Immunomodulatory drugs alone
or with immunotherapy represent
new treatment options for patients
with chronic lymphocytic leukemia.
Gracia Dayton. Not Quite Autumn. Watercolor on paper, 22 × 30 .
Immunomodulatory Drugs and Active Immunotherapy for
Chronic Lymphocytic Leukemia
Estrella Carballido, MD, Marays Veliz, MD, Rami Komrokji, MD, and Javier Pinilla-Ibarz, MD, PhD
Background: The last decade witnessed the emergence of several therapeutic options for patients with chronic
lymphocytic leukemia (CLL) for ﬁrst-line and relapsed settings. The vast majority of patients with relapsed or
refractory CLL carry poor prognostic features, which are strong predictors of shorter overall survival and resistance
to ﬁrst-line treatment, particularly ﬂudarabine-based regimens.
Methods: This article highlights the current role of immunomodulatory drugs (IMiDs) and active immunotherapy
as treatment options for this select group. The rationale of using IMiDs is discussed from the perspective of
lenalidomide as a novel active agent. Relevant clinical trials using IMiDs alone or in combinations are discussed.
New immunotherapeutic experimental approaches are also described.
Results: As a single agent, lenalidomide offers an overall response rate of 32% to 47% in patients with relapsed/
refractory disease. Recent studies have shown promising activity as a single agent in treatment-naive patients.
The combination of lenalidomide with immunotherapy (rituximab and ofatumumab) has also shown clinical
responses. Encouraging preclinical and early clinical data have been observed with different immunotherapeutic
Conclusions: The use of IMiDs alone or in combination with immunotherapy represents a treatment option for
relapsed/refractory or treatment-naive patients. Mature data and further studies are needed to validate overall
and progression-free survival. The toxicity proﬁle of lenalidomide might limit its use and delay further studies.
Immunotherapy offers another potential alternative, but further understanding of the immunogenicity of CLL
cells and the mechanisms of tumor ﬂare reaction is needed to improve the outcomes in this ﬁeld.
Prognostic factors, discussed in a separate article in this
issue (Sagatys EM, Zhang L; pp 18-25), are strong predic-
tors of progression-free survival (PFS) and overall survival
From the Department of Malignant Hematology at the H. Lee Mofﬁtt
Cancer Center & Research Institute, Tampa, Florida. (OS) in patients with newly diagnosed chronic lympho-
Submitted June 3, 2011; accepted September 1, 2011. cytic leukemia (CLL). Patients with poor prognostic
Address correspondence to Javier Pinilla-Ibarz, MD, PhD, Depart- features are more likely to be refractory to ﬁrst-line treat-
ment of Malignant Hematology, Mofﬁtt Cancer Center, 12902 Mag- ment, or they relapse early, requiring salvage therapies.
nolia Drive, Tampa, FL 33612. E-mail: Javier.Pinilla@mofﬁtt.org Although several alternative therapies are available, none
Dr Pinilla-Ibarz receives honoraria from GlaxoSmithKline Corp, offers a durable response. Even with current standard
Genentech Inc, and Cephalon Inc.
therapies, there are still clearly unmet needs. Therefore,
The other authors report no signiﬁcant relationship with the com-
panies/organizations whose products or services may be refer- the treatment of relapsed or refractory CLL has become a
enced in this article. challenge. For these reasons, considerable effort is aimed
54 Cancer Control January 2012, Vol. 19, No. 1
toward the development and use of different therapeutic duction and secretion of prosurvival cytokines. One such
agents for this population. cytokine is vascular endothelial growth factor (VEGF),
Immunotherapy is an appealing alternative and ap- which has autocrine and paracrine properties that di-
pears to offer a logical approach if we consider the biol- rectly affect the B-cell CLL (B-CLL) cell growth, apoptosis,
ogy of CLL. The tumor microenvironment and different and resistance to treatment. Tumor microenvironment
cytokines play an important role in the evolution of this is responsible for the accumulation of clonal malignant
disease. Immunomodulatory drugs (IMiDs) offer dif- B cells, particularly by promoting and housing increased
ferent biologic effects on cytokine and cell-mediated survival while avoiding apoptosis. The survival of the
responses. This article reviews the use of this new class tumor cells is also promoted by the ineffective immune
of drugs for the treatment of relapsed and refractory CLL response of the host in the milieu of tumor antigens.
and also discusses new immunotherapeutic approaches. These evasive strategies can be overcome by introducing
IMiDs that might help defeat tumor resistance. Taking
Immunomodulatory Drugs this into consideration, targeting the microenvironment
Thalidomide and Lenalidomide: as part of the treatment for CLL seems to be an attractive
Chemistry and Metabolism proposal. For these reasons, further understanding of the
Thalidomide [α-(N-phthalimido)glutarimide] is a syn- microenvironment and cytokines role is an important
thetic glutamic acid derivative, with an empirical formula part in guiding the selection and further discoveries of
(3 H10 N2 O4). It is formulated as a racemic mixture of new therapeutic agents.
two active enantiomers, S(−) and R(+). Both isoforms
S(−) and R(+) are considered to have teratogenic proper- Mechanisms of Action
ties.1 Thalidomide is an oral agent since it is not soluble Thalidomide, initially developed in the 1950s as an an-
in water. The mean plasma protein binding is 55% to ticonvulsant medication, offered insufﬁcient efﬁcacy.
66%, and it is metabolized by nonenzymatic hydrolysis Since sedation was a common effect of thalidomide, it
to different metabolites, which are then eliminated in was used as a sleeping and sedative drug. Thalidomide
the urine. The speciﬁc pharmacokinetic characteris- was also used as antiemetic treatment in pregnancy, but
tics of this drug in the setting of renal or hepatic dys- due to its detrimental teratogenic effects, the drug was
function are unknown. Thalidomide has a wide dosing withdrawn by the US Food and Drug Administration in
range, with daily doses of 50 mg to 800 mg, depending 1961. By serendipity, thalidomide was found to be an
on each particular disease. In searching for a less toxic effective treatment against erythema nodosum leprosum
and better tolerated drug, lenalidomide was created using because it helped reduce fevers and improve skin lesions
thalidomide as a backbone. Lenalidomide is a 4-amino- in patients with dermatologic and rheumatolic condi-
gultaramide derivative of thalidomide in which an amino tions.3 The effect of thalidomide on tumor necrosis factor
group was added to the fourth carbon of the phthaloyl alpha (TNF-α), a cytokine that regulates the inﬂammatory
ring of the parent compound.2 This modiﬁcation led to cascade (higher levels reported on CLL patients),4,5 is
an enhanced immunomodulatory potency drug with responsible for this beneﬁt. Despite the setbacks since its
less neurologic toxicity. As thalidomide, it also exists as a initial development, the beneﬁcial effects of thalidomide
racemic mixture of the active S(−) and R(+) forms. Like on the cytokines and its enhancement inﬂuence on the
thalidomide, lenalidomide is available in oral formula- immune system have prompted interest in the drug as
tion and is given every 21 to 28 days of monthly cycles. an option in treating hematologic malignancies.”6 Tha-
Since renal elimination predominates, adjusted doses are lidomide is now considered the ﬁrst immunomodulat-
recommended with impaired creatinine clearance. In ing agent. In an effort to improve efﬁcacy and reduce
contrast to thalidomide, lenalidomide lacks signiﬁcant toxicity, thalidomide analogs were developed using the
neurosedative toxicity; however, they share the risk of backbone of the thalidomide structure. Lenalidomide
venous thromboembolism. (CC-5013) and pomalidomide (CC-4047) are two of the
analogs that were later developed.
Rationale A few characteristics of thalidomide identiﬁed it as
An understanding of CLL pathogenesis is necessary to an attractive anticancer drug. Various potential antitumor
comprehend the logistics behind the use of IMiDs. A key mechanisms have been attributed to the IMiDs, starting
feature of CLL is the inability to undergo programmed with the discovery of its potent anti-inﬂammatory ac-
cell death and subsequent increased survival of mature tivities through the inhibition of the synthesis of TNF-α
B cells, mostly due to the aberrations in the apoptosis by activated monocytes.7 TNF-α is mainly produced by
pathway. The proliferation, differentiation, and apoptosis monocytes and macrophages, but lymphocytes, under
of healthy B lymphocytes are regulated directly by several stimuli, also produce it.8 This property allows the use
cytokines and growth factors. Data suggest that malig- of thalidomide for several diseases associated with in-
nant B cells can resist apoptosis through their ability to creased TNF-α, such as autoimmune deﬁciency disease
manipulate the microenvironment and through the pro- (AIDS), Kaposi sarcoma, and rheumatologic conditions.
January 2012, Vol. 19, No. 1 Cancer Control 55
Thalidomide and its analogs modulate cytokine antiangiogenic activity in vitro, and this is likely to con-
production, with inhibitory effects in inﬂammation and tribute to their antitumor effects in vivo.2,21 IMiDs might
stimulatory immune-mediated effects. This dual prop- help to minimize metastasis by reducing the expression
erty is through the suppression of TNF-α production of proangiogenic cytokines such as VEGF, by decreasing
(by enhancing the degradation of TNF-α mRNA)9 from blood-vessel density, and by affecting cell-adhesion mol-
endotoxin-stimulated monocytes and macrophages10 ecules.2 However, Andritsos et al22 reported that VEGF
and also through T-cell costimulation that induces in- serum concentrations remained unchanged, regardless of
creased cytokine production to enhance the immune response, in patients treated with lenalidomide.
response.2 Both lenalidomide and CC-4047 (pomalido- Hideshima et al23 have suggested that lenalidomide
mide) are 4-amino-glutaramide derivatives of thalidomide has proapoptotic effects that inhibit the proliferation of
that have been shown to be more potent TNF-α antago- B-CLL, according to in vitro tumor models. Lenalidomide
nists in endotoxin models.11,12 Lenalidomide is 50,000 effects on the bone marrow microenvironment modu-
times more potent in inhibiting TNF-α in vitro than is late the adhesive interactions and also alter tumor cell
its parent drug, thalidomide.11 Effects of lenalidomide growth, survival, and drug resistance.23 In discussing the
in the production of cytokines have been reported that effects of lenalidomide on tumor cell microenvironment,
support its role as an immune-modulating agent in this Chanan-Khan et al24 suggested that its antileukemic ef-
disease.13,14 Lenalidomide has been shown to increase fect is most likely from in vivo modulation of the tumor
circulating cytokines, particularly interleukin 6 (IL-6), microenvironment as is demonstrated from changes in
IL-10, IL-2, and TNF receptor-1 levels.14 the cytokine milieu and the cellular immune response.
