Abstract

The epidemiology, etiology, classification, and treatment of non-Hodgkin's lymphoma (NHL) are reviewed, and rituximab, a
newly available therapy, is discussed.

NHL comprises a group of lymphoproliferative disorders the frequency of which continues to rise. Although many classification
systems exist for identifying specific histological subtypes, NHL is generally divided into indolent (low-grade) and aggressive
(intermediate- and high-grade) forms. Low-grade NHL is characterized by a slowly progressive, continually relapsing course,
with eventual transformation to a more rapidly progressive form that is usually fatal. Several options are available for the
management of indolent NHL; none is curative. Rituximab, a human-mouse monoclonal antibody that targets the CD20 antigen
expressed in over 90% of B-cell NHLs, provides an alternative to conventional chemotherapy that is relatively safe and
effective. In a Phase III trial involving 166 patients with relapsed or refractory low-grade B-cell NHL, rituximab produced an
overall response of 48%, with 20 of 80 responders still in remission more than 36 months after treatment. Study results in
patients with bulky disease and those requiring retreatment have also been encouraging. Most adverse effects associated with
rituximab are mild to moderate. Infusion-related reactions occur more commonly during initial infusions and in patients with
evidence of increased tumor burden but can be effectively managed with premedication, supportive care, and adjusted infusion
rates. Hematologic effects are generally mild and transient, and adverse immune responses are rare.

Rituximab is an alternative to conventional chemotherapy for the treatment of relapsed or refractory low-grade or follicular
CD20-positive B-cell NHL.

Introduction

Non-Hodgkin's lymphoma(NHL) encompasses a heterogeneous group of lymphoproliferative disorders that vary in clinical
course, prognosis, and management depending on histological subtype, tumor bulk, and other patient-specific factors. Despite
the introduction of many effective treatments, overall survival time has not increased significantly. Several therapeutic
approaches, including monoclonal antibodies, are currently being examined either alone or in combination with other modalities
in order to identify effective, tumor-selective treatment regimens for prolonging survival in patients with NHL.

This article reviews the epidemiology, etiology, classification, and treatment of NHL and discusses a newly available
monoclonal antibody, rituximab (Rituxan, Genentech, Inc., and IDEC Pharmaceuticals), which has been approved for use in the
treatment of relapsed or refractory low-grade or follicular CD20-positive B-cell NHL.

Epidemiology

Between 1973 and 1991 the incidence of NHL increased by 73%, currently ranking it as the fifth most common malignancy in
the United States after prostate, breast, lung, and colon cancers.[1] This rise in NHL frequency has been influenced by the AIDS
epidemic, an aging population, and the widespread practice of organ transplantation.[2-4] The risk of NHL increases steadily from
childhood through the eighth decade of life; on average, patients are in their early 40s at diagnosis.[4,5] NHL is more common in
Caucasians than in African-Americans and Asian-Americans and in males than in females.[3-6] It is estimated that over 54,900
new cases of NHL and 26,100 NHL-related deaths will be documented in 2000, accounting for almost 5% of all cancers and
cancer-associated deaths.[1,5] The estimated 1-year survival rate is 70%, while the 5- and 15-year rates approximate 50% and
35%, respectively.[4] Owing to the substantial number of productive years lost, NHL ranks fourth among all cancers in the United
States in terms of societal economic impact.[2]

Etiology

Several etiologic factors in NHL have been described, including infectious agents, congenital and acquired immunodeficiency
syndromes, environmental exposures, and genetic factors (Table 1).[2,3,7,8] It has been suggested that no single factor is
responsible for NHL but rather that multiple factors probably interact in a multistep fashion to promote lymphomagenesis.[9] For
example, reactivation of latent Epstein-Barr virus in B cells of an individual who has AIDS or is receiving immunosuppressive
drug therapy after renal transplantation is believed to allow oncogene activation and unchecked lymphocyte proliferation.

Cytogenetic research has demonstrated that chromosomal reorganization may play an important role in the conversion of
normal cells to malignant lymphoma cells. A chromosomal translocation that juxtaposes the bcl-2 proto-oncogene on
chromosome 18 with the antibody heavy-chain gene on chromosome 14 has been observed in 50% of patients with NHL and in
up to 89% of those with follicular NHL.[10,11] This genetic rearrangement is responsible for overexpression of bcl-2, which
normally functions as a regulator of programmed cell death (apoptosis), resulting in prolonged inhibition of apoptosis and
promoting malignant transformation.[12,13]Expression of bcl-2 before and after treatment, as detected by the polymerase-chain-
reaction (PCR) test, has been assessed to determine its potential prognostic significance. Current studies indicate that bcl-2
negativity in blood [14] or bone marrow [15,16] after NHL treatment may be associated with significantly increased disease-free
survival time. Conversely, serial evaluations indicating consistent bcl-2 positivity, or the persistence of lymphoma, may be
useful in identifying patients most likely to relapse and for whom additional therapies may be considered.[17]
NHL arises from the malignant proliferation of B or T lymphocytes at various stages of differentiation; most cases (85%) are of
B-cell origin.[18] Classification systems based on morphological and immunologic characteristics of these cells have long been
relied on to establish a diagnosis, make a prognosis, and select treatment in a scientifically accurate and clinically relevant
manner. Clinicians have challenged the clinical usefulness of the multiple available histopathologic classification systems,
particularly in comparing clinical study data and applying the results to the formulation and selection of effective treatment
strategies.

