British Journal of Cancer (2006) 95, 1148 – 1154
`& 2006 Cancer Research UK All rights reserved 0007 – 0920/06 $30.00
`
`www.bjcancer.com
`
`A phase II clinical and pharmacodynamic study of temsirolimus in
`advanced neuroendocrine carcinomas
`
`I Duran1, J Kortmansky2, D Singh3, H Hirte1, W Kocha1, G Goss1, L Le1, A Oza1, T Nicklee1, J Ho1, D Birle1,
`GR Pond1, D Arboine1, J Dancey4, S Aviel-Ronen1, M-S Tsao1, D Hedley1 and LL Siu*,1
`1Department of Medical Oncology and Hematology, Princess Margaret Hospital Phase II Consortium, 610 University Avenue, Suite 5-718, Toronto, ON,
`Canada M5G 2M9; 2Memorial Sloan Kettering Cancer Center, New York, USA; 3University of Chicago, Chicago, USA and 4National Cancer Institute,
`Bethesda, USA
`
`Standard cytotoxic treatments for neuroendocrine tumours have been associated with limited activity and remarkable toxicity. A
`phase II study was designed to evaluate the efficacy, safety and pharmacodynamics of temsirolimus in patients with advanced
`neuroendocrine carcinoma (NEC). Thirty-seven patients with advanced progressive NEC received intravenous weekly doses of
`25 mg of temsirolimus. Patients were evaluated for tumour response, time to progression (TTP), overall survival (OS) and adverse
`events (AE). Twenty-two archival specimens, as well as 13 paired tumour biopsies obtained pretreatment and after 2 weeks of
`temsirolimus were assessed for potential predictive and correlative markers. The intent-to-treat response rate was 5.6% (95% CI
`0.6 – 18.7%), median TTP 6 months and 1-year OS rate 71.5%. The most frequent drug-related AE of all grades as percentage of
`patients were:
`fatigue (78%), hyperglycaemia (69%) and rash/desquamation (64%). Temsirolimus effectively inhibited the
`phosphorylation of S6 (P¼ 0.02). Higher baseline levels of pmTOR (phosphorylated mammalian target of rapamycin) (P¼ 0.01)
`predicted for a better response. Increases in pAKT (P¼ 0.041) and decreases in pmTOR (P¼ 0.048) after treatment were associated
`with an increased TTP. Temsirolimus appears to have little activity and does not warrant further single-agent evaluation in advanced
`NEC. Pharmacodynamic analysis revealed effective mTOR pathway downregulation.
`British Journal of Cancer (2006) 95, 1148 – 1154. doi:10.1038/sj.bjc.6603419 www.bjcancer.com
`Published online 10 October 2006
`& 2006 Cancer Research UK
`
`Keywords: mTOR; neuroendocrine carcinoma; phase II; pS6; temsirolimus
`
`

`
`

`
`Clinical Studies
`
`Neuroendocrine carcinomas (NEC) comprise a family of neo-
`plasms derived from the diffuse neuroendocrine system with a
`wide range of morphologic, functional and behavioural character-
`istics (Jensen and Doherty, 2005). Conventional chemotherapy has
`not shown a significant activity in advanced NEC, except for islet
`cell carcinomas (ICC) where streptozocin-based combinations
`with either 5-fluorouracil or doxorubicin have produced partial
`remissions in 40 – 60% of selected patients (Moertel et al, 1982;
`Moertel et al, 1992). However, carcinoid tumours (CT) seem to be
`quite chemo-resistant, with response rates of o10% and a median
`5-year survival of 18% (Oberg, 2001). Alternative treatments
`including a-interferon 2b and local hepatic therapies for isolated
`metastases, such as hepatic arterial bland or chemo-embolisation,
`resection and radiofrequency ablation, have not demonstrated
`significant impacts on overall survival (OS).
`
`*Correspondence: Dr LL Siu; E-mail: lillian.siu@uhn.on.ca
`Supported by clinical trial contracts from the US National Cancer
`Institute N01-CM-17107 and translational
`research fund contract
`number N01-CO-124001, subcontract number 22XS208-T08/P6171.
