Abstracts 1st Daniel den Hoed Day 2012
October 31st, 2012
Vs. 121018
9.05
Breast density in women with familial risk for breast cancer as indicator for the
use of mammography or MRI to screen for breast cancer (FaMRIsc): a
multicentre randomized controlled trial
Sepideh Saadatmand1, Emiel JT Rutgers2, Rob AEM Tollenaar3, Hermien M
Zonderland4, Margreet GEM Ausems5, Kristien BMI Keymeulen6, Margreet S SchloozVries7, Linetta B Koppert1, Eveline AM Heijnsdijk8, Caroline Seynaeve9, Cees Verhoef1,
Jan C Oosterwijk10, Inge-Marie Obdeijn11, Harry J de Koning8, Madeleine MA TilanusLinthorst1
This study was supported by funds of: The Dutch Cancer Society (DDHK 2009-4491),
ZonMw grant no. 200320002, A Sisters Hope and Stichting Coolsingel.
Department of Surgery, Erasmus University Medical Centre, Rotterdam
s.saadatmand@erasmusmc.nl
2
Department of Surgery, The Netherlands Cancer Institute, Antoni van Leeuwenhoek
Hospital, Amsterdam
3
Department of Surgery, Leiden University Medical Centre, Leiden4 Department of
Radiology, Academic Medical Centre, Amsterdam
5
Department of Medical Genetics, University Medical Centre, Utrecht 6 Department of
Surgery, Academic Hospital, Maastricht
7
Department of Surgery, Radboud University, Nijmegen
8
Department of Public Health, Erasmus University Medical Centre, Rotterdam
9
Department of Medical Oncology, Erasmus University Medical Centre-Daniel den Hoed
Cancer Centre, Rotterdam
10
Department of Genetics, University of Groningen, UMCG, Groningen
11
Department of Radiology, Erasmus University Medical Centre, Rotterdam
1
Background
To reduce mortality, women with a family history of breast cancer often start
mammography screening at a younger age than the general population. Breast density is
high in over 50% of women younger than 50 years. High breast density increases breast
cancer incidence, but decreases mammography sensitivity. Therefore, mammography
might not be the optimal method for breast cancer screening in young women. Magnetic
Resonance Imaging (MRI) has a higher sensitivity for invasive breast cancer than
mammography. However, MRI is expensive and is associated with more false-positive
results. The limitation of previous MRI screening studies is that they have a paired
design; all participants received both MRI and mammography. Therefore, it cannot
empirically be assessed in which stage tumours would have been detected by either test
alone. The aim of the Familial MRI Screening Study (FaMRIsc) is to compare the efficacy
of MRI screening to mammography for women with a familial risk. Furthermore, we will
assess the influence of breast density.
Methods/Design
In this Dutch multicentre, randomized controlled trial women with a cumulative lifetime
risk for breast cancer of ≥20%due to their family history, aged 30-55 years, are
1
randomized into two groups. Group 1 receives yearly mammography and clinical breast
examination (n=1000), and group 2 yearly MRI and clinical breast examination, and
mammography biennially (n=1000). Primary endpoints are the number and stage of the
detected breast cancers in each arm. Secondary endpoints are the number of falsepositive results in both screening arms. Analyses will also be performed with
mammographic density as stratification factor.
Discussion
Breast density may be a key discriminator for selecting the optimal screening strategy for
women < 55 years with familial breast cancer risk; mammography or MRI.
9.25
Tumor load of the Sentinel Node in Melanoma patients
Stijn van der Ploeg, Alexander van Akkooi, Alexander Eggermont, Kees Verhoef
Sentinel node biopsy (SNB) for primary melanoma is accepted worldwide as a diagnostic
procedure for patients with ≥ 1.00 mm primary melanoma or when having adverse
prognostic features. When SN positive, completion lymph node dissection (CLND) is
usually performed. Approximately 80% of CLND patients do not have non-sentinel node
(NSN) metastases, while the therapeutic benefit is unknown and morbidity rates are
high. Therefore, there is a search for patients who might safely be spared CLND.
Prognosis of SN positive patients is highly heterogeneous with five-year survival rates
ranging in different subgroups from approximately 30 to over 90%. The maximum size
(Rotterdam criteria), intranodal location (Dewar criteria) and penetrative depth (Starz
classification) of SN metastases are important criteria to stratify melanoma patients for
prognosis and risk of NSN metastases.1-3 Patients with < 0.1 mm metastases have
excellent prognosis and low NSN positivity rates, especially when located in the
subcapsular area.4 Their prognosis seems to be comparable to the prognosis of SN
negative patients, which have 5-year melanoma specific survival rates of approximately
90%. These patients might safely be spared CLND. Primary and SN tumor burden
parameters and patient co-morbidities should be taken into consideration when offering
CLND to an individual patient. In the future, prospective studies as the Multicenter
Selective Lymphadenectomy Trial-II and the EORTC 1208 (Minitub) will provide more
answers to whether CLND has a therapeutic benefit and to which patients might safely be
spared CLND.
References
1.
