Two Cases of γ-Heavy Chain Disease and a Review of the Literature

Gamma heavy chain disease (γ-HCD) is a rare lymphoproliferative disorder characterised by the production of a truncated immunoglobulin heavy chain. Fewer than 200 cases have been reported in the literature. In some cases, γ-HCD occurs with other lymphoid neoplasms. This study reports clinical, biochemical, haematological, and histological findings in two cases of γ-HCD. We describe newer biochemical diagnostic tools (HevyLite measurement, capillary electrophoresis, and immunotyping) that can aid in the characterisation of γ-HCD. The first case is an 88-year-old woman with γ-HCD. The second case is an 81-year-old woman who developed γ-HCD during treatment for Waldenstrom's macroglobulinemia. In the second patient, histopathology identified a separate clone responsible for the secretion of the gamma heavy chain. Studies on the clonal evolution of the disease may provide insight into therapeutic implications and the genomic complexity of the disease.


Introduction
Gamma heavy chain disease (c-HCD) is also called Franklin's disease, named after the author of the first report in 1964. e disease is defined as a neoplasm of lymphocytes, plasmacytoid lymphocytes, and plasma cells characterised by the production of an abnormally truncated gamma heavy chain protein that lacks associated light chains. Since Franklin first reported the case, fewer than 200 cases have been described in the literature. A wide variety of disorders have been associated with c-HCD. In some cases, c-HCD occurs with other lymphoid neoplasms or with a history of autoimmune disorder. c-HCD shows a wide clinical spectrum ranging from completely asymptomatic to progressively malignant forms.
Normal heavy chains not associated with light chains have not been detected in serum of healthy individuals. In c-HCD abnormally short truncated heavy chains can be isolated from patient's serum without associated light chains. In several instances where the gene encoding the shortened heavy chain gene has been characterised, the truncation originated from deletions and/or insertions within the rearranged variable region genes (V) that in addition harbor somatic point mutations. One possibility is that several types of oncogene translocations happen as a by-product of somatic hypermutation [1]. It has been established the c-HCD immunoglobulins are the products of aberrant biosynthesis, for example, deletions, splice site correction, and amino-terminal proteolysis [2]. Structural analysis of the defective gamma heavy chain in 23 patients showed some common features. In most cases, the variable region sequence was short and interrupted by large deletions, usually including the C H 1 domain. Normal sequence began at the hinge region or at the C H 2 domain [3].
Genetic subtypes of multiple myeloma (MM) have been identified which have different biological features and heterogeneity in clinical outcomes [4]. New gene sequencing techniques have yielded new insights into the pathogenesis and evolution of the disease and potential individualised treatment by novel targeted approaches [5].
We report two cases of c-HCD: one case in a patient with lymphadenopathy and a second case in a patient with a history of Waldenstrom's macroglobulinemia (WM). Further, we review previous case series on c-HCD. ese two cases were diagnosed using the techniques of capillary zone electrophoresis, immunofixation, and "HevyLite" measurement.
Use of newer commercially available techniques simplified the diagnosis of c-HCD. In one patient, there was no evidence of previous chemotherapy. e second patient developed c-HCD during treatment for WM.

