Coordinate Expression of Wilms' Tumor Genes Correlates with Wilms by byrnetown71


									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
                                                                                                               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
      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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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
             other        tissue
                                 and      tumor
                                                                                                                                                                                                       --i.                    (

                                                                                                                                                                                                                                                 ,4                      I
nents,    such       as muscle          (arrow-
heads) and squamous               epithelium                                                                                                                                                           -                                   e
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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

         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                  -

          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

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                                               HOMOTYPIC        WILMS    TUMORS


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                                              HETEROTYPIC       WILMS    TUMORS

             2.   Histograms
               in the homotypic

                        to an actin
                                                 relative      percentage
                                                            to those
                                                       In general,
                                                                         from normal
                                                                        WT1 expression
                                                                                                 of WIT1 and
                                                                                         of Wilms’
                                                                                            fetal kidney

                                                                                                  is highest
                                                                                                                                                                  . .
      homotypic        tumors     and significantly              lower     in heterotypic        tumors.      The
      tumors     have     been    placed        in descending        order    with   respect     to V/Il
      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,
                                                                                                                                                                                                                        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

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      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
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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.
      Tumor         stages were typically                   represented:              six in stage II,            References
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