- Email: [email protected]
Immune Signatures Associated With Clonal Isotype Switch After Autologous Stem Cell Transplantation for Multiple Myeloma
Original Study
Immune Signatures Associated With Clonal Isotype Switch After Autologous Stem Cell Transplantation for Multiple Myeloma Rebecca Ye,1 Sirisha Kundrapu,2 Stanton L. Gerson,3 James J. Driscoll,4 Rose Beck,2 Naveed Ali,5 Ola Landgren,6 Willem VanHeeckeren,7 George Luo,8 Nicolaus Kroger,9 Paolo Caimi,5,8 Marcos De Lima,5,8 Ehsan Malek5,8 Abstract A retrospective analysis of 177 patients with multiple myeloma undergoing autologous stem cell transplant found that 22% developed new and small concentrations of monoclonal protein after transplant that differed from that originally identified at diagnosis. This phenomenon had a benign nature and correlated with improved survival and more robust bone marrow immune reconstitution beyond the B-cell compartment. Background: High-dose chemotherapy and autologous stem cell transplantation (ASCT) are integral components of the overall treatment for patients with multiple myeloma (MM) aged 65 years. The emergence of oligoclonal immunoglobulin bands (ie, immunoglobulins differing from those originally identified at diagnosis [termed clonal isotype switch (CIS)]) has been reported in patients with MM after high-dose chemotherapy followed by autologous stem cell transplantation. However, the clinical relevance and the correlation with immune reconstitution remains unclear. Patients and Methods: Patients with MM who had undergone ASCT from 2007 to 2016 were included in the present study. The percentage of natural killer cells, B-cells, and T-cells was measured using flow cytometry in pre- and postASCT bone marrow samples. CIS was defined as the appearance of a new serum monoclonal spike on serum protein electrophoresis and immunofixation that differed from original heavy or light chain detected at diagnosis. Results: A retrospective analysis of 177 patients with MM who had undergone ASCT detected CIS in 39 (22%). CIS after ASCT correlated with improved progression-free survival (52.2 vs. 36.6 months; P ¼ .21) and overall survival (75.1 vs. 65.4 months; P ¼ .021). Patients with a relapse had an isotype that differed from a CIS, confirming the benign nature of this phenomenon. CIS was also associated with lower CD8 T-cell percentages and a greater CD4/CD8 ratio (2.8 vs. 0.2; P ¼ .001) compared with patients who did not demonstrate a CIS, suggestive of more profound T-cell immune reconstitution in this group. Conclusion: Taken together, our data have demonstrated that a CIS is a benign phenomenon and correlates with a reduced disease burden and enriched immune repertoire beyond the B-cell compartment. Clinical Lymphoma, Myeloma & Leukemia, Vol. 19, No. 5, e213-20 ª 2019 Elsevier Inc. All rights reserved. Keywords: Autologous stem cell transplantation, Clonal isotype switch, Immune reconstitution, Multiple myeloma, Myeloma microenvironment
1
Department of Medicine, New York University School of Medicine, New York, NY Department of Pathology, University Hospitals Cleveland Medical Center, Cleveland, OH 3 Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, OH 4 Division of Hematology and Oncology, The Vontz Center for Molecular Studies, University of Cincinnati College of Medicine, Cincinnati, OH 5 Adult Hematologic Malignancies and Stem Cell Transplant Program, Seidman Cancer Center, University Hospitals Cleveland Medical Center, Cleveland, OH 6 Myeloma Service, Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY 7 Division of Hematology and Oncology, University Hospital Cleveland Medical Center, Cleveland, OH 2
2152-2650/$ - see frontmatter ª 2019 Elsevier Inc. All rights reserved. https://doi.org/10.1016/j.clml.2018.12.022
8
Case Western Reserve University School of Medicine, Cleveland, OH Department of Stem Cell Transplantation, University Medical Center HamburgEppendorf, Hamburg, Germany 9
Submitted: Aug 3, 2018; Revised: Nov 19, 2018; Accepted: Dec 28, 2018; Epub: Jan 3, 2019 Address for correspondence: Ehsan Malek, MD, Adult Hematologic Malignancies and Stem Cell Transplant Program, Seidman Cancer Center, University Hospitals Cleveland Medical Center, Cleveland, OH 44106 E-mail contact: [email protected]
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Clonal Isotype Switch After ASCT Introduction Multiple myeloma (MM) is a malignancy of terminally differentiated plasma cells characterized by a decrease in both the number and the functionality of immune effector cells (ie, natural killer [NK] and T helper cells), an increase in immune suppressor cells (ie, regulatory T-cells and myeloid-derived suppressor cells), and osteoclast activation, leading to tumor progression, infection, and osteolytic bony lesions.1-3 During the past 2 decades, the advent of novel agents has led to significantly increased survival for patients with MM. However, the disease has remained nearly uniformly fatal through chemoresistance, the persistence of minimal residual disease (MRD), and frequent overt disease relapse. Modifications in the cellular and molecular interactions in the bone marrow (BM) during the course of the disease provide a permissive tumor microenvironment that favors the emergence of chemoresistant populations, leading to MRD persistence.4 High-dose melphalan with autologous hematopoietic stem cell transplantation (ASCT) is the standard of care for patients with MM and is aimed at achieving long-term remission.5 Robust post-ASCT immune system reconstitution has been shown to correlate with lower MRD and improved clinical outcomes.6 Although currently, no specific validated immune signature has been determined to predict superior survival among patients with MM, studies have reported on a clonal isotype switch (CIS) after ASCT.7 The phenomenon has been documented in the current data under a number of different names, such as oligoclonal immunoglobulins, abnormal protein bands, atypical serum immunofixation patterns, and secondary monoclonal gammopathy of undetermined significance (MGUS). To the best of our knowledge, previous studies have not investigated detailed BM immune cell subsets before and after ASCT. We hypothesized that a comprehensive immune profiling of BM T-cell, NK-cell, B-cell, and dendritic cell subpopulations would identify immune cell phenotypes associated with prolonged progression-free survival (PFS) and overall survival (OS) after ASCT. Examining immune profiling after ASCT would facilitate our understanding of immune reconstitution after ASCT and its correlation with the outcomes. Strategies to enhance selected populations after ASCT could improve the outcomes for patients with MM. MM cells originate after a light and heavy chain switch in B lymphocytes, as suggested by the lack of mutation in the variable regions of light or heavy chains.8,9 Therefore, the malignant plasma cell clones almost always produce a single unique monoclonal heavy and/or light chain with a constant isotype. This is represented on serum protein electrophoresis (SPEP) as an M-spike, which is considered the major disease marker monitored throughout the initial therapy and remission. A CIS results in a new serum Mprotein seen on SPEP, distinct from the M-protein pattern present at diagnosis, and can be associated with a good prognosis.7,10,11 A CIS often occurs after ASCT or even after induction chemotherapy.11 Some reports have suggested that MM relapse will present with the original monoclonal protein documented at diagnosis and not with the CIS protein.12 This would suggest that the transient CIS bands might represent part of the post-transplant immune reconstitution process and might even possess some anti-MM activity.13 To date, no studies have characterized the immune profile associated with CIS bands in patients undergoing ASCT. In the
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Table 1 Patient and Disease Characteristicsa Without CIS (n [ 138)
With CIS (n [ 39)
P Value
60.4
61.5
.43
Male
74 (54)
21 (54)
Female
64 (46)
18 (46)
White
107 (78)
29 (74)
Black
28 (20)
9 (23)
Other
3 (2)
1 (3)
Characteristic Median age, y Gender, n (%)
.39
Race, n (%)
.26
Monoclonal type, n (%)
.001
IgAk
10 (7)
8 (20)
IgAL
7 (5)
5 (13)
IgGk
70 (51)
10 (26)
IgGL
32 (23)
6 (15)
IgD
1 (1)
1 (3)
18 (13)
9 (23)
12.1
11.4
.19
Light chain Median hemoglobin, g/dL Median albumin, g/dL
3.9
3.9
.42
Median ALC before ASCT, 109/L
1.09
1.23
.37
Median AMC before ASCT, 109/L
0.5
0.55
.13
Median ALC/AMC ratio before ASCT
2.2
2.1
.34
Median plasma cell burden at diagnosis, %
40
50
.19
Biclonal disease at diagnosis, n (%)
3 (2)
1 (3)
.47
15 (17)
6 (23)
.11
I
43 (31)
8 (21)
II
54 (39)
15 (38)
III
41 (30)
16 (41)
I
16 (17)
5 (19)
II
61 (67)
16 (61)
III
13 (14)
3 (11)
High-risk cytogenetics,b n (%) ISS stage, n (%)
.21
R-ISS stage,b n (%)
.41
Response before ASCT, n (%)
.02
VGPR
32 (23)
20 (52)
PR
79 (57)
6 (17)
CR
27 (20)
12 (31)
Abbreviations: ASCT ¼ autologous stem cell transplantation; ALC ¼ absolute lymphocyte count; AMC ¼ absolute monocyte count; CIS ¼ clonal isotype switch; CR ¼ complete response; PR ¼ partial response; R-ISS ¼ revised International Staging System; VGPR ¼ very good partial response. a CIS occurrence correlated with monoclonal protein type and deeper therapeutic response. b Cytogenetic data were available for 90 patients without CIS and 26 patient with CIS.
