Vol.
`
`4,
`
`2419-2424,
`
`October
`
`/998
`
`Clinical
`
`Cancer
`
`Research
`
`2419
`
`of Cadherin-6
`Expression
`of Patients
`Prognosis
`Cell
`Carcinoma
`
`Diagnostic
`a Novel
`as
`E-Cadherin-absent
`
`Tool
`Renal
`
`with
`
`To Predict
`
`Downloaded from http://aacrjournals.org/clincancerres/article-pdf/4/10/2419/2069209/2419.pdf by guest on 14 June 2023
`
`essential
`RCC.
`
`role
`
`in
`
`cell-cell
`
`adhesion
`
`in
`
`E-cadhenn-absent
`
`INTRODUCTION
`the most
`is
`metastasis
`a distant
`of
`existence
`the
`In RCCs,2
`(1-4).
`survival
`patient
`predicting
`for
`factor
`powerful
`prognostic
`to a
`the
`primary
`tumor
`from
`cascade
`Although
`the metastatic
`adhe-
`loss
`of
`cell-cell
`controversial,
`distant
`organ
`still
`remains
`of
`this
`as
`a first
`step
`role
`an
`important
`is
`sion
`considered
`to
`play
`to pre-
`functions
`E-cadherin
`cascade
`In many
`carcinomas,
`(5).
`this mol-
`decreased
`expression
`of
`serve
`integrity,
`and
`epithelial
`presence
`of metastatic
`disease
`and
`ecule
`with
`the
`is
`associated
`with
`of patients
`(6-8).
`poor
`prognosis
`reported
`have
`(9)
`Although
`Katagiri
`et
`a!.
`value
`has
`a prognostic
`expression
`E-cadherin
`of
`approximately
`no
`more
`than
`in
`cadherin
`expressed
`is
`of RCCs
`the majority
`in fact,
`RCCs
`10). And
`renal
`proximal
`in
`the
`epithelia
`originate
`from
`the
`We
`have
`reported
`is not
`expressed.
`which
`E-cadherin
`( 10)
`mRNA
`of
`several
`lines
`expressed
`cell
`that
`RCC
`and
`cadherin-6,
`with
`N-cadherin,
`such
`as E-cadherin,
`than
`E-cadherin.
`Nouwen
`frequently
`two
`expressed
`more
`expression
`in
`the
`proximal
`(1 1)
`observed
`N-cadherin
`In
`our
`study
`on RCC
`epithelia
`of
`normal
`kidneys.
`expressions
`of N-cadherin
`however,
`we detected
`mRNA
`these
`expressions
`did
`the
`cell
`lines
`observed,
`and
`correlate
`with
`cellular
`morphology
`(10).
`Cadherin-6
`nal]y
`isolated
`from a hepatocellular
`carcinoma
`cell
`E-
`and
`P-cadherin,
`and
`shown
`it was
`that
`normal
`RCC
`cell
`lines
`expressed
`cadherin-6
`mRNA
`dem-
`which
`(12),
`(13).
`rat K-cadherin
`onstrated
`97% homology
`with
`the
`cDNA
`of
`Hence,
`we hypothesized
`that
`cadherin-6,
`rather
`than N-cadherin,
`might
`be
`an
`integral
`molecule
`in the
`cell-cell
`adhesion
`in RCC.
`In
`this
`study,
`we
`a newly
`developed
`anti-cadherin-6
`anti-
`body
`to investigate
`expression
`pattern
`and
`the
`localization
`of
`cadherin-6
`in RCC.
`In
`addition,
`we
`analyzed
`the
`prognostic
`value
`of
`cadherin-6
`in the
`particular
`case
`of
`patients
`with
`
`are
`
`E-
`of
`to
`in
`
`normal
`that
`in RCC,
`20%
`thought
`tubules
`previously
`cadherins,
`the
`latter
`
`cell
`
`a!.
`et
`tubular
`lines,
`in all of
`seem
`to
`not
`was
`origi-
`line
`lacking
`kidney
`and
`
`E-
`
`(9,
`
`used
`the
`
`Oosterwijk,
`Egbert
`Paul
`Bringuier,
`Pierre
`Berkel,
`van
`Wakka,
`Frans
`
`M.
`
`J. Debruyne,
`
`Shimazui,’
`Toru
`Hideyukiz
`Akaza,
`Hans
`Emiel
`Ruijter,
`Jeannette
`Oosterwijk
`Adrie
`Bokhoven,
`van
`Jack
`A. Schalken
`Department
`University
`Medicine,
`of Clinical
`Institute
`of Urology,
`Japan
`[T. S., H. A.],
`and Urological
`Tsukuba,
`Tsukuba
`305-8575,
`Research
`Laboratory
`[T. S., E. 0.,
`P. P. B., H. v. B.,
`J. 0. W.,
`A. v. B., F. M. J. D.,
`J. A. S.]
`and Department
`of Pathology
`[E. R.],
`6500 HB,
`the Netherlands
`University
`Hospital
`Nijmegen,
`Nijmegen
`
`and
`
`of
`
`ABSTRACT
`to be a
`is considered
`E-cadherin
`carcinomas,
`In many
`and
`patient
`survivals,
`prognostic
`marker
`for
`decreased
`its
`re-
`Among
`disease.
`with metastatic
`expression
`is associated
`only
`20% of
`tumors
`(RCCs),
`however,
`nal
`cell
`carcinomas
`higher
`percentage
`ex-
`whereas
`a much
`express
`E-cadherin,
`and
`cadherin-6
`(T.
`other
`press
`cadherins,
`N-cadherin
`e.g.,
`1996). Among
`Res.,
`et al., Cancer
`Shimazui
`56:
`3234-3237,
`has
`been
`in RCCs,
`cadherin-6
`these
`cadherins
`expressed
`tubules
`in the
`renal
`proximal
`identified
`as a major
`cadherin
`Hence,
`we have
`investigated
`and
`in the
`tumors
`themselves.
