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Original Article
A Randomised, Double Blind, Placebo-Controlled Pilot Study of Oral
Artesunate Therapy for Colorectal Cancer☆
,
☆☆
Sanjeev Krishna
a,
⁎, Senthil Ganapathi
b
, Irina Chis Ster
a
, Mohamed E.M. Saeed
c
,MattCowan
d
,
Caroline Finlayson
a
, Hajnalka Kovacsevics
a
,HerwigJansen
e,1
,PeterG.Kremsner
f
,
Thomas Efferth
c
, Devinder Kumar
b
a
Institute of Infection and Immunity, Department of Pathology, United Kingdom
b
Department of Surgery, St. George's,University of London, Cranmer Terrace, SW17 0RE, United Kingdom
c
Department of Pharmaceutical Biology, Johannes Gutenberg-University, Staudinger Weg 5, 55128 Mainz, Germany
d
Department of Gastroenterology, Surrey and Sussex Healthcare NHS Trust, East Surrey Hospital, Canada Avenue, Redhill, Sussex RH1 5RH, United Kingdom
e
Dafra Pharma nv, 2300 Turnhout, Belgium
f
Institut für Tropenmedizin, Universitätsklinikum Tübingen, Wilhelmstraße 27, D-72074 Tübingen, Germany
abstractarticle info
Article history:
Received 6 October 2014
Received in revised form 13 November 2014
Accepted 13 November 2014
Available online 15 November 2014
Keywords:
Colorectal cancer
Artesunate
Dihydroartemisinin
Ki67
Neutropaenia
Background: Artesunate is an antimalarial agent with broad anti-cancer activity in in vitro and animal experi-
ments and case reports. Artesunate has not been studied in rigorous clinical trials for anticancer effects.
Aim: To determine the anticancer effect and tolerability of oral artesunate in colorectal cancer (CRC).
Methods: This was a single centre, randomised, double-blind, placebo-controlled trial. Patients planned for cura-
tive resection of biopsy confirmed single primary site CRC were randomised (n = 23) by computer-generated
code supplied in opaque envelopes to receive preoperatively either 14 daily doses of oral artesunate (200 mg;
n = 12) or placebo (n = 11). The primary outcome measure was the proportion of tumour cells undergoing
apoptosis (significant if N7% showed Tunel staining). Secondary immunohistochemical outcomes assessed
these tumour markers: VEGF, EGFR, c-MYC, CD31, Ki67 and p53, and clinical responses.
Findings: 20 patients (artesunate = 9, placebo = 11) completed the trial per protocol. Randomization groups
were comparable clinically and for tumour characteristics. Apoptosis in N7% of cells was seen in 67% and 55%
of patients in artesunate and placebo groups, respectively. Using Bayesian analysis, the probabilities of an
artesunate treatment effect reducing Ki67 and increasing CD31 expression were 0.89 and 0.79, respectively.
During a median follow up of 42 months 1 patient in the artesunate and 6 patients in the placebo group devel-
oped recurrent CRC.
Interpretation: Artesunate has anti-proliferative properties in CRC and is generally well tolerated.
© 2014 Published by Elsevier B.V. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/3.0/).
1. Introduction
Colorectal cancer (CRC) contributes 9–10% of the annual global can-
cer burden in men (746,000 cases) and women (614,000 cases) (Ferlay
et al., 2012). In the UK, 110 new cases are diagnosed daily, with older
patients particularly at risk of death (UK CR, 2014) and with N50% of
newly diagnosed cases having locally advanced disease (T3/T4). Resec-
tion is the only curative treatment for non-metastatic CRC but this has to
be combined with neo-adjuvant chemo- and/or radio-therapy, to
downstage more advanced presentations.
Prognosis with best available treatments does not increase disease
free or overall survival beyond ~ 60% at 5 years after diagnosis. For
most patients, access to advanced treatment modalities is lacking, too
expensive to be widely available, or associated with significant morbid-
ity thereby further compromising their survival. There is therefore a
continuing and urgent need to develop new, cheap, orally effective
and safe CRC therapies. One approach is to study existing drugs that al-
ready have some anticancer properties in experimental settings, and to
assess their safety and efficacy in in vivo studies.
Artesunate is derived from artemisinin, which is extracted from
Artemisia annua L. and is a widely used antimalarial that can be ad-
ministered by oral, rectal and parenteral routes (Gomes et al., 2009;
Kremsner and Krishna, 2004; Kremsner et al., 2012; Nealon et al.,
2002; Hien et al., 1994, 1992; Jiang et al., 1982). Soon after the isolation
of artemisinin by a Chinese government's programme, the anticancer
properties of artemisinins were first reported (Efferth et al., 2007;
EBioMedicine 2 (2015) 82–90
☆Funding
☆☆ This study did not receive any specific funding. Drug and placebo was supplied by
Dafra pharmaceuticals.
