V O L U M E 2 3 d N U M B E R 2 3 d A U G U S T 1 0 2 0 0 5
`
`JOURNAL OF CLINICAL ONCOLOGY
`
`O R I G I N A L R E P O R T
`
`From the Mayo Clinic College of
`Medicine and Mayo Foundation,
`Rochester, MN; Scottsdale CCOP,
`Scottsdale, AZ; Carle Cancer Center
`CCOP, Urbana, IL; Metro-Minnesota
`Community Clinical Oncology Program,
`St Louis Park, MN; Iowa Oncology
`Research Association CCOP, Des
`Moines, IA; Wichita CommunityClinical
`Oncology Program, Wichita, KS; and
`Hematology & Oncology of Dayton Inc,
`Dayton, OH.
`
`Submitted January 24, 2005; accepted
`April 7, 2005.
`
`Supported in part by Public Health
`Service grants CA-25224,
`CA-37404, CA-15083, CA-63826,
`CA-35195, CA-35267, CA-35101,
`CS-35431, CA-35090, CA-35113,
`CA-35415, CA-60276, CA-35448,
`and CA-63848, and grant CA97274
`from the National Cancer Institute,
`Department of Heath and Human
`Services, Bethesda, MD.
`
`This study was conducted as a
`collaborative trial of the North Central
`Cancer Treatment Group and Mayo Clinic.
`Additional participating institutions include
`Medcenter One Health Systems,
`Bismarck, ND (Edward Wos, MD); Illinois
`Oncology Research Association CCOP,
`Peoria, IL (John W. Kugler, MD); Toledo
`Community Hospital Oncology Program
`CCOP, Toledo, OH (Paul L. Schaefer, MD);
`Scottsdale CCOP, Scottsdale, AZ (Tom R.
`Fitch, MD); Geisinger Clinic & Medical
`Center CCOP, Danville, PA (Albert
`Bernath, MD); and Ann Arbor Regional
`CCOP, Ann Arbor, MI (Philip J. Stella, MD).
`
`Terms in blue are defined in the glossary,
`found at the end of this issue and online
`at www.jco.org.
`
`Authors’ disclosures of potential
`conflicts of interest are found at the
`end of this article.
`
`Address reprint requests to Thomas E.
`Witzig, MD, Mayo Clinic, Stabile 628,
`200 First St SW, Rochester, MN 55905;
`e-mail: witzig@mayo.edu.
` 2005 by American Society of Clinical
`Oncology
`
`0732-183X/05/2323-5347/$20.00
`
`DOI: 10.1200/JCO.2005.13.466
`
`Phase II Trial of Single-Agent Temsirolimus (CCI-779)
`for Relapsed Mantle Cell Lymphoma
`Thomas E. Witzig, Susan M. Geyer, Irene Ghobrial, David J. Inwards, Rafael Fonseca, Paul Kurtin,
`Stephen M. Ansell, Ronnie Luyun, Patrick J. Flynn, Roscoe F. Morton, Shaker R. Dakhil,
`Howard Gross, and Scott H. Kaufmann
`
`A
`
`B
`
`S
`
`T
`
`R
`
`A
`
`C
`
`T
`
`Purpose
`Mantle cell lymphoma (MCL) is characterized by a t(11;14) resulting in overexpression of
`cyclin D1 messenger RNA. This study tested whether temsirolimus (previously known as
`CCI-779), an inhibitor of the mammalian target of rapamycin kinase that regulates cyclin
`D1 translation, could produce tumor responses in patients with MCL.
`Patients and Methods
`Patients with relapsed or refractory MCL were eligible to receive temsirolimus 250 mg intra-
`venously every week as a single agent. Patients with a tumor response after six cycles were
`eligible to continue drug for a total of 12 cycles or two cycles after complete remission, and
`were then observed without maintenance.
`Results
`Thirty-five patients were enrolled and were assessable for toxicity; one patient had MCL by
`histology but was cyclin D1 negative and was ineligible for efficacy. The median age was 70
`years (range, 38 to 89 years), 91% were stage 4, and 69% had two or more extranodal sites.
`Patients had received a median of three prior therapies (range, one to 11), and 54% were
`refractory to the last treatment. The overall response rate was 38% (13 of 34 patients;
`90% CI, 24% to 54%) with one complete response (3%) and 12 partial responses (35%).