IMiDs have a costimulatory effect on T-cell responses In in vitro models, lenalidomide inhibits the cell pro-
that include increased production of IL-2 and IFN-α by liferation of B-malignant cell lines by arresting cells in
increasing the proliferation of CD3- or IL-2–activated the G0-G1 phase.25 As discussed by Chanan-Khan et al,24
T cells, which activate natural killer (NK) cells enhanc- the downregulation of prosurvival pathways such as
ing tumor cell death.2,15-17 This costimulatory activity the phosphatidylinositol pathway provides lenalidomide
provides an immunologic adjuvant to promote an other- with its modulating antileukemic effects. Lapalombella et
wise ineffective immune response associated with al26,27 addressed this hypothesis in several reports. Their
malignancies.2 In most tumors, including CLL, an in- data support that lenalidomide induces the downregula-
creased number of T regulatory cells are present. The tion of CD20 surface antigen expression via the enhanced
expression of CD152 (cytotoxic T-lymphocyte–associ- internalization and the upregulation of CD40 expression
ated antigen 4 [CTLA4]) in T cells of patients with CLL on primary B-CLL cells, enhancing the efﬁcacy of the anti-
is particularly increased and can correlate with advanced CD40 antibody SGN-40. Lenalidomide also promotes the
disease and prognostic factors.18 Lenalidomide and upregulation of functional CD154 on CLL cells, which
pomalidomide strongly inhibit T regulatory cell prolif- may reverse the humoral immune defect characteristic
eration and suppressor function.19 Data from LeBlanc in the immunopathology of CLL. Given the modula-
et al17 demonstrated that lenalidomide activates CD28 tory effects of lenalidomide in the immune responses,
and overcomes the CTLA4 immunoglobulin blockade, as well as its antitumor effects, the novel use of IMiDs is
thereby conﬁrming that drug-induced costimulation is becoming more popular in various tumor types such as
mediated via the B7-CD28 pathway. multiple myeloma, myelodysplastic syndrome, renal cell
The effect of IMiDs in natural killer (NK) T cells was carcinoma,28 and prostate cancer.29,30
described by Davies et al16 when they reported that in the
in vivo setting, there is an increase in the number of NK Clinical Trials
cells in patients with multiple myeloma (MM) cells who Several clinical trials have been conducted to assess the
responded to lenalidomide, which was accompanied use of lenalidomide, either alone or in combinations, in
by an increase in IL-2 and IFN-α secretion. Hayashi et al20 patients with CLL. Different doses, regimens, and effects
demonstrated how the IMiDs augment NK-cell cyto- have been investigated (Table).
toxicity in myeloma cells, triggering NK-cell–mediated Chanan-Khan et al35 investigated high doses of le-
tumor cell lysis. These effects were produced via nalidomide in a nonrandomized phase II study that in-
the induction of IL-2 transcription and secretion in cluded patients with relapsed or refractory B-CLL. Among
T cells. In vitro data from NK cell modulation have not 64 patients assessed, 64% with advanced Rai stage III or
yet been demonstrated for in vivo models; however, IV and 51% who were refractory to ﬂudarabine received
this suggested the effect of NK cells in anti-MM immune 25 mg once daily for 21 days on a 28-day schedule. Pa-
responses. Lenalidomide antiangiogenesis properties tients were able to receive rituximab in the evidence of
were discovered and explored for cancer treatment, progressive or stable disease for 2 consecutive months.
mostly while the role of new blood vessel formation was The major overall response (OR) in this study was 47%,
deﬁned as a crucial component for tumor growth and me- with a complete response (CR) rate of 9%. A partial
tastasis. Preclinical data showed that IMiDs have a potent response (PR) was achieved in 38%. These data dem-
56 Cancer Control January 2012, Vol. 19, No. 1
onstrated that the antitumor activity of lenalidomide imbalance, uremia, and renal failure, caused 1 fatality. TFR,
was evident as early as day 8 of treatment, with 24 of which is associated with painful swelling of the lymph
34 patients (70.5%) demonstrating a decrease in their nodes and/or splenomegaly with or without fever and
peripheral-blood absolute lymphocyte count. Tumor rash, predominated as one of the most common nonhe-
lysis syndrome (TLS) and tumor ﬂare reaction (TFR) matologic toxicities, in addition to fatigue (86%).
were among the toxicities, accounting for 5% and 58%, A study by the Ferrajoli et al14 reported the clinical
respectively. TLS, which is characterized by electrolyte activity of lenalidomide (10 mg to 25 mg) in patients with
Table. — Selected Clinical Trials Using Lenalidomide Therapy Alone or in
Combination With Rituximab and Ofatumumab for Treatment of Chronic Lymphocytic Leukemia
Study Initial Dose/ No. of TLS TFR Hematologic Overall Partial Complete Overall Progression-
Regimen Patients All All Side Effects Response Response Response Survival Free
Grades Grades Grade 3/4 (%) (%) (%) (%) Survival
Badoux et al31 5 mg/d L 60 0b 52% Neutropenia 34% 65 43 10 88 60%*
Phase II escalated to Thrombocytopenia 12%
(elderly)a 25 mg/d Anemia < 1%
Chen et al32 2.5 mg/d 25 0 88% Neutropenia 72% 56 56 0 92 89%*
Phase IIa,c escalated to Thrombocytopenia 28%
10 mg/d Ld Anemia 20%
Maddocks et al33 2.5 mg/d 14 NR 14%f Neutropenia 35.7 % NR 10g 0 NR NR
Phase Ic escalated to Thrombocytopenia 7%
15 mg/d Le Anemia 14%
Wendtner et al34 2.5 mg/d 52 3.8% 44% Neutropenia 65% NR 11.5i NR NR 24.1 wks
Phase Ih escalated to Thrombocytopenia 33% (ITT) and
5 mg/d L until Anemia 9.6% 42.1 wks
MTD of 20 mg/d (responders)
Aue et al13 20 mg/d L 33 0 53% Neutropenia 56% NR 16j NR NR NR
Phase II lowered to Thrombocytopenia 30%
10 mg/d Anemia 15%
Chanan-Khan 5 mg/d 45 5% 58% Neutropenia 70% 47 38 9 NR NR
et al35 escalated to Thromobocytopenia 45%
Phase IIc 25 mg/d Anemia 18%
Lk + R
Ferrajoli et al14 10 mg/d 44 0 12%l Neutropenia 41% 32 25 7 73m NR
Phase II escalated to Thrombocytopenia 15%
25 mg/d L Anemia 3%
Ferrajoli et al36 10 mg/d 59 1.7% 37% Neutropenia 68% 64 39o 8 90 NR
Phase IIn L + R wkly Thrombocytopenia 22%
Veliz et al37 2.5 mg/d to 17 4.5% 27.2% Neutropenia 36.3% 67 NR 0 NR NR
Phase II 20 mg/d Thrombocytopenia 4.5%
Badoux et al38 10 mg/d 16p NR 13% Neutropenia 50% 63 50 13 NR NR
Phase II L+O Anemia 13%
L = lenalidomide, R = rituximab, O = ofatumumab, TLS = tumor lysis syndrome, TFR = tumor ﬂare reaction, NR = not reported, ITT= intention-to-treat population.
* estimated 2 years PFS
a Treatment-naïve, 7 fatalities reported.
b No grade 3/4 TLS, grade 1/2 not reported.
c One fatality.
d Protocol was amended; initial dose of lenalidomide 10 mg per day with 5 mg dose escalations to a target of 25 mg caused severe toxicities (TLS, fatal sepsis) in the ﬁrst 2
e Protocol was amended; initial dose of lenalidomide 25 mg caused TLS in 3 patients, 1 death and 1 grade 3 neutropenia with sepsis.
f Not including 3 patients who developed TLS with initial dose of 25 mg per day.
g Ten patients evaluated for response.
h Three fatalities.
I 58% had stable disease.
j A total of 80% had del(17p).
k Protocol was amended; initial dose was 25 mg per day that caused TLS in 2 patients.
l Averaged per cycle of treatment; 53% incidence of any grade.
m Overall survival rate 73% are alive with a median follow-up time of 14 months.
n Two fatalities during treatment and 6 deaths occurred after progression of disease during subsequent therapies.
o Plus 12% nodular PR.
p Twenty-six patients accrued, data of 16 were reported.
January 2012, Vol. 19, No. 1 Cancer Control 57
relapsed and refractory CLL. Of the 44 patients included were seen in 65%, 33%, and 9.6% of the patients, respec-
in this trial, 45% had advanced Rai stage (III and IV), 59% tively. TLS occurred in only 2 patients receiving the low-
had unfavorable cytogenetics, and 27% were refractory est dose of lenalidomide. However, 23 patients developed
to prior ﬂudarabine treatment. In this well-structured grade 1/2 TFR, and 5 patients developed grade 3 TFR.
study, plasma levels of angiogenic factors, inﬂammatory The best response obtained was PR and stable disease.
cytokines, and cytokines receptors at baseline on day 7 Six of the 52 patients had a PR, with a median time to
and day 28 were measured. In addition of showing an response of 18.7 weeks. Stable disease was seen in 58%
OR rate of 32%, with a CR rate of 7% (3 patients) CR and of the patients, regardless of lenalidomide dose (eg, 7 of
a 25% rate of PR (10 patients), the authors also demon- 30 patients with SD received the lowest dose, 2.5 mg).
strated an association between lenalidomide and im- These outcomes should be evaluated in the context of
mune activation, exempliﬁed by changes in the levels of a heavily pretreated population with high-risk features,
TNF-α and its soluble receptor TNF-R1, and increased where stable disease or PR might be signiﬁcant in the
levels of IL-6, IL-10, and IL-2. None of the 44 patients absence of alternative agents.
in this group had TLS; however, the incidence of TFR Similarly, after adjusting for dose and allowing for
at any grade was higher in patients with lymph nodes slow dose escalation, Chen et al32 accrued 25 treatment-
larger than 5 cm (53%) and had no correlation with the naive patients. Of these, 32% had 17p or 11q deletion,
OR rate. When comparing results of Chanan-Khan et 60% had ZAP70+, and 75% had unmutated IgVH. Mature
al35 with those of Ferrajoli et al,14 the incidence of grade data from this treatment-naive group of CLL patients
3/4 neutropenia was 70% vs 41%, respectively, as well were recently published. The hematologic toxicities
as 11% for grade 1/2 in the Ferrajoli study. The Chanan- included grade 3/4 neutropenia in 72%, with 5 cases
Khan study used a higher dose of lenalidomide of 25 of febrile neutropenia, and 28% developed grade 3/4
mg on a schedule of 3 weeks on and 1 week off, and the thrombocytopenia. A high rate of skin reactions (64%)
Ferrajoli study used 10 mg daily for 28 days as a starting was reported, described as maculopapular rash, nodular
dose. At this time, it is still not clear whether continuous and urticarial. Fatigue was also a predominant side ef-
vs intermittent exposure offers a better control of the fect (72%). TFR was evident in 88% of patients, and they
disease. Further studies with different schedules — 3 were treated with corticosteroids; however, no TLS was
weeks on and 1 week off vs 28 consecutive days using reported. A PR was reported in 56%, and 40% had stable
different doses — are needed to establish a better toler- disease. One patient progressed during treatment and
ated toxicity proﬁle. died of Richter’s transformation. The estimated 2-year
Sher et al39 reviewed the cases of relapsed/refractory OS rate was 92% (95% CI, 81%–100%), and the PFS rate
CLL patients with high-risk cytogenetics who were in- was 89% (95% CI, 74%–100%).
cluded in the phase II clinical trial of Chanan-Khan et al.35 While phase II data from Chanan-Khan et al40 and Fer-
Patients with del(11q)(q22.3) or del(17p)(p13.1) received rajoli et al14 proposed that doses up to 25 mg offer clinical
single-agent lenalidomide until disease progression, when activity, a recent report documented serious outcomes
rituximab was added. Durable responses with a median with higher doses of lenalidomide.22 Four patients with
PFS of 12.1 months were reported, with a clinical re- relapsed/refractory CLL were treated with lenalidomide
sponse reported in 6 of 16 patients (38%; 95% conﬁdence at 25 mg daily for 21 days in a 28-day cycle. Unacceptable
interval [CI], 15%–65%). Three patients achieved a CR toxicity was reported in 3 of the 4 patients, with 1 death
(19%) and 3 achieved a PR (19%). This subgroup analysis and serious TFR that required acute hospitalization in 2
conﬁrmed the data presented by Ferrajoli et al14 showing patients. The fourth patient developed sepsis and pulmo-
lenalidomide to have durable response in patients with nary and renal complications. These small series contrast
high-risk cytogenetics, with an OR rate of 52%. with data from earlier phase II trials14,40 discussed above.
