Fluorescent In Situ Hybridization on Isolated Tumor Cell Nuclei: A Sensitive Method for 1p and 19q Deletion Analysis in Paraffin-Embedded Oligodendroglial Tumor Specimens

Article (PDF Available)inModern Pathology 16(7):708-15 · August 2003with26 Reads
DOI: 10.1097/01.MP.0000076981.90281.BF · Source: PubMed
Abstract
In oligodendroglial neoplasms, losses of chromosomal material at 1p and 19q associate with chemosensitivity and prolonged survival. Thus, 1p/19q testing is increasingly proposed for use in brain tumor diagnosis and prognostic assessment. Fluorescent in situ hybridization (FISH) is a classic technique for investigation of 1p/19q status in paraffin-embedded tissues. A major limitation of this method is truncation of tumor cell nuclei complicating assessment of hybridization results. In our study, we analyzed 1p and 19q status in a series of 79 oligodendroglial neoplasms (49 oligodendrogliomas, 30 oligoastrocytomas, WHO: 57 Grade II, 22 Grade III tumors) and controls (gliotic brain tissue: n = 4, diffuse low-grade astrocytoma: n = 4) using FISH on isolated whole tumor cell nuclei, prepared as cytospin preparations, thus bypassing the problem of nuclear truncation. For interpretation of FISH results, we used consensus criteria as defined by the SIOP-Europe Neuroblastoma Study Group for analysis of peripheral neuroblastic tumors. FISH yielded interpretable results in 98.7% for 1p and 92.1% for 19q. Chromosome 1p/19q alterations comprised deletions (1p: 79.5%, 19q: 80%) and imbalances (1p: 11.5%, 19q: 12.9%). 1p aberrations were more frequent in oligodendroglioma than in oligoastrocytoma (100% versus 75.9%, P =.001). The frequency of 1p/19q alterations was not significantly different in WHO Grade II or Grade III tumors or in primary and recurrent tumors. We conclude that FISH on isolated cell nuclei, with application of the SIOP Europe Neuroblastoma consensus criteria, is a sensitive method for detection and interpretation of 1p and 19q aberrations in paraffin-embedded tissue specimens of oligodendroglial neoplasms.
Fluorescent In Situ Hybridization on Isolated Tumor
Cell Nuclei: A Sensitive Method for 1p and 19q Deletion
Analysis in Paraffin-Embedded Oligodendroglial Tumor
Specimens
Ellen Gelpi, M.D., Inge M. Ambros, M.D., Peter Birner, M.D., Andrea Luegmayr, Marcus Drlicek, M.D.,
Ingeborg Fischer, M.D., Reinhold Kleinert, M.D., Hans Maier, M.D., Michael Huemer, M.D.,
Brigitte Gatterbauer, M.D., Johann Anton, M.D., Karl Rössler, M.D., Herbert Budka, M.D.,
Peter F. Ambros, Ph.D., Johannes A. Hainfellner, M.D.
Institute of Neurology (EG, PB, IF, HB, JAH), Institute of Pathology (PB), and Department of Neurosurgery
(BG, KR), University of Vienna, Vienna; Children’s Cancer Research Institute (CCRI), St. Anna Children’s
Hospital (IMA, AL, PFA), Vienna; Institute of Pathology, LNK Wagner-Jauregg (MD), Linz; Ludwig
Boltzmann Institute of Neurooncology, Kaiser Franz Josef Hospital (MD, IF), Vienna; Institute of
Pathology, University of Graz (RK), Graz; Institute of Pathology (HM) and Department of Neurosurgery
(JA), University of Innsbruck, Innsbruck; and Department of Neurology, Christian Doppler Hospital (MH),
Salzburg, Austria
In oligodendroglial neoplasms, losses of chromo-
somal material at 1p and 19q associate with chemo-
sensitivity and prolonged survival. Thus, 1p/19q
testing is increasingly proposed for use in brain
tumor diagnosis and prognostic assessment. Fluo-
rescent in situ hybridization (FISH) is a classic tech-
nique for investigation of 1p/19q status in paraffin-
embedded tissues. A major limitation of this
method is truncation of tumor cell nuclei compli-
cating assessment of hybridization results. In our
study, we analyzed 1p and 19q status in a series of
79 oligodendroglial neoplasms (49 oligodendroglio-
mas, 30 oligoastrocytomas, WHO: 57 Grade II, 22
Grade III tumors) and controls (gliotic brain tissue:
n 4, diffuse low-grade astrocytoma: n 4) using
FISH on isolated whole tumor cell nuclei, prepared
as cytospin preparations, thus bypassing the prob-
lem of nuclear truncation. For interpretation of
FISH results, we used consensus criteria as defined
by the SIOP—Europe Neuroblastoma Study Group
for analysis of peripheral neuroblastic tumors. FISH
yielded interpretable results in 98.7% for 1p and
92.1% for 19q. Chromosome 1p/19q alterations
comprised deletions (1p: 79.5%, 19q: 80%) and im-
balances (1p: 11.5%, 19q: 12.9%). 1p aberrations
were more frequent in oligodendroglioma than in
oligoastrocytoma (100% versus 75.9%, P .001).
