Emergence of Oligoclonal Bands in Association with the use of Chemotherapy and Hematopoietic Stem Cell Transplantation

Abstract

Multiple myeloma (MM) is a rare malignant condition with an abnormal clonal proliferation of plasma cells in the bone marrow. Chemotherapy and Hematopoietic Stem cell transplantation (HCT) are the main modalities of myeloablative therapy. The study aimed to determine the frequency of oligoclonal bands (OB) in multiple myeloma patients receiving primary therapy alone with chemotherapy as well as patients undergoing HCT at a single institution. The clinical and laboratory records of 76 MM patients were reviewed who underwent HCT from January 2012 to January 2019. Another 74 cases receiving chemotherapy alone, were also reviewed. In total 85 patients were selected by the availability of at least 3 serial immunofixation electrophoresis(IFE) results in non-transplanted cases and 2 post-transplant IFE results in the HCT cases after attainment of very good partial response(VGPR). 40 patients were non transplanted cases while 45 patients underwent HCT. Oligoclonal bands emerged in twenty-four (28%) patients. 15% (6/40) of the patients treated without HCT and, 40% (18/45) of patients treated with HCT from their respective cohorts. To conclude, this is the first Indian report showing a higher frequency of oligoclonal response in patients in VGPR attained after hematopoietic stem cell transplantation versus chemotherapy. This difference could be due to a stronger immune reconstitution, or graft vs. host reaction, or autoimmune response to myeloma antigens and may not be an active disease process or relapse. However to determine the prognostic impact of OB further investigations and follow-ups are required.

Introduction

Multiple myeloma (MM) is a hematological malignancy characterized by uncontrolled clonal proliferation of abnormal plasma cells in the bone marrow [1, 2]. These plasmas cells produce unusually large numbers of monoclonal immunoglobulin or antibody with a typical isotype and light chain restriction that is when present in serum and/or urine is known as paraprotein or the M component [3]. This uniqueness in the restriction pattern is important in both diagnosis and monitoring. MM is a rare malignancy that accounts for approximately 10% of all hematologic malignancies [4]. MM is generally treatable but mostly incurable. The major treatment modalities include chemotherapy and hematopoietic stem cell transplantation. To monitor the course of the disease and its progression, there is the International Myeloma Working Group (IMWG) that has formulated uniform response criteria [4] and recommends certain serological diagnostic studies to be performed in the serum and urine including serum protein electrophoresis (SPE), immunofixation electrophoresis (IFE), a serum-free light chains (sFLC) assay along with immunohistochemistry or immunofluorescence studies [5–7]. With the advancement of these diagnostic modalities, the course of the disease, its progression, and treatment response can be monitored [8, 9].

Usually, a sharp band or the M band in electrophoresis is produced by monoclonal protein or immunoglobulin made of a heavy chain and a light chain. Recent studies [10–15] have reported incidences of Oligoclonal bands (OB) with varying frequencies from single digits to 73% following chemotherapy and/ or HCT. The phenomenon of oligoclonality is represented by the presence of an additional or abnormal band on SPE that is different from the initial monoclonal spike with different migration pattern; immunoglobulin isotype switching; multiple immunoglobulin subtypes at immunofixation detected in serum and/or urine with or without detectable primary immunoglobulin.

In this hospital-based study [16] a retrospective analysis of clinical history and laboratory records of serial SPE and serum IFE of MM patients is done to determine the frequency of oligoclonal bands after attainment of at least very good partial response (VGPR) either with chemotherapy alone or with haematopoietic stem cell transplantation.

Materials and Methods

This is an institution based retrospective observational study being carried out in the Department of Biochemistry, Tata Medical Centre, Kolkata, India.

Protein Electrophoresis

Semiautomated instrument Hydrasys 2 scan from Sebia was used to perform serum protein electrophoresis and immunofixation with high-resolution Agarose gel. Densitometry scan was done to quantify protein fractions and the M spike. For immunofixation electrophoresis, the samples were treated with antisera to heavy chains isotypes (α, γ, μ) and light chains isotypes (λ, κ) and assayed in Hydrasys 2 scan from Sebia.

Patient and Data Selection

Baseline characteristics including demographics, clinical history, laboratory records, and treatment records of 150 patients together with their final diagnosis of Plasma cell myeloma were reviewed. In total 76 cases underwent HCT from January 2012 to January 2019. Another 74 cases receiving chemotherapy alone, were also reviewed who came for follow up. The records of SPE and IFE were evaluated for the oligoclonal pattern after the initial attainment of at least VGPR. Only the patients in whom at least 3 serum IFE test reports were available were short-listed. In transplant patients undergoing HCT, a minimum of 2 serum IFE reports in the post-transplant phase were included.