Early-phase trials provoked signiﬁcant changes in the In vitro data also showed that lenalidomide-induced B-
dosage of lenalidomide. A phase I study in relapsed or cell activation corresponds to the degree of tumor ﬂare,
refractory CLL34 and a phase II study in treatment-naive showing a relationship between B-cell activation and
patients had protocol amendments secondary to fatali- lenalidomide toxicity.
ties, particularly TLS and neutropenic sepsis.32 Signiﬁcant Considering the toxicities associated with higher
side effects were reported with the initial doses (10 mg doses of lenalidomide, Maddocks et al33 presented at the
vs 25 mg), when 5 out of 18 patients developed TLS re- 2009 meeeting of the American Society of Hematology
sulting in 2 fatalities, thereby causing adjustments in the the preliminary data from a small phase I dose escalation
protocol.34 The redesigned phase 1 study34 included 52 study of lenalidomide in patients with relapsed/refracto-
patients, of whom 69% had bulky disease and 48% had ry CLL. In this trial, 14 patients received lenalidomide 2.5
high-risk disease [del(17p) and/or del(11q)]. A dose of mg per day escalated to 15 mg per day or to a maximal
2.5 mg was then used, allowing dose escalation with a tolerated dose of 25 mg (after an amendment was done
maximum of 25 mg daily after 28 days. Hematologic to the initial starting dose of 25 mg due to signiﬁcant
grade 3/4 neutropenia, thrombocytopenia, and anemia toxicity associated with this dose). Dose-limiting tox-
58 Cancer Control January 2012, Vol. 19, No. 1
icities included grade 4 thrombocytopenia in 1 patient, lenalidomide were studied to analyze the lymphocyte
grade 3/4 neutropenia in 2 patients, and grade 2 TFR in populations in the bone marrow and peripheral blood.
1 patient. TLS prophylaxis with dexamethasone was Further analysis of these data suggests that lenalidomide
incorporated in an effort to improve safety. The overall induces a functional reconstitution of the lymphocytes
effect of this dose adjustment will be evident once ma- in the peripheral blood and bone marrow, which might
ture data are available. be the key phenomenon necessary for the antileukemic
Aue et al13 reported a further change in the adminis- effect of lenalidomide.41
tration of lenalidomide, which was given in pulse dosages A review of these trials highlights the two particular
for 3 weeks followed by 3 weeks off. In this phase II nonhematologic toxicities of lenalidomide, which are
trial, high-risk patients with a poor prognosis were repre- also seen with the parent drug thalidomide. TLS and TFR
sented: 52% were Rai stage III-IV, 43% had del(17p), 15% occurred regardless of the dose, schedule, or Rai stage
had del(11q), 70% had bulky disease, 56% were ZAP70+, of CLL patients. Both were closely monitored in all the
and 64% expressed unmutated IgVH genes. Initial dose clinical trials presented. Using higher doses, TLS oc-
was reduced from 20 mg to 10 mg due to the toxicities curred in 2 of 45 patients, causing 1 fatality, and TFR
observed in other lenalidomide trials. Grade 3/4 neutro- occurred in 58% of patients.40 Most of the clinical data
penia, thrombocytopenia, and anemia occurred in 56%, showed that lenalidomide was associated with a sim-
30%, and 15% of cycles, respectively. While TLS was not ilar toxicity proﬁle regardless of the dose used. It is
seen, an increase in grade 3 deep venous thrombosis was important to emphasize the presentation of TFR since
reported in 5 patients. Infection complications included it can be mistaken for progression of disease. Fever,
grade 3 cytomegalovirus colitis, Pneumocystis carinii enlarged or worsening lymphadenopathy, and rash are
pneumonia, and candidemia. One patient died of strep- part of the constellation of symptom associated with
tococcal sepsis. Conversely, the hypothesis of achieving a TFR. In order to improve pain control in TFR, the use of
safer and more tolerable toxicity proﬁle was not obtained prophylactic corticosteroids, NSAIDs, or allopurinol was
with this alternative regimen since TFR was observed in administered to patients enrolled in the trials with good
78%, 48%, 38%, and 30% in cycles 1, 2, 3, and 4, respec- response.13,31-34 Among the hematologic toxicities, myelo-
tively. Among the patients evaluated for response, 16% suppression seems to be the most common side effect
achieved a PR, 58% showed stable disease, and 26% had associated with the use of this immunomodulatory agent.
progressive disease. Conversely, patients with del(17p)
and bulky disease appeared to have a remarkable PR rate Lenalidomide Plus Rituximab
of 80%; however, 27% of patients could not complete Since lenalidomide offers an OR rate of 32% to 47%,14,35
beyond 4 cycles. two trials were conducted to evaluate its combination
In 2010, Badoux et al31 designed a phase II trial with with rituximab in patients with relapsed or refractory
lenalidomide for elderly treatment-naive patients. Since CLL. Ferrajoli et al36 treated patients with rituximab
patients older than 65 years of age typically present with weekly for 1 cycle and then once every 4 weeks during
signiﬁcant comorbidities or borderline performance cycles 3 to 12 combined with lenalidomide at the dose
status, front-line chemotherapy or myelosuppressive of 10 mg per day starting on day 9 of cycle 1 and con-
therapy can be challenging. Taking these factors into tinuing daily for 12 cycles. Data suggest that this com-
consideration, lenalidomide was administered daily at 5 bination offers a superior treatment when compared to
mg and could be titrated up by 5 mg every 28 days to single-agent lenalidomide, with PR and OR rates of 39%
25 mg daily. Eighteen of the 60 patients had Rai stage III and 64%, respectively. Complete response was seen in
and IV disease, 33% had unfavorable cytogenetics (17p 8% of the subjects. The toxicity proﬁle was similar to
deletion or 11q deletion), and 55% had unmutated IgVH. single-agent lenalidomide, with hematologic complica-
Grade 3/4 hematologic toxicities included neutropenia tions and fatigue as common side effects. Grade 3 and
and thrombocytopenia in 34% and 12% of the cycles, 4 neutropenia and thrombocytopenia were reported in
respectively, and anemia was seen in < 1%. Only grade 68% and 22% of patients, respectively. Only 1 patient had
1/2 TFR was reported in 52% of patients. The presence of grade 3 TLS but 22 patients (37%) had grade 1/2 TFR.
17p deletion was associated with shorter PFS (statistically Our group recently presented preliminary data of
signiﬁcant) compared with other cytogenetic abnormali- a phase II clinical trial with the same combination.37
ties (median PFS 6 months vs not reached). There was a This study, which included patients with relapsed or
high tendency to achieve CR in patients with unmutated refractory mantle cell lymphoma and CLL, allowed dose
IgVH; however, this was not statistically signiﬁcant (P = escalation of the lenalidomide in 28-day cycle and weekly
.07). Seven fatalities occurred: 1 patient developed Rich- rituximab at 375 mg/m2 for 4 weeks starting on day 15
ter’s transformation and 2 died of unrelated malignancies. of cycle 1. Interim analysis showed that 42% of the pa-
This study showed that lenalidomide offered an OR rate tients with CLL had a PR and 50% had stable disease,
of 65%, including a CR rate of 10% and a PR rate of 43%. with a median duration of response of 18 and 12 months,
The results of 34 patients who completed 15 cycles of respectively. Unpublished data from this small trial
January 2012, Vol. 19, No. 1 Cancer Control 59
showed the same hematologic toxicities, predominantly with this incurable leukemia. Lenalidomide has been
a 36.3% rate of grade 2/3 neutropenia. TFR and TLS were shown to be an active IMiD that offers a signiﬁcant clinical
reported at 27% and 4.5%, respectively. response in patients with CLL who are heavily pretreated
Based on these two small studies, the combination of and carry unfavorable features. While lenalidomide has
lenalidomide and rituximab appears to offer a promising been shown to be an attractive alternative in CLL due
synergistic effect. Mature data and further studies are to its particular effect in the tumor microenvironment,
needed to conﬁrm and validate OS and PFS. larger studies are being conducted to improve our un-
derstanding of its mechanism of action. These promising
Lenalidomide Plus Ofatumumab data will also help evaluate the most effective dose and
The initial results of a phase II study evaluating the ef- schedule of this agent. In addition to its use in treating
ﬁcacy and tolerability of the combination of lenalido- CLL, lenalidomide is being explored as a novel agent for
mide and ofatumumab in patients with relapsed CLL other hematologic and solid tumor malignancies.