In 1982, the National Cancer Institute responded to this controversy by sponsoring the development of an International Working
Formulation (IWF) to provide an effective method of translation among the six classification systems available at that time.
[19]
Commonly used in clinical trials, the IWF has been widely accepted for its organization of NHL into 10 major subtypes (A-J)
on the basis of morphological characteristics (Table 2).[18,20,21] It groups these subtypes into three prognostic categories (low
grade, intermediate grade, and high grade), as determined by clinical course, treatment response, and survival rate. Low-
grade, or indolent, NHLs (subtypes A-C) account for approximately 40% of all B-cell NHLs. They are considered more favorable
because of their slow progression, the potential for spontaneous remission, and the relatively long estimated survival time (5-10
years or longer).[20,22] Low-grade NHLs respond well to initial treatment but characteristically relapse and are rarely curable.
[22,23]
The likelihood of histological conversion to a more aggressive type of NHL increases with time, reaching 60% and 100% at
8 and 16 years, respectively, and there is an attendant decrease in survival time.[20,21,23] Intermediate-and high-grade NHLs
progress more rapidly; without therapeutic intervention, survival is measured in months. Although the terms "indolent" (for low
grade) and "aggressive" (for intermediate grade and high grade) often are preferred in the clinical setting, most research
studies continue to use the IWF classification. This article uses both sets of terms.

In the early 1990s, an international panel of hematopathologists, the International Lymphoma Study Group, proposed the need
for an updated system for classifying lymphomas to reflect advances in diagnostic methods and chemotherapy regimens. The
result of this collaboration was the Revised European-American Classification of Lymphoid Neoplasms (REAL), an updated list
of "real" disease entities classified by the morphological, immunophenotypic, genetic, and clinical assessments routinely used
in clinical practice.[24] Table 2 includes the REAL classification nomenclature for comparison with the IWF categories. Despite
concerns about its complexity and clinical validity, which currently limit its wide-spread adoption among pathologists and
clinicians, the REAL system represents a comprehensive consensus based on expert experience, including encounters with
disease entities in daily practice.[18] A multicenter trial evaluating the REAL system in 1403 previously untreated patients found
that most of the major types of NHL could be diagnosed with at least 85% accuracy.[25]

Clinical application of the REAL or any other classification system should include an evaluation of prognostic factors (age,
performance status, stage, extranodal involvement, and serum lactate dehydrogenase [LDH] concentration) to be most useful
in clinical decision-making and estimating survival.[26] The extent and distribution of disease in patients with NHL can be
assessed by using the Ann Arbor Staging System, originally developed for use in Hodgkin's disease. Stage I NHL involves a
single lymphoid structure or lymph node region. In stage II, two or more lymph node regions on the same side of the diaphragm
are involved. Stage III NHL involves two or more lymphoid structures or lymph node regions on opposite sides of the
diaphragm. Extranodal disease is the hallmark of stage IV NHL.

Principles of Therapy

The treatment of NHL is based on tumor histology, disease extent (or stage), and patient-related factors such as age, functional
status, and the presence of other underlying diseases.[2] Since low-grade tumors are generally slow to progress, some clinicians
advocate delaying treatment until symptoms appear in order to limit chemotherapy-induced toxicity and resistance and to
preserve quality of life.[7,23] Moreover, clinical studies indicate that overall survival time is not compromised by adopting the
"watchful waiting" approach rather than treating aggressively. Spontaneous remission may even occur.[23,27,28] Disadvantages of
watchful waiting include the need for frequent monitoring, patient discomfort associated with visible enlargement of lymph
nodes, and potentially lower rates of complete remission with delayed chemotherapy.[28]

Patients with localized (stage I or II) indolent disease may be effectively treated with radiation therapy with or without adjuvant
chemotherapy.[7] However, most patients with indolent NHL have disseminated disease (stage III or IV) at diagnosis and require
immediate chemotherapy. Several treatment options are available, including oral alkylator therapy (chlorambucil or
cyclophosphamide with or without prednisone),[29] other chemotherapy regimens (e.g., CHOP [cyclophosphamide, doxorubicin,
vincristine, and prednisone], CVP [cyclophosphamide, vincristine, and prednisone], fludarabine alone or combined with
cyclophosphamide, and others),[29-32] combination chemotherapy plus interferon alfa,[33] and radiation therapy alone or combined
with chemotherapy.[29,34] Most treatments are associated with initially high overall response rates, but relapse is common, grade
3 and 4 adverse effects are frequent, and responses to subsequent treatment courses are generally less dramatic and briefer.
[22]
Relapses may be treated with a different combination of drugs or with high-dose chemotherapy followed by autologous bone
marrow or peripheral blood stem-cell transplantation, but it is still unclear whether overall survival is prolonged by this
intervention.[35]

An important treatment objective for patients with indolent NHL is to employ tumor-selective drugs that prolong survival with
minimal toxicity. Monoclonal antibody (MAb) therapy directed at tumor-specific cell-surface antigens provides a promising
alternative or adjunct to conventional chemotherapy. A century ago, Ehrlich [36] proposed his "magic bullet" theory for targeting
cancer cells with antibodies. His approach involved the production of immunoglobulin proteins (antibodies) in response to
repeated immunizations of animals with foreign cells, with resultant binding of these antibodies to cell-surface antigens and
induction of cell lysis and death. This process required the use of large animals to obtain adequate volumes of antiserum for
testing, which was further limited by variability in antibody specificity, affinity, and yield. Antibody technology did not advance
significantly until 75 years later, when Köhler and Milstein [37] described their hybridoma methodology for producing antibodies
from a single clone of cells, for which they were awarded the Nobel Prize in 1984. Their technology facilitated unlimited
production of murine-derived antibodies with improved affinity and specificity for targeted antigens. Drawbacks included poor
host effector-cell responses and immune system stimulation that resulted in the development of human antimouse antibodies
(HAMAs). Occurring in approximately 30% of patients treated with murine MAbs, HAMAs reduce the effectiveness of
subsequent infusions by binding MAbs, thus allowing their rapid clearance from the systemic circulation. [38,39]

