Analysis of Glioblastoma’s Peritumoral Brain Zone

Developing of Per-operative Analysis and New Therapeutic Targets

Jean-Michel Lemée

1,2

, Anne Clavreul

and Philippe Menei

1,2

Department of Neurosurgery, University Hospital of Angers, 4, rue Larrey, 49933, Angers Cedex 09, France

INSERM U1066, LUNAM University, 4, rue Larrey, 49933, Angers Cedex 09, France

1 STAGE OF THE RESEARCH

Our research topic is focused on the glioblastoma

(GB), an aggressive primary brain tumor, and

especially the study of its peritumoral brain zone

(PBZ). The aim of our study is to developp per-

operative analysis of GB-PBZ to guide the surgery

and to develop targeted therapies for post-operative

therapeutic management.

During the first part of my PhD, we studied the

heterogeneity of the glioblastoma’s peritumoral

brain zone (GB-PBZ) using the different techniques

used routinely to analyze tumor samples : radiology,

histology, flow cytometry, genomic, transcriptomic,

proteomic and cellular cultures. We identified a

specific profile of the GB-PBZ, distinct from both

the normal brain tissu and the glioblastoma’s

tumoral zone. This research will be the subject of a

publication, currently being drafted.

We also identified in this study several

alterations specific to the GB-PBZ. These alterations

of the GB-PBZ profile will be the subject of further

studies to define whether or not their expression may

be a prognosis or therapeutic response marker, or a

potential target for targeted therapies.

More, the proteomic study of the GB-PBZ

confronted us to a methodological problem: the

absence of a defined control sample for the

proteomic analysis of brain tumor samples. This

methodological problem was solved and was the

subject of a publication (Lemée et al. 2013).

2 OUTLINE OF OBJECTIVES

The second part of my PhD will be focused on two

main subjects: the development of per-operative

analysis of the GB-PBZ to guide the surgical

resection of the tumor, and the molecular analysis of

the GB-PBZ to develop new therapeutic targets for

GB treatment.

3 RESEARCH PROBLEM

The GB is the most frequent and aggressive primary

tumor of the central nervous system with an

incidence of 4.96/100.000/year, a poor prognosis

and the absence of a curative treatment.

One of the leading causes of this poor prognosis

is the systematical recurrence of the tumor,

occurring in 90% of cases from the margin of the

resection’s cavity (Sherriff et al., 2013); (Petrecca et

al., 2013). Few observations have been made on this

peritumoral zone defined by the brain tissues outside

the contrast enhancement on gadolinium T1

weighted MR-scan.

Most of them were post mortem observation and

indicated that microscopic tumor infiltration

extended a considerable distance beyond the low

densities areas on CT scan or the high-intensity

areas on T2 weighted MRI. This may offer a

potential explanation to the tumoral recurrence

around the resection’s cavity (Yamahara et al.,

2010); (Nagashima et al., 1999).

Infiltrating tumor cells have been isolated, by the

culture in a serum-free medium, of biopsy

specimens taken from the brain tissue surrounding

the resection cavity (Glas et al., 2010). These cells

display the alterations typical of GB, but can be

distinguished from tumor cells isolated from the

tumor core on the basis of their distinctive molecular

marker profiles and responses to drug and irradiation

challenges in vitro (Glas et al., 2010 #87).

Using a different type of culture medium, we

have isolated, from the brain tissue surrounding GB,

another unexpected population of cells from the

stroma that we have named glioblastoma associated

stromal cells (GASCs) (Clavreul et al., 2011). These

cells do not display the genomic alterations typical

of GB cells and resemble the cancer-associated

fibroblasts (CAFs) described in the stroma of

carcinomas.

These few studies showed that the peripheral

brain zone, although macroscopically and

Lemée J., Clavreul A. and Menei P..

Analysis of Glioblastoma’s Peritumoral Brain Zone - Developing of Per-operative Analysis and New Therapeutic Targets.

 2014 SCITEPRESS (Science and Technology Publications, Lda.)

radiologically considered as normal brain tissue, is

in fact distinct at different levels from both the

tumor zone of glioblastoma (TZ) and the normal

brain (NB). A better understanding of the

characteristics of the PBZ is critical to understand

the mechanisms of GB recurrence, optimize the

quality of the surgical resection and develop new

therapies.

