JOURNAL OF PATHOLOGY, VOL. 179: 162-168 (1996)

THE EXPRESSION PATTERN OF WILMS’ TUMOUR GENE (WTI) PRODUCT IN NORMAL TISSUES AND

PAEDIATRIC RENAL TUMOURS

PRAMILA RAMANI* AND JOHN K. COWELL?

*Department of Histopathology, GOS Hospital for Children NHS Trust, Great Ormond Street, London W C l N 3JH, U K.; t ICRF Oncology Group, Institute of Child Health, 30 Guiljord Street, London WC1 N 1 EH, U. K.

SUMMARY

W T l , the Wilms’ tumour suppressor gene located at chromosome llp13, plays an important role in the development of the kidney. It is mutated in 10 per cent of Wilms’ tumours (WTs) and their putative precursors called nephrogenic rests (NRs). A sensitive immunohistochemical technique was established to localize the WT1 gene product in archival normal tissues and paediatric renal tumour samples. Strong nuclear signal was seen in the various sites such as the kidneys, gonads, and decidua. Nuclear immunoreactivity of variable intensity was also seen in the skeletal muscle, smooth muscle of urinary bladder, ureter, and arteries. All 28 WTs (including the anaplastic variant) showed the WT1 gene product in a varying proportion of the blastema, epithelium, and stroma. The anaplastic nuclei in six WTs showed an intensity of staining comparable to their adjacent favourable histology counterparts. All the intralobar and perilobar types of NR demonstrated the WTl gene product. All three malignant rhabdoid tumours were positive, while three of four mesoblastic nephromas and a clear cell sarcoma were negative. These findings provide an insight into the interrelationships of these renal tumours.

KEY WORDS-paediatric renal tumours; Wilms’ tumour; nephroblastoma; WT1 gene product

INTRODUCTION

Wilms’ tumour (WT), or nephroblastoma, is the commonest renal tumour of childhood.’ Histologically it mimics the various stages of nephrogenesis, indicating aberrant differentiation of the metanephric blastema. Approximately 3 0 4 4 per cent of WTs show nephro- genic rests (NRs) in the adjacent renal parenchyma.2 These are considered to be the precursor lesions of WTs and may show various developmental fates. The precise location of a NR within the renal lobe, which is the developmental unit, probably reflects the timing of the genetic insult. There are two major topographical types of NR. Intralobar rests (ILNRs) are located deep within the parenchyma and are thus indicative of an earlier developmental disturbance. ILNRs are generally associ- ated with intralobar WTs, which recapitulate the entire spectrum of nephrogenesis and contain heterologous elements. Perilobar rests (PLNRs) occur at the periphery of the renal lobe and suggest a later developmental insult. PLNRs are associated with perilobar WTs, which are composed predominantly of blastema or epithelium and mimic the later phases of nephrogenesis, when differentiation of nephrons from blastema is the principal developmental event.2 The overall 4-year survival rate (stages 2 4 ) in the favourable histology WTs is 88 per cent, while that in anaplastic WTs is 54 per cent.3 Other renal tumours are rare and their histogenesis remains unclear.’ Mesoblastic nephroma (MN) has a good outcome, whereas clear cell sarcoma

Addressee for correspondence: P. Ramani, Department of Histo- pathology, Birmingham Children’s Hospital NHS Trust, Ladywood Middleway. Ladywood, Birmingham B16 8ET, U.K.

CCC 0022-3417/96/060162-07 1996 by John Wiley & Sons, Ltd.

(CCSK) and malignant rhabdoid tumour (MRTK) usually show a poor prognosis.’