`This study was presented in part at the AACR-NCI-EORTC International
`Conference, Philadelphia, USA, November 2005.
`Received 3 August 2006; revised 11 September 2006; accepted 11
`September 2006; published online 10 October 2006
`
`The mammalian target of rapamycin (mTOR) is a serine –
`threonine kinase that participates in the regulation of cell growth,
`proliferation and apoptosis through modulation of cell cycle
`progression (Vignot et al, 2005). Mammalian target of rapamycin
`regulates initiation of cap-dependent translation by phosphoryla-
`ting 4E-binding protein 1, releasing eukaryotic initiation factor
`4E (eIF4E) to bind with eIF4G of the 4E initiation protein complex.
`Mammalian target of
`rapamycin also modulates
`ribosomal
`function by phosphorylating p70S6 kinase which activates the
`ribosomal protein S6 (Podsypanina et al, 2001). Signalling through
`the PI3K/AKT/mTOR pathway leads to an increase in translation,
`particularly of proteins regulating cell cycle progression and
`metabolism. In cancer cells, aberrant activation of the pathway
`may occur
`through increased signalling via growth factor
`receptors, activating mutations/amplification of
`the pathway
`kinases or by loss of the tumour suppressor protein PTEN. The
`latter has been described in NEC (Wang et al, 2002). Temsirolimus
`(sirolimus 42-ester 2,2-bis hydroxymethyl propionic-acid; CCI-
`779) is a more water-soluble ester derivative of
`its parent
`compound sirolimus, selected for development as an anticancer
`agent based on its more favourable pharmaceutical characteristics
`and superior therapeutic index. Temsirolimus has already been
`tested in phase I and II trials with promising activity and good
`safety profile (Atkins et al, 2004; Baselga et al, 2004; Raymond
`et al, 2004; Chan et al, 2005). In phase I studies, rash and mucositis
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`Phase II study of temsirolimus in neuroendocrine tumours
`I Duran et al
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`ClinicalStudies
`
`were dose-limiting, and other adverse events (AE) observed
`include eczematous reactions, dry skin, herpes-type lesions, mild
`myelosupression, hypercholesterolaemia and hypertriglyceridemia
`(Atkins et al, 2004; Baselga et al, 2004; Raymond et al, 2004).
`The primary objective of this study was to examine the objective
`response rate (ORR) of temsirolimus in patients with recurrent
`or metastatic NEC. Secondary objectives were: (i) to assess drug
`toxicity; (ii) to determine through pharmacodynamic evaluations
`whether
`temsirolimus downregulates mTOR and modulates
`elements of the PI3K/AKT/mTOR pathway and (iii) to associate
`pretreatment molecular characteristics, and molecular changes
`between paired tumour biopsies, with clinical outcome.
`
`PATIENTS AND METHODS
`
`Eligibility
`
`Patients were eligible if they were 18 years of age or older and had
`histologically or cytologically confirmed NEC of either carcinoid or
`pancreatic ICC pathologies. Patients had to have documented
`progressive metastatic disease within 6 months of study entry.
`Previous chemotherapy, investigational agents, radioactive thera-
`pies and/or radiation were allowed if completed 44 weeks before
`study entry. Previous local
`therapy (e.g. bland or chemo-
`embolisation) was allowed if completed 46 weeks before study
`entry. Patients were required to have measurable disease, an ECOG
`performance status p2, normal serum cholesterol and triglyceride,
`adequate haematologic, hepatic, renal and cardiac functions and a
`life expectancy of 43 months. Patients had to have tumour lesions
`accessible for core biopsy, and must agree to undergo tumour
`biopsy before and 2 weeks after initiation of temsirolimus.
`
`Treatment
`
`Temsirolimus at 25 mg was administered as a 30-min intravenous
`infusion on a weekly schedule. Four weeks of treatment were
`considered as one cycle.
`
`Assessment of toxicity
`
`Adverse events were graded using the National Cancer Institute
`Common Terminology Criteria for Adverse Events v3.0.