was presented at the ASH 2010 meeting.38 Treatment
consisted on ofatumumab administered intravenously Active Immunotherapy
on a weekly basis for 4 weeks (300 mg in week 1, 1,000 CLL is characterized by immune deﬁciency with both
mg in week 2 and all subsequent doses), then monthly humoral and T-cell functional defects, thus representing
for months 2 through 6 and once every 2 months for a potential model in which to study immunotherapeu-
months 7 through 24. Lenalidomide was given at a dose tic approaches. Its slow growth allows time to gener-
of 10 mg daily starting on day 9 and continuing daily with ate an immune response against the tumor cells, and
a treatment duration of 24 months. Results for the ﬁrst tumor cells are easy to obtain in large numbers from
16 out of 40 planned patients who were on study for at the peripheral blood. Furthermore, B-CLL cells express
least 3 months have been reported. Four patients (25%) major histocompatibility complex (MHC) class I and II
were refractory to ﬂudarabine and all were previously in addition to the idiotype (Id), a tumor-speciﬁc epitope
treated with rituximab. Ten of 16 evaluable patients of the immunoglobulin B-cell receptor. Moreover, the
achieved a response: 2 CRs (13%) and 8 PRs (50%) for an chromosome abnormalities commonly present in CLL
OR rate of 63%. Four patients with stable disease were may encode altered self-proteins that could serve as target
continuing on treatment. The most common grade 3/4 antigens for immune recognition.43-46 Other observations
treatment-related adverse events were neutropenia (8 that support the use of immunotherapy in CLL include
patients, 50%) and anemia (2 patients, 13%). TFR was the physical association of CLL and T cells in secondary
limited to grade 1 in 2 patients (13%). Data from this lymphoid organs, which assures interactions between
study suggested that the combination of lenalidomide tumor-reactive T cells and tumor cells,47 spontaneous
and ofatumumab was well tolerated and is a therapeuti- remissions associated with heightened immune activity
cally active combination for patients with relapsed CLL. following viral infections,48 clinical responses following
treatment with immunomodulatory cytokines,49 and long-
Lenalidomide Plus Chemotherapy term disease-free survival after allogeneic bone marrow
In an effort to improve the efﬁcacy of IMiDs and obtain transplantation,50 possibly from a T-cell–mediated graft-vs-
better outcomes with their use, Brown et al42 initiated leukemia effect.51 Immune control of tumors, including
a small phase I trial that combined lenalidomide with CLL, is believed to be mediated mainly by CTLs that rec-
ﬂudarabine and rituximab for untreated patients with ognize tumor antigens. Cytotoxic CD8+ T cells are mainly
CLL. Low doses of lenalidomide (2.5 mg) were adminis- responsible for the destruction of epithelial tumors, but
tered daily for 21 days in 28-day cycles, with ﬂudarabine both CD4+ and CD8+ CTLs may kill B-cell tumors.52 The
25 mg/m2 on days 3 through 5 and rituximab 375 mg/ purpose of cancer vaccines is to increase the number
m2 on day 1. Due to signiﬁcant cytopenias and myalgias of these tumor-reactive CTLs and maintain their activity
associated with elevated creatine phosphokinase (CPK), long enough to clear tumor cells. In theory, cancer vac-
a side effect evident early in the trial, the lenalidomide cines should both promote tumor clearance and prevent
administration was reduced to every other day. Regard- relapse by providing long-term antitumor immunity.
less of the dose adjustment, persistent toxicities of my- A successful cancer vaccine requires tumor-associ-
elosuppression and TFR led to an early closure of the ated antigens (TAAs) in the cancer cell that can be pre-
trial. A 56% response rate was evaluated in 5 of the 9 sented with appropriate costimulatory signals to T cells
patients who had objective responses by intention to able to respond to these antigens. Activation of T cells
treat basis. Nevertheless, the results from this negative requires two signals delivered by antigen-presenting cells
trial showed the ﬁrst attempt to combine lenalidomide (APCs). The ﬁrst signal is mediated by the antigen MHC
with chemo-immunotherapy. interacting with the T-cell receptor; the second signal is
All these provocative trials have emerged in an effort provided by costimulatory molecules expressed by the
to improve outcomes, develop non-chemotherapy treat- APCs, such as IL-2, CD80, or CD86, that bind to CD28
ment options, and discover better alternatives for patients on the T cell. Dendritic cells (DCs) and macrophages
60 Cancer Control January 2012, Vol. 19, No. 1
are potent professional APCs in that they are capable of More recently, increased frequencies of CD4+/CD25hi
providing costimulatory signals. A major goal in tumor regulatory T cells (Treg cells) have been described as
immunotherapy is to mount a systemic CTL response of an additional mechanism that reduces immunity. A
the tumor-bearing host against TAAs. A number of TAAs study of 73 patients with B-cell CLL demonstrated sig-
have been identiﬁed and overexpressed in CLL, and many niﬁcantly increased frequencies of CTLA4+, Forkhead
functional studies have been conducted to verify the ex- box P3 (FOXP3+), glucocorticoid-induced TNF receptor-
istence of naturally occurring reactive T cells. These TAAs related protein (GITR+), CD62L+, TGF-β1+, and IL-10+
include ﬁbromodulin,53,54 the receptor for hyaluronic Treg cells in patients with CLL. The inhibitory function
acid-mediated motility (RHAMM/CD168),55 murine dou- of Treg cells was decreased or even abrogated in the
ble-minute 2 oncoprotein (MDM2),56 telomerase reverse majority of patients treated with regimens containing
transcriptase (hTERT),57 the oncofetal antigen-immature ﬂudarabine or cyclophosphamide, suggesting that the
laminin receptor protein (OFAiLRP),58 adipophilin,59 sur- use of these agents to reduce immunosuppression prior
vivin,60 KW1 to KW14,61 and the tumor-derived IgVH- to cancer immunotherapy may be a promising strategy.73
CDR3 region,62,63 which is speciﬁcally expressed by
the tumor cells as surface membrane immunoglobulins Vaccination Approaches
sharing idiotypic determinants. Tumor-associated CLL Multiple vaccination approaches as active immuno-
antigens are likely encoded by genes that are mutated or therapy for CLL have been investigated, including gene
overexpressed during oncogenesis and may be unique therapy, DC-based vaccines, whole modiﬁed tumor cell
to each patient. Despite intense research efforts, ac- vaccines (eg, trioma cell vaccines), tumor-speciﬁc Id vac-
tive immunotherapy has achieved limited success, in cines, and TAA-derived peptide vaccines.
part because of the uniqueness of CLL antigens that are
patient-speciﬁc, thus requiring an individually prepared Gene Therapy
vaccine for each patient. Several potential reasons may Since TAAs have not been molecularly identiﬁed in most
explain why patients fail to mount an effective T-cell–me- cases, vaccination protocols have been developed using
diated immune response against their disease. Despite whole autologous tumor cells genetically modiﬁed to
having a normal expression of MHC class I and class II express cytokines or costimulatory surface molecules.
molecules on the cell surface, B-CLL cells are poor APCs The transfer of an immunostimulatory gene into malig-
as they lack costimulatory and cell-adhesion molecules nant tumor cells and the use of these autologous cells
such as CD80, CD86, and CD54, which are essential in as vaccine have been extensively investigated in B-cell
producing an effective T-cell response.64 Additionally, malignancies. The immunostimulatory genes studied
upregulation of MHC class I expression by B-CLL cells include IL-2, IL-12,TNF-α, and granulocyte-monocyte col-
in response to interferon gamma (IFN-γ) was reduced. ony-stimulating factor (GM-CSF), as well as gene-encoding
This relative MHC class I expression defect of B-CLL cells immune accessory surface molecules such as CD80 and
may reduce their susceptibility to CTL lysis in response CD40-ligand (CD154).74
to immunotherapeutic approaches.65 In addition, CLL The malignant B-CLL cells express a range of tumor-
cells were resistant to FAS (CD95) ligand-mediated apop- associated and tumor-speciﬁc antigens, as well as high
tosis in vitro.66 Functional T-cell abnormalities have also levels of MHC class I and II molecules; however, they lack
been described, including inversion of the CD4/CD8 costimulatory molecules and are ineffective APCs. Their
ratio with an increase in absolute numbers of activated immunogenicity can be increased by manipulation of the
CD4 and CD8 cells.67,68 Additionally, there is an altered CD40/CD40 ligand (CD40L) pathway, in which CD40L
production of cytokines including IL-4, IFN-γ, as well interacts with CD40 on B-CLL target cells to increase
as downmodulation of CD154 (CD40 ligand), the zeta their antigen-presenting capacity through upregulation
chain of the T-cell receptor, and the costimulatory mol- of the costimulatory molecules CD80 and CD86 as well
ecule CD28.69,70 Furthermore, CLL cells are known to as adhesion molecules such as CD54.75,76 One such ap-
secrete transforming growth factor beta (TGF-β), a factor proach involves the introduction of CD40L into CLL
known to have potent immunosuppressive functions.71 cells. Several strategies can be utilized to accomplish this:
Gene expression proﬁles of peripheral blood T cells the use of adenovirus, recombinant adeno-associated
from previously untreated patients with B-CLL revealed virus, or herpes simplex virus vectors, as well as the
differentially expressed genes, mainly involved in cell molecular transfer from ﬁbroblasts that overexpress the
differentiation in CD4 cells and defects in cytoskeleton ligand, a nonviral electroporation-based gene delivery
formation, vesicle trafﬁcking, and cytotoxicity in the CD8 system, and a standard plasmid-carrying CD40L cDNA.77-81
cells of these patients.72 Suppressed T-cell function has CD40L also induces DC maturation in vivo, increasing
been described as a major hurdle for the development their ability to take up and process antigens.82 A phase
of clinically efﬁcient cancer immunotherapy. Inhibi- I clinical trial was performed to evaluate the response
tion of antitumor immune responses has been mainly to intravenous administration of autologous CLL B cells
linked to inhibitory factors present in cancer patients. transduced with a gene encoding murine CD154 us-
January 2012, Vol. 19, No. 1 Cancer Control 61
ing a replication-defective adenovirus vector.76 Eleven and can mitigate the resistance of p53-deﬁcient CLL cells
patients with progressive intermediate or high-risk CLL to anticancer drug therapy.
by the modiﬁed Rai criteria were enrolled in the study. Human interleukin 2 (hIL-2) has been shown to fur-
Four of the patients were previously treated with che- ther potentiate the immunogenicity of human CD40
motherapy. After a one-time bolus infusion of autologous ligand (hCD40L) in preclinical murine models.77 An
Ad-CD154-transduced leukemia cells, there was increased early-phase vaccination study of autologous B-CLL cells
or de novo expression of immune-accessory molecules that expressed both hCD40L and hIL-2 was conducted
on bystander, noninfected CLL cells in vivo. Patients in 9 patients who received 3 to 8 subcutaneous vaccina-
also developed high plasma levels of IL-12 and IFN-γ, as tions.85 The vaccine was administered without evidence
well as increased numbers of leukemia-speciﬁc T cells of signiﬁcant local or systemic toxicity. A B-cell CLL-
as demonstrated by an autologous enzyme-linked immu- speciﬁc T-cell response was detected in 7 patients. Three
nosorbent spot (ELISPOT) assay and mixed lymphocyte patients produced leukemia-speciﬁc immunoglobulins.
reactions. These biological effects were associated with Three patients had greater than a 50% reduction in the
reductions in lymphocyte count, spleen size, and lymph size of affected lymph nodes, although these responses
node size. The infusion was well tolerated; however, were transient. High levels of circulating CD4+/CD25+/
patients developed not only antileukemic immune re- LAG-3+/FoxP-3+ immunoregulatory T cells (Tregs) were
sponses, but also antimurine CD154 antibodies. To avoid present before, during, and after treatment. The authors
this, a phase I study was conducted in which patients speculated that the increase in Tregs might have limited
were infused with autologous CLL cells transduced ex the magnitude and duration of the antileukemic immune
vivo to express ISF35, a humanized, membrane-stable response since in vitro removal of these cells increased
CD154.83 Infusions were well tolerated and consistently the antileukemic T-cell reactivity.
