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					                                                                                 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:     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%
                                                                            Anemia 10%

 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

 patients enrolled.
 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
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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-
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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-
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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
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January 2012, Vol. 19, No. 1                                                                                                             Cancer Control 67

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