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Technical Advance |
From the Department of Pathology,*
University of
California, San Francisco, San Francisco, California; the Department of
Laboratory Medicine,
University of California,
San Francisco Cancer Center, San Francisco, California; and the
Departments of Laboratory Medicine and
Pathology,
Mayo Clinic, Rochester, Minnesota
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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These are compelling results to use the molecular analysis of chromosomes 1p and 19q to assist in the histological diagnosis of human gliomas and clinical management of affected patients. Several molecular methods are currently used to detect 1p/19q loss in oligodendrogliomas in both clinical and basic research settings. Allelic imbalance, sometimes referred to as loss of heterozygosity (LOH),3 detects reduction from heterozygosity to homozygosity at 1p and 19q loci.1,4-6 The routine use of LOH to detect alterations at specific loci is limited by the requirements of paired normal tissue from the same patient and the presence of heterozygosity in the normal tissue (an informative locus). Fluorescence in situ hybridization (FISH) and comparative genomic hybridization are established methods to detect copy number, but both are labor-intensive techniques.6-11 Real-time quantitative polymerase chain reaction (PCR) is currently also used to determine copy number at genomic loci but, to date, only single-copy sequence-specific probes have been used.12-16 A novel modification of this technique that utilizes a probe designed to bind to CA repeats, quantitative microsatellite analysis (QuMA), has recently been described.17,18 QuMA is a high-throughput real-time PCR assay that may be adaptable as a molecular diagnostic technique, as it can be performed on DNA derived from either frozen or paraffin-embedded tissue in as little as 2 hours. In the initial report,17 the template DNAs were in general prepared from fresh or frozen tissue samples. On a practical level, many tumor samples are available only as formalin-fixed, paraffin-embedded tissues from pathology archives. Accordingly in this study, we determined whether DNA extracted from paraffin-embedded tissues would be a substrate of sufficient quality for QuMA. We then applied QuMA to 41 cases of oligodendroglioma/oligoastrocytoma, and assessed its reliability by comparing the results in a subset of cases with a well-established molecular technique, FISH.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Oligodendrogliomas and oligoastrocytomas were obtained as routinely processed paraffin-embedded samples from clinical material at the University of California, San Francisco. For each case, histological assessment of the tumor tissue to be used was performed by a neuropathologist (KA). If at least 90% of the area of the tissue was interpreted as tumor, the sections were directly cut from the block into an Eppendorf tube for DNA isolation. If the proportion of tumor was <90%, 10 to 20 sections were cut on glass slides, and the tumor tissue was hand dissected from the normal tissue. Tumor DNA was prepared from paraffin-embedded tissues by digesting deparaffinized tumor sections for 3 to 5 days with proteinase K at 55°C (0.5 mg/ml in 100 mmol/L NaCl, 10 mmol/L Tris-HCl, pH 8.0, 25 mmol/L ethylenediaminetetraacetic acid, 0.5% sodium dodecyl sulfate), followed by a phenol:chloroform:isoamyl extraction and ethanol precipitation. Concentrations were determined on the ABI 7700 so that each DNA, when amplified with the reference locus, reached a threshold cycle equivalent to a 5-ng control DNA.
QuMA
All primer sets were used to perform amplifications in triplicate
on the ABI 7700 instrument (Applied Biosystems, Foster City, CA). The
probe for the detection of amplified product was a 21-bp oligomer
complementary to the microsatellite CA repeat [5' 6-carboxy
fluorescein (FAM)-TGTGTGTGTGTGTGTGTGTGT 3' 6-carboxy tetramethyl
rhodamine (Integrated DNA Technologies, Coralville, IA)], rendering it
capable of hybridizing to microsatellite loci spread throughout the
human genome. The flanking primers, thus, could be continuously changed
while the probe remained constant (Figure 1A)
. As FAM is released from the 5' end
of the probe by Taq polymerase, fluorescence is detected and
plotted as a function of PCR cycle number.12,19
Copy
number is determined from the PCR cycle number (Ct) at which DNAs reach
a threshold amount of fluorescence above background.