The frequency of 1p/19q alterations was not signif-
icantly different in WHO Grade II or Grade III tu-
mors or in primary and recurrent tumors. We con-
clude that FISH on isolated cell nuclei, with
application of the SIOP Europe Neuroblastoma con-
sensus criteria, is a sensitive method for detection
and interpretation of 1p and 19q aberrations in
paraffin-embedded tissue specimens of oligoden-
droglial neoplasms.
KEY WORDS: Chemosensitivity, Chromosome 1p,
Fluorescent in situ hybridization, Oligodendroglioma.
Mod Pathol 2003;16(7):708 –715
Oligodendroglioma is a morphologically distinct
type of diffuse glioma with variable clinical out-
come. In contrast to other types of gliomas, oligo-
dendroglioma is associated with generally good re-
sponse to adjuvant therapy (1–3). Various clinical,
radiological and histological features have been
suggested as prognostic factors. However, none of
these parameters predicts reliably response to ad-
juvant therapy (2, 4–11).
Recently, DNA losses on chromosomes 1p and
19q have been disclosed as common genetic alter-
ations in oligodendroglial neoplasms and much less
frequently in astrocytic neoplasms (12–18). Patients
Copyright © 2003 by The United States and Canadian Academy of
Pathology, Inc.
VOL. 16, NO. 7, P. 708, 2003 Printed in the U.S.A.
Date of acceptance: April 1, 2003.
This study was supported by the Scientific Funds of the Mayor of Vienna
(Project No. 2026) and by CCRI, St. Anna Children’s Hospital Vienna,
Vienna, Austria.
This study is a cooperative project of the Austrian Neuro-Oncology Net-
work (ANN, www.ann.at).
Authors EG and IMA contributed equally.
Address reprint requests to: Johannes A. Hainfellner, M.D., Institute of Neu-
rology, AKH 4J, POB 48, Währinger Gürtel 18-20, A-1097 Vienna, Austria; fax:
43-1-40400-5511; e-mail: johannes.hainfellner@akh-wien.ac.at.
DOI: 10.1097/01.MP.0000076981.90281.BF
708
with anaplastic oligodendrogliomas harboring a
1p/19q deletion were shown to have a more favor-
able prognosis and significantly better response to
chemotherapy as compared with patients with in-
tact 1p/19q status (1, 17, 1923). In patients with
diffuse low-grade gliomas, the 1p/19q status pre-
dicts prognosis (24) and the predictive value of
1p/19q status seems more reliable than histology
(25).
Therefore, it has been repeatedly suggested to
amend conventional histological evaluation of ana-
plastic oligodendroglioma and diffuse low-grade
gliomas with molecular analysis of chromosome
arms 1p and 19q (1, 3, 9, 19 22, 24 26). As a con-
sequence, 1p/19q testing is increasingly proposed
for use in the diagnostic setting. Various methods
are used for 1p/19q deletion analysis, in particular
PCR-based investigations (microsatellite analysis,
real-time PCR), comparative genomic hybridiza-
tion, and fluorescent in situ hybridization (FISH)
techniques (12, 14, 24, 2730). Consensus criteria
for assessment and interpretation of analytical re-
sults in brain tumors are, however, lacking. In con-
trast, in peripheral neuroblastic tumors consensus
criteria concerning methodology, evaluation of an-
alytical results and data interpretation of diagnostic
1p testing are well established (31, 32).