Therapy

The chemotherapy regimens were VTD (bortezomib, thalidomide, dexamethasone) / VRD (bortezomib, lenalidomide, dexamethasone) / VCD (bortezomib, cyclophosphamide, dexamethasone) TD(thalidomide, dexamethasone)/VD (bortezomib, dexamethasone)/ CTD(cyclophosphamide, thalidomide, dexamethasone) / KPD (carfilzomib, pomalidomide, dexamethasone) with bisphosphonates followed by maintenance therapy with lenalidomide / thalidomide / pomalidomide.

In the patients planned for HCT, myeloablative treatment with high dose melphalan followed by stem cell rescue was the usual regimen.

Response

MM patients who had at least a very good partial response (VGPR) after treatment were included for analysis.

Respose: VGPR	IMWG Criteria
	Monoclonal M-protein detectable by IFE but not on electrophoresis in serum and urine or ≥ 90% reduction in serum monoclonal M-protein plus urine M-protein level < 100 mg/24 h

Result Interpretation

The clinical biochemist recorded the type of restriction performed in serum IFE in terms of immunoglobulin light and/or heavy chain. The oligoclonal pattern/ band was characterized as the emergence of abnormal protein bands or as only one band different from the primary immunoglobulin and with or without detectable primary malignant monoclonal immunoglobulin [11, 13, 15]. The following were not taken as an indication of oligoclonal pattern: additional light chain bands of the same type as the original malignant immunoglobulin; and polyclonal response. The time of appearance of OB was noted in terms of months following HCT in post-transplant patients. A positive SPE was delineated by the presence of a quantifiable M spike. The M bands were quantified into large (> 3 g/dL), moderate (1.5–3 g/dL), and small M bands (< 1.5 g/dL) respectively. Further scaling of M protein band is introduced as ‘Faint’ and ‘Very Faint’.

Statistical Analysis

Baseline characteristics of the study population were assessed for the significance of differences between the two groups. Student's t-test has been used for continuous variables and categorical variables are evaluated with Fisher's exact test. The differences were considered significant with p value < 0.05.

The study received approval from the local ethics committee.

Results

A total of 85 patients with VGPR or better responses satisfied the inclusion criteria set for the study; 45 patients in the transplant group and 40 patients in the group treated with chemotherapy(conventional and/or high dose) alone. In total 24[28%] patients developed OB during the study period. For the 45 patients that received HCT, 18 patients [40%] exhibited an oligoclonal pattern in the post-transplant setting. In the 40 patients who did not receive stem cell transplantation, only 6 patients [15%] displayed an oligoclonal pattern followed by chemotherapy. Table 1 summarizes the baseline characteristics of the study population and Table 2 outlines the immunoglobulin class at the time of diagnosis in the OB positive patients. Most of the oligoclonal patterns developed within the six months of HCT. The main characteristics of patients who developed oligoclonal bands are shown in Table 3. From, the measures of central tendency it has been observed that the time of appearance of OB is earlier (around 3–6 months) for transplant patients compared to non-transplant patients (approximately 9 months). The pattern of progression of OB in two instructive patients is shown in Fig. 1a and b and Fig. 2a and b respectively, where Fig. 1 represents a transplant case, and Fig. 2 represent a non-transplant case.

Table 1.

Baseline Characteristic of patients with and without emergence of Oligoclonal bands [n = 85]

Characteristics	Non-emergence of OB n = 61 [72%]	Emergence of OB n = 24 [28%]	p value
Median Age at diagnosis in years (Range)	62 (42–83)	60 (44–70)	0.516
Male/Female	36/25	19/05	0.024
Patients with SCT n = 45 (%)	27/45 (60.0)	18/45 (40.0)	0.013
Patients with CT, n = 40 (%)	34/40 (85.0)	06/40 (15.0)	0.116

Table 2.

Paraprotein characterization at initial diagnosis in 24 Patients who subsequently developed OB

Ig type	No. of patients with HCT [n = 18]	No. of patients with CT [n = 6]	Total
Ig A-K	4	2	6
Ig A-L	1	0	1
Ig G-K	5	2	7
Ig G-L	4	1	5
K	1	1	2
L	3	0	3

Table 3.