⁎Corresponding author.
1
Deceased.
http://dx.doi.org/10.1016/j.ebiom.2014.11.010
2352-3964/© 2014 Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/).
Contents lists available at ScienceDirect
EBioMedicine
journal homepage: www.ebiomedicine.com
Author's personal copy
Krishna et al., 2008). Subsequently, many studies of artemisinins using
in vitro and animal models have confirmed their remarkable capacity
to exert broad anti-cancer effects (Efferth et al., 2007). They reduce
cell proliferation and angiogenesis and trigger apoptosis (Anfosso
et al., 2006; Efferth et al., 2001, 1996).
There have only been isolated case reports in humans of anti-cancer
effects of artemisinins (reviewed Krishna et al., 2008). These include
cases of metastatic uveal melanoma (Berger et al., 2005) laryngeal squa-
mous cell carcinoma (Singh and Verma, 2002) and pituitary macro-
adenoma (Singh and Panwar, 2006). An open-label Chinese study
treated non-small cell lung cancer patients and showed prolonged
time to cancer progression compared with controls when artesunate
was added to conventional treatment (Zhang et al., 2008), but no
benefit on mortality. An open-label pilot study of patients receiving
artesunate for advanced cervical cancer suggested that it was well toler-
ated and improved symptoms (Jansen et al., 2011). There has been a
phase II trial on the activity of artesunate in non-resectable tumours of
dogs (Rutteman et al., 2013)andefficacy of extracts of A. annua in 5 vet-
erinary sarcomas (Breuer and Efferth, 2014). This study examines anti-
CRC effects and tolerability of artesunate used as monotherapy in a
rigorous study design.
2. Methods
2.1. Ethics
The trial was approved by Wandsworth Ethics Committee (Wands-
worth UK, Ref: 08/H0803/3) and was registered (ISCRTN05203252).
2.2. Trial Design
This was a single-centre, double-blind, placebo-controlled trial with
balanced randomisation of patients (1:1) conducted at the St George's
University of London, UK and St. George's Healthcare NHS Trust.
2.3. Participants for Inclusion
Eligible participants were with biopsy confirmed single primary site
colorectal adenocarcinoma; aged 21–90 years; with all stages amenable
to surgical treatment and not requiring neoadjuvant treatment; with
planned curative resection; and with written, informed consent.
2.4. Exclusion Criteria
These were: contraindication to useof artesunate due to hypersensi-
tivity; pregnancy; history of hearing or balance problems; immunosup-
pression or concomitant medication known to interact with artesunate
(see below); weight b50 kg or N100 kg; severe anaemia (haemoglobin
b8 g/dL); other planned intervention, apart from standard of care;
inability to give informed consent; inability or unwillingness to take ef-
fective contraception in women of child-bearing age; chronic kidney
disease of NKF D/QOFI stage 3 or above (eGFR b60 mL/min); bilirubin
N2 of the upper limit of normal without haemolysis or known chronic
liver disease.
2.5. Recruitment
Recruitment was at St George's Healthcare NHS Trust in London
from 9 March 2009 to 15 October 2012.
2.6. Interventions
Patients received two weeks of experimental medication (artesunate
or placebo) just before surgery and standard care. Artesunate (Arinate®
100 mg) was manufactured by Famar Italia S.p.A and matching pla-
cebo tablets were manufactured by MPF in The Netherlands under a
manufacturing licence in accordance with EU cGMP certified by
Dafra Pharma (Belgium). Study medication was packaged, labelled
and certified by B&C CliniPack (Belgium) and was in pack sizes of
30× 100mg andwasreceived,storedanddispensedbythePharmacy
at St George's Healthcare NHS Trust.
The dose of artesunate for the study was 200 mg orally, daily for
fourteen days, with medication stopped 48–72 h prior to surgery.
Medication was provided in blister packs with one patient box pro-
vided 14 doses, sufficient for the duration of the study.
There was nodelay in surgery if patients entered into this study, nor
any otherchange in clinicalmanagement, and the 62 day rule (requiring
treatment within this time period after confirmation of diagnosis) was
strictly adhered to.
2.7. Outcomes
The primary endpoint of the trial was the presence or absence of
significant apoptosis in the epithelial cells of the tumour specimen de-
fined as N7% of cells with apoptotic features.
Secondary outcomes included seven immunohistochemical stains
applied to the paraffin-embedded tumour specimens and quantified
in both epithelial cells and fibroblasts: vascular endothelial growth fac-
tor (VEGF), c-MYC status and EGF-receptor status; microvessel density
determining the quantity of the cluster of differentiation 31 (CD31) pro-
tein; proliferative activity assessed with Ki67 staining and p53 tumour
suppressor protein expression. Each stain in each patient was generally
evaluated in 6 microscopic areas with a semiautomatic system (in a few
cases 7 or 8 areas were evaluated, and in some –especially for fibro-
blasts –measurements less than 6 or no areas could be evaluated).
