A single amino acid substitution converts a histidine decarboxylase to an imidazole acetaldehyde synthase

Article abstract of DOI:10.1016/j.abb.2020.108551

Histidine decarboxylase (HDC; EC 4.1.1.22), an enzyme that catalyzes histamine synthesis with high substrate specificity, is a member of the group II pyridoxal 5′-phosphate (PLP) -dependent decarboxylase family. Tyrosine is a conserved residue among group II PLP-dependent decarboxylases. Human HDC has a Y334 located on a catalytically important loop at the active site. In this study, we demonstrated that a HDC Y334F mutant is capable of catalyzing the decarboxylation-dependent oxidative deamination of histidine to yield imidazole acetaldehyde. Replacement of the active-site Tyr with Phe in group II PLP-dependent decarboxylases, including mammalian aromatic amino acid decarboxylase, plant tyrosine/DOPA decarboxylase, and plant tryptophan decarboxylase, is expected to result in the same functional change, given that a Y-to-F substitution at the corresponding residue (number 260) in the HDC of Morganella morganii, another group II PLP-dependent decarboxylase, yielded the same effect. Thus, it was suggested that the loss of the OH moiety from the active-site Tyr residue of decarboxylase uniquely converts the enzyme to an aldehyde synthase.

Full text of DOI:10.1016/j.abb.2020.108551

Neoplasia . Vol. 7, No. 10, October 2005, pp. 883 – 893 883
RESEARCH ARTICLE
www.neoplasia.com
Patients with High-Grade Gliomas Harboring Deletions
of Chromosomes 9p and 10q Benefit from
1
Temozolomide Treatment
,
y,2
y,2
z
z
y
§
Silke Wemmert* , Ralf Ketter , J o¨ rg Rahnenf u¨ hrer , Niko Beerenwinkel , Martin Strowitzki , Wolfgang Feiden ,
b
z
#
Christian Hartmann , Thomas Lengauer , Florian Stockhammer , Klaus D. Zang*, Eckart Meese*,
Wolf-Ingo Steudel , Andreas von Deimling and Steffi Urbschat*
y
b
,y
y
*
Neurosurgery, Saarland University, Homburg/Saar D-66421, Germany; Max-Planck-Institute for Informatics,
Institute of Human Genetics, Saarland University, Homburg/Saar D-66421, Germany; Department of
z
§
Stuhlsatzenhausweg 85, Saarbr u¨ cken D-66133, Germany; Institute of Neuropathology, Saarland University,
b
Homburg/Saar D-66421, Germany; Institute of Neuropathology, Charit e´ , Universitaetsmedizin Berlin, Berlin
#
D-13353, Germany; Department of Neurosurgery, Charit e´ , Universitaetsmedizin Berlin, Berlin D-13353, Germany
Abstract
Surgical cure of glioblastomas is virtually impossi-
ble and their clinical course is mainly determined by
the biologic behavior of the tumor cells and their
response to radiation and chemotherapy. We inves-
tigated whether response to temozolomide (TMZ)
chemotherapy differs in subsets of malignant glio-
blastomas defined by genetic lesions. Eighty patients
with newly diagnosed glioblastoma were analyzed
with comparative genomic hybridization and loss of
heterozygosity. All patients underwent radical resec-
tion. Fifty patients received TMZ after radiotherapy
clinical course is determined by the biologic behavior of
the tumor, including growth rate and response to radiation
and chemotherapy.
Neuropathologic diagnosis is the most valuable tool for
the classification of human tumors. However, within neuro-
pathologic entities, morphologic and immunohistochemical
analyses usually cannot predict prognosis and response to
therapy. However, molecular genetic analysis has succeeded
in determining distinct genetic subgroups that may exhibit
different biologic behavior [1–3].
Trials on the effects of systemic chemotherapy on survival
and recurrence in adults with high-grade gliomas showed
extended survival times, but there was no evidence that the
effect of chemotherapy depended on age, sex, histology,
Karnofsky performance status (KPS), or extent of resection [4].
However, patients of young age and with complete resection
have a considerably better prognosis [5–7]. Temozolomide
(TMZ), an orally administered second-generation imidazote-
trazine, has been demonstrated to increase the survival time
of glioma patients [8]. Phase II studies of TMZ (versus procar-
bazine) showed that TMZ has an acceptable safety profile
and can improve the quality of life [8–10].
(
TMZ group) and 30 patients received radiotherapy
alone (RT group). The most common aberrations de-
tected were gains of parts of chromosome 7 and losses
of 10q, 9p, or 13q. The spectrum of genetic aberrations
did not differ between the TMZ and RT groups. Patients
treated with TMZ showed significantly better survival
than patients treated with radiotherapy alone (19.5
vs 9.3 months). Genomic deletions on chromosomes
9
and 10 are typical for glioblastoma and associated
with poor prognosis. However, patients with these
aberrations benefited significantly from TMZ in uni-
variate analysis. In multivariate analysis, this effect was
pronounced for 9p deletion and for elderly patients
with 10q deletions, respectively. This study demon-
strates that molecular genetic and cytogenetic analy-
ses potentially predict responses to chemotherapy in
patients with newly diagnosed glioblastomas.
Abbreviations: CGH, comparative genomic hybridization; GBM, glioblastoma multiforme;
KPS, Karnofsky performance status; LOH, loss of heterozygosity; MGMT, methylguanine
methyltransferase; MRI, magnetic resonance imaging; sGBM, secondary glioblastoma
multiforme; SSC, sodium saline citrate; ST, survival time; TMZ, temozolomide; WHO, World
Health Organization
Neoplasia (2005) 7, 883–893
Address all correspondence to: Steffi Urbschat, PhD, Neurooncology, Institute of Human
Genetics, Saarland University, Homburg/Saar D-66421, Germany.