Characteristics of patients with oligoclonal bands

Patient no	Age	Sex	Primary CT	HCT	Original M spike	OB band	Time of appearance (in months,M)	Response evaluation
1	56	F	TD	Auto, Yes	IGA-K	IGA-K,IGG-K, FREE -K	60 M	VGPR
2	56	M	VTD	Auto, Yes	L	IGG-L	2 M	VGPR
3	70	M	VTD	Auto, Yes	K	IGG-K, FREE K	6 M	VGPR
4	60	M	VTD,VRD,CTD	Auto, Yes	IGA-K	IGA-K, IGA-L	3 M	VGPR
5	66	F	VCD	Auto, Yes	IGG-L	IGG-K	18 M	VGPR
6	48	M	VCD	Auto, Yes	IGG-L	IGG-L, IGG-K	6 M	VGPR
7	61	M	VCD	Auto, Yes	IGG-L	IGG-K	3 M	VGPR
8	54	M	VTD	Auto, Yes	IGG-K	L	18 M	VGPR
9	61	F	VTD	Auto, Yes	L	IGG-K, IGG-L	3 M	VGPR
10	52	M	VTD	Auto, Yes	IGG-K	IGG-K, IGG-L	3 M	VGPR
11	57	M	VCD,VRD	Auto, Yes	IGG-K	L	4 M	VGPR
12	45	M	VCD	Auto, Yes	IGA-K	IGG-L	2 M	VGPR
13	66	M	VTD	Auto, Yes	L	IGG-K	12 M	VGPR
14	59	M	VTD	Auto, Yes	IGG-L	IGG-L, MULTIPLE BANDS FREE K	3 M	VGPR
15	65	M	VTD	Auto, Yes	IGG-K	IGG-K, FREE L	6 M	VGPR
16	44	M	VRD	Auto, Yes	IGA-L	IGG-K,FREE L	12 M	VGPR
17	54	M	VTD,VRD	Auto, Yes	IGG-K	IGG-K, IGG-L	2 M	VGPR
18	66	M	VTD,KPD	Auto, Yes	IGA-K	IGG-L, IGG-K, IGA-K, FREE K	3 M	VGPR
19	64	M	Yes, VRD	No	IGA-K	K	8 M	VGPR
20	69	M	Yes, VTD	No	IGG-L	IGG-K, IGG-L	9 M	VGPR
21	59	M	Yes, VD	No	IGG-K	L	10 M	VGPR
22	47	M	Yes, VTD	No	IGG-K	IGG-K, L	12 M	VGPR
23	63	F	Yes, VTD	No	K	IGG-K	9 M	VGPR
24	63	F	Yes, VTD	No	IGA-K	IGG-K,IGA	9 M	VGPR

VTD bortezomib, thalidomide, dexamethasone, VRD bortezomib, lenalidomide, dexamethasone, CTD Cyclophosphamide, thalidomide, dexamethasone, VCD bortezomib, cyclophosphamide, dexamethasone TD thalidomide, dexamethasone VD bortezomib, dexamethasone, KPD carfilzomib, pomalidomide, dexamethasone, M Months

Fig. 1.

Transplant case with pre and post-transplant IFE patterns (a) IgA–κ (b) IgG–λ+IgG–κ+IgA–κ and additional κ light chains

Fig. 2.

Non transpant case: before and after chemotherapy IFE patterns (a) IgG–κ (b) Faint λ

For the transplant case Fig. 1, initially the IFE showed IgA Kappa restriction pattern Fig. 1a. Following the appearance of OB Fig. 1b, IFE was reported as: “IgA Kappa,IgA lambda,IgG lambda restrictions seen. Addendum:Two distinct kappa bands observed; one seen close to the beta region and the other in the gamma region”.

Figure 2 represents a non-transplant case. Here the initial IFE showed IgG Kappa restriction pattern Fig. 2a. After OB appearance Fig. 2b, IFE was reported as: “A faint restriction noted in the lambda lane. Addendum: This band is a slow moving band in the gamma region”.

The change in M protein concentration and FLC results were also observed under this study, which is reported as below:

• FLC ratio: Out of 24 patients; 20 patients (> 80%) exhibited decrease in FLC ratio. Thus a decrement in FLC ratio may be concluded.

• M protein concentration: Out of 24; 11 patients exhibited increase and 13 patients exhibited decrement values. However, more number of patients (Sample size) is needed for any kind of correlation to establish.