Dewar DJ, Newell B, Green MA, et al. The microanatomic location of metastatic
melanoma in sentinel lymph nodes predicts nonsentinel lymph node involvement.
J Clin Oncol 2004; 22(16):3345-9.
2.
Starz H, Siedlecki K, Balda BR. Sentinel lymphonodectomy and s-classification: a
successful strategy for better prediction and improvement of outcome of
melanoma. Ann Surg Oncol 2004; 11(3 Suppl):162S-8S.
3.
van Akkooi AC, de Wilt JH, Verhoef C, et al. Clinical relevance of melanoma
micrometastases (<0.1 mm) in sentinel nodes: are these nodes to be considered
negative? Ann Oncol 2006; 17(10):1578-85.
4.
van der Ploeg AP, van Akkooi AC, Rutkowski P, et al. Prognosis in patients with
sentinel node-positive melanoma is accurately defined by the combined rotterdam
tumor load and dewar topography criteria. J Clin Oncol 2011; 29(16):2206-14.
2
9.45
Neoadjuvant chemotherapy in patients with resectable colorectal liver
metastases: Do all patients benefit?
Ninos Ayez1, Dirk Grunhagen1, Jeroen de Jonge1,2, Jan N.M. Ijzermans2, Alexander M.M.
Eggermont3, Cornelis Verhoef1
Division of surgical Oncology, Erasmus University MC, Daniel den Hoed Cancer Center,
Rotterdam
2
Division of transplantation and Hepatobiliary Surgery, Erasmus University MC,
Rotterdam
3
Institut de Cancérologie Gustav Roussy, Villejuif, France
c.verhoef@erasmusmc.nl
1
Introduction
The combination of chemotherapy (CTx) and hepatic resection is increasingly accepted as
an effective treatment for patients with colorectal liver metastases (CRLM). However,
controversy remains regarding the sequence of surgery and CTx and whether all patients
with CRLM benefit from peri-operative CTx. We investigated the survival of neoadjuvant
CTx in patients with resectable CRLM stratified by the clinical risk score (CRS) described
by Fong et al..
Methods
Patients who underwent liver resection for CRLM between January 2000 and December
2009 were analyzed. We compared survival rates of patients with and without
neoadjuvant CTx. None of the patients received adjuvant CTx after surgery. The CRS
includes five risk factors and defines two risk groups: low CRS (0-2) and high CRS (3-5).
Results
In total 365 patients (64% male) were included, median age 63 years (range 30-86).
Prior to liver resection, 221 patients had a low CRS (65 received neoadjuvant CTx) and
144 patients had a high CRS (88 received neoadjuvant CTx). Median follow up 36 months
(range 0-131). The 5-year disease free survival 27% and the 5-year overall survival was
45%. In the low CRS group, there was no significant difference in median overall survival
(OS) between patients with and without CTx (65 months [range 39-91] vs. 56 months
[range 43-69], p=0.622). In the high CRS group, there was a significant difference in
median OS between patients with and without CTx (67 months (range 41-96) vs. 33
month (range 29-37), p=0.005).
Conclusion
In our series, patients with a high CRS benefit from neoadjuvant CTx. In patients with a
low CRS neoadjuvant CTx was not beneficial.
10.05
Approaches to approve liposomal chemotherapy
Ann L.B. Seynhaeve1 and Gerben A. Koning2
1.Vascular Development and Response Group and 2. Innovative Targeting Group,
Laboratory Experimental Surgical Oncology, Section Surgical Oncology, Department of
Surgery, Erasmus MC, Rotterdam, The Netherlands (g.koning@erasmusmc.nl)
Liposomes have proven successful in the delivery of chemotherapeutic agents, and are
currently applied clinically in the treatment of various types of cancer. Liposomes offer
the advantage of a high drug payload, decreased drug toxicity and enhanced drug
accumulation at tumor sites. Increased accumulation is due to the relatively leaky tumor
vasculature that allows liposome extravasation. However, between different types of
3
tumors and even within a tumor, vascular permeability and thus liposome extravasation
may differ greatly. Furthermore, upon accumulation of liposomes in the tumor area, drug
bioavailability is not guaranteed. At present these are the major issues for clinically used
liposomal drugs like liposomal doxorubicin (Doxil ® / Caelyx ®).
By using intravital optical imaging in mice bearing dorsal skin-fold window chambers with
implanted tumors we have been able to demonstrate that tumor-accumulated
doxorubicin-containing liposomes are internalized by tumor cells but only slowly release
their contents intracellularly.
To improve liposomal drug delivery to solid tumors we apply two main strategies. First,
we aim to improve liposome extravasation and penetration into tumor tissue by inducing
tumor vasculature hyperpermeability. For this we apply tumor necrosis factor alpha (TNF)
or mild hyperthermia. Second, we aim to improve liposome-mediated delivery of
chemotherapeutic drugs by improving drug bioavailability. Several different approaches
are studied including targeting, modulating cell membrane permeability and heattriggered drug release. For specific targeting of tumor cells or tumor vasculature we
couple specific targeting ligands to the surface or drug-loaded liposomes. This results in
increased specificity and intracellular drug delivery.