2.8. Blood Samples
Three blood samples were taken: (1) at baseline, (2) after one
week of medication (following protocol amendment for enhanced
safety monitoring) and (3) after ending the two week medication
(just before surgery). In each sample the safety measures included
assay of potassium, sodium, creatinine, urea, albumin, alkaline phospha-
tase, ALT, bilirubin, haemoglobin, platelet count and white cell count.
Carcinoembryonic antigen (CEA), was monitored where available in pa-
tients at baseline and after randomization.
2.9. Secondary Outcomes
These were measures of safety and tolerability (both clinical and
laboratory) according to conventional criteria assessed by comparing
baseline blood test results and those during or after treatment and anti-
cancer efficacy (with markers described above).
2.9.1. Changes to Outcomes
There were no changes to predefined endpoints.
2.10. Sample Size
An indicative sample size calculation, given thepioneering nature of
this pilot study, was carried out on the primary outcome before starting
the trial based on the assumptionthat colorectal cancer is unlikely to ex-
hibit significant apoptosis if untreated. Most patients in the placebo
group (more than 95%) were anticipated to have less than 7% of cells
with apoptotic features. The majority of patients (greater than 60%) in
the artesunate group were anticipated to have significant apoptosis.
This large difference was derived from published baseline estimates of ap-
optotic indices (Yamamoto et al., 1998; Ikenaga et al., 1996; Bendardaf
et al., 2003). With equal group sizes a sample size of 2*11 was estimated
to have 80% power and accepting a Type I error of 5% for superiority, bear-
ing in mind that in most pilot studies the aim is to demonstrate proof-of-
concept (Arain et al., 2010) rather than exclusively test a hypothesis.
83S. Krishna et al. / EBioMedicine 2 (2015) 82–90
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2.11. Randomisation
Subjects were randomised to receive either artesunate or placebo in
equal numbers. Randomisation was performed using a computer-
generated code, and results supplied in opaque and sealed envelopes
by Dafra Pharma. After enrollment and allocation of the next study
number in the series, participants were given their randomization
pack by a pharmacist. Copies of the key to the randomisation codes
were held by theClinical Trials Pharmacist only and allocation and con-
cealmentsteps were performedin Belgium. The code wasnot opened to
investigators, patients, data collectors until the data collection ended,
histological results had been analysed and datasets were locked.
2.12. Sequence Generation and Allocation Concealment Mechanism
Study medications were pre-packed in blister packs and consecu-
tively numbered for each participant according to the randomisation
schedule. Each participant was assigned an order number once they
had consented to the study and after eligibility checks, and they re-
ceived the medication pack with the corresponding randomization
number.
2.13. Blinding
It became necessary to unblind the allocation to 2 participants
during the course of the study at the request of the MHRA after receipt
of notification of Adverse Events. Blinding was maintained for all inves-
tigators and codes were supplied by the Sponsor's office (SGUL) to the
MHRA.
2.14. Data Collection and Structure
Immunohistopathological data generated multiple measurements
per individual as the number of slides differed according to the sizes
of the tumours. Each measurement represents an estimate of staining
of cancer cells found on a slide with 0 denoting no staining observed
on a section. Therefore, the dataset inherits a hierarchical structure
with patients at level one and within individual measurements as
level two. With the exception of the three individuals (Fig. 1)there
are no records considered missing for statistical analysis.
2.15. Exploratory Statistical Analysis
The nature of all data variables have been graphically assessed and
summarised accordingly with means/medians standard deviation for
continuous data or proportions for binary data. Correlations were ex-
plored with Spearman's coefficient.
2.16. Immunochemistry Results and Inferential Data Analysis
A random effects (variance components) model was employed for
immunochemistry data to capture their variabilities correctly, given
their inherent hierarchical structures.
Patients were followed-up with CEA measurements every 6 months
and annual CT scans for disease recurrence. Time since surgery to the
first disease recurrence has been modelled with survival analysis.
Patient CRC06 has been included in survival analysis and although
there were no samples obtained for immunochemistry, the patient
was known to have survived. Patient CRC13 was initially randomised
Assessed for eligibility (n= 47)
I
A
Excluded (n= 30)
Not meeting inclusion criteria (n= 24)
Declined to participate (n= 6)
Analysed (n= 11)
Excluded from analysis (n= 0)
Lost to follow-up (n= 0)
Discontinued intervention (n= 0)
Allocated to intervention (n= 11)
Received allocated intervention (n= 11)
Did not receive allocated intervention (n= 0)
Lost to follow-up (n= 0)
Discontinued intervention (n= 0)
Allocated to intervention (n= 12)
Received allocated intervention (n= 10)
Did not receive allocated intervention
(travelled elsewhere for surgery) (n= 2)
Analysed (n= 9 )
Excluded from analysis (no cancer in
resected bowel) (n= 1)
Allocation
Follow-Up
Randomized (n= 23)
Enrollment
♦
♦
♦
♦
♦
♦
♦
♦
Analysis
Fig. 1. Patientflow diagram. Assessment for eligibility was recorded after6 patients had been randomised, so that these added to the 17 patientsrandomised after screening give the total
number of randomised patients (n = 23).