Keywords: CGH, LOH, glioblastoma multiforme, survival, TMZ.
E-mail: hgsmu r@ uniklinik-saarland.de
1
This work was supported by the Hedwig-Stalter Foundation (S. Wemmert), research
advancement training program HOMFOR A/2003/25 (R. Ketter), and BMBF grant 01GR0453
(
J. Rahnenf u¨ hrer). The work at the Max-Planck Institute for Informatics has been per-
formed in the context of the BioSapiens Network of Excellence (EU contract no. LSHG-CT-
003-503265).
2
2
Introduction
S. Wemmert and R. Ketter contributed equally to this work.
Treatment of diffuse gliomas still remains one of the
most disappointing tasks in oncology. Surgical cure of
these infiltrating brain tumors is virtually impossible. The
Received 22 April 2005; Revised 8 June 2005; Accepted 10 June 2005.
Copyright D 2005 Neoplasia Press, Inc. All rights reserved 1522-8002/05/$25.00
DOI 10.1593/neo.05307

8
84
Deletions of 9p and 10q in Temozolomide Therapy
Wemmert et al.
Numerous studies revealed that the most common somatic
been linked to specific genetic alterations, namely LOH on
1p or combined LOH on 1p and 19q, and the absence of
homozygous deletion of the CDKN2A tumor-suppressor
gene on 9p21 [19,35]. Apart from these data on the effect
of genetic changes on the overall prognosis of gliomas, there
is no information at the moment on the significance of further
genetic changes on therapy response. Therefore, we ana-
lyzed a series of TMZ-treated patients in comparison to a
retrospective, conventionally treated control group with
newly diagnosed glioblastoma with respect to the above-
mentioned typical chromosomal alterations in glioblastomas.
The aim of this study was to determine whether spe-
cific genetic markers predict response to TMZ chemother-
apy and may serve as parameters for the rational design
of chemotherapy.
chromosomal changes in malignant gliomas are complete or
partial loss of chromosome 10 and gain of chromosome 7.
Various molecular genetic alterations have been identified,
including the amplification of EGFR, CDK4, and MDM2, as
well as the deletion of tumor-suppressor genes like TP53
(
17p), RB (13q), CDKN2A (9p), CDKN2B (9p), PTEN (10q),
and DMBT1 (10q) [2,11–15]. These tumor-suppressor genes
play crucial roles in the regulation of cell proliferation and
apoptosis. The TP53 gene product, p53, is involved in the
regulation of cell repair, apoptosis, and cell cycle. Cyclin-
dependent kinases (cdk), such as CDK4 and their inhibitors,
p16 and p15, proteins from CDKN2A and CDKN2B, and
pRB, are key regulators of the cell cycle. The gene products
of CDKN2A/B locus on 9p also participate in the TP53 path-
way through a protein encoded by an alternate reading
arf
frame, p14 , which binds to the p53/MDM2 complex and
inhibits MDM2-mediated degradation of p53. Therefore,
homozygous deletion of the CDKN2A/B locus affects both
Rb and TP53 pathways [16].
Materials and Methods
Patients
In recent years, studies have identified a correlation be-
tween alterations on chromosome 10q and shorter survival
in patients with high-grade glioma. Tada et al. [3] reported
significantly shorter survival rates of patients with glioblas-
toma multiforme (GBM) with loss of heterozygosity (LOH) on
In total, 80 cases of newly diagnosed glioblastomas
operated on during the period of 1997 to 2003 were studied
(Table 1). The patients were treated in two centers: 48 pa-
tients in the Department of Neurosurgery of the Saarland
University and 32 patients in the Department of Neuro-
surgery, Charit e´ , University Berlin. Patients eligible for this
nonrandomized study were 18 to 70 years of age, with a
histologically proven GBM (World Health Organization [WHO]
grade IV astrocytoma) [2] and a KPS of 70 or better. Patients
with renal, hepatic, or bone marrow impairment; HIV infec-
tion; prior chemotherapy; or stereotactic biopsy were ex-
cluded. All patients underwent radical resection followed
by radiotherapy within 4 weeks of surgery. Radiotherapy
consisted of fractionated focal irradiation at a dose of 1.8 to
2 Gy per fraction, given once daily 5 days per week over a
period of 6 weeks, for a total dose of 60 Gy. Radiotherapy
was delivered to the gross tumor volume with a 2-cm margin
volume for the clinical target volume on a preoperative
magnetic resonance imaging (MRI).
1
0q containing the PTEN/MMAC1 gene, and in anaplastic
astrocytoma patients with LOH on 10q in the region contain-
ing DMBT1. Several authors [3,17–19] detected poorer sur-
vival rates associated with LOH on 10q in glioblastomas,
but showed that PTEN mutation is only marginally asso-
ciated with survival [17,20].
A further candidate on chromosome arm 10q is MGMT.
The MGMT gene encodes for the DNA repair enzyme
6
O -alkylguanine-DNA-alkyltransferase, which is responsible
for protecting cells from alkylating agents. The enzyme ef-
ficiently removes methyl adducts at the O-6 position of gua-
nine, which is an important target of alkylating agents [21].
It could been shown that the sensitivity to alkylating agents
like BCNU correlates inversely to MGMT activity [22,23]. The
responsiveness to BCNU is associated with an increase in
overall survival rate [24]. Further on, the presence of aber-
rant promoter hypermethylation of MGMT was associated
with loss of the MGMT protein, in contrast to retention of
protein in the majority of tumors without hypermethylation
Two groups of patients were defined. The first group in-
cluded patients from March 1997 to April 1999, treated after
radical resection with radiotherapy alone (control group). In
the second group, patients from the time period from April
[
25]. Further clinical trials suggested that methylation of the
Table 1. Patient Characteristics.