Next, we have identified a series of short-chain sphingolipids (SCS) that are able to
temporarily modulate tumor cell membrane permeability and improve doxorubicin cell
membrane traversal. Co-delivery of these SCS and doxorubicin in a liposomal formulation
increased intracellular drug delivery and strongly improved anti-tumor activity. Finally,
we apply hyperthermia not only to increase levels of liposome extravasation but also to
trigger drug release from thermosensitive liposomes. Increased liposome extravasation
was observed in several tumor models and triggered drug release improved therapeutic
efficacy.
In conclusion, using strategies to enhance liposome extravasation and to improve
bioavailability of liposomal drugs in the tumor the therapeutic activity of liposomal drugs
can be strongly enhanced.
10.55
Anne-Joy de Graan
Internal Oncology, Erasmus MC, Rotterdam
Pharmacological approaches to individualize taxane therapy in solid tumors
The chemotherapeutic agents docetaxel and paclitaxel (taxanes) are known for their
small therapeutic window and for their large interindividual variability in metabolism and
toxicity profile. Several factors may influence the systemic exposure or the toxicity profile
for these drugs, including genetic factors (i.e., polymorphisms in genes coding for drug
transporters), comedication (i.e., use of strong CYP3A inhibitors), and environmental
factors (i.e., smoking). Because of the small therapeutic window, an alteration in drug
exposure may easily lead to unexpected toxicities or suboptimal therapeutic effects in
individual patients with cancer. In three different studies we aimed to elucidate factors
that influence taxane therapy for a more personalized approach to taxane therapy
In the first study
we investigated the contribution of organic anion transporting
polypeptides to the disposition of docetaxel using an array of in vitro and in vivo model
systems (de Graan et al, Clin Cancer Res. 2012 Aug 15;18(16):4433-4440). Our results
indicate the existence of at least two potentially redundant uptake transporters in the
human liver with similar affinity for docetaxel (OATP1B1 and OATP1B3) that regulate the
initial, rate-limiting step in the elimination of docetaxel. In view of the established
exposure–toxicity relationships for docetaxel, our results suggest that caution is
warranted if docetaxel has to be administered together with agents that potently inhibit
both these transporters.
4
In the second study, we investigated the influence of cigarette smoking on taxane
therapy (de Graan et al, Clin Cancer Res. 2012 Aug 15;18(16):4425-32. Epub 2012 May
29). Before, smoking was identified as a factor influencing the pharmacokinetics and
pharmacodynamics of cytotoxic drugs metabolized by CYP3A but was not studied
thoroughly for taxanes before; therefore, knowledge of the effects of smoking on taxane
therapy is important for the further individualized treatment of docetaxel and paclitaxel
treatment. In this study we showed that cigarette smoking does not alter the
pharmacokinetic determinants of docetaxel and paclitaxel. However, smokers treated
with docetaxel and paclitaxel have less neutropenia and leukopenia and further research
is warranted to elucidate this potential protective effect.
In the third study we studied predictive pharmacokinetic and pharmacogenetic
determinants for the development of neurotoxicity in paclitaxel treatment (de Graan et
al, Annual meeting of European Society of Medical Oncology, abstract 1667). In an
exploratory cohort treated with paclitaxel, incidence of neurotoxicity and PK parameters
were determined. Genetic variants tested in the pharmacokinetic pathway of paclitaxel
were CYP3A4*22, CYP2C8*3, CYP2C8*4, and ABCB1 3435 C>T. The association between
CYP3A4*22 and neurotoxicity found in the exploratory cohort was studied in an
independent validation cohort. In this study, female CYP3A4*22 carriers were at
increased risk of developing neurotoxicity during paclitaxel therapy. Also, there is a clear
linear relationship between paclitaxel exposure and severity of paclitaxel-induced
neurotoxicity.
11.15
FOXA1 EXPRESSION: REGULATED BY EZH2 AND ASSOCIATED WITH FAVORABLE
OUTCOME TO TAMOXIFEN IN ADVANCED BREAST CANCER
Esther A. Reijm1, Maurice P.H.M. Jansen1, Jean Helmijr1, Charlotte Soler2, Renée
Beekman3, Frank Gerritse1, Wendy Prager-van der Smissen1, Kirsten Ruigrok-Ritstier1,
Wilfred Van IJcken4, Mary Stevens2, Marcel Smid1, Maxime P. Look1, Marion E. Meijer-van
Gelder1, Anieta M. Sieuwerts1, Stefan Sleijfer1, John A. Foekens1, Els M.J.J. Berns1
Department of Medical Oncology1, Erasmus MC – Daniel den Hoed, Rotterdam, The
Netherlands
Department of Cell Biology2, Erasmus MC, Rotterdam
Department of Immunology3, Erasmus MC, Rotterdam
Erasmus Center for Biomics4, Erasmus MC, Rotterdam
Background
Enhancer of Zeste Homolog 2 (EZH2) is a member of the polycomb complex 2 and serves
as a histone methyltransferase. Through trimethylation of histone 3 lysine 27, EZH2
causes ‘genome wide’ epigenetic transcription silencing. Cyclin-dependent kinase 1
(CDK1/CDC2) regulates EZH2 function through phosphorylation of threonines 345 and
487 in a cell cycle-dependent manner.
