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Prepublished online as a Blood First Edition Paper on December 27, 2002; DOI 10.1182/blood-2002-10-3162.
CLINICAL OBSERVATIONS, INTERVENTIONS, AND THERAPEUTIC TRIALS
From the Department of Biomedical Sciences and Human
Oncology, Section of Internal Medicine and Clinical Oncology,
University of Bari Medical School, Bari, Italy, and Division of
Experimental Oncology I, Centro di Riferimento Oncologico, Aviano
(Pordenone), Italy.
A controlled study has been carried out to assess the efficacy of
rituximab, a chimeric antibody that binds to the B-cell surface antigen
CD20, in 20 patients with mixed cryoglobulinemia (MC) and hepatitis C
virus (HCV)-positive chronic active liver disease, resistant to
interferon Mixed cryoglobulinemia (MC) is a chronic immune
complex-mediated disease.1 Cryoglobulins are cold
precipitable immunoglobulins that are accounted for by 2 or more
immunoglobulin isotypes, with (type II) or without (type III) a
monoclonal component.2 MC is frequently associated
with the development of vascular, renal, and neurologic
lesions.3 Oligoclonal or monoclonal B-cell expansions are
significant molecular features of bone marrow4 and
liver.5 It is estimated that almost 10% of patients with
MC progress to frank B-cell non-Hodgkin lymphoma
(NHL).6
The association between hepatitis C virus (HCV) and MC has been
rendered evident since the recognition of serologic markers of HCV
infection.7-9 In Italy, more than 80% of MC patients are infected with HCV.10 The primary role of HCV in the
mechanism of cryoprecipitation is mainly suggested by its selective
concentration in cryoglobulins.11,12
Based on the close correlation between HCV infection and MC, treatment
with interferon This has led us to search for innovative therapeutic strategies with
the aim of reducing or depleting the B-cell clonal expansion that
sustains production of IgM rheumatoid factor (RF) molecules. One such
approach involves the use of monoclonal antibodies directed to CD20
antigen, a transmembrane protein expressed on pre-B lymphocytes and
mature lymphocytes.19 Rituximab, a humanized murine
monoclonal antibody of this kind, is highly effective for in vivo
B-cell depletion.20 Peripheral blood B lymphocytes become
undetectable after a single infusion and recover 6 to 9 months after
discontinuation of treatment.21 Rituximab was originally
approved for the treatment of low-grade B-cell non-Hodgkin lymphoma
(NHL).22 It has since become a promising therapeutic
approach for diffuse large B-cell lymphomas, mantle cell
lymphoma,23 hairy cell leukemia,20 and
chronic lymphocytic leukemia.24 It has also been used in several other hematologic disorders including pure red cell aplasia and
hemolytic anemia,25 primary cold agglutinin
disease,26 posttransplantation
B-lymphoproliferative disorders,27 Waldenström macroglobulinemia,28 and idiopathic thrombocytopenic
purpura.29
The present report summarizes the results of a 4-dose rituximab course
in HCV+ MC patients who failed to respond to IFN- Patients
Exclusion criteria were: (1) pregnancy; (2) concomitant serious illness
likely to preclude completion of the study; (3) hepatic failure
characterized by history of ascites, bleeding, esophageal varices,
hepatic encephalopathy, bilirubin level more than 3 mg/dL, serum
albumin level less than 3 g/dL, and prothrombin time 3 seconds longer
than that of controls; (4) leukocyte count less than
3 × 109/L or hemoglobin level less than 10 g/dL; and (5)
positive test for antibodies to HIV.
The study was approved by the Institutional Ethical Committee and
written informed consent was obtained from all patients. Rituximab was diluted in normal saline up to a maximum
concentration of 1 mg/mL. Patients were scheduled to receive 4 intravenous infusions of 375 mg/m2 once a week over a
period of 1 month. Premedication with acetaminophen and diphenhydramine
as well as sufficient hydration was recommended. Infusion was started
at 50 mg/h during the first hour and increased to a final 400 mg/h if
well tolerated. The use of concomitant corticosteroids was limited to
the treatment of severe allergic reactions.
Baseline evaluation included disease history and stage, actual signs
and symptoms, and previous medication. Physical examination, laboratory
values, adverse events, and formulation of stereotyped questions30 were recorded weekly throughout the treatment.