`The median time-to-progression in all patients was 6.5 months (95% CI, 2.9 to 8.3 months),
`and the duration of response for the 13 responders was 6.9 months (95% CI, 5.2 to 12.4
`months). Hematologic toxicities were the most common, with 71% (25 of 35 patients) having
`grade 3 and 11% (four of 35 patients) having grade 4 toxicities observed. Thrombocytopenia
`was the most frequent cause of dose reductions but was of short duration, typically resolving
`within 1 week.
`Conclusions
`Single-agent temsirolimus has substantial antitumor activity in relapsed MCL. This study dem-
`onstrates that agents that selectively target cellular pathways dysregulated in MCL cells can
`produce therapeutic benefit. Further studies of this agent in MCL and other lymphoid malig-
`nancies are warranted.
`J Clin Oncol 23:5347-5356.  2005 by American Society of Clinical Oncology
`
`INTRODUCTION
`
`lymphoma (MCL) is an in-
`Mantle cell
`curable, aggressive B-cell non-Hodgkin’s
`lymphoma (NHL) that represents approx-
`imately 8% of cases of NHL. The disease
`usually presents in an advanced stage (III
`or IV), and involvement of extranodal sites
`such as the gut, bone marrow, and periph-
`
`eral blood are common. There is a male
`predominance, and most patients are older
`adults. The characteristic tumor cell immu-
`nophenotype is CD20⫹, CD10⫺, CD5⫹,
`and CD23⫺, with monoclonal light chain
`expression on the cell surface. MCL is a
`unique subtype in that the tumor cells
`have a t(11;14)(q13;q32) chromosomal
`translocation that juxtaposes the cyclin D1
`
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`Witzig et al
`
`gene on chromosome 11 to the immunoglobulin heavy
`chain enhancer region on chromosome 14.1-3 The tran-
`scription enhancers on 14q32, now linked to the cyclin
`D1 gene, result in the characteristic overexpression of cy-
`clin D1 in the MCL tumor cells.
`There is currently no standard therapy for newly di-
`agnosed or relapsed MCL. Many regimens have been dem-
`onstrated to be highly active in producing responses,4-17
`but relapse typically occurs, and patients usually die of
`their disease, with a median survival of 3 to 4 years. It
`is clear that new treatments are needed for MCL.
`Even though cyclin D1 mRNA is constitutively ex-
`pressed in MCL, it is potentially subject to translational
`regulation by a pathway (Fig 1) involving the mammalian
`target of rapamycin (mTOR).18,19 Activated receptor tyro-
`sine kinases and activated ras proteins enhance the cata-
`lytic activity of the lipid kinase phosphatidylinositol-3
`kinase (PI3K), which converts phosphatidylinositol-4,5-
`bisphosphate
`(PIP2)
`to
`phosphatidylinositol-3,4,5-
`trisphosphoate (PIP3). PIP3 activates the protein kinase
`phosphoinositide-dependent kinase 1 (PDK1), which,
`along with a second kinase such as integrin-linked kinase
`(ILK), contributes two phosphorylations required for
`maximal Akt activity. Akt then phosphorylates a number
`
`of substrates, including tuberous sclerosis (TSC) protein 2
`(TSC2), which in its unphosphorylated state is complexed
`with TSC protein 1 (TSC1) and acts as a GTPase activating
`protein that diminishes activation of the small guanine
`nucleotide binding protein Rheb. When the TSC1/TSC2
`complex is inactivated by Akt, Rheb remains in a GTP-
`bound state that activates mTOR, a protein kinase that
`regulates mRNA translation by phosphorylating two crit-
`ical
`substrates, eukaryotic initiation factor (eIF) 4E
`(eIF4E) binding protein (4E-BP1) and p70S6 kinase.20,21
`Previous studies have shown that eIF4E is a component
`of a helicase complex that binds to the cap structure
`at the 5# end of mRNAs and enhances the ability of
`ribosome-eIF complexes to scan the mRNA in search of a
`translation initiation site.22 The ability of eIF4E to bind
`to and participate in this helicase complex is inhibited
`when 4E-BP1 is bound. This inhibitory interaction is
`possible only when 4E-BP1 is unphosphorylated and is
`abrogated when 4E-BP1 is sequentially phosphorylated
`by mTOR and other kinases.22,23 At the same time, mTOR-
`mediated phosphorylation activates p70S6K, enabling
`its phosphorylation of ribosomal protein S6 and possibly
`other substrates, thereby enhancing the translation of
`messages with 5# terminal oligopyrimidine tracts.18,22
`
`Activated Ras
`
`PI3K
`
`PIP2
`
`PIP3
`
`PTEN
`ILK
`
`PDK1
`
`Akt
`
`pAkt
`
`ppAkt
`
`TSC1/TSC2
`
`pTSC2
`+
`TSC1
`
`Rheb.GTP
`
`Rheb.GDP
`
`eIF4E.4EBP-1 complex
`
`P 4E-BP1
`+
`eIF4E
`
`mTOR
`
`p70S6k
`
`P p70S6k
`
`Protein S6
`
`P S6
`
`Rapamycin
`+
`FKBP12
`
`Rapamycin .FKBP12
`
`Cyclin D1 mRNA
`
`Cyclin D1 protein
`
`Fig 1. Current understanding of the mamma-
`lian target of rapamycin (mTOR) pathway and
`mechanism of action of rapamycin.