Downregulation of EZH2 is associated with increased expression of the estrogen receptor
(ER) and favorable outcome to tamoxifen in metastatic breast cancer (Reijm et al. Breast
Cancer Res Treat (2011) 125: 387-394; Reijm et al. Breast Cancer Res Treat (2012)
133: 937-47).
The binding of ER to its target genes is enhanced by Forkhead Box A1 (FOXA1), a pioneer
factor that unwinds chromatin to make DNA accessible for transcription factors.
The aim of this study is to investigate the potential association between EZH2, CDC2 and
FOXA1 and their relation with endocrine treatment outcome in patients with advanced
breast cancer.
5
Experimental design
EZH2, CDC2 and FOXA1 mRNA levels were analysed for the molecular subtypes using
available gene expression profiles of 344 primary breast cancer specimens of lymphnode-negative (LNN) untreated patients. The mRNA expression levels of these genes
were also related to outcome after first-line tamoxifen using microarray profiles of 109
ER-positive (ER+) tumors and qPCR data of an independent set of 230 ER + tumors of
patients with advanced disease treated with tamoxifen.
For functional analysis of EZH2, cell lines were generated with different short hairpin
knockdown-constructs for EZH2. These cell lines were evaluated for their mRNA and
protein expression levels and with chromatin immunoprecipitation (ChIP) followed by
qPCR and/or next generation sequencing (NGS; ChIPseq).
Results
In the 344 LNN breast cancer patients, EZH2 was highly expressed in breast tumors of
the basal subtype. In contrast, FOXA1 was almost not expressed in this subtype, but
highly in the luminal A and B subtypes. High FOXA1 levels were related to a prolonged
time to progression (TTP) after first-line tamoxifen in the 109 ER+ primary tumors
(HR=0.56 [0.37-0.85] P=0.006) whereas increased expression of CDC2 was associated
with poor outcome (OR=0.45 [0.31-0.65], P<0.001) and TTP (HR=1.53 [1.29-1.81],
P<0.001) in 230 ER+ tumors.
In the functional analyses we obtained 50-70% EZH2 knockdown at protein level in the
breast cancer cell lines MCF7 (ER+/PR+/FOXA1+), SUM52PE (ER+/PR-/FOXA1+), and
MM231 (ER-/PR-/FOXA1-). In both EZH2-knockdowns of MCF7 and MM231 CDC2 protein
levels were decreased with 32% (P<0.001) and 20% (P=0.001) compared to the
parental cell lines, respectively.
ChIP followed by qPCR demonstrated higher H3K27 trimethylation levels in parental cell
lines compared to EZH2-knockdowns at the ER-locus for MM231 and at the PR-locus for
SUM52PE and MM231. ChIPseq evaluation of the MM231 EZH2 knockdown identified 200
loci with a significant decrease in read numbers for H3K27me3 compared to the parental.
Interestingly, amongst these 200 loci the FOXA1-locus was identified. These results
suggest FOXA1 as an EZH2-target since its expression was not seen in the parental
MM231.
Conclusion
High EZH2 and CDC2 but low FOXA1 mRNA levels are associated with poor outcome after
first-line tamoxifen therapy in advanced breast cancer. Functional studies indicate that
EZH2 might not only regulate the expression of ER, as previously shown, but also of
FOXA1. Although CDC2 activates EZH2 through its phosphorylation, our findings point
towards the influence of EZH2 on CDC2 expression levels. These results suggest that
EZH2, CDC2 and/or FOXA1 could identify patients at risk for tamoxifen therapy failure
and these genes might be putative targets for therapy.
11.35
Dendritic cell-based immunotherapy in combination with metronomic
cyclophosphamide in patients with mesothelioma.
Joachim G Aerts1,2, Robin Cornelissen1, Margaretha Lambers1, Henk C Hoogsteden1,
Joost P Hegmans1
Department of Pulmonary Medicine, Erasmus Medical Centre Rotterdam
Department of Pulmonary Diseases, Amphia Hospital, Breda
1
2
6
Introduction
Dendritic Cells (DC) are extremely potent antigen presenting cells capable of inducing a
CD8+ cytotoxic T cell reaction. In an earlier study on autologuous tumor lysate pulsed
DC based immune therapy in mesothelioma (MM) we showed that we were able to induce
an anti-tumor response. However it is known that the induced anti-tumor immunity in
cancer patients is created in a unfavorable highly suppressive tumor environment.
Regulatory T-cells (Tregs) are prominent cells in this suppressive environment. We have
previously showed the presence of Tregs in patients with MM. In a murine model on MM,
depletion of Treg with metronomic cyclophosphamide (CTX) could enhance the antitumoral immune response induced by DC vaccination1.
The aim of the present study was to determine whether metronomic CTX was able to
lower Treg in patients . Secondary endpoints are specific anti-tumor response activity
and overall survival.