present study, we postulated that the occurrence of a CIS would correlate with a more robust reconstitution of the immune system after ASCT.
Rebecca Ye et al Patients and Methods Patients and Therapies All patients with MM who had undergone ASCT from 2007 to 2016 at the University Hospital Cleveland Medical Center were studied. Those patients who had undergone allogeneic stem cell transplantation were excluded. The institutional review board approved the present retrospective study. Data collection was performed by reviewing the electronic medical records and cellular therapy database. Patients who did not have flow cytometry data available were excluded. The parameters included the patient characteristics, induction therapy, mobilization regimen, and graft characteristics, including CD34þ cell dose, myeloid and erythroid progenitor cells (multipotential colony forming unit [CFU] or CFU-GM [granulocyte, monocyte] and erythroid burst-forming unit). Myeloma staging was reported using the International Staging System. Our institutional ASCT protocol includes peripheral blood CD34þ cell collection after mobilization with filgrastim (Amgen, Thousand Oaks, CA) with or without plerixafor (Sanofi Genzyme, Cambridge, MA). Filgrastim was continued throughout apheresis. Erythroid burst-forming unit and CFU-GM assays were performed using a fresh collected graft for all patients. The transplant day was counted as day 0, and all the days were calculated from this reference point. The conditioning regimen included amifostine 740 mg/m2 intravenously on days 2 and 1 and melphalan 200 mg/m2 or 140 mg/m2 intravenously (ie, according to patient age and organ dysfunction) on day 1, as previously described.14 Daily filgrastim was administered from day þ1 and was continued until neutrophil engraftment, defined by 3 consecutive days of an absolute neutrophil count > 0.5/mL.
CIS Identification Monoclonal proteins and quantitative immunoglobulins were assessed at approximately day þ100 and with a frequency of 2 to 8
times annually thereafter. The presence of a CIS was screened by reviewing all SPEP, immunofixation (IFE), and urine protein electrophoresis, and serum light chain assays before and after ASCT. A CIS was defined as the appearance of a new serum monoclonal spike on SPEP and IFE that differed from the original heavy or light chain detected at diagnosis. Serum IFE was performed using the Sebia Hyrdrasys agarose gel electrophoresis apparatus (Sebia, Norcross, GA) according to the manufacturer’s recommendations. Centrifuged plasma was run on alkaline agarose gels. Antibodies against IgG, IgA, IgM, and kappa and lambda light chains were applied.
Immune Cell Subset Recovery Ethylenediaminetetraacetic acideanticoagulated fresh BM aspirate (1 mL) from each subject was immunophenotyped, using a 6color direct immunofluorescence technique previously described in detail.15 We used antibodies from Becton Dickinson (Franklin Lakes, NJ) and the BD FACSDiva platform. The percentage of NK cells (CD56þCD3 and CD16þCD3), T-cells (CD3þCD56), B-cells (CD19þ), and T-cell (CD4þCD8þ) subpopulations were measured using flow cytometry in pre- and post-ASCT fresh BM aspirates.
Statistical Analysis The start date for all time-dependent variables was the day of transplant (day 0). OS was calculated from day 0 to the date of death and censored at the date of the last follow-up visit. The Kaplan-Meier method was used to assess OS and PFS. Two-tailed log-rank tests were used to compare the OS and PFS curves. Factors with P < .2 on univariate analysis were included in the multivariate analysis, which was performed using the Cox regression hazard model. All statistical tests were 2-sided, and P < .05 was considered to indicate statistical significance. All analyses were performed using SAS, version 9.2 (SAS Institute, Cary, NC).
Figure 1 Induction Regimen Distribution Between Patients With and Without Clonal Isotype Switch (CIS) After Autologous Stem Cell Transplantation. The Larger Proportion of Patients With CIS Had Received a Revlimid-containing Induction Regimen Before Transplantation (30.4% vs. 11.2%; P [ .001)
Abbreviations: CyborD ¼ cyclophosphamide, bortezomib, dexamethasone; K ¼ Kyprolis (carfilzomib); R ¼ Revlimid (lenalidomide); V ¼ Velcade (bortezomib).
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Clonal Isotype Switch After ASCT Table 2 Transplantation Characteristics and Posttransplantation Immunoprofilinga Characteristic Median pre-ASCT weight, kg Median pre-ASCT BMI, kg/m2 Median weight change, kg Median weight change, % Median pre-ASCT BM CD4/CD8 ratio Median pre-ASCT CD8 cell, % Median pre-ASCT B cell, % Median pre-ASCT NK cell, % Median total nucleated cells, 108/kg Median infused CD34þ cell dose, 106/kg Median myeloid precursors (CFU-GM), 104 Median erythroid precursors (BFU-E), 104 Mucositis, n (%) Median mucositis grade Median mucositis length, d Median length of hospital admission, d Median post-ASCT ALC, 109/L Median post-ASCT AMC, 109/L Median post-ASCT ALC/AMC ratio Median post-ASCT CD4/CD8 ratio Median post-ASCT CD8 cells, % Median post-ASCT B cells, % Median post-ASCT NK cells, % Maintenance therapy, n (%)
Without CIS (n [ 138)
With CIS (n [ 39)
P Value
84.6 30.5 3.7 4 1.3
81 28.4 4.65 6 3
.131 .320 .313 .278 .066
29 24 19 9.92
28 28 22 9.82
.430 .233 .369 .485
4.95
5.16
.380
105.2
109
.343
189.4
182.6
.288
63 (46) 1.70 5 15
14 (35) 1.94 7.5 16
.256 .323 .279 .315
1.03 0.46 2.53 0.2 43 16 17 85 (61)
1.345 0.445 2.84 2.8 28 32 21 23 (58)
.167 .345 .143 .001 .015 .056 .291 .437
Abbreviations: ALC ¼ absolute lymphocyte count; AMC ¼ absolute monocyte count; ASCT ¼ autologous stem cell transplantation; BM ¼ bone marrow; BMI ¼ body mass index; CIS ¼ clonal isotype switch; CFU-GM ¼ colony-forming unit granulocyte, macrophage; EBFU ¼ erythroid burst-forming unit; NK ¼ natural killer. a CIS occurrence correlated with more robust lymphocyte subset recovery after transplantation.