MGMT promoter is predictive for better outcome in patients
with malignant gliomas treated with alkylating agents such
as TMZ [26–28].
Patients
TMZ (n = 50)
Conventionally
Treated (n = 30)
Gains of chromosome 7 are known to be associated with
shorter patient survival in anaplastic astrocytomas and low-
grade astrocytomas [29,30], but, to our knowledge, no cor-
relation between additional copies of chromosome 7 and
survival in GBM has been found so far. However, EGFR
amplification is considered to be an unfavorable marker for
survival [31,32]. Further indicators of poor prognosis are
LOH on 9p [17,33] and p16 mutations [34].
Age (years)
Median
Range
53
26–72
17
58
24–77
3
Still alive
Gender
Male
33
17
20
10
Female
KPS
100
90
27
19
4
6
16
2
8
0
0
Chemosensitivity and prolonged overall survival of pa-
tients with anaplastic oligodendroglioma have recently
7
6
Neoplasia . Vol. 7, No. 10, 2005

Deletions of 9p and 10q in Temozolomide Therapy
Wemmert et al.
885
1
999 to November 2003 were included. This group received,
10p with markers D10S1172, D10S1159, D10S527, and
D10S506; chromosomal arm 10q with markers D10S1419,
D10S1171, D10S523, D10S1765, D10S1143, D10S 1173,
D10S520, D10S521, D10S1141, D10S503, D10S1165,
D10S1439, D10S505, D10S1134, and D10S1248; chromo-
somal arm 13q with markers D13S326, D13S887, D13S788,
and D13S773; and chromosomal arm 9p with markers
D9S759, D9S925, D9S1121, and D9S319. Nucleotide se-
quences and mapping information were retrieved from the
Human Genome Database (www.gdb.org) or the Coopera-
tive Human Linkage Center (www.chlc.org) database files.
Polymerase chain reaction (PCR) was performed in a final
volume of 10 ml containing 10 ng of DNA, 50 mM KCl, 10 mM
Tris–HCl (pH 8.3), 200 mM deoxynucleotides, 0.1% gelatin,
and 20 pmol of each primer. Taq polymerase (Gibco BRL/
Life Technologies, Karlsruhe, Germany) was used and the
after radical resection, the abovementioned radiotherapy
regime and an adjuvant chemotherapy consisting of TMZ
(
TMZ group). TMZ was administered as a test dosage of
2
2
50 mg/m body surface area per day (750 mg/m total dose
1
per cycle) on days 1 to 5 in the first cycle because of its
dose-limiting toxic effect resulting in thrombocytopenia. The
2
following cycles were performed at a dosage of 200 mg/m
2
per day (1000 mg/m total dose per cycle). Treatment cycles
were repeated every 28 days. Altogether, eight cycles were
carried out. Patients were seen at least every 3 months in
the first 2 years. At each follow-up visit, clinical performance
status, neurologic status, and MRI were recorded.
Specimens of resected tumors were immediately frozen
and stored at ꢀ80jC, or fixed in formalin and embedded in
paraffin. All patients gave written informed consent for the
use of the tumor samples for genetic analysis.
MgCl
concentration ranged from 1.0 to 2.0 mM, depending
2
on the primer pair. Initial denaturation at 95jC for 3 minutes
was followed by 29 cycles on a thermocycler (Biometra,
Goettingen, Germany), denaturation at 95jC for 30 seconds,
annealing at temperatures ranging from 52jC to 64jC for
40 seconds, and extension at 72jC for 30 seconds. A final
extension step of 10 minutes at 72jC was added. PCR prod-
ucts were separated on 8% denaturating acrylamide gels
and visualized by silver staining [39]. LOH was scored as
previously described [38].
Comparative Genomic Hybridization (CGH)
DNA was obtained using standard protocols. Reference
DNA from the blood of a healthy donor and tumor DNA
from frozen tumor tissues were labeled with biotin and digoxi-
genin by standard nick translation (Roche Diagnostics, Mann-
heim, Germany). Six hundred nanograms of each tumor
and reference DNA was hybridized together with COT1
DNA (Roche Diagnostics) to normal chromosome meta-
phase spreads from peripheral blood lymphocytes pre-
pared following standard procedures. After 3 to 4 days of
hybridization at 37jC, posthybridization washes were per-
formed at a stringency of 50% formamide/2ꢁ standard sa-
line citrate (SSC), 2ꢁ SSC, and 0.1ꢁ SSC at 45jC. Tumor
DNA was visualized with fluorescein isothiocyanate (Vector
Laboratories, Burlingame, CA) and reference DNA with rho-
damine (Roche Diagnostics). Fluorescence images were
captured using a fluorescence microscope Olympus AX 70
Statistical Analyses
Subgroups of patients were defined by genetic status,
therapy, and age (old patients, > 53 years; young patients,
V 53 years). We chose this classification because the median
age was 54 years. Comparisons between groups were per-
formed by Kaplan-Meier curves and Cox regression analysis.
(Olympus, Hamburg, Germany) with a cooled charged-coupled
device camera. Image processing was performed by use
of ISIS (MetaSystems, Altlussheim, Germany). Average ratio
profiles were determined from analyses of 10 to 15 meta-
phases. The thresholds used for ratio profiles were 1.2 for
gain and 0.8 for loss.
Because of suppression with COT1 DNA, the fluores-
cence intensities were not representative at chromosome
regions with tandem repetitive DNA clusters (i.e., at the
heterochromatic blocks on chromosomes 1, 9, 16, and Y, at
the centromeric regions, and along the short arms of acro-
centric chromosomes). These areas were excluded from
evaluation. Chromosome 19 and the chromosomal segment
1
p34-pter were also excluded from the analysis because
results for 19 and 1p34-pter have been observed to be prone
to artifacts in our and others’ laboratories [36].
