Clonal X-inactivation analysis of human tumours using the human androgen receptor gene (HUMARA) polymorphism: a non-radioactive and semiquantitative strategy applicable to fresh and archival tissue.
ABSTRACT Assessment of clonality of cellular proliferations is important in experimental and clinical cancer research. X-chromosome inactivation studies are widely used to assess clonality, but most assays require relatively large amounts of high molecular weight DNA. Two PCR-based strategies, the phosphoglycerate kinase (PGK) and the human androgen receptor (HUMARA) clonality assays allow studies of small tissue samples. The HUMARA assay was adapted to non-radioactive analysis taking advantage of an automated sequencer providing high resolution of alleles and immediate quantitation. This assay was validated by comparison with X-inactivation patterns obtained by Southern analysis with the probes M27 beta and PGK. Fifteen gastrointestinal carcinomas, 25 benign goiter nodules and normal peripheral leukocytes of 27 individuals (12 who were under 15 years and 15 over 80 years) were analysed. Furthermore, DNA extracted from formalin-fixed paraffin-embedded tissue (FPT) was analysed with the two PCR-based methods and compared with X-inactivation patterns determined by Southern analysis of high molecular weight (HMW) DNA. This modified HUMARA assay is reliable in most patients; as with other clonality assays, constitutive skewing in normal tissue precludes clonal analysis in some individuals. Extremely skewed X-inactivation patterns were found in normal peripheral leukocytes of 7 out of 15 old females (over 80 years) and in 1 of 12 of the young females tested (under 15 years). Comparison of results obtained with HMW and FPT DNA yielded consistent results for the HUMARA assay whereas the PGK PCR assay was much less reliable. The HUMARA assay thus permits studies of selected areas of tissue sections without significant stromal components, allowing correlation of histological and genotype findings in fresh and archival specimens.
- Journal of Allergy and Clinical Immunology - J ALLERG CLIN IMMUNOL. 01/2005; 115(2).
Article: Prevalence of mutations in TSHR, GNAS, PRKAR1A and RAS genes in a large series of toxic thyroid adenomas from Galicia, an iodine deficient area in NW Spain F Palos-Paz1, O Perez-Guerra1, J Cameselle-Teijeiro3, C Rueda- Chimeno5, F Barreiro-Morandeira4, J Lado-Abeal1,2� and the Galician Group for the Study of Toxic Multinodular Goitre� � Galician Group for the Study of Toxic Multinodular Goitre
Molecular and Cellular Probes (1997) 11, 217–228
Clonal X-inactivation analysis of human tumours using the
human androgen receptor gene (HUMARA) polymorphism:
a non-radioactive and semiquantitative strategy applicable
to fresh and archival tissue
Peter Kopp,1,6Rolf Jaggi,5Andreas Tobler,3,5Bettina Borisch,2
Madeleine Oestreicher,5Leah Sabacan,6J. Larry Jameson6and Martin F. Fey4,5∗
1Department of Internal Medicine,2Institute of Pathology,3Central Hematology
Laboratory,4Institute of Medical Oncology, Inselspital,5Laboratory for Clinical and
Experimental Research, University of Berne, CH-3010 Berne and6Center for
Endocrinology, Metabolism and Molecular Medicine, Northwestern University,
Chicago, IL, USA
(Received 13 November 1996, Accepted 14 January 1997)
Assessment of clonality of cellular proliferations is important in experimental and clinical cancer
research. X-chromosome inactivation studies are widely used to assess clonality, but most assays
require relatively large amounts of high molecular weight DNA. Two PCR-based strategies, the
phosphoglycerate kinase (PGK) and the human androgen receptor (HUMARA) clonality assays
allow studies of small tissue samples. The HUMARA assay was adapted to non-radioactive analysis
taking advantage of an automated sequencer providing high resolution of alleles and immediate
quantitation. This assay was validated by comparison with X-inactivation patterns obtained by
Southern analysis with the probes M27? and PGK. Fifteen gastrointestinal carcinomas, 25 benign
goiter nodules and normal peripheral leukocytes of 27 individuals (12 who were under 15 years
and 15 over 80 years) were analysed. Furthermore, DNA extracted from formalin-fixed paraffin-
embedded tissue (FPT) was analysed with the two PCR-based methods and compared with X-
inactivation patterns determined by Southern analysis of high molecular weight (HMW) DNA.
