Oligoclonal gammopathy: An analysis of 253 cases

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Background
Oligoclonal gammopathy (OG) is a clinical condition characterized by the production of at least 2 separate monoclonal components (M-proteins) detectable in serum or urine. Their presence might be the result of the proliferation of more than 1 clone of pathological plasma cells, or the result of the production of distinct proteins by 1 specific clone. 1,2 Despite the seemingly low probability of the first alternative, it has already been confirmed by the analyses of mutational profiles that 2 distinct populations of neoplastic plasma cells may exist in an individual patient. 1,3 Most of the previously published literature concerns biclonal gammopathy (BG). However, the term 'triclonal gammopathy' (TG) is also used. 4,5 Other names, such as 'biclonal paraproteinemia' or 'double gammopathy' have already been relegated to history. 1 However, pathologists might refer to the manifestation of biclonal gammopathy as 'double gammopathy manifestation'. 2 At least 2 distinct monoclonal proteins can be identified in 1-6% of gammopathies. [6][7][8] The specific types of gammopathy include biclonal gammopathy of undetermined significance (BGUS), 6 together with asymptomatic and symptomatic multiple myeloma (MM). The last one likely develops from a previously diagnosed monoclonal gammopathy of undetermined significance (MGUS) 4 and other plasma cell dyscrasia, such as light chain amyloidosis. However, the spectrum of hematologic diagnoses identified in patients with OG is not limited to plasma cell dyscrasias. Other underlying abnormalities include lymphoid malignancies (e.g., chronic lymphocytic leukemia, diffuse large B-cell lymphoma or follicular lymphoma) or myeloid malignancies (e.g., acute myeloid leukemia, acute prolymphocytic leukemia or myelodysplastic syndrome). 6 According to the published data, the clinical picture and response to therapy in patients with biclonal myeloma are similar to those observed in patients with monoclonal myeloma. 2,9 However, it is still unknown whether the presence of 2 nd or further monoclonal proteins affects the incidence or aggressiveness of potential relapse. Based on these results, authors have recommended identical treatment approaches for both groups of patients. 9 Unfortunately, most of the data on biclonal and triclonal gammopathies come from case reports. [10][11][12][13] Hence, more research is still needed on this subject to determine if there are any specific differences between the conditions.

Objectives
This study aimed to assess the differences between patients in whom OG was recognized during initial diagnosis (further termed as 'primary OG') and the remaining patients who had OG diagnosed later ('secondary OG').
In secondary OG, we evaluated the time at which the abnormality developed. Then, we evaluated the underlying hematopoietic disorders and possible differences between biclonal and triclonal gammopathies. Moreover, we assessed whether there are differences in the contribution of various monoclonal proteins as indicators of biclonality and triclonality.

Study design
In this retrospective study, we searched a database of a large, 1000-bed hospital Serum Protein Electrophoresis (SPEP) Laboratory for results containing at least 2 distinct peaks of monoclonal proteins verified with immunofixation (OG). Their presence was the only inclusion criterion in our study. Next, we extracted clinical data from the hospital records of the patients to be included in the study.

Setting
The analysis concerned the results of serum immunofixation of patients who had this test performed during the period from January 2017 to December 2020, and relevant clinical data from all available patient records of those who met the criterion of OG. Data were collected from January 2021 to November 2021.

Participants
We identified 253 patients who met the criterion of OG (137 males and 116 females, 54.2% and 45.8%, respectively) and subsequently analyzed their hospital records. Then, we identified a group of 13 patients with primary OG, that is OG at initial presentation prior to any treatment. The remaining 240 patients had secondary OG, in whom the 2 nd peak of monoclonal protein developed later during observation and/or treatment.

Variables
The data available for this study included the patient's age and sex (available for all patients, n = 253), types of monoclonal proteins (2 or more) detectable in the patient's serum (available for all patients diagnosed during the 4-year period from January 2017 to December 2020 (n = 253)), the type of hematopoietic disorder they were diagnosed with (available for n = 154 (60.9%)), the date of diagnosis (available for n = 86 (34%), the period from 2005 to 2020), Durie-Salmon stage for myeloma patients (available for n = 69 (27.3%)), and specific cytogenetic alterations (n = 16 (6.3%)).

Data sources
All included data were collected exclusively from the mentioned sources, namely laboratory results and patient hospital records.

Study size
The patients were chosen as participants of the study only based on the results of immunofixation. No other criteria were used for the selection of the participants. The study size of 253 was chosen to maximize the chances of identifying primary OG patients (the smaller portion of the overall cohort) and to minimize the period of observation (4 years).

Quantitative variables
Quantitative variables were analyzed and compared in terms of means, medians and 95% confidence intervals (95% CIs).

