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Vol. 3, 855-864, December 1992 Cell Growth & Differentiation 855
Coordinate Expression of Wilms’ Tumor Genes Correlates
with Wilms’ Tumor Phenotypes’
Herman Yeger,2 Catherine Cullinane, Ann Flenniken, tissue counterparts (1, 2). Thus, it is expected that the
Susan Chilton-MacNeilI, Christine Campbell,3 normal function of some tumor associated genes is to
Annie Huang, Laura Bonetta, Max J. Coppes,3 determine the differentiation phenotype of specific cell
Paul Thorner, and Bryan R. G. Williams3 types.
Classical Wilms’ tumor, or human nephroblastoma,
Departments of Pathology [H. Y., C. Cu., P. T.], Genetics [A. F.,
S. C-M., C. Ca., A. H., L. B., B. R. G. W.J, and Hematology-Oncology
manifests the morphological features of aberrant nephro-
[M. J. C.], The Hospital For Sick Children, Toronto, Ontario, genesis, characterized by persistent blastema, dysplastic
CanadaM5G 1X8 tubules, pseudoglomeruloid structures, and a substantial
supporting mesenchyme or stroma (1, 3). The propor-
tions of these components vary from infrequent to abun-
Abstrad dant within and among individual tumors. Atypical mes-
The cloning and molecular charaderization of two enchymal derived components such as striated muscle,
putative tumor genes, WT1 and Win, from the cartilage, and even bone are frequently observed (1, 3).
chromosome 1 1 p 1 3 region has provided a means of In addition, precursor lesions have been noted in 30 to
evaluating their role in the generation of Wilms’ tumor 44% of kidneys removed due to the presence of a Wilms’
heterogeneity. A series of 29 tumors were analyzed for tumor (4). From a review of the pathogenesis of these
WT1 and WIT1 expression by Northern blot or RNase tumor types, Beckwith and his colleagues (5) have re-
protedion analyses, and results were compared with cently proposed a new classification scheme for Wilms’
tumor histopathology. Tumors were scored for the tumors based on the distribution and histology of these
percentage of mesenchymal and epithelial derived precursor lesions, essentially NR.4 The classification takes
tissue components. Homotypic tumors comprised into account lobular position of the NR, proliferative
blastema, tubular epithelium, and a fibroblast-like status, and age at occurrence. ILNR occur with metach-
mesenchyme. In addition to these tissue components, ronous contralateral Wilms’ tumors and in association
the group of tumors designated as heterotypic also with aniridia and Denys-Drash syndrome, whereas PLNR
contained edopic cell phenotypes such as muscle and occur with synchronous bilateral Wilms’ tumors and in
squamous epithelium. The analyses suggest that association with hemihypertrophy and/or BWS. At the
heterotypic differentiation patterns occur when WT1 cellular level, ILNR occur with heterotypic tissues like
and WIT1 expression is low relative to normal fetal muscle and cartilage, whereas PLNR occur with the hom-
kidney. In situ hybridization using antisense RNA otypic blastema and epithelial tubule phenotype. One
probes showed that WT1 and WIT1 were concordantly possible interpretation of these observations is that, since
expressed in normal fetal kidney and in the blastema of ILNR present chronologically earlier than PLNR, ILNR
arise from genetic events earlier in development of the
tumors. The ratio of WT1:WIT1 expression remained
relatively constant in homotypic tumors but deviated nephron.
Cytogenetic studies (6, 7) carried out on patients with
significantly in heterotypic tumors. These results
suggest that expression patterns of the WT1 and WIT1 WAGR syndrome laid the groundwork for the subse-
quent identification of candidate Wilms’ tumor genes
genes can be closely correlated to Wilms’ tumor
within the chromosome 11p13 locus (8-15). The WTJ
histopathology.
gene was identified on the basis of its deletion in a
sporadic Wilms’ tumor and encodes a protein with four
Introdudion C2H2-type zinc finger motifs (8, 16). The WT1 gene is
The morphological heterogeneity exhibited by most tu- encoded in 10 exons, with two alternative splice sites
mors complicates both histopathological classification accounting for the insertion of 17 amino acids and 3
and tumor diagnosis. The basis for this heterogeneity amino acids in the generation of four possible transcripts
probably reflects aberrant regulation of the same contin- (17, 18). WT1 likely encodes a transcriptional regulatory
uum of cell differentiation usually manifested by normal protein which, based on its developmental and tissue
distribution, is postulated to be involved in genitourinary
development (8, 19-21). The sequence homology of the
zinc finger portions of WTJ and EGR1 , the demonstration
Received 4/14/92.