Hematologic and physical examinations were then performed monthly up to
12 months. Laboratory evaluation at baseline included complete
hemogram, serum chemistry profile, direct and indirect Coombs test, and serologic tests for hepatitis B virus (HBV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), and HIV. Immunology included C3 and C4
fractions of complement, RF activity, and immunoglobulin concentration. Neurologic assessment included strength evaluation,31
electromyography, motor and sensory nerve conduction velocity, and
short-latency somatosensory-evoked potentials.
Mononuclear cells were isolated from fresh blood by Lymphoprep
separation (Nycomed Pharma, Oslo, Norway), washed and stained with
fluorochrome-conjugated antibodies against CD19, CD20, CD3, CD4, CD8
(fluorescein isothiocyanate [FITC]-labeled; Becton-Dickinson, San
Diego, CA), CD5 and HLA-DR (phycoerythrin [PE]-conjugated; Immunotech, Marseille, France). Samples were analyzed in single or
double immunofluorescence on a FACScan instrument (Becton-Dickinson) before and after each rituximab infusion and then monthly up to 12 months.
The primary efficacy was the objective response rate, that is, the
proportion of patients achieving a complete response (CR) at any time.
CR was based on previously established criteria17 and
defined as reduction of cryocrit to less than 75% of the initial value
associated with at least 2 of the following: disappearance of purpura,
arthralgia, and weakness and improvement of neuropathy. Relapse from CR
was defined as an increase of the cryocrit to more than 50% of the
decrement value, along with deterioration of one or more clinical parameters.
An adverse event was defined as any adverse change from the patient's
baseline condition, whether related to the treatment or not. Each event
after rituximab infusion was graded according to the National Cancer
Institute Common Toxicity Criteria grading system.32
Grades 3 and 4 events plus grade 2 infections were recorded in detail,
grade 1 and 2 events were not.
The results were expressed as means ± SD. Differences between
dichotomous variables were analyzed by Fisher exact test. Continuous variables were compared by Student t test. Multivariate
logistic regression was used to analyze the response to treatment. Rate of remission was calculated with life-table analysis.
Cryoprecipitate preparation was carried out as specified
elsewhere.13 Cryoprecipitates diluted in 0.5 M NaCl were
fractionated by high-resolution gel electrophoresis to type
cryoglobulins. Individual monoclonal bands were identified by
immunofixation after electrophoresis using a cellulose acetate strip
impregnated with antibodies specific for heavy and light chains (Dako,
Copenaghen, Denmark).
HCV RNA in unfractionated sera, supernatants, and corresponding
cryoprecipitates was determined using a reverse
transcription-polymerase chain reaction (RT-PCR) test (Amplicor HCV;
Roche Diagnostic Systems, Branchburg, NY) and quantified with the
Quantiplex HCV RNA kit (Chiron, Emeryville, CA) based on the
quantitative branched DNA signal amplification
assay.33
HCV genotypes were determined with the line probe assay (Inno-Lipa HCV;
Innogenetics, Zwijnaarde, Belgium). This identifies HCV types and
subtypes. Measures to prevent contamination were used at all times.
Anti-HCV antibodies directed to structural and nonstructural proteins
were detected in unfractionated serum samples, cryoprecipitates and
corresponding supernatants using a microparticle enzyme immunoassay (AxSYM HCV 3.0; Abbott, Rome, Italy). Results were calculated as
optical density (OD) sample/OD cutoff and were considered positive when
the ratio was greater than 1.1.