`cadherin-6
`expres-
`the
`relationship
`between
`and
`prognosis
`RCC.
`Expression
`with
`sion
`in tumor
`cells
`in 43 patients
`immu-
`was
`detected
`cadherin-6,
`E-cadherin,
`and
`a-catenin
`nohistochemically
`and
`evaluated
`microscopically
`as normal,
`heterogeneous,
`absent.
`Normal,
`heterogeneous,
`and
`ab-
`or
`sent
`expression
`cadherin-6
`were
`observed
`in 19,
`16,
`and
`8
`of
`of 43
`cases,
`respectively.
`Coexpression
`of E-cadherin
`and
`cadhenn-6
`was detected
`in only
`10 cases. Among
`30 tumors
`in which
`E-cadherin
`expression
`was
`absent,
`24
`expressed
`cadherin-6.
`In addition,
`the
`expression
`pattern
`of a-catenin
`correlated
`more
`highly
`with
`that
`of
`cadherin-6
`than
`it did
`E-cadherin
`0.025).
`In survival
`anal-
`(P
`0.0003
`with
`versus
`aberrant
`expression
`of cadherin-6
`correlated
`with
`poor
`yses,
`survivals
`both
`among
`all patients
`in those
`and
`(P
`0.0009)
`RCC (P
`0.0008).
`results
`These
`with
`E-cadhenn-absent
`suggest
`that
`cadhenn-6
`is
`a major
`cadherin
`playing
`
`an
`
`of
`
`cadherin-absent
`
`RCC.
`
`MATERIALS
`
`Forty-three
`RCC
`
`AND METHODS
`from
`snap-frozen
`specimens
`the
`at
`underwent
`nephrectomies
`who
`with
`patient
`the
`of
`were
`studied.
`Details
`Nijmegen
`Hospital
`I . Age
`in Table
`and
`follow-up
`investigations
`are
`shown
`age
`the
`average
`bution
`ranged
`from
`17 to 80
`years,
`and
`time
`surgery
`was
`56.0
`years.
`Histological
`evaluations
`
`obtained
`
`patients
`University
`profiles
`distri-
`at
`the
`were
`
`in part
`the
`by
`be hereby marked
`1734
`solely
`
`to
`
`of
`
`at Department
`of Tsukuba,
`Phone
`
`and
`
`of
`1-1-1,
`Fax:
`
`2 The
`
`abbreviation
`
`used
`
`is: RCC,
`
`renal
`
`cell
`
`carcinoma.
`
`8/4/98.
`4/26/98; revised 7/23/98; accepted
`Received
`defrayed
`of publication
`of
`this
`article
`were
`The
`costs
`of page
`charges.
`This
`article must
`therefore
`payment
`in
`accordance
`with
`18 U.S.C.
`Section
`advertisement
`fact.
`this
`indicate
`be addressed,
`should
`reprints
`for
`I To whom requests
`University
`of Clinical Medicine,
`Urology,
`Institute
`Tennodai,
`Tsukuba-city,
`Ibaraki,
`305-8575,
`Japan.
`81-298-533223.
`
`1 of 6
`
`OnCusp
`Ex. 1026
`
`

`

`2420 Cadherin-6
`
`and Prognosis
`
`in RCC
`
`Age
`(yr)
`
`Case
`
`Table
`
`1
`
`Patient
`
`characteristics
`
`and
`
`treatments
`
`Pathological
`
`stage”
`
`Immunostaining’
`
`Prognosis”
`
`Sex
`
`Grade
`
`pT
`
`pN
`
`M
`
`Treatment”
`Metastases
`pV______________________________________
`
`bone
`mediastinum
`
`Lung,
`Lung,
`
`Lung
`
`Lung
`
`Adrenal
`Ileocecal
`
`gland
`
`LND,
`
`1L2
`
`Cx
`
`IFN, RT
`
`colectomy
`
`Ovary,
`
`liver,
`
`bone
`
`IFN,
`
`RT
`
`Bone
`
`IFN
`
`Lung,
`
`bone
`
`pneumox,
`
`RT
`
`Cd6
`
`H
`H
`H
`H
`(+)
`(+)
`H
`(+)
`(+)
`H
`(-)
`
`Ec
`
`(-)
`
`(-)
`H
`(-)
`(+)
`(-)
`
`(-)
`(-)
`
`(-)
`
`(-)
`
`(-)
`
`H
`
`(-)
`
`(-)
`
`(-)
`(-)
`
`(+)
`(+)
`(-)
`(+)
`(-)
`(-)
`
`a
`
`(+)
`
`H
`H
`H
`(+)
`(+)
`
`(+)
`(+)
`
`(+)
`
`(+)
`
`(+)
`
`H
`
`(+)
`
`(+)
`
`H
`H
`
`(+)
`(+)
`H
`(+)
`H
`(+)
`
`Month
`
`A/D
`
`8
`68
`72
`18
`15
`84
`
`25
`74
`
`69
`
`7
`
`82
`
`5
`
`68
`
`77
`
`9
`66
`
`60
`66
`
`65
`60
`8
`67
`
`60
`
`D
`D
`D
`D
`A
`A
`
`D
`A
`
`A
`
`D
`
`A
`
`D
`
`A
`
`A
`
`D
