treatment of MGRS

MGRS treatment depends on the underlying clone. The main objective of the therapy is directed at renal improvement and preventing the recurrence post transplantation. If a patient presents in early stages of CKD, chemotherapy offers the maximum benefit. If the patient presents with late stage CKD, the objective of chemotherapy would be to achieve complete hematological remission before transplantation to mitigate the incidence of recurrence. 


The Study

In the KI Reports article Clinical Outcomes of MGRS without Detectable Clones, the authors ask  the question, how are outcomes different in patients with MGRS disease processes without detectable clones?


Methods

The current study is a single center retrospective cohort from Brigham and Women’s Hospital. Patients who underwent renal biopsy between January 1, 2010 and December 31, 2022 and reported monoclonal immunoglobulin or paraprotein deposition in biopsy are identified in the study. 


Immunoglobulins or light chains are identified

  1. either in glomeruli or tubular basement membranes by immunofluorescence.

  2. As electron dense deposits in mesangial, subendothelial, or subepithelial locations and/or tubular basement membranes.


Out of 493 kidney biopsy cases of monoclonal immunoglobulin deposition, 29 cases of MGRS, without detectable monoclonal protein or clone, were included in the study. The criteria used for MGRS in the study are


  1. Negative serum protein electrophoresis and Immunofixation electrophoresis.

  2. Normal serum free light chain assay adjusted for glomerular filtration rate.

  3. No clones identified in serum, urine and bone marrow.

  4. Absence of Haematological conditions like plasma cell dyscrasias.


Includes incidence of complete remission, partial remission and the need for renal replacement therapy. Complete remission is defined as proteinuria <0.5g/g Creatinine with less than 20% decline in eGFR. Partial remission is defined <3 g/gCr proteinuria but with more than 50% reduction in proteinuria from the baseline.

 

Results

Out of the 29 cases incorporated in the study, 2 were in transplanted kidneys and the remaining in native kidneys. The median age of diagnosis was 58 years and nearly half of them were males. The median amount of proteinuria was 4.6 grams on spot urine protein-to-creatinine ratio or in 24-hour urine collection. More than 50% of cases had membranoproliferative glomerulonephritis as the major histopathological pattern. Other injuries manifested as mesangioproliferative, membranous and proliferative glomerulonephritis. A majority of the cases had mild interstitial fibrosis and tubular atrophy (IFTA).


There were four categories of treatment regimens allocated to patients depending on the level of proteinuria and renal function. 


1. Conservative treatment group (28%) (lower proteinuria) which included renin angiotensin aldosterone blockade and no MGRS specific treatment.Less than 50% of these patients of the conservative group achieved complete remission


2. Plasma Clonal directed therapy (21%) (chemotherapy directed at plasma cells) which included bortezomib, daratumumab, combination of cyclophosphamide, bortezomib and dexamethasone.


3.Lymphocyte Clonal directed therapy (24%) (chemotherapy directed at lymphocyte cells) which included rituximab based regimens.


4.Non-clone directed therapy(28%) which included glucocorticoids, oral cyclophosphamide, and mycophenolate mofetil.


Few of the patients had switched to another treatment subset in case of failure to respond to the initial therapy.


The following figure summarizes the treatment regimen.


Kidney Outcomes

The type of therapy did not have a significant impact on proteinuria reduction after 1 year. The type of therapy instituted depended upon the amount of proteinuria and renal function.


The primary driver for treatment response appeared to be the amount of interstitial fibrosis and tubular atrophy (IFTA) on the biopsy. It was observed in the current study that patients with more than 25% IFTA had worse outcomes. Repeat kidney biopsy was considered in cases of worsening proteinuria or renal function. It was determined that repeat biopsy enabled individualization of further therapy by either decreasing or intensifying the immunosuppression depending. 


Here is the beautiful VA summarizing the study by Denisse Arellano MD.

visual abstract on clinical outcomes of MGRS without detectable clones

Detection of clones facilitates clone directed therapy, and hence better prognostication.Very few studies are available in the literature about consensus of treatment in cases of MGRS with undetectable clones. A retrospective case series by Gumber et al  prescribed plasma clonal or lymphocytic clonal directed therapy. Guiard et al, in this retrospective study used lymphocyte clone directed therapy and non clone directed therapy with optimal favorable outcomes whereas patients on conservative therapy progressed to end stage renal disease. Kousios et al also emphasized use of lymphocytic clone directed therapy predominantly. Fermand recommendations for treatment of MGRS are widely postulated. In the absence of clones, combination chemotherapeutic agents like cyclophosphamide, bortezomib and dexamethasone are preferred. B cell clones are treated with alkylating agents like cyclophosphamide, immunomodulatory drugs like thalidomide and proteasome inhibitors like bortezomib.


We have come a long way from melphalan to combination of daratumumab, cyclophosphamide, bortezomib and dexamethasone with autologous stem cell transplantation in management of amyloidosis. Lymphoplasmacytic clones are treated with rituximab based regimens.


Renal transplantation in MGRS is associated with a high rate of recurrence. It is indicated after achieving hematological remission. Recurrence also depends on the type of monoclonal protein as injury is specific to the monoclonal protein. Light chain amyloidosis is late to recur compared to Light chain proximal tubulopathy and PGNMID. 


Summary

MGRS are a set of diverse renal conditions which often is challenging to diagnose and manage. The diagnosis should aim at detection of monoclonal immunoglobulin and clones  MGRS in native and graft kidney is undoubtedly taxing, more so if we don't encounter the culprit clone. Individualized therapy based on the amount of proteinuria and renal function promise satisfactory outcomes. Certain entities like that of PGNMID would confront the clinicians with inability to detect clones. Thorough means of clonal detection is possible with the availability of more efficient modalities like mRNA thorough output sequencing on bone marrow samples. Lymphocytic and plasma clonal directed therapies are commonly used regimens in cases of undetectable clones. Patients on conservative management are monitored periodically to check if they satisfy the criteria for clonal directed therapies. There should be a low threshold for kidney biopsy which would help tailor the future course of immunosuppression.


AcademicCME (www.academiccme.com) is accrediting this educational activity for CE and CME for clinician learners. Please go to https://academiccme.com/kicr_blogposts/ to claim credit for participation.

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