of V/Ti protein binding to an EGR1 consensus binding
1 This work was supported by a grant from the National Cancer Institute
of Canada (NCIC) with funds from the Canadian Cancer Society to H. Y.
and B. R. G. W.; a Steve Fonyo NCIC studentship to A. H.; an Ontario
Graduate studentship to L. B.; and a TerMeulen Fonds award from the
Royal Netherlands Academy of Arts and Science to M. J. C. 4 The abbreviations used are: NR, nephrogenic rests; PLNR, perilobar NR;
2 To whom requests for reprints should be addressed, at Department of ILNR, intralobar NR; BWS, Beckwith-Wiedemann syndrome; WAGR,
Pathology, The Hospital For Sick Children, 555 University Avenue, Wilms’ tumor-aniridia-genitourinary abnormalities-mental retardation;
Toronto, Ontario, Canada M5G 1X8. kb, kilobase(s); bp, base pair(s); LOH, loss of heterozygosity.
Present address: Department of Cancer Biology, Cleveland Clinic Foun- S B. R. G. Williams, C. Campbell, P. Huang, and L. Bonetta, unpublished
dation Research Institute, Cleveland, OH 44195. observations.
856 Histopathogenesis of Wilms’ Tumor
sequence (22), and the ability of two of the potential results of our studies support a molecular basis for the
transcripts to repress transcription via binding to EGR1 histopathological differences observed among Wilms’
consensus binding sequences (23) suggest multiple func- tumors.
tional roles for WTJ transcripts in genitourinary devel-
opment. Pelletier et a!. (24, 25) have described two
Results
Wilms’ tumor cases and 10 patients with Denys-Drash
syndrome (26), all of whom carry heterozygous germ- Histopathology. The majority of (27 of 29) of the tumor
line mutations in WTJ. In each case, the wild WTJ allele specimens contained varying combinations of blastema,
was absent in the tumor DNA. Since both groups of tumor epithelial structures, and tumor stroma (Fig. 1A;
patients presented with a spectrum of genital abnormal- Table 1 ). In general, blastema predominated, followed
ities, it is evident that abnormal WT1 expression levels by epithelial tubules and stroma. Only two Wilms’ tu-
may contribute to development of genitourinary abnor- mors, WiT-6 and WiT-34, lacked any significant epithelial
malities or Wilms’ tumorigenesis. or stromal tumor component, respectively. Pseudoglom-
The status of the WT1 gene in the majority of sporadic erular structures and primitive glomeruloids were found
Wilms’ tumors not involving genitourinary abnormalities in 21 Wilms’ tumors. These structures appeared to be
remains unclear. Data supporting WT1 as a Wilms’ pre- randomly distributed among the different tumors.
disposition gene have been reported for a small number Seventeen of the 29 tumors were designated as horn-
(<5%) of sporadic tumors showing structural re- otypic (Table i) and 1 2 of 29 as heterotypic (Table 1).
arrangements within the WTJ gene (10, 27-33). A case The most frequently occurring heterotypic elements
of bilateral Wilms’ tumor showing an 1 1-kb intragenic were muscle (rhabdomyomatous) and squamous epithe-
deletion in WT1, in which loss of the wild-type allele in hum (Fig. i B). Five of the 1 1 (45%) rhabdomyornatous
the tumor was accompanied by loss of heterozygosity of cases showed concordant development of muscle with
both 1 1 p and 1 1 q DNA markers in one tumor and just squamous epithelium. The combination of muscle and
1 1 p markers in the other, further supports this contention bone elements was seen in two cases, and one case each
and provides evidence for multiple mechanisms whereby had cartilage or neuronal (ganglion) elements.
loss of the wild-type allele may occur (29). However, for Anaplasia was noted in six cases (four focal and two
the majority of Wilms’ tumors, the predisposing gene diffuse) and nephroblastomatosis (NR) was seen in four
rearrangements have not been identified. cases. The anatomical origins of the NR were not
Additional loci are involved in non-i 1 p associated determined.
familial Wilms’ tumors (13, 34) and BWS associated Overall, several trends were noted in the data. First,
Wilms’ tumors (35, 36). LOH studies on WAGR patients the homotypic group showed a higher cumulative per-
with a constitutional deletion within 1 1p1 3 (37) and in a centage of blastema plus epithelium, whereas the het-
subgroup of sporadic Wilms’ tumors (38, 39) showed erotypic group showed a higher percentage of stroma.