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To normalize for differences in the amount of total input DNA,
amplification at a reference locus was performed once per plate in
triplicate for each individual DNA. The maximum number of additional
test loci that could therefore be analyzed on a single 96-well plate
for one DNA is 31. To suppress the effect of possible deviations from
the diploid state at a single locus in unstable tumor DNAs, the
reference chosen was a multiplex PCR or amplification of a pool of
microsatellite primer pairs in a single well, rather than any
individual locus. For these experiments, six primer sets
for the reference pool were chosen from chromosomes that typically
remain unaltered during the course of glioma
tumorigenesis:20-22
2q (D2S385), 3p (D3S1554), 5q
(D5S643), 8q (D8S1800), 12q (D12S1699), and 21p (D21S1904) (Table 1)
. The final concentration of the pooled
primers in the reference PCR wells is 0.4 µmol/L for each of the 12
primers and reached Ct at one to four cycles before that of the test
loci used in this study.
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was dependent on the mean 
Ct (values between 1 to 4) and the SD
(<0.25) (Figure 1B)
derived from amplification of normal DNA for 8 to
10 unrelated individuals. Genomic DNAs to calculate the average Ct
were isolated from paraffin-embedded normal brain tissue. To minimize
potential amplification variability of a locus between different
individuals, loci were selected that: 1) are 
150 bp in length, 2)
contain the full 10.5 CA repeats of the probe, and 3) have an
efficiency of amplification that is >90% (Figure 2, A and B)
. PCR efficiency (E) can be
determined from the slope of a four-point curve where the log of the
input DNA mass is plotted against the threshold cycle (Ct). The
equation of this line is derived from XCt =
X0(1 + E)Ct, where
XCt = DNA quantity at the threshold
(constant) and X0 = quantity of input DNA. The
slope of this line, S, is equal to -1/log(1 + E) or E =
10(-1/S) - 1. Amplification of 5 ng for these
experiments falls within the linear range of the plot of Ct against log
X0 and reaches threshold at cycle numbers <32.
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The Ct values for each set of triplicates were averaged. The Ct of
the pooled reference was subtracted from the Ct for each locus to
obtain the Ct [Ct = Ct (microsatellite) - Ct
(reference)]. Ct values were determined for loci in tumor samples
and a set of 10 normal genomic DNAs. The average of the 10 Ct values
[Ct (normal)] measured from the normal DNAs was calculated once
for each locus in this study and used in the subsequent calculations
for all experiments performed on a single ABI 7700. The relative copy
number of a locus in tumor DNA to normal DNA =
2-Ct, where Ct = Ct (tumor) -
Ct (normal). Because the copy number is 2 in normal diploid DNA, the
relative copy number multiplied by 2 yields copy number in the unknown
sample at the locus amplified (Figure 1B)
.
To determine whether the copy number calculated differs significantly
from normal DNA so as to be called a loss or a gain, the pooled SD for
all loci in normal DNAs was used to create a tolerance interval (TI)
with a confidence of 95%.17
TI = 2 x 2 to the
power ±[2.28 x the square root of {
i
(ni - 1) x
SDi2/i(ni - 1)}] where
ni= number of normals analyzed per microsatellite locus and
2.28 is a two-sided tolerance limit factor for a total of 167
measurements [i (ni -
1)].23
Based on this TI, copy numbers <1.58 were
considered to be losses whereas those >2.53 were considered to be
gains.