Microsatellite Analysis for LOH
DNA from frozen tumor tissues and, if not available, DNA
from paraffin-embedded tumor sections (n = 24) was sepa-
rated using previously published protocols [37,38].
The following regions were examined for allelic losses by
nonradioactive microsatellite analysis: chromosomal arm
Figure 1. Kaplan-Meier estimates for patients with GBM treated with TMZ
and radiotherapy (TMZ; solid lines) versus the untreated control group
(radiotherapy alone; dashed lines) had significantly higher overall survival.
Censored data (patients still alive) are plotted as hash marks.
Neoplasia . Vol. 7, No. 10, 2005

8
86
Deletions of 9p and 10q in Temozolomide Therapy
Wemmert et al.
Table 2. Clinical Characteristics and CGH Results of TMZ-Treated and Conventionally Treated High-Grade Gliomas.
Case
Number
Tumor Age/Sex Chromosomal Imbalances by CGH
ST (months) Gains
Losses
TMZ-treated high-grade gliomas
1
2
1
1
3
782
65
sGBM
GBM
GBM
GBM
sGBM
26/M
46/F
54/M
49/M
37/M
24.2
23.1
33.8
23.8
14.3
19p, X
None
16p13.1p13.3
10q25.3qter, 16p12p13.3
16p, Y
349
099
69
X
7, 12q13.2q13.3
1q, 5p15.1pter, 7q31.3qter, 8q21.1qter, 9p,
1p31.3p32.3, 1q32.1q41, 10q
1p33p36.1, 4q21.1qter, 5q21qter, 10q21.3qter,
11p12p15.3, 13q12.1q31, 15q14q21.2, 19
4p16pter, 10q24.1qter, 13q21.3qter
10q, 13q12.3q22, Y
10p, 12p, 15q25qter, 18p11.2pter
1
1
6
1
1
1
1
4
1
534
106
62
GBM
GBM
GBM
GBM
sGBM
GBM
sGBM
GBM
GBM
62/M
70/M
70/M
56/M
40/F
47/M
39/F
58/F
31/F
18.4
19.8
13.4
19.2
11.2
29.4
17.4
16.9
11.9
7q11.2q21, 19p13.1p13.3, X
7q21.1qter, 12q13.1q14, 19p
3q24q26.3, 7p11.1p12, 7q31.1q34, 18q12.1q21.1, X
7q34qter, 12p13.1pter
4p16, 8p22pter, 10q, 16p11.2p13.2, Y
10q21.3qter, 22q13.1q13.3
2p23pter, 3p24.2p26, 21q21qter
10q21.1qter
326
515
707
691
97
19p
7, 12q13.3q14
None
7p11.2p13, amp 1p36pter
2q34q37.1, 3p26pter, 3q23.2qter, 4p11p16,
1p35p36.1, 4q27q32, 9p21, 9q21.2q32, 14
6q12q21, 9p23p24
3q11.1q25.2, 5p, 13q12.3qter, 16q, 20q11.2qter, Y
460
4
1
q11q13.3, 8p22pter, 9, 15q22.3qter, 18q12.3qter,
9q11q13.1, 20p, 22
896
GBM
51/M
13.0
7
6q22.2qter, 9p21pter, 10p12.2pter, 10q23.1qter,
1
3q13qter, 14, 16q13qter, 22q12.2
1
6
1
405
43
GBM
GBM
sGBM
53/M
54/F
31/M
7.4
15.3
6.9
4q31.3q33
4p16pter, 7p11.1p13, 7q32qter, 16p12pter, 19q, 22
None
8p23.1pter, 16p, 17, 19q13.2qter
10, 13q14.3q22.1
5p15.1pter, 6q16.3qter, 8p23.2pter, 9p13pter
795
1
8q12.1qter
3q13.1q25.3, 9p
T4789
T5958
GBM
GBM
38/M
63/F
44.1
14.4
8p, Xq23q28
2q22q35, 3p11.1p14.1, 3p25pter, 3q13.1q21,
1p32.3pter, 10, 12q23q24.3, 16p, 17p, 17q24qter,
18p11.2, 19, 22, Y
3
5
1
q24q26.3, 4p14p15.3, 4q21.2q34, 5p14,
q13.1q22, 6q14q23.1, 7, 8q22.3, 11p13p15.3,
1q14.1qter, 13q14.1q33, 14q12q22, 20p12pter,
amp 7p11.1p11.2
7p13p22, 7q11.1q22, 19p, X, amp 7p11.1p13
7
84
GBM
GBM
GBM
52/M
53/M
53/M
22.5
11.2
15.1
4p16, 8p22pter, 9p11p13, 9p23pter, 10p13pter,
10q11.2q21.2, 16p
1p21p31.3, 3p, 3q23q26.3, 10, 13, 17p11.1p13, 18, 22
T6002
5, 6, 7, 12p, 12q11q14, 12q24.1qter, 15, 19, 20,
amp 7p11.1p11.2
3p11.1p14.1, 3q11.1q26.3, 7, 9q, 21q21qter, X
1536
1p33p36.1, 4p15.1pter, 10, 12p12.1pter, 12q11q21.3,
1
2
5, 17q12q23, 18p11.1p11.2, 19q13.3qter,
0p11.1p11.2
1
6
940
GBM
GBM
54/F
41/M
17.3
26.2
5p13.3p15.2, 7p15.3pter, 18p
6q12q15, 6q25.1qter, 7, 12q14q21.1, X
8q22.1q24.1, 10, 12q14q23, Xp11.4pter, Xq13q21.3
4p16, 9p11p23, 10, 11q12q14.3, 15q11.1q22.2,
2
3q26.1q36.3, 10, X
0p11.1p11.2, Y
5
9
96
47
GBM
GBM
GBM
GBM
32/M
46/F
45/M
37/M
21.1
17.7
8.6
7, 12q13.3q21.1, 17p11.1p12, 19, Y
7, 15q24q26.1, 18p, 19, 20q
9p21p23, 10q21.1q25.2
1p36pter, 10, 13q13q21.1, 17p12pter, 22q13.1qter, X
6q25.2qter, 7q36
XXL