Isolation of human MAbs soon followed, although difficulties in mass production and screening have resulted in few clinical
studies evaluating their activity. Advances in genetic engineering recently allowed the production of mouse-human, humanized,
and human antibodies, which more effectively interact with human effector cells, have a longer half-life, and have a greatly
reduced potential for immunogenicity compared with murine MAbs.[40,41]

In addition to the antibody source, careful consideration of antigenic characteristics is important in identifying the best
immunotherapy for a specific cancer and in reducing the agent's toxic potential. Antigens found solely on the cancer cell
surface are termed tumor-specific antigens and are desirable targets for MAb therapy. The preferred absence of antigens on
normal cells and stem cells reduces toxic effects on normal tissues and allows repopulation of depleted cells after treatment.
Other desirable antigenic characteristics are a high density of tumor surface expression, stability of the antigen on the cell
surface, and a biological function necessary for tumor cell survival.[42]

B-cell lymphomas are considered ideal models for intervention with MAb therapy owing to the expression of several well-
defined surface antigens, including the CD20 antigen (Figure 1).[43] The CD20 antigen is a membrane-embedded protein
expressed on the cell surface in more than 90% of B-cell lymphomas and some chronic lymphocytic leukemias.[44] This antigen
has several characteristics considered ideal for improving tumor selectivity and reducing host toxicity. It is expressed on normal
B-cell precursors and mature B cells but not on stem cells or plasma cells.[45] Absence of the CD20 antigen from plasma cells
and stem cells is advantageous because it allows uninterrupted immunoglobulin (IgG) production and repopulation of B cells
after treatment-induced depletion. Some cell-surface antigens are shed into the blood-stream, thus interfering with the binding
of therapeutic antibodies to tumor cells; this is not the case with CD20. Moreover, CD20 is not altered or internalized upon
antibody binding, a property that enhances antibody-dependent cell-mediated cytotoxicity.[46] Finally, CD20 may play an
important role in the regulation of cell growth by functioning as a calciumion channel, such that MAb binding to CD20 alters
transmembrane calcium movement associated with the cell cycle.[47]

Figure 1.
Surface-antigen targets for monoclonal antibody therapy in lymphoma.
Reproduced from reference 43, with permission from Glennie MJ.

(Enlarge
Image)

ntibodies are Y-shaped structures consisting of two identical heavy chains and two identical light chains connected by disulfide
bonds (Figure 2). The proteolytic cleavage of antibodies by papain produces two antigen-binding fragments (Fab fragments)
and a crystallizable fragment (Fc fragment). The upper arms of the Y, containing both variable-sequence and constant-
sequence regions, form the Fab pieces. The Fc piece, which is recognized by specific receptors on human effector cells,
consists of the portions of the heavy chains that make up the stem of the Y.

Figure 2.
Diagram of structure of rituximab. Rituximab is a chimeric antibody of
the immunoglobulin G1 kappa type with murine anti-CD20 variable-
sequence regions (filled areas) and human constant-sequence regions
(Enlarge
(open areas).
Image)
The chimeric structure of rituximab comprises human IgG 1 and kappa-chain constant regions and heavy- and light-chain
variable regions from a murine antibody to CD20 (Figure 2).[40] The murine variable regions selectively bind to the CD20 antigen
expressed on the surface of both normal B lymphocytes and most B-cell lymphomas. The presence of a human constant region
allows rituximab to bind to Fc receptors on human effector cells (e.g., lymphoma cells, macrophages, and neutrophils) to
mediate both complement- dependent cytotoxicity and antibody-dependent cell-mediated cytotoxicity and decreases its
immunogenicity.[40,49] In vitro, rituximab has been shown to inhibit cell proliferation, induce apoptosis, and sensitize lymphoma
cells to the effects of certain chemotherapy agents.[69,70] This synergistic interaction suggests the potential for enhanced
treatment efficacy when rituximab and chemotherapy are combined and encourages further investigation in patients with
relapsing disease. After rituximab administration, antibody-coated B cells are rapidly depleted from peripheral blood, lymph
nodes, and bone marrow.[40]

Pharmacokinetic analysis of data from 166 patients treated with rituximab 375 mg/m 2 once weekly for four doses over 22 days
revealed a steady rise in preinfusion and postinfusion serum antibody levels with each rituximab dose.[71] Moreover, detectable
antibody levels were still present one, three, and six months after the end of treatment. Consistent with these findings, the
elimination half-life of rituximab increased from a mean ± S.D. of 76.3 ± 31.1 hours after the first infusion to 205.8 ± 95.0 hours
after the fourth and final infusion. Clearance was 38.2 mL/hr after the first dose and 9.2 mL/hr after the fourth. Prolongation of
rituximab's half-life is probably due to the absence of circulating CD20-positive B cells, which bind antibodies during initial
infusions. It is also likely that prior rituximab saturation of involved lymphnode sites would increase antibody availability and
decrease clearance.

Several important serum concentration- response relationships have been observed.[49,51,55,71] First, antibody levels were
significantly higher in patients who responded to treatment than in nonresponders, becoming significant by the
second preinfusion and fourth postinfusion determinations and persisting throughout the assessed posttreatment
period. In addition, serum antibody levels were inversely correlated with baseline lymphocyte counts, as well as with
two measures of baseline tumor bulk. It is believed that serum antibody accumulation and long-term persistence are
major contributors to the achievement and maintenance of a therapeutic response to rituximab therapy. Finally, serum
rituximab levels were lower in patients with IWF type A NHL, which may be related to a higher tumor burden or
reduced CD20 expression. These findings suggest a potential benefit of using rituximab in higher dosages or over
prolonged periods in patients with bulky disease and those not responding to initial treatment. Clinical Trials

Data from clinical trials of rituximab alone or combined with chemotherapy in adults with indolent or aggressive forms of B-cell
NHL are summarized in Table 4.