LOH changes in only the 1 1 p1 5 region, implicating both
Secondly, muscle elements dominated within the heter-
loci in the development of at least a subgroup of Wilms’ otypic group. Finally, all three cases of bilateral Wilms’
tumors. Devilee et a!. (40) showed loss of heterozygosity tumors fell into the homotypic group. Two of the three
only on 1 i p for Wilms’ tumor (38% of informative cases)
bilateral cases were associated with other syndromes.
and not on chromosomes 3p, 5q, i3q, 17p, 18q, and
Molecular Analysis.
Northern blot analysis for WT!
22q, which are frequently involved in breast and colon
and RNase protection analysis for W!T1 were performed
carcinomas. Furthermore, loss of heterozygosity studies
on the 29 Wilms’ tumors described in Table i The results .
have implicated both chromosome 1 1 p and i 6q in
are presented in histogram format as Fig. 2 and represent
Wilms’ tumorigenesis (41-43) and tumor suppression
percentage expression levels relative to that in human
(44). Thus, we are left to consider two other gene loci
fetal kidney (set at iOO%) and as normalized against an
that can contribute not only to the initiation of Wilms’
actin signal. Within the homotypic group of Wilms’ tu-
tumors but to the observed histopathological variations.
mors, 12 of i 7 expressed relatively high to moderate
To further complicate the genetic picture of Wilms’
tumor, a second gene, WIT!, lies proximal to WTJ and is
amounts of WT! and WIT! mRNA and 5 of 17 expressed
low to barely detectable amounts of WT! and WIT!. In
transcribed divergently. Although no point mutations
have been found in W!T1 to date, the proximity of the contrast, in the heterotypic group of Wilms’ tumors, only
two genes and their coordinate pattern of expression (14, 1 of 1 2 tumors expressed WT! at a level greater than
15) have led us to believe that WTJ and W!T1 may share 40% of normal fetal kidney, whereas the remainder ex-
at least some transcriptional regulatory elements and pressed significantly lower amounts of WT! transcript.
therefore WIT! may also play a role in Wilms’ tumorigen- Three of i 2 tumors expressed moderate to high amounts
esis. Preliminary evidence has been provided that both of WIT! transcript but with comparably lower amounts
WT1 and WIT! are coordinately expressed in Wilms’ of WT!. Overall expression of WT! and WIT! was mark-
tumors (14). Since this preliminary analysis of WT! and edly less than that in the homotypic group. As expected,
WIT! expression in Wilms’ tumors suggested coordinated the WiT-i 3 tumor, which has been shown previously to
expression of the genes, we asked whether there was a be homozygously deleted for the WT! and WIT! genes
relationship between gene expression and tumor phe- (15, 43), did not express either mRNA. An example of a
notype. We now report on a series of Wilms’ tumors that Northern blot and RNase protection analysis demonstrat-
have been examined histopathologically and analyzed ing the relative expression levels of WT! and WIT! in a
for their expression of WT! and WIT!. A strong correla- subset of the tumors is shown in Fig. 3. In summary, the
tion has been found between differential expression of analyses show that 1 3 tumors, all but one of them horn-
these genes and the frequency and distribution of the otypic had relatively high to moderate expression,
various mesenchymal and epithelial components. The whereas most of the tumors in the heterotypic group and
Cell Growth & Differentiation 857
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. ‘.*
: -,-
- --.
p- . .:
‘:4
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i--, .- -.
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fig. 1. a, the homotypic pattern
, ..
of Wilrns’ tumor histology. BIas- -, .-
tema (B) and tubules (T) are or-
ganized into nodules sur-
rounded by tumor stroma (Se.
x66. b, heterotypic components
in Wilms’ tumor. In addition to
blastema,
stroma,
tubules,
other tissue
and tumor
compo-
--i. (
;.:
,4 I
nents, such as muscle (arrow-
heads) and squamous epithelium - e
(Sq) are found
stromal cells. x66.
surrounded by
--
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, :.-!: .f.