T6044
7p15.1pter, 7q11.1q31.3, amp 2p22pter
18p11.1p11.2, 19p, X
7.6
Conventionally treated high-grade gliomas
8
1
1
2
61
GBM
GBM
GBM
GBM
70/F
46/M
62/M
39/M
22.7
65.8
5.6
2p21p23, 7p12, 12q14q21.3, 13q21.3q32
13q21.1q32
6q11q14, 7, 19p, X, Y
4p15.3pter, 8p22pter, 16p, 19p, 20q13.1qter
1p34pter, 17q12q21.3, 19q, 22, Y
10, 14, 15, 18
856
028
046
39.8
1p13.3p31.1, 1q25q41, 3p24.1pter, 3p11.1p13,
1p35pter, 10, 11, 12q22qter, 13q12.1q14.1,
13q32qter, 15q21.3qter, 17, 19, 20q11.2qter, 22
3
4
8
2
q11.2q13.3, 3q24q26.3, 4p11p15.2, 4q12q13.3,
q22q28, 5p13.3pter, 5q12q23.3, 6q12q23.1, 7,
p22pter, 9p, 14q11.2q24.1, 18p11.3pter, 20p13,
1q11.1q22.1
T6025
GBM
73/M
6.3
1p34.3p36.1, 1q31q43, 3p14.1p21.1, 7,
2p24pter, 4p15.1p15.3, 5p15.2pter, 8p21.3pter,
8q22.1q24.1, 10, 12q15q21.3, 13q14.3qter, 14,
15q21.3qter, 20p12pter
1
1
6p11.1p13.2, 17p11.1p12, 17q, 18p11.1p11.3,
9p, Y, amp 7p11.1p12
3
93
GBM
GBM
GBM
65/M
61/M
76/F
4.6
3.3
1
1p, 3, 7, 12q11q13.2, 12q23q24.3, 16p, 17q, 19,
0q, 22. amp 7p11.1p12
1p24.1pter, 11q11q14.1, 16p, 18p11.1p11.2, 19,
0q, 22q12.3q13.3, Xp, Xq11.1q21.2
4q32qter, 5p14pter, 5q23.3q35.1, 6p23pter,
1q32.2qter, 9p, 10, 13q22q31, 21q11.1q21
2
T5954
9p13pter, 13, 14q11.1q12, 18q12.1qter
17q11.2q21.3, 19p
2
1
6
819
3
6
1
q22.3qter, 7q33q36, 8q23q24.2, 13q21.3q33,
8q12.2qter
GBM
GBM
GBM
77/F
24/F
57/M
10.9
4.8
7p11.1p12, 7q11.1q11.2
5p, 5q11.2q23.1, 6p23pter, 6q15q22.3, 9p23pter,
1
8p11.3pter
3p25pter, 3q26.3qter, 6p23pter, 8q22.1qter,
p21pter, 11q23.3qter, 13q21.3q31, 20q13.2qter
3p, 9p13p24, 9q22.1q31, 10, 21q22
T6052
32
5, 7, 12, 18, 19p, amp 12p13.1, 18q11.1q11.2
4, 5, 7, 8q22.1q24.3, 14q11.1q12, 14q24.3q31,
9
8
8.9
17q24q25, 19, 20, 22, X
(continued on next page)
Neoplasia . Vol. 7, No. 10, 2005

Deletions of 9p and 10q in Temozolomide Therapy
Wemmert et al.
887
Table 2. (continued )
Case
Number
Tumor
Age/Sex
Chromosomal Imbalances by CGH
ST (months)
Gains
Losses
Conventionally treated high-grade gliomas
GBM
T4795
66/M
0.2
4p15.1p16, 6q24q27, 7p15.1p22, 18q12.3q23,
10, 11p, 11q11q23.1, 13, 14, 19, 22, X, Y
20p13
838
T4803
GBM
GBM
56/w
59/M
1.3
9.4
7, 16, 17q12q21.3, 19, X, amp 7p11.2p12
1p32.1p36.3, 7, 9q32q34.2, 12q13.1q14,
9p21p24, 10, 13, 14, 22
10, 14
15q22.1q24, 17, 20q11.1q13.1
H549
GBM
38/M
5.5
3q21q23, 3q26.2qter, 7q21.3q31.1,
amp 12q11q14
19p, 20q11.2q13.1, X
6q21q23.3, 10q, 11p, 11q11, 13q12.1qter, 14
H321
H147
H281
N111
sGBM
GBM
GBM
48/M
48/M
56/M
24/M
18.2
7.5
5p15.2pter, 9p21pter, 10q, Y
10p13pter, 10q11.2q21.3
None
7q11.1q11.2, 19, 20q11.1q13.1
7p11.1p12
1p32pter, 11q13, 17, 19, 20, 22
12.5
17.4
sGBM
6q16q23
sGBM, secondary glioblastoma multiforme; M, male; F, female; ST, survival time; amp, amplification.
In the univariate Cox proportional hazards model, for a
given genetic status, the main effect of therapy on survival
was tested. In the first multivariate Cox model, for a given
genetic status, main effects of therapy, age, gender, and KPS
were estimated. In the second multivariate Cox model, main
effects of therapy and age and an interaction effect between
therapy and age were estimated. All effects were quantified
by hazard ratio estimates with 95% confidence intervals
Genetic status, treatment, gender, and age Clinical data,
CGH, and LOH results of patients in the TMZ and control
groups are summarized in Tables 1–3. Both groups showed
a similar genetic pattern. The most frequent genetic aber-
rations were complete or partial losses of chromosome
10 (63%) and complete or partial gains of chromosome
7 (58%). Further deletions were detectable on 9p (29%)
and 13q (35%) (Figure 2).