Monotherapy

The clinical activity of rituximab in indolent NHL was initially demonstrated in two Phase I dose-escalation studies.[49,50] In these
trials, single doses of up to 500 mg/m 2 and four weekly doses of 125, 250, or 375 mg/m 2 administered to patients with
histologically confirmed disease were associated with favorable clinical responses and had no dose-limiting toxicities. Overall
response rates (ORRs), including those for patients receiving only single doses, ranged from 33% to 40%. In the absence of
dose-limiting toxicities, the highest dose tested for weekly administration for four weeks (375 mg/m 2 ) was selected for Phase
II and III study in patients with relapsed or refractory indolent NHL.[49,51,52]

In the pivotal, Phase III, open-label, single-group, multicenter study, McLaughlin et al.[52] evaluated 166 adults with relapsed or
unresponsive CD20-positive indolent or follicular NHL for tumor response to rituximab. Eligible patients had progressive
disease with no more than four relapses, a good performance status (World Health Organization status 0, 1, or 2), and
adequate renal, hepatic, and hematologic function at baseline. Excluded were patients with bulky disease (the presence of any
lesion measuring >10 cm in diameter), a count of circulating lymphocytes of >5000/mm 3 , AIDS-related lymphoma, CNS
involvement, or serious nonmalignant disease and patients who had received treatment within the preceding three weeks.

The average age of the study participants was 58 years (range, 22-79 years); 45% exceeded 60. The median time since
diagnosis was 4.1 years (range, 0.5-25 years). Thirty-three patients had small lymphocytic lymphoma; 67, follicular small
cleaved cell lymphoma; 53, follicular mixed small cleaved cell and large cell lymphoma; 3, other low-grade variants; and 10,
follicular large cell lymphoma. The patients had received a median of three prior treatments, including chemotherapy (97%),
radiation therapy (25%), and bone marrow or stem-cell support (14%). Twenty-two patients (13%) had demonstrated resistance
to all previous therapies, while 45 (27%) were unresponsive to the most recent course of chemotherapy. All patients received
four weekly doses of rituximab 375 mg/m 2 by i.v. infusion after pretreatment with oral acetaminophen and diphenhydramine.

Primary efficacy outcomes included complete and partial responses to the study treatment, as determined from comprehensive
physical and laboratory examinations, radioisotopic imaging studies, and bone marrow biopsies. A complete response (CR)
was defined as the resolution of all signs and symptoms of lymphoma, including bone marrow clearing, for at least 28 days. A
partial response (PR) was at least a 50% reduction in total lesion size without evidence of disease progression for 28 days.
Time to progression (TTP) of disease was measured as the time between the first infusion and evidence of progression.

The need for standardized response criteria to facilitate the evaluation of treatment effects and allow valid comparisons among
agents was recently addressed by an international panel of NHL experts.[72] This panel emphasized the importance of defining
the response to lymphoma therapies in terms of reduction in the size of lymph nodes or nodal masses. Therefore, for a CR to
be achieved in the McLaughlin et al. study, lymph nodes had to regress to 1.0 cm in their greatest transverse diameter.[73]

In the intention-to-treat analysis, CRs were observed in 6% of the 166 patients and PRs in 42%, for an ORR of 48%. A similar
ORR of 50% was achieved in the 151 evaluable patients. Of 75 nonresponders, 56 had a decrease in measurable disease
during the study period; the mean decrease in the sum of the products of perpendicular measurements of lesions was 32%.
[52]
Median TTP among responders was 13.2 months, which exceeds the 8-10 months typically observed after salvage
chemotherapy in patients with relapsing or refractory NHL.[53] Twenty of 76 responders remained in remission at 19.3-36.7+
months. Adverse events related to rituximab were usually of grade 1 (mild) or 2 (moderate) and occurred either during
treatment or within 30 days after it ended. After the first infusion, 55% of patients receiving rituximab had no further adverse
events for the remainder of the study.

A univariate analysis showed that a poor response to rituximab was related to the presence of any of the following clinical
features: small lymphocytic histology (IWF lymphoma type A), bone marrow involvement, involvement of two or more
extranodal sites, and, in a subset of 118 patients with follicular disease, PCR-documented absence of bcl-2 gene re-
arrangement in peripheral blood or bone marrow cells before treatment. [52] Additionally, the response rate tended to be lower
among patients demonstrating resistance to their last prior course of chemotherapy, defined as failure to achieve a CR or a PR,
than among those whose disease was sensitive to chemotherapy (36% versus 53%, p = 0.06). Factors not associated with a
reduced response rate were advanced age, elevated LDH or Beta 2 -microglobulin, and bulky disease. Stepwise logistic
regression analysis identified the following factors as significantly related to a reduced response to rituximab: small lymphocytic
histology (p < 0.001), resistance to the most recent chemotherapy course (p = 0.018), and bcl-2 negativity of peripheral blood
cells at baseline (p = 0.024).

The rate of response to rituximab was significantly higher among patients who had previously undergone autologous bone
marrow transplantation or peripheral blood stem-cell transplantation than among those who had not (78% versus 43%, p <
0.05). Higher response rates were also observed among patients with fewer prior relapses, such that patients with one, two,
and three previous relapses had ORRs of 57%, 46%, and 38%, respectively (p < 0.05).