Statistical analyses
Comparative analyses were performed using age to segregate the patients. The arranged cohorts were compared with the use of Welch's t-test, with a statistical significance threshold of p < 0.05. The normal distribution of the data in the analyzed cohorts was verified using Shapiro-Wilk test, with a threshold of p < 0.05 necessary to reject the hypothesis of normality.
Interestingly, free light chains were found in 29 combinations (n = 18 with λ and n = 11 with κ), 12 of which were found among patients with TG (n = 29). The patient with 4 distinct M-proteins also had a detectable free light chain band.
The distribution of M-protein types with their categorization into dominant and secondary M-proteins is shown in Table 1. Analogous results, selectively for TG cases, are shown in Table 2. An individual case of OG with 4 distinct monoclonal proteins displayed the combination of IgGλ+free λ chain+IgGκ+IgMλ.
We further investigated whether there is a specific time preference for the development of secondary oligoclonality after the initial diagnosis of monoclonal gammopathy (Fig. 2). As shown, the curve is biphasic, with faster development of biclonality during the first 30 months after the diagnosis and a slower rate at later timepoint. However, there is no specific timepoint in which the risk of oligoclonality development is increased compared to other timepoints, or a time when it no longer occurs. specific combinations -see Table 1 3 distinct M-proteins (n = 29) specific combinations -see Table 2 The development of oligoclonality occurred on average after 28.8 months (95% CI: 21.3-36.3 months). Notably, approx. 75% of cases occurred within 30 months, a cutoff point chosen to separate different phases of the Kaplan-Meier curve. Among the patients with an available diagnosis (n = 154), there were 133 patients with BG, 20 patients with TG and 1 patient with 4 M-proteins. In BG patients, it was possible to distinguish subgroups solely with plasma and lymphoplasma cell dyscrasia (n = 111), and classify these patients based on another accompanying disorder (n = 22). Furthermore, in the subgroup of patients with another disorder, it was possible to distinguish those with lymphoid (n = 11), myeloid (n = 8), other neoplasia (n = 1), and non-neoplastic accompanying diseases (n = 2). However, the subgroup with plasma cell dyscrasia included MM (n = 86 (64.7%)), OG of uncertain significance (n = 11), MM + amyloidosis (n = 7), Waldenström's macroglobulinemia (MWalden) (n = 4), light chain deposition disease (n = 1), amyloid light-chain (AL) amyloidosis (n = 1), and plasmacytic leukemia (n = 1). Interestingly, OG with other lymphoid malignancies included chronic lymphocytic leukemia (CLL) (n = 4), marginal zone lymphoma (MZL) (n = 3), diffuse large B-cell lymphoma (DLBCL) (n = 2), small lymphocytic lymphoma (SLL) (n = 1), and mantle cell lymphoma (MCL) (n = 1). Oligoclonal gammopathy cases with myeloid malignancy included acute myeloid leukemia (AML) (n = 5), myelodysplastic syndrome (MDS) (n = 2) and chronic myeloid leukemia (CML) (n = 1). There was also a case of OG with adenocarcinoma (n = 1). Finally, there were individual cases of OG patients with primary autoimmune thrombocytopenia and autoimmune hemolytic anemia (Fig. 3).
Among 20 patients with TG and available diagnoses, there were 14 cases of plasma cell dyscrasias, including MM (n = 13 (65%)) and MWalden (n = 1). There were also 3 cases of lymphoid neoplasias: CLL (n = 1), SLL (n = 1) and splenic lymphoma (n = 1); 2 cases of myeloid neoplasias: AML (n = 1) In each combination, the dominant protein is written first, followed by the secondary protein. Apart from included combinations, there were miscellaneous combinations of M-proteins. These combinations often included free light chains (κ or λ), free heavy chains (IgG, IgA or IgE) and immunoglobulins (IgEλ or IgDλ) as components.
and MDS (n = 1); and 1 case of OG resulting from a non-neoplastic disorder, namely glomerulonephritis with vasculitis. The patient with 4 distinct M-proteins was diagnosed with chronic myelomonocytic leukemia (CMML), a unique diagnosis throughout the entire cohort.
Diagnoses were available for all patients with primary OG (n = 13) and in all cases, plasma cell or lymphoplasma cell dyscrasia was detected, where the most common was MM (n = 11 (84.6%)). Among them, there was 1 case of MM + amyloidosis and 1 MM + light chain deposition disease. The remaining diagnoses were MWalden and OG of uncertain significance.
Regarding the age at diagnosis (data available for n = 86), the patients with primary OG (n = 12) were significantly    Table 3.
The analysis of Durie-Salmon stages in OG myeloma patients (data available for n = 69 (27.3%)) revealed the following results: IA -n = 6, I -n = 1, IB -n = 0, IIA -n = 12, II -n = 2, IIB -n = 3, IIIA -n = 25, III -n = 0, IIIB -n = 20. Therefore, the majority of patients developed oligoclonality in the advanced stages of myeloma, sometimes years after the primary diagnosis of monoclonal gammopathy. Conversely, in patients with primary OG (data available for n = 6), 2 patients were classified to stage IA, 2 patients to IIA, 1 to IIIA, and 1 to IIIB. For myeloma patients with 3 detectable clones, Durie-Salmon staging was available for 8 out of 13 cases. The results were as follows: 1 patient with IA, 1 patient with IIA, 3 patients with IIIA, and 3 patients with IIIB stage. While no statistical comparison was possible with these numbers, the data did not reveal major differences between the BG and TG cohorts.
The cytogenetic analysis was only performed in 16 out of 253 patients but revealed 10 individuals with karyotype abnormalities, as follows: t (4,14) in 2 patients and del(17) + del(1), IGH/FGFR3 fusion with rearrangements of IGH, t(11, 14), hyperdiploid, del(17p−), deletion of TP53, monosomy of 17p, and partial monosomy of 17p + partial deletion of TP53, each in 1 patient. Among patients with primary OG, cytogenetic alterations were not found. Finally, the patient with monosomy of chromosome 17p had 3 detectable M-proteins in serum.