`In this
`diagram, arrows pointing downward or curved
`arrows pointing to the right indicate activation,
`whereas curved arrows pointing leftward or
`lines ending in crossbars indicate inhibition. The
`phosphorylation of S6 was studied before
`and after
`temsirolimus on clinical samples
`from patients in this study (see Fig 4).
`PI3K, phosphatidylinositol-3 kinase; PIP2,
`phosphatidylinositol-4,5-bisphosphate;
`PIP3,
`phosphatidylinositol-3,4,5-trisphosphoate; PDK1,
`phosphoinositide-dependent kinase 1;
`ILK,
`integrin-linked kinase; TSC, tuberous sclerosis;
`4E-BP1, eIF4E binding protein; eIF4E, eukary-
`otic initiation factor 4E.
`
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`Temsirolimus for Mantle Cell Lymphoma
`
`Collectively, these events markedly enhance translation of
`a small but important group of messages, including those
`encoding c-myc, ornithine decarboxylase, and cyclin D1,
`as well as ribosomal proteins themselves.18,22,24,25
`mTOR activity is modulated by mitogenic signals,
`which are transmitted through a signal transduction path-
`way involving PI3K, Akt, and TSC1 and TSC2 (Fig
`1).18,19,26,27 In addition, mTOR-mediated signaling is
`also subject to modulation by the macrocyclic lactone ra-
`pamycin and its derivatives.19,26,27 Once these agents bind
`to the 12 kDa cytosolic FK506-binding protein FKBP12,
`the resulting rapamycin-FKBP12 complexes bind to a spe-
`cific site near the catalytic domain of mTOR and inhibit
`phosphorylation of mTOR substrates by a mechanism
`that remains somewhat poorly understood.27 As a con-
`sequence, translation of messages that require mTOR sig-
`naling is inhibited. This mechanism is thought to be
`responsible for the immunosuppressive effects of rapamy-
`cin as well as its putative antineoplastic activity.
`Temsirolimus (also known as CCI-779), a dihy-
`droester of rapamycin that is suitable for intravenous
`use, is currently undergoing testing in solid tumor patients
`as a potential antineoplastic agent.28-31 In view of the role
`of cyclin D1 in MCL, we conducted a phase II trial of
`single-agent
`temsirolimus
`for patients with relapsed
`MCL to learn if therapy that specifically targeted this
`pathway could result in tumor responses.
`
`PATIENTS AND METHODS
`A single-stage phase II study with an interim analysis was con-
`ducted to assess the proportion of previously treated MCL patients
`who achieved a partial response (PR) or better after treatment with
`temsirolimus. This study was conducted through the North Cen-
`tral Cancer Treatment Group (NCCTG) cooperative group and
`was approved by the institutional review boards of each treatment
`site. Patients were eligible for this trial if they had previously re-
`ceived therapy and had relapsed or were refractory to their last
`treatment. There was no limit on the number of prior therapies.
`Central pathology review confirmed the diagnosis of MCL based
`on morphology and phenotype. In addition, all tumors were pos-
`itive for cyclin D1 by immunohistochemistry or demonstrated
`t(11;14)(q13;q32)/immunoglubulin H fusion by fluorescence in
`situ hybridization. Patients were required to have measurable dis-
`ease with a lymph node or tumor mass $ 2 cm or malignant lym-
`phocytosis with an absolute lymphocyte count $ 5,000; a life
`expectancy of $ 3 months; Eastern Cooperative Oncology Group
`performance status of 0, 1, or 2; absolute neutrophil count (ANC)
`$ 1,000; platelets $ 75,000; hemoglobin $ 8 g/dL; serum creati-
`nine # 2⫻ the upper limit of normal (ULN); serum bilirubin
`# 1.5 ULN; serum cholesterol # 350 mg/dL; and triglycer-
`ides # 400 mg/dL. Patients could not have had CNS involvement
`or HIV infection.