84 S. Krishna et al. / EBioMedicine 2 (2015) 82–90
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to artesunate but was included in the survival analysis as placebo as s/he
did not receive any drug (as per protocol analysis). Patient CRC21 was
deemed as missing and sensitivity analysis including them in either
artesunate or placebo groups is provided. The Cox proportional hazard's
(PH) model has been applied to investigate the hazard ratio of disease
recurrence for artesunate compared with placebo. and their pointwise
95% confidence intervals are provided for each treatment group.
2.17. Statistical Inference
Model based Bayesian analysis and classical frequentist approaches
have been applied as appropriate. Frequentist statistical significance is
conventionally associated with p-values lessthan 0.05 with uncertainty
of parameters assessed by the 95% confidence intervals (CI). Parameter
estimates in Bayesian inference are summarised by their posterior
means and the corresponding 95% credible intervals (CrI). Initially, no
prior knowledge was assumed for the parameter that quantifies the
treatment effect, i.e. the difference between the group means in terms
of immunochemistry measurements and the inference that has been
drawn. If prior information exists, then it is appropriate to assess how
the parameter values change based on this evidence. Ki67 and CD31
were the only stains with prior anti-CRC information available (Li
et al., 2007; Jansen et al., 2011). A sensitivity analysis was conducted
with Ki67. Statistical software used included OpenBUGS (Thomas
et al., 2006) STATA (StataCorp. 2013. Stata Statistical Software: Release
13. College Station, TX: StataCorp LP) and R (Team RDC, 2011).
3. Results
3.1. Participant Flow
Fig. 1 summarises patient flows. 12 patients were randomised to re-
ceive artesunate and 11 to placebo. 2 patients did not receive artesunate
despite randomization (one travelled for surgery outside the UK and
could not be followed up, and the drug expiry date for the relevant
batch had been reached when the other patient attended). One
artesunate recipient could not be evaluated for the primary endpoint
Table 1
Baseline demographic, clinical and laboratory characteristics.
Artesunate
N=12
Mean (SD)
Placebo
n=11
Mean (SD)
Numbers
a
(artesunate/
placebo)
Demographics 3
Age (y) 69 (11) 66 (14) 12/10
Gender (%, F) 0.58 0.64
Ethnicity (% Caucasians) 0.83 0.82
Height (m) 1.70 (0.12) 1.62 (0.08) 10/9
Weight (kg) 74 (16.7) 75 (66, 85) 11/10
Biochemistry
Sodium (mmol/L) 140 (1)
a
139 (2.28) 11/11
Albumin (g/L) 38 (5.7) 36 (5.7) 11/11
ALT (U/L) 23 (8) 24 (10)
a
11/11
Bilirubin (μmol/L) 9 (3) 8 (3) 9/11
Creatinine (μmol/L) 73 (23) 65 (15) 9/11
Urea (mmol/L) 5 (2) 5 (1) 9/11
Haematology
Haemoglobin (g/dL) 11.5 (2) 12.2 (2)
a
9/11
White Cell count (/L) 5.3 (2.8) 6.7 (2) 9/11
Platelet Count (/L) 370000 (20000)
a
31300 (78000) 8/11
Dukes' stage
A22
B53
C1 2 6
C2 1 1
a
Numbers of patients contributing to each value are indicated in the last column.
Table 2
Predicted “grand means”of immunohistochemistry results. Results are presented on a linear scale by treatment groups with their 95% credible intervals following a Bayesian analysis
which takes into account the variability within individual measurements as well as that of between different individuals in the cluster. The probability of an effect is the probability that
the difference between the grandmeans in the two groups (artesunate–placebo) is greaterthan 0 (and is not a p-value).*For Ki67 all corresponding estimates with informative priorsare
presented in after sensitivity analysis, with skeptical priors presented first followed by informative priors. Results for some epithelial stains were correlated with each other in placebo
recipients (EGFR and c-MYC; r = 0.664; 0.026) and for artesunate recipients (EGFR and p53, r = 0.68; p = 0.04, and Tunel and p53 r = 0.87; p = 0.0025).