`A
`
`A
`A
`
`D
`A
`D
`A
`
`A
`
`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
`29
`30
`31
`32
`33
`34
`35
`36
`37
`38
`39
`40
`41
`42
`43
`
`44
`17
`57
`42
`77
`63
`60
`61
`67
`66
`57
`27
`65
`47
`61
`46
`35
`74
`67
`54
`56
`69
`52
`68
`48
`65
`80
`47
`46
`80
`69
`55
`59
`25
`66
`62
`28
`49
`55
`62
`51
`72
`73
`
`F
`M
`M
`M
`F
`M
`F
`M
`M
`F
`F
`F
`M
`M
`M
`M
`F
`M
`M
`M
`M
`F
`M
`M
`M
`M
`F
`M
`M
`M
`M
`M
`M
`F
`M
`F
`F
`M
`M
`F
`M
`M
`F
`
`3
`3
`2
`3
`2
`2
`2
`1
`2
`2
`I
`1
`2
`1
`2
`1
`3
`1
`2
`2
`2
`1
`I
`3
`1
`2
`2
`1
`2
`2
`2
`2
`2
`2
`2
`2
`2
`2
`1
`2
`2
`1
`2
`
`3B
`3
`2
`4
`3B
`2
`3B
`2
`3B
`4
`2
`3A
`3B
`2
`3B
`3A
`3B
`2
`3B
`2
`3B
`2
`3A
`3B
`2
`2
`2
`3A
`3A
`3A
`3B
`3A
`2
`2
`2
`3B
`2
`3A
`2
`3B
`3A
`3B
`3C
`
`1
`3
`0
`0
`0
`0
`0
`0
`I
`0
`0
`1
`0
`0
`X
`X
`0
`0
`X
`0
`I
`0
`0
`0
`0
`0
`X
`0
`0
`0
`3
`0
`1
`0
`0
`0
`0
`I
`0
`0
`0
`0
`0
`
`1
`1
`0
`1
`0
`0
`1
`0
`1
`1
`0
`1
`0
`0
`0
`1
`0
`0
`0
`0
`1
`0
`0
`1
`0
`0
`0
`0
`1
`0
`0
`0
`1
`0
`0
`0
`0
`1
`0
`1
`0
`0
`0
`
`1
`0
`0
`1
`1
`0
`1
`0
`0
`1
`0
`1
`1
`0
`0
`0
`1
`0
`2
`0
`1
`0
`0
`2
`0
`0
`0
`0
`0
`0
`1
`0
`0
`0
`0
`1
`0
`1
`0
`1
`0
`2
`2
`
`Lung,
`
`bone
`
`Pancreas,
`
`kidney
`
`Bone
`
`Lung
`
`Lung
`
`Nx
`Nx,
`Nx
`IFN,
`Nx,
`Nx,LND
`Nx
`Nx,
`Nx
`Nx
`Nx,
`Nx
`LND,
`Nx,
`Nx,Cx
`Nx
`Nx, LND,
`Nx,IFN
`Nx,LND
`Nx
`Nx,IVC
`Nx
`Nx,
`Nx
`Nx
`IVC,
`Nx,
`Nx,LND
`Nx(partial)
`Nx
`Nx
`Nx,
`Nx
`Nx,LND
`Nx,LND
`Nx,RT
`Nx
`Nx,LND
`Nx,LND
`Nx,LND
`Nx,
`LND,
`Nx,LND
`Nx
`LND
`Nx,
`IVC
`Nx,
`Nx, IVC
`
`LND,
`
`(-)
`H
`(+)
`H
`(+)
`(-)
`(+)
`(-)
`H
`H
`(+)
`(+)
`H
`(+)
`(-)
`(+)
`H
`H
`H
`(-)
`(+)
`(+)
`(+)
`H
`(+)
`(-)
`H
`(+)
`(-)
`(+)
`(+)
`(+)
`
`pneumox
`
`1L2,
`
`IFN
`
`RT,
`
`IL2,
`
`IFN
`
`tumor;
`primary
`a pT,
`nephrectomy;
`I, Nx,
`cadherin-6;
`‘, Cd6,
`‘I A, alive; D, dead.
`
`regional
`pN,
`lymph
`LND,
`Ec, E-cadherin;
`
`lymph
`node
`a,
`
`nodes;
`dissection;
`a-catenin;
`
`metastasis;
`distant
`M,
`IFN,
`interferon-a;
`normal;
`(+).
`(-),
`
`pV,
`RT,
`absent;
`
`invasion.
`venous
`pneumox,
`irradiation;
`H, heterogeneous.
`
`pneumonectomy.
`
`(-)
`
`(-)
`(+)
`(-)
`(-)
`(+)
`(-)
`
`(-)
`
`(-)
`(+)
`(+)
`(-)
`(+)
`(-)
`
`(+)
`
`(-)
`(-.-)
`
`(-)
`(-)
`
`(-)
`(+)
`
`(+)
`
`H
`(+)
`H
`(+)
`(+)
`H
`
`H
`H
`(+)
`(+)
`(+)
`(+)
`(+)
`
`(+)
`
`H
`(+)
`
`H
`(+)
`
`(+)
`(+)
`
`4
`58
`24
`64
`56
`30
`
`28
`10
`61
`53
`42
`34
`42
`
`21
`
`13
`36
`
`18
`29
`22
`12
`
`D
`A
`D
`A
`A
`D
`
`D
`A
`A
`D
`A
`D
`A
`
`A
`
`D
`A
`
`A
`A
`A
`A
`
`Downloaded from http://aacrjournals.org/clincancerres/article-pdf/4/10/2419/2069209/2419.pdf by guest on 14 June 2023
`
`an
`
`RpR265
`the
`protein
`was
`immunogen;
`against
`
`the
`
`vector;
`purified
`(c)
`purified
`
`(b)
`in an
`female
`protein,
`
`glutathione
`the
`affinity
`column
`BALB/c
`mice
`and
`the
`
`S-transferase
`and was
`were
`3rd
`
`fusion
`as
`used
`immunized
`boost,
`spleen
`
`after
`
`cells
`
`were
`
`fused
`
`with
`
`SP2/0
`
`mouse
`
`myeloma
`
`cells;
`
`and
`
`(d)
`
`were
`specific
`a
`as
`cadherin-6
`with
`was
`
`hybridomas
`vealed
`against
`comigrating
`munostaining
`procedure
`strated
`expressed
`
`and
`screened,
`producing
`clone
`a
`recognized
`that
`on Western
`cadherin-6
`out
`according
`also
`carried
`Immunohistochemically,
`(8).
