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Association of chromosome 10 losses and negative prognosis in oligoastrocytomas.

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19. Southard-Smith EM, Angrist M, Ellison JS, et al. The
Sox10Dom mouse: modeling the genetic variation of
Waardenburg-Shah (WS4) syndrome. Genome Res 1999;9:
215–225.
20. Maquat LE, Carmichael GG. Quality control of mRNA function. Cell 2001;104:173–176.
Association of Chromosome
10 Losses and Negative
Prognosis in
Oligoastrocytomas
Lorena Bissola, PhD,1 Marica Eoli, MD,2
Bianca Pollo, MD,2 Bianca Maria Merciai, PhD,3
Antonio Silvani, MD,2 Ettore Salsano, MS,1
Carmelo Maccagnano, MD,1
Maria Grazia Bruzzone, MD,1
Anna Maria Fuhrman Conti, PhD,3
Carlo Lazzaro Solero, MD,4 Sergio Giombini, MD,4
Giovanni Broggi, MD,4 Amerigo Boiardi, MD,2
and Gaetano Finocchiaro, MD1
Oligoastrocytomas are mixed gliomas harboring different
genetic alterations and with heterogeneous clinical evolution. We have looked for correlations between genetic
losses and clinical evolution in 34 oligoastrocytomas. Loss
of heterozygosity (LOH) with different microsatellite
markers was studied on chromosomes 1p, 10q, 17p, and
19q. LOH on 1p was found in 44% of the tumors, on 10q
in 24%, on 17p in 18%, and on 19q in 38%. LOH on 1p
and 19q was combined in 29% of the patients. LOH on
1p was associated with significantly longer overall survival
(p ⴝ 0.0092) and LOH on 10q with shorter overall survival (p ⴝ 0.0206). The observation that LOH on 10q predicts a short survival in oligoastrocytomas is novel and
provides further evidence that genetic analysis may help to
predict the clinical evolution of different gliomas, giving a
more rationale basis to therapeutic options.
Ann Neurol 2002;52:842– 845
Oligoastrocytomas are composed of a mixture of neoplastic cells resembling atypical astrocytes and oligodendrocytes. The pathological criteria for tumoral diagnosis are not univocal. Genetic alterations of these
mixed gliomas include loss of heterozygosity of chromosomes 1p and/or 19q, typical of oligodendrogliomas, and mutations of the p53 tumor suppressor gene,
located on chromosome 17p, frequent in astrocytomas.1– 6
Oligoastrocytomas may respond to chemotherapy
but less frequently than oligodendrogliomas.7–9 Indeed
oligoastrocytomas are heterogeneous for their clinical
evolution, and this often makes their clinical management uncertain. Thus, there is hope that a wider
knowledge of their genetic alterations may help to
characterize these tumors thoroughly, giving a more
rational basis to their treatment. With this aim, we
have examined 34 oligoastrocytomas for allelic losses
on chromosomes 1p, 19q, 17p, and 10q (where
PTEN and possibly other tumor suppressor genes are
located10), and we have correlated our findings with
their clinical evolution.
The results show that significant correlations exist
and stress the positive and the negative role that chromosome 1p and 10q losses, respectively, may play as
prognostic factors.
Patients and Methods
Tumor Specimens and DNA Extraction
Tumor and corresponding blood samples were obtained
from 34 patients treated at the Istituto Nazionale Neurologico “C. Besta,” Milano, between 1992 and 2000. Tumor
classification, in agreement with World Health Organization
(WHO) guidelines,11 was performed by two neuropathologists of our institution. To make the diagnosis, we identified
at least 25% of the neoplastic cells as astroglial or oligodendroglial. Tumors included 9 oligoastrocytomas (OAs, WHO
grade II) and 25 anaplastic oligoastrocytomas (AnOAs,
WHO grade III). DNA was extracted from frozen tissues
(n ⫽ 30) or from paraffin-embedded material (n ⫽ 4) using
established methods.12 Control DNA was extracted from
blood lymphocytes.
Clinical Parameters
1
From the Departments of Experimental Neurology and Diagnostics and 2Clinical Neurology, Istituto Nazionale Neurologico Besta;
3
Department of Biology and Genetics for Medical Sciences, University of Milan; and 4Department of Neurosurgery, Istituto Nazionale
Neurologico Besta, Milan, Italy.
Received Jun 14, 2002, and in revised form Aug 23. Accepted for
publication Aug 23, 2002.