(
CIs). All P values were calculated with two-sided tests.
Subgroups of patients were defined by their genetic
pattern. In the univariate model, patients with deletions of
9p and 10q, respectively, did associate with better prognosis
under treatment (Figure 3; Table 4). No correlation with
treatment was observed for gains of chromosomes 7p and
12, losses of chromosome 13, and gender. Due to the me-
thodical limitations of LOH, only deletions could be detected.
A positive effect on survival was observed for better KPS and
younger age (Table 5).
Median survival rates were calculated using the Kaplan-
Meier method. Association between dichotomous variables
was tested by chi-square analysis.
LOH data were summarized per chromosomal arm. LOH
was defined as present if at least one loss on the respective
chromosomal arm was detected. For chromosome arm 10q
with a large number of primer, LOH was defined as present
if the majority of the considered regions were lost.
In the multivariate Cox proportional hazards models, for
the genetic subgroups, the main effects of TMZ treatment,
gender, KPS, and age, as well as an interaction effect be-
tween treatment and age, were estimated. Patients with de-
letions of 9p showed a positive correlation between TMZ
chemotherapy and survival also in multivariate analysis,
whereas gender, age, and the interaction of age and treat-
ment had no effect (Table 6). Patients with deletions on 10q
and higher age had a poorer prognosis (P = .0076). However,
the negative effect of the genetic status and age was com-
pensated by TMZ treatment (P = .0042) (Table 7).
Results
Univariate and Multivariate Prognosis Analyses
Treatment, gender, and age In total, 60 patients had died
and 20 were alive during the last follow-up (20 censored
data). Median follow-up was 17.2 months and median age at
diagnosis was 54 years.
Median overall survival for the TMZ group (n = 50; median
age, 53 years) was 19.5 months and for the control group
(
n = 30; median age, 58 years) was 9.3 months. Univariate
LOH analysis of the CDKN2A region, as well as LOH on
chromosome arm 10q and the MGMT region, respectively,
produced results similar to that of CGH (Tables 6 and 7;
Figures 4 and 5). The only difference was the association of
high KPS and better prognosis in patients with losses of
9p and 10q, in contrast to the corresponding CGH data.
analysis showed that TMZ chemotherapy was significantly
associated with longer survival rates (P = .00063; Figure 1).
Multivariate analysis indicated that the strongest factors
associated with survival were chemotherapy and age.
Younger patients survived longer than older patients
(
> 53 years of age) in the control group (P = .024). However,
for older patients, a prolonged survival time under adjuvant
chemotherapy treatment was detectable (P = .00014), but not
for younger patients (P = .96). There was no significant
difference in survival time by age in the TMZ-treated
group (P = .83).
Discussion
Today, the aim of chemotherapy in patients with glio-
blastomas is palliation and improvement of health-related
quality of life (HQL), as well as prevention of neurologic
Neoplasia . Vol. 7, No. 10, 2005

8
88
Deletions of 9p and 10q in Temozolomide Therapy
Wemmert et al.
Table 3. LOH Results and Comparison with CGH Data of TMZ-Treated and Conventionally Treated High-Grade Gliomas.
y
y
y
y
p16 LOH/Inf
Tumor
ST (months)
CGH 10q*
PTEN LOH/Inf
MGMT LOH/Inf
CGH 13q*
RB LOH/Inf
CGH 9p*
TMZ-treated high-grade gliomas
1
1
795
534
6.9
40.9
55.3
39.9
19.2
11.9
35.3
20.2
11.2
19.8
16.9
13.4
24.2
56.8
23.8
29.4
21.1
14.4
51.6
15.1
12.0
17.7
8.6
0
1
0
0
1
0
1
1
0
1
0
1
0
0
1
1
1
2/2
1/4
0/2
1/4
1/4
0/5
3/4
3/3
1/1
1/4
1/3
2/2
3/3
0/5
4/4
5/5
3/3
1/4
0/5
4/4
0/0
5/5
1/1
1/1
5/5
0/5
2/2
1/2
1/1
0/1
1/1
0/1
5/5
0/4
2/3
6/6
4/4
6/6
4/4
0/5
0/1
3/3
1/2
0
0
0
0
0
1
1
1
0
1
0
0
0
0
0
0
0
0/3
0/2
0/2
0/2
0/3
2/2
0/3
2/2
1
0
1
1
0
0
0
0
0
0
1
0
0
0
0
0
0
3/3
0/2
1/1
2/2
0/2
0/2
3/3
0/1
1/1
0/2
2/4
T4789
1
1
1
6
3
1
1
4
6
1
1
1
1
5
3
1
1
9
9
691
326
460
43
0/2
1/5
69
3/4
515
106
97
2/2
2/5
2/6
4/5
0/1
0/2
0/3
0/2
0/3
0/1
0/1
0/1
0/1
0/3
1/1
0/1
0/3
62
0/3
2/2
0/2
1/2
1/2
0/3
2/3
0/1
0/1
782
349
099
707
96
4/6
3/4
4/4
98
777
536
0
1
4/4
4/5
0
0
47
1
1
1
1
0
1
0
0
1
0
0
0
2/3
0/2
XXL
4/5
7
1
84
921
22.4
3.4
0/2
0/2
0/3
0/1
0/1
0/1
0/2
0/1
1/2
0/3
N529/02
N20/02
21.4
24.8
18.6
13.5
13.6
24.5
9.0
4/4
N690/02
N1082/02
N1421/01
N934/02
N1124/01
N1507/01
N363/02
N449/03
N1443/02
N528/03
N438/03
N202/03
N412/03
N759/01
N193/03
N1618/02
1/2
0/1
16.0
18.9
8.2
0/3
0/1
2/4
0/3
1/3
2/3
2/2
1/2
0/2
1/2
0/2
0/2
0/2
0/1
0/4
3/3
15.1
6.7
0/4
0/3
0/2
0/3
1/3
1/2
8.9
10.8
9.0
2/2
31.5