`border
`staining
`in the
`cell-cell
`cadherin-6
`alone
`10;
`
`the
`
`re-
`
`was
`clone
`2B6-D8
`antibody
`a monoclonal
`band
`single
`l25-kDa
`Im-
`la).
`blot
`(Fig.
`previous
`to our
`demon-
`2B6-D8
`line
`that
`SKRC-33
`cell
`Fig.
`In
`addition,
`lb).
`
`a
`
`(Ref.
`
`performed
`staged
`Four
`cerous
`
`and
`normal
`areas
`Serial
`chemically
`catenin
`nm,
`and
`developed
`tathione
`human
`ment
`herin-6
`
`was
`
`sections.
`on H&E
`according
`were
`graded
`kidney
`samples
`were
`of
`nephrected
`kidneys.
`immunohisto-
`were
`frozen
`sections,
`4
`p.m thick,
`Japan),
`anti-a-
`stained
`using
`HECD-l
`(Takara,
`serum
`(8),
`and
`2B6-D8
`for E-cadherin,
`a-cate-
`mouse
`cadherin-6,
`respectively.
`2B6-D8
`newly
`as
`a mouse
`monoclonal
`antibody
`glu-
`S-transferase
`(GST)
`fusion
`extracellular
`of
`a
`cadherin-6
`follows:
`l233-bp
`frag-
`as
`(a)
`(nucleotides
`323-1555
`corresponding
`cad-
`cDNA)
`generated
`using
`PCR
`into
`
`The
`
`pathologically
`were
`tumors
`classification
`to
`TNM
`also
`obtained
`from
`
`(14).
`noncan-
`
`was
`against
`
`the
`domain
`nucleotide
`to
`human
`and was
`cloned
`
`2 of 6
`
`OnCusp
`Ex. 1026
`
`

`

`Clinical
`
`Cancer
`
`Research
`
`2421
`
`in
`
`the
`
`E-cadherin-
`
`cadherin-6
`
`the
`than
`not
`
`with
`those
`
`with
`cadherin-6
`of
`P. 0.007
`and
`x2’
`the
`expression.
`In
`patients
`normal
`that
`of
`with
`statistically
`significant
`with
`patients
`absent
`cadherin-6
`strongly
`Additionally,
`Fig.
`3b).
`analysis
`revealed
`
`E-Cadherin-
`correlated
`
`P.
`
`10.15;
`survival
`expres-
`abnormal
`(P =
`E-cad-
`correlated
`the Cox
`the
`ex-
`
`that
`
`cantly
`absent
`
`absent
`
`correlated
`group
`Statistical
`RCC.
`
`(x2(cid:1)
`
`and
`
`stage
`tumor
`with
`independent
`0.006)
`although
`analyses,
`E-cadherin
`sion
`of
`the
`expression,
`Fig.
`4a).
`0.265;
`expression,
`hem
`patient
`survival
`with
`proportional
`hazards
`
`33
`
`: 4ll6kDa
`
`6(cid:1)
`
`E _:(cid:1)=.(cid:1)116kDa
`
`(cid:1)97.4kDa
`
`b
`
`(cid:1) a
`
`expression
`shows
`
`Cadherin-6
`I
`Fig.
`blot
`analysis
`Western
`line
`expressed
`cadherin-6
`E-cadherin.
`cadherin-6
`b,
`contact
`site
`imunofluorescence
`body,
`2B6-D8.
`
`In
`by
`
`that
`(125
`
`In a,
`line.
`cell
`carcinoma
`cell
`in a renal
`cell
`carcinoma
`renal
`cell
`the SKRC-33
`not
`kDa)
`but
`kDa)
`and
`a-catenin
`(105
`the
`cell-cell
`at
`expression
`is
`restricted
`staining
`using
`a monoclonal
`anti-
`
`of
`that
`with
`P. 0.0006).
`11.8;
`Subgroup
`in
`the
`Analyses
`The
`expression
`pattern
`(x2. 7.30;
`grade
`of E-cadherin
`prognosis
`of
`was
`better
`difference
`was
`For
`the
`restricted
`expression
`of
`(P.
`0.0008;
`regression
`
`pattern
`
`of
`
`cadherin-6
`
`had
`
`a
`
`higher
`
`risk
`
`ratio
`
`in
`
`prog-
`
`the
`
`pathological
`
`parameters
`
`for
`
`patients
`
`with
`
`RCC
`
`pression
`
`nosis
`(Table
`
`than
`3).
`
`as
`
`Downloaded from http://aacrjournals.org/clincancerres/article-pdf/4/10/2419/2069209/2419.pdf by guest on 14 June 2023
`
`and
`
`considered
`gene
`different
`skin,
`
`DISCUSSION
`It
`is generally
`suppressor
`invasion
`with
`many
`patients
`and
`prostate,
`breast,
`17).
`Katagiri
`expression
`RCC.
`(70-80%)
`
`(9)
`
`et
`a!.
`is associated
`However,
`it has
`demonstrated
`
`E-cadherin
`that
`correlates
`with
`carcinomas,
`colon,
`and
`have
`reported
`with
`a better
`been
`shown
`absent
`E-cadherin
`
`e.g.,
`
`gastric
`that
`prognosis
`(9,
`l0)that
`expression
`
`functions
`the
`survival
`bladder
`tumors
`cancers
`(5-7,
`16,
`normal
`E-cadherin
`of patients
`with
`most
`RCCs
`
`an
`of
`
`and
`
`only
`
`20%
`the
`
`exhibited
`normal
`
`normal
`expression
`
`expression
`E-cadherin
`rate
`of E-cadherin
`
`with
`compared
`as
`in other
`carcinomas.