`Patients were treated with a flat dose of 250 mg of tem-
`sirolimus diluted in 250 mL of normal saline and delivered in-
`travenously (IV) over 30 minutes. Patients were pretreated with
`diphenhydramine 25 to 50 mg IV. Treatment was weekly, and
`4 weeks was considered to be one cycle. A CBC was performed
`
`each week, and the full dose of temsirolimus was delivered if
`the platelet count was $ 50,000 and the ANC $ 1,000, and if
`there were no grade 3 or 4 nonhematologic toxicities (National
`Cancer Institute Common Toxicity Criteria, version 2). Patients
`who did not meet the retreatment criteria had the dose held until
`recovery, followed by a stepwise dose modification to 175, 125,
`75, or 50 mg. Patients were not to receive prophylactic WBC
`growth factors to maintain dosing but could receive them at
`physician discretion if neutropenia developed. Erythropoietin
`treatment for anemia was also permitted.
`Patients were restaged after one cycle and every three cycles
`thereafter or at physician discretion. Responses were categorized
`using the International Workshop Criteria.32 Patients who pro-
`gressed anytime or those patients with stable disease after six cycles
`went off study. Patients who had a complete remission (CR) or PR
`at 6 months were to receive two cycles after CR or for a total of 12
`months if there was a PR and they were then observed without
`further therapy.
`
`Statistical Design
`This trial was designed to test the null hypothesis that the
`true overall response rate (ORR) was at most 5%. The smallest
`ORR that would indicate that this regimen was worth further
`study in this relapsed MCL patient population was 20%. The de-
`sign was generated based on the parameters and assumptions of
`a two-stage Simon min max design, but where accrual was not
`suspended for the interim analysis. This study design required
`a maximum of 32 assessable patients, where the interim analysis
`was performed after 18 patients had been accrued and followed
`up for at least 24 weeks for response. An additional three patients
`were accrued to this cohort (for a maximum of 35 patients over-
`all) to account for the possibilities of ineligibility, withdrawal
`from study before drug administration, or major violations.
`However, only the first 32 assessable patients were used to eval-
`uate the decision criteria for this design. At least one response in
`the first 18 assessable patients needed to be observed in the in-
`terim analysis to continue accrual. At the time of the final ana-
`lyses, a total of four or more responses were required to indicate
`that this regimen warrants further evaluation in this patient
`population. The proportion of responses was calculated, and the
`90% exact binomial CI for the true ORR was calculated (with
`all eligible patients accrued), assuming that the number of re-
`sponses was binomially distributed.
`Duration of response (DR) was defined as the time from the
`date of documented response to the date of progression. Patients
`who went off treatment due to other reasons (eg, adverse reac-
`tions, refusal of further treatment) were censored at that time.
`Time to progression (TTP) was defined as the time from regis-
`tration to the date of progression. Patients who died without dis-
`ease progression were censored at the date of their last evaluation.
`If a patient died without documentation of disease progression,
`the patient was considered to have had disease progression at the
`time of death unless there was sufficient documented evidence to
`conclude that progression did not occur before death. Time
`to discontinuation of active treatment was defined as the time
`from registration to the date the decision was made to take
`the patient off active treatment. Patients who were still receiving
`treatment at the time of these analyses were censored at the date
`of their last evaluation. Overall survival (OS) was defined as the
`time from registration to death resulting from any cause. The dis-
`tributions of these time-to-event end points were each estimated
`using the Kaplan-Meier method.33
`
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`Witzig et al
`
`Tissue Culture and Exposure to Rapamycin
`in Vitro
`The MO2058 human line, which was established from a
`patient with prolymphocytic leukemia and which contains the
`t(11;14)(q13;q32) translocation associated with Cyclin D1 activa-
`tion,34 was propagated at 37°C in RPMI 1640 medium containing
`10% heat-inactivated fetal bovine serum, 2 mmol/L L-glutamine,
`100 units/mL penicillin G, and 100 ␮g/mL streptomycin. To
`establish conditions for detecting an effect of rapamycin on
`downstream signaling of mTOR, cells were treated with various
`rapamycin (Sigma, St Louis, MO) concentrations for 24 hours,
`washed three times with ice-cold serum-free RPMI 1640
`10 mmol/L HEPES (pH 7.4), solubilized in buffered 6M guani-
`dine hydrochloride under reducing conditions, and prepared
`for electrophoresis, as previously described.35
`To determine whether mTOR signaling was inhibited in
`MCL tumor cells in situ, circulating mantle cells were purified
`from the peripheral blood of eight patients at four to five time
`points, which typically included: before therapy, 24 hours after
`administration of dose 1, 48 hours after dose 1, before dose 5,
`and before dose 12. At each time point, 1 to 2 ⫻ 106 CD19⫹ cells
`were purified by magnetic bead selection, washed and solubilized
`under strongly denaturing conditions as describe above. Fur-
`ther characterization of an additional aliquot of these cells by
`flow cytometry confirmed that they were typically ⬎ 90% CD19⫹.