3.1 Per-operative Study of

Glioblastoma’s Peritumoral Brain

Zone

The gross total resection of the GB is an important

prognostic factor of overall survival and tumoral

progression-free survival for the patient (Chaichana,

2013).

The difficulty of achieving a gross total resection

in GB lays in the infiltrative nature of the tumor,

with the presence a small tumoral infiltration in the

surroundings of the tumor. To avoid this we cannot

take security margins around the tumor during the

resection, because of the major risk of aggravating

the already impaired neurological status of the

patient, without a significant benefit for him. In this

condition, a macroscopically complete resection is

the optimal surgical treatment we can propose.

Thus, the development of new techniques of

neurosurgery, or the adaptation of the existing ones,

to allow a fast, easy, accurate and reproducible

analyse of the GB-PBZ during the surgery is

essential to ensure a complete resection of the tumor

without impairing the patient’s neurological status.

Technological advances in this field may be quickly

transferred to clinic and will have a critical impact

on the patients’ therapeutic management, functional

outcome and survival.

3.2 Development of New Therapeutic

Targets

Despite the development of new therapies, the

prognosis of GB remains poor with a mean survival

of 15 months with an optimal therapeutic

management. The identification and the validation of

new therapeutic targets are essential for the future of

adjuvant GB’s therapy in an era of personalized

medicine.

The future of GB therapy will be the

development of targeted therapies for custom-made

treatment after specific tumor profiling. One lead is

the identification of targets for existing targeted

therapies like Herceptin in Her2+ breast cancer or

vemurafenib in B-Raf proto-oncogene V660E muted

lung cancer and melanoma. The second option may

be the identification of specific target for GB and

GB-PBZ allowing the development of new targeted

therapies through the use of lipidic nanocapsules

(LNC). Different chemotherapeutic agents can be

encapsulated in these particles and thus focused

delivery will allow us to increase the dose of

therapeutic agent without increasing the potential

side effects of chemotherapy agents.

4 STATE OF THE ART

4.1 Glioblastoma’s Therapeutic

Management

GB is the most frequent and aggressive primary

tumor of the central nervous system. The

mechanisms of recurrence remain unclear but the

infiltration of the PBZ by GB cells may be

considered as the explanation of the high rate of

local recurrences.

The gold standard for GB’s therapeutic

management is a surgical gross total resection

followed by radiotherapy and chemotherapy with

temozolomide, according to the protocol described

by Stupp (Stupp, 2009). However, even with this

optimal treatment, the prognosis remains poor with a

progression-free survival of 7 months and a mean

survival of 15 months.

4.2 Per-operative Study of the

Peritumoral Brain Zone

As mentioned before, the quality of the surgical

resection of the GB is critical for its therapeutic

management making the realization of per-operative

quality controls of the surgical resection essential.

Actually, the gold standard of GB surgery is to

perform a macroscopically complete resection,

stopping the surgery when no tumoral tissue is left in

the surgical cavity, without taking any security

margin. But if the complete resection of the tumor

jeopardizes the neurological status of the patient, a

subtotal resection is performed instead. Numerous

techniques have been used to attempt to develop a

better per-operative control of the resection’s

quality.

The actual techniques used in per-operative GB-

PBZ study are the per-operative imaging and the

per-operative fluorescence. Per-operative imaging

consist in the realization during the surgery of a

brain scanner using a CT- or a MR-scan to ensure

the complete resection of the tumor. Due to the cost

AnalysisofGlioblastoma'sPeritumoralBrainZone-DevelopingofPer-operativeAnalysisandNewTherapeuticTargets

of a dedicated scanner or MRI in the operating

room, this technique is still confidential and only

available in a few centers.

The use of per-operative fluorescence allows to

see with a specific light the tumoral cells stained in

red, after the ingestion of a 5-amino levulinic acid

analog by patients 5 hours prior to surgery. This

simplify the completion of the surgical resection by

showing clearly where are located the tumoral

remnants on the wall of the resection’s cavity. This

technique is still under evaluation in France and is

actually the object of a national clinical trial

(RESECT).