`
`to
`
`the
`fact
`attributed
`be
`probably
`this
`can
`above,
`As mentioned
`an origin
`presumably
`are
`which
`tubules,
`proximal
`that
`the
`renal
`Although
`N-cadherin
`has
`E-cadherin.
`not
`express
`of RCC,
`do
`adhesion
`proteins
`expressed
`in
`the
`cell
`of
`as one
`been
`identified
`( 1 1), Tani
`( 18)
`have
`demon-
`the
`renal
`proximal
`tubules
`a!.
`do
`expression
`not
`correlate
`with
`strated
`E-
`and N-cadherin
`that
`unclear
`remains
`whether
`not
`it
`tumor
`in RCC.
`Thus,
`grade
`or
`aberrant
`expression
`the
`cadherin
`molecules
`expressed
`in RCC
`is
`associated
`with
`prognosis
`of
`patients.
`Recently,
`Paul
`(19)
`demonstrated
`that
`cadherin-6
`is
`expressed
`renal
`in
`a!.
`proximal
`tubular
`epithelia
`and
`that RCC
`and
`its aberrant
`expres-
`sion
`seem
`to
`correlate
`dedifferentiation
`and
`progression
`RCC.
`In this
`expression
`the
`between
`correlation
`focus
`on
`the
`with
`RCC.
`of patients
`the
`prognosis
`cadherin-6
`and
`coex-
`each
`cadherin,
`of
`expression
`When
`looking
`at
`the
`observed
`in only
`10
`was
`cadhenn-6
`pression
`of E-cadherin
`and
`up-regulation
`of E-
`by
`explained
`be
`may
`of
`43
`tumors.
`This
`from
`arising
`renal
`of RCC
`carcinogenesis
`cadherin
`during
`the
`RCCs
`some
`may
`is
`that
`proximal
`tubules.
`Another
`explanation
`coexpresses
`that
`nephron
`be
`derived
`from
`the
`segment
`of
`the
`up-regulation
`if
`the
`both
`E-cadherin
`and
`cadherin-6.
`Even
`in RCC,
`phenomenon
`E-cadherin
`expression
`is
`an
`aberrant
`associated
`be
`seems
`homogeneous
`cell
`border
`expression
`(9).
`of patients
`prognosis
`with
`less metastatic
`disease
`and
`better
`in the
`expression
`For
`this
`reason,
`we
`focused
`on cadherin-6
`aberrant
`group,
`subgroup
`with
`E-cadherin-absent
`tumors.
`In this
`disease
`cadherin-6
`expression
`correlated
`with
`distant
`metastatic
`not
`(2 of
`(10
`of
`16) whereas
`normal
`cadherin-6
`expression
`did
`14).
`This
`finding
`suggested
`that
`cadherin-6
`acted
`a metastasis
`
`et
`
`of
`poor
`
`with
`
`study,
`
`we,
`
`therefore,
`
`extended
`
`their
`
`research
`
`pattern
`
`et
`
`of
`
`to
`
`of
`
`of
`its
`
`to
`
`as
`
`mouse
`renal
`
`as
`
`the
`
`distinguish
`to
`used
`was
`(15).
`previously
`evaluated
`sections
`were
`cells
`in
`pattern
`cancer
`the
`antigen
`expression
`absent
`if
`there
`(i.
`e.,
`or heterogeneous
`and
`negative
`
`by
`was
`was
`was
`
`a
`
`e.,
`(i.
`areas),
`
`RC-3,
`antibody,
`monoclonal
`described
`tubules
`as
`proximal
`tumor
`expressions
`on
`Protein
`microscope.
`If
`staining
`light
`exclusively
`at
`cell-cell
`borders,
`was
`scored
`normal.
`If
`reactivity
`complete
`absence
`of
`immunoreactivity)
`positive
`if
`the
`tumor
`was
`composed
`of
`abnormal.
`antigen
`expression
`was
`scored
`as
`cadherin-6
`to compare
`Statistical
`analyses
`were
`performed
`patients
`in
`restricted
`with
`expression
`pathological
`parameters
`cad-
`with
`E-cadherin-absent
`The
`correlation
`between
`by x2 test.
`herin-6
`and
`tumor
`grade
`were
`evaluated
`Survival
`curves
`were
`the Kaplan-Meier
`method,
`by
`and
`the
`differences
`assessed
`by
`log-rank
`were
`the
`test.
`The
`clinical
`these molecules
`was
`analyzed
`the Cox
`proportional
`model.
`
`RCC.
`and
`stage
`constructed
`in
`survival
`relevancy
`of
`hazards
`
`by
`
`RESULTS
`in the Normal
`Molecules
`Adhesion
`of Cell
`Expression
`in distal
`tubules
`restricted
`was
`Kidney.
`E-cadherin
`expression
`was
`expressed
`cadherin-6
`through
`collecting
`tubules,
`whereas
`loop.
`E-cadherin
`expression
`proximal
`tubules
`and
`Henle’s
`thin
`sites
`seen
`was
`at
`all
`of
`the
`lateral
`cell-cell
`contact
`with
`hand,
`exception
`of
`the
`basal
`membrane.
`On
`other
`cadherin-6
`expression
`was
`located
`at
`the
`basolateral
`membranes
`along
`with
`a-catenin
`expression
`(Fig.
`2a-d).
`speci-
`in RCC
`molecule
`each
`Details
`of
`the
`expression
`of
`showed
`1 . Only
`1 1 tumors
`are
`summarized
`in
`Table
`mens
`borders,
`at
`cell-cell
`the
`of
`expression
`E-cadhenn
`normal
`tumors,
`expression
`in 30
`of 32 E-cadherin-aberrant
`whereas,
`heterogeneous,
`Normal,
`was
`completely
`absent.
`E-cadherin
`in
`19,
`were
`observed
`expression
`of
`cadherin-6
`absent
`cases,
`respectively.