FISH
FISH was performed as previously described.6 Briefly, 5-µm sections from paraffin-embedded tumors were deparaffinized, dehydrated in an ethanol series, microwaved on high power for 10 minutes in 10 mmol/L citrate buffer, pH 6.08, and digested in pepsin (4 mg/ml in 0.9% NaCl, pH 1.5) at 37°C for 15 minutes. Bacterial artificial chromosomes (BACs) from 1p36, 1q24, 19p13, and 19q13.3 were directly labeled with fluorescent nucleotides, and just before hybridization, probes and target DNA were simultaneously denatured at 80°C for 5 minutes. Hybridizations were performed at 37°C overnight, and subsequent washes were performed at 45°C as follows: 1.5 mol/L urea/0.1x standard saline citrate, twice for 15 minutes; 2x standard saline citrate, 5 minutes; 2x standard saline citrate/0.1% Nonidet P-40, 5 minutes. After hybridization, the mean number of signals scored for each of the four probes was determined after counting 300 to 500 nuclei. A ratio between p and q arms was calculated for both chromosome 1 and chromosome 19. In general, for this set of tumors, the ratios (0.5 to 1.0) simply reflected loss or retention. A ratio of 0.85 or less was scored as a loss. A gain was scored on either chromosome when >15% of nuclei exhibited more than two signals for either a single probe or both.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To test whether PCR would be quantitative in paraffin DNA samples,
serial dilutions were prepared from isolated normal and tumor DNAs from
paraffin sections and subjected to real-time quantitative PCR at a
single microsatellite locus (Figure 2A)
. Over a range of 1 ng to 50 ng,
the starting DNAs reached threshold as predicted from the equation
Xn = X02n where n =
Ct and efficiency is 100% (Figure 2B)
. For example, the 20 ng and 5 ng
quantities differed by exactly two cycles at the threshold, reflecting
the fourfold difference in DNA mass.
Our analysis encompassed the pattern of loss at microsatellites along
the entire 160 cM of 1p. Copy numbers at five 1p loci were determined
for 41 tumors and are presented in Figure 3
. In these experiments, 1p loss (as
defined by copy number <1.58 in two or more sequential loci) was
detected in 28 of 41 (68%) cases where an oligodendroglial component
was histologically apparent. The entire 1p arm was lost in 23 cases.
Loss limited to the two distal markers (D1S468 and D1S214) occurred in
two cases (10896 and 7876) and interstitial deletions that encompassed
the region previously identified were detected in two cases (8503 and
307). For 8503, the marker D1S228 at 32.4 cM was also retained.
Deletion of consecutive markers in a nonoverlapping region of 1p
(D1S2783 and D1S514) with retention of distal markers was found only in
a single tumor (283). In addition to the losses detected, two cases
(185 and 262) appeared to have extensive regions of gain along
chromosome 1.
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To gain insight into the alterations that appear in conjunction with chromosome 1p loss in our patient sample, QuMA was performed at loci on chromosomes 10q, 19q, and 19p. Deletion of chromosome 1p is prevalent in oligodendrogliomas that have sustained 19q loss,6,10,20 and chromosomal abnormalities of 10q have been associated with the development of higher grade gliomas, including glioblastomas.20,24,25 A single site on 19p, not known to be lost at high frequency, was included as an internal control.20 QuMA, as other molecular methods, detected frequent combined 1p/19q loss in oligodendroglial tumors of all grades. Interestingly, loss of two 10q markers was not observed in any tumors with whole arm loss of chromosome 1p/19q, whereas loss of a single 10q marker did occur in two cases. Specifically, 10q loss (both markers) was seen in 0 of 20 cases with whole arm 1p/19q loss, but 3 of 16 cases without whole arm 1p/19q loss. Although this was not quite statistically significant (P = 0.078, Fisher’s exact test), these results are consistent with previous data showing that these events do not generally occur concurrently.10
FISH
FISH analysis was performed with molecular probes from chromosomes
1p and 19q on a subset of paraffin-embedded tumors to test the accuracy
of QuMA against an accepted molecular technique. An example of an
oligodendroglioma showing loss of 1p with concordant results by FISH
and QuMA is shown in Figure 4
. Copy
number changes detected by QuMA and FISH were compared in 23 tumors (45
chromosomal arms) (Figure 5)
. Concordance
between the two methods with respect to the scoring of loss or gain was
evident at 37 of 45 chromosomal arms tested. We were primarily
interested in the detection of 1p/19q deletions that may be indicative
of responsive tumors. With respect to such deletions, FISH performed
with a probe from 1p36 identified only a single deletion of chromosome
1p that QuMA did not detect (283). In this region of the chromosome,
QuMA did, however, uncover a partial 1p deletion in one case (8503)
that was not detectable by FISH (Figure 5)
. An additional case (14670)
displayed a potential loss of the most distal marker (D1S468) by QuMA
that was not concordant with FISH. In these cases, the deletions
defined by the QuMA markers may not encompass the region containing the
FISH probe. Overall, using FISH as a gold standard, the sensitivity for
the detection of loss by QuMA for 1p and 19q was 92% and 94%,
respectively. The specificity for the detection of loss for 1p and 19q
was 80% and 100%, respectively.