Address correspondence to Dr Finocchiaro, Unit of Neuro-Oncology
and Gene Therapy, Istituto Nazionale Neurologico Besta, Via Celoria
11, 20133 Milano, Italy. E-mail: gaetano.finocchiaro@libero.it
842
© 2002 Wiley-Liss, Inc.
Clinical data included age, gender, date of surgical resection
and pathology of the specimen, extent of surgery (assessed by
comparison of preoperative computed tomography/nuclear
magnetic resonance with scans taken within 24 hours after
surgery and 2 months later), Karnofsky performance score
(assessed on the seventh day after surgery), treatment (chemotherapy and/or radiation therapy), time of tumor progression (defined as the time of the first sign of radiological progression13), time of the last follow-up, and status of the
patient (living/deceased) at the time of the last follow-up.
Clinical and neuroradiological controls were performed every
6 months at our institution. Because many patients were
alive at the last follow-up, time to tumor progression (TTP)
6-carboxyfluorescein (6-FAM), 2⬘,7⬘,4,7 tetrachlorofluorescein (TET), or 4,7,2⬘,4⬘,5⬘,7⬘ hexachlorofluorescein (HEX)
fluorochromes. Polymerase chain reaction products were analyzed on an ABI Prism 377 (Applied Biosystems, PerkinElmer). After electrophoresis, data were collected with the
Gene Scan program for fragment analysis (ABI Prism; Perkin
Elmer, Applied Biosystems, Foster City, CA).
Allelic imbalance was evaluated by comparing polymerase
chain reaction products from tumor and normal DNA in
neighboring lanes. The ratios of the peak heights of the two
alleles was calculated in blood (N1/N2) and tumor (T1/T2)
DNA. Allelic imbalance resulted from the ratio of normal to
tumor signal (N1/N2 over T1/T2). LOH values less than or
equal to 0.5 indicate the loss of the longer allele in the tumor
whereas LOH values greater than or equal to 1.5 indicate the
loss of the shorter allele.
Statistical Analysis
The associations between molecular genetic data and the histological subtyping were assessed using the ␹2 test. Because
in five patients genetic data were available only from surgery
Fig 1. (top) Oligoastrocytoma showing distinct components
with oligodendroglial and astrocytic differentiation (H & E,
⫻40). (bottom left) Oligodendroglioma area of the same tumor, displaying the typical cells with clear cytoplasm and a
network of branching capillaries (H & E, ⫻100). (bottom
right) Same tumor with area of anaplastic astrocytoma, showing high cellularity, nuclear atypia, and marked mitotic activity (H & E, ⫻100).
Fig 2. (A) Kaplan–Meier analysis showing overall survival of
patients with loss of heterozygosity (LOH) on 1p versus others.
(B) Kaplan–Meier analysis showing overall survival of patients
with LOH on 10q versus others. (C) Kaplan–Meier analysis
showing overall survival of patients with grade III tumors and
LOH on 1p versus others. (D) Kaplan–Meier analysis showing
overall survival of patients with grade III tumors and LOH
on 10q versus others. (E) Kaplan–Meier analysis showing
overall survival of patients with grade II tumors and LOH on
1p versus others.
was considered in addition to the survival time (ST) to study
their prognosis.
The 34 patients (20 men and 14 women) had a mean age
of 40 years at the diagnosis (range, 20 – 68 years). Most of
them (18 AnOA and 4 OA) were treated by chemotherapy
with procarbazine-cyclohexylchloroethylnitrosurea-vincristine
(PCV) or bischloroethylnitrosourea and cisplatin and radiotherapy after the first surgery. At recurrence, all patients received PCV chemotherapy and radiotherapy, if not performed before.
Microsatellite Analysis for Loss of Heterozygosity on
Chromosomes 1p, 19q, 10q, 17p
To identify allelic losses, we used these microsatellite markers: D1S508, D1S2743, D1S457 on chromosomes 1p36.3,
1p32, and 1p13, respectively; D19S219, D19S412 on chromosome 19q13.3; D10S562, D10S212 on chromosomes
10q22 and 10q26, respectively; and D17S1353 on chromosome 17p13 (close to TP53 gene) and D17S520 on chromosome 17p12.