;w. d’. s” tr
4’ : ‘ .#
--,- --
- ..‘- #{149}
#{149}.#{149}$‘-
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.-
Table 1 Wilms tumors grouped according to percentage fractions of blastema, epithe(iuni. and necrosis and scarring
The presence or absence of heterotypic components, glomeruloid structures, and nephrogenic rests is noted. Anaplasia i s either foal r diffuse when
present. Heterotypic cell types are: M, muscle; Sq. squamous epithelium; B, bone; C, cartilage; N, neuronal.
. , . . . Heterotypic Necrosis/
Wilms tumor /o blastema /o tubular epithelium /o stroma ± glomeruloids ± NR Anaplasia
elements sarring
Homotvpic
WiT-32 35 60 5 + - - 20 -
pWiT-1O 10 85 5 + - - <5 Focal
pWiT-31 50 40 10 + - + 5 -
W,T21b 45 50 5 + - - <5 -
pWiT-12”(Blooms( 80 15 5 + - + 10 -
pWIT-25 70 20 10 + - - 5 -
WIT-40 65 10 25 - - - 30 -
WiT-34 70 30 0 + - - 5 Focal
pWiT-7 70 20 10 + - - 10 Diffuse
W1T-36 50 50 <5 + - - 50 Diffuse
pWiT-30 70 15 15 + - - 15 -
pWiT-52 55 40 5 - - - 10 -
pWiT-18 60 10 30 - - - SO -
WiT28b (Henii(’ 80 15 5 - - - 40 -
WjT49b 20 40 40 + - + 10 Focal
WiT-56 40 50 10 - - - <5 -
WiT-35” 15 25 60 + - - 15 -
Heterotypic
pWiT-1 40 20 40 + M, B - 40 -
pWiT-27 30 35 35 + M - 10 -
pWiT-19 60 25 15 + M - <5 -
pWiT-15’ 45 45 10 + M, B, C + 5 -
pWiT-24” 30 10 60 + M, Sq - 60 -
pWIT9d 35 15 50 - M, Sq - 5 -
W1T-45 40 10 50 + M, Sq - 35 -
pWiT-26 70 5 25 + M - 10 -
WiT-38’ 15 20 65 - M, Sq - 50 -
pWiT-6 60 0 40 - M, Sq - 10 -
pWiT-5’1 20 10 70 + M, N - 25 -
pWiT-13 45 15 40 + Sq - 10 -
p-tumor tissue acquired prechemotherapy.
b Bilateral Wilms’ tumors.
( WiT-28 (Hemi), patient with documented hemihypertrophy.
d Where percentage total of blastema and epithelium is 50, and where percentage of mesenchyme is 50.
858 Histopathogenesis of Wilms’ Tumor
51) U WTi
Ki 2345
z 0 win
;!
0
0
HOMOTYPIC WILMS TUMORS
a
I
, .
U WT1
0 win
. :1.
z
b
HETEROTYPIC WILMS TUMORS
Fig.
WT1
2. Histograms
in the homotypic
Densitometric
normalized
values
relating
to an actin
were
versus
related
signal.
relative percentage
heterotypic
to those
In general,
expression
groups
from normal
WT1 expression
of WIT1 and
of Wilms’
fetal kidney
tumors.
is highest
and
in
. .
homotypic tumors and significantly lower in heterotypic tumors. The
tumors have been placed in descending order with respect to V/Il
C
expression values. All low expressing tumors showed a detectable but
not necessarily measurable quantity of WT1 or WIT1 expression, with the
fig. 3. Molecular analysis of WT1 and WIT1 expression in Wilms’ tumors.
exception of WiT- 1 3 which had no expression of either gene.
a, Northern blot analysis of WT1 mRNA expression in a series of Wilms’
tumors and in comparison to normal human fetal kidney (K); the 3-kb
WT1 transcript is indicated (arrowhead). b, RNase protection analysis for
WIT! in the same series of Wilms’ tumors. A protected band of 175 bp
5 of 1 7 in the homotypic group expressed at relatively is indicated (arrowhead). c, the Northern blot was reprobed with an actin
low levels. probe to correct for differences in loading of the RNA. a-c: tumors: 1,
WiT-34; 2, WiT-40; 3, WiT-38; 4, WiT-36; 5, WiT-35; M, markers.