`Coexpression
`of E-cadherin
`of 43
`was
`detected
`10 cases
`(Fig.
`c and
`d). Of
`in
`3,
`herin-6
`E-cadherin
`expression
`was
`absent,
`14 showed
`in which
`and
`16 showed
`abnormal
`expression
`of cadherin-6
`(Table
`and J). In
`3,
`addition,
`expression
`of
`a-catenin
`b,
`e,
`
`the
`
`in
`
`the
`
`8
`
`of
`and
`and
`16,
`cad-
`and
`30 tumors
`normal
`2, Fig.
`signifi-
`
`a,
`
`3 of 6
`
`OnCusp
`Ex. 1026
`
`

`

`2422 Cadherin-6
`
`and
`
`Prognosis
`
`in RCC
`
`t(cid:1).
`
`(cid:1);-
`
`-
`
`‘..
`
`.,
`
`/,
`
`r
`
`.
`
`,-
`
`%_.s..,
`
`.(cid:1)d
`
`(cid:1).
`
`Expression
`2
`Fig.
`catenin
`in the
`and
`Renal
`proximal
`ney.
`by
`RC-3
`stained
`and
`cadherin-6
`(a)
`at
`the
`basolateral
`ithelium
`but
`cadherin
`(b).
`pressed
`the
`the renal
`distal
`(b).
`
`of Cadherin
`normal
`kid-
`tubules
`(‘0
`express
`a-catenin
`(c)
`the
`site
`of
`ep-
`not
`express
`E-
`do
`E-cadherin
`is cx-
`cell-cell
`border
`in
`tubular
`epithelia
`
`at
`
`.
`.(cid:1) (cid:1) (cid:1)
`(cid:1)(cid:1) (cid:1) (cid:1) .t
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`
`#{149}‘(cid:1)(cid:1)‘
`
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`
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`
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`
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`
`.-(cid:1)LJ
`
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`
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`
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`
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`
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`
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`
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`
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`
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`
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`
`.(cid:1)‘,.
`
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`
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`:
`#{149}.
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`
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`
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`
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`
`combinations
`Several
`3
`Fig.
`pattern
`between
`expression
`of
`E-cadherin
`cadherin-6
`RCC
`specimens.
`observed
`shows
`normal
`cell-
`Case
`6 RCC
`staining
`of
`cad-
`cell
`border
`(a) with
`absent
`E-cad-
`herin-6
`hem
`(b). In
`case
`both
`32,
`cadherin-6
`and
`E-cadherin
`(c)
`are
`homogeneously
`(d)
`at
`pressed
`the
`cell-cell
`Heterogeneous
`cadherin-6
`staining
`is observed
`in case
`(f)
`whereas
`E-cadherin
`is
`sent.
`
`cx-
`border.
`
`(e)
`38,
`ab-
`
`(cid:1)(cid:1).
`
`-‘(cid:1)-
`
`4.
`
`.,(cid:1)(cid:1)
`(cid:1) (cid:1)(cid:1)
`(cid:1)(cid:1) (cid:1) (cid:1)(cid:1)
`
`.
`
`“ rr4
`
`‘I
`
`4..
`
`.(cid:1)
`
`(cid:1)(cid:1)
`
`,(cid:1).
`
`4
`
`(cid:1)t(cid:1)_;’
`
`.-
`
`Li
`
`-
`
`. j-.;
`
`4 of 6
`
`OnCusp
`Ex. 1026
`
`(cid:1)
`(cid:1)
`(cid:1)
`(cid:1)
`(cid:1)
`(cid:1)
`(cid:1)
`(cid:1)
`(cid:1)
`(cid:1)
`(cid:1)
`(cid:1)
`(cid:1)
`(cid:1)
`(cid:1)
`(cid:1)
`(cid:1)
`(cid:1)
`(cid:1)
`

`

`Table
`
`2
`
`Correl
`
`ation
`
`betwee
`
`n E-cadherin
`
`and
`
`c adherin-6
`
`i n RCC
`
`Table
`
`3
`
`Cox
`
`Clinical
`
`Cancer
`
`Research
`
`2423
`
`hazards
`proportional
`E-cadherin-absent
`
`model
`RCC
`
`in the patients
`
`with
`
`E-Cadherin
`
`Cadherin-6
`
`Normal
`
`Heterogeneous
`
`Absent
`
`Total
`
`Normal
`Heterogeneous
`Absent
`
`Total
`
`6
`3
`2
`
`11
`
`0
`1
`1
`
`2
`
`13
`12
`5
`
`30
`
`19
`16
`8
`
`43
`
`Values
`
`Cadherin-6
`Stage
`Nodal
`Distant
`Grade
`
`status
`metastasis
`
`x2
`
`4.93
`0.57
`0.037
`0.012
`0.16
`
`P
`
`0.027
`0.45
`0.85
`0.91
`0.69
`
`Hazards
`
`ratio
`
`12.66
`3.46
`0.860
`0.831
`1.39
`
`p=0.0008
`
`I
`
`I
`
`I
`
`I
`
`I
`
`I
`
`III
`
`111rIosrl1aIc...dII.ri,I6(cid:1)Icadherin6
`
`(n*14)
`
`abnormal
`
`cadhsrin
`
`6(nzl6)
`
`b r
`
`ate
`
`p=O.265
`
`(cid:1)dh.(cid:1)(cid:1)i(cid:1)E.cadherin(nz1
`
`I)
`
`abnormal
`
`E-cadherln
`
`(na32)
`
`I .0
`
`0.8
`
`0.6
`
`0.4
`
`0.2
`
`a
`
`Survival
`rate
`
`10
`
`08
`
`0.6
`
`0.4
`
`0.2
`
`0.0
`
`Downloaded from http://aacrjournals.org/clincancerres/article-pdf/4/10/2419/2069209/2419.pdf by guest on 14 June 2023
`
`0
`
`12
`
`24
`
`36
`
`48
`
`60
`
`72
`
`84
`
`96
`
`:
`
`12
`
`24
`
`36
`
`48
`
`60
`
`72
`
`months
`
`84
`
`96
`months
`
`4
`
`Fig.