This modified HUMARA assay is reliable in most patients; as with other clonality assays, constitutive
skewing in normal tissue precludes clonal analysis in some individuals. Extremely skewed X-
inactivation patterns were found in normal peripheral leukocytes of 7 out of 15 old females (over
80 years) and in 1 of 12 of the young females tested (under 15 years). Comparison of results
obtained with HMW and FPT DNA yielded consistent results for the HUMARA assay whereas the
PGK PCR assay was much less reliable. The HUMARA assay thus permits studies of selected areas
of tissue sections without significant stromal components, allowing correlation of histological and
genotype findings in fresh and archival specimens.
1997 Academic Press Limited
KEYWORDS: tumour clonality, human androgen receptor polymorphism, PCR, X-inactivation
∗ Author to whom all correspondence should be addressed at: Institute of Medical Oncology, Inselspital, University of Berne, CH-
3010 Berne, Switzerland.
1997 Academic Press Limited
P. Kopp et al.218
chain reaction (PCR) provided that X-linked poly-
morphic sequences and methylation-sensitive sites
are adjacent on a small DNA fragment suitable for
amplification. Both the PGK and the HUMARA gene
polymorphismsare usefulin thisrespect.10–14ThePGK
assay requires amplification of a DNA segment of
about 600bp, encompassing both the polymorphic
BstXI site and the methylation-sensitive HpaII cutting
sites. In the HUMARA locus, the polymorphic CAG
repeat is very close to the differentially methylated
sites, and amplification of a segment of approximately
300bp is sufficient.
In this study, we established a semiquantitative
non-radioactive HUMARA clonality assay using
fluorescently labelled PCR products. HUMARA X-
inactivation patterns detected by this assay were val-
idated by comparison with Southern blot analyses
using the M27? and PGK probes in a variety of
relevant DNA sources. Secondly, the PCR-based PGK
and HUMARA clonality assays were applied to the
DNA from malignant tumours.
Assessment of clonality in human tumours is of direct
relevance to theories of carcinogenesis and has prac-
tical implications for diagnosis and follow-up of dis-
ease.1,2Clonal X-chromosome inactivation analysis
in females permits studies of the clonality of tumours
regardless of their tissue origin and histological type.
The principle is based on the Lyon hypothesis: in
each female somatic cell either the paternal or the
maternal X-chromosome is randomly inactivated in
The prerequisites for clonal X-inactivation analysis
include the ability to distinguish the paternal and
maternal X-chromosome copies using DNA poly-
morphisms in heterozygous females, and the dif-
ferentiation of active or inactive alleles based upon
differences in DNA methylation which can be de-
tected by methylation-sensitive restriction endo-
nucleases (e.g. HpaII or HhaI).1,2,4Alternatively, the
analysis of X-chromosomal gene expression at the
mRNA or protein levels can be used provided that
the polymorphism is located in a coding sequence,
since only genes on the active X-chromosome will
Restriction fragment polymorphisms suitable for X-
inactivation analysis have been found in the phos-
phoglycerate kinase (PGK) and the hypoxanthine
phosphoribosyl transferase genes, but their use is
limited by relatively low rates of heterozygosity (33
or 29%, respectively).4A highly polymorphic X-chro-
mosomal probe, M27? (DXS255), detects a variable
number tandem repeat polymorphism, and about
90% of all women are informative at this locus. The
M27? polymorphism permits assessment of tumour
clonality in most females.2,5–7However, the use of
this probe can be problematic for several reasons:
constitutional methylation patterns may be tissue-
specific; skewed/non-random X-inactivation may
occur in normal blood leukocytes; and the method
requires relatively large amounts of high molecular
weight (HMW) DNA to perform Southern analysis.7–9
The detection of a highly informative CAG tri-
nucleotide repeat polymorphism adjacent to dif-
chromosomal androgen receptor gene (HUMARA)
provides an important alternative to these methods.