The Wilms’ tumour gene, WTI, is a tumour- suppressor gene located at chromosome 11 band ~ 1 3 . ~ Its product appears to function as a transcription factor which represses the action of several growth-promoting genes in in vitvo transfection assays5 and is critical to the development of the kidneys.6 WT1 expression is restricted in a temporal fashion to a few fetal and adult tissue^.^^^-' In WTs its expression is seen in the malig- nant counterparts of those elements which express it during normal de~elopment .~ l 3 A similar frequency and type of WT1 mutations in NRs and adjacent WTs have provided evidence in favour of the NRs being genetic precursors of WTs.14 The distinctive phenotype and clinical behaviour of anaplastic WTs indicate that they may be genetically different from the favourable histol- ogy WTs. Only single examples of NR’O and anaplastic WT12 have been studied by in situ hybridization for WTl mRNA expression. WT1 mRNA has not been detected in the cell lines or samples from the other renal tumours, either by Northern blot analyses or by in situ hybridization.’5-18 A recent study examining WT1 gene product included only a small number of favourable histology WTs.” There are no studies examining WTl gene product in NRs, anaplastic WTs, and other rare renal tumours.

We used a sensitive immunohistochemical technique for detecting WT1 gene product in formalin-fixed, routinely-processed tissue. An affinity-purified poly- clonal antibody against the human WT1 gene product has been characterized in fetal and postnatal tissues. We report here its immunoreactivity in the various components of the two major categories of WT. In

Received 24 March 1995 Accepted 5 December 1995

WT1 GENE PRODUCT IN PAEDIATRIC RENAL TUMOURS 163

Table ILNuclear immunoreactivity of WTl gene product in fetal tissues

Tissue Sites Staining intensity

Kidney

Gonads and ducts Testis

Wolffian ducts

Ovary

UterUS

Fallopian tube

Spleen

Bone marrow

Thymus Lymph node Lungs

Heart

Arteries Intestine

Liver

Pancreas Adrenal Smooth muscle Skeletal muscle

Brain Spinal cord Nerves

Loose (uninduced) blastema Condensed metanephrogenic blasteina Renal vesicle, comma- and S-shaped bodies Visceral layer of Bowman’s capsule (podocytes) Proximal and distal tubules, loops of Henle Collecting duct and pelvic epithelium

Germinal epithelium and Sertoli cells Germ cells and Leydig cells Rete testis Mucosal lining Stroma Germinal epithelium Granulosa cells Germ cells Pelvic mesothelium (visceral) Myometrium Endometrium Mucosal lining Stroma Mesothelial lining Connective tissue cells Megakaryocytes Myeloid cells Erythroid cells Thymocytes Lymphocytes Pleural mesothelium (visceral pleura) Acini Pericardial mesothelium (visceral layer) Endocardium Myocardium Smooth muscle Mucosal lining Muscularis propria Peritoneal mesothelium Hepatocytes and bile ducts Myeloid prccursors Acini and ducts Cortex and medulla Ureter and bladder wall Tongue Neck Choroid plexus Ependymal lining Peripheral nerves Ganglion cells Ventral horn cells

0 2 3 3 0 0

3 0 2 0 1 3 2 0 3 2 0 0 2 3 2 2 1 0 0 0 3 0 2 0 1 2 0 2 3 0 1 0 0 2 2 2 3 2 0 0 3

~~ ~

Intensity of staining relative to glomcrular podocytes and gi-aded as 3=strong, ?=moderate, 1 =weak, O=iiegativc

addition. the distribution of WT1 gene product has been compared in the favourable histology and anaplastic types of WT as well as in rare childhood renal tumours, with a view to clarifying their relationship to WT.

and were obtained following therapeutic termination of pregnancy. Postnatal kidneys, ureter, urinary bladder, gonads, heart, tongue, decidua, placenta, spleen, liver, and bone marrow were also examined.

MATERIALS AND METHODS Patients and tumouv samples-The clinical features of the 28 cases of WT, together with the various compo- nents of the tumours, are summarized in Table 11. There were 11 males and 17 females. The median age of the cases (n=10) with perilobar WTs (without anaplasia) was 41 months (range 8-120 months), while that of the

Tissues and patients Normal rissue -The fetal tissues used to study the

distribution of the WT1 gene product are shown in Table I . These ranged from 15 to 20 weeks in gestation

164 P. RAMANI AND J. K. COWELL

Table II-WTs: clinical and histological features

'%) tissue components Case no. Alsex C T Blastema Tubule Glom St roma Heterotopic Anaplasia, NR