11.0
10.3
0/3
3/3
1/2
Conventionally treated high-grade gliomas
1
1
1
2
6
856
394
819
046
3
58.5
0.5
0
0/3
5/5
0/4
4/4
0/6
3/3
0/4
0/3
4/4
0/5
2/4
1/3
3/4
3/3
4/4
1/3
2/2
0/2
0/3
1/2
0/2
3/3
0
0/1
2/2
0/3
0
0/3
3/3
0/2
0/1
0/1
0/3
0/3
1/2
2/2
0/2
2/2
0/2
2/3
2/3
1/1
1/1
1/1
0/2
0/3
2/4
2/5
1.0
0
1
0
1
0
1
1
1
0
1
0
0
0
0
1
0
0
0
1
0
0
1
1
0
39.8
10.9
9.4
T4803
N111
0/3
0/1
0/2
0/4
0/3
0/1
1/2
0/2
0/1
1/1
0/2
0/1
0/2
0/3
17.4
18.2
4.6
2
3
1
064
93
3/3
4/4
028
5.6
5.8
N28/99
2/6
3/4
N1106/00
N113/99
N600/98
N1047/00
N982/00
N823/01
N894/00
N860/01
N1320/99
N231/00
N358/00
39.0
9.5
16.7
0.7
5/5
14.4
14.4
21.3
21.3
4.7
1/2
2/7
12.4
11.2
1/2
0/2
1/1
*
1, deletion; 0, no deletion determined by CGH.
y
LOH at available informative markers.
Neoplasia . Vol. 7, No. 10, 2005

Deletions of 9p and 10q in Temozolomide Therapy
Wemmert et al.
889
deterioration. Multimodal therapy approaches had limited
success in the treatment of patients with malignant glioma
12.1 months, respectively [43,44], whereas patients with
TMZ chemotherapy and radiotherapy had an overall sur-
vival of 13.4 to 16 months [23,43,44]. The survival time of
our TMZ-treated group is better than that reported in the
papers before, but is in line with the study of Hegi et al. [28].
They reported a median survival of 21.7 months for patients
treated with radiation and chemotherapy when defining sub-
groups according to the MGMT promoter methylation status.
Several clinical and histopathologic features are known
to be of prognostic significance for survival in patients with
glioblastomas. Favorable features include young age and
[
40–42]. The present studies demonstrate that TMZ chemo-
therapy after prior radiotherapy significantly prolongs survival
among patients with newly diagnosed glioblastomas. Their
median overall survival time was 19.5 months, compared to
9
.3 months of patients with radiotherapy alone. Due to the
retrospective nature of our study, patient selection cannot
be excluded. However, the outcome of patients with radio-
therapy alone in our study compares to the outcome in the
literature. These patients had an overall survival of 7.7 and
Figure 2. Overview of genetic imbalances of (A) TMZ-treated GBMs and (B) conventionally treated GBMs. Lines on the left represent losses, and lines on the right
represent gains; amplifications are in bold.
Neoplasia . Vol. 7, No. 10, 2005

8
90
Deletions of 9p and 10q in Temozolomide Therapy
Wemmert et al.
Table 5. Univariate Analysis of Effects of Predictors on Overall Survival.
Predictor
Hazard (95% CI)
P Value (Two-Sided)
TMZ
Sex
Age
KPS
0.409 (0.245 –0.683)
1.03 (0.589–1.79)
1.03 (1.00–1.05)
.00063
.93
.017
0.955 (0.923 –0.988)
.0074
elderly patients, may be caused by the presence of con-
comitant diseases [45]. For gliomas, to our knowledge, there
are no appropriate results concerning this matter.
In our present study, young age also correlated with
better prognosis, but interestingly, the negative impact of
age on survival was compensated by the TMZ treatment.
Recent studies suggest that there are genetic subtypes
of diffuse gliomas associated with survival time [3,29], and
that their clinical course as well as their response to radia-
tion and chemotherapy are primarily determined by the bio-
logic behavior of the tumor cells. Attention has already been
drawn to gliomas with LOH on 1p and 19q. These alterations
are typical for oligodendroglial tumors [46–49] and have
been proposed to be a powerful independent factor for
prolonged survival time and favorable response to chemo-
therapy in WHO grade III anaplastic oligodendrogliomas
[
35,50]. These genomic alterations are also observed in
GBM, however, at lower frequencies, with approximately
0% for LOH 1p and up to 30% for LOH 19q [19,51], but in-
1
dicating better survival [19].
Uniform numerical and/or structural alterations affecting
chromosomes 7, 9, 10, 12, and 13 represent the most com-
mon genetic abnormalities in high-grade gliomas [11–13,51]
despite genetic heterogeneity [52,53]. Several investigators
have noted that LOH on 9p and 10q is associated with shorter
survival time in these tumors [3,17–19]. The candidate genes
CDKN2A and CDKN2B on the short arm of chromosome 9,
and PTEN and DMBT1 on 10q are discussed to influence
survival time. LOH around PTEN has been associated with
Table 6. Subgroup Analysis of Differential TMZ, Age, Gender, and KPS
Effects Depending on Genetic Aberration with Respect to Survival Assessed
by Cox Proportional Hazard Regression.
Figure 3. Kaplan-Meier estimates of overall survival, according to genetic
alteration and assignment to TMZ and radiotherapy or radiotherapy alone.