Results Patient Characteristics and Frequency of CIS in PostASCT Setting The analysis included 177 patients with MM who had undergone ASCT during the study period and had flow cytometry data available. The median age was 60.7 years (range, 36-74 years) at the time of ASCT. The median follow-up period for the surviving patients was 38 months (range, 2-101 months). A CIS was detected in 39 patients (22%). The patient and disease characteristics are listed in Table 1. Seventeen patients (46%) had only 1 new monoclonal protein. However, 10 (25%) had developed 4 monoclonal bands throughout the post-ASCT follow-up period. The newly detected monoclonal proteins were < 0.5 g/dL in 34 patients (87%). The median interval to the occurrence of a CIS was 7.1 months (range, 1.9-32 months). The nature of the CIS band heavy and light chains was mostly IgG (68%) and kappa light chain (65%), respectively.
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Age, gender, MM risk stratification, International Staging System stage, and BM plasma cell burden were not associated with the occurrence of a post-ASCT CIS. The distribution of induction regimen between the 2 cohorts is illustrated in Figure 1. None of the patients had received anti-MM monoclonal antibodies. A significantly greater incidence of CIS was found in patients who had received lenalidomide before ASCT (30.4% vs. 11.2%; P ¼ .001), patients with IgA myeloma (21% vs. 7%; P ¼ .01), and patients who had achieved a very good partial response before ASCT (52% vs. 23%; P ¼ .023). Also, a CIS occurred more frequently in patients without suppressed uninvolved immunoglobulin (92% vs. 8%; P ¼ .001).
ASCT Characteristics Others have reported statistically significant differences in the frequency of CIS occurrence after ASCT compared with patients who had not undergone ASCT (eg, Mayo Clinic data: 22.7% of 458 patients who had undergone ASCT compared with 1.6% of 1484 patients who had not undergone ASCT; P < .001).16 These findings raise the question of whether the degree of immunotoxicity associated with ASCT might lead to different humoral rebounds after ASCT, causing CIS. Therefore, we assessed different ASCT parameters that affect or reflect post-ASCT immunosuppression, such as melphalan dose, graft composition, oral mucositis, weight loss, and length of neutropenic fever period associated with ASCT (Table 2). Graft characteristics, including the collected total nucleated cells, infused CD34þ cells, and myeloid and erythroid precursors, were comparable in the CIS cohort compared with the patients with no CIS (Table 2). Similarly, a melphalan dose of 200 mg/m2 compared with 140 mg/m2 as the conditioning regimen (26% vs. 21%; P ¼ .321), ASCT-associated mucositis length or severity, and the length of the hospital admission were associated with a similar frequency of CIS (Table 2).
Immune Subset Recovery in Patients With CIS Our group, and others, have shown the significance of peripheral lymphocytes and monocytes as prognostic factors in MM.17,18 We found no difference between the pre- or post-ASCT peripheral absolute lymphocyte or monocyte count in patients who had experienced post-ASCT CIS and those who had not (Table 2). Similarly, no significant difference was found in the CD4, CD8, NK, or B-cell percentages or in the CD4/CD8 ratio in the pre-ASCT BM samples from patients with or without CIS. The appearance of post-ASCT CIS was associated with lower CD8 T-cell percentages (43% in patients without CIS vs. 28% in patients with CIS; P ¼ .015) and a higher CD4/CD8 ratio (0.2 vs. 2.8; P ¼ .001; both with differences that were statistically significantly), suggestive of faster T-cell compartment reconstitution in this patient group (Figure 2). Patients with a CIS showed a trend toward faster B-cell recovery compared with patients without CIS. However, this difference did not reach statistical significance (16% vs. 32%; P ¼ .056). No statistically significant difference was found between the NK cell percentages in the post-ASCT BM in either group (17% vs. 21%; P ¼ .291).
OS and PFS The presence of post-ASCT CIS correlated significantly with improved PFS (52.2 vs. 36.6 months; P ¼ .21) and OS (75.1 vs.
Rebecca Ye et al Figure 2 Immune Subset Recovery in T-, B-, and Natural Killer (NK) Cell Compartment Before and After Autologous Stem Cell Transplantation (ASCT) in Patients With and Without Clonal Isotype Switch (CIS). (A, B) Although No Significant Difference Was Found Between the CD8D Cell Percentage or CD4D/CD8D Ratio Before ASCT, Patients With CIS Developed Higher CD4D/CD8D Ratios With Lower CD8D Percentages After ASCT. *Statistically Significant Difference Between the 2 Groups. (C, D) NK-cell and B-lymphocyte Subset Recovery After ASCT in Patients With and Without CIS After ASCT
65.4 months; P ¼ .021; Figure 3A,B). Age, cytogenetics, response category, presence of CIS, and low lactate dehydrogenase were shown to influence PFS on univariate analysis. Cytogenetics, lactate dehydrogenase, and CIS presence were also significantly associated with the MM response on multivariate analysis. The median interval between the pre- and post-ASCT BM assessment was 92 days (range, 79-123 days; Table 1). All patients who had experienced a relapse had an isotype different from that of the CIS, highlighting the benign nature of this phenomenon.
Discussion MM has generally been characterized by the abnormal production of a single heavy and/or light chain isotype by the malignant clone.19 In the present study, we defined the monoclonal protein signature in the peripheral blood for patients who had undergone ASCT and showed that the CIS pattern is associated with the emergence of certain BM immune phenotypes in the post-ASCT setting.20 This BM immune repertoire is suggestive of a robust immune reconstitution beyond the B-cell compartment. Our data
are consistent with the benign nature of the CIS and that the CIS is not a source of relapse. A previously reported retrospective analysis of the same phenomena from our institute by Manson et al,20 who had studied patients who had received ASCT from 2000 to 2009, demonstrated a significant OS benefit for patients with a delayed occurrence of a new monoclonal protein compared with the rest of the patients. The lower percentage of CIS in their cohort might have resulted from the lower usage of novel agents in 2000 to 2009 compared with that in 2007 to 2015.11 Multiple groups have correlated the occurrence of a CIS with superior survival among patients with MM who have undergone ASCT or allogeneic stem cell transplantation.21,22 However, the origin of the new monoclonal bands remains largely unknown. This phenomenon has been described under different names, such as secondary MGUS,16,20 oligoclonal or abnormal protein bands,23,24 atypical serum immunofixation patterns,25 immunoglobulin isotype switch, and immunoglobulin class switch,23 reflecting the ambiguity around its pathogenesis. Gene sequencing of heavy chain variable region in 7 patients with post-ASCT CIS did not show a clonal
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Clonal Isotype Switch After ASCT Figure 3 Outcomes of Patients With or Without a Clonal Isotype Switch (CIS). Progression-free Survival (PFS) and Overall Survival (OS) Comparison Between 2 Groups Illustrating Superior Outcomes in Patients With CIS
Abbreviations: CI ¼ confidence interval; HR ¼ hazard ratio.