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

45lM 35lF 44lF 461M 19lM

120lF 12lM 8/M

91lF 39lF 60lM 30lM 17lF 40lF 53lF 6lF

76lM 35/M

9/F 5/F

35lM 501F 301F 28lF 39lF 39lF 96lF 151M

n Y

Y n Y n

n Y Y n Y Y Y

Y Y Y Y Y Y Y n Y n Y Y

n

11

n

PL PL PL PL PL PL PL PL PL PL PL IL IL IL IL IL IL IL I L IL IL IL IL IL IL IL IL IL

80 10 90 5 60 20 60 20 95 0 90 0 0 95 0 95

70 20 75 5 60 30 40 30 60 10 40 30 60 10 35 5 0 20 5 10

90 5 60 20

0 10 5 25 5 15

50 30 15 15 60 20 60 20 35 35

0 10 0 5 0 20 0 20 0 5 0 10 0 5 0 5 5 5 0 20 0 10

10 20 5 25 5 25 5 25 0 60

20 60 0 85 0 5

10 10 0 90 0 70 5 75 0 20 5 65 0 20 0 20 5 25

0 0 0 0 0 0 0 0 0 0 0 ca

srn, ca sk, ca, m u

sq sm, sk, sq

sk fat sk

sk, fa t srn, sk sm, sk sm, sk

ca sk

sk, ca sk

sm, sq

Uiiilateral PLNR Bilateral PLNR

Anaplasia-diffuse, PLNR

Bilateral PLNR Bilateral ILNR

Unilateral combined NR Unilateral combined NR

Bilateral WT, bilateral PLNR Bilateral WT, bilateral PLNR Bilateral W T , bilateral PLNR

Anaplasia-focal Anaplasia-diffuse, PLNR

Anaplasia-diffuse Anaplasia-diff use Anaplawa-diffuse

A=age at diagnosis (in months); F=female; M =male; C=chemotherapy prior to resection; y=yes; n=no; T=tumour type; PL=perilobar; IL=intralobar; NR=nephrogenic rest; glom=glomeruloids; sm=smooth; sk=skeletdl; mu=mucinous; sq=squamous; ca=cartilage.

cases (n= 12) with intralobar WTs was 32 months (range 5-76 months). Eighteen cases had chemotherapy prior to nephrectomy. The median age of the cases (n=6) with focal and diffuse anaplasia was 39 months (range 15-96 months) and without anaplasia (n=22) 35 months (range 5-120 months). All the stages were represented (data not shown). Case 8 showed familial inv01vement.l~ The WTs from some of these cases have been the sub- ject of a previous study.20 None of the cases showed hemihypertrophy, aniridia, or other WT-predisposing syndromes. Using the single-strand conformation poly- morphism technique, deletions/mutations in WT1 have not been detected in the constitutional cells or tumours from these patients (unpublished observations). The median age of MN cases (n=4) was 2 months (range 0.5-2 months) and that of MRTK cases ( n = 3 ) was 14 months (range 2-30 months). The boy with the CCSK was 36 months old.

Three cases (Nos21, 22, and 23) had synchronous bilateral WTs diagnosed on biopsies. Following chemo- therapy, unilateral nephrectomy was performed. Thus there were 28 nephrectomy specimens showing 11 peri- lobar and 17 intralobar WTs. All the intralobar WTs contained varying proportions of heterologous elements. One WT showed focal while five showed diffuse anaplas- tic nuclear changes.

Eleven of the 28 WTs (39 per cent) showed associated NRs. These NRs were single or multiple. Six cases (including three with bilateral WTs) had bilateral NRs. These were diagnosed on biopsies or local excisions (single or multiple) of NRs in the contralateral kidney. All the NRs were categorized with regard to their topographical location (Table 11) and putative develop- mental fates.2%2' A single NR often displayed a mixture of sclerosing with hyperplastic or sclerosing with adenomatous subtypes.