Marker Artesunate Placebo P of an effect Difference or change Sensitivity analysis (prior)
Mean 95% CrI Mean 95% CrI
Epithelial
cMYC 45 (27, 53) 43 (27, 64) 0.59 2.6 (−22, 26)
CD31 8 (3,12) 5 (2,9) 0.79 2.6 (−4, 9)
EGFR 32 (12, 58) 27 (9, 45) 0.66 5.4 (−24, 32)
p53 25 (5, 45) 18 (3,35) 0.72 7 (−20, 30)
Tunel 18 (4,32) 20 (7,32) 0.4 −2(−20, 17)
VEGF 38 (21, 54) 43 (40, 75) 0.45 −5(−28, 17)
Ki67* 33 (13, 52) 49 (31, 66) 0.11 −16 (−42, 10) N (0, 1000)
Non-informative
34 (14, 53) 48 (31, 65) 0.13 −15 (−40, 10) N (0, 100)
Vaguely informative
38 (22, 54) 44 (29, 59) 0.23 −6(−22, 10.1) N (0, 10)
Informative
32 (12, 51) 49 (32, 66) 0.08 −18 (−42, 8) N (−15, 1000)
Non-informative
32 (13, 51) 49 (32, 66) 0.08 −18 (−42, 8) N (−15, 100)
Vaguely informative
32 (17, 49) 48 (33, 63) 0.03 −15 (−31, 0) N (−15, 10)
Informative
Fibroblast
c-MYC 7 (−7, 21) 19 (7,32) 0.10 −12 (−30, 7)
CD31 13 (−2, 26) 11 (0, 23) 0.58 1 (−17, 19)
EGFR 0.1 (−0.5, 0.7) 0.4 (0, 0.9) 0.21 −0.3 (−1, 1)
Ki67 5 (−3, 12) 10 (3,16) 0.16 −5(−15, 5)
p53 0.7 (−0.8, 2.1) 0.9 (−0.3, 2) 0.40 −0.2 (−2.1, 1.2)
Tunel 3 (0, 5) 4 (1,6) 0.31 0.8 (−4, 3)
VEGF 0.2 (−0.02, 0.5) 0.2 (0.1, 0.3) 0.52 0.02 (−0.3–0.3)
85S. Krishna et al. / EBioMedicine 2 (2015) 82–90
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because no tumour was identified by histology after operation. Recruit-
ment ended after the planned numbers were randomised.
3.2. Baseline Data
Baseline demographic and clinical characteristics are summarised in
Table 1. There is comparability between groups, including for Dukes'
staging.
3.3. Primary Outcome
55% of placebo recipients and 67% of artesunate treated patients
achieved the primary outcome (patients in whom the proportion of ap-
optotic cells was N7%). In designing this trial, it was assumed that only
≤1/11 patients receiving placebo would have N7% of tumour cells
displaying apoptosis. The unpredicted higher baseline values in placebo
recipients precluded detection of an artesunate effect in the primary
outcome. Staining results for Tunel as a continuous variable are in
Table 2.
3.4. Secondary Outcomes
3.4.1. Immunohistochemistry Analyses
Table 2 presents analysis of immunohistochemistry results. A ran-
dom effects model scaled linearly provides the posterior distributions
of the group means, their 95% CrI and their estimated differences be-
tween groups. The estimated posterior distributions of differences be-
tween treatment groups lie on both sides of 0 for most measurements
suggesting that the two treatment groups do not differ markedly for
most analysed markers.
Interestingly, the probability of a reduction in Ki67 staining after
artesunate is 89–92% (Table 2; a result that is 1–0.11, because Ki67 is
downregulated) using a non-informative prior on the Ki67 difference
between groups and resulting in a posterior difference of −16 (−42,
10) for treatment. A similar result is confirmed if the confidence around
the parameter value is increased as illustrated in Fig. 2a. With an opti-
mistic informative prior for artesunate of −15 (N (−15,10)), the prob-
ability of an artesunate effect increases to 97% (1–0.03; Table 2), by
altering the parameter distributions as summarised in Fig. 2b.
To complete this analysis, we included a skeptical informative prior
for this parameter (N (0, 10)). Despite this, the probability of an
artesunate effect remains at a probability of 0.77 with a true mean
value estimated at −16 (−22,10; Table 2). CD31 also provided a high
probability (0.79) for a treatment effect. Representative immuno-
stainings are shown in Fig. 3.
3.4.2. Survival Analysis
During a median follow up of 42 months, there were 6 recurrences in
the placebo group and 1 recurrence in an artesunate recipient. Fig. 4 il-
lustrates results from a Cox's proportional hazards model. The hazard
ratio of first disease recurrence is 0.16 (95% CI (0.02, 1.3)) in the
artesunate group compared with placebo. The survival beyond 2 years
in the artesunate group is estimated at 91% (95% CI (54%, 98%)) whilst
surviving the first recurrence in the placebo group is only 57%(95% CI
(28%, 78%)). A full sensitivity analysis for patient CRC21 is given in Sup-
plementary Table 1 and suggests that if this patient was in the
artesunate group without recurrence at 3 years, then a p-value for an
artesunate effect on survival would be p = 0.07 (95% CI 0.02, 1.21).