Most adverse events were grade 1 or 2 infusion-related symptoms that developed mainly during the first infusion. The most
frequently reported infusion-related symptoms were fever, chills, nausea, urticaria, fatigue, headache, and a sensation of
swelling of the tongue or throat. These reactions generally occurred within 30 minutes to two hours of the beginning of therapy
and resolved with slowing or interruption of the infusion and institution of supportive care. More severe events (grades 3 and 4)
were less frequent and included arrhythmia, neutropenia, leukopenia, thrombocytopenia, hypotension, and bronchospasm.

Similar efficacy outcomes have been observed in patients with bulky disease. In a study by Davis and colleagues,[54] 31 patients
with relapsed or refractory CD20-positive low-grade or follicular B-cell lymphoma (IWF types A, B, C, and D) and at least one
tumor larger than 10 cm were given four weekly doses of rituximab 375 mg/m 2 . Most patients (68%) had stage III or IV
disease at the time of diagnosis, and the median number of previous treatments for lymphoma was three. Among 28
assessable patients, the ORR was 43%, with 1 CR (4%) and 11 PRs (39%). The median time to response was 50 days, similar
to that for patients without bulky disease. Responders had a median TTP of 8.1 months (4.5-18.6+ months), with a median
duration of response of 5.9 months (2.8-12.1+ months). The average decrease in tumor size was 76% in patients
demonstrating a partial response, while a 26% reduction was observed in patients with stable disease. Subgroup analysis
revealed a significantly better outcome in patients with IWF lymphoma types B, C, and D than in those with type A (ORR of
55% versus 0%, p < 0.005). Although adverse effects were most commonly infusion related and grade 1 or 2, a higher rate of
grade 3 or 4 toxicity was observed than among patients without bulky disease.[54] For this reason, the investigators
recommended that patients with bulky NHL be monitored carefully during rituximab treatment.

An advantage of rituximab is its ability to produce favorable response rates upon retreatment.[55] Among 57 evaluable patients
with relapsed low-grade or follicular NHL previously responding to rituximab, a second course of rituximab (375 mg/m 2 weekly
for four doses) produced an ORR of 40%, including 6 CRs (~10%) and 17 PRs (~30%). The median time to response (49 days)
was not different from that seen in studies in which patients took rituximab for the first time.[51] Median TTP and response
duration had not yet been reached at 16.7+ and 15.0+ months, respectively. Infusion-related adverse effects, including fever,
chills, asthenia, pruritus, and throat irritation, were observed most commonly but were generally limited to the initial infusion.
These findings are consistent with safety data for patients previously unexposed to rituximab who had relapsed or refractory
disease upon initial treatment.[52] Repeated courses of rituximab may provide an effective alternative to myelosuppressive
chemotherapy regimens, but prospective randomized studies are needed to document continued benefit.

The effects of extended rituximab treatment have also been examined in Phase II trials involving patients with either indolent
(low-grade or follicular B-cell) or aggressive (large-cell or mantle-cell) forms of NHL who had relapsed or had failed to respond
to primary therapy.[56,57] In an open-label, single-group, multicenter study, Piro et al.[56] observed 5 CRs (14%) and 16 PRs (46%)
(ORR, 60%) associated with eight weekly infusions of rituximab 375 mg/m 2 in 35 assessable patients (median age, 55 years)
with CD20-positive low-grade or follicular NHL (IWF types A, B, C, and D). For the 21 evaluable responders, median TTP and
duration of response of 19.4+ and 13.4+ months, respectively, had not yet been reached. Further support for extended
rituximab therapy included an average 42% reduction in maximum tumor size in patients with stable disease. Consistent with
other studies, patients with IWF type A NHL did not respond as well to rituximab as did those with types B, C, and D (14%
versus 69%). Similarly, response rates were lower in patients with bulky disease: 68% for those with lesions of <5 cm; 45%, 5-7
cm; and 25%, >7 cm. Again, adverse events were primarily infusion related, decreasing in frequency after the initial dose. The
frequency and severity of adverse effects were not substantially different from those observed during four weeks of treatment.[51]

In a multicenter study, Coiffier et al.[57] evaluated the clinical effects of extended rituximab treatment in patients with
intermediate- or high-grade CD20-positive NHL (IWF types D-H). Fifty-four patients, including 9 previously untreated patients
older than 60 years and 45 patients of any age with progressive disease following one or two chemotherapy regimens, were
randomly assigned to receive eight weekly doses of 375 mg/m 2 (group A, n = 28) or one dose of 375 mg/m 2 followed by
seven doses of 500 mg/m 2 at weekly intervals (group B, n = 26). At the end of 16 weeks, ORRs were 32% and 31% for the
intention-to-treat patients in groups A and B, respectively. Since these responses were not significantly different, they were
pooled, resulting in an ORR of 31%, with 5 CRs (9%) and 12 PRs (22%). Overall, the median time to response was 56 days
(range, 18-115 days). However, median response time was shorter in group B (41.5 days; range, 18-90 days) than in group A
(80 days; range, 27-115 days). Outcomes were better among patients without prior treatment, those having a first relapse, and
those having a second relapse; response rates in these subgroups were 33%, 41%, and 80%, respectively, versus 22% among
patients with primary refractory disease. Other factors associated with a favorable response included a low tumor burden
(ORR, 46%, with the largest lesion measuring <5 cm) and a diffuse large B-cell lymphoma histology (ORR, 37%). Although the
ORRs achieved with an eight-week course of rituximab were generally lower than those associated with salvage
chemotherapy, the significant activity against aggressive B-cell lymphomas warrants further investigation of rituximab combined
with standard chemotherapy. Most adverse effects were infusion related (fever, rigors, hypotension, hypothermia, and edema)
and mild to moderate. Most adverse effects were associated with first infusions; however, adverse effects with subsequent
infusions occurred more commonly in the higher-dosage group.