relationship to the original malignant clone isotype,26 highlighting nonmalignant B cells as the likely origin of CIS. A related question is which conditions will lead to the expansion of the nonmalignant plasma cell clone secreting a new monoclonal protein in the post-ASCT setting. Multiple studies have demonstrated that greater rates of MGUS correlate with immune system hyperactivation in patients with autoimmune disease,27,28 infection, or inflammatory or allergic disorders29 and after myeloablative regimens in recipients of an allograft when humoral reconstitution has occurred in a clonally deregulated pattern.30 Our finding that CIS is associated with an expanded T- and, possibly, B-cell compartment supports the hypothesis that CIS is the result of robust immune reconstitution after ASCT. Furthermore, our results have confirmed those
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from other reports of increased polyclonal B-cell content in post-ASCT BM as a predictor of better survival.31 The nonmalignant nature of the CIS has been supported by our results and those from others.32 In contrast, the findings from all those with disease relapse differed from the CIS heavy and/or light chain in nature and CIS had decreased or disappeared with the development of relapse. This finding suggests a clonal competition between the malignant clone and the nonmalignant clone responsible for the CIS. In addition, the European Blood and Marrow Transplantation criteria have indicated that the presence of a different monoclonal protein in the absence of the original MM protein should be considered a complete response.32 We preferred to use the term “CIS” instead of “secondary MGUS” to differentiate
Rebecca Ye et al this monoclonal gammopathy from the more commonly known MGUS as a premalignant condition.33 Our data should be interpreted in the context of new knowledge regarding clonal tides in those with sequential relapses associated with different clones emerging over time, as a result of Darwinian mutational branching, and other reports of a monoclonal protein signature in the peripheral blood of patients with MM.34 The interplay between the clone responsible for CIS and emerging new and hybrid malignant clones remains to be defined. Campbell et al19 showed that smaller clones, represented as an accessory monoclonal protein band (ie, 10-20 times smaller than a major monoclonal band) will be detected at the diagnosis of biclonal MM. The present study and similar studies that relied on SPE/IFE to detect the CIS had an inherent limitation in their methods. These studies were mostly able to identify the clones that resulted in a different heavy or light chain than that of the original MM clone. Therefore, new clones that secrete the same M protein as the original MM clone have been missing.16 Most CISs occurred with clones that made IgG and kappa, just as in our cohort and as reported by others.20,22,35 In contrast, IgG kappa has been the most frequent nature of the malignant clone responsible for the bulk of initial disease for newly diagnosed MM. Therefore, one could hypothesize that a slow and small increase in the IgG kappa M-protein after ASCT in an asymptomatic patient with initial IgG Kappa disease might represent CIS, which will correlate with long remission and survival, rather than early post-ASCT relapse, which would denote clinically high-risk disease.36 Future studies are required to measure the disease burden using next generation sequencing, which could overcome the limitations of SPEP. Furthermore, these findings should be considered using response-adaptive strategies with MRD testing to gauge anti-MM therapy aimed at curative measure.34,37 Overall, the CIS is not an uncommon phenomenon after ASCT in patients with MM. Thus, practitioners should be aware that the occurrence of CIS should not be mistaken for biochemical relapse. Therapeutic monoclonal antibodies such as daratumumab and elotuzuma have been emerging as new elements in anti-MM therapy, and they can be detected as a low IgG kappa monoclonal protein in SPEP 6 months after therapy.38 Therefore, when interpreting newly identifed monoclonal proteins in patients with treated MM, CIS should not be confused with low-level monoclonal bands caused by these antibodies. The new assays to mitigate the interference from these antibodies will be able to differentiate CIS from pharmacologic monoclonal proteins.39 In the present study, we found that CIS occurrence correlated with a low disease volume and enriched immune repertoire. This setting could be optimal for modulation by upcoming immunebased therapies, such as checkpoint inhibitors, for further suppression or potential elimination of MRD in the post-ASCT setting. Therefore, CIS might serve as an immune biomarker that will help to define a subset of patients most likely to benefit from immunotherapy.17 Also, efforts to better understand how specific immune subpopulations trigger and regulate the immune response to tumors might improve the long-term control of MM. Further research to define whether CIS has an underlying antitumor effect is also needed.
Clinical Practice Points CIS has been associated with better immune reconstitution after
transplantation in patients with MM. The occurrence of a CIS correlated with superior PFS after
transplantation in patients with MM.
Acknowledgments E.M. was supported in part by an institutional K12 Paul Calabresi Career Development Award. O.L. was supported in part by the Memorial Sloan Kettering Core Grant (P30 CA008748) from the National Cancer Institute (Rockville, MD).
Disclosure J.D. has received research funding from Takeda/Millennium, Onyx/Amgen, Bristol-Myers Squibb, and Sanofi Oncology. E.M. has served on the advisory board or been a consultant or speaker for Takeda, Celgene, Sanofi, Amgen, and Janssen. The remaining authors have stated that they have no conflicts of interest.
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Clonal Isotype Switch After ASCT 19. Campbell JP, Heaney JLJ, Pandya S, et al. Response comparison of multiple myeloma and monoclonal gammopathy of undetermined significance to the same anti-myeloma therapy: a retrospective cohort study. Lancet Haematol 2017; 4: e584-94. 20. Manson GV, Campagnaro E, Balog A, et al. Secondary MGUS after autologous hematopoietic progenitor cell transplantation in plasma cell myeloma: a matter of undetermined significance. Bone Marrow Transplant 2012; 47:1212-6. 21. Schmitz MF, Otten HG, Franssen LE, et al. Secondary monoclonal gammopathy of undetermined significance after allogeneic stem cell transplantation in multiple myeloma. Haematologica 2014; 99:1846-53. 22. Zou D, An G, Zhu G, et al. Secondary monoclonal gammopathy of undetermined significance is frequently associated with high response rate and superior survival in patients with plasma cell dyscrasias. Biol Blood Marrow Transplant 2014; 20:319-25. 23. Zent C, Wilson CS, Tricot G, et al. Oligoclonal protein bands and Ig isotype switching in multiple myeloma treated with high-dose therapy and hematopoietic cell transplantation. Blood 1998; 91:3518-23. 24. de Larrea CF, Cibeira MT, Elena M, et al. Abnormal serum free light chain ratio in patients with multiple myeloma in complete remission has strong association with the presence of oligoclonal bands: implications for stringent complete remission definition. Blood 2009; 114:4954-6. 25. Mark T, Jayabalan D, Coleman M, et al. Atypical serum immunofixation patterns frequently emerge in immunomodulatory therapy and are associated with a high degree of response in multiple myeloma. Br J Haematol 2008; 143:654-60. 26. Guikema JE, Vellenga E, Veeneman JM, et al. Multiple myeloma related cells in patients undergoing autologous peripheral blood stem cell transplantation. Br J Haematol 1999; 104:748-54. 27. Krumbholz M, Derfuss T, Hohlfeld R, Meini E. B cells and antibodies in multiple sclerosis pathogenesis and therapy. Nat Rev Neurol 2012; 8:613-23. 28. Brito-Zerón P, Retamozo S, Gandía M. Monoclonal gammopathy related to Sjögren syndrome: a key marker of disease prognosis and outcomes. J Autoimmun 2012; 39:43-8. 29. Brown LM, Gridley G, Check D, Landgren O. Risk of multiple myeloma and monoclonal gammopathy of undetermined significance among white and black
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30. 31.