- Fig. I-WTI gene product in (A) a 16-week fetal kidney showing signal in the condensed (induced) blastema and presumptive podocytes of Lhe developing glomeruli. The collecting tubules and the loose blastema are negative; and (B) a postnatal kidney showing strong nuclear signal in the podocytes and parietal cells of the Bowman's capsule and the sclerosing tubules of PLNR

Fig. 2-WTI gene product in (A) blastema and gloiiieruloid but not in type B tubules of favourable histology WT; (B) skeletal muscle; (C) blastema of anaplastic WT: (D) stroma of anaplastic WT; (E) tubules of ILNR but not in adipocytes

WT1 GENE PRODUCT IN PAEDIATRK RENAL TUMOURS 165

166 P. RAMANI AND J. K. COWELL

lmmunohistochemistvy

Sections 4 pm thick were cut from a representative formalin-fixed, paraffin-embedded block from each case. After dewaxing and inhibition of endogenous peroxi- dase, sections were immersed in sodium citrate buffer (pH 6.0) and pretreated using hydrated autoclaving (in a domestic pressure cooker) for 2 min.22 They were incubated with the WTI antibody (1:600, Santa Cruz Biotechnology, CA, U.S.A.) for 2 h. Between successive washes with phosphate-buffered saline, sections were incubated for 35 min each with biotinylated swine anti-rabbit (1:200, Dako) followed by Strept ABC (1:500, Dako). The colour reaction was generated by incubating the slides in diaminobenzidine for 10 min and sections were counterstained in haematoxylin for 1 min.

The WT1 antibody is polyclonal and affinity-purified. It was raised in rabbits against a peptide 18 amino acids in length, mapping at the carboxy-terminus of the human Wilms’ tumour protein. Two batches of the antibody were tested in all the cases.

As negative controls for each fetal tissue and renal tumour, additional sections were taken through the procedure with omission of the WT1 antibody, or incubated with the antibody preabsorbed with the immunizing peptide (Santa Cruz Biotechnology, CA, U.S.A.). A section of a 20-week-old fetal kidney served as a positive control.

RESULTS

Normal tissue The distribution of the WT1 gene product in the fetal

tissues including kidney (Fig. 1A) is shown in Table I. The nuclear signal is in keeping with its function as a transcription f a ~ t o r . ~ , ~ In the postnatal tissue, strong nuclear labelling was seen in the visceral (podocytes) and parietal layer cells of the Bowman’s capsule (Fig. IB). The postnatal gonads showed a similar pattern of expression to the fetal gonads. The trophoblast of the placenta was negative but the decidual cells showed strong nuclear staining. Some of the myeloid cells in the spleen showed nuclear signal, as did the visceral peri- toneum. In these sites, a similar pattern and intensity of immunostaining were observed with both batches of the antibody.

The smooth muscle cells of the ureter, urinary blad- der, tunica media of the umbilical cord vessels and renal arteries, and the myocytes in the tongue and heart also displayed nuclear immunoreactivity. However, variabil- ity in the pattern and intensity of staining was observed between the two batches.

No reactivity was seen when the procedures were carried out with the omission of the primary antibody or with the antibody preabsorbed with the immunizing peptide. Weak to moderate intensity of cytoplasmic reactivity was seen in the fetal and postnatal renal tubules (proximal and distal convoluted tubules, loops of Henle, and collecting ducts), skeletal and smooth muscle cells, and hepatocytes. This was abolished when sections were incubated with the preabsorbed antibody.

Renal tumouvs All the WTs, including those with anaplastic nuclear

changes, showed nuclear immunoreactivity to the WT1 antibody in varying proportions of blastema, tubules, and stroma. There was variability in the intensity of staining in the same component of a WT. The intensity of staining was strongest in the neoplastic glomerular podocytes, similar to the non-neoplastic podocytes present in the non-tumour kidney, and was used for comparison with other components.

Strong nuclear staining was observed in 70-90 per cent of the blastema (Fig. 2A) and in 60-80 per cent of the type A tubules,23 which are derivatives of the con- densed or induced blastema and are reminiscent of the renal vesicles in the developing kidney. In the mono- morphous epithelial type of WT, all the tubules showed strong staining. Nuclear staining was not observed in type B tubules,23 which are derivatives of the ureteric bud and collecting ducts. In 30 per cent of the cases, only weak cytoplasmic staining of these tubules was observed. Patchy weak to strong nuclear signal was seen in the undifferentiated spindle-cell, myxoid, and fibro- blastic stroma in 40 per cent of the cases with both batches of the antibody.