FISH is a classic technique for molecular cytoge-
netic testing on paraffin-embedded tissues (33).
FISH is not limited by age of material and does not
require normal autologous or pooled normal refer-
ence tissue. Binding of probes can be controlled
microscopically. However, one major limitation is
nuclear truncation in tissue sections complicating
evaluation and interpretation of hybridization re-
sults. To circumvent the problem of signal trunca-
tion, an improved FISH technique, that is, FISH on
isolated whole tumor cell nuclei, has been devel-
oped for chromosome 1p analysis in germ cell tu-
mors. Side-by-side comparison of both methods
has proven that FISH on isolated tumor cell nuclei
is superior as compared with conventional FISH on
tissue sections (32). FISH on isolated tumor cell
nuclei has meanwhile replaced conventional FISH
on tissue sections not only in germ cell tumors but
also in other tumor types, most notably peripheral
neuroblastic tumors (32, 34). Studies on oligoden-
droglial neoplasms have continued to use conven-
tional FISH on tissue sections. Therefore, we inves-
tigated applicability of FISH on isolated tumor cells
on a large series of oligodendroglial neoplasms and
assessed FISH results according to consensus crite-
ria for 1p analysis in peripheral neuroblastic tu-
mors, as defined by the International Society of
Pediatric Oncology Europe (E-SIOP) Neuroblas-
toma Group (31).
MATERIALS AND METHODS
A total of 89 paraffin-embedded archival tumor
specimens diagnosed as low- and high-grade oligo-
dendrogliomas and oligoastrocytomas were col-
lected from the five major Austrian neuropatholo-
gy/neurooncology units (Vienna, Graz, Innsbruck,
Salzburg, and Linz). Age of the material ranged
between 2 and 23 years. Control tissues comprised
four cases of gliotic brain tissues (neurosurgically
resected specimens of temporal lobe/Ammons
horn sclerosis) and 4 cases of diffuse low-grade
astrocytoma. Histological slides, 4
m in thickness,
were deparaffinized in xylol. Hematoxylin and eo-
sin (HE) staining and immunohistochemistry for
glial fibrillary acidic protein (GFAP, polyclonal an-
tibody Z0334; DAKO, Glostrup, Denmark) and epi-
thelial membrane antigen (EMA, monoclonal anti-
body E 29, DAKO) were performed according to
standard protocols (35). Immunolabeling was visu-
alized with the ChemMate kit (DAKO) and diami-
nobenzidine as chromogene. The tumor specimens
were reclassified according to WHO 2000 criteria
(36). Accordingly, we categorized those tumors as
oligodendrogliomas which displayed uniform oli-
godendroglial morphology, and we categorized
those tumors as oligoastrocytomas that contained
distinct components with oligodendroglial and as-
trocytic differentiation (Fig. 1). We used the anti-
GFAP immunostaining pattern as support for the
distinction of oligodendroglioma versus oligoastro-
cytoma. We considered a ring-like perinuclear
gliofibrillary immunostaining pattern as a char-
acteristic oligodendroglial feature, and we consid-
ered immunolabeling of a fibrillary tumor matrix as
a characteristic feature of an astrocytic component.
We used anti-EMA immunostaining for initial
screening of the cases to exclude immunoreactive
cell surface structures, a feature that would indicate
clear cell ependymal neoplasms.
For FISH analysis, we marked representative tu-
mor areas on HE stained sections. Corresponding
areas were identified on the paraffin blocks, and
core tissue biopsies of 1.5 mm in diameter were
taken from each block using a Yamshidi needle. The
same procedure was used for sampling of control
tissues. We prepared isolated cell nuclei of tumor
and control samples as described previously (32). In
detail, the paraffin-embedded tissue samples and
1-mm-diameter glass pellets were put into small
bags of nylon gauze. The material was then depar-
affinized with xylene for 1.53 hours, followed by an
ethanol row with decreasing concentrations (100%,
90%, 70%, and 35% for 1 h, respectively). After
transfer into distilled water, the deparaffinized tis-
sue specimens were incubated with 0.1% protease
(Sigma P8038, St. Louis, MO) in 0.1 N Tris-HCl at
37° C for 310 minutes and was agitated occasion-
FISH for 1p/19q in Oligodendroglioma (E. Gelpi et al.) 709
ally. Afterwards, the digestion was stopped on ice.