3.4.3. CEA Levels
Six artesunate and 4 placebo recipients had CEA levels measured be-
fore and after trial medication (and before resection, classified as re-
duced, stable or increased). No patients with artesunate had increased
CEA levels whereas 3 patients in the placebo group had increased values
(p = 0.03, Fisher's exact test).
3.5. Adverse Events
Six patients (26% for the ITT population) had adverse events (2 se-
vere, Table 3 and Supplementary Table 1). Two adverse events possibly
related to study drug are described in detail. In the remaining 4 cases, 2
complications (anastomotic leaks after surgery) were considered
unlikely to be related to artesunate, and one case of iron-deficiency
anaemia (with no neutropaenia) was attributed to underlying disease.
There was one report of nausea. Detailed descriptions of 2 cases of
neutropaenia are given below and illustrated in Fig. 5.
CRC04: An 81 yo 51 kg female presented with anaemia and a change
in bowel habit, and was discovered to have a large, annular ascending
colon, polypoidal, carcinoma that was not producing obstruction on colo-
noscopy. She was randomised to receive artesunate. There was no evi-
dence of metastatic spread on staging scans (CEA = 3 μg/L). Her mid-
treatment review was unremarkable. She returned for surgery and was
found to be anaemic and neutropenic (Fig. 4a). She was transfused, mak-
ing this a Grade 3 adverse event according to CTCAE criteria (v4.0; http://
% positive
Placebo Artesunate
-40
-20
0
20
40
60
80
100
02
03
05
08
10
12
15
19
20
22
23
01
04
06
07
09
11
13
14
16
17
18
21
CRC Patients
Staining (%)
Probability density
0
.01
.02
.03
.04
.05
-80 -60 -40 -20 020 40
Treatment effect
a
b
Fig. 2. 2a Ki67 staining in treatment groups. Individual results for Ki 67 epithelial cell
staining (% positive), the grand means in each treatment group as predicted by the ran-
dom effects model and individual predications are displayed. Interquartile intervals are
presented for the raw measurements and the 95% credible intervals correspond to the in-
dividual and group predicted means. This analysis has been carried out under missing at
random assumption (MAR) with large uncertaintyaround the individualmeans of missing
individuals, as expectedand shown for 3 individuals randomisedto receive artesunate but
not able to be analysed fo r reasons given in results. These results correspond to non-
informative prior assumption with regards to the differencebetween the two groups. 2b
Sensitivity analysisto various prior information on thedifference betweenthe two groups
with respect to Ki67 —the analysis has been justified by published experimental results.
The probability that the difference between artesunate and placebo is negative remains
high even under a skeptical prior of no effect (0.77).
86 S. Krishna et al. / EBioMedicine 2 (2015) 82–90
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evs.nci.nih.gov/ftp1/CTCAE/CTCAE_4.03_2010-06-14_QuickReference_
5x7.pdf). Her neutropenia recovered the following day.
She underwent a laparoscopic right-sided hemicolectomy with an
uneventful recovery. She was offered post-operative chemotherapy in
light of her moderately differentiated pT3, Dukes' stage B adenocarcino-
ma, which she declined. This was defined as a non-severe adverse event
as there was no prolongation in hospitalization.
CRC07: A 79 yo 50 kg lady presented with anaemia, rectal bleeding
and a change in bowel habit in the preceding few months. She had a
history of endometrial carcinoma and underwent a total abdominal
hysterectomy and bilateral salpingo-oophorectomy followed by radio-
therapy 11 years before.
Colonoscopy confirmed an adenocarcinoma with impassable stric-
ture at the splenic flexure. Staging CT scan of the chest, abdomen and
pelvis excluded metastasis. She was randomised to receive artesunate.
Her CEA was 212 μg/L but fell steeply following artesunate (to a nadir
of 56 μg/L, Fig. 4b) and no other intervention. She had a persistent
thrombocytosis, but she developed anaemia and leucopenia, which
A
B
C D
E F
G H
Fig. 3. Immunohistochemical staining of biomarkers in colorectal cancer. (a) Detection of apoptotic cells by the Tunel assay, (b) Ki67, (c) p53, (d) EGFR, (e) c-MYC, (f) CD31, (g) VEGF,
(h) negative control (without primary antibody). Magnification: ×250. For determination of protein expression the UltraVision polymer detection method (kit from Thermo Fisher Sci-
entific GmbH, Dreieich, Germany) was used as detailed in Supplementary methods. The immunostained slides were scannedby Panoramic Desk (3D Histotech Pannoramic digital slide
scanner,Budapest, Hungary)and interpreted(Quantification of immunostained slides) bypanoramic viewersoftware (NuclearQuant and membraneQuant, 3DHISTECH) in which positive
stainednucleus or membrane werecounted in each definedannotated area. Evaluation parameters includednumber of overall detected objects(nucleus or membrane)in each annotated
area, average of positivity and intensity.Nuclear stainings(Ki67, p53, c-MYC,TUNEL) were quantified using the Nuclear Qant software and Membrane-bound and cytosolic stainings were
quantified by the MembraneQuant software (3D histoQuant). Results are in Table 2.