One of the primer pairs was 5⬘end-labeled with
Bissola et al: Chromosome 10 in Mixed Gliomas
843
Table. Statistical Analysis of Clinical and Genetic Parameters
in 34 Patients with Grade II or Grade III Oligoastrocytomas
Characteristic
PFS ( p)
ST ( p)
Univariate analysis
Age (ⱕ40 vs ⬎40 yr)
0.38
0.60
WHO II vs III
0.50
0.30
LOH 1p
0.01
0.01
LOH 19q
0.22
0.38
LOH 1p and 19q
0.27
0.27
LOH 10q
0.04
0.02
LOH 17p
0.90
0.85
KPS (ⱕ80 vs ⬎80)
0.50
0.20
Entity of surgery
0.20
0.05
CHT ⫹ RT vs no treatment
0.13
0.20
Multivariate analysis
LOH 10q 0.030 (RR, 0.315;95% CI, 0.111–0.895) for
PFS
LOH 10q 0.017 (RR, 0.222;95% CI, 0.064–0.764) for
ST
LOH 1p 0.014 (RR, 4.220;95% CI, 1.323–13.470) for
PFS
LOH 1p 0.017 (RR, 13.112;95% CI, 1.566–109.782)
for ST
Entity of surgery 0.035 (RR, 3.887;95% CI, 1.101–
13.721) for ST
PFS ⫽ progression-free survival; ST ⫽ survival time; WHO ⫽
World Health Organization; LOH ⫽ loss of heterozygosity; KPS ⫽
Karnotsky performance score; CHT ⫽ chemotherapy; RT ⫽ radiotherapy; RR ⫽ relative risk; CI ⫽ confidence interval.
on a tumor relapse, progression-free survival and survival
time were analyzed in only 29 specimens obtained from the
first surgery.
Progression-free survival was defined as the time from surgery to the first sign of radiological progression.13 ST was
defined as the time between tissue acquisition and patient
death. Survival distribution was estimated by Kaplan–Meier
analysis and compared among patient subsets with log-rank
tests. The following variables were investigated: age (younger
than 40 or 40 years or older), histological grade (II and III),
chemotherapy and radiotherapy versus no treatment, LOH
on chromosomes 1p, 19q, 17p, and 10q, entity of surgery
(total/subtotal vs partial resection), Karnofsky performance
score (⬍80 vs ⱖ80). A multivariate analysis and a Cox proportional hazards regression model analysis were performed
on these variables to investigate their independent prognostic
role. The statistical analysis was performed using software
StatView 4.5 (Abacus Concepts in Berkeley, CA).
Results and Discussion
In all tumors, at least one marker for chromosome was
informative. LOH was detected in 30 of 34 patients
(88%). Allelic losses on 1p occurred in 15 of 34 (44%;
OA: 3/9, AnOA: 12/25), and allelic losses on 19q in
13 of 34 (38%; OA: 4/9, AnOA: 9/25). LOH on 10q
was seen in 8 of 34 tumors, all anaplastic (0/9 OA and
8/25 AnOA; p ⫽ 0.037). LOH on 17p was found in 6
of 34 patients (18%; 2/9 OA and 4/25 AnOA). Consistently with previous reports3,5 genetic losses on 1p
844
Annals of Neurology
Vol 52
No 6
December 2002
and 19q were associated in 10 of 34 patients (29%;
OA: 2/9, AnOA: 8/25; p ⫽ 0.005). In two other patients, LOH on 10q was associated with LOH on 17p
or LOH on 19q, respectively. In the remaining tumors,
the genetic alterations were mutually exclusive.
Half of the tumors were located in the frontal lobe
(n ⫽ 17), eight in the temporal lobe, seven in the temporal lobe plus another lobe (mixed temporal), one in
the parietal lobe, and one in the parietal and frontal
lobes. In agreement with recent observations,14,15 only
4 of 17 tumors with 1p and/or 19q LOH were in the
temporal lobe ( p ⫽ 0.0111, ␹2 test). Tumors with 10q
and/or 17p LOH were more frequent in the temporal
lobes ( p ⫽ 0.0730, ␹2 test). Patients with temporal
tumors were older than the others (44.4 vs 37.2 years).
Histologically, tumors were either biphasic (n ⫽ 14)
or mixed (n ⫽ 20). Of tumors with 1p and/or 19q
losses (n ⫽ 17), nine were biphasic and eight were
mixed. The oligodendroglial component was prevalent
in 10 of 17 patients and the astroglial component was
prevalent in 3 patients, and in 4 patients there was
equilibrium. Of tumors with 10q and/or 17p losses
(n ⫽ 13), eight were mixed and five were biphasic.