followed by reductions in blood lymphocyte counts and Due to the complexities and expense of manufactur-
lymphadenopathy. After infusion, circulating CLL cells ing viral vectors, as well as their lingering safety concerns,
had enhanced or de novo expression of CD95, DR5, p73, further studies have investigated the transduction of B-
and Bid, which enhanced their susceptibility to death- CLL cells using nonviral gene delivery methods such as
receptor–mediated or drug-induced apoptosis, including electroporation, a physical means of transferring CD40L
CLL cells with del(17p). Two patients who had CLL with and IL-2 plasmids to produce vaccines with similar bio-
del(17p) had subsequent chemoimmunotherapy and logical properties in vitro and in vivo.81 A vaccine clini-
responded well to treatment, suggesting that the vac- cal trial using this strategy was conducted in which 7
cine might enhance the susceptibility of CLL cells with patients received a total of 6 subcutaneous injections
del(17p) to chemoimmunotherapy. of autologous transduced cells using electroporation
To examine the p53 dependency of the acquired in the presence of DNA plasmids encoding hCD40L or
latent sensitivity to Fas-mediated apoptosis following hIL-2.86 Following vaccination, all patients had stable
activation by CD154, investigators examined the in vitro leukemia counts and 1 patient had a transient decrease
responses to CD154 of CLL cells that did or did not have of more than 50% in local adenopathy. However, using
functional p53.84 They found that CD154 induces CLL the National Cancer Institute response criteria, no CRs or
cells to express p53 and the p53-target genes CD95, DR5, PRs were observed. Compared with adenoviral vaccines,
p21, and Bid. Also, CLL cells still lacking functional p53 electroporation generally provides a simpler and more
were induced to express Bid and to acquire sensitivity rapid means of preparing IL-2/CD40L-expressing B-CLL
to CD95-mediated apoptosis following co-culture with vaccines, but the cells express higher levels of IL-2 and
CD154-bearing cells. Such treatment also induced p73, lower levels of secondary costimulator molecules.87 Ad-
a p53-related transcription factor regulated by c-Abl ki- ditionally, the administration of the transduced cells via
nase, and enhanced the sensitivity of CLL cells lacking subcutaneous injection limited the number of CLL cells
functional p53 to ﬂudarabine. The transduction of CLL that could be exposed to the transfected CLL cells, there-
cells with an adenovirus encoding p73 also induced Bid by minimizing the bystander effect seen with intravenous
and CD95 and enhanced the sensitivity of p53-deﬁcient infusion of autologous adenovirus-CD40L–transduced
CLL cells to ﬂudarabine. However, inhibition of c-Abl CLL cells.74 A second clinical trial is currently evaluating
with imatinib suppressed CD154-induced expression of a prolonged vaccination using 18 deltoid injections of
p73, p73-induced expression of Bid, and CD95 and also this vaccine strategy over 52 weeks (NCT00458679).88
blocked the sensitization of p53-deﬁcient CLL cells to IL-12 is a potent cytokine that stimulates T cells
CD95-mediated or ﬂudarabine-induced apoptosis. Con- and natural killer cells. Animal studies of vaccination
versely, CLL cells transduced with an imatinib-resistant using a murine B lymphoma cell line transfected with a
c-Abl mutant could be induced by CD154 to express replication-defective retrovirus encoding IL-12 induced
p73 and Bid even when treated with imatinib. These T-cell–mediated antitumor immunity in mice.89 TNF-α
results indicate that CD154 can sensitize leukemia cells is a cytokine that can directly induce apoptosis of some
to apoptosis via the c-Abl–dependent activation of p73 tumors and can stimulate DC maturation and function.
62 Cancer Control January 2012, Vol. 19, No. 1
Given the systemic toxicity of TNF-α, gene therapy strate- CD4- and CD8-mediated T-cell responses, suggesting that
gies that allow local expression of TNF-α in the tumor mi- that this approach might be a potent new strategy in the
croenvironment are being developed. The costimulatory treatment of CLL.95 Based on a study showing several
molecules CD80 and CD86 bind to CD28 on T cells and different clones of leukemia-reactive CD4 and CD8 cells
provide a second positive signal to T cells for activation present in CLL patients,96 it has been speculated that
and proliferation. Animal studies with transduced cell a vaccination approach using whole tumor cells was
lines have conﬁrmed in vivo activity.90,91 GM-CSF is a he- preferable to a single deﬁned antigen. Additionally, this
matologic growth factor that also induces DC maturation approach minimizes the potential selection of tumor
and activation. In vitro studies of murine T-cell leukemia antigen escape variants.97 Compared to using DCs alone,
transduced with retrovirus-encoding GM-CSF induced the use of DCs electrofused with CLL B cells (fusion
tumor-speciﬁc immunity. However, none of these vac- hybrids) produced higher levels of speciﬁc cytotoxic T-
cine approaches have been investigated in clinical trials cell responses to tumor cells. Additionally, the cytokine
of CLL patients. response induced by Apo-DCs (DCs that had endocyted
An alternative vaccine strategy, called TRICOM, was CLL apoptotic bodies) was signiﬁcantly higher than
created to enhance the immunogenicity of CLL cells that induced by DCs fused with tumor cells. This study
via infection with vectors encoding for three essential showed that endocytosed apoptotic tumor cells induced
costimulatory molecules: CD80, lymphocyte function- a signiﬁcantly stronger T-cell response than DC hybrids
associated antigen 3 (LFA-3), and intercellular adhesion induced, making this strategy a better candidate for vac-
molecule 1 (ICAM-1). A recombinant-modiﬁed vaccinia cine production.98 A clinical study evaluated the poten-
virus strain Ankara (MVA), which is a highly attenuated, tial of allogeneic monocyte-derive DCs obtained from
replication-impaired virus variant, was successful in in- normal donors pulsed ex vivo with tumor cell lysates or
fecting and delivering the simultaneous expression of apoptotic bodies to stimulate antitumor immunity in pa-
the three human costimulatory molecules in TRICOM on tients with B-CLL in early stages.99 Nine patients with Rai
the surface of CLL cells. Cytotoxic T lymphocytes, gen- stage 0 and 1 CLL were vaccinated 5 times with a mean
erated in vitro by stimulation of autologous T cells with number of 32 × 106 stimulated DCs administered intra-
MVA-TRICOM–infected CLL cells, showed cytotoxicity dermally once every 2 to 3 weeks. No signs of autoim-
against unmodiﬁed, uninfected CLL cells. These ﬁndings munity were detected, and only mild local skin reactions
suggest that the use of CLL cells infected ex vivo with were noted. A decrease in peripheral blood leukocytes
MVA-TRICOM or via direct injection of MVA-TRICOM in and CD19+/CD5+ leukemic cells was observed during
patients with CLL had potential for the immunotherapy the treatment period. A signiﬁcant increase of speciﬁc
of CLL.92 Furthermore, following incubation with irradi- cytotoxic T lymphocytes against RHAMM/CD168 was
ated MVA-TRICOM–modiﬁed CLL cells, allogeneic and detected in 1 patient after DC vaccination. A second cel-
autologous CD4+ and CD8+ T cells expressed signiﬁcantly lular vaccination study was conducted by the same group
higher levels of CD80, ICAM-1, and LFA-3. This increase using autologous DCs pulsed ex vivo with tumor cell
was shown to be the result of physical acquisition from lysates.100 Twelve patients with early-stage CLL received
the APCs, and puriﬁed T cells that acquired costimulatory up to 8 intradermal vaccinations. Five patients showed
molecules from MVA-TRICOM–modiﬁed CLL cells were a decrease in peripheral blood leukocytes and CD19+/
able to stimulate the proliferation of untreated T cells. CD5+ leukemic cells, 3 showed a stable disease, and 4
These results demonstrated for the ﬁrst time that T cells progressed despite DCs vaccination. A signiﬁcant in-
from CLL patients can acquire multiple costimulatory mol- crease in speciﬁc cytotoxic CD8+ T lymphocytes against
ecules from autologous CLL cells and can then act as APCs the leukemia-associated antigens RHAMM or ﬁbromodu-
themselves. Given the immunodeﬁciencies characteristic lin was detected in 4 patients following DC vaccination.
of CLL, enhancing the antigen-presenting function of CLL In patients with a clinical response, an increase of IL-12
cells and T cells simultaneously could be a distinct advan- serum levels and a decrease of the frequency of CD4+/
tage in the effort to elicit antitumor immune responses.93 CD25+/FOXP3+ T regulatory cells were observed. These
Clinical trials evaluating this vaccine approach for patients results justify further investigation of this immunothera-
with CLL have not yet been conducted. peutic approach. A phase I/II clinical trial using Apo-DC
vaccination for the treatment of previously untreated CLL
DC-Based Vaccines patients is ongoing.97
As previously discussed, CLL cells are poor APCs. One
way of improving immune response to antigens is to use Whole Modiﬁed Tumor Cell-Based Vaccines
more powerful APCs such as DCs. This strategy involves The difﬁculty in ﬁnding appropriate tumor antigens has
loading DCs with peptides derived from TAA, tumor ly- led to innovative vaccination approaches in CLL. The use
sates, RNA, or DNA from tumors or fusing DCs with tumor of polyvalent cellular vaccines using whole modiﬁed tu-
cells.94 The use of DCs transfected with in vitro ampliﬁed mor cells would allow multiple antigens to be ingested by
B-CLL mRNA elicited both HLA class I and II CLL-speciﬁc APCs. The most potent strategy is the trioma approach,
January 2012, Vol. 19, No. 1 Cancer Control 63
which is based on immunization with lymphoma cells sociated with enhanced antitumor T-cell activity in vitro
modiﬁed to express an antibody against an internaliz- were observed in 5 of the 18 patients. Stable disease was
ing and activating surface molecule (Fc receptor) on observed in 6 patients, and disease progression appeared
APCs.101 This approach is based on redirection of the to be unaffected in the remaining patients. Toxicity was
tumor-speciﬁc immunoglobulin Id toward professional minimal. This vaccination method appears worthy of
APCs, thereby overcoming the inefﬁcient presentation further investigation and may be a potential alternative
on the parental transformed B cell. In this method, ma- to a “watch and wait” strategy for selected CLL patients.105
lignant B cells are fused to a xenogeneic hybridoma cell In further attempts to increased immunogenicity of
line that secretes an antibody against a surface receptor CLL cells, a phase I clinical study of intradermal vaccina-
of APCs, resulting in trioma cells. These cells express tion with irradiated autologous CLL cells with Bacillus
tumor-derived antigens and have anti-APC speciﬁcity. The Calmette-Guérin (BCG) as an adjuvant was conducted
trioma cell binds to the Fc receptor of an APC, resulting in in 17 patients with previously untreated early-stage CLL.