87S. Krishna et al. / EBioMedicine 2 (2015) 82–90
Author's personal copy
was noted on the day of her planned surgery (Fig. 4c). Her pre-
surgical screening carried out 5 days before surgery also showed
anaemia and leucopenia. Her surgery was delayed and after ap-
propriate expert consultation and bone marrow examination her
anaemia was treated with blood transfusions and her persistent
neutropenia with G-CSF, making this also a Grade 3 adverse event
according to CTCAE criteria.
Bone marrow aspiration showed normal erythropoiesis, with dys-
plastic granulopoiesis and maturation arrest. Some myeloid precursors
were vacuolated with nucleocytoplasmic asynchrony, but without ex-
cess myeloblasts. Megakaryocytes were plentiful. There was a slight in-
crease in plasma cells. Findings were consistent with drug induced
myelosuppression.
The neutrophil count rose after 2 days of G-CSF, which was
stopped. The patient underwent a left hemi-colectomy with en
bloc resection of a small segment of involved small bowel and pri-
mary colonic and small bowel anastamosis 11 days after artesunate,
without any post-operative complications, and a fall in platelet
count (Fig. 4c). The adenocarcinoma was staged as a Dukes' C1 and
T4abN2M0. The tumour was found to be poorly differentiated signet
ring type adenocarcinoma with extramural vascular invasion. She
was advised to have adjuvant chemotherapy as the risk of recur-
rence was ~50%. She declined and opted for surveillance alone.
After 3 years of follow up she is confirmed to be symptom and dis-
ease free and continues to lead an independent life.
4. Discussion
This is the first randomised, double blind study to test the anti-CRC
properties of oral artesunate. Escape from apoptosis is a hallmark of tu-
mour cells (Fiandalo and Kyprianou, 2012), with higher apoptotic indi-
ces being associated with more aggressive CRCs (Alcaide et al., 2013).
The pre-defined primary endpoint (proportion of patients with N7%
Tunel positive staining of tumour cells) after artesunate treatment
was not informative, perhaps because an unexpectedly high proportion
(55%) of placebo recipients exceeded the pre-defined threshold. Never-
theless, several secondary endpoints have given encouraging results,
despite limitations of a small study size and an inherent variability in
quantitating immunohistochemical markers.
Artesunate has a very high probability (0.97, calculated with an in-
formative prior in Bayesian analysis, Fig. 2b) of effect on Ki67 staining
of tumour cells. This is consistent with a high probability of artesunate
effect on Ki67 staining of fibroblasts (0.84; Table 2). Ki67 is a marker
of tumour cell proliferation whose upregulation is associated with a
poorer prognosis in colorectal cancer. Other markers of tumour biology
were also affected by artesunate, although with lower probabilities (for
example, 0.79 probability for increased CD31 expression). In one case
(Fig. 4b) there was a ~ 75% fall in circulating CEA levels after 2 weeks
of artesunate treatment alone.
The recurrence-free survival probability was also higher after
artesunate compared with placebo (at 3 years 0.89 compared with
0.5; Fig. 3) although confidence intervals for these estimates overlap
(HR 0.16,p = 0.091, Supplementary Table 1) because of the small num-
bers of patients and therefore events included in this study. Till this
analysis, there have been no deaths in artesunate recipients (despite
some patients having relatively poor prognosis), and 3 deaths in place-
bo recipients.
Two patients who were at the lower weight limit for inclusion in this
study (50 kg, giving an effective dose of 4 mg/kg of artesunate/day)
developed leucopenia (Fig. 4). In one case this reversed shortly after
stopping artesunate, whereas in the other G-CSF may have hastened
recovery. Bone-marrow examination suggested a toxic effect of arte-
sunate. These findings are consistent with the recent observations in
malaria of a dose-dependent neutropenia with artesunate (N4 mg/kg)
(Bethell et al., 2010), although bone marrow examinations have not
been carried out before. We instituted mid-treatment monitoring for
neutropenia after observations on malaria but did not note this compli-
cation in other patients. Artesunate associated leucopenia may be dose-
dependent in cancer patients as it is in malaria, and although delayed
haemolysis has been observed after artemisinin use (Rolling et al.,
2014, 2012) it was not a complication in our patients. In future studies,
it may be safer to restrict daily dose of artesunate to b4 mg/kg and to
monitor for haematological complications. A recent publication on
an artesunate dose-finding study in metastatic breast cancer disease
suggests that 200 mg once a day can be tolerated for up to 3 weeks
(Ericsson et al., 2014).