This polymorphism is informative in about 90% of
females, and the assay has been shown to be suitable
for assessing tumor clonality in fresh or formalin-fixed
tissue DNA.10,11This opens new avenues to explore
blot analysis, and allows one to correlate directly
clonality and morphology at the microscopic level.
Such assays are now feasible with the polymerase
MATERIALS AND METHODS
Fifteen gastrointestinal carcinomas (oesophagus, gast-
ric and colon) shown to be monoclonal on Southern
blot analysis with the M27? and/or PGK probes were
selected. In all of these patients, normal mucosa was
used as a control. Sixty nine samples were analysed
(three to six per patient). Twenty five macroscopically
distinct goiter nodules previously characterized in
terms of clonality with the probe M27? were also
pheral blood leukocytes were included as controls.
High molecular weight (HMW) DNA was extracted
by standard protocols from fresh tissue and from
peripheral blood leukocytes of 27 healthy females
(12 under 15 years and 15 over 80 years) with normal
Formalin-fixed paraffin-embedded tissue (FPT) ob-
tained from histological slides of three malignant B
cell lymphomas and three gastrointestinal cancers
(one oesophagus, one gastric and one colon car-
cinoma) was studied with the PGK and HUMARA
PCR assays. All of these neoplasms had previously
been shown to be of monoclonal origin by PGK
Southern analysis of HMW DNA extracted from fresh
tumour tissue. The lymphomas also showed clonal
immunoglobulin gene rearrangements without any
detectable germline fragments.17
in the humanX-
Clonal X-inactivation of human tumours219
Several protocols described previously were ad-
apted for extraction of DNA from FPT.18–22Tumour
tissue was fixed in phosphate-buffered formalin (4%)
regions of cells of about 5–10mm2were scraped with
sterile blades or pipette tips directly from the slide
into Eppendorf tubes under a stereomicroscope. For
very small samples, tissue flakes were suspended in
mineral oil to facilitate collection.23The tissue flakes
were deparaffinized in xylene, washed with absolute
ethanol, dried under vacuum and digested with pro-
teinase K (final concentration 200ng?l−1) in lysis
buffer (10m Tris pH7·5, 10m NaCl, 10m EDTA)
overnight at 37°C. The proteinase K was inactivated
by boiling. After extraction with Tris-saturated phenol
and chloroform/isoamylalcohol (25:24:1, v/v), DNA
was precipitated with ice-cold absolute ethanol and
solubilized in demineralized water. Samples which
were not amplified efficiently were purified further
with 25% Chelex (Bio-Rad).11
cases, three times. To allow assessment of different
allele sizes, internal size marker standards (Rox 350)
were included. The inclusion of these size markers
also facilitated the differentiation of stutter peaks
(corresponding to the shadow bands on auto-radio-
graphy) from the main peaks representing the alleles.