Methods
A non-randomized study was performed in patients with MM who were non progressive
after standard chemotherapy. A pleurectomy/decortication (P/D) was performed before
DC vaccination in case that was considered best interest of the patient. According to our
previous study, 3 doses of pulsed autologuous DC were re-injected every 2 weeks.
Patients were treated with 100mg CTX/day for 7 days starting 9 days before every
vaccination. After the 3rd vaccination again 7 days of CTX were taken.
Results
9 patients were enrolled. Mean age 60 (range 35-78yrs). Data on Treg levels will be
presented at the conference. Response according to modified RECIST and survival are
presented in the table
No
Respons to
chemotherapy
Pleurectomy/
Decortication
1
2
3
4
5
6
7
8
9
SD
SD
SD
PR
SD
SD
PR
SD
PR
No
No
No
No
No
Yes
Yes
Yes
Yes
Response to
DC
immunotherapy
SD
SD
SD
CR
SD
SD
PD
PR
SD
Survival
(Months)
24
25
14
12
14
20
12
26
37
alive
alive
alive
alive
SD = stable disease, PR = partial response, CR = complete response, PD = progressive
disease.
No major side effects were determined during the study
Conclusion
CTX can be safely administered during DC immunotherapy. The results on Tregs will be
presented at the conference. The results on overall survival confirm the data found from
our earlier study on DC immunotherapy in patients with mesothelioma.
1. Hegmans, Veltman, Lambers, de Vries, Figdor, Hendriks, Hoogsteden, Lambrecht,
Aerts. Am J Respir Crit Care Med 2010;181:1282.
7
12.05
Structured reporting of multiparametric MRI for prostate cancer: accuracy and
interobserver variability
R.J. van Soest, R.S. Dwarkasing, G. van Leenders, H. Teertstra, P. de Koekkoek, S.
Heijmink, M. Wildhagen, H.G. van der Poel, E.W. Steyerberg, C.H. Bangma
Departments of Urology, Radiology, Pathology and Public Health, Erasmus MC Rotterdam.
Departments of Urology and Radiology, the Netherlands Cancer Institute / Antoni van
Leeuwenhoek hospital Amsterdam.
Introduction and objective
Recently a structured reporting system (PI-RADS) for multiparametric magnetic
resonance imaging (mpMRI) has been reported. We aimed to determine the accuracy and
interobserver variability of structured reporting of mpMRI in detecting prostate cancer.
Materials and methods
Twenty-four consecutive patients with biopsy-proven prostate cancer underwent mpMRI
(3 Tesla with endorectal coil) using a standardized protocol before radical prostatectomy.
Three independent radiologists (2 experienced, 1 not experienced in prostate MRI),
blinded for pathology results, retrospectively reported the MRI’s. Lesions were scored
according to the PI-RADS reporting system in which the prostate is divided into 16
regions. One urogenital pathologist analyzed all specimens, outlined tumor location on a
glass cover, and reported a TNM classification. Per-region the likelihood of tumor
presence reported by the radiologist was compared with the presence of tumor in
histopathology. Receiver operating characteristic (ROC)-curves were used to analyze the
accuracy of tumor detection. Area under the curve (AUC)s were statistically compared
using non-parametric bootstrap resampling. Kappa-values were calculated to determine
inter-rater agreement.
Results
Prostate cancer was present in 153(41.6 %) and absent in 215(58.4%) of the 368
regions at histopathology. The AUC for detecting prostate cancer was 0.69(CI 0.68-0.70)
and 0.72(CI 0.71-0.73) for the experienced radiologists. The AUC for the less
experienced radiologist was 0.66(CI 0.64-0.68). For inter-rater agreement experienced
readers 1 versus 3 showed a kappa-value of 0.47 (0.43-0.52). Less experienced reader 2
versus experienced reader 3 showed a kappa-value of 0.38 (CI 0.33-0.42). Reader 1
versus 2 showed a kappa-value of 0.57 (0.53-0.62).
Conclusion
Structured reporting of mpMRI using the PI-RADS scoring system is only useful for
detecting prostate cancer in the hands of an experienced radiologist. Interobserver
variability needs to be improved before implementing the PI-RADS system in the clinic.
12.25
Suppressive tumor-infiltrating T cells in patients with liver cancer
Alexander Pedroza Gonzalez, Dept. of Gastroenterology and Hepatology, Erasmus MC,
Rotterdam
Hepatocellular carcinoma (HCC) and liver metastases from colorectal cancer (LM-CRC)
are the main malignancies affecting the liver and they are among the most common
cancers and leading causes of cancer mortality. The current therapeutic options for both
malignancies are limited and they cannot prevent the recurrence of the disease. Novel
immunotherapeutic options, like dendritic cell (DC) vaccination, have limited effects
because of immunoregulatory mechanisms that constrain the tumor-specific T cell
response within the tumor. Recently, we described accumulation of CD4+FoxP3+
8
regulatory T cells (Tregs) at the tumor site of patients with liver cancer. Tumorinfiltrating Tregs (TiTregs) are activated and potent suppressors of anti-tumor immunity.