`
`Dose modifications
`
`Dose modifications of temsirolimus were based on haematologic
`and non-haematologic toxicities at the time of every weekly dose.
`Upon recovery of toxicity within a maximum delay of 3 weeks,
`temsirolimus may be re-started with a dose reduction. Stepwise
`dose modifications from 25 to 20, 15 and 10 mg were allowed, but
`doses once reduced cannot be re-escalated.
`
`Response assessment
`
`Radiological imaging was repeated every 8 weeks to assess for
`tumour response until disease progression, completion of study
`treatment or discharge of patient from study. Tumour responses
`were evaluated according to standard RECIST criteria (Therasse
`et al, 2000). Objective responses were confirmed by central
`independent radiological review.
`
`Correlative studies
`
`Archival tissues Archival paraffin slides were stained for PTEN,
`p53, pAKT, pS6 and pmTOR (phosphorylated mTOR) by
`immunohistochemistry. Slides were pretreated and incubated with
`primary antibody (Appendix 1), followed by biotin-conjugated
`secondaries and HRP-Streptavidin labelling reagent (ID Labs Inc.,
`
`London, Ontario, Canada). Two pathologists, who were blinded to
`clinical outcome (M-ST. and SA-R), evaluated independently for
`each marker: (i) the percentage of stained cells, which was
`converted to a four-tiered system (0¼ 0; 1¼ p10%; 2¼ 11 – 50%
`and 3¼ X51%), and (ii) the intensity of staining (0 – 3). The sum
`of both values was the specimen’s score (range 0 – 6). The scores
`from the two pathologists were then averaged. PTEN was scored as
`either ‘lost’ or ‘retained’.
`
`Computerised image analysis for paired tumour biopsies Tumour
`biopsies taken before and after 2 weeks post-treatment with
`temsirolimus were collected into 10% neutral buffered formalin,
`fixed overnight and transferred in 70% ethanol for processing into
`paraffin blocks. Four micrometre thick sections were cut onto
`Surgipath x-trat slides. Slides were pretreated by either pepsin
`digestion or microwave retrieval and then incubated in primary
`antibody overnight inside a moist chamber (Appendix 1). This was
`followed with Cy3- or Cy5-conjugated secondaries (Jackson
`ImmunoResearch, West Grove, PA, USA) for immunofluorescence.
`Paired biopsies were stained for pAKT, pS6, pmTOR and peIF4G.
`Biopsy slides were imaged with a laser scanning TISSUEscope
`(Biomedical Photometrics, Waterloo, Ontario, Canada) using 2 mm
`per pixel resolution. All images were analysed blinded using MCID
`Elite software (Imaging Research Inc., St Catharines, Ontario,
`Canada). A threshold was set to select for positive staining of a
`specific marker of
`interest. Results were expressed as the
`percentage of positively stained areas in square microns within
`the tumour regions, and the staining intensity reported as mean
`optical density (IOD) in grey levels.
`
`Statistical analysis
`
`The primary endpoint was objective tumour response rate
`(complete response (CR) or partial response (PR)). Secondary
`endpoints included toxicity, stable disease (SD) rate, response and
`SD duration, time to progression (TTP) and OS. A two-stage phase
`II design was used. The treatment combination would be assumed
`to be inactive if the objective response was at most 5% and active if
`it was at least 25%, with a¼ 0.05 and b¼ 0.10. After completion of
`accrual of 15 evaluable patients to stage I, while no CR or PR was
`observed, there were 10 patients who fulfilled the criteria for SD.
`After plotting out patient’s pretreatment and post-treatment
`tumour progression rate, it was observed that at least three out
`of these 10 patients had a significant decrease in this parameter
`after starting temsirolimus, along with improvement
`in their
`disease-related symptoms. As a result of these findings, it was
`hypothesised that temsirolimus may have antitumour activity in
`this tumour type but possibly not reliably evaluated by conven-
`tional RECIST criteria. The protocol was amended and additional
`patients accrued to stage II.