uptake, processing, and presentation of the Id. In a mouse The investigators hypothesized that inducing apoptosis
model, vaccination with trioma cells conferred long- in irradiated leukemic cells would increase the anti-
lasting, T-cell–dependent tumor immunity and was able genicity of malignant cells by enhanced presentation of
to eradicate established lymphomas.102 In a preclinical tumor antigens, without requiring the precise identiﬁ-
study, malignant cells from 11 patients with B-CLL were cation of antigenic targets.106 Proliferation studies did
fused to an anti-Fc receptor hybridoma.103 In 7 cases, not show any signiﬁcant activation of speciﬁc T cells.
trioma cells could successfully be generated from B-CLL However, hematologic improvement (> 25% reduction
cells. Stimulation of autologous lymphocytes with tri- in leukocyte count) was observed in 5 of the 17 patients,
oma cells induced a leukemia-speciﬁc T-cell response stabilization of disease in 5 patients, and no response to
in vitro. Furthermore, DCs pulsed with trioma cells ef- immunotherapy in 7 patients. Additionally, a signiﬁcant
fectively activated T lymphocytes against CLL in vitro. increase of the lymphocyte doubling time was noted in
In this study, activation of T cells was more pronounced 7 of 9 patients, suggesting that cellular immunotherapy
after stimulation with trioma-pulsed DCs compared with might prolong disease progression and the need for
stimulation with trioma cells in the presence of APCs, chemotherapy. Further investigation using this vaccine
and overexpressed antigens associated with malignant method is warranted.
transformation, such as BCL-2, MDM2, and ETV5, serve as
targets for those T cells.101 Immune escape by antigen Id Vaccines
loss or mutation is less likely to occur if immunity is Normal B cells express an immunoglobulin with unique
directed against altered self-proteins that are involved variable region sequence in the heavy and light chains
in malignant transformation. Therefore, vaccines based that together form the antigen-binding site. During ma-
on modiﬁed tumor cells such as triomas show prom- lignant transformation, this Id is maintained by the ma-
ise for immunotherapy of CLL and other malignancies. lignant clone and therefore can be regarded as a TAA.
Polyvalent vaccines originally designed as individualized Animal studies have shown an effective humoral and
therapeutics may be more broadly applicable, at least cellular mechanism against Ids at inducing tumor regres-
in patients showing similar antigen patterns. Clinical sion.107 A potential limitation of this approach is the fact
studies will determine the effectiveness and safety of that Id vaccines must be custom-made for each patient.
trioma-induced immunity. Furthermore, one study showed that CTL responses gen-
A different approach that used altered tumor cells as erated against naive immunoglobulin-derived peptides
an antigen source was investigated by subjecting blood were weak.62 These limitations may explain why no
from 25 patients with CLL to a combination of oxida- clinical trials have been reported in patients with CLL
tive physicochemical stressors in a blood treatment unit. using this approach.
It was hypothesized that this treatment would release
antigen-binding heat shock proteins and free radicals that TAA-Derived Peptide Vaccines
would activate APCs and increase the immunogenicity Generating a T-cell–mediated response targeted at the
of the CLL cells in vivo. The treated blood sample was TAA represents a novel therapeutic approach for patients
intramuscularly reinjected into the patients twice weekly with CLL. Vaccination with TAA-derived peptides might
for 6 weeks.104 After vaccination, an increase in CLL- allow exact monitoring of T-cell responses to these par-
reactive T-cell levels was seen in patients with preexisting ticular antigens by different methods including ELISPOT
CLL-reactive T cells, and there was an inverse correlation assays, ﬂow cytometry analysis of intracellular IFN-γ, or
between disease stage and anti-CLL T-cell reactivity. A tetramer staining.108 As previously noted, several TAA
subsequent phase I/II clinical trial was conducted to have been identiﬁed and are overexpressed in CLL. En-
evaluate the feasibility, safety, and efﬁcacy of autologous couraging results in vitro, as well as the safety, feasibil-
vaccines made from oxidized tumor cells in 18 patients ity, and economic advantage of peptide vaccination in
with earlier-stage CLL.105 Partial clinical responses as- patients with other hematologic malignancies, provided
64 Cancer Control January 2012, Vol. 19, No. 1
a solid basis to start clinical peptide vaccination trials cytes. J Exp Med. 1991;173(3):699-703.
8. Pawelec G, Schaudt K, Rehbein A, et al. Differential secretion of
in B-CLL. Among the identiﬁed TAAs in CLL, RHAMM/ tumor necrosis factor-alpha and granulocyte/macrophage colony-stimulating
CD168 has already been tested in clinical trials. The factors but not interferon-gamma from CD4+ compared to CD8+ human T
cell clones. Eur J Immunol. 1989;19(1):197-200.
tumor-restricted expression of RHAMM/CD168 was ﬁrst 9. Moreira AL, Sampaio EP, Zmuidzinas A, et al. Thalidomide exerts its
described in myeloid malignancies, multiple myeloma, inhibitory action on tumor necrosis factor alpha by enhancing mRNA degra-
and breast cancer.109-112 It plays a role in the formation of dation. J Exp Med. 1993;177(6):1675-1680.
10. List A, Kurtin S, Roe DJ, et al. Efﬁcacy of lenalidomide in myelodys-
the mitotic spindle apparatus and in the signal transduc- plastic syndromes. N Engl J Med. 2005;352(6):549-557.
tion cascade Ras-Raf-MEK-ERK.109 Following the detec- 11. Muller GW, Chen R, Huang SY, et al. Amino-substituted thalidomide
analogs: potent inhibitors of TNF-alpha production. Bioorg Med Chem Lett.
tion of speciﬁc CTL responses in B-CLL patients against 1999;9(11):1625-1630.
RHAMM/CD168, a phase I clinical trial of RHAMM-de- 12. Muller GW, Corral LG, Shire MG, et al. Structural modiﬁcations of
thalidomide produce analogs with enhanced tumor necrosis factor inhibitory
rived R3 peptide vaccination was initiated in patients activity. J Med Chem. 1996;39(17):3238-4320.
with previously untreated CLL.113 Six HLA-A2+ CLL 13. Aue G, Soto S, Valdez J, et al. Phase II trial of pulse dosed lenalido-
mide in previously treated chronic lymphocytic leukemia. Blood (ASH An-
patients were vaccinated 4 times at biweekly intervals. nual Meeting Abstracts). 2010;116:1383.
Four patients exhibited reduced white blood cell counts 14. Ferrajoli A, Lee BN, Schlette EJ, et al. Lenalidomide induces com-
plete and partial remissions in patients with relapsed and refractory chronic
during vaccination. In 5 of the 6 patients, R3-speciﬁc lymphocytic leukemia. Blood. 2008;111(11):5291-5297.
CD8+ T cells were detected with the corresponding 15. Corral LG, Haslett PA, Muller GW, et al. Differential cytokine modula-
tion and T cell activation by two distinct classes of thalidomide analogues
peptide/HLA-A2 tetrameric complex. Vaccination was that are potent inhibitors of TNF-alpha. J Immunol. 1999;163(1):380-386.
also associated with the induction of regulatory T cells 16. Davies FE, Raje N, Hideshima T, et al. Thalidomide and immuno-
modulatory derivatives augment natural killer cell cytotoxicity in multiple my-
in 4 patients; however, in this study, no correlation be- eloma. Blood. 2001;98(1):210-216.
tween initial Treg frequencies and either immunologic 17. LeBlanc R, Hideshima T, Catley LP, et al. Immunomodulatory drug co-
stimulates T cells via the B7-CD28 pathway. Blood. 2004;103(5):1787-1790.
or clinical responses was found. Peptide vaccination 18. Galustian C, Meyer B, Labarthe MC, et al. The anti-cancer agents
was safe and could elicit to some extent speciﬁc CD8+ lenalidomide and pomalidomide inhibit the proliferation and function of T
regulatory cells. Cancer Immunol Immunother. 2009;58(7):1033-1045.
T-cell responses against the tumor antigen RHAMM. 19. Motta M, Rassenti L, Shelvin BJ, et al. Increased expression of
CD152 (CTLA-4) by normal T lymphocytes in untreated patients with B-cell
chronic lymphocytic leukemia. Leukemia. 2005;19(10):1788-1793.
Conclusions 20. Hayashi T, Hideshima T, Akiyama M, et al. Molecular mechanisms
Clinical trials using lenalidomide as a single agent or whereby immunomodulatory drugs activate natural killer cells: clinical ap-
plication. Br J Haematol. 2005;128(2):192-203.
in combination with immunotherapy in patients with 21 Dredge K, Horsfall R, Robinson SP, et al. Orally administered lenalido-
relapsed and refractory CLL have shown promising mide (CC-5013) is anti-angiogenic in vivo and inhibits endothelial cell migra-
tion and Akt phosphorylation in vitro. Microvasc Res. 2005;69(1-2):56-63.
response rates. More recently, compelling data from 22. Andritsos LA, Johnson AJ, Lozanski G, et al. Higher doses of le-
phase II trials have demonstrated signiﬁcant activity in nalidomide are associated with unacceptable toxicity including life-threaten-
ing tumor ﬂare in patients with chronic lymphocytic leukemia. J Clin Oncol.
chemotherapy-naive patients. In this setting, lenalido- 2008;26(15):2519-2525.
mide appears to offer a survival beneﬁt and improve PFS. 23. Hideshima T, Chauhan D, Shima Y, et al. Thalidomide and its ana-
logs overcome drug resistance of human multiple myeloma cells to conven-
Even after reviewing and analyzing these provoca- tional therapy. Blood. 2000;96(9):2943-2950.
tive results, the toxicity proﬁle associated with the use 24. Chanan-Khan A, Porter CW. Immunomodulating drugs for chronic
lymphocytic leukaemia. Lancet Oncol. 2006;7(6):480-488.
of lenalidomide makes its use challenging. Because of 25. Verhelle D, Corral LG, Wong K, et al. Lenalidomide and CC-4047
its potential treatment complications, particularly tumor inhibit the proliferation of malignant B cells while expanding normal CD34+
progenitor cells. Cancer Res. 2007;67(2):746-755.
ﬂare reaction and tumor lysis syndrome, this drug should 26. Lapalombella R, Yu B, Triantaﬁllou G, et al. Lenalidomide down-
be used only in a clinical trial setting. Other immuno- regulates the CD20 antigen and antagonizes direct and antibody-dependent
cellular cytotoxicity of rituximab on primary chronic lymphocytic leukemia
therapeutic approaches are also showing some beneﬁt cells. Blood. 2008;112(13):5180-5189.
in patients with high-risk CLL for whom chemotherapy 27. Lapalombella R, Andritsos L, Liu Q, et al. Lenalidomide treatment
promotes CD154 expression on CLL cells and enhances production of anti-
regimens are no longer an option. bodies by normal B cells through a PI3-kinase-dependent pathway. Blood.