For checking of digestion, 10
L of 4'-6-diamidino-
2-phenylindole-2HCl (DAPI; 0.002
L/mL PBS) and
10
L of cell suspension were put on a slide, cov-
ered by coverslip and examined under the fluores-
cence microscope. Cytospins on glass slides were
prepared by centrifugation of 50100
L cell sus-
pensions (8 min, 500 rpm) with the help of a
FIGURE 1.
Representative histology of oligodendrogliomas and oligoastrocytomas. (A: HE, 100; inset a: HE; magnification, 170).
Oligodendrogliomas are characterized by a uniform oligodendroglial, that is, clear cell morphology, whereas (B: HE, 100) oligoastrocytomas are
composed of distinct astrocytic (inset a: HE, 170) and oligodendroglial (inset b: HE, 170) components.
710 Modern Pathology
SHANDON-Cytospin 2 cytocentrifuge (Pittsburgh,
PA). The cytospin preparations were air-dried for
224 hours and fixed with 4% buffered formalde-
hyde for 10 minutes. The samples were immedi-
ately processed for FISH or stored before FISH in
sealed boxes at 20° Cor80° C. 1p analysis was
performed with the paracentromeric probe D1Z1
(1q12) (37) and subtelomeric probe D1Z2 (1p36.3;
both Q-BIOgene, Heidelberg, Germany) in a
double-color FISH approach as described previ-
ously. For 19q analysis, probes 19pter and 19q13.3
were used (both Vysis, Bergish-Gladbach, Germa-
ny). For visualization, the probes were either di-
rectly fluorescence labeled or biotin/digoxigenin-
labeled probes were detected with fluorescence-
labeled antibodies.
Assessment and interpretation of FISH results
were performed according to guidelines defined by
the International Society of Pediatric Oncology (E-
SIOP Neuroblastoma Study Group) for studies of
peripheral neuroblastic tumors (31). In each case,
hybridization signals of 200 tumor cell nuclei
were assessed. The ratios between the numbers of
paracentromeric and subtelomeric regions of chro-
mosomes 1p and 19q were calculated.
Deletion: ratio of paracentromeric and subtelo-
meric signals 2/1, partially in conjunction with
4/2, 3/1, 4/1 ratios. This hybridization pattern in-
dicates the presence of an LOH.
Imbalance: disproportion of the ratio of paracen-
tromeric and subtelomeric signals, number of sub-
telomeric signals 1(3/2, 4/3, 4/2, 5/3, etc.). Such
a result can indicate but does not prove an LOH.
Clarification of this specific question requires com-
plementary techniques, such as PCR or Southern
blot analysis.
No deletion, no imbalancenormal result: equal
ratio of paracentromeric and subtelomeric signals
2/2, 4/4.
Unclear result: result is not interpretable because
of material (too little tissue, calcifications, etc.) or
technical reasons (enzymatic overdigestion).
Statistical testing was performed using
2
test and
statistic as appropriate. Two-tailed P values .05
were considered as statistically significant.
RESULTS
On histological review, we classified 49 speci-
mens as pure oligodendrogliomas and 30 speci-
mens as oligoastrocytomas. Ten tumors were ex-
cluded from further analysis because histology
revealed features of clear cell ependymoma in 3
cases, subependymoma in one case, astrocytoma in
two cases, primitive neuroectodermal tumor
(PNET) in one case, glioblastoma with focal oligo-
dendroglial features in one case, and unclassifiable
clear cell gliomas in two cases. The cohort of oligo-
dendroglial neoplasms included 59 primary and 20
recurrent tumors. Fifty-seven specimens fulfilled
criteria of Grade II, and 22 cases, Grade III, accord-
ing to current WHO grading criteria.
We performed 1p analysis in 79/79 tumor cases
and 19q analysis in 76/79 tumor cases. In addition,
we performed 1p/19q analysis in 8/8 controls. FISH
showed intact 1p and 19q status (2:2 ratio) in 4/4
gliotic brain tissue samples and in 4/4 diffuse as-
trocytomas, used as controls. Results in the cohort
of oligodendroglial neoplasms are summarized in
Table 1. FISH yielded interpretable results in 98.7%
(78/79) for 1p and in 92.1% (70/76) for 19q. In the
remaining cases, results were unclear, because of
material (too little tissue, too low tumor cell content
in tissue, calcification of the tissue) or methodology
(enzymatic overdigestion). In the cohorts with in-
terpretable results (discounting the cases with un-
clear results), 79.5% (62/78) had 1p deletion and
11.5% (9/78) had 1p imbalance, 80.0% (56/70) had
19q deletion, and 12.9% (9/70) had 19q imbalance.