Discussion

In the recent past, discovery of various immunomodulatory drugs and haematopietic stem cell transplantation has lead to a considerable rise and variability in the incidence of oligoclonal patterns, starting from single digit value till up to 73% [3, 11–13, 15].

To the best of our knowledge, this is the first Indian study to report the emergence of oligoclonality in myeloma patients after myeloablative therapies including chemotherapy as well as HCT. In our study, oligoclonality emerged in 28% of patients achieving VGPR or better response. The rate of oligoclonal response was more in patients who underwent HCT [40%] as compared to non-transplanted patients[15%] which is in concordance with the previous studies [17–22]. Moreover, it takes lesser time for the oligoclonal bands to appear in patients who underwent transplant as compared to patients receiving chemotherapy solely.The underlying pathophysiology or the biochemical mechanism associated with the emergence of oligoclonal bands and its prognostication on patient health and survival is the focus of constant research.

The appearance of OB phenomenon may have multiple postulates. Pathological changes may include immunoglobulin class switching by the malignant plasma cell clone, emergence of a new additional malignant clone, discrepancy in the B-cell maturation process [17, 20–23], or infections. OB may also reflect thapeutic bone marrow response and recovery in the due course of treatment in the form of immune reconstitution; graft vs. host reaction or autoimmune response to myeloma antigens, especially in HCT [26–28]. As reported in previous studies [24, 25], a preponderance of IgG-κ oligoclonal immunoglobulin was observed in our study. This isotype predominance may be a result of preferential B-cell proliferation in response to a strong immune reconstitution response to therapy.

Despite the mechanism responsible, the interpretation of oligoclonal bands is challenging for the biochemists due to variability in banding pattern. Recent studies have revealed [10, 11, 29, 30] that the emergence of oligoclonal bands interferes with electrophoresis interpretation as well as degrades the performance of Serum Free Light Chain Assays. This may ultimately interfere with the line of the treatment plan. However, with the advancement of MM treatment, the increasing incidence of OB can be attributed to better treatment responses but its effect on overall survival need to be studied in future. In a previous study by Fijisawa et al. [13], they concluded that the development of OB exclusively indicated favorable responses but its mere presence could not validate a better survival. In another similar study [3], the results were conflicting where the authors had claimed the phenomenon of oligoclonality with a favorable impact on progression-free survival.

Strengths and Limitations of the Study

To our knowledge this is the first Indian study reporting the incidence of oligoclonality in multiple myeloma patients with a very good partial response after hematopoietic stem cell transplantation versus those who were treated with chemotherapy alone. This study provided an insight to the clinicians and biochemists about interpretation of oligoclonality to monitor the treatment response in the MM patients.

However, limitations like prognostic impact of oligoclonality on overall survival or progression-free survival could not be predicted due to the limited sample size, poor follow-up of the patients owing to financial strains, ignorance or death in a developing country like India. Also, to establish any definite correlation with the M protein needs a larger sample size and long term follow-up.

Conclusion

In conclusion, this study is the first of its kind in the Indian cohort evaluating oligoclonal response against different treatment modalities. The observed higher frequency of oligoclonal bands in patients receiving hematopoietic stem cell transplantation could be likely due to a stronger immune reconstitution. Periodic noting of the location and type of restriction pattern may be useful in the differentiation of oligoclonal bands. Advanced resolution protein separation techniques are needed to effectively allow the identification of abnormal bands. This study intensifies the need for further investigations and long term follow-up to study the prognostic impact of IFE oligoclonality on overall survival.

Abbreviations

MM

Multiple myeloma

HCT

Hematopoietic stem cell transplantation

IFE

Immunofixation electrophoresis

VGPR

Very good partial response

OB

Oligoclonal bands

SPE

Serum protein electrophoresis

Authors’ Contribution Statements

All authors were involved in the conception, design, write-up, and revision of this article. CP and SC planned the study and collected the data. CP, SC, SC carried out the analysis. SC and CP wrote, edited, and revised the manuscript.

Funding

The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not- for-profit sectors.

Data Availability

Data are available upon reasonable request. The data sets used and analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical Approval

This study was approved by the institutional ethical committee of the TATA MEDICAL CENTER, KOLKATA, INDIA. All personal identifiers (name, employer, contact) were removed from the data set, and analyses were carried out at the institution level.

Consent to Participant

Patients and/or the public were not involved in the design, or conduct, or reporting, or dissemination plans of this research.