Allele intensity was calculated from the peak area
which is proportional to the molar amount of PCR
product from a given allele. For each sample, the
allele ratio in the non-HpaII-digested aliquot (allele
1/allele 2) was divided by the allele ratio in the HpaII
predigested sample (allele 1/allele 2). In normal tissue
this ratio is referred to as the control ratio, in tumour
ratio (lesion ratio/control ratio) was determined in
order to adjust for preferential amplification of one
of the two alleles whichmay occur in cell populations
with constitutive skewing.24A sample was defined to
be monoclonal if the corrected ratio indicated that
one of the paternal alleles was represented in an
excess of 50% or more in comparison to the other
allele (corrected ratio Ζ0·33, or [3).24Tissues of
polyclonal composition with random X-inactivation
would be expected to show ratios equal or close to
Quantitative HUMARA PCR assay
The principle of the HUMARA assay has been out-
lined in several recent publications.8,10,24Samples of
250ng of DNA were digested overnight at 37°C in
20?l reactions with RsaI (10U) alone, or RsaI (10U)
and HpaII (20U) followed by inactivation of the
enzymes by heating at 95°C for 10min. Digested
DNA (50ng) was included in a 50?l PCR reaction
containing 50mmol KCl, 10mmol Tris (pH8·3),
150?mol of each deoxynucleotide-triphosphate,
1·5mmol MgCl25% dimethylsulfoxide and 0·1pmol
of each primer:
M27? X-inactivation assay
X-inactivation analysis of genomic HMW DNA with
the probe M27? (DXS255), a 2·3kb EcoRI fragment
from an anonymous single copy genomic clone at
Xp11.22,25was performed by Southern blotting and
the results were expressed as allelic cleavage ratios
PGK X-inactivation assays by Southern blotting
Southern blot analysis of HMW DNA was performed
as described using a 0·8kb BamHI/EcoRI fragment
from clone pSPT/PGK kindly provided by Dr. B.
Vogelstein.4,6Normal mucosa was analysed and com-
pared with tissue from the gastrointestinal tumours,
but no normal tissue was available in the lymphomas.
For clonality assessment using FPT DNA, a con-
ventional PGK PCR technique, as well as a nested
PCR with several different sets of PGK gene-specific
fragments of 495 to 619bp. Positive amplification
products were obtained in only 50 to 60% of the
primers increased the success of the PCR by 10 to
20%. The hot start PCR technique26further improved
The cycling conditions were: 1 cycle of 96°C
(2·5min), 60°C (45s), 72°C (45s); 18 to 28 cycles of
96°C (45s), 60°C (45s), 72°C (45s); and 1 final cycle
of 96°C (2·5min), 60°C (45s), 72°C (2·5min). An
aliquot (1?l) of the PCR reaction was diluted to 1:20
to 1:50. The sample was loaded onto a 6% denaturing
gel (0·4mm), electrophoresed at 800 V/40 mA/28W
and analysed using the Genescan672 Software.
Each sample was analysed at least twice, and in most
P. Kopp et al.220
the efficiency of amplification, giving reproducibly
positive results in 57 of 59 separate rounds of ex-
periments, and was therefore chosen as our standard
protocol.12DNA (2–500ng) was predigested with
2–40U HpaII (Boehringer Mannheim, Germany)
overnight at 37°C. PCR was performed with the hot
start technique with wax beads according to the
CT, USA). Negative controls containing all reagents
but no template DNA were run with each PCR am-
plification. The final concentrations of reagents in-
dicated below are calculated for a total volume of
100?l. The lower layer reagent mix (25 ?l) contained
2·5?l 10× buffer II, 2·5m MgCl2, 200? of each
dNTP, 0·2pmol of each primer. The primers had the
following sequences (A1, B113and A2, B212):
BstXI overnight at 37°C. The DNA was precipitated,
washed with ethanol, dried and resuspended in 10 ?l
H2O, and electrophoresed in ethidium bromide-
stained 1·5% agarose gels. Depending on the primer
pair, the expected fragment size varied between 495
and 619bp. Although 1U of HpaII should theo-
retically cut 1?g of DNA to completion prior to PCR,
the extreme sensitivity of the PCR could lead to
amplification of small numbers of uncut allele copies
which would produce false polyclonal patterns in
truly monoclonal tumours. As a control, DNA was
predigested with MspI, the isoschizomer of HpaII,
which cleaves the site CCGG irrespective of its
methylation status. Thus, all fragments should be
cleaved and PCR should fail to provide any am-
plification products. DNA samples were also pre-
digested with an excess of BstXI prior to PCR which
would preclude amplification of polymorphic alleles
containing a BstXI site.
