Rezumat ENGLEZA (1) treatment strategies for... · 2021. 2. 26. · í .(< :25'6 jolreodvwrpd...
Transcript of Rezumat ENGLEZA (1) treatment strategies for... · 2021. 2. 26. · í .(< :25'6 jolreodvwrpd...
MINISTRY OF EDUCATION AND RESEARCH University of Medicine and Pharmacy of
Craiova DOCTORAL SCHOOL
Phd THESIS
ABSTRACT
NEW TREATMENT STRATEGIES FOR
GLIOBLASTOMA MULTIFORME IN VITRO
SCIENTIFIC COORDINATOR:
PROF.UNIV.DR. DRICU ANICA
PhD student:
FOLCUȚI CĂTĂLIN-ALEXANDRU
CRAIOVA
2020
TABLE OF CONTENTS
1. BRAIN TUMORS. OVERVIEW .................................................................................. 1
2. GLIOBLASTOMA ..................................................................................................... 1
2.1. Tumor heterogeneity and recurrence (classical, proneural, neural and mesenchymal subtypes) ........................................................................................ 1
2.2. The genetic and molecular factors in glioblastoma ........................................ 1
2.2.1. Epithelial growth factor receptor (EGFR) ................................................. 1
2.2.2. PDGFR ...................................................................................................... 2
2.2.3. PTEN ......................................................................................................... 2
2.2.4. MGMT ...................................................................................................... 2
2.2.5. IDH1/2 ...................................................................................................... 2
2.2.6. β-arrestin .................................................................................................. 2
2.3. GBM therapy ................................................................................................... 2
3. PERSONAL CONTRIBUTION .................................................................................... 3
3.1. WORK HYPOTHESIS AND GENERAL OBJECTIVES ............................................. 3
3.2. RESULTS AND DISCUSSION .............................................................................. 4
3.2.1. Meta-analytic investigation of dendritic cell vaccination and viral therapy in patients with malign glioma. ............................................................ 4
3.2.2. The effect of EGFR inactivation on malign glioma cells viability ............. 4
3.3. STUDY OF Β-ARRESTINE 1 TRANSFECTION INFLUENCE ON MALIGN GLIOMA CELL PROLIFERATION AND ON THE RESPONSE OF E TREATMENT ....................... 5
4. CONCLUSIONS ........................................................................................................ 9
BIBLIOGRAPHY ……………………………………………………………………………………………………..9
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KEY WORDS: glioblastoma, therapy, arrestin, temozolamide
1. BRAIN TUMORS. OVERVIEW
Brain tumors are the most severe form of tumors, considering the fact they are
malign cell masses that affect vital systems in neurological balance regulation (1). They
are considered to be primary, classified according to the type of cell that generated them
and secondary – cerebral metastases (2).
Gliomas are the most frequent type of central nervous system tumors, representing
almost 80% of primary brain tumors. These types of tumors can develop at any age, with a
peak in the 5th-6th decade. Glial tumors are characterized by an important intratumoral
heterogeneity, proliferative potential and tumor recurrence (1). Taking into consideration
these properties, the overall survival is less that 15 months, despite the latest therapeutic
approaches (3).
The study of histological characteristics of gliomas is crucial in identifying the
molecular mechanisms underlying the actual limitations of therapy and their resistance to
chemotherapy and radiotherapy.
2. GLIOBLASTOMA
The overall survival in glioblastoma, the most aggressive, invasive and
undifferentiated brain tumor, is 14,6 months (4), despite modern multimodal therapeutic
modalities.
2.1. Tumor heterogeneity and recurrence (classical, proneural, neural and
mesenchymal subtypes)
Glioblastoma is known for its’ heterogeneity regarding signaling pathways, tumor
site, phenotype, genetics, epigenetics and molecular properties.
2.2. The genetic and molecular factors in glioblastoma
2.2.1. Epithelial growth factor receptor (EGFR)
Specialized studies from the last decade have shown that over 50% of GBM present
EGFR mutations. The most common is EGFR amplification (approximately 40% of EGFR
amplification are EGF mutant) (3).