`
`Immunoblotting
`Aliquots containing protein from 5 ⫻ 105 immunopurified
`B cells were subjected to electropheresis on sodium dodecyl sulfate
`(SDS)-polyacrylamide gels containing 5% to 12% acrylamide,
`transferred to nitrocellulose, and probed under previously de-
`scribed conditions36 with polyclonal antibodies that recognize
`the following antigens: phospho-Ser235/236 ribosomal protein S6,
`S6, phospho-Thr389 p70S6K and p70S6K (all from Cell Signaling
`Technology, Beverly, MA). Antigen-antibody complexes were
`detected using peroxidase-coupled secondary antibodies (KPL,
`Gaithersburg, MD) and enhanced chemiluminescence reagents
`(Amersham Pharmacia Biotech, Piscataway, NJ) as described.36
`Blots were reprobed with antibody to heat shock protein 90 (David
`Toft, Mayo Clinic, Rochester, MN) as a loading control.
`
`RESULTS
`
`Patient Characteristics
`A total of 35 patients were enrolled onto this trial by
`the NCCTG sites from April 2002 to October 2003. One
`patient was declared ineligible after pathology review in-
`dicated that although the histology was consistent with
`MCL, the cyclin D1 stain was negative. The patients tended
`to be older adults with a median age of 70 years (range, 38
`to 89 years). Most patients (91%) had stage IV disease and
`were heavily pretreated with a median number of three
`prior therapies (mean, four therapies; range, one to 11
`therapies). The majority of patients had failed to improve
`on rituximab, an alkylator agent such as cyclophospha-
`mide, and an anthracycline such as doxorubicin. More
`than half of the patients had received a purine nucleoside
`analogue. Twenty-nine percent of patients (10 of 35) had
`an elevated lactate dehydrogenase at baseline. Additional
`
`baseline characteristics of these patients are presented
`in Table 1.
`
`Clinical Outcomes
`The ORR was 38% (13 of 34 patients; 90% CI, 24% to
`54%) with one CR and 12 PR. The tumor responses oc-
`curred rapidly, with a median time to response of 1 month
`(range, 1 to 8 months) (Fig 2). Eight responses occurred
`after one cycle, three were documented after three cycles,
`and one each after the evaluations at 4 and 8 months,
`respectively. In addition, the patient who was ineligible
`obtained a PR with temsirolimus.
`The patient who achieved a CR received a total of six
`cycles; three patients who achieved a PR completed 12
`cycles; and one patient completed six cycles and went to
`observation with stable disease. The other nine patients
`who achieved PR received a mean of six cycles (median,
`6.5; range, 3 to 10). One patient with a PR remains on
`treatment;
`the other eight patients with PR stopped
`drug before 12 cycles for various reasons: progression
`on temsirolimus (two patients), adverse events (three
`patients), and refusal of further treatment (three patients).