Of the heterologous elements, nuclear signal was observed in the smooth and skeletal myocytes (Fig. 2B). However, a difference in the number and staining intensity of myocytes was evident between the two batches of antibody. Some cells comprising the squamous epithelium showed nuclear reactivity, while the mucinous epithelium, cartilage, and adipose tissue were negative with both batches of the antibody.

All the anaplastic WTs showed strong staining of a large proportion of the blastemal component with both batches of the antibody. Comparable intensity of stain- ing was seen in the cells with and without anaplastic nuclear changes (Figs 2C and 2D).

Strong nuclear immunoreactivity was seen in both of the topographical types of NR, with both batches of the antibody. The blastema of a dormant NR showed a moderate intensity of immunoreactivity, as did its coun- terpart in the adjacent WT. The majority of the tubules and glomeruli comprising the sclerosing or regressing type were positive (Fig. IB), as were the tubules com- prising the adenomatous type of NRs (Fig. 2E). Adipose tissue, the heterologous element in an ILNR, was negative (Fig. 2E).

The spindle cells in three of four MNs were non- reactive to both batches of WT1 while in one, weak nuclear staining was observed with one batch of the antibody. The podocytes in the entrapped glomeruli of all four MNs showed strong nuclear immunoreactivity. The tumour cells comprising CCSK were uniformly negative. All three MRTKs demonstrated variable intensity of nuclear and weak to moderate cytoplasmic immunoreactivity in 60-80 per cent of the tumour cells.

DISCUSSION

This study, to our knowledge, is the first demonstra- tion of the WTl gene product in formalin-fixed,

WTI GENE PRODUCT IF PAEUIATRIC RENAL TIJMOURS 167

paraffin-embedded tissue. Using the hydrated auto- claving method of antigen retrieval,22 we established an effective immunohistochemical technique to examine its distribution. Its nuclear localization is in accord with its presumed function as a transcription factor that is critical to kidney and gonadal development.’ The sites of expression i n the fetal and postnatal tissues are generally in agreement with previous studies using different techniques.’

Both batches of the antibody showed immuno- reactivity to muscle, albeit patchy and of variable intensity. The WT1 mRNA has not been reported here p r e v i o ~ s l y , ~ . ~ or has been detected only by the sensitive RNA-PCR assay.7 While cross-reactivity of these com- mercially available polyclonal antisera cannot be entirely excluded, discordant levels of the proto- oncogene BCL-2 mRNA and protein levels have been noted in normal lymphoid tissue.24 One explanation for the identification of the WTI protein in a wider spectrum of cell types may be the marked sensitivity of our technique, together with precise localization due to better appreciation of morphology inherent in fixed tissues. The cytoplasmic location of the WTI gene product in some tissues is not surprising, as it is well documented for other transcription factors such as c-mwyr gene product in formalin-fixed normal and tumour tissue.25,26

We confirmed the presence of the WTl gene product in the blastemal and epithelial components of all the WTs, including both the perilobar and the intralobar varieties. These findings are consistent with previous morphological studies.*-I3 Various groups used North- ern blot analyses to show that the highest levels of the WTl mRNA are generally present in the homologous rather than the heterologous type of tumours, thus correlating with the amount of blastemal and tubular components.”.” In the present study, demonstration of the WTI protein suggests that at least one, presum- ably functional WT/ allele is retained in these WTs.

The presence of the WTl gene product in the undif- ferentiated stromal and some heterologous elements extends the observation that WTl expression is seen in the malignant counterparts of those elements that express it during normal development.8 l 3 In all the in situ hybridization studies to date,8.10 WTI mRNA has not been detected in the stromal component of WTs, nor have recent morphological studies demonstrated the WT1 protein in the stroma.’.13 The origin of the mesen- chymal stroma in WTs has generated some debate and it has been proposed that it is derived from the induced nietanephric blastema.I0 Since the WT1 gene product is present in both of these structures, our study suggests that a part of the stroma, at least, may be derived from this blastema. A recent cytogenetic and fluorescence in situ hybridization study also provides support to our findings.”’ Variability in the intensity of WTl expression in the three histological components, as well as patchy stromal expression, may provide an explanation for the wide variation in the levels of WTl transcripts which has been noted in WTs using Northern blot a n a l y s i ~ . ~ . ’ ~ . ~ ~ - ~ ~