(
A) Survival curves for patients with loss of 9p (thick lines) and without loss of
p (thin lines). (B) Survival curves for patients with loss of 10q (thick lines)
9
and without loss of 10q (thin lines), determined by CGH. The Kaplan-Meier
estimates for overall survival indicate that the patients with losses of 9p and
Alteration Variable
Hazard Ratio CGH
and LOH (95% CI)
P Value CGH
and LOH (Two-Sided)
10q had significantly longer overall survival rates under TMZ treatment.
Loss 9p
TMZ
0.065 (0.0043–0.98) .048
0
.12 (0.031 –0.47)
.0024
.64
.95
Age (> 53 years)
Gender
1.63 (0.21 –12.76)
1.04 (0.33 –3.25)
0.81 (0.23 –2.87)
good KPS [2,7,19]. For lymphomas, it was already suggested
that increased perioperative and postoperative morbidity
and mortality, as well as diminished tolerance to therapy of
.75
.47
0
.61 (0.16 –2.34)
KPS (z 90)
1.32 (0.21 –8.30)
.086 (0.017–0.43)
0.32 (0.13 –0.80)
0.74 (0.30 –1.85)
1.20 (0.46 –3.17)
.76
0
.003
.014
.52
Loss 10q TMZ
Age (> 53 years)
Table 4. Univariate Analysis of the TMZ Effect on Overall Survival.
.71
2
.39 (1.00 –5.73)
0.77 (0.21 –2.87)
.46 (0.13 –1.61)
0.41 (0.075 –2.28)
.051
.77
Alteration
Hazard Ratio CGH
and LOH (95% CI)
P Value CGH and
LOH (Two-Sided)
Gender
0
.22
.31
KPS (z 90)
Loss 9p
0.55 (0.0064–0.48)
.0085
.0015
.0071
.27
0
.028 (0.0048–0.16) .000068
0
.16 (0.053–0.5)
0.31 (0.134–0.729)
.63 (0.28–1.43)
Loss 10q
In the multivariate Cox model, all predictors are included and only main
effects are estimated.
0
Neoplasia . Vol. 7, No. 10, 2005

Deletions of 9p and 10q in Temozolomide Therapy
Wemmert et al.
891
Table 7. Subgroup Analysis of Differential TMZ and Age Effects Depending
on Genetic Aberration with Respect to Survival Assessed by Cox Proportional
Hazard Regression.
Alteration/ Variable
Normal
Hazard Ratio CGH
and LOH (95% CI)
P Value CGH and
LOH (Two-Sided)
Loss 9p
TMZ
0.037 (0.0017–0.78) .034
0
.49 (0.054–4.51)
0.81 (0.081–8.09)
.31 (0.47–39.36)
.53
.86
.20
.45
.23
.79
.10
.0076
Age (> 53 years)
TMZ ꢁ age
4
3.57 (0.13–99.15)
0.20 (0.015–2.71)
0.85 (0.25–2.91)
(
> 53 years)
Loss 10q
TMZ
5.64 (0.71–44.57)
Age (> 53 years) 10.47 (1.87–58.65)
33.56 (3.67–307.35) .0019
0.040 (0.0044–0.36) .0042
0.017 (0.0014–0.21) .0015
TMZ ꢁ age
> 53 years)
(
shorter survival time [3,18]; however, mutations of PTEN did
not affect prognosis for survival [20,34].
Esteller et al. [25] were able to show that aberrant MGMT
promoter hypermethylation was associated with loss of
MGMT protein, in contrast to retention of protein in the ma-
jority of tumors without aberrant hypermethylation. Recent
studies suggested that methylation of the MGMT promoter
is predictive for good outcome in patients with glioblastomas
treated with alkylating agents [24,26–28]. Our data support
the results mentioned above because MGMT is included in
the deleted regions on 10q. The results for our control group
showed that deletions on chromosome 10 indicate a trend
to shorter survival. The same results are observed for LOH of
MGMT, whereas LOH of PTEN showed no association with
survival as reported before [17,20]. Interestingly, patients
with deletion on 10q benefited significantly from adjuvant
chemotherapy in univariate analysis.
Figure 5. Microsatellite analysis for LOH on chromosome 10.
Therefore, deletion as well as inactivation by hypermeth-
ylation of MGMT will predict responses to alkylating chemo-
therapy, probably in a dose-related manner. Further on,
multivariate analysis showed that patients benefited signifi-
cantly from the therapy especially if they belonged to the
older age group (Table 7). Hence, our data suggest that the
negative effect of age was compensated by TMZ treatment.
Homozygous deletion of CDKN2A gene mapping on chro-
mosome 9p21 has been reported as an indicator of poor
prognosis and resistance to chemotherapy for patients with an-
aplastic oligodendroglioma. However, homozygous CDKN2A
losses were observed exclusively in tumors without LOH
on 1p [35]. In glioblastomas with oligodendroglial compo-
nents, similar results were found [54,55].
An association of tumor necrosis and microvascular pro-
liferation with 9p deletion and CDKN2A alterations was
observed in oligodendrogliomas. The higher vascularization
of tumors harboring 9p deletion may explain the reasons for
better response to chemotherapy [56]. This may also hold
true for glioblastomas. However, this effect has never been
proven in trials.
To our knowledge, this is the first report demonstrating an
effect of 9p deletion on chemotherapy response in glioblas-
tomas. The unfavorable prognostic factor of this alteration
[
15,17,33,34,57] was confirmed by CGH and LOH analyses
in our control group. Further on, the effectivity of TMZ is
Figure 4. Microsatellite analysis for LOH on chromosome arm 9p.
Neoplasia . Vol. 7, No. 10, 2005
enhanced by deletion of 9p by CGH and LOH of CDKN2A,

8
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Deletions of 9p and 10q in Temozolomide Therapy
Wemmert et al.
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9
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Acknowledgements
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