4.3 Therapeutic Targets in GB

Actually, there are no validated or available drugs

targeted therapies available for GB treatment. GBs

are currently under a specific study to identify

potential targets for known and commercially

available targeted therapies in AcSE study.

For now, we didn’t dispose of a specific marker

of GB or GB-PBZ tumoral tissue for target specific

delivery of chemotherapeutic agents.

5 METHODOLOGY

5.1 Per-operative Study of the

Glioblastoma’s Peritumoral Brain

Zone

In the search of new means to study the GB-PBZ,

we will study several techniques routinely used in

medical practice in specialities other than the

Neurosurgery.

5.1.1 Second Harmonic Imaging Microscopy

and Two Photon-excited Fluorescence

First, we decided to study two modern techniques of

microscopy: the Second Harmonic Imaging

Microscopy (SHIM) and the Two Photons-Excited

Fluorescence (TPEF). SHIM is an efficient non-

destructive method to study in vivo tissues without

involving molecular excitation. TPEF is a technique

similar to the immunofluorescence, but less

susceptible to lead to phototoxicity and

photobleaching. SHIM reacts specifically to non-

centrosymmetric molecules such as collagen,

myosin and microtubules. This is an interesting lead

because normal brain cells do not produce collagen

whereas GB cells produce collagen in their

extracellular matrix using it as a support for cell

migration.

TPEF is a living imaging fluorescence that

allows deep study of tissue up to 1mm. TPEF can

excite fluorescent dyes with far less toxicity than the

other fluorescent methods and has a high definition

thank to a strong suppression of background signal

due to infrared photon absorption.

Preliminary Study. In collaboration with the Dr

Denis Gindre, of the Department of Physics of the

University of Angers, we did a preliminary study on

paraffin embedded sections of brain tumor to assess

the feasibility of the study.

The obtained images showed a differential SHIM

signal between the different brain zones, with the

absence of SHIM signal in the central tumoral

necrosis, a strong signal in tumoral zone and an

intermediary signal in the GB-PBZ (Figure 1). These

data are in favour of the continuation of this study

on a wider panel of brain tumor samples.

Adaptation of the Technique. However, before

continuing in scanning the samples, we will have to

solve a problem specific to the brain tumor and the

SHIM, the representativity of the sample.

Indeed, the SHIM and TPEF techniques allow the

scanning of a square of 100x100 μm. This is an

insufficient size to obtain a representative SHIM and

TPEF profile of a GB sample, which is by definition

a heterogeneous tumor with foci of necrosis and

tumoral proliferation.

Dr. Gindre and one of his PhD student are

currently creating a program which will scan random

squares of 100x100 μm of the tumor sample under

different polarization angle to create an mean image

of the SHIM and TPEF signals of the lesion in order

to obtain a representative SHIM and TPEF profiles

of the lesion.

Creating an Atlas. The next objective, when the

acquisition of a representative SHIM and TPEF

profiles of the studied samples will be validated, will

be to create an atlas of the SHIM and TPEF profiles

of the different types of normal brain and brain

tumor samples, using a collection of brains’ paraffin

embedded sections with an histologically confirmed

diagnosis.

The constitution of the atlas of SHIM and TPEF

signals of brain tumors samples will allow us to

assess the samples obtained per-operatively, based

on the profile obtained and its comparison with the

atlas.

Per-operative Testing. The final step will be to

perform extemporaneous analysis of the tumor

samples obtained during surgery to perform an

BIOSTEC2014-DoctoralConsortium

Figure 1: Illustration of the SHIM and TPEF acquisition of different GB area. The peritumoral brain zone from

glioblastoma (GB-PBZ), the interface between florid tumor and peripheral brain and the central necrotic zone of the tumor.

immediate diagnosis of the tumor sample, which can

take up to 10 days with classical pathological

analysis, and also assess samples of the border of the

resection’s cavity to confirm the absence of tumoral

cells and thus the complete resection of the tumor.

5.1.2 Other Potential Leads for

Per-operative Study of the GB-PBZ

We are planning to try the optic coherence

tomography (OCT) to study the 3D conformation

and surface of the resection’s cavity to ensure an

optimal resection of the tumor. This opportunity

comes from the commercialization of smaller,

portable and usable in an operating theatre.