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. Molecular Diagnosis of Oligodendrogliomas
In general, gliomas that displayed many of the "classic"
oligodendroglial features, such as round nuclei, perinuclear halos, and
chicken-wire vasculature, tended to be those in which whole arm 1p/19q
loss had occurred. There were, however, cases in which the molecular
genetics provided useful or unexpected information. Examples of
correlations of histopathology with molecular analysis are shown in
Figure 6
. The histology of a classic
oligodendroglioma (Figure 6A)
with 1p/19q loss is contrasted with that
of the tumor in Figure 6B
. Although this tumor (2470) has some
characteristics of anaplastic oligodendroglioma including a monomorphic
appearance and lack of discrete tumor cell cytoplasm, it lacked classic
features, such as round nuclei and perinuclear halos, but did show
1p/19q loss by QuMA. In Figure 6C, a
tumor, diagnosed as
oligodendroglioma on the basis of uniformly round nuclei, was found to
have 1p/19q intact. Finally, six out of nine oligoastrocytomas had
sustained loss of 1p/19q. Discrete regions of oligodendroglial and
astrocytic morphology in one of these six cases is shown in Figure 6, D and E
, respectively.
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Chromosome 1p losses that accompany glial tumorigenesis are
thought to signal the presence of a tumor suppressor gene(s). Many of
the tumors with 1p loss in this study exhibited deletion of the entire
chromosomal arm, but QuMA exposed partial deletions in four gliomas
that may be useful in mapping the location of a putative tumor
suppressor. In Figure 7
, QuMA results
define a common region of deletion from D1S468 to D1S2736 that is
consistent with previous data from FISH and LOH analyses showing loss
at 1p36.6
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our primary goal in this study was to develop a clinical assay to be used for the efficient detection of 1p/19q genetic losses in human oligodendrogliomas. Combined 1p/19q loss determined by QuMA was specific for tumors containing an oligodendroglial component as only a single astrocytic tumor displayed 1p loss (data not shown). Using FISH as the standard, QuMA was concordant at 37 of the 45 chromosomal arms tested. Only one case of 1p loss identified by FISH (283) was not detected by QuMA. As the FISH probe used in the study maps between the two telomeric QuMA markers used in this study (D1S214 and D1S468), it is possible that a small deletion may exist on chromosome 1p that was not identified by QuMA because of the location of our markers. However, the copy numbers determined by QuMA for these loci were 1.61 and 1.64, respectively, which was on the border of our cutoff for loss (1.58). Conversely, in two cases (14670 and 8503), small 1p deletions were identified by QuMA but not by the FISH probe used in this study. It is possible that the region of deletion in these two tumors does not include the region targeted by the FISH probe. Overall, the results indicated concordance between QuMA and FISH detection of loss at 1p and 19q. The fact that QuMA identified 10q loss as an infrequent occurrence with 1p/19q loss, consistent with other studies of oligodendroglioma, provides additional evidence that it is a valid technique to detect genetic loss.