32.
33. 34. 35. 36. 37. 38.
39.
male United States veterans with prior autoimmune, infectious, inflammatory, and allergic disorders. Blood 2008; 111:3388-94. Pageaux G-P, Bonnardet A, Picot MC, et al. Prevalence of monoclonal immunoglobulins after liver transplantation: relationship with posttransplant lymphoproliferative disorders. Transplantation 1998; 65:397-400. Byrne E, Naresh KN, Giles C, Rahemtulla A. Excess bone marrow B-cells in patients with multiple myeloma achieving complete remission following autologous stem cell transplantation is a biomarker for improved survival. Br J Haematol 2011; 155:509-11. Bladé J, Samson D, Reece D, et al. Criteria for evaluating disease response and progression in patients with multiple myeloma treated by high-dose therapy and haemopoietic stem cell transplantation. Br J Haematol 1998; 102:1115-23. Landgren O, Kyle RA, Pfeiffer RM, et al. Monoclonal gammopathy of undetermined significance (MGUS) consistently precedes multiple myeloma: a prospective study. Blood 2009; 113:5412-7. Landgren O, Giralt S. MRD-driven treatment paradigm for newly diagnosed transplant eligible multiple myeloma patients. Bone Marrow Transplant 2016; 51:913-4. Fujisawa M, Seike K, Fukumoto K, et al. Oligoclonal bands in patients with multiple myeloma: its emergence per se could not be translated to improved survival. Cancer Sci 2014; 105:1442-6. Caillon H, Dejoie T, Le Loupp A-G, et al. Difficulties in immunofixation analysis: a concordance study on the IFM 2007-02 trial. Blood Cancer J 2013; 3:e154. Mailankody S, Korde N, Lesokhin AM, et al. Minimal residual disease in multiple myeloma: bringing the bench to the bedside. Nat Rev Clin Oncol 2015; 12:286-95. Beck R, Tang F, Math S, Schmotzer C. Investigating the pattern of detection of therapeutic monoclonal antibodies elotuzumab and daratumumab by routine serum protein electrophoresis and immunofixation in patients with myeloma. Am J Clin Pathol 2018; 149(suppl 1):S7-8. McCudden C, Axel A, Slaets D, et al. Assessing clinical response in multiple myeloma (MM) patients treated with monoclonal antibodies (mAbs): validation of a daratumumab IFE reflex assay (DIRA) to distinguish malignant M-protein from therapeutic antibody. J Clin Oncol 2015; 33(suppl):8590.
Immune Signatures Associated With Clonal Isotype Switch After Autologous Stem Cell Transplantation for Multiple Myeloma Rebecca Ye,1 Sirisha Kundrapu,2 Stanton L. Gerson,3 James J. Driscoll,4 Rose Beck,2 Naveed Ali,5 Ola Landgren,6 Willem VanHeeckeren,7 George Luo,8 Nicolaus Kroger,9 Paolo Caimi,5,8 Marcos De Lima,5,8 Ehsan Malek5,8 Abstract A retrospective analysis of 177 patients with multiple myeloma undergoing autologous stem cell transplant found that 22% developed new and small concentrations of monoclonal protein after transplant that differed from that originally identified at diagnosis. This phenomenon had a benign nature and correlated with improved survival and more robust bone marrow immune reconstitution beyond the B-cell compartment. Background: High-dose chemotherapy and autologous stem cell transplantation (ASCT) are integral components of the overall treatment for patients with multiple myeloma (MM) aged 65 years. The emergence of oligoclonal immunoglobulin bands (ie, immunoglobulins differing from those originally identified at diagnosis [termed clonal isotype switch (CIS)]) has been reported in patients with MM after high-dose chemotherapy followed by autologous stem cell transplantation. However, the clinical relevance and the correlation with immune reconstitution remains unclear. Patients and Methods: Patients with MM who had undergone ASCT from 2007 to 2016 were included in the present study. The percentage of natural killer cells, B-cells, and T-cells was measured using flow cytometry in pre- and postASCT bone marrow samples. CIS was defined as the appearance of a new serum monoclonal spike on serum protein electrophoresis and immunofixation that differed from original heavy or light chain detected at diagnosis. Results: A retrospective analysis of 177 patients with MM who had undergone ASCT detected CIS in 39 (22%). CIS after ASCT correlated with improved progression-free survival (52.2 vs. 36.6 months; P ¼ .21) and overall survival (75.1 vs. 65.4 months; P ¼ .021). Patients with a relapse had an isotype that differed from a CIS, confirming the benign nature of this phenomenon. CIS was also associated with lower CD8 T-cell percentages and a greater CD4/CD8 ratio (2.8 vs. 0.2; P ¼ .001) compared with patients who did not demonstrate a CIS, suggestive of more profound T-cell immune reconstitution in this group. Conclusion: Taken together, our data have demonstrated that a CIS is a benign phenomenon and correlates with a reduced disease burden and enriched immune repertoire beyond the B-cell compartment. Clinical Lymphoma, Myeloma & Leukemia, Vol. 19, No. 5, e213-20 ª 2019 Elsevier Inc. All rights reserved. Keywords: Autologous stem cell transplantation, Clonal isotype switch, Immune reconstitution, Multiple myeloma, Myeloma microenvironment
1
Department of Medicine, New York University School of Medicine, New York, NY Department of Pathology, University Hospitals Cleveland Medical Center, Cleveland, OH 3 Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, OH 4 Division of Hematology and Oncology, The Vontz Center for Molecular Studies, University of Cincinnati College of Medicine, Cincinnati, OH 5 Adult Hematologic Malignancies and Stem Cell Transplant Program, Seidman Cancer Center, University Hospitals Cleveland Medical Center, Cleveland, OH 6 Myeloma Service, Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY 7 Division of Hematology and Oncology, University Hospital Cleveland Medical Center, Cleveland, OH 2
2152-2650/$ - see frontmatter ª 2019 Elsevier Inc. All rights reserved. https://doi.org/10.1016/j.clml.2018.12.022
8
Case Western Reserve University School of Medicine, Cleveland, OH Department of Stem Cell Transplantation, University Medical Center HamburgEppendorf, Hamburg, Germany 9
Submitted: Aug 3, 2018; Revised: Nov 19, 2018; Accepted: Dec 28, 2018; Epub: Jan 3, 2019 Address for correspondence: Ehsan Malek, MD, Adult Hematologic Malignancies and Stem Cell Transplant Program, Seidman Cancer Center, University Hospitals Cleveland Medical Center, Cleveland, OH 44106 E-mail contact: [email protected]