28. Stebbing J, Benson C, Eisen T, et al. The treatment of advanced renal
References cell cancer with high-dose oral thalidomide. Br J Cancer. 2001;85(7):953-958.
1. Eriksson T, Björkman S, Roth B, et al. Intravenous formulations of 29. Aragon-Ching JB, Dahut WL. The role of angiogenesis inhibitors in
the enantiomers of thalidomide: pharmacokinetic and initial pharmacody- prostate cancer. Cancer J. 2008;14(1):20-25.
namic characterization in man. J Pharm Pharmacol. 2000;52(7):807-817. 30. Ning YM, Gulley JL, Arlen PM, et al. Phase II trial of bevacizumab,
2. Bartlett JB, Michael A, Clarke IA, et al. Phase I study to determine thalidomide, docetaxel, and prednisone in patients with metastatic castra-
the safety, tolerability and immunostimulatory activity of thalidomide ana- tion-resistant prostate cancer. J Clin Oncol. 2010;28(12):2070-2076.
logue CC-5013 in patients with metastatic malignant melanoma and other 31. Badoux XC, Keating MJ, Wen S, et al. Lenalidomide as initial therapy
advanced cancers. Br J Cancer. 2004;90(5):955-961. of elderly patients with chronic lymphocytic leukemia. Blood. 2011;118(13):
3. Melchert M, List A. The thalidomide saga. Int J Biochem Cell Biol. 3489-3498.
2007;39(7-8):1489-1499. 32. Chen CI, Bergsagel PL, Paul H, et al. Single-agent lenalidomide in
4. Foa R, Massaia M, Cardona S, et al. Production of tumor necrosis the treatment of previously untreated chronic lymphocytic leukemia. J Clin
factor-alpha by B-cell chronic lymphocytic leukemia cells: a possible regulatory Oncol. 2011;29(9):1175-1181.
role of TNF in the progression of the disease. Blood. 1990;76(2):393-400. 33. Maddocks K, Lapalombella R, Blum KA, et al. Preliminary results of
5. Ferrajoli A, Keating MJ, Manshouri T, et al. The clinical signiﬁcance a phase I study of lenalidomide in relapsed chronic lymphocytic leukemia.
of tumor necrosis factor-alpha plasma level in patients having chronic lym- Blood (ASH Annual Meeting Abstracts). 2009;114 3446.
phocytic leukemia. Blood. 2002;100(4):1215-1219. 34. Wendtner CM, Hillmen P, Mahadevan D, et al. Final results of a mul-
6. Ribatti D, Vacca A. Therapeutic renaissance of thalidomide in the ticenter phase 1 study of lenalidomide in patients with relapsed or refractory
treatment of haematological malignancies. Leukemia. 2005;19:1525-1531. chronic lymphocytic leukemia. Leuk Lymphoma. 2011 Nov 15. Epub ahead
7. Sampaio EP, Sarno EN, Galilly R, et al. Thalidomide selectively in- of print.
hibits tumor necrosis factor alpha production by stimulated human mono- 35. Chanan-Khan A, Miller KC, Musial L, et al. Clinical efﬁcacy of lenalid-
January 2012, Vol. 19, No. 1 Cancer Control 65
omide in patients with relapsed or refractory chronic lymphocytic leukemia: associated antigens in chronic lymphocytic leukemia by SEREX. Blood.
results of a phase II study. J Clin Oncol. 2006;24(34):5343-5349. 2002;100(6):2123-2131.
36. Ferrajoli A, Badoux XC, O’Brien S, et al. The Combination of Lenalid- 62. Harig S, Witzens M, Krackhardt AM, et al. Induction of cytotoxic T-
omide and Rituximab Induces Complete and Partial Responses In Patients cell responses against immunoglobulin V region-derived peptides modiﬁed
with Relapsed and Refractory Chronic Lymphocytic Leukemia. Blood (ASH at human leukocyte antigen-A2 binding residues. Blood. 2001; 98(10):2999-
Annual Meeting Abstracts). 2010;116:1395. 3005.
37. Veliz M, Santana R, Lancet JE, et al. Phase II study of lenalidomide 63. Trojan A, Schultze JL, Witzens M, et al. Immunoglobulin framework-
in combination with rituximab for patients with CD5+/CD20+ hematologic derived peptides function as cytotoxic T-cell epitopes commonly expressed
malignancies who relapse or progress after rituximab. Interim analysis. in B-cell malignancies. Nat Med. 2000; 6(6):667-672.
Blood (ASH Annual Meeting Abstracts). 2009;114:2376. 64. Buhmann R, Nolte A, Westhaus D, et al. CD40-activated B-cell
38. Badoux X, O’Brien S, Wierda WG, et al. Combination of ofatumumab chronic lymphocytic leukemia cells for tumor immunotherapy: stimulation
and lenalidomide in patients with relapsed chronic lymphocytic leukemia: of allogeneic versus autologous T cells generates different types of effector
initial results of a phase II trial. Blood (ASH Annual Meeting Abstracts). cells. Blood. 1999;93(6):1992-2002.
2010;116:2464. 65. Juffs H, Fowler N, Saal R, et al. B cell chronic lymphocytic leukae-
39. Sher T, Miller KC, Lawrence D, et al. Efﬁcacy of lenalidomide in pa- mia cells have reduced capacity to upregulate expression of MHC class I in
tients with chronic lymphocytic leukemia with high-risk cytogenetics. Leuk response to interferon-gamma. Pathology. 2004;36(1):69-76.
Lymphoma. 2010;51(1):85-88. 66. Williams JF, Petrus MJ, Wright JA, et al. fas-mediated lysis of chronic
40. Chanan-Khan AA, Ersing N, Krammer D, et al. Pro-Apoptotic effect lymphocytic leukaemia cells: role of type I versus type II cytokines and au-
of lenalidomide (L) in patients with chronic lymphocytic leukemia (CLL) is tologous fasL-expressing T cells. Br J Haematol. 1999;107(1):99-105.
possibly mediated through interruption of the phosphatidylinositol pathway. 67. Foa R, Giovarelli M, Jemma C, et al. Interleukin 2 (IL 2) and in-
Blood (ASH Annual Meeting Abstracts). 2006;108:2102. terferon-gamma production by T lymphocytes from patients with B-chronic
41. Badoux X, Reuben J, Lee B, et al. Lenalidomide therapy is associ- lymphocytic leukemia: evidence that normally released IL 2 is absorbed by
ated with normalization of lymphocyte populations and increase in immuno- the neoplastic B cell population. Blood. 1985;66(3):614-619.
globulin levels in elderly patients with chronic lymphocytic leukemia. Hae- 68. Tötterman TH, Carlsson M, Simonsson B, et al. T-cell activation and
matologica. 2009;94(suppl 3):S94. Abstract 10.31. subset patterns are altered in B-CLL and correlate with the stage of the dis-
42. Brown JR, Abramson J, Hochberg E, et al. A phase I study of lenalid- ease. Blood. 1989;74(2):786-792.
omide in combination with ﬂudarabine and rituximab in previously untreated 69. Rossi E, Matutes E, Morilla R, et al. Zeta chain and CD28 are poorly
CLL/SLL. Leukemia. 2010;24(11):1972-1975. expressed on T lymphocytes from chronic lymphocytic leukemia. Leukemia.
43. Meinhardt G, Wendtner CM, Hallek M. Molecular pathogenesis of 1996;10(3):494-497.
chronic lymphocytic leukemia: factors and signaling pathways regulating 70. Cantwell M, Hua T, Pappas J, et al. Acquired CD40-ligand deﬁciency
cell growth and survival. J Mol Med. 1999;77(2):282-293. in chronic lymphocytic leukemia. Nat Med. 1997;3(9):984-989.
44. Lens D, De Schouwer PJ, Hamoudi RA, et al. p53 abnormalities in 71. Schuler M, Tretter T, Schneller F, et al. Autocrine transforming growth
B-cell prolymphocytic leukemia. Blood. 1997;89(6):2015-2023. factor-beta from chronic lymphocytic leukemia-B cells interferes with prolif-
45. Johnston JB, Daeninck P, Verburg L, et al. P53, MDM-2, BAX and BCL- erative T cell signals. Immunobiology. 1999;200(1):128-139.
2 and drug resistance in chronic lymphocytic leukemia. Leuk Lymphoma. 72. Görgün G, Holderried TA, Zahrieh D, et al. Chronic lymphocytic leu-
1997;26(5-6):435-449. kemia cells induce changes in gene expression of CD4 and CD8 T cells. J
46. Bullrich F, Rasio D, Kitada S, et al. ATM mutations in B cell chronic Clin Invest. 2005;115(7):1797-1805.
lymphocytic leukemia. Cancer Res. 1999;59(1):24-27. 73. Beyer M, Kochanek M, Darabi K, et al. Reduced frequencies
47. Harris NL. Low-grade B-cell lymphoma of mucosa-associated lym- and suppressive function of CD4+CD25hi regulatory T cells in patients
phoid tissue and monocytoid B-cell lymphoma. Related entities that are with chronic lymphocytic leukemia after therapy with ﬂudarabine. Blood.
distinct from other low-grade B-cell lymphomas. Arch Pathol Lab Med. 2005;106(6):2018-2025.
1993;117(8):771-775. 74. Wierda WG, Kipps TJ. Gene therapy and active immune therapy
48. Ribera JM, Vinolas N, Urbano-Ispizua A, et al. “Spontaneous” com- of hematologic malignancies. Best Pract Res Clin Haematol. 2007;20(3):
plete remissions in chronic lymphocytic leukemia: report of three cases and 557-568.
review of the literature. Blood Cells. 1987;12(2):471-483. 75. Dilloo D, Brown M, Roskrow M, et al. CD40 ligand induces an anti-
49. Ziegler-Heitbrock HW, Schlag R, Flieger D, et al. Favorable re- leukemia immune response in vivo. Blood. 1997;90(5):1927-1933.
sponse of early stage B CLL patients to treatment with IFN-alpha 2. Blood. 76. Wierda WG, Cantwell MJ, Woods SJ, et al. CD40-ligand (CD154) gene
1989;73(6):1426-1430. therapy for chronic lymphocytic leukemia. Blood. 2000;96(9):2917-2924.
50. Pavletic ZS, Arrowsmith ER, Bierman PJ, et al. Outcome of alloge- 77. Takahashi S, Rousseau RF, Yotnda P, et al. autologous antileukemic
neic stem cell transplantation for B cell chronic lymphocytic leukemia. Bone immune response induced by chronic lymphocytic leukemia B cells express-
Marrow Transplant. 2000;25(7):717-722. ing the CD40 ligand and interleukin 2 transgenes. Human Gene Therapy.