In several cases, apparent gains in 1p or 19q were detected by either method but were not concordant. In three cases in which QuMA detected a gain that was not corroborated by FISH (8503, 185, and 262), 10 to 15% of nuclei in these specimens displayed gains by FISH but were not scored as a gain. Tumor heterogeneity may explain some of these discrepancies, although tumor DNA for QuMA and sections for FISH originated from the same paraffin blocks. In 8503, the primer sets used for QuMA may flank a small interstitial deletion that is detectable by the FISH probe. In one tumor (185), 9 of 11 loci across three chromosomal arms appeared to show gain by QuMA that was not corroborated by FISH. Because QuMA at a specific locus is dependent on comparison to a reference pool, it is possible that the reference pool does not represent a copy number of 2 in this tumor because of alterations sustained in the chromosomes where the reference pool primers reside. Copy numbers calculated using this pool as the reference locus could therefore be misleading in occasional cases.
A potential limitation of QuMA is the requirement for sufficient tumor DNA to perform the assay. As the assay is currently set up, no more than 15 ng of DNA is necessary for analysis per locus in addition to the reference pool. In our experience, obtaining sufficient DNA was a difficulty encountered primarily with older paraffin samples (>10 years), and the most recent cases (<2 years), easily yielded sufficient DNA quantities (µg) of high quality (data not shown). This suggests that for some tumor samples, especially those obtained 10 years ago, and with small amounts of tumor, another method, such as FISH, may be more appropriate. However, little difficulty with respect to DNA isolation was encountered in the majority of recent cases. A second potential limitation of QuMA is contaminating genomic DNA from normal cells, which can dilute signal from tumor genomic DNA. It has been previously shown that loss can still be detected by QuMA in samples containing as much as 30% of normal DNA.17 In addition, the use of microdissection, to increase the relative proportion of tumor cells from the paraffin section, can alleviate this problem in most cases.
Real-time PCR is dependent on copy number rather than polymorphism, and
for this reason, all loci, theoretically, should be informative in each
sample. However, under the standard cycling conditions, only some
primer pairs will amplify with PCR efficiency close to 100% and yield
a consistent Ct over a panel of normal DNAs. The standard deviations
of the average Cts across the 10 normal DNAs did vary from 0.1 to
0.4, and those that yielded standard deviations >0.25 (or >0.5 of a
copy), were unacceptable for analysis. In many of these cases,
sufficient sequence was available so that alternative primer sets could
be chosen for a locus. For example, microsatellite D19S596 was
particularly important to study because it lies within a common region
of loss on 19q defined by other laboratories.5,6,26-29
However, initial experiments indicated that the primer set described in
the National Center for Biotechnology Information database was not
suitable for QuMA (multiple bands appeared on amplification of genomic
DNA indicating nonspecific amplification). An alternative primer pair
based on the known sequence flanking the CA repeat of this locus was
designed that was ultimately successful for quantitation of copy
number. This approach could be used for other repeats in regions of
interest that do not amplify consistently in initial experiments.
QuMA, like FISH, will not detect genetic events that do not result in
change in copy number, specifically loss of one allele followed by
duplication of the remaining allele. Previous reports have demonstrated
a high correlation between LOH and FISH on chromosome 1p in
oligodendrogliomas indicating that for gliomas, in general, 1p deletion
events will be assessed by methods that detect copy
number.6
Primer sets may also lead to false conclusions
about change in copy number in a specific tumor. For example, where
polymorphisms or acquired mutations interfere with primer sets, loss
may be thought to occur. Alternatively, a single locus may appear to be
retained or amplified in contrast to copy numbers of flanking loci. For
example, in two cases (4541 and 11654), D1S214 was retained whereas the
other four markers, including one telomeric and three centromeric to
D1S214, showed loss. For both of these tumors, analysis was performed
at additional loci (12 total). QuMA detected loss at all of these
markers, leaving D1S214 as the sole retained marker in these tumors
(data not shown). Several possibilities can explain these data. First,
the region encompassed by D1S214 is retained in these tumors. Second,
normal alleles for a specific locus may exist in the human population
in which some feature of the allele (size of the repeat element, for
example) may affect amplification to an extent that the resultant Ct
deviates significantly from the average of a panel of normals. In
essence, this repeat would represent a noninformative locus in the
analysis of the corresponding tumors. Third, a genetic event that
resulted in a small region of gain has occurred at this site, and it
appears to be retention. Finally, because the TI was calculated with
95% confidence, we expect that 5% of the numbers may be scored
incorrectly. An advantage of QuMA is that many loci can be analyzed
rapidly and efficiently along a chromosomal arm such that, similar to
LOH analyses, divergent copy numbers require evaluation in the context
of data from flanking markers.