In the total group (including cases with unclear
results), 56/76 (73,7%) had 19q deletion, and 9/76
(11,8%) had 19q imbalance. Aberrations in 1p were
always present in oligodendrogliomas but not in all
oligoastrocytomas (100% versus 75.9%, exact
2
test:
TABLE 1. Analysis of 1p and 19q Status in Oligodendrogliomas and Oligoastrocytomas
Oligodendroglioma Oligoastrocytoma
Primary
Tumors
Recurrent
Tumors
WHO Grade
II
WHO Grade
III
Total
n%n%n%n%n%n%n%
1p deletion 42 85,7 20 66,7 45 76,3 17 85 44 77,2 18 81.9 62 78,5
1p imbalance 7 14,3 2 6,7 7 11,9 2 10 5 8,8 4 18,1 9 11,4
1p normal 0 0 7 23,3 6 10,1 1 5 7 12,3 0 0 7 8,9
1p unclear 0 0 1 3,3 1 1,7 0 0 1 1,2 0 0 1 1,2
49 30 59 20 57 22 79
19q deletion 37 75,7 19 70,4 40 71,5 16 80 42 77,8 14 63,6 56 73,7
19q imbalance 6 12,2 3 11,1 6 10,7 3 15 4 7,4 5 22,7 9 11,8
19q normal 2 4,1 3 11,1 5 8,9 0 0 4 7,4 1 4,6 5 6,6
19q unclear 4 8,2 2 7,4 5 8,9 1 5 4 7,4 2 9,1 6 7,9
19q not done 0 0 3 3 0 3 0 3
49 30 59 20 57 22 79
FISH for 1p/19q in Oligodendroglioma (E. Gelpi et al.) 711
P .001). Figure 2 shows representative FISH
results.
There was a tight positive association of 1p and
19q alterations (Table 2): 96.9% (63/65) of the cases
with 1p alteration had in addition 19q aberration.
Only 5.7% (4/70) had either isolated 1p or 19q ab-
erration. Statistical testing illustrated the high de-
gree of association (kappa: 0.569; P .001)
The frequencies of 1p and 19q alterations were
not significantly different in primary and recurrent
tumors (Table 1): 1p aberrations occurred in 89.7%
(52/58) of primary tumors and 95% (19/20) of re-
current tumors (P .670; exact
2
test). 19q aber
-
rations occurred in 90.2% (46/51) of primary tu-
mors and in 100% (19/19) of recurrent tumors (P
.313). There was also no significant difference in the
frequencies of 1p and 19q aberrations in WHO
Grade II and Grade III tumors (Table 1): 1p aberra-
tions occurred in 87.5% (49/56) of Grade II tumors
and in 100% (22/22) of Grade III tumors (P .182).
19q alterations occurred in 92% (46/50) of Grade II
tumors and in 95% (19/20) of Grade III tumors (P
1.000).
DISCUSSION
DNA gains and losses are common alterations in
a host of tumors, and some of these DNA alter-
ations correlate with clinical outcome and therapy
response (38). In case of primary brain tumors,
DNA losses on chromosomes 1p and 19q associate
with the oligodendroglial phenotype. Clinicopatho-
logical correlation analysis has disclosed that 1p
and 19q aberrations in oligodendroglial neoplasms
are predictive for patient survival and chemore-
sponsiveness (1, 3, 7, 16, 20, 2224, 29, 39). There-
fore, determination of the 1p/19q status is increas-
ingly proposed to be integrated in the diagnostic
setting, amending histology (26). Among various
techniques, PCR-microsatellite analysis is com-
monly used for 1p and 19q investigations (2830).
This technique is relatively cheap and fast. How-
ever, a major limitation is the need for paired blood
samples. In addition, for this kind of studies, the
tumor cell content must not be 60%. Leukocyte
preparation in parallel to brain tumor processing
and freezing of tumor samples for molecular anal-
ysis has not yet become standard in many neuro-
pathology laboratories or is not possible because of
lack of dedicated personnel and laboratory space.