The upper layer reagent mix (75?l) consisted of
10× buffer II, 5U Taq polymerase, and DNA in
amounts varying from 1ng to 1?g. The cycling con-
ditions were as follows: 25–30 cycles at 95°C (1min),
step and a terminal extension step of 5min each. A
25?l aliquot of the amplification solution was then
placed in a new reaction tube and digested with 20U
Analysis of constitutional DNA with the
quantitative HUMARA PCR assay
Constitutional patterns of X-inactivation at the HU-
MARA locus were analysed using DNA from peri-
pheral leukocytes of 27 females (Table 1). The
polymorphism was informative in 26 (96%) of the
individuals. Eighteen showed random or only slightly
assay and the HUMARA assay of normal peripheral leukocytes from old (>80
years) and young (<15 years) female individuals
Comparison of X-inactivation analysis with the M27? Southern blot
∗M27? (ACR) and ∗∗HUMARA results outside the random range (moderately to
severely skewed ratios, Ζ0·33, [3). M27? results are derived in part from our previous
Clonal X-inactivation of human tumours 221
skewed ratios indicating polyclonal leukocyte popu-
lations. Eight women showed a skewed pattern, i.e.
a marked non-random distribution of the two alleles
defined as a predominance of one allele over the
other of [50%. Seven of these eight individuals were
elderly females ([80 years). When comparing the
M27? with the HUMARA results, concordant patterns
were noted in 18 of 26 cases. In six cases, the M27?
pattern was skewed and the HUMARA result was
random whilst the reverse was seen in two cases. Of
15 patients with gastrointestinal tumours, 12 were
informative for the HUMARA polymorphism; all 12
samples of normal gastrointestinal mucosa exhibited
with polyclonality and these results were confirmed
in the M27? and/or PGK assays (Table 2).
the results were fully concordant, revealing un-
equivocal monoclonality of the tumours and random
X-inactivation patterns in the corresponding mucosa
with either assay (Table 2). Cases No. 8 and No. 13
were exceptions since M27? analysis demonstrated
high ACRs in the tumour, suggesting a clonal origin,
consistent with polyclonality.
Clonal analysis of nodules from benign
The HUMARA assay was also applied to the study of
nodules from multinodular goiters where clonality
cannot easily be established by morphology alone.27
The clonal composition was examined in 25 nodules
from nine females with multinodular benign goiters
which had been studied previously with M27? and
PGK probes by Southern blotting.15In some cases,
several different nodules from the same patient were
studied (Table 3, Fig. 1). Two of the patients in-
formative for M27? (No. 1 and No. 9) were not
informative at the HUMARA locus. The HUMARA
results were consistent with M27? data in all but one
sample (No. 8.3) which showed a monoclonal M27?
pattern and a polyclonal result in the HUMARA assay
(Table 3). Patient 6 was informative for the HUMARA
Comparative clonal analysis of gastrointestinal
The HUMARA PCR assay was next compared with
standard Southern blot analyses with M27? and PGK
probes in 15 gastrointestinal carcinomas (Table 2).
Two patients (No. 4 and No. 6) were not informative
for the HUMARA polymorphism, and PCR was un-
successful in one case (No. 5). In 12 patients, the
HUMARA results could be directly compared with
either M27? or PGK analysis, or both. In 10 patients,
HMW DNA from gastrointestinal cancers and corresponding normal mucosa
Comparison of X-inactivation patterns detected by M27?, PGK and the HUMARA Genescanassay in fresh
ACR, allelic cleavage ratio; ni, not informative. Ratios: Ratio of non-HpaII-digested aliquot (allele 1/allele 2) divided by ratio of the
HpaII predigested aliquot (allele 1/allele 2). In normal tissue this ratio is referred to as the control ratio, in tumour tissue as the lesion
ratio. For tumour tissue, a corrected ratio (lesion ratio/control ratio) was determined in order to adjust for preferential amplification of
one of the two alleles. Monoclonal pattern=corrected ratio Ζ0·33, [3.