`The Kaplan – Meier method was used to estimate survival
`outcomes. Time to progression was measured from the first date
`a patient received study medication until the date of tumour
`progression. Progression-free survival (PFS) was estimated from
`the first date a patient received study medication until the date of
`progression, or death; OS was measured from the first date a
`patient received study medication until the date of death or last
`date the patient was known to be alive.
`Molecular marker levels before temsirolimus treatment and
`their changes measured on study were investigated as predictors of
`objective tumour response, TTP and OS, using Spearman rank
`correlation coefficients and Cox proportional hazards regression.
`tests were two-sided and P-values of 0.05 or less were
`All
`considered statistically significant.
`This study was a collaborative effort between three consortia, led
`by the Princess Margaret Hospital Phase II Consortium. Local
`institutional
`review board approvals were obtained at all
`participating centres.
`
`& 2006 Cancer Research UK
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`British Journal of Cancer (2006) 95(9), 1148 – 1154
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`The intent-to-treat response rate for the entire study cohort is
`2/36¼ 5.6% (95% CI 0.6 – 18.7%) and tumour control (SDþ PR)
`rate is 23/36¼ 63.9% (95% CI 46.2 – 79.2%). Response outcomes
`were similar between the CT and ICC histologies, with PR rates of
`4.8 and 6.7%, respectively.
`
`TTP Five patients remain on study and continue to receive
`treatment as of January 2006. Of the 31 patients who have come off
`treatment,
`the reasons
`for discontinuation were: PD (15),
`symptomatic PD (four), death (one), physician discretion (two),
`AE (seven) and patient withdrawal (two). Median TTP is estimated
`to be 6.0 months (95% CI 3.7-not reached); 6-month progression-
`free rate is estimated to be 48.1% (95% CI 33.0 – 70.1%) and 1-year
`progression-free rate is estimated to be 40.1% (95% CI 23.8 –
`67.4%) (Figure 2).
`
`Survival At the time of reporting, 11 patients have died. Median
`follow-up on the 25 patients alive at last follow-up is 13.9 months
`(range 2.8 – 22.6 months), minimum follow-up is 6.9 months.
`Median OS has not been reached; 6-month survival rate is
`estimated to be 91.6% (95% CI 82.9 – 100.0%) and 1-year survival
`rate is estimated to be 71.5% (95% CI 57.1 – 89.5%) (Figure 3).
`Patients with ICC appeared to have slightly better TTP and OS but
`statistical comparisons were not made for this subgroup analysis
`(Table 2).
`
`Toxicity
`
`Safety and tolerability data are available for 213 treatment cycles,
`with a median number of four cycles delivered per patient (range
`1 – 21), AE deemed by the investigator as at least possibly related to
`
`Phase II study of temsirolimus in neuroendocrine tumours
`I Duran et al
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`
`RESULTS
`
`Patients
`
`A total of 37 patients were accrued to the study from January 2004
`to July 2005. One patient did not receive any treatment owing to
`progressive disease before treatment initiation and was consider-
`able ineligible. Thirty-six patients received at least one dose of
`temsirolimus and were evaluated for safety. Patient characteristics
`are listed in Table 1.
`
`Efficacy
`
`Tumour response Two patients, one with CT and one with ICC,
`achieved a confirmed PR. One of them progressed after 18 cycles
`(11.4 months after first observation of PR) and the other came off
`study owing to unrelated cardiac disease (4.5 months after first
`observation of PR). A third patient had an unconfirmed PR at the
`end of cycle 8 and discontinued therapy not owing to toxicity.
`Twenty additional patients had SD of at least 2 months’ duration
`and among these, 10 patients continued treatment beyond six
`cycles. Ten patients progressed on temsirolimus without ever
`achieving an objective response. Eight of
`these patients had
`radiological evidence of disease progression, one had symptomatic
`progression during cycle 1, and one patient died of disease before
`end of cycle 2. Figure 1 shows the maximum percentage of target
`tumour lesion(s) reduction compared to baseline as assessed by
`the RECIST criteria, listed by individual study patients.
`As serum markers such as chromogranin A or 5HIAA were not
`mandated in this protocol and not collected in all patients, the
`biochemical response could not be assessed.