`(a; P
`significantly
`
`Survival
`0.265).
`
`curves
`Among
`lower
`than
`
`demonstrate
`patients
`that
`
`of
`
`with
`those
`
`no SD in survival
`E-cadherin-absent
`showing
`normal
`
`rates
`tumors,
`cadherin-6
`
`patients
`between
`survival
`the
`expression
`
`showing
`tumors
`with
`with
`tumors
`those
`rate
`of
`0.0008).
`(b; P =
`
`and
`
`normal
`showing
`
`abnormal
`abnormal
`
`expression
`cadherin-6
`
`of E-cadherin
`expression
`
`was
`
`with
`study
`
`a!.
`
`cadherin-6
`of
`Expression
`in RCC.
`molecule
`suppressor
`grade
`tumor
`an
`increase
`of
`decreased
`cantly
`These
`(19).
`by
`Paul
`as
`in
`the
`much
`et
`parameters
`and
`pathological
`cadherin-6
`between
`cancer
`group.
`in the E-cadherin-absent
`pronounced
`with
`correlated
`expression
`significantly
`Cadherin-6
`cadherin-6
`that
`in our
`series.
`This
`suggested
`nm expression
`complex
`forming
`a
`functional
`cell
`adhesion
`have
`been
`investigation
`as
`in RCC,
`it did
`in our
`previous
`a-catenin
`in
`vitro
`study
`(19)
`revealed
`in an
`immunoprecipitation
`(10).
`Paul
`et a!.
`that
`and
`a-
`in RCC
`(cid:1)3-catenin
`are
`coprecipitated
`with
`cadherin-6
`to be
`likely
`suggesting
`that
`the
`binding
`to catenins
`is
`cell
`lines,
`other
`the
`for
`proper
`functioning
`of
`cadherin-6.
`On
`important
`cadherin-6
`tumors
`expressed
`E-cadherin
`nor
`hand,
`some
`neither
`31,
`and
`40).
`but
`express
`a-catenin
`11,
`19,
`26,
`did
`tumors
`(i.e.,
`N-cadherin,
`that
`This
`fact may
`indicate
`other
`cadherins,
`e.g.,
`a-catenin
`a cell
`expressed
`and
`formed
`adhesion
`complex
`with
`(20). Additional
`immunohistochemical
`studies
`will
`be needed
`clarify
`the
`diversity
`of
`cadherin
`in RCC.
`expression
`specifically
`conclusion,
`we
`revealed
`that
`cadherin-6
`is
`In
`in the
`epithelial
`cells
`renal
`proximal
`tubules
`and
`of
`expressed
`in most
`RCCs.
`Thus,
`cadherin-6
`could
`be
`considered
`retained
`the major
`cadherins
`expressed
`in RCC,
`and
`its expression
`one
`of
`be
`considered
`to have
`greater
`prognostic
`value
`than
`tumor
`could
`in
`or
`grade
`this
`disease.
`These
`immunohistochemical
`stage
`observations
`constitute
`the
`first
`report
`on the
`prognostic
`value
`cadhenn-6
`for
`patients
`with
`RCC.
`
`signifi-
`stage
`and
`relationships
`were
`highly
`
`a-cate-
`may
`with
`
`to
`
`is
`
`of
`
`REFERENCES
`
`1. Fuselier,
`the Ochsner
`445-448,
`2. Nurmi,
`operative
`
`S. L.,
`H. A., Guice,
`medical
`institution
`1983.
`
`and Brannan,
`experience
`
`W. Renal
`( 1945-1978).
`
`carcinoma:
`cell
`J. Urol.,
`130:
`
`J. Prognostic
`M.
`findings.
`Br.
`
`factors
`J. Urol.,
`
`56:
`
`in renal
`270-275,
`
`carcinoma.
`1984.
`
`An
`
`evaluation
`
`of
`
`B., Dosoretz,
`3. Bassil,
`nodes
`and metastasis
`450-454,
`1985.
`
`and
`E.,
`classification
`
`Jr. Validation
`G. R.,
`Prout,
`ofrenal
`cell
`carcinoma.
`
`the
`of
`J. Urol.,
`
`tumor,
`134:
`
`and
`
`Am.
`
`K.,
`T.
`
`P.,
`Sperber,
`Joshi,
`M.,
`A.,
`Renal
`carcinoma:
`cell
`4. Golimbu,
`factors.
`Urology,
`prognostic
`291-301,
`1986.
`survival
`and
`27:
`H., Oka,
`H., Tahara,
`H., Miyata,
`M., Kobayashi,
`Shiozaki,
`5.
`Iihara, K., Doki, Y., Hirano,
`S., Takeichi,
`M.,
`and Mori,
`Tamura,
`S.,
`of
`immunoreactive
`E-cadherin
`adhesion
`molecules
`in human
`Expression
`1991.
`J. Pathol.,
`17-23,
`cancers.
`139:
`H. E.,
`Schaafsma,
`P. P.,
`R.,
`Isaacs, W. B., Bringuier,
`6. Umbas,
`H. F., Oosterhof,
`G. 0. N., Debruyne,
`F. M. J., and Schalken,
`Karthaus,
`J. A. Decreased
`E-cadherin
`expression
`is associated
`with
`poor
`prognosis
`in patients
`with
`prostate
`cancer.
`Cancer
`Res.,
`3929-3933,
`1994.
`54:
`
`7. Bringuier,
`Debruyne,
`noreactivity
`mors.
`Cancer
`
`P., Umbas,
`P.
`J.,
`and
`F. M.