P. Kopp et al.222
Comparative clonal X-inactivation analysis of nodules from benign multinodular goiters
Lesion ratio Final ratio
ni Not informative
Constitutive skewing of normal tissue
ACR, allelic cleavage ratio; I, larger PGK BstXI allele; II, smaller PGK BstXI allele; ni, not informative. Ratios: see Methods.
Monoclonal pattern=corrected ratio Ζ0·33, [3. ∗Hypermethylation=consistent with monoclonal pattern.7†Monoclonal pattern with
the M27? assay, polyclonal pattern with the HUMARA assay.
polymorphism and showed extreme skewing in nor-
mal tissue (Fig. 2a). Disparate methylation patterns at
the HUMARA locus in different normal tissues were
observed in two patients (No. 4 and No. 7): normal
thyroid tissue showed random X-inactivation (ratios
of 1·7 and 1·32, respectively) whereas peripheral
blood mononuclear cells showed severe skewing (ra-
tios 0·12 and 7·25, respectively) (Fig. 2b).
detectable even after prolonged exposure. In three
tumours, the smaller PGK allele was methylated/
inactive and resistant to HpaII digestion, while in the
other three tumours, the larger allele was inactive.
In the three gastrointestinal cancers, histologically
normal mucosa showed polyclonal X-inactivation
patterns. These samples were subjected to repeated
PGK and HUMARA PCR analyses using HMW DNA
from fresh material, and FPT DNA extracted from the
respective histological sections (Table 4).
In the three lymphomas, the PGK PCR assay was
repeated several times using HMW DNA (case 1: 25
separate runs; case 2: 32 separate runs; case 3: 5
separate runs). Using HMW DNA, the monoclonal
it yielded a false polyclonal pattern. In three further
runs, a monoclonal pattern was produced by PGK
PCR, but active and inactive alleles were reversed
with respect to the Southern blot result. The same
problems were encountered when studying degraded
FPT DNA from these same lymphoma samples and
the three gastrointestinal tumours. A total of 59 PGK
PCR reactions were performed on the tumour samples
(2–25 per case; Table 4) and 42 of these yielded false
Clonal analysis of severely degraded DNA from
formalin-fixed histological sections of
malignant tumours with the PGK and the
HUMARA PCR assays
Three lymphomas and three gastrointestinal adeno-
carcinomas were selected for these experiments. The
lymphoma biopsy sections used for DNA extraction
contained >90% malignant cells, and the gastro-
intestinal cancers contained >70% adenocarcinoma
cells. All tumour samples showed an unambiguous
monoclonal pattern by Southern blot analysis of fresh
HMW DNA with the PGK probe (lymphomas: Fig. 3;
gastrointestinal tumours: not shown). No auto-
Clonal X-inactivation of human tumours 223
7.1 to 7.4 the shorter allele was markedly reduced in intensity after HpaII digestion, in nodules 7.5 to 7.7 the longer
allele was reduced after HpaII digestion (marked by ∗). All nodules were found to be monoclonal with the HUMARA
assay as well as with the probe M27? and the PGK probes (see Table 2). In the normal tissue both alleles were reduced
to a similar degree after HpaII digestion.
Clonal analysis with the HUMARA assay of seven goiter nodules from patient number 7 (Table 2). In nodules
polyclonal patterns. After predigestion with either
MspI or BstXI (which should impede amplification
of the target PGK fragment) small amounts of PCR
amplicons could still be detected (data not shown).