`
`Clinical Studies
`
`PD patients
`SD or PR patients
`
`40
`
`20
`
`0
`
`–20
`
`–40
`
`Percent change from baseline
`
`Figure 1 Maximal percentages of tumour reduction for target lesion(s)
`by RECIST criteria (Note: some patients with PD progressed owing to new
`or increasing non-target lesions, or by symptomatic progression).
`
`Progression-free estimate
`95% confidence interval
`
`1.0
`
`0.8
`
`0.6
`
`0.4
`
`0.2
`
`0.0
`
`Proportion progression-free
`
`0
`
`3
`
`6
`
`9
`12
`Months
`
`15
`
`18
`
`21
`
`Figure 2 Time to progression for entire study cohort.
`
`%
`
`58
`42
`
`44
`53
`3
`
`58
`42
`
`6
`58
`14
`11
`72
`25
`
`39
`25
`19
`8
`8
`
`Patients N¼ 36
`
`56
`36 – 77
`
`Table 1 Patient characteristics
`
`Age (years)
`Median
`Range
`
`Sex
`Female
`Male
`
`ECOG PS
`0
`1
`2
`
`Type of tumour
`Carcinoid
`Islet cell carcinoma
`
`Prior treatment
`Adjuvant chemotherapy
`Palliative chemotherapy
`Radiotherapy
`Bland embolisation
`Surgery
`Octreotide
`
`No. of prior chemotherapy regimens
`0
`1
`2
`3
`4
`
`No.
`
`21
`15
`
`16
`19
`1
`
`21
`15
`
`2
`21
`5
`4
`26
`9
`
`14
`9
`7
`3
`3
`
`British Journal of Cancer (2006) 95(9), 1148 – 1154
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`& 2006 Cancer Research UK
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`ClinicalStudies
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`Phase II study of temsirolimus in neuroendocrine tumours
`I Duran et al
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`1151
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`pmTOR and pAKT) and tumour response, TTP or survival.
`Despite being nonstatistically significant,
`the loss of PTEN
`expression was associated with a trend towards a shorter TTP
`(P¼ 0.07).
`Paired baseline and post-treatment biopsies were obtained from
`23 patients, and 13 paired-samples were evaluable. The best
`responses of these 13 patients were one PR, eight SD, three PD and
`one nonevaluable. Baseline expression levels of pAKT, pS6,
`pmTOR and peIF4G were determined and compared with
`expression levels following 2 weeks of treatment. Temsirolimus
`effectively inhibited the phosphorylation of S6 (P¼ 0.02) (Figure 4)
`and higher baseline levels of pS6 showed a trend towards being
`predictive of a better response (P¼ 0.097). Higher baseline levels
`response (P¼ 0.01).
`of pmTOR were predictive of
`tumour
`Increases in the expression of pAKT (P¼ 0.041), and decreases
`in pmTOR (P¼ 0.048) after 2 weeks on treatment, were associated
`with an increased TTP.
`A discrepancy was noted in the predictive abilities of pS6 and
`pmTOR between freshly procured pretreatment specimens vs
`paraffin-embedded archival specimens. However, only seven
`patients had both archival specimens and pretreatment tumour
`biopsies that were evaluable. Hence, statistical comparisons were
`not performed.
`
`DISCUSSION
`
`Neuroendocrine carcinomas, generally subcategorised into CT and
`ICC, often pursue an indolent clinical course. However, patients
`ultimately will become symptomatic either as a result of increasing
`tumour bulk or hormonal hypersecretion. Somatostatin analogues
`have proven successful in ameliorating symptoms of the carcinoid
`syndrome but its benefit in survival is unclear (Saltz et al, 1993).
`Streptozocin and DTIC-based regimens have been tested with only
`modest activity and may also be associated with significant toxicity
`(Moertel and Hanley, 1979; Engstrom et al, 1984; Moertel et al,
`1992; Bukowski et al, 1994; Rivera and Ajani, 1998; Cheng and
`Saltz, 1999; Ramanathan et al, 2001; McCollum et al, 2004; Sun
`et al, 2005). As conventional
`systemic approaches
`remain
`insufficient and highly toxic, there is an obvious need for novel
`therapies in this tumour population.