In general, a higher expression of WT! Correlated
positively with the presence of a significant (>50%) con-
tent of blastema and epithelial derivatives. It will be
noted, however, that in the WiT-13 tumor, which lacks the same cells. Cross-sections of the fetal kidney dis-
expression of both WT! and WIT! mRNA, there are the played the various stages of nephrogenesis including
classical histopathological features of blastema, epithelial condensing metanephric mesenchyme, S-tubular vesicle,
tubules, and a tumor stroma. The presence of a small
and early glomerular stages. WT! expression was local-
number of heterotypic squamous elements placed WiT-
ized to the condensed blastema, the presumptive po-
13 in the heterotypic group. The presence of squamous
docyte layer of the S shaped bodies, and continued to
epithelium in the absence of heterotypic mesenchymal
be expressed in the immature glomeruli (Fig. 4, a and b).
derived elements such as muscle, bone, or cartilage when
The hybridization signal was relatively low in the con-
both WT! and WIT! are not expressed may be of histo-
genetic significance. densing blastema and most intense in the S shaped
In situ hybridization was used to examine and compare bodies. In the more mature glomeruli within the interior
the expression patterns of the WT! and WIT! genes in of the kidney, the hybridization signal progressively de-
normal fetal kidney and in tumors representing high, low, creased. No WT! expression was detected in the ureteric
or no expression by RNA analysis. We also wanted to bud, tubules, or supporting stroma (Fig. 4, a and b). The
determine whether WT! and WIT! were expressed in WIT! expression pattern paralleled that of WT!; however,
c#{128}’ll
Grovi,’th & Ditterentiation 859
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fig. 4. Localization by in situ hybridization of WT1 (a and h) and LVT! ft and d( tr,inscripts within a (r(iss-section of normal fetal kidney. ‘(‘(ti(ins were
photographed with bright-field illumination (Id!) to depict the morphology and with dark-field illumination (right( to show hvbridiiation signals. The
blastema (B), S-tubules (5), and glomeruli (C) show expression ot 1)0th A’Tl and NIT!. The strom,i (! ) and uret(’ri( bud (!‘( do mit t’\press ‘Tl ,3nd
WIT! . Note a consistently higher (- 10-fold) expression of AT 1 over SIT 1 in these tissu(’ comp(inents. The “‘TI .10(1 ‘IT I sense priil)(’s sv’re ifl( luded
in all experiments and gave no labeling above background. x46.
the intensity of the hybridization signal appeared to be WIT! or WT1. The relatively high percentage (60%) of
approximately one-tenth that of WT! (Fig. 4, c and d). stroma coupled with variable blastemal expression could
In situ hybridization performed on the three cases of have accounted for an overall assessment of low WIT!
Wilms’ tumor confirmed the Northern and RNase pro- and WT! expression. In contrast, low expressing tumors
tection data but with an interesting addition. In tumor WiT-28 and WiT-56 contained only small amounts of
tissue, the hybridization signals for both WT! and WIT! stroma.
were localized to blastema and epithelial elements and In summary, the presence of WT! and WIT! expression
were not found in the stroma. In contrast to normal fetal in Wilms’ tumors approximating normal fetal kidney 1ev-
kidney, tumor tubules expressed WT1 and WIT!. Tumor els correlates positively with the absence of heterotypic
WiT-7, a homotypic tumor with essentially high levels of differentiation and the dominant classical blastemal/epi-
WT! and WIT! expression by quantitative RNA analyses, thelial phenotype. The exception was WiT-28, from a
showed an intense WT! signal in the blastemal tissue and patient with hemihypertrophy whose tumor exhibited
only a background signal in the stroma (Fig. 5, a and b). low WT! and WIT! mRNA levels but a predominance of
As in the normal fetal kidney tissue, the signal intensity blastema and epithelium. Since, the hemihypertrophy
of WIT! relative to WT! was approximately one-tenth associated BWS locus resides within the 11p15.5 region
(Fig. 5, c and d). Tumor W1T-13 is a tumor which has a (1 9, 20), the anomalous phenotype o WiT-28 may reflect
homozygous deletion of the 1 1 pi 3 region encompassing the interaction of WT! and/or WIT! with a second puta-
the WT! and WIT! genes. WiT-i3 therefore provides a tive Wilms’ tumor locus at 1 1 p1 5.5 or with other genes
negative control for the antisense RNA probes for WT! in this region.
(Fig. 5, c and 1) and WIT! (Fig. 5, g and h).