51. Horowitz MM, Gale RP, Sondel PM, et al. Graft-versus-leukemia re- 2001;12(6):659-670.
actions after bone marrow transplantation. Blood. 1990; 75(3):555-562. 78. Biagi E, Dotti G, Yvon E, et al. Molecular transfer of CD40 and OX40
52. Gitelson E, Hammond C, Mena J, et al. Chronic lymphocytic leuke- ligands to leukemic human B cells induces expansion of autologous tumor-
mia-reactive T cells during disease progression and after autologous tumor reactive cytotoxic T lymphocytes. Blood. 2005;105(6):2436-2442.
cell vaccines. Clin Cancer Res. 2003; 9(5):1656-1665. 79. Tolba KA, Bowers WJ, Hilchey SP, et al. Development of herpes
53. Mayr C, Bund D, Schlee M, et al. Fibromodulin as a novel tumor- simplex virus-1 amplicon-based immunotherapy for chronic lymphocytic leu-
associated antigen (TAA) in chronic lymphocytic leukemia (CLL), which kemia. Blood. 2001;98(2):287-295.
allows expansion of speciﬁc CD8+ autologous T lymphocytes. Blood. 80. Wendtner CM, Koﬂer DM, Theiss HD, et al. Efﬁcient gene transfer of
2005;105(4):1566-1573. CD40 ligand into primary B-CLL cells using recombinant adeno-associated
54. Mikaelsson E, Danesh-Manesh AH, Luppert A, et al. Fibromodulin, virus (rAAV) vectors. Blood. 2002;100(5):1655-1661.
an extracellular matrix protein: characterization of its unique gene and pro- 81. Li LH, Biagi E, Allen C, et al. Rapid and efﬁcient nonviral gene deliv-
tein expression in B-cell chronic lymphocytic leukemia and mantle cell lym- ery of CD154 to primary chronic lymphocytic leukemia cells. Cancer Gene
phoma. Blood. 2005;105(12):4828-4835. Ther. 2006;13(2):215-224.
55. Giannopoulos K, Li L, Bojarska-Junak A, et al. Expression of 82. Bennett SR, Carbone FR, Karamalis F, et al. Help for cytotoxic-T-cell
RHAMM/CD168 and other tumor-associated antigens in patients with B-cell responses is mediated by CD40 signalling. Nature. 1998;393(6684):478-480.
chronic lymphocytic leukemia. Int J Oncol. 2006; 29(1):95-103. 83. Wierda WG, Castro JE, Aguillon R, et al. A phase I study of immune
56. Mayr C, Bund D, Schlee M, et al. MDM2 is recognized as a tumor- gene therapy for patients with CLL using a membrane-stable, humanized
associated antigen in chronic lymphocytic leukemia by CD8+ autologous T CD154. Leukemia. 2010; 24(11):1893-1900.
lymphocytes. Exp Hematol. 2006; 34(1):44-53. 84. Dicker F, Kater AP, Prada CE, et al. CD154 induces p73 to overcome
57. Counter CM, Gupta J, Harley CB, Leber B, Bacchetti S. Telomerase the resistance to apoptosis of chronic lymphocytic leukemia cells lacking
activity in normal leukocytes and in hematologic malignancies. Blood. 1995 functional p53. Blood. 2006;108(10):3450-3457.
85(9):2315-2320. 85. Biagi E, Rousseau R, Yvon E, et al. Responses to Human CD40
58. Siegel S, Wagner A, Kabelitz D, et al. Induction of cytotoxic T-cell Ligand/Human Interleukin-2 Autologous Cell Vaccine in Patients with B-Cell
responses against the oncofetal antigen-immature laminin receptor for the Chronic Lymphocytic Leukemia. Clin Cancer Res. 2005;11(19 Pt 1):6916-
treatment of hematologic malignancies. Blood. 2003;102(13):4416-4423. 6923.
59. Schmidt SM, Schag K, Müller MR, et al. Induction of adipophilin- 86. Fratantoni JC, Li L, Liu LN, et al. A practical approach for achieving
speciﬁc cytotoxic T lymphocytes using a novel HLA-A2-binding peptide that clinical immunotherapy of CLL with hCD40L- and hIL-2 expressing autolo-
mediates tumor cell lysis. Cancer Res. 2004;64(3):1164-1170. gous tumor cells. Blood (ASH Annual Meeting Abstracts). 2005;106:450.
60. Granziero L, Ghia P, Circosta P, et al. Survivin is expressed on CD40 87. Okur FV, Yvon E, Dotti G, et al. Vaccination Strategies for Patients
stimulation and interfaces proliferation and apoptosis in B-cell chronic lym- with B-CLL. Blood (ASH Annual Meeting Abstracts). 2008;112:2106.
phocytic leukemia. Blood. 2001;97(9):2777-2783. 88. ClinicalTrials.gov. U.S. National Institute of Health. www.clinicaltri-
61. Krackhardt AM, Witzens M, Harig S, et al. Identiﬁcation of tumor- als.gov. Accessed September 15, 2011.
66 Cancer Control January 2012, Vol. 19, No. 1
89. Nishimura T, Watanabe K, Yahata T, et al. Application of interleukin 113. Giannopoulos K, Dmoszynska A, Kowal M, et al. Peptide vaccination
12 to antitumor cytokine and gene therapy. Cancer Chemother Pharmacol. elicits leukemia-associated antigen-speciﬁc cytotoxic CD8+ T-cell responses
1996;38(suppl):S27-S34. in patients with chronic lymphocytic leukemia. Leukemia. 2010;24(4):798-
90. Stripecke R, Skelton DC, Pattengale PK, et al. Combination of CD80 805.
and granulocyte-macrophage colony-stimulating factor coexpression by
a leukemia cell vaccine: preclinical studies in a murine model recapitulat-
ing Philadelphia chromosome-positive acute lymphoblastic leukemia. Hum
Gene Ther. 1999;10(13):2109-2122.
91. Vereecque R, Buffenoir G, Preudhomme C, et al. Gene transfer of
GM-CSF, CD80 and CD154 cDNA enhances survival in a murine model of
acute leukemia with persistence of a minimal residual disease. Gene Ther.
92. Palena C, Foon KA, Panicali D, et al. Potential approach to immuno-
therapy of chronic lymphocytic leukemia (CLL): enhanced immunogenicity
of CLL cells via infection with vectors encoding for multiple costimulatory
molecules. Blood. 2005;106(10):3515-3523.
93. Litzinger MT, Foon KA, Sabzevari H, et al. Chronic lymphocytic leu-
kemia (CLL) cells genetically modiﬁed to express B7-1, ICAM-1, and LFA-3
confer APC capacity to T cells from CLL patients. Cancer Immunol Immuno-
94. Ridgway D. The ﬁrst 1000 dendritic cell vaccines. Cancer Invest.
95. Müller MR, Tsakou G, Grünebach F, et al. Induction of chronic lym-
phocytic leukemia (CLL)-speciﬁc CD4- and CD8-mediated T-cell responses
using RNA-transfected dendritic cells. Blood. 2004;103(5):1763-1769.
96. Rezvany MR, Jeddi-Tehrani M, Wigzell H, et al. Leukemia-associat-
ed monoclonal and oligoclonal TCR-BV use in patients with B-cell chronic
lymphocytic leukemia. Blood. 101(3):1063-1070.
97. Palma M, Adamson L, Hansson L, et al. Development of a dendritic
cell-based vaccine for chronic lymphocytic leukemia. Cancer Immunol Im-
98. Kokhaei P, Rezvany MR, Virving L, et al. Dendritic cells loaded with
apoptotic tumour cells induce a stronger T-cell response than dendritic cell-
tumour hybrids in B-CLL. Leukemia. 2003;17(5):894-899.
99. Hus I, Rolinski J, Tabarkiewicz J, et al. Allogeneic dendritic cells
pulsed with tumor lysates or apoptotic bodies as immunotherapy for pa-
tients with early-stage B-cell chronic lymphocytic leukemia. Leukemia.
100. Hus I, Schmitt M, Tabarkiewicz J, et al. Vaccination of B-CLL pa-
tients with autologous dendritic cells can change the frequency of leukemia
antigen-speciﬁc CD8+ T cells as well as CD4+CD25+FoxP3+ regulatory T
cells toward an antileukemia response. Leukemia. 2008;22(5):1007-1017.
101. Kronenberger K, Nössner E, Frankenberger B, et al. A polyvalent
cellular vaccine induces T-cell responses against speciﬁc self-antigens
overexpressed in chronic lymphocytic B-cell leukemia. J Immunother.
102. Mocikat R, Selmayr M, Thierfelder S, et al. Trioma-based Vaccina-
tion against B-Cell Lymphoma Confers Long-Lasting Tumor Immunity. Can-
cer Research. 1997;57(12):2346-2349.
103. Wahl U, Nössner E, Kronenberger K, et al. Vaccination against B-
cell Chronic Lymphocytic Leukemia with Trioma Cells. Clin Cancer Res.
104. Gitelson E, Hammond C, Mena J, et al. Chronic Lymphocytic Leu-
kemia-reactive T Cells during Disease Progression and after Autologous Tu-
mor Cell Vaccines. Clin Cancer Res. 2003;9(5):1656-1665.
105. Spaner DE, Hammond C, Mena J, et al. A phase I/II trial of oxidized
autologous tumor vaccines during the “watch and wait” phase of chronic
lymphocytic leukemia. Cancer Immunol Immunother. 2005;54(7):635-646.
106. Hus I, Kawiak J, Tabarkiewicz J, et al. Immunotherapy with irradiated
autologous leukemic cells in patients with B-CLL in early stages. Oncol Rep.
107. Campbell MJ, Carroll W, Kon S, et al. Idiotype vaccination against
murine B cell lymphoma. Humoral and cellular responses elicited by
tumor-derived immunoglobulin M and its molecular subunits. J Immunol.
108. Greiner J, Li L, Ringhoffer M, et al. Identiﬁcation and characterization
of epitopes of the receptor for hyaluronic acid-mediated motility (RHAMM/
CD168) recognized by CD8+ T cells of HLA-A2-positive patients with acute
myeloid leukemia. Blood. 2005;106(3):938-945.
109. Greiner J, Ringhoffer M, Taniguchi M, et al. Receptor for hyaluro-
nan acid-mediated motility (RHAMM) is a new immunogenic leukemia-
associated antigen in acute and chronic myeloid leukemia. Exp Hematol.
110. Greiner J, Ringhoffer M, Taniguchi M, et al. Characterization of sev-
eral leukemia-associated antigens inducing humoral immune responses in
acute and chronic myeloid leukemia. Int J Cancer. 2003;106(2):224-231.
111. Crainie M, Belch AR, Mant MJ, Pilarski LM. Overexpression of the
receptor for hyaluronan-mediated motility (RHAMM) characterizes the ma-
lignant clone in multiple myeloma: identiﬁcation of three distinct RHAMM
variants. Blood. 1999;93(5):1684-1696.
112. Assmann V, Marshall JF, Fieber C, et al. The human hyaluronan
receptor RHAMM is expressed as an intracellular protein in breast cancer
cells. J Cell Sci. 1998;111(Pt 12):1685-1694.
January 2012, Vol. 19, No. 1 Cancer Control 67