Discussion
This study analyzed the problem of secondary OG and provided a comparison of BG and TG for the first time. We observed that the development of both biclonality and triclonality is frequently a late event in the evolution of MGUS, often observed when MGUS progresses to MM. Furthermore, most cases of so-called primary OG had symptomatic MM. It suggests that they underwent an evolution from monoclonal gammopathy during undiagnosed disease and not as a primary event. Furthermore, our data suggest that the spectrum of both biclonality and triclonality is very large and every theoretically possible combination of M-proteins may be observed.
We failed to associate any specific event in a disease course (transformation, treatment, progression, remission, stabilization) with the development of oligoclonality (the detection of the 2 nd or subsequent clone). Therefore, we performed a time analysis with the Kaplan-Meier curve (Fig. 2). Some studies postulate potential emerging factors of OG, such as autologous hematopoietic stem cell transplantation. 6 However, there are no prospective cohort studies that have analyzed this aspect of the disease. Therefore, the etiology of OG remains unknown and no contributing factors have been identified.
There are 2 major possible mechanisms for developing oligoclonality. One possibility is that 2 or more clonally different plasma cell dyscrasias co-occur in the same patient. The other is that BG and TG represent an evolution of the primary clone with subclones derived from cells that underwent additional mutational events. Recent analysis with next-generation sequencing has revealed that aberrations in the TP53 signaling pathway are responsible for the occurrence of multiple, synchronous primary cancers. 14 Moreover, and specifically for OG, the cases of 2 (or more) monoclonal immunoglobulins that contain distinct light chains (κ and λ) might be considered truly biclonal, since no molecular mechanism of changing light chain expression has been described so far. 15 Such a statement has already been made by some authors 3 and our study reveals numerous cases of oligoclonal gammopathies where Mproteins include immunoglobulins with different light chains. This phenomenon seems to occur too frequently to be just a coincidence without any underlying mutational background. Lastly, polymerase chain reaction (PCR) and immunofluorescence (IF) analyses of clonal relationship in patients with BG imply that 2 independent clones can coexist synchronously in individual patients, even when the clones produce immunoglobulins with the same type of light chain (κ and λ). 16 We failed to identify major differences between primary and secondary OG, except for primary OG patients of more advanced age. The similarities between both groups (primary compared to secondary OG) have been observed regarding the dominant diagnosis. In both groups, MM or MM + comorbidities were the dominant diagnoses, which remains consistent with earlier findings. 4 We have also failed to identify significant differences between patients with biclonal compared to triclonal gammopathies when comparing age, underlying disorder and the dominant combinations of monoclonal proteins that are present. In the only other analysis of TG concerning 6 cases, there were 2 cases of MWalden, 1 case of non-Hodgkin lymphoma, 1 case of polycythemia vera, and 2 cases without any hematopoietic disease. 17 However, the same analysis revealed that 64.6% of TGs were detected in lymphoproliferative diseases, based on all known cases. 17 The IgG occurred the most often in combinations of monoclonal proteins in all groups. This is not surprising, since this serum protein is also detectable in the majority of monoclonal gammopathy cases (followed by IgM and IgA). 4,18 The additional 3 rd Mprotein in patient serum was often a free light chain. Furthermore, free light chains tended to be detectable as a part of a triclonal combination (12/29), as opposed to biclonal combinations (17/223). The reasons for the presence of additional free light chains in patients with TG (especially the patients with primary OG) remain unknown, and this phenomenon requires further research and clarification.
While data concerning cytogenetic alterations are limited (10 out of 16 tested patients), they only identified changes that have already been found in MM cases. 23 However, they appear to be quite common in OG cases.

Limitations
The main limitation of the study was the availability of clinical data, such as diagnoses. The lack of data resulted from the fact that OG is a rare disorder. Moreover, we did not identify any comprehensive disease mechanism underlying OG.

Conclusions
The diagnosis of patients with primary OG is more often established in males in older age (approx. 10 years older) than in patients with secondary OG. The detection of secondary OG often (in approx. 75% of cases) occurs up to 30 months after the initial diagnosis. Moreover, there are laboratory and clinical findings that are specific to patients with primary OG and TG cases. Despite the findings of this study, OG remains a poorly understood disorder and more research, especially prospective studies, is necessary to support these conclusions. It will take more time for the detailed pathogenesis of the disease to be established.