`The intent-to-treat response rate of 5.6% and median TTP of 6.0
`months observed in our study compares favourably with other
`targeted therapies tested in this tumour population. A recently
`reported phase II study of gefitinib in 96 patients with progressive
`NEC (55 with CT and 42 with ICC) revealed a 6-month-PFS of 51%
`for the former and 28% for the latter. However, objective responses
`were only seen in one out of 40 patients (2.5%) in the CT group
`(Hobday et al, 2005). A phase II study with the multi-targeted
`tyrosine kinase inhibitor sunitinib involving 112 patients (41 CT
`and 61 ICC), reported an ORR of 8.8%, with a median TTP of 40
`weeks and a high percentage of SD (Kulke et al, 2005). Efficacy
`varied by tumour histology with an ORR of 2% (1/41) among CT vs
`13% (8/61) among ICC. In another phase II study where 44 patients
`with CT were randomised to bevazucimab or pegylated interferon,
`the former showed superior activity (4/22 vs 0/22 confirmed PR),
`reduction in tumour perfusion and improvement in PFS at 18
`weeks (96% vs 68%) (Yao et al, 2005). However, neither of these
`two latter trials required evidence of progressive disease before
`study entry, therefore the patient populations are likely different
`from ours.
`Regarding histology, in our study temsirolimus appears to be
`slightly more active in ICC than in CT, as reported with other
`therapies (Moertel and Hanley, 1979; Engstrom et al, 1984; Sun
`et al, 2005; Kulke et al, 2006). While our small sample size
`precludes any definitive conclusions, it is possible that CT and ICC
`will manifest different sensitivities to different targeted agents, as
`is the situation with cytotoxic agents.
`
`Survival estimate
`95% confidence interval
`
`1.0
`
`0.8
`
`0.6
`
`0.4
`
`0.2
`
`0.0
`
`Proportion alive
`
`0
`
`3
`
`6
`
`9
`
`12
`Months
`
`15
`
`18
`
`21
`
`Figure 3 Overall survival for entire study cohort.
`
`Table 2 Efficacy outcomes by histology
`Islet cell n¼ 15
`n (%)
`
`Carcinoids n¼ 21
`n (%)
`
`Best response
`PR
`SD
`PD
`Non-evaluable
`PR+SD
`
`Time to progression
`Median (months)
`6-month (%)
`1-year (%)
`
`Overall survival
`Median (months)
`6-month (%)
`1-year (%)
`
`Status at last follow-up
`Alive
`
`Receiving treatment
`
`1 (6.7)
`9 (60.0)
`4 (26.7)
`1 (6.7)
`10 (66.7)
`
`10.6
`51.6
`25.8
`
`1 (4.8)
`12 (57.1)
`6 (28.6)
`2 (9.5)
`13 (61.9)
`
`6.0
`45.3
`45.3
`
`Not reached
`93.3
`85.6
`
`Not reached
`90.5
`60.7
`
`11 (73.3)
`
`14 (66.7)
`
`2 (13.3)
`
`3 (14.3)
`
`study drug and that have occurred in more than 10% of the cycles
`are shown in Table 3. Overall, treatment with temsirolimus was
`well tolerated. The most frequent AE of all grades, at least possibly
`related to study treatment, were: fatigue (78% of patients; 53% of
`cycles), hyperglycaemia (69% of patients; 54% of cycles) and rash/
`desquamation (64% of patients; 50% of cycles). Severe AE that
`were grade X3 and at least possible attribution were uncommon.
`Seven patients developed pneumonitis considered as possibly
`related to temsirolimus, three of whom required drug discontinua-
`tion. Other observed AE include two patients who had grade 5
`events. One died from a pneumothorax and bronchospasm in cycle
`2 unlikely related to temsirolimus and the other died from
`pulmonary embolism 5 days after removal from study, deemed to
`be possibly drug related.