Combination Therapy

Indolent disease. Progressively lower response rates with repeated courses of chemotherapy, the potential for a synergistic
effect between rituximab and chemotherapy agents, and minimal overlapping toxicities form the rationale behind ongoing
studies of combination therapy in patients with NHL.[58,59] In a Phase II, open-label, single-group, multicenter study, the clinical
efficacy of rituximab combined with CHOP was evaluated in 40 patients (median age, 48.5 years; range, 29-77 years) with
histologically confirmed CD20-positive low-grade or follicular NHL.[58] Thirty-one patients had not received prior treatment. The
patients received CHOP at three-week intervals for a total of six cycles plus six i.v. infusions of rituximab 375 mg/m 2 . To
maximize rituximab's potential for chemosensitization, synergy with chemotherapy agents, and activity in minimal disease, the
first and second doses of rituximab were given before the first CHOP cycle as induction immunotherapy; the third and fourth
doses were administered before the third and fifth CHOP cycles, respectively; and the last two doses were given after the last
CHOP cycle as "mop-up" therapy for residual disease. An ORR of 95% was observed in the intention- to-treat patients; 22
(55%) had CRs and 16 (40%) had PRs. ORRs for subgroups with extranodal disease, an age of 60 years, elevated LDH,

bone marrow involvement, or bulky disease (5-10 cm) reached 100%, with CRs of 45% or higher among all patients but those
with bulky disease (29%). Overall, the median time to response was 47 days. Of 38 assessable patients, 28 (74%) remained in
remission after a median of 29 months. At one center, prospective assessment of bcl-2 status by PCR testing identified
baseline positivity in 8 of 18 patients; 7 of them converted to negative status after treatment ended and were considered to
have achieved complete remission on the basis of standard staging evaluations. Five patients remained bcl-2 negative for at
least 24 months. Adverse effects of the combination treatment were similar to those of CHOP alone, except for rituximab's
infusion-related toxicities.

Aggressive disease. In a study of 31 patients with previously untreated intermediate- or high-grade NHL (IWF types D-H), the
combination of rituximab and CHOP produced an ORR of 96%, with 19 CRs (63%) and 10 PRs (33%).[59] Serious toxicities
reported for 156 of 186 cycles included grade 3 neutropenia (16 cycles) and grade 4 neutropenia (17 cycles) and did not differ
from those occurring with CHOP alone. Most patients (74%) had advanced disease (stage III or IV), and the median age was
49 years. All the patients received the combination regimen at 21-day intervals for a total of six cycles; rituximab 375 mg/m 2
was given on day 1, followed 48 hours later by CHOP.

Safety Issues

In studies involving more than 500 patients receiving rituximab alone for NHL, over 90% of the adverse effects reported were
grade 1 or 2. [49,51,52,54,57] The frequency of adverse events related to rituximab was highest with the first infusion and declined
substantially with subsequent infusions (Figure 3).[49,52,56,57] Brief episodes of infusion-related effects were reported most
commonly. Hematologic abnormalities were relatively infrequent and were generally mild and reversible. Despite the profound
B-cell depletion associated with rituximab's mechanism of action, immune system complications were negligible. Rare but
serious cardio-pulmonary events have been reported among patients with a large number of circulating tumor cells
(>25,000/mm 3) or preexisting cardiac or pulmonary disease.
 Figure 3.

Adverse events occurring in 166 patients receiving rituximab. Most

events were grade 1 or 2 and decreased in frequency with additional
infusions. Reproduced from reference 52, with permission.
(Enlarge
Image)

Although dose-ranging studies have not identified dose-related toxicities, in one study the overall frequency of rituximab-related
adverse events was higher with 500-mg/m 2 doses than with 375-mg/m 2 doses.[57] Other studies suggest that patients with a
higher tumor burden, for example patients with bulky disease or many circulating malignant cells, may be at greater risk for
more serious adverse events.[54,74] Adverse-effect frequency and severity have not been shown to be greater in patients older
than 60 years or in patients being retreated with rituximab.[52,55] In patients receiving rituximab combined with CHOP, the
frequency of adverse effects was similar to that observed with CHOP alone.[58,59]

Infusion-Related Reactions

Infusion- related reactions, typically occurring during the first 30 minutes to two hours of the first rituximab infusion, have been
reported frequently.[49-52,54-57] These infusion-related reactions are characterized by one or more of the following: fever, chills,
rigors, asthenia, nausea, rash, pruritus, angioedema, mild to moderate hypotension, headache, and bronchospasm. In clinical
studies, reactions were successfully managed by temporarily slowing or stopping the infusion and by giving supportive
medications, such as diphenhydramine, acetaminophen, and vasopressors. Bronchospasm was effectively reversed with
inhaled bronchodilators. These symptoms usually resolved within hours of infusion-rate adjustment, allowing the infusion to be
completed at half the previous rate without further events. In clinical trials in general, 80% and 40% of patients had infusion-
related events during the first and subsequent infusions, respectively.[68] In the pivotal trial by McLaughlin et al.,[52]adverse effects
with the first dose of rituximab were observed in some 75% of patients; however, 55% of patients had no toxic effects during
the second, third, and fourth infusions.

Treatment with acetaminophen and diphenhydramine before each infusion is recommended to reduce the frequency and
severity of adverse effects. Corticosteroids should not be routinely administered because they may interfere with rituximab's
activity. Supportive care with i.v. infusions of 0.9% sodium chloride injection and bronchodilator therapy may be required; these
and other resuscitative treatments should be kept at the bedside.

