Patients with glioblastoma (GM) have a median overall survival of approximately 15
months.Standard therapy for GM encompasses maximum surgical resection followed by
radiation and chemotherapy using temozolomide (TMZ) (1). Regardless of initial tumor
response, tumor recurrence is inevitable, after which survival drops to less than 6
months. GM tailored approaches targeting oncogenes that might drive the growth of the
bulk of primary tumors, have been unsuccessful so far in clinical trials(2), creating a
large unmet need warranting new approaches to overcome intrinsic and acquired resistance
to current treatment schedules. The objective of this research is to establish primary
patient derived organoid cultures from GM to study mechanisms that contribute to
aggressive tumor growth and treatment resistance in primary and recurrent GM.
1. Inter-and intratumoral heterogeneity in glioblastoma. Tumor tailored approaches for
GM are being hampered by inter-and intratumoral heterogeneity of both
microenvironment and genomic alterations in GM cells. It has been shown that tumors
are composed of multiple clones harboring distinct genetic alterations (3-7). The
clonal evolution model posits that tumor formation is initiated in a cell of origin
and is followed by the subsequent accumulation of multiple genetic and epigenetic
alterations, leading to tumor cell survival and growth advantage (8). Divergent
genetic alterations in early transformed cells give rise to a variety of clones
under the selective pressure of the tumor microenvironment (3-7). An important
microenvironmental stressor is intratumoral hypoxia, which is frequent in GM and a
negative prognostic and predictive factor associated with reduced survival (9,10).
Emerging evidence implicates a subpopulation of tumor cells with characteristics of
normal stem cells so-called glioma stem cells (GSC) in intrinsic and acquired
treatment resistance. GSC are endowed with specific properties including high tumor
initiating ability, unlimited self-renewal potential and capacity for multipotent
differentiation, generating a diverse progeny(11). GSC are marked by common stem
cell markers including CD133+, SOX2, Olig1, and have been shown to reside in the
perivascular region as well as in hypoxic areas. GSC are expanded under
hypoxia12depend on glycolysis (13,14).
Combined with their low proliferation, increased DNA repair, high anti-oxidant
activity and among others, make GCS more resistant to conventional treatments
(radiation and temozolomide) then non-GSC (15,16) {Jamal, 2012 #52}.
This implies that GSCs form an important driver of GM recurrence after
chemoradiation. There are currently no effective treatments to eliminate glioma stem
cells. Blocking hypoxia signalling in tumors (inhibiting self-renewal and survival
of GSC cells)(12,17) and blocking the NOTCH stem cell pathway (rendering GSCs
sensitive to radiation(18) and TMZ(19-21)), seem to promising but drugs interfering
with these pathways have not passed beyond early phase clinical trials yet(22).
Current standard of care of newly diagnosed glioblastoma is multimodal and consists
of surgery, radiation therapy and TMZ, an alkylating agent modifying purine bases of
DNA (O6-guanine; N7-guanine and N3-adenine). The addition of TMZ to radiotherapy has
increased the overall survival of GM patients significantly, but only up to 14.6
months(1). Intratumoral hypoxia has been shown to decrease therapeutic efficacy of
RT and chemotherapy(23). Hypoxic GM cells are genetically unstable and show
increased MGMT expression and thereby resistance to alkylating TMZ chemotherapy(24).
In non-GSCs, MGMT promoter methylation is a predictive marker for response to TMZ
treatment(25,26). However the interpretation of the MGMT methylation assay is
complex, since the extent of MGMT promoter methylation in GM is heterogeneous and
the level of heterogeneity is underestimated, since only GM biopsies or fragmented
GM tissue is being analysed(27,28). Importantly, MGMT is also expressed in the
normal brain endothelial cells and in immune cells including tumor infiltrating
cells(29). Thus depending on the extent of normal tissue contamination in GM
biopsies the level MGMT methylation may also differ.
Since intra-tumoral heterogeneity in MGMT expression cannot be objectivated using
currently available diagnostics and since undertreatment of patients should be
prevented at all times, at present most patients diagnosed with ´de novo´ GM,
receive TMZ; although there seems to be little benefit of TMZ in GMs with MGMT
unmethylated glioma cells.
Designing new treatment protocols for newly diagnosed glioblastoma is quite complex,
since diagnostic tools, predicting inter-and intra-GM heterogeneity of MGMT promoter
methylation status (or level of MGMT expression), are lacking. Additionally, the
level of MGMT expression, needed for significant TMZ response and improvement of
clinical outcome, is not known. Thus since most GMs are defined by both MGMT
methylated and MGMT unmethylated tumor clones, combining TMZ with drugs that target
non-methylated glioma cells/glioma stem cells and/or microenvironment might be
necessary.