Molecular analysis of B-cell clonality
For each sample 0.5 to 1 µg DNA was processed in the PCR analysis for B-cell clonal expansion using a seminested protocol of amplification, according to well-established procedures.35 The upstream primer was complementary to the third framework V region (Fr3, 5'-ACACGGC[C/T][G/C]TGTATTACTGT-3') of the IgH gene; the downstream primer was directed to an outer conserved region of the IgH J (5'-TGAGGAGACGGTGACC-3') in the first round of amplification and to an inner conserved sequence of the same J region in the second round (5'-GTGACCAGGGTNCCTTGGCCCCAG-3'). DNA was amplified for 30 cycles in the first round and for 20 cycles in the second. Each cycle consisted of 94°C for 45 seconds, 50°C for 45 seconds, and 72°C for 30 seconds, with an additional extension interval of 5 minutes at 72°C after the last cycle. The sensitivity of the technique was checked by the amplification of serial dilutions of DNA from clonal B cells admixed with DNA from polyclonal B cells. The detection threshold was estimated to be about 0.5% to 1%.36 For each experiment, a control was included using primers for the
PCR products were analyzed by electrophoresis in 5% agarose gel (Seaken LE, FMC Bioproduct, Rockland, ME) in TBE buffer (100 mM Tris [tris(hydroxymethyl)aminomethane], 100 mM boric acid, 2 mM EDTA [ethylenediaminetetraacetic acid]), stained with ethidium bromide, and optically evaluated by UV transillumination. Clonal expansion was indicated by one or more dominant bands within the predicted size and a fully polyclonal pattern by a smear with no specific dominant bands. A monoclonal B-cell expansion was defined as 1 or 2 (if both alleles were rearranged) discrete narrow band(s) within the predicted size. Distinction of biclonal from monoclonal biallelic rearrangements was based on the results of subsequent sequence analyses, in that a nonfunctional rearrangement of 1 of the 2 alleles (ie, one dominant band) was detected in the case of a monoclonal disorder, whereas both dominant bands were representative of a functional IgH rearrangement in biclonal disorders. An oligoclonal picture was represented by 3 or more discrete bands. Cloning and sequencing analyses PCR products amplified from lymphocyte DNA were run on agarose gel in Tris-borate-EDTA buffer and stained with ethidium bromide, according to standard procedures. Individual bands were excised from the gel, cloned, and sequenced. DNA was purified using the QIAEX II Gel extraction kit (Qiagen, Hilden, Germany), ligated into a pGem-T cloning vector (Promega, Madison, WI), and transfected into Escherichia coli DH5 -competent cells. Transfected cells were plated onto
Luria-Bertani (LB)-ampicillin agar plates containing X-gal and
isopropylthiogalactose. Uncolored colonies were selected at random and
cultured. Plasmid DNA was purified with the Wizard Plus Minipreps DNA
Purification System (Promega).
Sequence reactions were carried out on an ABI Prism 310 Genetic Analyzer (Perkin Elmer, Foster City, CA). All sequences were confirmed by sequencing in both directions with primers T7 and SP6. Ten or more clones were sequenced for each dominant band. Sequences were considered to be related if they shared the same CDRH3 regions, but had differences in the number of point mutations. Where a sequence showed similarity to more than one DH region, all possible DH regions in the CDRH3 were identified and those that could be assigned without overlap, and with the smallest number of nucleotides in between, were used.
The main initial laboratory features and histologic findings of
the 4 men and 16 women enrolled in this study are summarized in Table
1. Cryoglobulins were classified as mixed
type II in 13 (65%) and type III in 7 (35%).
CR was achieved by 16 (80%) patients. Mean length of disease, HCV
genotype 1b distribution, and mean age prior to treatment were not
different between responders and nonresponders (12.4 ± 7.7 versus 10.3 ± 6.9 years; 56.2% versus 50%; 56.8 ± 13.6 versus 59.4 ± 12.8 years). Average cryocrit values were also similar (8.6% ± 6.4%
versus 10.3% ± 7.9%). The mean cryocrit value regressed to 2.1% ± 2.6% in the responders. In 3 patients (18.7%), cryoglobulins stably
disappeared throughout the observation period. As depicted in Figure
1, the probability of CR 2 months after
therapy was 62.5% (10 of 16). The overall response rate occurred
within the next 5 months.
Frequency of organ-related signs and symptoms before and after
rituximab treatment is reported in Table
2. Purpura was present in 14 of the 16 responders and rapidly disappeared in 12 (87.5%). Rapid disappearance
was also noted for cutaneous urticarial manifestations, whereas
features and extension of livedo reticularis remained unchanged.
Interestingly, long-lasting leg ulcers showed a consistent reduction
followed by dramatic healing in 3 of 5 patients. Peripheral neuropathy,
occurring in 12 patients (75%) at the time of enrollment, improved in
6 (50%). Positive changes in motor conduction were recorded,
associated with improvement of motor and sensory nerve conduction. No
changes of muscular strength were noticed. Weakness and arthralgia were
no longer recorded in 7 and 8 patients, respectively. A dramatic
improvement of muscular pain and morning stiffness was reported. On the
contrary, no significant improvement of renal functional parameters was
detected in the only patient with kidney involvement. Finally, both
frequency and size of hepatomegaly and mean serum transaminase levels
were substantially unchanged.