`
`Table 1. Patient Characteristics
`Characteristic
`No. of Patients
`
`Age, years
`Median
`Range
`Sex, male
`Performance status
`0
`1
`2
`Tumor stage
`1
`2
`3
`4
`Bone marrow involvement
`‘‘B’’ symptoms
`No. of extranodal sites
`0
`1
`2
`3
`4
`5
`6
`0-1
`$ 2
`Disease status
`Relapsed
`Refractory
`No. of prior therapy treatments
`Mean
`Median
`Range
`Type of prior therapy
`Rituximab
`Alkylator
`Anthracycline
`Purine nucleoside analog
`Platinum analog
`Radiotherapy
`Stem-cell transplantation
`
`70
`38-89
`
`4
`3
`1-11
`
`26
`
`19
`12
`4
`
`1
`1
`1
`32
`27
`5
`
`3
`8
`12
`8
`3
`0
`1
`11
`24
`
`16
`19
`
`31
`33
`29
`20
`10
`8
`4
`
`%
`
`74
`
`54
`34
`12
`
`3
`3
`3
`91
`77
`14
`
`9
`23
`34
`23
`9
`0
`3
`31
`69
`
`46
`54
`
`89
`94
`83
`57
`29
`23
`11
`
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`Temsirolimus for Mantle Cell Lymphoma
`
`A
`
`Pre-CCl779
`
`After One Cycle
`
`B
`
`Fig 2. Computed tomography scans
`from two patients who had marked
`tumor response after one cycle (four
`doses) of temsirolimus (CCI779). (A)
`Patient with a large perigastric lym-
`phomatous mass;
`(B) patient with
`bulky paratracheal and left axillary
`adenopathy.
`
`(range, 6.7 to 24.6⫹). Overall, 30 patients have had disease
`progression, and 22 patients have died. No patients have
`had documented death without disease progression.
`
`Safety and Tolerability
`All 35 patients were included in the analysis of safety
`and tolerability. Patients tolerated the 30-minute infusion
`of temsirolimus without significant toxicity. All severe
`(grade 3 or greater) toxicities experienced by these patients
`
`3
`6
`9
`12
`15
`18
`Months From Study Registration
`
`21
`
`100
`
`80
`
`60
`
`40
`
`20
`
`0
`
`% Progression-Free
`
`Fig 3. Time to progression after temsirolimus in all 34 patients.
`
`Fourteen additional patients progressed on temsirolimus
`without ever achieving a response. Six patients went off
`study without tumor response or progression due to ad-
`verse reactions (three patients), refusal of further treat-
`ment (one patient), treatment with alternative therapy
`for MCL (one patient), and other medical problems
`(one patient). Those patients who refused further treat-
`ment or who went off for other medical problems discon-
`tinued this treatment regimen largely due to low-grade
`adverse events and a perceived decline in quality of life.
`The median time to discontinuation of treatment was
`3.7 months (95% CI, 3 to 6.2 months).
`Dose reductions were necessary in all but four pa-
`tients. Overall, nine patients were able to receive 250
`mg weekly for at least the first cycle of treatment, with
`a median of 2.5 cycles at this full dose (range, 1 to 8
`cycles); the other patients required dose reductions in
`the first cycle. Of the six patients who received more
`than one cycle at the full dose level, two eventually re-
`quired a dose reduction in subsequent cycles. Across
`all patients, the median dose received per month on
`study was 525 mg, with 564 mg in responding patients
`and 525 mg in nonresponders.
`The median time to progression (Fig 3) was 6.5
`months (95% CI, 2.9 to 8.3 months). The median overall
`survival was 12 months (95% CI, 6.7 months to not yet
`reached). The median duration of response for the 13 re-
`sponders was 6.9 months (95% CI, 5.2 to 12.4 months).
`The median follow-up on living patients was 11 months
`
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`
`that were considered at least possibly related to temsiroli-
`mus are presented in Table 2. Thrombocytopenia was the
`cause of most dose reductions and was rapidly reversible
`with drug delays of typically only 1 week. Only three patients
`required platelet transfusions, and four patients required
`RBC transfusions. Thirteen patients experienced grade 3
`infections without concomitant neutropenia, two patients
`had febrile neutropenia, and three had infection (grade 3)
`with neutropenia. One patient developed a right lower
`motor neuron facial palsy (Bell’s palsy), and mental status
`changes and underwent a magnetic resonance imaging
`scan and cerebral spinal fluid analysis that did not reveal ev-
`idence of involvement with MCL. The conclusion was that
`this was idiopathic Bell’s palsy, and it eventually resolved.