Case Report
2.1. Case 1. An 88-year-old woman presented with a change in the bowel habit. A colonoscopy showed some diverticular disease.
e CT scan showed splenomegaly and some lymphadenopathy particularly in the region of the splenic hilum. e liver, kidney, pancreas, and adrenals were normal. She had a past history of osteopenia, type II diabetes, and fragility fracture. She was taking vitamin B12, vitamin D, and bisphosphonates.
ere was no history of sweating, weight loss, bruising, or recent infections. Her biochemistry and hematology at diagnosis and 3 months after diagnosis are summarised in Tables 1 and 2.
Gel protein electrophoresis and immunofixation ( Figure 1(a)) and capillary zone electrophoresis and immunotyping (Figures 2(a) and 2(b)) (Sebia, UK) identified 2 c-heavy chains. Both methods were negative for kappa and lambda light chains. Differing sensitivities of heavy and light chain reagents can cause false-negative results for light chain immunofixation, and the results were confirmed by a second gel electrophoresis (Helena, UK) method. Urine immunofixation identified a c-heavy chain (Figure 3(a)).
Total IgG (determined by immunoturbidimetry on the Binding Site SPA-plus analyser) was elevated at 38.7 g/L (RR 7-16), with decreased levels of IgA and IgM (Table 1). IgG1 subclass levels were elevated, and other subclass levels were lower than the reference range (Table 3). Serum-free light chain ratios (determined by the Binding Site method, UK) were within the reference range (Table 4). HevyLite (Binding Site, UK) measurements are specific for individual heavy chains with either kappa or lambda light chains and can provide more specific information than individual immunoglobulin quantitation. Observed increased heavy chain pair ratios (e.g., IgMkappa/IgMlambda) can be indicative of clonal expansion. IgGkappa/IgGlambda ratios were within the reference range confirming the absence of clonal expansion of IgGkappa/IgGlambda intact immunoglobulins (Table 5).
In a previous study, HevyLite reagents which did not recognise intact IgG immunoglobulin ratios ((IgGkappa + IgGlambda)/total IgG) were used as indirect measures of heavy chain fragmentation [6]. e ratio of 0.04 in this patient was less than the average value of 0.18 found in the previous study [6].
Bone marrow biopsy showed subtle infiltrates by lowgrade B-NHL, associated with groups of clonal plasma cells, which expressed neither kappa nor lambda chain but stained with IgG heavy chain (Figures 4(a)-4(g)). Lymphatic infiltrate was surrounded by a rim of CD138+ plasma cells.
ough there was equal expression of kappa and lambda light chains, the sum of kappa-and lambda-positive cells appeared to be much less than the number of CD138+ cells which indicated that most plasma cells do not express light chains.
e patient is currently monitored on a 3-monthly basis.

Case 2. Patient 2 is an 81-year-old lady under treatment
for WM (IgMkappa paraprotein). Details of her treatment are given in Figure 5. Capillary zone electrophoresis prior to the development of c-HCD is shown in Figure 6(a). Gamma heavy chain developed during treatment and an unusually diffuse c-heavy chain band was identified by gel electrophoresis ( Figure 1(b)) and capillary zone electrophoresis (Figures 6(b) and 6(c)) (Sebia, UK) and confirmed by a second gel electrophoresis method (Helena, UK). Urine immunofixation identified the c-heavy chain and kappa light chain ( Figure 3(b)). Her biochemistry and hematology prior to the development of c-HCD and during follow-up are given in Tables 1  and 2. HevyLite measurements confirmed an IgMkappa paraprotein, and ratios of IgGkappa/IgGlambda were within the reference range, confirming increased IgG measurements were due to the IgG heavy chain (Table 5). e ratio of ((IgGkappa + IgGlambda)/total IgG) was 0.096. e serum-free light chain ratio was elevated secondary to the presence of IgMkappa paraprotein (Table 4). Her total IgG levels were increased at 35.1 g/L, at diagnosis of c-HCD, with suppressed IgA and an IgMkappa paraprotein of 10.7 g/L (Tables 1 and 2). IgG1 subtype was elevated, and other IgG subtypes were either decreased or at the lower reference range (Table 3). She is currently under observation for ibrutinib treatment.
Her bone marrow results are shown in Figures 7(a) and 7(b) and in the Supplementary Material (available here). Lymphoma cells were CD5−, CD20+, and partially CD79a +, CD10+, CD23+, CD56+, and CD138+. A second CD56+ plasma cell clone most likely to be the c-heavy chain producer was identified. Bone marrow histology identified lymphoplasmacytic lymphoma/WM, which appeared as IgM/kappa plus another population of scattered plasma cells, occasionally expressing IgG and/or lambda.