For 1p/19q testing, such laboratories have to resort
to methods that work on paraffin-embedded brain
tumor samples and which do not require paired
blood samples. Fulfilling this criterion, FISH is a
good method for 1p/19q testing on paraffin-
FIGURE 2.
FISH analysis of chromosomal arm 1p on isolated cell
nuclei. Representative hybridization results are shown in two-cell nuclei
(DAPI stain), respectively. A, normal result in a non-neoplastic control
case with an equal 2:2 ratio of paracentromeric (green) and
subtelomeric (red) signals. B, deletion status in an oligodendroglial
neoplasm with a 2:1 ratio of paracentromeric (green) and subtelomeric
(red) signals. C, imbalance status in an oligodendroglial neoplasm with
4:2 and 5:3 ratios of paracentromeric (green) and subtelomeric (red)
signals. (AC: original magnification, 3,000)
TABLE 2. Combinations of 1p and 19q Status in
Oligodendroglial Neoplasms
1p
Deletion
1p
Imbalance
1p
Normal
1p
Unclear
19q deletion 55 0 1 0 56
19q imbalance 2 6 1 0 9
19q normal 2 0 3 0 5
19q unclear 3 3 0 0 6
19q not done 0 0 2 1 3
62 9 7 1
712 Modern Pathology
embedded tissue samples (22, 24, 30). A further
advantage of the FISH technique as compared with
PCR is the possibility of investigating tumor tissue
at the cellular level. Thus, correlation with mor-
phology and immunocytology, if needed, can be
performed. Tumor cell content can be 60% (the
limit for LOH studies), and paired blood samples
are not needed allowing the use of archival
material.
Conventional FISH analysis is performed on his-
tological sections and, therefore, fraught with the
problem of nuclear truncation due to cutting of cell
nuclei. Assessment of FISH results on sections is a
time consuming procedure requiring statistic eval-
uation of several hundred tumor cell nuclei in each
case. For taking nuclear truncation into account, a
numerical cutoff of signal ratios has to be defined.
In cases with results close to the cutoff value, 1p
and 19q may not be unequivocally classifiable as
normal or abnormal. This problem is in particular
grave in tumors composed of large tumor cells (e.g.,
peripheral neuroblastic tumors) with an inherently
higher rate of nuclear truncation on histological
sections (32, 33, 40, 41). FISH on isolated cell nuclei
circumvents the problem of nuclear truncation. In
contrast to FISH on histological sections, tumor cell
nuclei are isolated mechanically and enzymatically
from paraffin-embedded tissue specimens. FISH is
then performed on cytospin preparations of the
tumor cell nuclei. In case of peripheral neuroblastic
tumors, FISH on isolated cell nuclei has proven
superior and has replaced conventional FISH on
histological sections (31, 32, 34). To our knowledge,
this method has been used so far only in a small
series of brain tumors (42). Our study on a large
series of oligodendroglial neoplasms shows that
FISH on isolated cell nuclei of oligodendroglial neo-
plasms is a sensitive method for 1p and 19q analy-
sis. Using E-SIOP criteria, the yield of interpretable
results is 98.7% for 1p and 92.1% for 19q.
The majority of our cases with abnormal 1p/19q
displayed a 2:1 ratio of paracentromeric and subte-
lomeric FISH signals, definitely indicating an LOH
status. However, a portion of tumor specimens
showed other abnormal ratios with subtelomeric
signals 1, for instance, 3:2, 4:3, 5:3 ratios (11.5%
for 1p and 12.9% for 19q). Such ratios can imply but
do not prove an LOH. Therefore, the E-SIOP Neu-
roblastoma Study Group has designated these ra-
tios as FISH imbalance (31). Previous FISH studies
analyzing 1p/19q status in brain tumors have not
discriminated between deletion and imbalance sta-
tus. It will be important to clarify in future prospec-
tive studies the 1p and 19q status in oligodendro-
glial neoplasms with FISH imbalance using FISH
and PCR techniques in parallel. Only if FISH imbal-
ance in oligodendroglial neoplasms consistently
represents an LOH status, FISH can be used as sole
technique for 1p/19q analysis. If FISH imbalance
includes oligodendroglial neoplasms with and
without a deletion status, PCR analysis will be man-
datory as complementary technique, as is the case
in peripheral neuroblastic tumors. Clarification of
this issue is important for translation of FISH-based
1p/19q testing on brain tumors into common clin-
ical use.