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Clonal Isotype Switch After ASCT Introduction Multiple myeloma (MM) is a malignancy of terminally differentiated plasma cells characterized by a decrease in both the number and the functionality of immune effector cells (ie, natural killer [NK] and T helper cells), an increase in immune suppressor cells (ie, regulatory T-cells and myeloid-derived suppressor cells), and osteoclast activation, leading to tumor progression, infection, and osteolytic bony lesions.1-3 During the past 2 decades, the advent of novel agents has led to significantly increased survival for patients with MM. However, the disease has remained nearly uniformly fatal through chemoresistance, the persistence of minimal residual disease (MRD), and frequent overt disease relapse. Modifications in the cellular and molecular interactions in the bone marrow (BM) during the course of the disease provide a permissive tumor microenvironment that favors the emergence of chemoresistant populations, leading to MRD persistence.4 High-dose melphalan with autologous hematopoietic stem cell transplantation (ASCT) is the standard of care for patients with MM and is aimed at achieving long-term remission.5 Robust post-ASCT immune system reconstitution has been shown to correlate with lower MRD and improved clinical outcomes.6 Although currently, no specific validated immune signature has been determined to predict superior survival among patients with MM, studies have reported on a clonal isotype switch (CIS) after ASCT.7 The phenomenon has been documented in the current data under a number of different names, such as oligoclonal immunoglobulins, abnormal protein bands, atypical serum immunofixation patterns, and secondary monoclonal gammopathy of undetermined significance (MGUS). To the best of our knowledge, previous studies have not investigated detailed BM immune cell subsets before and after ASCT. We hypothesized that a comprehensive immune profiling of BM T-cell, NK-cell, B-cell, and dendritic cell subpopulations would identify immune cell phenotypes associated with prolonged progression-free survival (PFS) and overall survival (OS) after ASCT. Examining immune profiling after ASCT would facilitate our understanding of immune reconstitution after ASCT and its correlation with the outcomes. Strategies to enhance selected populations after ASCT could improve the outcomes for patients with MM. MM cells originate after a light and heavy chain switch in B lymphocytes, as suggested by the lack of mutation in the variable regions of light or heavy chains.8,9 Therefore, the malignant plasma cell clones almost always produce a single unique monoclonal heavy and/or light chain with a constant isotype. This is represented on serum protein electrophoresis (SPEP) as an M-spike, which is considered the major disease marker monitored throughout the initial therapy and remission. A CIS results in a new serum Mprotein seen on SPEP, distinct from the M-protein pattern present at diagnosis, and can be associated with a good prognosis.7,10,11 A CIS often occurs after ASCT or even after induction chemotherapy.11 Some reports have suggested that MM relapse will present with the original monoclonal protein documented at diagnosis and not with the CIS protein.12 This would suggest that the transient CIS bands might represent part of the post-transplant immune reconstitution process and might even possess some anti-MM activity.13 To date, no studies have characterized the immune profile associated with CIS bands in patients undergoing ASCT. In the
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Table 1 Patient and Disease Characteristicsa Without CIS (n [ 138)
With CIS (n [ 39)
P Value
60.4
61.5
.43
Male
74 (54)
21 (54)
Female
64 (46)
18 (46)
White
107 (78)
29 (74)
Black
28 (20)
9 (23)
Other
3 (2)
1 (3)
Characteristic Median age, y Gender, n (%)
.39
Race, n (%)
.26
Monoclonal type, n (%)
.001
IgAk
10 (7)
8 (20)
IgAL
7 (5)
5 (13)
IgGk
70 (51)
10 (26)
IgGL
32 (23)
6 (15)
IgD
1 (1)
1 (3)
18 (13)
9 (23)
12.1
11.4
.19
Light chain Median hemoglobin, g/dL Median albumin, g/dL
3.9
3.9
.42
Median ALC before ASCT, 109/L
1.09
1.23
.37
Median AMC before ASCT, 109/L
0.5
0.55
.13
Median ALC/AMC ratio before ASCT
2.2
2.1
.34
Median plasma cell burden at diagnosis, %
40
50
.19
Biclonal disease at diagnosis, n (%)
3 (2)
1 (3)
.47
15 (17)
6 (23)
.11
I
43 (31)
8 (21)
II
54 (39)
15 (38)
III
41 (30)
16 (41)
I
16 (17)
5 (19)
II
61 (67)
16 (61)
III
13 (14)
3 (11)
High-risk cytogenetics,b n (%) ISS stage, n (%)
.21
R-ISS stage,b n (%)
.41
Response before ASCT, n (%)
.02
VGPR
32 (23)
20 (52)
PR
79 (57)
6 (17)
CR
27 (20)
12 (31)
Abbreviations: ASCT ¼ autologous stem cell transplantation; ALC ¼ absolute lymphocyte count; AMC ¼ absolute monocyte count; CIS ¼ clonal isotype switch; CR ¼ complete response; PR ¼ partial response; R-ISS ¼ revised International Staging System; VGPR ¼ very good partial response. a CIS occurrence correlated with monoclonal protein type and deeper therapeutic response. b Cytogenetic data were available for 90 patients without CIS and 26 patient with CIS.
present study, we postulated that the occurrence of a CIS would correlate with a more robust reconstitution of the immune system after ASCT.
Rebecca Ye et al Patients and Methods Patients and Therapies All patients with MM who had undergone ASCT from 2007 to 2016 at the University Hospital Cleveland Medical Center were studied. Those patients who had undergone allogeneic stem cell transplantation were excluded. The institutional review board approved the present retrospective study. Data collection was performed by reviewing the electronic medical records and cellular therapy database. Patients who did not have flow cytometry data available were excluded. The parameters included the patient characteristics, induction therapy, mobilization regimen, and graft characteristics, including CD34þ cell dose, myeloid and erythroid progenitor cells (multipotential colony forming unit [CFU] or CFU-GM [granulocyte, monocyte] and erythroid burst-forming unit). Myeloma staging was reported using the International Staging System. Our institutional ASCT protocol includes peripheral blood CD34þ cell collection after mobilization with filgrastim (Amgen, Thousand Oaks, CA) with or without plerixafor (Sanofi Genzyme, Cambridge, MA). Filgrastim was continued throughout apheresis. Erythroid burst-forming unit and CFU-GM assays were performed using a fresh collected graft for all patients. The transplant day was counted as day 0, and all the days were calculated from this reference point. The conditioning regimen included amifostine 740 mg/m2 intravenously on days 2 and 1 and melphalan 200 mg/m2 or 140 mg/m2 intravenously (ie, according to patient age and organ dysfunction) on day 1, as previously described.14 Daily filgrastim was administered from day þ1 and was continued until neutrophil engraftment, defined by 3 consecutive days of an absolute neutrophil count > 0.5/mL.
CIS Identification Monoclonal proteins and quantitative immunoglobulins were assessed at approximately day þ100 and with a frequency of 2 to 8
times annually thereafter. The presence of a CIS was screened by reviewing all SPEP, immunofixation (IFE), and urine protein electrophoresis, and serum light chain assays before and after ASCT. A CIS was defined as the appearance of a new serum monoclonal spike on SPEP and IFE that differed from the original heavy or light chain detected at diagnosis. Serum IFE was performed using the Sebia Hyrdrasys agarose gel electrophoresis apparatus (Sebia, Norcross, GA) according to the manufacturer’s recommendations. Centrifuged plasma was run on alkaline agarose gels. Antibodies against IgG, IgA, IgM, and kappa and lambda light chains were applied.
Immune Cell Subset Recovery Ethylenediaminetetraacetic acideanticoagulated fresh BM aspirate (1 mL) from each subject was immunophenotyped, using a 6color direct immunofluorescence technique previously described in detail.15 We used antibodies from Becton Dickinson (Franklin Lakes, NJ) and the BD FACSDiva platform. The percentage of NK cells (CD56þCD3 and CD16þCD3), T-cells (CD3þCD56), B-cells (CD19þ), and T-cell (CD4þCD8þ) subpopulations were measured using flow cytometry in pre- and post-ASCT fresh BM aspirates.