Rituximab dramatically reduced the number of peripheral blood cells
expressing CD20 antigen. The immunophenotype of lymphocyte subsets was
always determined during and up to 12 months after treatment. The
number of CD20+ and CD19+ cells in addition to
that of CD20+/CD5+ and
CD3 The dynamics of B-cell depletion and recovery was similar in the responders and nonresponders. The percentage of CD20+ cells dropped in both responders (13.2% ± 8.8%) and nonresponders (11.2% ± 3.3%) to less than 1% after the first infusion. Recovery of B-cell count began from 6 months in responders (4% ± 2.2%) and nonresponders (5.6% ± 1.4%). CRs were correlated with patients' characteristics to identify factors
associated with a favorable response. Immunologic parameters were
serially determined in sera, cryoprecipitates, and supernatants (Table
3). No significant differences between
responders and nonresponders were found for serum baseline levels of
IgM, RF activity, IgG, and C3 and C4 fractions.
After fractionation, as compared with the supernatants, almost half of IgM molecules were retained in the cryoprecipitates in the responders, whereas they were significantly more concentrated in the nonresponders (P = .03). In step with the decrement of cryocrit levels, responders showed a significant decline of mean serum IgM levels (P = .002), whereas no significant changes were noted in the cryoprecipitates, suggesting that reduction of IgM did not modify the amount of cryoprecipitating molecules. In contrast, in nonresponders mean serum IgM levels did not change and significant increment in the cryoprecipitates was documented (P = .02). As compared with the supernatants, RF activity was significantly more concentrated in the cryoprecipitates in both responders (P = .04) and nonresponders (P = .003). A decrement was documented in the sera (P = .15) and in cryoprecipitates (P = .02) in the responders after rituximab treatment. In the nonresponders, a significant decrement of RF activity in the sera was accompanied by an increment in the cryoprecipitates (P = .006). Mean serum IgG levels, on the other hand, were significantly enriched in the supernatants of the responders (P = .002) and nonresponders (P =.046). After rituximab therapy mean decrement of about 40% was noted in the cryoprecipitates (P = .20) in responders, whereas no changes occurred in the nonresponders. Complement-related factors were also considered. C3 and C4 protein mean levels were significantly less represented in the cryoprecipitates as compared with supernatants in both responders (P = .0002 and P = .0006, respectively) and nonresponders (P = .008 and P = .001, respectively). No significant variations were noticed at the different time points. Influence of rituximab on circulating viral load and anti-HCV titers is
reported in Table 4. Mean HCV RNA
baseline levels were similar in the 2 groups. After fractionation, HCV
RNA was significantly enriched in the cold-precipitated fractions as
compared with corresponding supernatants in the responders
(P = .02) and the nonresponders (P = .01).
Surprisingly, despite substantial reduction of cryocrit levels, HCV RNA
increased to a significant extent in the responders. At the end of
follow-up, a sustained increment of HCV RNA was achieved in
unfractionated sera (P = .02), supernatants
(P = .04), and cryoprecipitates (P = .016). These results were similarly validated when HCV RNA was normalized to
cryoprecipitating proteins. After rituximab treatment, HCV RNA
concentration in the cryoprecipitates significantly differed from
baseline values (808 311 ± 712 865 IU/mg versus 569 277 ± 495 250
IU/mg, P = .02) whereas no significant changes were noted in the nonresponders.
Anti-HCV reactivity titered semiquantitatively was initially similar in
the 2 groups. After cold precipitation, the titers were less
represented in the cryoglobulins compared with the supernatants in the
responders (P = .019) and the nonresponders
(P = .011). In the responders, they were significantly
reduced in the supernatants by an average of 13%
(P = .04) and more than 70% in the cryoprecipitates (P = .02). Regression analysis pointed to a highly
significant correlation between decrement of anti-HCV titers in the
cryoprecipitates and reduction of cryocrit levels (r = 0.81,
P < .005). In sharp contrast, anti-HCV titers were
virtually unchanged in the nonresponders. PCR directed to
IgH VDJ genes on DNA extracted from peripheral blood
cells was performed to analyze B-cell clonal restriction and determine
the persistence or disappearance of originally expanded clones in 11 responders and the 4 nonresponders before and after each infusion
(Figure 2). Gel agarose analysis showed
that 2 and 6 responders had monoclonal (1 or 2 bands) and oligoclonal
(
VH-CDR3 nucleotide sequences of dominant clonotypes in
responders and nonresponders are reported in Table
5. Sequence analyses were carried out in 5 responders and 3 nonresponders. In responders, initially expanded B-cell clones declined and disappeared, and new and
distinct clonotypes appeared at varying time points, even in patients
with a monoclonal pattern.