TiTregs in both types of liver tumors are characterized by higher expression of the
glucocorticoid-induced tumor necrosis factor receptor (GITR), the inducible T-cell
costimulator (ICOS) and the cytotoxic T lymphocyte-associated antigen 4 (CTLA-4)
compared to Tregs present in tumor-free liver tissue and blood. Importantly, treatment
with soluble GITR ligand or blocking antibodies to CTLA-4 induces a decrease in the
suppression mediated by the activated TiTregs and restores the proliferative capacity and
cytokine production of conventional CD4+CD25- T cells. Our data not only provide new
insight into the pathogenesis of malignancies affecting the liver, but also identify
potentially novel therapeutic targets.
13.45
A unique mechanism of aberrant expression of the EVI1 gene in AML with
chromosome 3q26 abnormalities
Mathijs Sanders & Stefan Groeschel, Dept. Of Hematology, Erasmus MC, Rotterdam
The genomic basis for structural variations underlying acute myeloid leukemia with
inv(3)(q21;q26.2) or t(3;3)(q21;q26.2) is not well established. This subtype of AML is
clinically characterized by a fatal disease course and can be distinguished molecularly by
the aberrant activation of the proto-oncogene EVI1. Putative regulatory sequences of the
housekeeping gene RPN1 at 3q21.3 are suspected to cause overexpression following
their chromosomal rearrangement to the EVI1 locus at 3q26.2. However, a hotspot
region of 3q breakpoints has not been defined yet, nor have the involved DNA elements
been sufficiently mapped with high resolution. To overcome this, we have applied
chromosome 3q capture-sequencing to 50 cases of inv(3)/t(3;3) AML and show that
breakpoint junctions display a common feature of genomic organization. Breakpoints
form a unique cluster at the 3q26/EVI1 locus, uniquely distinguishing inv(3) from t(3;3)
cases. Notably, we find that endogenous retroviral elements are recurrently involved in
the chromosomal rearrangement. Epigenetic modification of the retroviral repeat
sequences on the rearranged allele as well as transcriptional hyperactivity initiating at
their locus is associated with overexpression of EVI1 in these AML cases. We provide
evidence for these retroviral elements to be involved in the pathogenesis of this high-risk
type of myeloid leukemia.
14.15
Functional activation of human NCR+ innate lymphoid cells
Ferry Cornelissen, Patricia Aparicio-Domingo and Tom Cupedo
Department of Hematology, Erasmus University Medical Center, Rotterdam
RORgt innate lymphoid cells (ILC) are intensively investigated for their pivotal role in
homeostasis and pathogenesis in mucosal tissues. A considerable number of RORgt ILC
found in the intestine and tonsil secrete the effector cytokine IL-22 and express natural
cytotoxicity receptors (NCR), which were initially described to be activating receptors on
NK cells. All known ILC activators described so far are soluble molecules but the role of
these activating receptors on ILC is still unclear. Using NCR signaling antibodies in
conjunction with the TLR2 synthetic ligand Pam3Cys, we show functional activation of
human tonsilar RORC+ ILC as measured by IL-22 protein. Most of the natural occurring
NCR ligands are unknown, however the tumor ligand B7H6 is shown to bind NKp30. We
analyzed different intestinal epithelial cell lines and B7H6 transcript levels could be
measured at varying levels. In an attempt to recreate more physiological conditions,
B7H6 expressing intestinal epithelial cells lines were used in a co-culture system
9
withuman tonsilar RORC+ ILC. Full activation of RORC+ ILC is observed after addition of
TLR2 co-stimulatory signals, revealing a novel mechanism of ILC activation through
membrane bound ligands. This highlights the importance of a local regulation of Rorgt
ILC activation in mucosal tissues. Furthermore, we observed an important role for NCR
ligation on ILC since activation is ameliorated upon disruption of NCR signaling.
14.35
Deconstructing niche contributions to oncogenesis in the hematopoietic system
Marc Raaijmakers, Dept. of Hematology, Erasmus MC, Rotterdam
Deconstructing niche contributions to oncogenesis in the hematopoietic system
Oncogenesis has long been considered a predominantly cancer-cell autonomous process.
Contributions of
micro-environmental ‘niche’ cells remain poorly explored and niche factors have yet to
enter the clinical arena as prognostic variables or therapeutic targets. We have
established proof of concept in mouse models that disruption of well-defined
hematopoietic stem cell niche cells is implicated in the initiation and leukemic evolution
of myelodysplastic syndrome (MDS), the principle premalignant disease of the
hematopoietic system. Here, we will present data from transgenic MDS mice supporting
the view that preneoplastic cells may change their environment in such a way that the
latter starts collaborating in leukemic evolution. We will further outline ongoing work with
the aim to translate these concepts to human disease, proposing whole transcriptome
RNA-sequencing of biologically equivalent niche populations in preclinical models and a
large prospective cohort of MDS patients, followed by functional validation of candidate
‘leukemia-promoting’ niche factors in genetic models.