Statistical Analysis The start date for all time-dependent variables was the day of transplant (day 0). OS was calculated from day 0 to the date of death and censored at the date of the last follow-up visit. The Kaplan-Meier method was used to assess OS and PFS. Two-tailed log-rank tests were used to compare the OS and PFS curves. Factors with P < .2 on univariate analysis were included in the multivariate analysis, which was performed using the Cox regression hazard model. All statistical tests were 2-sided, and P < .05 was considered to indicate statistical significance. All analyses were performed using SAS, version 9.2 (SAS Institute, Cary, NC).
Figure 1 Induction Regimen Distribution Between Patients With and Without Clonal Isotype Switch (CIS) After Autologous Stem Cell Transplantation. The Larger Proportion of Patients With CIS Had Received a Revlimid-containing Induction Regimen Before Transplantation (30.4% vs. 11.2%; P [ .001)
Abbreviations: CyborD ¼ cyclophosphamide, bortezomib, dexamethasone; K ¼ Kyprolis (carfilzomib); R ¼ Revlimid (lenalidomide); V ¼ Velcade (bortezomib).
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Clonal Isotype Switch After ASCT Table 2 Transplantation Characteristics and Posttransplantation Immunoprofilinga Characteristic Median pre-ASCT weight, kg Median pre-ASCT BMI, kg/m2 Median weight change, kg Median weight change, % Median pre-ASCT BM CD4/CD8 ratio Median pre-ASCT CD8 cell, % Median pre-ASCT B cell, % Median pre-ASCT NK cell, % Median total nucleated cells, 108/kg Median infused CD34þ cell dose, 106/kg Median myeloid precursors (CFU-GM), 104 Median erythroid precursors (BFU-E), 104 Mucositis, n (%) Median mucositis grade Median mucositis length, d Median length of hospital admission, d Median post-ASCT ALC, 109/L Median post-ASCT AMC, 109/L Median post-ASCT ALC/AMC ratio Median post-ASCT CD4/CD8 ratio Median post-ASCT CD8 cells, % Median post-ASCT B cells, % Median post-ASCT NK cells, % Maintenance therapy, n (%)
Without CIS (n [ 138)
With CIS (n [ 39)
P Value
84.6 30.5 3.7 4 1.3
81 28.4 4.65 6 3
.131 .320 .313 .278 .066
29 24 19 9.92
28 28 22 9.82
.430 .233 .369 .485
4.95
5.16
.380
105.2
109
.343
189.4
182.6
.288
63 (46) 1.70 5 15
14 (35) 1.94 7.5 16
.256 .323 .279 .315
1.03 0.46 2.53 0.2 43 16 17 85 (61)
1.345 0.445 2.84 2.8 28 32 21 23 (58)
.167 .345 .143 .001 .015 .056 .291 .437
Abbreviations: ALC ¼ absolute lymphocyte count; AMC ¼ absolute monocyte count; ASCT ¼ autologous stem cell transplantation; BM ¼ bone marrow; BMI ¼ body mass index; CIS ¼ clonal isotype switch; CFU-GM ¼ colony-forming unit granulocyte, macrophage; EBFU ¼ erythroid burst-forming unit; NK ¼ natural killer. a CIS occurrence correlated with more robust lymphocyte subset recovery after transplantation.
Results Patient Characteristics and Frequency of CIS in PostASCT Setting The analysis included 177 patients with MM who had undergone ASCT during the study period and had flow cytometry data available. The median age was 60.7 years (range, 36-74 years) at the time of ASCT. The median follow-up period for the surviving patients was 38 months (range, 2-101 months). A CIS was detected in 39 patients (22%). The patient and disease characteristics are listed in Table 1. Seventeen patients (46%) had only 1 new monoclonal protein. However, 10 (25%) had developed 4 monoclonal bands throughout the post-ASCT follow-up period. The newly detected monoclonal proteins were < 0.5 g/dL in 34 patients (87%). The median interval to the occurrence of a CIS was 7.1 months (range, 1.9-32 months). The nature of the CIS band heavy and light chains was mostly IgG (68%) and kappa light chain (65%), respectively.
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Age, gender, MM risk stratification, International Staging System stage, and BM plasma cell burden were not associated with the occurrence of a post-ASCT CIS. The distribution of induction regimen between the 2 cohorts is illustrated in Figure 1. None of the patients had received anti-MM monoclonal antibodies. A significantly greater incidence of CIS was found in patients who had received lenalidomide before ASCT (30.4% vs. 11.2%; P ¼ .001), patients with IgA myeloma (21% vs. 7%; P ¼ .01), and patients who had achieved a very good partial response before ASCT (52% vs. 23%; P ¼ .023). Also, a CIS occurred more frequently in patients without suppressed uninvolved immunoglobulin (92% vs. 8%; P ¼ .001).
ASCT Characteristics Others have reported statistically significant differences in the frequency of CIS occurrence after ASCT compared with patients who had not undergone ASCT (eg, Mayo Clinic data: 22.7% of 458 patients who had undergone ASCT compared with 1.6% of 1484 patients who had not undergone ASCT; P < .001).16 These findings raise the question of whether the degree of immunotoxicity associated with ASCT might lead to different humoral rebounds after ASCT, causing CIS. Therefore, we assessed different ASCT parameters that affect or reflect post-ASCT immunosuppression, such as melphalan dose, graft composition, oral mucositis, weight loss, and length of neutropenic fever period associated with ASCT (Table 2). Graft characteristics, including the collected total nucleated cells, infused CD34þ cells, and myeloid and erythroid precursors, were comparable in the CIS cohort compared with the patients with no CIS (Table 2). Similarly, a melphalan dose of 200 mg/m2 compared with 140 mg/m2 as the conditioning regimen (26% vs. 21%; P ¼ .321), ASCT-associated mucositis length or severity, and the length of the hospital admission were associated with a similar frequency of CIS (Table 2).
Immune Subset Recovery in Patients With CIS Our group, and others, have shown the significance of peripheral lymphocytes and monocytes as prognostic factors in MM.17,18 We found no difference between the pre- or post-ASCT peripheral absolute lymphocyte or monocyte count in patients who had experienced post-ASCT CIS and those who had not (Table 2). Similarly, no significant difference was found in the CD4, CD8, NK, or B-cell percentages or in the CD4/CD8 ratio in the pre-ASCT BM samples from patients with or without CIS. The appearance of post-ASCT CIS was associated with lower CD8 T-cell percentages (43% in patients without CIS vs. 28% in patients with CIS; P ¼ .015) and a higher CD4/CD8 ratio (0.2 vs. 2.8; P ¼ .001; both with differences that were statistically significantly), suggestive of faster T-cell compartment reconstitution in this patient group (Figure 2). Patients with a CIS showed a trend toward faster B-cell recovery compared with patients without CIS. However, this difference did not reach statistical significance (16% vs. 32%; P ¼ .056). No statistically significant difference was found between the NK cell percentages in the post-ASCT BM in either group (17% vs. 21%; P ¼ .291).
OS and PFS The presence of post-ASCT CIS correlated significantly with improved PFS (52.2 vs. 36.6 months; P ¼ .21) and OS (75.1 vs.