This is the first study of the WT1 gene product in NRs and we have demonstrated it in all the samples,

irrespective of the presumed developmental fate or topo- graphical location. The single previously reported case of nephroblastomatosis (panlobar type) which has been examined by in situ hybridization comprised only epi- thelial components and these revealed WT/ mRNA. The NRs examined in our series included all three components. The blastema and epithelium showed an intensity of signal comparable to their adjacent neoplas- tic counterparts. Adipose tissue, the usual heterologous component of I L N R s , ~ was, as in WTs, negative for WTl protein. Identical WTI mutations have been detected in 10 per cent of WTs and associated NRs, indicating that both share a clonal origin.I4 The presence of the WT/ protein in WTs and adjacent NRs is not inconsistent with the view that NRs are the genetic precursors of WTs.I4 WTi mutations in NRs represent an earlier genetic event,21,31 with WT genes at other loci contributing to the formation of a clinically detectable WT.4331

This study also demonstrated WTI gene product in all the anaplastic WTs. Only one anaplastic WT tumour sample has been examined previously using in situ hybridization.’* WTI mRNA was observed in the blastema and tubules but not in the stroma12 and no comment was made on the comparative signal inten- sities of the two phenotypes. Our findings are signifi- cant not only because the blastemal component showed the WTl protein, but also because the intensity of staining was comparable in the adjacent individual components of favourable and anaplastic WTs. There- fore our data do not suggest that the WT1 expression accounts for the differences in the phenotypc or the poor outcome of the anaplastic WTs. Mutations in the tumour-suppressor gene p53 are seen mainly in the anaplastic variety4 and it is possible that this mechan- ism plays a role in tumour progression from the favourable to its anaplastic form and contributes to its chemoresistance .4,.3

WT1 gene product has not been reported previously in MRTK and its occurrence raises the possibility of a histogenetic relationship with WT. However, at a phenotypic and clinical level, MRTK is a distinct entity.I A larger number of MRTK tumour samples need to be investigated at a molecular and immunohis- tochemical level to determine the precise relationship of these two renal tumours. The WT1 gene product was not detected in three MNs, although one tumour did display a weak nuclear signal. This is in accordance with the lack of WTI mRNA expression by Northern blot analy- ses.17,18 Although our study comprised only one case of CCSK, the findings are consonant with the report utilizing iii situ hybridization on two CCSKs. I 6 While firm conclusions regarding the histogenesis of these rare renal tumours cannot be drawn from the expression pattern of one developmental gene, our data provide some insight into their relationship with WTs.

ACKNOWLEDGEMENTS

We would like to thank Santa Cruz Biotechnology, Inc. for the gift of the WT1 control peptide, Dr P. LanXing for helpful discussions, and the oncologists and

168 P. RAMANI AND J. K. COWELL

the surgeons who were involved in the care of these patients. Our special thanks to the financial support from the Fitton Trust, MS Association, J. Sainsbury, Headley Trust, Pamela Champion Foundation, and Ince and c O . 2 and all the other friends and supporters. J. Bruce Beckwith provided the inspiration for this study.

15. Garvin AJ, Re JJ, Tarnowski BI, Hazen-Martin DJ, Sens DA. The (3401 cell line, utilized for studies of chromosomal changes in Wilms’ tumour, is derived from a rhabdoid tumour of the kidney. Am J Puthol 1993; 142: 375-380.

16. Yun K. Clear cell sarcoma of the kidney expresses insulin-like growth factor-I1 but not WTI transcripts. Am J Puthol 1993; 142 3947.

17. Tomlinson GE, Argyle JC, Velasco S, Nisen PD. Molecular characteriza- tion of congenital mesoblastic nephroma and its distinction from Wilms’ tumor. Curzcer 1992 7 0 2358-2361.

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