Liver recurrence was commonest in our patients, followed by perito-
neal and ovarian sites, suggesting that seeding is mainly haematogenous
and trans-peritoneal. As patients had clear circumferential and longitudi-
nal margins at surgery, and detectable metastases were not identified at
Placebo Artesunate
Surv(1yrs)=0.78(95%CI(0.46, 0.92))
Surv(2yrs)=0.63(95%CI(0.32, 0.83))
Surv(3yrs)=0.45(95%CI(0.18, 0.70))
0
.2
.4
.6
.8
1
0
200
400
600
800
1000
1200
1400
Days (12 patients)
Probability of surviving reoccurence
Surv(1yrs)=0.96(95%CI(0.74, 0.99))
Surv(2yrs)=0.93(95%CI(0.61, 0.99))
Surv(3yrs)=0.89(95%CI(0.47, 0.98))
0
.2
.4
.6
.8
1
0
200
400
600
800
1000
1200
1400
Days (10 patients)
Fig. 4. Survival recurrence curves predicted by Cox proportional hazards model. Patient
CRC21 was assumed to be missing completely at random (please see Supplementary
Table 1 for a full sensitivity analysis). In the placebo group 2 patients died within a year
(108, 170) days leaving 10 (83%) in the study, another 2 within the next year (383, 663
days) leaving 8 (66%) in the study and the other two died within the third year of the follow
up (749 and 990, respectively) leaving 50% patients beyond the third year. The only death in
the artesunate group happened after 552 days leaving 9 patients (90%) surviving beyond the
third year. These crude estimates support the estimates from the data above.
Table 3
Adverse events. For treatment allocations, please see Fig. 2a.
Study number Event Due to pre-existing illness Related to study drug Serious? Study treatment Outcome
CRC 004 Neutropaenia and anaemia No Possibly N Resolved
CRC 007 Neutropaenia and anaemia No Possibly Y Stopped Resolved
CRC 017 Nausea, but no vomiting No Possibly N Continued unchanged Resolved
CRC 018 Anastomotic leak No Not related Y Resolved
CRC 019 Anastomotic leak No Unlikely Y Stopped Resolved
CRC 022 Anaemia Yes Unlikely N Continued unchanged Resolved
88 S. Krishna et al. / EBioMedicine 2 (2015) 82–90
Author's personal copy
randomisation, it is likely that micrometastases spread through vascular
invasion (VI) caused recurrence. PreviousexperiencesuggeststhatVIpre-
dicts decreased survival in CRC (Ganapathi et al., 2011; Liang et al., 2007;
Talbot et al., 1980; Betge et al., 2012). Patients with cryptic dissemination
of CRC may benefit from systemic neo-adjuvant therapy and artesunate
may be particularly suitable because it does not usually delay surgery. It
also reduces liver metastases in an animal model (Li et al., 2007).
These observations provide critical information for the design of fur-
ther studies. In assessing its neo-adjuvant properties, we have also ex-
amined artesunate's mechanisms of action in human CRC. Artesunate
does not restore apoptosis in tumour cells in our study, but rather de-
creases the expression of a Ki67. Ki67 is also an important marker of
prognosis in CRC unlike CD31, which is increased in expression. These
findings are consistent with uncontrolled observations made in cervical
cancer (Jansen et al., 2011), where decreased Ki67 staining was also ob-
served, although decreased CD31 staining of blood vessels in that study
contrasts with our observations. Detailed laboratory observations on
anti-cancer mechanisms of artesunate such as on proliferation (Efferth
et al., 2007, 2003; Yeung et al., 2013) and expression of tumour cell
markers (Konkimalla et al., 2009; Efferth et al., 2004; Konkimalla and
Efferth, 2010) (including for angiogenesis) can now be interpreted in
light of in vivo observations. Larger clinical studies with artesunate
that aim to provide well tolerated and convenient anticancer regimens
should be implemented, and may provide an intervention where
none is currently available, as well as synergistic benefits with current
regimens.
Funding and Acknowledgements
Artesunate and placebo were donated by Dafra Pharma (Turnhout,
Belgium). We thank Dr. Jan Poliniecki for sample size estimations and
statistical support to the Data Safety Monitoring Committee, Mr.
Nikolaos Katsoulas for clinical support and Mrs.Doris Rohr for technical
assistance with immunohistochemistry. This study did not receive any
direct funding. MEMS was funded by a PhD stipend from the National
Research Council, Sudan. This study is dedicated to the memory of
Yasmin Krishna.
Author contributions
SK and HJ conceived the study and designedit together with PGK,DK
and TE; ICS carried out statistical analysis, SG, MC, DK and SK recruited
and managed patients; HK managed the study; CF provided clinical histo-
pathological analysis; TE and MEMS analysed immunohistochemistry; SK
wrote the first draft to which all authors contributed to produce the final
version. No author declares a conflict of interest.
Appendix A. Supplementary Data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.ebiom.2014.11.010.
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