Persistence of B-cell clonal expansions was demonstrated in the nonresponders. Their single B-cell clones exhibited identical rearrangement at different time points and were highly resistant and refractory to rituximab. Amino acid length and composition determine the ability of CDRH3 to bind antigen with higher affinity. In this data set (data not reported) we noticed CDRH3 ranging from 13 to 25 amino acids in responder patients and from 14 to 17 in nonresponders. There was no difference in gene usage between the 2 groups. D2, D3, D4, D5, and D6 families were represented in the responders, and D3, D4, and D6 in the nonresponders. No selective use of JH fragment was noted (JH2-JH6 gene segments were used), although JH4 was the most represented. Twelve months after discontinuation of rituximab, the response
maintenance rate was 75% (Figure 3).
Four patients (25%) had a relapse, with the estimated time to relapse
exceeding 7 months. Clinically, relapse was characterized by
reappearance of purpura and recurrence of weakness and arthralgias.
Simultaneous increment of cryocrit and IgM RF molecules were also
demonstrated. Interestingly, anti-HCV antibody titers reached
pretreatment levels in the cryoprecipitate.
The present results indicate that rituximab reduces cryoglobulin
levels and significantly improves cutaneous vasculitis and several
clinical and immunologic parameters in patients who had been resistant
to IFN- The rituximab schedule was borrowed from earlier trials in which this antibody was used to treat relapsed or refractory low-grade or follicular B-cell NHLs.21 This study, the largest to date, confirms and extends recent limited observations in MC.37-39 We considered rituximab to be effective treatment of MC because 80% of patients were responders. More than 60% showed rapid improvement of general signs. Nonresponders, on the contrary, did not improve at all and this suggests that an early clinical response may predict its efficacy. Modality of the response was mainly related to changes in the cryoprecipitates modifying the biologic characteristics of cold-precipitable material. We monitored components of cryoglobulins before and after rituximab and demonstrated that their precipitation was strictly related to a number of factors, namely, decrease of Ig concentration, decline of RF activity, and reduction of anti-HCV titers. The negative relation between cryocrit levels and circulating HCV load raises some important issues about the mechanisms of cryoglobulin precipitation. Cold-dependent insolubility of HCV particles and proteins seems the result of IgM RF activity, which acts as incomplete cryoglobulin, in that it precipitates at low temperature in the presence of IgG with specific anti-HCV reactivity. The dynamics of cold-dependent insolubility demonstrates that the addition of an irrelevant IgG to an IgM RF/HCV proteins mixture was unable to precipitate the protein. This implies that a potentially functional RF repertoire may be positively selected by IgG anti-HCV antibodies. At variance from the immediate and dramatic decline in viral load after rituximab in EBV-infected patients,40 our data are consistent with a sustaining effect of anti-CD20 antibody on HCV virogenesis. In responders, in step with reduction of cryocrit levels, increment of viral load has been documented. Sequential measurements of HCV RNA levels, quantified with branched DNA signal amplification assay, showed that they progressively increased after rituximab. Twelve months later, they increased by approximately twice the baselines. Importantly, these results deeply differed from those obtained in nonresponders, in which HCV RNA levels were unchanged over sequential time points. Because B lymphocytes are thought to harbor HCV,41 it could be argued that the HCV RNA increment reflects virus shedding through rituximab-induced B-cell cytotoxicity. However, this is not a plausible explanation because, despite the B-cell depletion in the nonresponders, no significant variations of HCV RNA levels were noted. Thus, it can be envisaged that the impact of rituximab on immunoglobulin levels, that is, IgG anti-HCV antibodies, is an effective mechanism capable of modulating and interfering with HCV replication. Decline of anti-HCV titers leads HCV to avoid immune pressure and favors its replication. This points to the presence of immunoglobulin with neutralizing properties in immune response in HCV chronic carriers.42 However, it must be emphasized that selective increments of HCV RNA did not match significant changes of hepatocytolytic activity or deterioration of liver function, as defined by sequential measurements of serum transaminases and the clinical findings. This further supports the concept that HCV is not