The astroglial component prevailed in 6 of 13 patients
and the oligodendroglial in 3 patients, and in 4 patients there was equilibrium. One representative tumor,
with 10q LOH, is shown in Figure 1.
All patients had a minimum follow-up of 15
months. The loss of 1p was a statistically significant
predictor of longer progression-free survival and overall
survival (log-rank, p ⫽ 0.0136 and p ⫽ 0.0092, respectively; Fig 2A), whereas loss of chromosome 10q
was associated significantly with a shorter progressionfree and overall survival (log-rank, p ⫽ 0.0441 and
p ⫽ 0.0206, respectively; see Fig 2B). Smith and colleagues did not find such association.8 The difference
may be explained by the lower number of patients who
they examined and by heterogeneity of the histological
diagnosis, which may affect genetic findings, such as
18q loss 14 of 19 (74%) for Smith and colleagues and
13 of 34 (38%) in this report.
1p losses maintained a prognostic significance even if
only grade III tumors were considered ( p ⫽ 0.006 and
p ⫽ 0.0450 for TTP and ST, respectively, of patients
LOH of 1p vs the others; see Fig 2C). The same was
true for 10q losses ( p ⫽ 0.012 and p ⫽ 0.0370 for
TTP and ST, respectively, of patients LOH of 10q vs
the others; see Fig 2D). A trend toward prolonged survival of patients with 1p LOH was also present among
grade II tumors (see Fig 2E). As mentioned before, 10q
LOH was not found in grade II tumors.
To assess correlations between genetic data and the
treatment, we considered only 22 patients who were
treated homogeneously. Their radiological follow-up
was based on magnetic resonance imaging (n ⫽ 20) or
computed tomography scan (n ⫽ 2). In 18 of 22 pa-
tients, the tumor was stable during the treatment (ie,
for approximately 8 months), 1 of 22 regressed partially and 3 of 22 progressed. LOH on 1p and/or 19q
was found, 16 of 19 tumors with no progression and 0
of 3 with progression ( p ⫽ 0.0023, ␹2 test). In agreement with the radiological data, the TTP of patients
with 1p and/or 19q losses was longer than the others
( p ⫽ 0.0171). In the 12 patients who were untreated
or partially treated, TTP differences between these
groups were not significant.
Among the clinical parameters that may affect survival the entity of surgery (ie, gross total vs incomplete
resection of the mass) influenced the prognosis (logrank test, p ⫽ 0.05), as also shown previously.16,17
When this variable was entered into a multivariate Cox
analysis, however, LOH on 1p and 10q remained independent prognostic factors (Table).
Overall, the data indicate that the study of molecular
genetic alterations should be included in the management of oligoastrocytomas, because it may give a relevant prognostic information complementing the histopathological analysis and helping to define a rationale
treatment of these tumors.
This work was supported by a grant from the Associazione Italiana
per la Ricerca sul Cancro (G.F.).
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N-Acetylglutamate Synthase
Deficiency and the
Treatment of
Hyperammonemic
Encephalopathy
Orly Elpeleg, MD,1 Avraham Shaag, MSc,1
Efrat Ben-Shalom, MD,1 Tal Schmid, MD,1
and Claude Bachmann, MD2
Carbamylphosphate synthase is the first enzymatic reaction of the urea cycle. Its activator, N-acetylglutamate, is
synthesized from acetyl-CoA and glutamate in a reaction
catalyzed by N-acetylglutamate synthase (NAGS). We
have identified the putative human NAGS gene and re-
From the 1The Metabolic Disease Unit, Shaare-Zedek Medical
Center, Faculty of Medicine, Hebrew University, Jerusalem, Israel;
and 2Central Clinical Chemistry Laboratory, University Hospital,
Lausanne, Switzerland.
Received Jun 14, 2002, and in revised form Jul 23 and Aug 27.
Accepted for publication Aug 27, 2002.
Published online Nov 22, 2002, in Wiley InterScience
(www.interscience.wiley.com). DOI: 10.1002/ana.10406
Address correspondence to Dr Elpeleg, Metabolic Disease Unit,
Shaare-Zedek Medical Center, Jerusalem 91031, Israel. E-mail:
elpeleg@cc.huji.ac.il
Elpeleg et al: N-Acetylglutamate Synthase Deficiency
845
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