`
`Pharmacodynamic analysis
`
`Twenty-two patients had archival specimens evaluable for analysis
`of baseline molecular markers. No significant association was seen
`between any of the pretreatment markers tested (PTEN, p53, pS6,
`
`& 2006 Cancer Research UK
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`British Journal of Cancer (2006) 95(9), 1148 – 1154
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`Phase II study of temsirolimus in neuroendocrine tumours
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`Table 3 Drug-related adverse events occurring in 410% of treatment cycles
`
`Adverse event
`
`Patients n (%)
`
`Cycles n (%)
`
`Patients n (%)
`
`Cycles n (%)
`
`Grade 1 – 2
`
`Grade 3 – 4
`
`112 (53)
`
`111 (52)
`112 (53)
`61 (29)
`37 (17)
`
`92 (43)
`68 (32)
`58 (27)
`71 (33)
`71 (33)
`28 (13)
`35 (16)
`35 (16)
`33 (15)
`
`71 (33)
`47 (22)
`35 (16)
`40 (19)
`28 (13)
`31 (14)
`
`104 (48)
`76 (36)
`40 (19)
`38 (18)
`
`35 (16)
`
`0 (0)
`
`1 (3)
`1 (3)
`1 (3)
`0 (0)
`
`8 (22)
`2 (6)
`1 (3)
`2 (6)
`1 (3)
`1 (3)
`0 (0)
`4 (12)
`0 (0)
`
`0 (0)
`0 (0)
`0 (0)
`3 (9)
`0 (0)
`0 (0)
`
`1 (3)
`0 (0)
`0 (0)
`0 (0)
`
`0 (0)
`
`0 (0)
`
`1 (0.5)
`2 (1)
`1 (0.5)
`0 (0)
`
`24 (11)
`7 (3)
`1 (0.5)
`3 (1)
`1 (0.5)
`1 (0.5)
`0 (0)
`15 (7)
`0 (0)
`
`0 (0)
`0 (0)
`0 (0)
`3 (1)
`0 (0)
`0 (0)
`
`3 (1)
`0 (0)
`0 (0)
`0 (0)
`
`0 (0)
`
`Pharmacodynamic evaluations in paired biopsies, confirmed,
`for the first time in this patient population, that temsirolimus
`effectively downregulates the phosphorylation of S6, and that
`higher baseline levels of pS6 and pmTOR seem to predict for a
`better response. These results are consistent with those reported by
`Cho et al (2005) which analysed predictive markers of temsiro-
`limus in advanced renal cell carcinomas, confirming the value of
`pS6 (Cho et al, 2005). In this study, we have selected an antibody
`against pmTOR (ser2448) with high specificity, and this may
`explain the finding of pmTOR as a predictive marker of response.
`Other interesting pharmacodynamic findings include the rising
`trend in AKT phosphorylation noted after
`treatment with
`temsirolimus. Two alternative pathways that induce AKT activa-
`tion may explain this finding (Figure 5). RAPTOR (regulatory
`associated protein of mTOR) and RICTOR (rapamycin-insensitive
`companion of mTOR) are key partnering proteins which complex
`with mTOR and modulate its functions. Activation of AKT through
`the mTOR – RICTOR complex could explain our observation
`(Hresko and Mueckler, 2005; Sarbassov et al, 2005). Additionally,
`AKT phosphorylation has been described through a feedback loop
`of the PI3K/AKT/mTOR pathway from the insulin-like growth
`(O’Reilly et al, 2006). Further
`factor
`I
`receptor
`(IGF-IR)
`pharmacodynamic analysis revealed a positive association between
`increases in pAKT and decreases in pmTOR with a more
`prolonged TTP. These exploratory findings require confirmation
`with larger series. The evaluation of archival specimens searching
`
`28 (78)
`
`21 (58)
`19 (53)
`18 (50)
`13 (36)
`
`17 (47)
`13 (36)
`15 (42)
`15 (42)
`20 (55)
`6 (17)
`9 (25)
`8 (22)
`13 (36)
`
`26 (72)
`10 (28)
`13 (36)
`11 (30)
`10 (28)
`13 (36)
`
`22 (61)
`16 (44)
`12 (33)
`9 (25)
`
`7 (19)
`
`Constitutional symptoms
`Fatigue
`
`Hematologic
`Anaemia
`Lymphopenia
`Thrombocytopenia
`Leukocytes
`
`Metabolic
`Hyperglycaemia
`Hypercholesterolaemia
`Hypertriglyceridaemia
`ALT
`AST
`ALP
`Hypoalbuminaemia
`Hypophosphataemia
`Creatinine
`
`Gastrointestinal
`Mucositis
`Dysgeusia
`Nausea
`Diarrhoea
`Constipation
`Anorexia
`
`Dermatologic
`Rash (desquamation)
`Rash (acneiform)
`Dry skin
`Pruritus
`
`Pulmonary
`Pneumonitis
`
`Clinical Studies
`
`Correlative markers
`
`Pre-treatment
`
`Post-treatment
`
`200 ♯m
`
`Pre Post
`pS6
`
`Figure 4 Pre- and post-treatment liver biopsies. Tissue sections were
`first
`immunofluorescence-labelled for S235/236-S6 ribosomal protein,
`imaged, and then restained with H&E. The grey scale images of pS6 are
`unenhanced, at original resolution.