The homotypic WiT-35 tumor expressed a low amount
of WIT! and a still lower level of WT! mRNA (Fig. 2). In Discussion
situ hybridization revealed that clusters of normal ex- In this report, we have quantitated the major tissue
pressing tumor blastema coexisted with adjoining low components present in Wilms’ tumors in order to assess
expressing tumor blastema (Fig. 6, a and b). Heteroge- the relationship between pathology and WT1 and WIT!
neity of WIT! and WT! expression within this region was expression at the mRNA level. A markedly decreased
noted, whereas adjacent stromal areas did not express expression for 1)0th WTJ and WIT! was observed in those
860 II istopathog’ni’i it \\i)ru’ T tiniiir
St.., ,,
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I ig. ‘. In situ hhridii,ition of ‘ViT -7 Ii (I) md Vii .1 ) (e- h) for (\I)ression ot 14Tl i, Li, e, ,mfl(l I ) an(l (‘,‘lTl (, (I, g, and h). In VViT-7, the tumor
l)l,lSt(’i’fl,l (B) t’x1)rt’ss’d both VT I 10(1 Vi(T 1 in .i r,ilio (‘quivalent to that seen in fetal kidi’y (Fig. 4). The stroma ( St ( is negative. In WiT- 1 3, a tumor
hiimi)LygousIv (l(’)(’t(’(l for L’T I 01(1 Vi’(T I , no expressii)n is observ(’(l ti)r #{188}T ,in(l
1 V’IT I . a, ( , e, ,In(l g. brighttield; h, d. t, and h, dark-field niicrographs.
X66.
.
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I ig. 6. In ifii hvhridii,ition i)I \ViT- 3 ti)r (VT I i md h( and L’lT 1 ( an(I (I). L’i/Tl md Vi’IT 1 expression ixcurs focally within a blastenia (B) zone. Both
l)(iSitiI ,lfl(l il(’g,ilIv(’Iv expr(’ssing I)I,istenla cells ( i)e\ist in a(Ija( ent ,Ire,ls. The slron#{236}a(St ( is nonexpressing. Ag,iin, VIT 1 expression is significantly
higher thin that (it Vi(T I . a ,ln(l ( , bright-field; b ail ri, (l,trk-fiel(I mi(rogr,Iphs. X66.
Cell Growth & Differentiation 861
tumors exhibiting heterotypic components, mainly mus- in general, the Northern and RNase protection data were
cle and squamous epithelium. In this study, we could paralleled by the in situ hybridization studies, one excep-
demonstrate that tumors composed of >90% blastema tion, WiT-35, brought up the question whether other
plus tubular epithelium (e.g., WiT-56) showed low or factors affect expression of WT! and WIT!. In WiT-35,
nondetectable WT! and WIT! expression, whereas oth- blastemal foci contained both high expressing and low
ers (e.g., WiT-36) with significant necrosis and scarring, expressing cells. These two cell populations were indis-
showed normal expression levels for both genes. Thus, tinguishable at the histological level but were easily dis-
WT! and WIT! mRNA levels do not simply reflect the cerned from the nonexpressing stroma. Thus, it is possi-
presence of blastemal and epithelial components. This ble that WT! and WIT! gene expression can turn off prior
point is further strengthened by the fact that the only to morphological differentiation, indicating that a com-
tumor in which we can be certain both transcripts are mitment to an alternate differentiation pathway (e.g.,
completely absent, WiT-i3, which is homozygously de- stroma) has been made. It is conceivable that expression
leted for the WT! and WIT! genes, is also the only levels must be maintained sufficiently high and in a
heterotypic tumor in this group with no muscle compo- critical ratio to allow, for example, podocytic differentia-
nent but with otherwise classical Wilms’ tumor histology. tion, to proceed. Interruption of WT! and WIT! expres-
In a recent report by Gerald et a!. (45), 27 Wilms’ sion during this commitment phase could result in the
tumors of determinable histology were found to express evolution of a variety of histopathological variants. How
elevated levels of WT! These tumors were noted to lack
. WT! and WIT! are controlled at the transcriptional level
heterologous elements, represented by striated muscle, is yet to be determined, but it has been suggested that
bone, and cartilage, whereas WT! expression varied over WT! may regulate its own transcription (20). Expression
a 100-fold range. In the group of tumors possessing of WT! in tumor epithelial tubules suggests a relaxation
heterologous elements, WT! was expressed in signifi- of restricted gene expression in Wilms’ tumors and may
cantly lower amounts. In a similar type of study, recently contribute to a loss of structural tissue organization and
reported by Miwa et a!. (46) on 20 cases of Wilms’ cell-cell interactions. On the other hand, the finding in
tumors, a positive correlation was shown between high this study that WIT! expression parallels that of WT! in
expression of WT! transcripts and predominance of bIas- normal kidney and diverges in tumors and the possibility
tema, being highest in undifferentiated variants. Again, that transcriptional regulation of both genes is coordi-
predominantly stromal tumors expressed low or unde- nately controlled suggest a mechanism whereby disrup-
tectable amounts of WT! transcripts. Our results in gen- tion of this regulatory function could lead to a more
eral confirm the observations of Gerald et a!. (45) and permissive differentiation state and relaxed expression of
Miwa et a!. (46) but in addition suggest that there may WT!. Since WT! can encode four possible transcripts,
be exceptions in the relationship between expression of specific combinations or ratios of transcripts may dictate
WT! and the differentiation of blastema and epithelium patterns of tissue differentiation. Northern analyses have
in Wilms’ tumors. only indicated that WT! mRNAs can be transcribed in
Our in situ hybridization results demonstrate that WT! Wilms’ tumors in relative abundance. Whether these
and WIT! are developmentally and coordinately ex- transcripts produce functional proteins remains to be
pressed in both normal kidney and tumor tissues. The in determined. Aside from its tumor suppressor role, WT!