Rebecca Ye et al Figure 2 Immune Subset Recovery in T-, B-, and Natural Killer (NK) Cell Compartment Before and After Autologous Stem Cell Transplantation (ASCT) in Patients With and Without Clonal Isotype Switch (CIS). (A, B) Although No Significant Difference Was Found Between the CD8D Cell Percentage or CD4D/CD8D Ratio Before ASCT, Patients With CIS Developed Higher CD4D/CD8D Ratios With Lower CD8D Percentages After ASCT. *Statistically Significant Difference Between the 2 Groups. (C, D) NK-cell and B-lymphocyte Subset Recovery After ASCT in Patients With and Without CIS After ASCT
65.4 months; P ¼ .021; Figure 3A,B). Age, cytogenetics, response category, presence of CIS, and low lactate dehydrogenase were shown to influence PFS on univariate analysis. Cytogenetics, lactate dehydrogenase, and CIS presence were also significantly associated with the MM response on multivariate analysis. The median interval between the pre- and post-ASCT BM assessment was 92 days (range, 79-123 days; Table 1). All patients who had experienced a relapse had an isotype different from that of the CIS, highlighting the benign nature of this phenomenon.
Discussion MM has generally been characterized by the abnormal production of a single heavy and/or light chain isotype by the malignant clone.19 In the present study, we defined the monoclonal protein signature in the peripheral blood for patients who had undergone ASCT and showed that the CIS pattern is associated with the emergence of certain BM immune phenotypes in the post-ASCT setting.20 This BM immune repertoire is suggestive of a robust immune reconstitution beyond the B-cell compartment. Our data
are consistent with the benign nature of the CIS and that the CIS is not a source of relapse. A previously reported retrospective analysis of the same phenomena from our institute by Manson et al,20 who had studied patients who had received ASCT from 2000 to 2009, demonstrated a significant OS benefit for patients with a delayed occurrence of a new monoclonal protein compared with the rest of the patients. The lower percentage of CIS in their cohort might have resulted from the lower usage of novel agents in 2000 to 2009 compared with that in 2007 to 2015.11 Multiple groups have correlated the occurrence of a CIS with superior survival among patients with MM who have undergone ASCT or allogeneic stem cell transplantation.21,22 However, the origin of the new monoclonal bands remains largely unknown. This phenomenon has been described under different names, such as secondary MGUS,16,20 oligoclonal or abnormal protein bands,23,24 atypical serum immunofixation patterns,25 immunoglobulin isotype switch, and immunoglobulin class switch,23 reflecting the ambiguity around its pathogenesis. Gene sequencing of heavy chain variable region in 7 patients with post-ASCT CIS did not show a clonal
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Clonal Isotype Switch After ASCT Figure 3 Outcomes of Patients With or Without a Clonal Isotype Switch (CIS). Progression-free Survival (PFS) and Overall Survival (OS) Comparison Between 2 Groups Illustrating Superior Outcomes in Patients With CIS
Abbreviations: CI ¼ confidence interval; HR ¼ hazard ratio.
relationship to the original malignant clone isotype,26 highlighting nonmalignant B cells as the likely origin of CIS. A related question is which conditions will lead to the expansion of the nonmalignant plasma cell clone secreting a new monoclonal protein in the post-ASCT setting. Multiple studies have demonstrated that greater rates of MGUS correlate with immune system hyperactivation in patients with autoimmune disease,27,28 infection, or inflammatory or allergic disorders29 and after myeloablative regimens in recipients of an allograft when humoral reconstitution has occurred in a clonally deregulated pattern.30 Our finding that CIS is associated with an expanded T- and, possibly, B-cell compartment supports the hypothesis that CIS is the result of robust immune reconstitution after ASCT. Furthermore, our results have confirmed those
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from other reports of increased polyclonal B-cell content in post-ASCT BM as a predictor of better survival.31 The nonmalignant nature of the CIS has been supported by our results and those from others.32 In contrast, the findings from all those with disease relapse differed from the CIS heavy and/or light chain in nature and CIS had decreased or disappeared with the development of relapse. This finding suggests a clonal competition between the malignant clone and the nonmalignant clone responsible for the CIS. In addition, the European Blood and Marrow Transplantation criteria have indicated that the presence of a different monoclonal protein in the absence of the original MM protein should be considered a complete response.32 We preferred to use the term “CIS” instead of “secondary MGUS” to differentiate
Rebecca Ye et al this monoclonal gammopathy from the more commonly known MGUS as a premalignant condition.33 Our data should be interpreted in the context of new knowledge regarding clonal tides in those with sequential relapses associated with different clones emerging over time, as a result of Darwinian mutational branching, and other reports of a monoclonal protein signature in the peripheral blood of patients with MM.34 The interplay between the clone responsible for CIS and emerging new and hybrid malignant clones remains to be defined. Campbell et al19 showed that smaller clones, represented as an accessory monoclonal protein band (ie, 10-20 times smaller than a major monoclonal band) will be detected at the diagnosis of biclonal MM. The present study and similar studies that relied on SPE/IFE to detect the CIS had an inherent limitation in their methods. These studies were mostly able to identify the clones that resulted in a different heavy or light chain than that of the original MM clone. Therefore, new clones that secrete the same M protein as the original MM clone have been missing.16 Most CISs occurred with clones that made IgG and kappa, just as in our cohort and as reported by others.20,22,35 In contrast, IgG kappa has been the most frequent nature of the malignant clone responsible for the bulk of initial disease for newly diagnosed MM. Therefore, one could hypothesize that a slow and small increase in the IgG kappa M-protein after ASCT in an asymptomatic patient with initial IgG Kappa disease might represent CIS, which will correlate with long remission and survival, rather than early post-ASCT relapse, which would denote clinically high-risk disease.36 Future studies are required to measure the disease burden using next generation sequencing, which could overcome the limitations of SPEP. Furthermore, these findings should be considered using response-adaptive strategies with MRD testing to gauge anti-MM therapy aimed at curative measure.34,37 Overall, the CIS is not an uncommon phenomenon after ASCT in patients with MM. Thus, practitioners should be aware that the occurrence of CIS should not be mistaken for biochemical relapse. Therapeutic monoclonal antibodies such as daratumumab and elotuzuma have been emerging as new elements in anti-MM therapy, and they can be detected as a low IgG kappa monoclonal protein in SPEP 6 months after therapy.38 Therefore, when interpreting newly identifed monoclonal proteins in patients with treated MM, CIS should not be confused with low-level monoclonal bands caused by these antibodies. The new assays to mitigate the interference from these antibodies will be able to differentiate CIS from pharmacologic monoclonal proteins.39 In the present study, we found that CIS occurrence correlated with a low disease volume and enriched immune repertoire. This setting could be optimal for modulation by upcoming immunebased therapies, such as checkpoint inhibitors, for further suppression or potential elimination of MRD in the post-ASCT setting. Therefore, CIS might serve as an immune biomarker that will help to define a subset of patients most likely to benefit from immunotherapy.17 Also, efforts to better understand how specific immune subpopulations trigger and regulate the immune response to tumors might improve the long-term control of MM. Further research to define whether CIS has an underlying antitumor effect is also needed.
Clinical Practice Points CIS has been associated with better immune reconstitution after
transplantation in patients with MM. The occurrence of a CIS correlated with superior PFS after
transplantation in patients with MM.
Acknowledgments E.M. was supported in part by an institutional K12 Paul Calabresi Career Development Award. O.L. was supported in part by the Memorial Sloan Kettering Core Grant (P30 CA008748) from the National Cancer Institute (Rockville, MD).
Disclosure J.D. has received research funding from Takeda/Millennium, Onyx/Amgen, Bristol-Myers Squibb, and Sanofi Oncology. E.M. has served on the advisory board or been a consultant or speaker for Takeda, Celgene, Sanofi, Amgen, and Janssen. The remaining authors have stated that they have no conflicts of interest.
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