`
`British Journal of Cancer (2006) 95(9), 1148 – 1154
`
`& 2006 Cancer Research UK
`
`PAR-ZORT-000888
`
`NOVARTIS EXHIBIT 2174
`Par v Novartis, IPR 2016-00084
`Page 5 of 7
`
`

`
`Phase II study of temsirolimus in neuroendocrine tumours
`I Duran et al
`
`1153
`
`Nutrients
`
`Growth factors (e.g. VEGF)
`
`ClinicalStudies
`
`PI3K
`
`Akt
`
`PDK1
`
`
`
`PPPP
`
`Cytoplasm
`
`PTEN
`
`
`PPP
`
`PPP
`
`TSC2-1
`
`Rheb
`
`Rapamycin
`/FKBP12
`
`mTOR
`RAPTOR
`
`Cell cycle progression
`Cell proliferation
`Angiogenesis
`
`
`
`??
`
`mTOR
`RICTOR
`
`
`
`PPP
`
`p70S6K
`
`
`PPP
`4E-BP1
`
`
`
`??
`
`IGF-IR
`
`Protein translation
`(e.g. cyclinD1,
`ribosomes, translation
`machinery, VEGF)
`
`Figure 5 PI3K/AKT/mTOR pathway showing the mTOR protein complexes, mTOR/RAPTOR and mTOR/RICTOR, and the feedback loop involving IGF-
`IR. Arrows indicate activation; bars indicate inhibition.
`
`for prognostic factors only showed a trend towards shorter TTP in
`those patients with loss of PTEN. The discordance between the
`results from the archival and the freshly procured pretreatment
`specimens in our study could be due to differences in the genetic
`profile of primary and recurrent/metastatic tumours;
`tumour
`heterogeneity; and different protocols for specimen handling.
`Differences in time and speed of tissue fixation after biopsy or
`resection may significantly affect the phosphorylated states of
`signalling molecules.
`In conclusion,
`temsirolimus appears to have only modest
`activity with a manageable toxicity profile in advanced NEC.
`The results of this study do not warrant further investigation
`of this drug as a single agent in this patient population. Evaluation
`of
`temsirolimus,
`in combination with other targeted agents,
`such as a multi-kinase inhibitor or an antiangiogenic compound,
`
`should be considered. The loss of PTEN expression could
`represent a poor prognostic marker for NEC. Pharmacodynamic
`analysis in paired tumour biopsies reflected effective mTOR
`pathway downregulation and identified possible predictive
`factors. Evaluations in larger populations are needed to confirm
`these findings.
`
`ACKNOWLEDGEMENTS
`
`SAR is a fellow of the CIHR training program for clinician scientist
`in molecular oncology pathology [ICR/CIHR Training Grant STP-
`53912]. The authors would like to thank Drs Mark Minden and
`Karen Yee for their assistance with the manuscript.
`
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