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2.2.2. PDGFR
PDGFR overexpression is present in ~23% cases of GBM, having unfavorable
repercussions on patients prognosis and survival with IDH1 mutation.
2.2.3. PTEN
40% of GBM cases have shown an early diminution of PTEN expression (5-7), and
in 50-70% of recurrent GB, respectively in 50-90% of primary GB was found a loss of
chromosome 10 heterozygosis (8-9).
2.2.4. MGMT
MGMT methylation determine a growth in tumor cell sensitivity to the action of
alkylant agents like TMZ. In low grade gliomas, the role and clinical impact of MGMT
methylation status is still in scientific debate.
2.2.5. IDH1/2
Recent studies have shown that the most frequent mutation in gliomas is IDH1
R132H (10). Also, there have been observed IDH mutations in 80-90% of grade II and III
gliomas (10-11).
2.2.6. β-arrestin
A recent in vitro study conducted on GBM tumor cells has shown a growth of their
sensitivity to TMZ through β-arrestine1 gene activation (12). The exact role of β-arrestine
in gliomagenesis, proliferation and therapeutic response are still insufficient.
2.3. GBM therapy
GBM is still a therapeutic challenge despite multimodal and interdisciplinary
approaches (13). The classic therapeutic methods are surgery, chemotherapy and
radiotherapy. The new generation of therapeutic modalities consists in molecular target
therapy, immunotherapy, viral therapy, adoptive therapy, dendritic cell vaccination and
genetic therapy.
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3. PERSONAL CONTRIBUTION
3.1. WORK HYPOTHESIS AND GENERAL OBJECTIVES
OBJECTIVE NO. 1 Meta-analytic investigation of viral therapy effect comparing to immune therapy
with dendritic cells in malign gliomas.
OBJECTIVE NO. 2
In this study we analyzed the potential of EGFR-targeting on malign glioma cells
viability.
OBJECTIVE NO. 3 Study of β-arrestine 1 transfection influence on cell proliferation and on the response
of malign gliomas to the e treatment.
3.2. RESULTS AND DISCUSSION
3.2.1. Meta-analytic investigation of dendritic cell vaccination and viral therapy
in patients with malign glioma.
3.2.1.2. OS and PFS meta-analysis in dendritic cell vaccination
Our systematic analysis integrated 9 specialized studies. In these studies, were
included 357 patients, from which 104 patients were in experimental groups and 253 in
control groups. Of these, 207 patients were newly-diagnosed with HGG (70 patients in
experimental group and 137 patients in control group) and 150 patients were diagnosed
with recurrent HGG (34 patients in experimental group and 116 in control group).
Regarding therapy combined with dendritic cell vaccination, a PFS improvement was
noticed (HR 0,49), with CI 95% 0,21-1,16. Statistically, results of dendritic cell
vaccination therapy of HGG patients was not significant (p=0,10).
3.2.1.3. OS and PFS meta-analysis of viral therapy
In the four studies integrated in the systematic analysis of viral therapy we identified
642 newly-diagnosed HGG patients, the experimental group included 237 patients, and
control group included 405 patients. In total, the OS meta-analysis integrated 4 studies,
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and PFS meta-analysis included 3 studies. In three studies we observed an OS progress,
the most notable one being described by Wheeler et al (14) (HR 0,72, 95% CI: 0,54-0,97).
On the other hand, Rainov (15) reported a benefit for the patients from the control group
(HR 1.08, 95% CI: 0,81-1,46).
3.2.2. The effect of EGFR inactivation on malign glioma cells viability
3.2.2.1. The growth pattern of untreated HGG cells
In this study we analyzed the proliferation of 11 HGG cell line in 7 days.
Fig. 1. The growth pattern of HGG cells
3.2.2.2. The effect of EGFR inactivation on HGG cells
In this study we investigated the effect of AG556 on 11 HGG cells in order to
conclude on the cytotoxicity of this agent. HGG cells were exposed to different
concentrations of AG556: 10 µM, 20 µM and 30 µM. The cell proliferation rates were
measured in day 3 and day 7. We observed that the most significant cytotoxic effect was
reached at the concentration of 30 µM, with a diminish of survival malign cells with
approximately 17% in day 3 (Fig 2). This value was constant and there were no significant
changes the rest of the experiment, including day 7 of AG 556 treatment (Fig.3).