`A possible relationship to temsirolimus could neither be
`established nor eliminated. The patient who experienced
`blurred vision was diagnosed with retinitis due to reacti-
`vation of cytomegalovirus (CMV) infection. The patient
`
`Table 2. Grade 3 and 4 Toxicity (adverse events considered
`at least possibly related to temsirolimus) Was Observed
`in 91% (32 of 35) of Patients
`No. of Patients
`
`Toxicity type
`
`Grade 3
`
`Grade 4
`
`General
`Fatigue
`Weight loss
`Hematologic
`Thrombocytopenia
`Neutropenia
`Lymphopenia
`Anemia
`Infection
`Infection — no ANC
`Infection with ANC
`Febrile neutropenia
`Gastrointestinal
`Abdominal pain
`Diarrhea
`Anorexia
`Mucositis
`Dysphagia
`Metabolic
`Hypercholesterolemia
`Hypertriglyceridemia
`Hyperglycemia
`Hypokalemia
`Creatinine
`Hypertension
`Neurologic
`Muscle weakness
`Low consciousness
`Motor neuropathy
`Cranial neuropathy
`Blurred vision
`Headache
`Pulmonary
`Pneumonitis
`Hypoxia
`Dermatologic
`Rash
`Pruritis
`Maximum overall toxicity grade
`
`4
`3
`
`22
`8
`2
`9
`
`6
`2
`1
`
`1
`4
`0
`2
`1
`
`1
`1
`3
`1
`1
`1
`
`1
`0
`1
`1
`1
`1
`
`1
`1
`
`3
`1
`26
`
`0
`0
`
`1
`2
`0
`0
`
`0
`0
`1
`
`0
`0
`1
`0
`0
`
`0
`0
`0
`0
`0
`0
`
`0
`1
`0
`0
`0
`0
`
`0
`0
`
`0
`0
`6
`
`NOTE. Maximal overall toxicity grade refers to the number of patients
`that had the respective grade toxicity across all toxicity types.
`Abbreviation: ANC, absolute neutrophil count.
`
`had a history of CMV retinitis before enrollment onto this
`study, but the infection was not evident at the time of study
`entry. One 70-year-old female patient achieved a PR after 8
`months of temsirolimus. Throughout the last several months
`of temsirolimus therapy, she developed micrographia, de-
`creased arm swing with walking, and difficulty initiating
`gait consistent with Parkinson’s disease. She had a family
`history of Parkinson’s disease, but no symptoms before
`study participation. Four months after discontinuing tem-
`sirolimus, her symptoms were stable, and after neurologic
`consultation, the diagnosis of Parkinson’s disease versus
`Parkinsonism secondary to medication was obtained. She
`has responded well to carbidopa/levodopa.
`The most common adverse events of all grades were
`thrombocytopenia (100%), hyperglycemia (91%), anemia
`(66%), neutropenia (77%), increased triglycerides (77%),
`mucositis (71%), fatigue (66%), infection without concom-
`itant neutropenia (63%), rash (51%), nausea (49%), weight
`loss (46%), AST elevations (43%), abnormal sense of taste
`(43%), loss of appetite (40%), hypercholesterolemia (40%),
`and sensory neuropathy (37%). No grade 5 events (ie,
`deaths on treatment) were reported. One patient with
`a PR had weight loss due to the dysgeusia that was consid-
`ered grade 3, and could not restart temsirolimus. Although
`mucositis was common, all but two cases were grade 1 or 2.
`
`Pharmacodynamics
`To develop an assay for mTOR inhibition that could
`be applied in clinical MCL samples, we initially treated
`MO258 cells with varying concentrations of rapamycin
`in vitro and probed whole cell lysates for phosphorylation
`of substrates downstream of mTOR using commercially
`available antiphosphoepitope antibodies. Blotting for
`phospho–4E-BP1 in this cell line and others proved dif-
`ficult (data not shown). In contrast, as reported by
`others,37,38 we observed that phospho-S6 (Fig 4A) and,
`with somewhat more difficulty, phospho-p70S6 kinase
`could be demonstrated. Moreover, inhibition of S6 phos-
`phorylation was readily detectable at 0.1 nmol/L rapamy-
`cin and essentially complete at 1 nmol/L (Fig 4A).
`When this assay was applied to MCL samples from
`the blood of patients receiving temsirolimus, phosphory-
`lation of S6 was more readily detectable than phosphory-
`lation of p70S6K. Examination of serial samples revealed
`two distinct patterns. First, as illustrated in Figure 4B for
`one patient, S6 phosphorylation was inhibited after tem-
`sirolimus treatment in three patients (compare, lanes 2
`and 3 with lane 1). Of these three patients, one responded
`to therapy, one was stable, and one progressed without
`ever responding. In contrast, as illustrated in Figure 4C
`for one patient, there was no evidence that S6 phosphor-
`ylation was inhibited in circulating MCL cells from two
`other patients. One of these patients had a PR; the other
`progressed on therapy.