The majority of our patients received adjuvant
radio- and chemotherapy after first operation.
These treatments have DNA damaging effects. A
possible effect could be the increase of
polyploidization of tumor cells and, as a conse-
quence, an increase of the rate of FISH imbalance
status (e.g., 4/2 instead of 2/1, or 6/2 instead of 3/1).
Comparing the frequencies of FISH imbalance in
primary and recurrent tumors, we did not detect a
significant difference. Thus, tumor recurrency and
adjuvant treatment seem not to diminish the diag-
nostic yield of FISH.
In our cohort of oligodendroglial neoplasms, we
found a tight positive association of 1p and 19q
aberrations. Only 5.7% (4/70) showed either iso-
lated 1p or 19q alteration and 96.9% (63/65) of the
cases with 1p alteration had in addition 19q alter-
ation. 1p and 19q losses were also tightly associated
in other, previously published series of oligoden-
droglial neoplasms (12, 16, 18, 20, 24, 27, 30, 36, 43,
44). On the basis of the 1p/19q status, stratification
of adjuvant therapy of anaplastic oligodendrogli-
oma has been proposed (20, 26). According to this
proposal, initial chemotherapy but without radio-
therapy should be reserved for patients with com-
bined 1p/19q losses, whereas patients with isolated
1p loss should be treated with combined chemo-
therapy and radiotherapy. However, oligodendro-
gliomas with isolated 1p loss are rather rare. As
survival analysis has been performed so far only in
relative small patient cohorts, it has not been
proven unequivocally whether there is indeed a
significant difference of prognosis and/or response
to adjuvant therapy in patients with isolated 1p loss
as compared with patients with combined 1p/19q
loss. This issue needs to be clarified before stratifi-
cation of adjuvant treatment on basis of 1p/19q
status can be translated into common clinical use.
This will require investigations of large prospective
patient cohorts using carefully validated laboratory
techniques for 1p/19q analysis.
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FISH for 1p/19q in Oligodendroglioma (E. Gelpi et al.) 715
    • "I-FISH with specific DNA probes in paraffin-embedded tissues is a classical technique for analysis of chromosomal aberrations in brain tumour cells. However, one major limitation of this method is nuclear truncation in tissue sections, complicating evaluation and interpretation of hybridization results (Gelpi et al., 2003). To circumvent the problem of signal truncation, we analysed the 1p and 19q status in 16 samples of oligodendroglial neoplasms (4x oligodendrogliomas , grade II; 10x anaplastic oligodendrogliomas , grade III; 2x anaplastic oligoastrocytomas, grade III) using I-FISH on isolated whole tumour cell nuclei, prepared from fresh non-fixed tumour tissue samples, resuspended in media and processed using a standard cytogenetic procedure. "
    [Show abstract] [Hide abstract] ABSTRACT: In oligodendroglial brain tumours, losses of chromosomal material of the short arm of chromosome 1 and long arm of chromosome 19 have been shown to predict responsiveness to chemotherapy and prolonged patients' survival. Therefore, the correct diagnosis of these genetic alterations in tumours of oligodendroglial origin is particularly important. To detect deletions of 1p36 and/or 19q13.3 in oligodendroglial cells we used dual-colour I-FISH with locus-specific DNA probes. I-FISH was performed on isolated whole cell nuclei, prepared from fresh non-fixed tumour tissue samples resuspended in media and processed using a standard cytogenetic procedure, thus bypassing the problem of nuclear truncation. We examined 16 patients with histologically proved oligodendrogliomas (5x oligodendroglioma, 9x anaplastic oligodendroglioma, 2x anaplastic oligoastrocytoma). The results of molecular cytogenetic analyses were correlated with morphological and clinical findings. Molecular cytogenetic analyses were successful in 15 patients and, due to a non-adequate tissue specimen, were uninformative in one patient only. Combined deletions 1p36/19q13 were proved in 13 patients. However, in six of them additional genetic alterations typical for high-grade astrocytoma were found, which could have negative influence on the prognosis. One patient had isolated deletion of 1p36 and another had a normal genetic pattern without any chromosomal alterations. In summary, I-FISH on isolated cell nuclei is a powerful tool for detecting chromosomal aberrations in tumour cells. A systematic molecular cytogenetic analysis may advance diagnosis, prognostic stratification, and targeted treatment of patients with brain tumours.