2. Glioblastoma recurrence Even if a macroscopically complete resection of a GM can be
achieved, tumor cells remain at the resection site. It has been shown that GM cells
have a high capacity for dissemination. Invading tumor cells escape at the periphery
of the tumor mass and diffusely infiltrate the normal brain parenchyma. Deeply
infiltrated tumor cells are more likely to escape surgery and what is not known is
whether infiltration is a property of a more resilient cell population that
initiates and drives tumor recurrence. Even after postoperative chemotherapy and
radiation outside of the field of surgery to reduce infiltrative tumor cells, almost
all GM recur, mostly around the resection cavity. If a gross total resection cannot
be performed; primary radiation therapy and chemotherapy are also able to reduce
clonal diversity, but are thus insufficient to prevent recurrence.
This implies that tumor cells resistant to multiple therapies persist in the brain
parenchyma around the tumor cavity after gross total resection or in the remaining tumor
after chemoradiation which are responsible for tumor repopulation, making them a critical
target to overcome tumor recurrence. Genomic analysis of GM has shown that dominant
clones in recurrent tumors are composed of clones representative of the primary tumor as
well as new clones with little resemblance to the original tumor(3,30,31,32,33).
Consequently targets identified based on the analysis of the primary tumor may not be
informative to identify the best molecular targets to prevent recurrence(5).
After failure of first line GM treatment (radiation, TMZ), recurrence seems to be
accompanied with a phenotypic shift of GSCs to the mesenchymal (MES) subtype (loss of
CD133; gain of BMI1, SOX2 and CD44)(34-38). Such cells are more aggressive, invasive and
angiogenic than primary tumor derived GSCs, mostly the proneural (PN) subtype (CD133+,
CD15+)(39-41). MES GSC also show a higher expression of the inflammasome genes, such as
IL6, IL8, IL1B1C and CXCL2, reinforcing the notion that interplay with the
microenvironment and plays an important role in recurrence and progression(42,43). GM
specific clinical trials are being developed, using immune modulators. It has been shown
that MSH (mutS homologue) mutations correlate with TMZ resistance, as they are neither
found in pre-treatment GM nor radiotherapy post-treatment GM, but were detected in
approximately half of recurrent GM patients treated with TMZ and radiotherapy. This
strongly indicates that MSH6 alterations are associated with resistance to alkylating
agent therapy. Thus GM patients who initially respond to TMZ may acquire MMR defective
hypermutator phenotype(44-46). It is well established that an intact mismatch repair and
base excision repair (BER) contribute to effective TMZ cytotoxicity. Pharmacological
inhibition of BER using PARP inhibitors either alone or in combination with TMZ has shown
promise in clinical trials. However, acquired resistance to PARP inhibitors is observed
through up-regulation of base excision repair and homologous recombination repair to
compensate for diminished BER (47,48).
In conclusion, understanding the therapy induced genetic and epigenetic alterations of
the remaining tumor cells and GSCs and also the impact of standard of care to the
microenvironment /niche, in which recurrence occurs, will guide the development of new
treatments.
In this project we will use patient derived glioma stem cell organoids(49), mimicking de
novo GM and its intratumoral heterogeneity. Also these patient-derived organoids (PDO)
organoids will be used to study acquired temozolomide resistance and thus can be used for
identifying novel targeted agents.
Tumor cell heterogeneity in MGMT expression and its relevance for TMZ response will also
be addressed using these organoids, using single cell proteomics and IHC for MGMT, GSC
markers and exome sequencing (for common driver mutations) to identify those populations
that clonally expand under TMZ (RT) selection and those that disappear.
Another interesting feature we will explore using PDO is the possibility to analyze the
supernatants for circulating tumor DNA (ctDNA) or exosomes (secreted vesicles which
contain RNA, small non-coding RNA, proteins as well as DNA) before during after acquired
TMZ resistance which might lead to the identification of pharmacological response and
predictive biomarkers. Such biomarkers 'liquid biopsy' may become useful to measure
treatment response in blood or lumbar fluid of GM patients and enable dose-modification
(escalation or deintensification or termination).
The development of the PDOs (costs of materials) will be funded (607061 PI M. Vooijs,
MAASTRO and KWF grant Alpe D'Huzes PI M.Vooijs, MAASTRO. There are no additional costs
associated with obtaining the tumor tissue.