14.55
Disruption of circadian rhythmicity in liver metastasis of colorectal cancer
S.A. Huisman1, T.M. van Ginhoven1, H.P. Roest1, F. Tamanini2, J.N.M. IJzermans1, G.T.J.
van der Horst2, R.W.F. de Bruin1
Departments of Surgery1 and 2Genetics Erasmus University Medical Center, Rotterdam,
The Netherlands
Background
We have recently shown that short term fasting improves acute stress resistance and
protects against acute oxidative damage in kidney and liver. In our global gene
expression analysis from fasted mice, the most significantly upregulated gene was Per2.
Apart from improved stress resistance, regulation of clock genes may be an important
mechanism by which fasting induces its beneficial effects. Other data from our lab show
that fasting in mice is able to suppress growth of colorectal liver tumours. Since the
circadian clock plays an important role in cell cycle progression and carcinogenesis, a
more thorough understanding of the circadian performance of tumours may help in
elucidating the role of the clock in tumorigenesis and tumour growth. Therefore, we
investigated the 24-hour expression levels of key circadian clock genes in colorectal liver
metastasis and adjacent healthy liver tissue in the mouse.
Materials and methods
Male BALB/c mice were injected with C26 colorectal carcinoma cells into the spleen.
Three weeks after inoculation, a hepatectomy was performed at 6 different timepoints
over a 24-hour period (n=4 per timepoint) to compare circadian rhythmicity in tumor and
adjacent healthy tissue. RNA was isolated and RT-PCR performed on 5 clock genes: Rev-
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erbα, Per2, Per1, Dbp, and Bmal1 and a clock independent gene induced by feeding
(Acss2).
Results
Healthy liver tissue showed normal 24-hour oscillations of all 5 clock genes, consistent
with normal circadian rhytmicity. In colorectal liver metastasis however, 24-hour
oscillations were completely absent for all clock genes except Per2. Per2 oscillations were
similar in tumor and adjacent liver tissue, although expression levels in tumor tissue
were lower. Since Per2 may also be induced by feeding, we determined the expression of
Acss2, which is independent of circadian rhythm. Acss2 levels rose in response to
feeding, followed by a rise in Per2, suggesting that the increased expression of Per2 was
induced by feeding.
Conclusion
Our data demonstrate the absence of synchronized clocks in hepatic metastasis of C26
coloncarcinoma in vivo. The difference between tumor and healthy tissue in this model
may provide new clues to further understand the biology, and optimize treatment of
hepatic metastasis.
15.30
Hyperthermia-induced homologous recombination deficiency provides novel
anti-cancer treatment opportunities
Berina Eppink1, Przemek M. Krawczyk2, Jeroen Essers1,3,4, Jan Stap2, Hanny Odijk1, Alex
Zelensky1, Thomas Soullié5, Joost Rens5, Timo L.M. ten Hagen5 and Roland Kanaar1,3 .
Department of Cell Biology & Genetics, Cancer Genomics Center, 3Department of
Radiation Oncology, 4Department of Vascular Surgery, 5Department of Surgical Oncology,
Erasmus Medical Center, PO Box 2040, 3000 CA, Rotterdam
1
2
Center for Microscopical Research, Department of Cell Biology & Histology, University of
Amsterdam, 1105 AZ, Amsterdam
DNA double-strand break repair mechanisms are intensively investigated because of their
relevance in many anti-cancer strategies that are based on the toxicity of DNA doublestrand breaks such as induced by ionizing radiation. DNA repair mechanisms protect
(cancer) cells from the genotoxic effects of DNA double-strand breaks, reducing the
effectiveness of treatment. Recently we have demonstrated that hyperthermia in a
clinically-achievable range (41-42.5°C) represses one of the DNA double-strand break
repair pathways; homologous recombination (HR) and induces degradation of one of its
major proteins BRCA2. Defective HR sensitizes cells to PARP-1 inhibition and is currently
exploited in clinical treatment of HR-deficient tumors. We demonstrated that
hyperthermia can be used to sensitize innately HR-proficient tumor cells to PARP-1
inhibitors. Our results, obtained from cell lines and in vivo tumor models, enabled the
design of novel therapeutic strategies involving localized on-demand induction of HR
deficiency. As a prelude to the application of our findings in clinic, we use different
cellular models to test whether the clinically applied combination therapy of hyperthermia
and cisplatinum (CPT) can be improved by the addition of a PARP-1 inhibitor. Our
preliminary results suggest that therapy combining hyperthermia with CPT may be
augmented by the addition of a PARP-1 inhibitor. This brings us one step closer to the
clinic combining hyperthermia with PARP-inhibitors in our so-called ‘induced synthetic
lethality approach’.
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15.50
Hyperthermia in head and neck tumors
MM Paulides, Dept. of Radiation Oncology, Erasmus MC, Rotterdam
Treatment of advanced head and neck (H&N) tumors using the currently existing
treatment strategies is difficult, i.e. poor treatment outcome is accompanied by major
toxicity. Early, promising results reported for superficial hyperthermia treatment of
cervical lymphnodes, as well as the absence of increased heat-related radiation toxicity,
initiated us to develop technology for deep hyperthermia in the H&N region.