Discussion
Heavy chain diseases are rare B-cell lymphoproliferative disorders that are characterised by the production of immunoglobulin heavy chains without associated light chains. c-HCD has a variety of clinical presentations. e Mayo Clinic reported a series of 23 patients of whom 70% had an underlying lymphoplasma cell proliferative disorder, 13% an autoimmune disorder, and 4% no underlying disease. Pathological diagnosis of underlying disease reported in the 23 patients was lymphoplasma cell proliferative disorder, lymphoma, chronic lymphocytic leukemia, plasmacytoma, and plasma cell proliferative disorder.
e disease was associated with lymphadenopathy (34%), hepatomegaly (4%), splenomegaly (30%), or bone marrow involvement (30%). e median survival of the 23 patients was 7.4 years (range 1 month to more than 21 years) [7]. In a further series of 13 patients, 8 patients showed a lymphoplasmacytic neoplasm that was difficult to classify. In the remaining 5 patients, biopsy identified welldefined neoplasms: splenic diffuse red pulp and small B-cell lymphoma, splenic marginal zone lymphoma, MALT lymphoma, lymphoplasmacytic lymphoma/WM, and chronic NK cell lymphocytosis. In the 13 cases, the study reported the involvement of the lymph nodes (n � 7), spleen (n � 2), bone marrow (n � 8), and other extranodal tissues (n � 3). Patients showed a female predominance (85%) with frequent occurrence of autoimmune disease (69%) [8]. Iijima et al. [9] report a case c-HCD and T-cell large granular lymphocytic leukemia. c-HCD can exist in the absence of clinical symptoms similar to other forms of monoclonal gammopathy of undefined significance (MGUS) [10].
In our patients, two methods, capillary zone electrophoresis and gel electrophoresis, identified a gamma heavy chain with no corresponding light chain. Results from this report and previous reports [11] suggest that capillary zone   electrophoresis and immunotyping are able to detect and characterise c-HCD in serum. c-Heavy chain was identified in the urine of both patients, suggesting that the lowermolecular weight of the truncated heavy chain allowed renal clearance. In a modification of the immunofixation method, Gulli et al. [12] preincubated patient sera with anti-kappa and anti-lambda to precipitate intact immunoglobulins, except for free heavy chain, prior to immunofixation. e intact IgGkappa/IgGlambda (HevyLite) measurement confirmed the presence of gamma heavy chain in the absence of associated kappa or lambda light chains. In a previous study, HevyLite assays showed that in 10/15 patients, substantial portions of IgG were not quantitated by antibodies to intact IgGkappa and IgGlambda immunoglobulins [6].
Serum IgG subtypes showed an increase in IgG1 subclass in both patients. In a previous report, serum IgG measured by nephelometry on the Beckman Coulter Immage analyser was below the reference range in a patient with c-HCD [11]. Total IgG and IgG1 subclasses measured using the binding site reagent were increased in our patients. is has been previously illustrated by other groups [13]. e discrepant values observed between case reports can be the result of differences in analytical techniques, loss of antigenic domains due to truncation of the heavy chains, or analytical variation due to different detection antibody specificities.
ere have been several advances in the diagnosis of multiple myeloma. New techniques may further enhance the diagnosis of c-HCD. Capillary electrophoresis with immunotyping is able to characterise free heavy chains in serum, demonstrating the absence of intact IgG immunoglobulins by the HevyLite assay and the absence of serum-free light chains by serum-free light analysis are further valuable tools for diagnosis. Serum/urine electrophoresis and immunofixation are analytical tools in the workup of c-HCD patients.
Alpha (α), c, and mu (µ) heavy chain diseases have been described. α-HCD, the most common of heavy chain diseases, appears to be a condition affecting primarily the secretory IgA system and mainly the digestive tract. α-HCD may present as a continuous sequence of events from an apparently reversible hyperplastic process to an overt neoplastic proliferation [14]. µ-HCD is the least common of the heavy chain diseases. Patients present with lymphoplasma cell proliferative disorder,    Case Reports in Hematology and index of suspicion is high in patients with vacuolated plasma cells in the bone marrow [3]. e mechanism for the pathogenesis of c-HCD is unclear. Bone marrow histology of the first patient with isolated c-HCD suggested the presence of a clonal plasma cell which selectively secreted c-heavy chain in the absence of kappa or lambda light chain. Genomic events that underlie the progression of MGUS to MM have been difficult to dissect. Both bone marrow microenvironment and genomic instability play a role in MM pathogenesis. Bone marrow biopsy in the second patient, with WM and c-HCD, identified two possible malignant clones. It is worth noting that this patient's treatment included R-CVP, cladribine, and velcade. It is plausible that the treatment might have increased the transformation of the c-HCD clone by increasing the instability of the MM cells. Clonal evolution in MM before and after therapy can follow several patterns. Branching clonal evolution involves one or more subclones that emerge at a later time point. Current therapeutic approaches have shortcomings, and understanding of MM tumorogenesis and progression may have implications for genomically guided precision treatment [15].

Conclusions
In summary, we report two cases of c-HCD. e findings also provide information for practical guidance for the laboratory evaluation of c-HCD. In the first case, c-HCD developed as the presenting lymphoid malignancy. Bone marrow biopsy revealed an IgG heavy chain secreting clone. e second case was a composite lymphoid neoplasm of WM and c-HCD. c-HCD developed during treatment for WM. Bone marrow biopsy revealed the presence of two neoplastic lymphoid cell lines. e review of the literature   suggests further studies are indicated to develop an understanding of the clonal evolution of c-HCD.