`
`5352
`
`JOURNAL OF CLINICAL ONCOLOGY
`
`Downloaded from ascopubs.org by 12.40.227.4 on March 9, 2018 from 012.040.227.004
`
`Copyright © 2018 American Society of Clinical Oncology. All rights reserved.
`
`IPR2018-00685
`Celgene Ex. 2047, Page 6
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`

`

`Temsirolimus for Mantle Cell Lymphoma
`
`B
`Time (days)
`36
`
`29
`
`0
`
`1
`
`2 38 89
`
`P S6
`
`Hsp90
`
`1
`
`2
`
`3
`
`4
`
`5
`
`P p70S6K
`
`p70S6K6
`
`P S6
`
`S6
`
`HSP90
`
`A
`Rapamycin
`
`36
`
`29
`
`29
`24
`
`97
`
`85
`66
`
`Fig 4. Effect of treatment on down-
`stream phosphorylations in vitro and
`in vivo. (A) MO258 cells were treated
`for 24 hours with diluent (0.1% etha-
`nol, lane 1) or, 0.1, 0.3, 1, or 3 nmol/L
`rapamycin (lanes 2 to 5). Dotted line
`indicates location at which irrelevant
`lanes have been spliced from the
`blots. (B, C) Circulating mantle cells
`from two patients were subjected to
`immunoblotting for phosphorylated S6
`or, as a loading control, HSP90. Lane 1,
`pretreatment;
`lanes 2 to 5, post-
`treatment.
`In B, S6 phosphorylation
`was inhibited after a single dose of
`temsirolimus; the d38 sample showed
`S6 phosphorylation and the d89
`sample inhibition. In C, the signal for
`S6 phosphorylation, albeit weaker at
`baseline,
`is not abolished by tem-
`sirolimus treatment.
`
`1
`
`2
`
`3
`
`4
`
`5
`
`C
`Time (days)
`36
`
`0
`
`1
`
`2 28
`
`29
`
`116
`97
`85
`66
`
`P S6
`
`Hsp90
`
`1
`
`2
`
`3
`
`4
`
`DISCUSSION
`
`The treatment of MCL has remained problematic despite
`the availability of
`standard chemotherapy,
`stem-cell
`transplantation, and monoclonal antibody therapy with
`rituximab. Each of these modalities can produce tumor
`responses in MCL, but the disease typically recurs and
`requires additional therapy. There is no treatment regimen
`that can be considered the definitive treatment of choice
`at this time for patients with new, untreated MCL. Most
`patients are treated with combinations of rituximab and
`chemotherapy—often HyperCVAD (rituximab, hyper-
`fractionated cyclophosphamide, vincristine, doxorubicin,
`and dexamethasone),17 R-CHOP (cyclophosphamide,
`doxorubicin, vincristine, prednisone, and rituximab),16
`or a purine nucleoside analogue and rituximab. Patients
`who are eligible for high-dose therapy with stem-cell sup-
`port are increasingly transplanted in first remission.39,40
`Mantle-cell lymphoma remains a difficult disease to treat
`once it has relapsed, and patients are typically treated with
`multiple regimens because of the short time to progression
`between treatments. An improved understanding of the
`biology of MCL and the availability of new classes of
`chemotherapy agents led us to hypothesize that drugs
`
`that target the machinery that controls cyclin D1 expres-
`sion might have antitumor activity in MCL. In the study
`reported herein, temsirolimus was tested as a single agent
`in a cohort of heavily pretreated patients with MCL to learn
`if inhibition of mTOR would produce tumor responses.
`The study demonstrated an ORR of 38% (13 of 34 patients),
`with a median duration of response of 6.9 months and a me-
`dian TTP for all patients of 6.5 months. Tumor responses
`tended to occur rapidly, with most patients demonstrating
`a response by 3 months. Only two of the 13 responders had
`their initial response documented beyond 3 months.
`Consistent with previous studies showing that tem-
`sirolimus can cause thrombocytopenia,41 this toxicity
`was the most common side effect in the present trial.
`Several features of the trial design may have predisposed
`patients to this side effect. First, patients could enter the
`protocol with grade 1 thrombocytopenia ($ 75,000)
`and could receive 100% of the temsirolimus dose if the
`platelet count was $ 50,000 (grade 2). Second, the patients
`enrolled on this study were heavily pretreated. Third, most
`patients had marrow infiltrated with MCL cells, reducing
`marrow reserve.
`Because of the inabilit