In marked contrast to the PGK PCR technique, X-
inactivation patterns detected by the HUMARA assay
were consistently reproducible with both HMW and
FPT DNA with the HUMARA method in independent
runs (Fig. 3) with the exception of one sample where
two of four reactions showed a false polyclonal pat-
tern (case 4, Table 4). The patterns obtained clearly
demonstrate reduction of one of the alleles after HpaII
digestion in the three lymphomas included in this
series (Fig. 3). However, the calculated ratios of the
lesional tissue did not reach values which are in-
4, No. 1–3). Most likely, this is due to the absence of
normal tissue as control in these three samples and
thus the inability to calculate a corrected ratio. In the
gastrointestinal tumours, we were able to include
range in 19 of 21 runs with two false polyclonal
reactions. A direct comparison of the HUMARA assay
performed with HMW and FPT DNA from an oeso-
phageal cancer is shown in Fig. 4. In virtually all
of these samples, it is apparent that skewing of X-
inactivation patterns in the clonal tumour samples is
not as pronounced in the HUMARA assay as in the
Southern blots. Despite PCR conditions within the
linear range, this phenomenon is probably due to
amplification of alleles from stromal tissue which are
not picked up by the comparatively less sensitive
The HUMARA clonality assay can be performed ef-
ficiently by using an automated sequencer and ap-
P. Kopp et al.224
296 316280300 320
The normal tissue showed a non-random X-inactivation pattern with near total reduction of the longer allele after HpaII
digestion. Although the longer allele was even further reduced in nodular tissue, extreme non-random inactivation in
the normal tissue impeded an accurate analysis in this patient. (b) Comparison of clonal analysis with the HUMARA
assay in normal thyroid tissue and peripheral leukocytes of patients number 4 and 7 (Table 2). In contrast to the thyroid
tissue where the two alleles were reduced to a similar extent, the pattern found in peripheral leukocytes indicates a
non-random X-inactivation with reduction of the shorter allele in patient number 4 and the longer allele in patient
(a) Clonal analysis with the HUMARA assay of normal and nodular thyroid tissue of patient number 6 (Table 2).
assessment of the X-inactivation patterns in a variety
of poly- and monoclonal tissue samples. In order to
validate this assay, HMW DNA from three types of
material was used: (1) normal peripheral blood leu-
kocytes from females of two age categories; (2) mono-
clonal cancers and homologous normal control tissue
results with this approach were mostly consistent with
clonal X-inactivation analyses on these same samples
with established M27? and PGK Southern blot tech-
niques. The modified HUMARA assay used in this
study has several advantages over the original HU-
MARA technique. It is more rapid since after com-
pletion of PCR, the results are obtained within 1 to
4h. The inclusion of a fluorescently labelled primer
eliminates the need for radioactivity and subsequent
autoradiography. Another important advantage is the
tometry of autoradiographs unnecessary.
The PCR-based clonality assays are of special in-
terest for the analysis of DNA from small samples and
degraded archival material.28The PGK PCR assay has
been applied to HMW DNA from haematologic cell
shal et al. used the HUMARA assay for the analysis of
both HMW and FPT DNA from several types of solid
tumours.11We initially hoped that the PGK PCR assay
Clonal X-inactivation of human tumours 225
– H+ H
– H + H
– H+ H
with FPT DNA, the PGK Southern analysis with HMW DNA. Completely monoclonal patterns were obtained with the
PGK probe (right panel). The results obtained with the HUMARA assay also suggest a monoclonal composition with
preferential reduction of one allele (left panel), but skewing of the alleles is not as marked as in the PGK Southern
Clonal analysis of three B cell non-Hodgkin’s lymphomas (a, b, and c). The HUMARA assay was performed
noted a high rate of false polyclonal patterns in mono-
clonal malignant tumours with the PCR assay re-
gardless of whether HMW or degraded FPT DNA was
normal stromal cells present in the tumour samples
and/or amplification of unmethylated alleles from the
prior to PCR. Conversely, some polyclonal tissue
lysis, most probably due to the formation of het-
eroduplexes through reannealing of strands with and
eroduplexes resist BstXI digestion after PCR am-
the BstXI site. This latter problem does not occur in
the HUMARA assay. Although the PGK PCR clonality
fail to reflect the true clonal composition of a tissue
sample. In contrast to PGK PCR, the results generated
FPT DNA were reproducible and consistent with the
reference methods in most cases.