Also, one other possibility, depending on the

identification of specific targets of GB tumoral cells

will be a colorimetric staining of the resection’s

cavity walls with a reagent that will change colour

when in contact with a specific biomarker present in

GB cells.

5.2 Development of New Therapeutic

Targets

5.2.1 Validation of the Proteins

We identified from our multimodal analysis of the

GB-PBZ potential biomarkers suitable as therapeutic

target.

First, we will perform a double validation of the

presence of these proteins by immunochemistry

(IHC) and western blot analysis on the samples from

the patients used for multimodal analysis of the GB-

PBZ, whose profiles are known.

5.2.2 Study of the Biomarkers in Tumor

Samples

After the validation of the proteins, we will assess

the expression of these proteins in a cohort of

patients from the national clinico-biological

collection of GB, coordinated by Pr Menei. Crossing

the data obtained with the epidemiologic

characteristics and the evolution of the patients will

allow us to determine if the presence (or absence) of

these biomarkers have an impact on the survival or

the therapeutic response of patients with a GB.

A first cohort of 50 patients from the clinico-

biological collection and tumors samples will be

analysed using IHC.

If the results are concluding for this first cohort,

the analysis will be extended to the whole

population of the clinico-biological collection and be

done routinely on new patients included in the

national database.

AnalysisofGlioblastoma'sPeritumoralBrainZone-DevelopingofPer-operativeAnalysisandNewTherapeuticTargets

5.2.3 Study of the Biomarkers in Blood

Samples

The proteins identified in the PBZ are components

of the brain cells exosomes, and thus can be assayed

in blood samples from patients.

We look forward to this possibility because

blood samples are easy to obtain and allow repetitive

and reproductive measure of biomarkers blood level

in GB patients during their post-surgical evolution

and treatment.

We will assess the presence of these biomarkers

in the peripheral blood of GB patients using an

enzyme-linked immuno-assay (ELISA) technique.

As with the analysis of the tumor samples, a

preliminary study will be performed on a small

cohort of patients from the clinico-biological

collection and will be extended to the whole cohort

if the first results are positives.

5.2.4 Biomarkers as Therapeutic Targets

We will study the potential role of the identified

biomarkers as therapeutic target in GB treatment. A

step next will be to develop monoclonal antibodies

targeted against these proteins to see if the specific

neutralization of one of these proteins can improve

the survival or the therapeutic response of a

population of murine GB model.

If we identify a significant improvement in terms

of survival after GB implantation in a murine model

of the disease, we will begin a human clinical trial.

5.2.5 Biomarkers as Targets for Vectorized

Therapies

One other axis of development will be the use of

vectorized LNC aimed against the proteins identified

in the GB-PBZ. Production of LNC, encapsulation

of chemotherapeutic agents and targeted delivery of

these nanocapsules are a field of expertise of my

laboratory, which possess an international

recognition in this domain.

We aim to produce LNC targeted against the

identified biomarkers to use them as therapeutic

agent against GB. In a first phase, we will produce

LNC containing fluorochromes to assess the

distribution of the LNC in a murine model of GB.

Then, if the results are positive, we will produce

LNC containing a chemotherapeutic agent, and

assess in the same murine model of GB the impact

of the LNC injection in the functional prognosis and

survival of the mice, with always as first aim the

transfer of these researches to the clinical field in

human.

6 EXPECTED OUTCOME

At short-term, during the next year, the results of the

SHIM and TPEF imaging analysis of brain tumor

samples will be the subject of a publication in a high

impact review of Neuro-Oncology. Also the

validation of the biomarkers in the brain tumor and

the peripheral blood samples of the patients will be

the subject of a publication in a high impact review

in Neuro-Oncology.

The use of per-operative OCT or colorimetric

staining of the tumor are planned for the end of my

PhD thesis or will be the subject of my post-doctoral

studies.

The use of the proteins of interest as therapeutic

target or as target for chemotherapy-loaded LNC

will be the subject of further studies in my

laboratory, implicating Pharmacists and Galenists.

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AnalysisofGlioblastoma'sPeritumoralBrainZone-DevelopingofPer-operativeAnalysisandNewTherapeuticTargets