situ hybridization studies on normal fetal kidney con- may function to suppress unscheduled differentiation,
firmed the previous (47) localization of WT! expression but, as demonstrated by the WiT-13 tumor, it cannot be
to blastema, S-tubular, and early glomerular stages in the only determinant for differentiation. Certainly, the
nephrogenesis. The identical spatial distribution and ap- presence of a second Wilms’ tumor gene on 1 1p15.5
parent constant ratio (‘-lO:l of WT! :WIT!) argue strongly associated with the BWS syndrome (35, 36) and a puta-
in favor of their coregulation, and taken together with tive third Wilms’ tumor locus on 16q (27, 43) suggests
the close proximity of their transcriptional start sites, additional mechanisms for regulating nephrogenesis and
support the hypothesis that WT! and WIT! may share for circumventing the consequences from loss of WT! or
transcriptional regulatory elements (14). This coordinated functional inactivation.
pattern of WT! and WIT! expression suggests a role for Are there other factors that could interact with WT!/
both genes during the glomerular developmental phase WIT! to determine the etiology and histopathology of
in nephrogenesis. Since the expression levels of both Wilms’ tumors? Recent reports from two other laborato-
genes were lowest in blastema and highest in the 5- ries suggest that the PAX2 gene (48, 49) may play a role
tubular forms and then dropped off progressively as in the development of Wilms’ tumor. Expression of hu-
glomeruli matured but were absent from tubules, it is man PAX2, encoding a paired-box containing protein and
likely that their role is in regulating glomerular differen- potential transcription factor (50), was not attentuated in
tiation and possibly functional maturation, and not de- the nephrogenic rests of residual normal juvenile kidney
velopment of the other epithelial tubular cells of the tissue adjacent to a Wilms’ tumor and in the tumor itself
nephron. Together with the studies presented here, evi- (49). In normal kidney, expression of PAX2 was observed
dence has been provided for the restriction of not only in the progenitors, leading to differentiation of epithelial
WTJ but also WIT! expression to progenitor cells that tubules and collecting ducts, in contrast to WT!, which
generate podocytes in normal kidney. is restricted to glomerular differentiation. In addition to
Pritchard-Jones and Fleming (21) recently reported this already complex picture is added the growing body
that, by in situ hybridization, WT! was expressed in of evidence suggesting that imprinting may play a signif-
blastema, immature tubules, and pseudoglomeruli of icant role in determining the expression pattern of genes
Wilms’ tumors, but not in stromal or heterologous ele- during organ and tissue development (51). In fact, two
ments. We observed a similar expression pattern not genes, IGF2 and H!9, which map in the same region of
only for WT! but also for WIT! in our tumors. Although, 11p15 as the putative BWS locus, may be potential can-
862 Histopathogenesis of Wilms’ Tumor
didate genes involved in overgrowth and tumor devel- terms of the percentage of different cell phenotypes
opment (52, 53). If imprinting contributes to the control (Table 1). Tissue phenotypes included blastema, epithe-
of gene expression, then we might expect to find specific hum (tubular, pseudoglomeruli, or glomeruloid and squa-
patterns of expression from the 1 1 p loci dependent upon mous), and mesenchymal derivatives (muscle, cartilage,
their parental origin. The preferential loss of the maternal bone). All tumors except one (WiT-34) showed a variable
chromosome 1 1 and retention of the paternal chromo- amount of a fibroblastic stroma in addition to the other
some 1 1 with further duplication, as shown by restriction components. The degree of necrosis, a common feature
fragment length polymorphism analysis in the majority of in Wilms’ tumors, and scarring (most likely a posttreat-
Wilms’ tumors exhibiting LOH (54-57) and in BWS (58), ment effect), the presence of focal or diffuse anaplasia,