cytopathic for the cells it infects and that immune response plays the major role in the hepatocyte damage.43 PCR has been increasingly used for the rapid detection of gene rearrangements during B-lymphocyte development.44 It can be assumed that the consensus primers currently used amplify the CDRH3 of most B cells, but the products of a small clonal population may be obscured by the smear of polyclonal B cells in the sample. Indeed, we found B-cell polyclonality in a minority of patients at baseline, indicating that B-cell response is mainly not polyclonal and that selected antigens are preferentially recognized. Sequential IgH VDJ analyses demonstrated disappearance/deletion of individual B-cell clones in patients who responded to rituximab. On the contrary, persistence and stability of initially expanded clonotypes were found in nonresponders, suggesting that their B-cell clones are less sensitive to rituximab. Clonal susceptibility to rituximab may depend on other mechanisms and additional regulatory factors expressed on B cells, for example, levels of CD20 antigen45 and expression of complement inhibitors,46 failure of Fc receptor-dependent mechanism,47 or disturbed signals leading to apoptosis.48 Disappearance/deletion of B-cell clones and appearance of different clones in responders demonstrate that selected antigens are preferentially recognized as part of a limited host response to a pathogen capable of undergoing spontaneous long-term mutations that heavily contribute to the viral burden.49 Clonal disappearance/deletion never occurred in nonresponders and their HCV RNA concentration was stable at different time points. Sequencing of VH genes illustrated the role of antigens prior to or during rituximab treatment. Analysis of CDRH3 segments pointed to positive selection in responders, who displayed ongoing somatic mutations. The absence of selection in nonresponders suggests that clonal persistence may be antigen-independent. Alternatively, it can be envisaged that affinity of antibody toward the antigens is high enough to avoid a positive ongoing mutation, or else superantigens that bind immunoglobulin receptor via framework regions provide negative selection pressure on these regions without positive selection pressure on CDRH3 regions.50 Compared with the general population, the occurrence of B-cell NHL
remains higher in MC patients, even in those who clear HCV-RNA
following IFN- Enhanced viremia occurring after this treatment, however, is a
potentially harmful outcome, although no significant variations of
serum transaminases or deterioration of liver disease were noted in our
series. To reduce HCV replication, association of rituximab with
effective antiviral agents, including IFN-
Submitted October 18, 2002; accepted December 15, 2002.
Prepublished online as Blood First Edition Paper, December 27, 2002; DOI 10.1182/blood-2002-10-3162.
Supported in part by Associazione Italiana per la Ricerca sul Cancro (AIRC; Milan, Italy) and by a grant from the Italian Ministry of University and Scientific and Technological Research, National Project "Chronic liver damage induced by hepatitis C virus." F.A.T. is recipient of a Fondazione Italiana per la Ricerca Sul Cancro (FIRC) fellowship.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Franco Dammacco, Department of Biomedical Sciences and Human Oncology, Section of Internal Medicine and Clinical Oncology, University of Bari Medical School, Policlinico, Piazza G. Cesare 11, 70124 Bari, Italy; e-mail: dimoclin{at}cimedoc.uniba.it.
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with an anti-CD20
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Abstract
Introduction
-actin gene, which resulted in a single band of 300 base pair (bp).
In all cases, a 
HLA-DR+ decreased distinctly against the
baseline values and remained low for 6 to 7 months. T-cell counts
(CD3+, CD4+, CD8+) were not modified.
3 bands) B-cell expansions, respectively, whereas polyclonality was
found in the remaining patients. The 4 nonresponders displayed monoclonality at all times. Despite dramatic reduction of the number of
circulating B cells, IgH VDJ PCR revealed persistence of B-cell
clonotypes; one responder with a monoclonal picture changed to
oligoclonal at day 7, the other remained unchanged. Four responders
with initial oligoclonality displayed monoclonal restriction (2 at day
7, 1 at day 21, and 1 at day 28). The fifth responder presented a
polyclonal picture at day 14 and became again oligoclonal at day 28;
the sixth became polyclonal at day 21 and again oligoclonal at day 28. The 3 patients with polyclonal feature changed into oligoclonal at day
7; 2 became monoclonal at day 28, whereas in the other, transition to
polyclonality occurred at day 28.