Tumor Lysis Syndrome

Tumor lysis syndrome is a constellation of metabolic abnormalities that may occur in malignancies such as NHL, owing to
spontaneous or drug-induced lysis of tumor cells.[75,76] Associated hyperuricemia, hyperphosphatemia, hypocalcemia,
hyperkalemia, and azotemia have been reported to occur rarely within 12-24 hours after the first infusion of rituximab.
[68]
Hypovolemia, high tumor burden, elevated serum LDH, renal insufficiency, and large numbers of circulating malignant B cells
(>25,000/mm 3 ) may be important predisposing factors for tumor lysis syndrome.[68,75,76] Therefore, before rituximab therapy is
started, patients at risk should receive adequate hydration and prophylactic allopurinol.

Serious Adverse Effects

Although no evidence of tumor lysis syndrome, severe infusion-related effects, or cardiac abnormalities was observed during
the Phase III trial of McLaughlin et al.,[52] deaths associated with rituximab administration have occurred in the postmarketing
setting at a rate of 0.04-0.07% (4-7 deaths per 10,000 patients treated).[68] Events occurred within 24 hours of (in most cases)
the initial infusion and included hypoxia, pulmonary infiltration, adult respiratory distress syndrome, myocardial infarction,
ventricular fibrillation, and cardiogenic shock. Four reports of new arrhythmias and one report of angina followed by myocardial
infarction have been documented during rituximab infusion.[68] One patient discontinued treatment because of ventricular and
supraventricular tachycardia. Patients with preexisting cardiac and pulmonary conditions should be carefully monitored during
and after rituximab infusions.

Byrd and colleagues [74] reported a unique infusion-related reaction associated with rapid blood clearance of tumor cells. Five
patients (median age, 68 years) with hematologic malignancies (one with NHL, two with chronic lymphocytic leukemia, and two
with prolymphocytic leukemia) and leukocyte counts exceeding 70,000/mm 3 reportedly had significant bronchospasm,
hypoxia, fever, or rigors immediately after initiation or rate adjustment of a rituximab 375-mg/m 2 i.v. infusion. Infusions were
begun at rates of 25 or 50 mg/hr and were adjusted according to the manufacturer's recommendations.[68] All five patients had a
rapid reduction in white blood cell and platelet counts soon after rituximab administration and had laboratory test abnormalities
consistent with a mild tumor lysis syndrome, including elevations in serum uric acid, potassium, and phosphate; increased LDH;
and hypocalcemia. All the patients required hospitalization for treatment of these adverse effects.
Alternative infusion methods may be necessary for patients with a high peripheral-blood tumor burden to minimize adverse
effects, prevent hospital admission, and avoid costly drug waste. For example, an initial infusion rate of 25 mg/hr can be
considered for patients with >25,000 circulating lymphocytes per cubic millimeter at baseline. In their own practice, Byrd et al.
[74]
reported administering a small dose (100 mg) of rituximab on the first day, preceded by adequate hydration, standard
premedication, and allopurinol, and then giving the remainder of the 375- mg/m 2 dose on the following day as tolerated.

An additional postmarketing report described laboratory test-substantiated cytokine-release syndrome during the first rituximab
infusion in patients with lymphocyte counts greater than 50,000/mm 3 and B-cell chronic lymphocytic leukemia or leukemic
variants of other low-grade B-cell NHLs.[75] Eleven heavily pretreated patients with relapsed NHL were enrolled and scheduled
to receive four weekly 375-mg/m 2 i.v. infusions of rituximab. Baseline lymphocyte counts ranged from 200 to 294,300/mm 3 .
The first patient, who had a baseline lymphocyte count of 89,300/mm 3 , developed severe cytokine release syndrome during
the first infusion (375 mg/m 2 over 10 hours), with symptoms including fever, chills, and vomiting. Grade 4 thrombocytopenia, a
dramatic increase in serum LDH, and increased serum aminotransferases were also evident. Consequently, instead of a single
375-mg/m 2 infusion on day 1 of treatment, subsequently treated patients with >10,000 circulating lymphocytes per cubic
millimeter received 50 mg of rituximab on day 1, followed by 150 mg on day 2 and the remainder of the 375-mg/m 2 dose on
day 3. Each dose was infused in 1000 mL of 0.9% sodium chloride injection at a starting rate of 50 mL/hr. Nonetheless, severe
adverse effects occurring with the first infusion continued to occur in patients with high baseline lymphocyte counts. The
subjects were retrospectively separat-ed into two groups based on baseline lymphocyte count. Patients with a baseline
lymphocyte count of >50,000/mm3 had significantly more grade 3 and 4 adverse effects than did patients with a count of
<50,000/ mm3 (10 total versus 0, p = 0.0017). Moreover, 90 minutes after the first infusion was started, they exhibited
significantly higher mean serum concentrations of tumor necrosis factor- and interleukin-6. The investigators

recommended careful consideration before rituximab is administered to patients with high counts of circulating lymphocytes.
Furthermore, they suggested that it might be advantageous to reduce lymphocytes to <50,000/mm3 with conventional
chemotherapy before administering rituximab.

Hematologic Effects

Among the 166 patients receiving rituximab during the Phase III study, the rates of hematologic events (anemia,
thrombocytopenia, leukopenia, and neutropenia) ranged from 1% to 7%.[52] Twenty-four events were grade 1 or 2, three were
grade 3, and one was grade 4. Of the late adverse effects reported (31 days to one year after treatment), hematologic effects
were most common. Thirteen neutropenias (5 of them grade 3 and 1 grade 4), 10 leukopenias (1 of them grade 3 and none
grade 4), and 1 red-cell aplasia were reported. Eighty-six percent of the patients demonstrated no cytopenias throughout the
study or follow-up periods. Results were similar in Phase II trials evaluating rituximab alone in patients with relapsing low-grade
or follicular and aggressive forms of NHL.