Based on extensive simulation studies, we designed the HYPERcollar for hyperthermia
treatment of H&N tumors. At present, hyperthermia with the HYPERcollar is a standard
addition to sensitize the tumor to the relatively low dose of radiotherapy that can still be
given to recurrent H&N tumors in previously irradiated areas. A phase II study to
investigate the potential of hyperthermia in primary tumors is under development.
Patient-specific hyperthermia pre-treatment planning (HTP) has shown essential for this
tumor location and has been applied from the beginning. In addition, real-time HTP
assisted treatment has been implemented recently. For accurate treatment planning
simulations we implemented an extensive quality assurance program. HTP is further
being used to investigate treatment confounders and ways to reduce their influence.
Our quest for reducing - and accounting for – improvement of heating technology and
techniques is still ongoing. Based on analyses of treatment confounding parameters, we
are developing means to further improve simulation accuracy. Accuracy of predictions
accelerates our HTP based adaptive treatment strategy and provides an encouraging
basis for the introduction of novel dynamic heating strategies, e.g. time-modulated
hyperthermia. For the near future, we plan to study if non-invasive temperature
measurements using a magnetic resonance (MR) scanner can replace the current
invasive probes.
16.10
Photodynamic Therapy in Head and Cancer
D. J. Robinson
Center for Optical Diagnostics and Therapy, Department of Radiation Oncology, Erasmus
Medical Center, Rotterdam. The Netherlands
In Head & Neck cancer, photodynamic therapy (PDT) using mTHPC (Foscan) is used as a
primary treatment for superficial squamous cell carcinoma and in the palliation of
recurrences of these tumours. The response to PDT depends on the presence of 3
components; light, photosensitiser and oxygen in sufficient quantities to ablate tissue in
the treatment volume. If any one is missing, there is no biological effect. Studies have
shown that inter-and intra-subject differences in parameters such as tissue optical
properties, uptake/synthesis of photosensitiser and tissue response to PDT can lead to
wide variations in the dose delivered during PDT.
In superficial lesions approximately 10-15% of tumours do not respond despite being
adequately illuminated. In these tumours we have been able to correlate the absence of
photosensitiser within the treatment volume, measured using quantitative spectroscopy,
with the lack of clinical response. This is the first time we have ever been able to predict
that these small but significant numbers of treatments fail. These quantitative
measurements of tumour tissue have been combined with additional measurements form
the skin surrounding the injection site of the photosensitiser Foscan and indicate that an
ineffective injection (one that photosensitises the vein at the injection site leads to very
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low concentrations of systemic photosensitiser and no clinical response. Patients in which
this occurred were retreated using another injection site.
Also in the majority of treatment sessions we have been able to use the photobleaching
of the Foscan to confirm effective therapy. The success of our translational approach
means that quantitative fluorescence and reflectance spectroscopy have been
implemented in the routine clinical workflow for patients undergoing Foscan PDT.
16.30
Randomized Phase 3 Study On The Assessment Of Late Toxicity By Comparing
IMRT High Dose External Beam Radiotherapy Only With External Beam
Radiotherapy Combined With HDR Or PDR Brachytherapy In Patients With
Intermediate/high Risk Prostate Cancer
Shafak Aluwini, Dept. Of Radiation Oncology, Erasmus MC, Rotterdam
Purpose
There is by now accumulating evidence that high radiation dose (≥ 75 Gy) is necessary
for tumour control when treating intermediate and high risk prostate cancer. To treat
safely at high doses with external beam therapy, 3-Dimensional Conformal Radiotherapy
(3D-CRT) or Intensity Modulated Radiotherapy (IMRT) techniques must be used. In this
way using the IMRT with a validated position verification protocol as standard of care, the
toxicity can be limited while the dose to the prostate is escalated. However, despite the
use of IMRT the incidence rates for GI and GU toxicity are still high and the adverse
effect of IMRT dose distribution pattern on the long term has to be evaluated. Another
way of delivering high dose to the prostate, but limiting the dose to the neighbouring
organs is with brachytherapy. For treating intermediate risk prostate cancer with
brachytherapy, brachytherapy and IMRT are combined. IMRT is used to deliver an
elective dose to the prostate and seminal vesicles. Brachytherapy is used as a boost.
Study design
Randomized, prospective, phase III study
Patient population
Patients with prostate cancer, belonging to the intermediate- and partially high-risk
profile are candidates for the study, provided brachytherapy can be performed.
Intervention
Patients will be randomized into two groups. One group will be treated with high dose
external beam radiotherapy using the IMRT technique (standard treatment). The other
group will be treated with external beam radiotherapy (IMRT) combined with HDR or PDR
brachytherapy as boost.
Main study endpoints
Primary endpoint: the incidence of late gastro-intestinal and genito-urinary toxicity
(grade ≥ 2 RTOG) during 3 years of follow-up after treatment completion.
Secondary endpoints: Incidence of acute toxicity, bDFS, RFS, OS, QOL, costs and costeffectiveness (all costs of treatment and during 5 years of FU after treatment
completion).
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