This type of genetic analysis may be particularly useful for clinical cases in which the histological features are ambiguous. Neuropathologists often differ with respect to the distinction of astrocytomas and oligodendrogliomas. Complicating matters are the oligoastrocytomas, which are a mixed subtype of glioma that histologically exhibit both oligodendroglial and astrocytic features. Some cases of oligoastrocytoma contain well-defined alternating sections of oligodendroglioma or astrocytoma within a single tumor. Although demarcated regions may exist within a tumor, these cases have been shown to still be genotypically clonal and furthermore, they either display genetic alterations typical of one or the other, but never both.30,31 For example, mutation of p53 is common in astrocytic tumors but rarely occurs in oligodendrogliomas. In oligoastrocytomas where 1p/19q loss has occurred, p53 mutation, as in oligodendrogliomas, is not generally observed.31 Nine of the tumors analyzed were diagnosed as mixed oligoastrocytomas (both grade 2 and grade 3), and in six of these cases, 1p/19q loss was found. One of the oligoastrocytomas, tumor 48, was diploid at most loci tested and in this respect, was genotypically similar to tumors of astrocytic lineage. Interestingly, this tumor exhibited positive immunohistochemical staining for p53 which often represents mutation of the gene.32
In addition to the clinical ramifications of QuMA, this technique may
facilitate the identification of tumor suppressor genes that may be the
targets of recurring 1p and 19q losses in
oligodendrogliomas.4,6,26-29,33
QuMA, like LOH, has the
resolving power to detect small deletions, including homozygous
deletions,17
and may help in mapping the location of the
putative tumor suppressors. Partial deletions of 1p (loss of more than
one consecutive marker) were detected in four gliomas. The smallest
common region of deletion is defined by markers D1S468 and D1S2736
(Figure 7)
. An additional tumor, 14670, showed a possible loss of the
most distal marker, D1S468. These data, in the context of the FISH
results, raise two interesting possibilities regarding the location of
a putative tumor suppressor gene(s). The first is that completely
nonoverlapping deletions exist between 14670 and 8503. The FISH probe
lies between markers D1S214 and D1S468, and in both tumors, the probe
was retained. Therefore, the region complementary to the FISH probe may
not be contained within the minimal region of deletion for either
tumor. The second possibility is that the deletions in 14670 and 8503
each end within the BAC used for FISH. In this scenario, hybridization
by the BAC is still possible because sufficient sequence that is
complementary to the probe is retained in each tumor. Finally, tumor
283 exhibited a partial deletion by QuMA that did not overlap with
regions of loss in the other four tumors (Figure 3)
. Nonoverlapping
regions of 1p loss have also been observed in another series of
oligodendroglial tumors.33
The ease of QuMA makes it feasible to survey a tumor genome in a clinical setting in a rapid and efficient manner using paraffin material obtained from the pathology laboratory. It is hoped that this technique may be applied to a number of tumor types to supplement histological diagnosis to refine prognostic and therapeutic decisions for specific patients.
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Footnotes |
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Supported by Robert Wood Johnson Foundation grant 033349 (to K. A.), the National Institutes of Health grants CA50905 and CA85778 (to R. B. J.), and American Cancer Society grant IRG-97-150-01.
Accepted for publication December 18, 2000.
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