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Fig. 2, 3. The effect of AG556 inhibitor on EGFR inactivation in 11 HGG cell line at
3, respectively 7 days of treatment.
3.3. STUDY OF Β-ARRESTINE 1 TRANSFECTION INFLUENCE ON
MALIGN GLIOMA CELL PROLIFERATION AND ON THE RESPONSE OF E
TREATMENT
3.3.1. The TMZ treatment effect on malign glioma cells
Our experiment tested the cytotoxic effect of the alkylating agent TMZ on two
different malign cell lines: 18 HGG and U-343MGa. We analyzed the decrease of malign
cell proliferation at 24h, 48h and 72h after treating them with different concentrations of
the alkylating agent (200 µM, 250 µM and 300 µM).
0
50
100
150 11
7days
050
100150
U-343MGa
24h0
50100150
U-343MGa
48h
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Fig. 4, 5, 6. The effect of TMZ treatment on U-343MGa cell line at 24h, 48h and 72h
24 hours from the beginning of the treatment with 200 µM, we observed a decrease
of the cell viability with 17,32%. 250 µM concentration determined a 17,88% decrease
and the maximum concentration determined the highest cytotoxicity of 20,17% (Fig.4).
Regarding the prolonged treatment at 48h, we recorded a significant increase of the
cytotoxic effect in U-343MGa cell line, as follows: at 300 µM we observed the most
important cytotoxic effect (45,3%) (Fig 5). At 72 h from the TMZ treatment initiation,
tumor cells cytotoxicity was reduced comparing with the one recorded at 48 hours (Fig 6).
Regarding the second cell line, line 18 HGG, treated with the same concentrations of
alkylating agent for 24, 48 and 72 hours, we observed similar values with U-343MGa cell
line (Fig 7, 8, 9).
0
50
100
150U-343MGa
72h
0
50
100
150
18HGG
48h
7
Fig. 7, 8, 9. Efectul tratamentului cu TMZ la 24, 48, 72 h în linia celulară 18 HGG
3.3.2. Study of β-arrestine-1 transfection influence on the response of TMZ
treatment in U-343MGa cell line
Fig. 10, 11, 12. β-arrestine 1 transfection influence on the response of temozolomide treatment in U-343MGa cell line at 24, 48 and 72 hours
After 24 hours of treatment, β-arr1 transfection countered by ~4% the effect of 200
µM TMZ treatment, but this result cannot be considered statistically significant (p>0.05)
(Fig. 10). The results showed that U-343MGa cell line proliferation recorded a significant
0
50
100
15018HGG
72h
-20
30
80
130
180
Control TMZ200µM
TMZ250µM
TMZ300µM
U-343MGa 24h treatment
Untransf…
-20
30
80
130
180
Control TMZ200µM
TMZ250µM
TMZ300µM
U-343MGa 48h treatment
Untransf…
0
50
100
150
200
Control TMZ200µM
TMZ250µM
TMZ300µM
U-343MGa 72h treatment
Untransfe…
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growth after 48 hours from β-arr1 transfection (26%), comparing with the control group.
(p≤0.05) (Fig. 11). 72 hours from the beginning of TMZ treatment in high dose, the β-arr1
did not prevent cytotoxicity (p≥0.05) (Fig. 12).
3.3.3. Study of β-arrestine 1 transfection influence on the response of
temozolomide treatment in 18 HGG cell line
β-arrestine 1 transfection caused the decrease of 18 HGG cell viability and presented
a moderate influence on the cytotoxic effect of TMZ treatment.
Fig. 13, 14, 15. The influence of β-arr transfection on TMZ treatment at 24, 48 and 72 hours
020406080
100120 18HGG 24h treatment
020406080
100120
Control TMZ200µM
TMZ250µM
TMZ300µM
18 HGG 48h treatmentUntransfected
020406080
100120
Control TMZ200µM
TMZ250µM
TMZ300µM
18 HGG 72h treatment
Untransfected
β1-Arestin transfected
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4. CONCLUSIONS
Our analysis demonstrated that the therapeutic approach based on dendritic cell
vaccination shows an improvement of patients with newly-diagnosed (HR=0.65) and
recurrent (HR=0.63) high grade glioma overall survival and PFS (HR=0.49) comparing to
viral therapy.