could dictate the overall pattern of gene expression. For and the presence of nephroblastomatosis (typified by
one of our homotypic cases with classical triphasic his- NR) were recorded. A minimum of 4 and up to 20
tology, WiT-28, from a patient exhibiting hemihypertro- different tissue samples per tumor were reviewed, and
phy, in which WT! and WIT! are expressed at low levels, sections from these blocks were scored for the presence
this second locus on 1 1 p1 5.5 may have influenced of heterologous elements. Overt necrotic areas were
expression from the 1 1 p1 3 locus. However, in cases like avoided. The histopathology of these tumors was desig-
WiT-13, in which WT! and WIT! are homozygously nated as homotypic if containing only blastema, tubules,
deleted and both chromosomes 1 1 have been retained and a fibroblastic stroma, and heterotypic if containing
(59), determination of tumor histology from an alternate any or several of the ectopic tissue elements of squamous
locus is postulated. Thus, it is hypothetically feasible that
epithelium, muscle, cartilage, bone, and rarely neural
the etiology and ultimate histogenesis of a particular
corn ponents.
Wilms’ tumor are the result of the balanced expression
Molecular Analyses. The preparation of RNA with the
from these three loci or as further modified by inactiva-
method described by Chornczynski and Sacchi (62) and
tion of any one of these genetic inputs. Until the other
procedures for Northern hybridization and RNase pro-
genes have been identified, present data suggest that the
tection have been previously reported (14). WT! was
WT!/WIT! complex contributes significantly to both the
detected with a 1.8-kb fragment of 3lEl and WIT! with
etiology and histopathology of Wilms’ tumors.
a 175-bp probe representing nucleotides 1 to 175 (14).
Densitometric scanning of the X-ray films was utilized to
Materials and Methods quantitate WIT! by RNase protection and WT! by North-
Patients and Tissues. From January 1 982 to December em RNA signals and expressed relative to fetal kidney
1 989, 29 cases of Wilms’ tumor collected at The Hospital (14); these data were normalized against an actin probe
For Sick Children, Toronto, Ontario, Canada, were found signal and are presented in histogram form with the
to be suitable for a study to determine the relationship expression in fetal kidney set at 100%.
between tumor histopathology and expression from the In Situ Hybridization. In situ hybridization was per-
WT! and WIT! genes. Twelve of these Wilms’ tumors formed as previously described by Flenniken and Wil-
had been studied previously with respect to immunohis- hams (63) and Davis et a!. (64). RNA probes were labeled
tochemical and lectin histochemical characterization of with [35S]UTP (410 Ci/mmol; Amersham). The antisense
the primary tumors and their biological characteristics and sense WT! probes were prepared using the 1 41 9-bp
when xenografted in nude mice (60, 61). Fetal kidney
BamHI-EcoRl fragment (excised from complementary
tissue from the 12- to 20-week gestational period was DNA 31E1; 14). The antisense and sense WIT! probes
obtained as previously described (14).
were synthesized using the 2019-bp complementary
Twenty-four tumors presented with unilateral Wilms’
DNA (GB16; 14). All probes were lysed to approximately
tumor and 5 with bilateral Wilms’ tumor (Table 1). None
1 50 bp by alkaline hydrolysis according to Hogan et a!.
of the patients had aniridia and/or genitourinary abnor-
(65). Hybridization and washing procedures were carried
malities. One patient (WiT-28, Table 1) had been diag-
out as described by Wilkinson et a!. (66).
nosed with hemihypertrophy and another (WiT-12j with
Bloom’s syndrome. The Wilms’ patients collected at The
Hospital For Sick Children ranged in age from 3 months Acknowledgments
to 1 3 years and 4 months at first diagnosis. There were The authors thank Joyce Woolley for secretarial assistance and Mike Starr
12 males and 17 females. Those patients yielding tumor for photography.
samples prior to chemotherapy are indicated in Table 1.
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