Immunologic Effects

Despite a rapid depletion of peripheral blood B cells during rituximab therapy, serum IgG concentrations remained within
normal limits in most patients studied.[52,56-58] Recovery of B-cell populations was evident at 6 months and complete within 9-12
months after rituximab therapy. Moreover, B-cell depletion was not associated with clinically significant infectious complications;
most infections reported were minor and resolved with appropriate treatment.[51,52,54] Counts of T cells and natural killer cells were
unaffected. Human antichimeric antibodies were reported in <1% of all patients studied but were not associated with abnormal
signs and symptoms. These antibodies have not been found to compromise retreatment.[52,55,68]

Dosage and Administration

Thorough evaluation of the patient before rituximab administration is necessary to document the presence of any underlying
cardiac disorders, particularly hypertension, angina, or an arrhythmia. The clinician must review the patient's current
medications and consider withholding scheduled antihypertensive medications for 12 hours before infusing rituximab in order to
avoid enhanced hypotensive effects. Frequent blood pressure monitoring is critical during the first dose of rituximab. Similarly,
hydration status should be assessed to reduce risks of infusion-related hypotension and laboratory abnormalities promoting the
development of tumor lysis syndrome. Patients with histories of cardiac arrhythmia or angina require close monitoring for
associated signs and symptoms, since these conditions may be exacerbated during rituximab therapy.

Health care professionals prescribing and administering rituximab should be carefully instructed about the importance of
premedication with acetaminophen and diphenhydramine before each infusion. The routine use of corticosteroids is not
recommended.

Clinicians should also be knowledgeable about infusion-related reactions to ensure their effective and timely management.
Guidelines on adjusting the infusion rate should be reviewed and strictly followed to minimize these reactions. In the Phase III
trial, infusion times averaged 5.2 and 3.3 hours for initial and subsequent infusions, respectively.[52]Lower infusion rates (e.g., 25
mg/hr) and split doses may be advisable for patients at risk for severe infusion-related reactions. Medications and fluids for the
emergent management of infusion-related reactions, including i.v. fluids (0.9% sodium chloride injection), epinephrine,
antihistamines, inhaled bronchodilators, and corticosteroids, should be readily available. Meperidine hydrochloride 12.5-25 mg
i.v. may be administered for the treatment of infusion-induced rigors. Staffing patterns in outpatient clinics may require changes
in anticipation of prolonged infusions or adverse events in certain patients. Scheduling of first infusions in the morning will allow
the patient to receive the entire infusion.

The recommended dosage of rituximab in patients with low-grade or follicular NHL is 375 mg/m 2 infused i.v. at weekly
intervals for a total of four doses. In a majority of patients, this can be accomplished in an outpatient clinic over a 22-day period.
Dosage schedules were identical in patients requiring retreatment.

Just before administration, rituximab should be diluted with 5% dextrose in water or 0.9% sodium chloride injection to a final
concentration of 1-4 mg/mL. A 1-mg/mL dilution is preferable to facilitate adjustments in the infusion and to avoid adverse
effects potentially caused by inadvertently rapid administration. Rituximab should not be mixed with other medications or i.v.
fluids because there is a lack of compatibility data. Prepared infusions are stable in polyvinyl chloride or polyethylene bags at 2-
8 °C (36-46 °F) for 24 hours and at room temperature for an additional 12 hours. Unused portions of undiluted drug must be
discarded because of the absence of a preservative.

Thirty to 60 minutes before each rituximab infusion, acetaminophen 650-1000 mg and diphenhydramine hydrochloride 50-100
mg should be administered to help prevent infusion- related effects. Rituximab may be infused through either a central or
peripheral i.v. catheter but should never be given by i.v. push or bolus injection, owing to the risk of potentially serious infusion-
related adverse effects. Before beginning each infusion, the administering clinician must prime the i.v. tubing with drug-
containing solution to guarantee that active drug rather than other solution is being infused from the beginning. The first infusion
should be initiated at 50 mg/hr with the rate increased by 50 mg/hr every 30 minutes as tolerated until a maximum rate of 400
mg/ hr is reached. Subsequent infusions may be started at 100 mg/hr, with 100-mg/hr increases every 30 minutes as tolerated
until the maximum rate (400 mg/hr) is attained. In patients with a large tumor burden (white blood cell count, >25,000/ mm 3 ),
an initial infusion rate of 25 mg/hr should be considered. Since stability data indicate a 12-hour limit at room temperature,
splitting the dose into two infusion bags may be useful in managing adverse events when long infusion times are anticipated
and may reduce the costs associated with unused drug.

Because of the potential for additive hypotensive effects, antihypertensive medications may need to be withheld for 12 hours
before initiating rituximab infusions. If serious infusion- related reactions (bronchospasm, hypotension) are observed, the
infusion should be stopped until symptoms resolve completely. The infusion may be resumed at half the original rate and the
rate increased as tolerated.

Conclusion

Rituximab is an alternative to conventional chemotherapy for the treatment of patients with relapsed or refractory low-grade or
follicular CD20-positive B-cell NHL. An over-all response rate of 48% has been reported in these patients. Adverse reactions
tend to be mild and occur most commonly during the first infusion and in patients with a large tumor burden. Study results for
patients with bulky disease or receiving a second course of rituximab therapy are encouraging. Data on the durability of
responses to rituximab, given alone or in combination with conventional chemotherapy in patients with indolent or aggressive
NHL, are eagerly awaited.
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American Journal of Health-System Pharmacy. 2001;58(3) © 2001 American Society of Health-System Pharmacists
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