    Full-text · Article · Feb 2006
    • "Normal rabbit immunoglobulin-G was substituted for the primary antibody as the negative control, at a concentration where immunostaining of control slides gave faint cytoplasmic staining. As described previously (Gelpi et al., 2003), fluorescent in situ hybridization (FISH) on isolated tumor nuclei extracted from paraffin-embedded tissue samples was performed using paracentromeric probe D1Z1 (1q12) and subtelomeric probe D1Z2 (1p36.3) for 1p. "
    [Show abstract] [Hide abstract] ABSTRACT: Expression of hypoxia-related tissue factors in 1p-aberrant oligodendroglial neoplasms diminishes patient outcome. Differentiated embryo-chondrocyte expressed gene 1 (DEC1) has been described as novel hypoxia-related tissue factor. In our study, we assessed the expression of DEC1 in 1p aberrant oligodendroglial neoplasms and its association with necrosis and expression of hypoxia-inducible factor 1alpha (HIF-1alpha), carbonic anhydrase-9 (CA9), and vascular endothelial growth factor-mRNA (VEGF). 44 primary and 16 recurrent oligodendroglial neoplasms with 1p-aberrations were investigated immunohistochemically for the expression of DEC1, HIF-1alpha, and CA9. Expression of VEGF was investigated using in situ hybridization. DEC1 expression was correlated with necrosis and with expression of HIF-1alpha, CA9, and VEGF. DEC1 was expressed in tumor cell nuclei, and occasionally in nuclei of endothelial cells, and glial and neuronal cells of surrounding brain tissue. High expression (>10% of tumor cells immunolabeled) of DEC1 was found in 56 cases, low expression (<10% of tumor cells immunolabeled) was found in 3 cases. In 1 case no expression of DEC1 was evident. DEC1 expression showed no topographical association with necrosis or expression of HIF-1alpha, CA9, or VEGF. DEC1 expression is found in the majority of 1p-aberrant oligodendroglial neoplasms and does not correlate with necrosis or expression of HIF-1alpha, CA9, VEGF. Thus, immunohistochemical analysis of DEC1 expression is in our hands not suitable for detection of tissue hypoxia in this type of primary brain tumor.
    Full-text · Article · Nov 2005
    • "Cytological preparation avoid the problem of nuclear truncation whereas on paraffin sections thresholds varying from 30% to 45% are defined to retain a deletion [29]. Another way to bypass the problem of nuclear truncation has been recently described performing FISH on isolated whole tumor cell nuclei preparations [30]. The threshold to affirm a deletion in that conditions is near the one used in culture (2%). "
    [Show abstract] [Hide abstract] ABSTRACT: Among diffuse gliomas, oligodendrogliomas may account for 25% of cases. They have a better prognosis and chemosensitivity as compared to astrocytomas. Genetic studies have shown a correlation between oligodendrocyte phenotype and presence of 1p/19q deletions. In addition, these deletions are of prognostic value. The aim of the present study was to describe a new method to detect 1p/19q deletions when little tumoral material is available (stereotactic biopsies (SBs)). Since smears (cytological preparations) are routinely done for intraoperative diagnosis of gliomas, we have searched for 1p/19q deletions by FISH in a series of 30 patients with a glioma. In 14 cases, loss of heterozygosity (LOH) analysis was also performed in order to validate our method. We found that FISH analysis on frozen smears was a simple, rapid and reliable method to detect 1p/19q deletions and a good concordance was found with LOH data (85%). The main advantages of FISH analysis on frozen smears are the following. First, it requires little material and can be easily done in the case of SBs. Second, it has a higher sensitivity than LOH especially in infiltrative areas of gliomas. Third, it allows detection of a codeletion 1p/19q in a single tumor cell. In contrast, LOH analysis is easier to interpret and can detect smaller and partial deletion whose pronostic significance remains to be defined. In conclusion, these two techniques can be used to investigate 1p/19q status in gliomas. The appropriate choice of one or other of these two techniques will depend on the specific questions that need to be answered.
    Full-text · Article · Jul 2004
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