In our experiment, the EGFR inactivation with cytotoxic agent AG556 determined a
proportional decrease of cell proliferation with the inhibitor dose. This effect was
observed at 3 days of treatment, with no significant changes at the prolonged 7 days of
treatment.
Β-arr1 transfection determined a growth of cell proliferation in U-343MGa and
countered the temozolomide cytotoxic effect.
Β-arr1 transfection also determined the decrease of cell viability in 18 HGG cell line
and presented a moderate influence on the temozolomide cytotoxic effect.
BIBLIOGRAPHY
1. Rajesh Y., Ispita Pal, Payel Banik et al. Insights into molecular therapy of glioma:
current challenges and next generation blueprint. 2017; 38:591-613.
2. Chen L., Zou X. Wang Y. et al. Central nervous system tumors: a single center
pathology review or 34,140 cases over 60 years. BMC Clin Pathol. 2013; 13:1-4.
3. Simge Y, Anna K, Ihsan S et al. Management of patients with high-grade glioma. EMJ
Oncol. 2014; 2:91-99.
4. Farina H, Kanza M, Kahkashan P et al. Glioblastoma Multiforme: a review of its
epidemiology and pathogenesis through clinical presentation and treatment. Asian Pac J
Cancer Prev. 2017; 18(1):3-9.
10
5. Smith JS, Tachibana I, Passe SM, et al. PTEN mutation, EGFR amplification, and
outcome in patients with anaplastic astrocytoma and glioblastoma multiforme. J Natl
Cancer Inst. 2001; 93(16):1246–1256.
6. Srividya MR, Thota B, Shailaja BC, et al. Homozygous 10q23/PTEN deletion and its
impact on outcome in glioblastoma: a prospective translational study on a uniformly
treated cohort of adult patients. Neuropathology. 2011; 31(4):376–383.
7. Carico C, Nuno M, Mukherjee D, et al. Loss of PTEN is not associated with poor
survival in newly diagnosed glioblastoma patients of the temozolomide era. PLoS One.
2012; 7(3):e33684.
8. Chenn A, Walsh CA. Regulation of cerebral cortical size by control of cell cycle exit in
neural precursors. Science. 2002; 297(5580):365-369.
9. Fraser MM, Zhu X, Kwon CH, Uhlmann EJ, Gutmann DH, Baker SJ. Pten loss causes
hypertrophy and increased proliferation of astrocytes in vivo. Cancer Res. 2004;
64(21):7773–7779.
10. Hartmann C, Meyer J, Balss J, Capper D, Mueller W, Christians A, et al. Type and
frequency of IDH1 and IDH2 mutations are related to astrocytic and oligodendroglial
differentiation and age: a study of 1,010 diffuse gliomas. Acta Neuropathol. 2009;
118:469–74.
11. Cohen AL, Holmen SL, Colman H. IDH1 and IDH2 mutations in gliomas.Curr
Neurol Neurosci Rep. 2013; 13:345.
12. Lan T, Wang H, Zhang Z, Zhang M, Qu Y, Zhao Z, Fan X, Zhan Q, Song Y, Yu C.
Downregulation of beta-arrestin 1 suppresses glioblastoma cell malignant progression
vis inhibition of Src signaling. Experimental cell research; 2017; 357(1):51-58.
13. Kesari S. Understanding glioblastoma tumor biology: the potential to improve
current diagnosis and treatments. Semin Oncol. 2011; 38:2-10.
14. Wheeler, L. A. et al. Phase II multicenter study of gene-mediated cytotoxic
immunotherapy as adjuvant to surgical resection for newly diagnosed malignant
glioma. Neuro-oncology. 2016; 18:1137–1145.
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15. Stragliotto, G. et al. Effects of valganciclovir as an add-on therapy in patients with
cytomegalovirus-positive glioblastoma: a randomized, double-blind, hypothesis-
generating study. International journal of cancer. 2013; 133:1204–1213.