noteworthy. (1) In normal peripheral blood leukocyte
DNA, the HUMARA assay detected extreme skewing
particularly in elderly females as previously reported
It could reflect clonal descent from a single, possibly
transformed cell, or may be a physiological phe-
nomenon increasingly seen at advanced age. Whilst
this problem is of intrinsic biological interest, it limits
the use of the HUMARA assay for clonality studies of
system.30(2) In a given individual, tissue-specific
methylation in normal tissues of different origin may
also lead to difficulties of interpreting X-inactivation
P. Kopp et al.226
Comparison of the PGK Southern blot, PGK PCR and the HUMARA PCR assays using HMW and FPT DNA
High grade non-
All 3 consistent
All 3 consistent
Low grade non-
All 3 consistent
All 3 consistent
Low grade non-
All 3 consistent
All 3 consistent
All 3 consistent
All 3 consistent
All 3 consistent
0·4: 2 fpoly
All 3 consistent
All 4 consistent
All 3 consistent
All 3 consistent
All 3 consistent
All 4 consistent
All 3 consistent
All 3 consistent
I, smaller PGK allele inactive; II, larger PGK allele inactive; consistent, PCR result consistent with result of PGK Southern blot; fmono, false monoclonal with respect to Southern blot result; fpoly, false
polyclonal with respect to Southern blot result. Numbers in the PCR columns relate to the number of separate repetitive PCR experiments performed on the respective sample. PGK Southern results indicated as
ACR; PCR HUMARA results indicated in bold type as lesional ratios in the tumour samples.
Clonal X-inactivation of human tumours 227
HMW DNA FPT DNA
FPT DNA from normal mucosa and tumour tissue were analysed in parallel. The tumour tissue is monoclonal with a
corrected ratio of 0·13 in HMW DNA and of 0·12 in FPT DNA.
Clonal analysis with the HUMARA assay of an oesophageal cancer: HMW DNA extracted from fresh tissue and
patterns.9We have previously reported this phe-
nomenon with the M27? system6and now found sim-
ilar results in some of our cases using the HUMARA
assay. (3) Three tumours clearly shown to be mono-
clonal by M27? Southern analysis appeared poly-
clonal in the HUMARA assay. This is probably due to
PCR amplification of alleles from stromal tissue which
are not picked up by the comparatively less sensitive
Southern analyses. (4) Because of the relatively fre-
quent skewing in normal tissues and preferential am-
plification of one of the alleles, the assay requires
normal control samples from the same patient.
the HUMARA assay is a valuable amendment to the
currentarmentarium ofclonal X-inactivationanalysis.
informative with the important added advantage that
the assay is readily amenable to automated PCR ana-
lysis providing increased speed and quantitation. The
assay is useful for clonal analysis of abnormal cellular
proliferation including many types of solid tumours
Since the HUMARA assay requires only very small
be dissected at the microscopical level, and the assay
allows direct histological and immunohistochemical
correlation with molecular clonality assessment. Se-
lective assessment of clonality in different zones of a
tumour is an interesting approach to study clonal
tumour heterogeneity31,32or to analyse patch size in
normal and neoplastic tissue.33However, this study
clearly identifies some important limitations of clo-
cellularproliferation derivedfrom thehaematopoietic
system and we confirm our previous observation that
non-random X-inactivation patterns are particularly
pronounced in older females. Nevertheless, the HU-
MARA assay is the only assay currently available that
permits reproducible PCR-based X-inactivation ana-
lysis from very small samples or severely degraded
DNA from archival tissue blocks.
We thank Prof H. Studer (Berne, Switzerland) for his sup-
port. This work was supported by grants 31-28744.90
(B.B.), 31-43458.95 (A.T.) and 31-28579.90 (M.F.F.) of the
Swiss National Foundation and the ‘‘Stiftung fu ¨r Krebsbek-
a ¨mpfung’’ (M.F.F.). P. K. is the recipient of a postdoctoral
fellowship of the Schweizerische Stiftung fu ¨r Biologisch-
P. Kopp et al.228
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