`correlates
`Res.,
`
`R.,
`Schalken,
`with
`poor
`3241-3245,
`
`53:
`
`H. E., Karthaus,
`Schaafsma,
`J. A. Decreased
`E-cadherin
`survival
`in
`patients
`with
`1993.
`
`H.
`
`bladder
`
`F. M.,
`immu-
`tu-
`
`J., Akaza,
`C. F.
`Jansen,
`L. A.,
`J. A., Giroldi,
`Schalken,
`T.,
`Shimazui,
`8.
`P. P. Prognostic
`value
`F. M. J., and Bringuier,
`K., Debruyne,
`H., Koiso,
`and -y-catenins
`and
`pl2O(cid:1))
`molecules
`(a-,
`(cid:1)3-
`in
`of
`cadherin-associated
`Res.,
`4154-4158,
`1996.
`Cancer
`bladder
`tumors.
`56:
`R., and Tomita,
`Y. E-cadherin
`9. Katagiri,
`A., Watanabe,
`significance
`in metastasis
`and
`renal
`cell
`cancer
`and
`its
`Cancer,
`376-379,
`1995.
`71:
`
`expression
`survival.
`
`Br.
`
`in
`J.
`
`5 of 6
`
`OnCusp
`Ex. 1026
`
`

`

`S. 0.
`Wamaar,
`antigens:
`to renal
`Am.
`J. Pathol.,
`
`analysis
`Immunohistochemical
`application
`in the diagnosis
`301-309,
`1986.
`123:
`
`of monoclonal
`of
`renal
`cell
`
`antibody
`carcinoma.
`
`Rimm,
`16.
`J. H.,
`D. L., Sinard,
`E-cadherin
`expression
`in breast
`Shimoyama,
`Y., Hirohashi,
`17.
`Y., Takeichi,
`and Abe,
`human
`epithelial
`tissues
`and
`1989.
`
`M.,
`
`and Morrow,
`cancer.
`Lab.
`S., Hirano,
`0. Cadherin
`carcinomas.
`
`J. S. Reduced
`Invest.,
`72:
`S., Noguchi,
`cell-adhesion
`Cancer
`Res.,
`
`a-catenin
`and
`506-512,
`1995.
`M., Shimosato,
`molecules
`2128-2133,
`
`49:
`
`in
`
`Downloaded from http://aacrjournals.org/clincancerres/article-pdf/4/10/2419/2069209/2419.pdf by guest on 14 June 2023
`
`Tani,
`18.
`Expression
`carcinoma
`407-414,
`
`L., Kangas,
`Laitinen,
`T.,
`of E- and N-cadherin
`cell
`lines
`in vitro
`and
`1995.
`
`L.,
`in renal
`in their
`
`and
`V-P.,
`Lehto,
`carcinomas,
`cell
`xenografts.
`Int.
`
`Virtanen,
`in renal
`J. Cancer,
`
`I.
`cell
`64:
`
`R., Ewing,
`Paul,
`19.
`K. R., Wheelock,
`molecule
`specifically
`cell
`carcinoma.
`Cancer
`
`M.
`
`C. M.,
`J., and
`expressed
`Res.,
`
`J. C., Marshall,
`Robinson,
`Isaacs, W. B. Cadherin-6,
`in the proximal
`renal
`2741-2748,
`1997.
`
`57:
`
`F. F.,
`a cell
`tubule
`
`Johnson,
`adhesion
`and
`renal
`
`Liponen,
`20.
`prognosis
`1992.
`
`P. K. Review
`in transitional
`
`of
`
`cytometric
`bladder
`
`the
`in
`methods
`cancer.
`Eur. Urol.,
`
`cell
`
`assessment
`177-183,
`21:
`
`of
`
`2424
`
`Cadherin-6
`
`and Prognosis
`
`in RCC
`
`P. P., Oosterwijk,
`in renal
`cell
`
`and
`E.,
`carcinoma.
`
`L. A., Bringuier,
`T., Giroldi,
`10. Shimazui,
`cadherin
`expression
`Schalken,
`J. A. Complex
`Cancer
`Res.,
`56:
`3234-3237,
`1996.
`I., and Dc Broe, M. E.
`der Biest,
`I I. Nouwen,
`E.
`J., Dauwe,
`S., Van
`of cell-adhesion
`molecules
`N-
`Stage-
`and
`segment-specific
`expression
`CAM,
`A-CAM
`and L-CAM
`in the
`kidney.
`Kidney
`Int.,
`147-158,
`44:
`1993.
`12.
`hashi,
`mentary
`carcinoma
`
`Terasaki,
`analysis
`sequence
`2206-221
`Suzuki,
`M.,
`M.,
`Shen, Q., Sugimura,
`encoding
`K-cadherin,
`in fetal
`kidney
`and
`1994.
`
`M.,
`T., Kitajima,
`human
`cadherin-6
`and
`its expression
`1, 1995.
`Igarashi,
`H.,
`
`Hiro-
`and
`comple-
`in human
`
`E.,
`H., Kiyokawa,
`I. Isolation
`and Kino,
`a
`novel
`rat
`cadherin
`carcinoma.
`Cancer
`
`kidney
`
`Shimoyama,
`S.
`Isolation
`DNA for
`cells.
`
`M.,
`
`Y., Gotoh,
`of
`sequence
`the full
`coding
`Cancer
`Res.,
`55:
`
`of
`
`Y-Y.,
`13. Xiang,
`Naito,
`Y., Ohtawara,
`of
`complementary
`preferentially
`Res.,
`54:
`
`Tanaka,
`Y.,
`DNA
`expressed
`3034-3041,
`
`Cancer.
`Against
`Union
`International
`14.
`and L-H. Sobin
`In: P. Hermanek
`cancer).
`Malignant
`Tumors,
`4th ed., pp. 136-138.
`15. Oosterwijk,
`E., Ruiter,
`D.
`J., Wakka,
`J. W.,
`Jonas,
`U., Fleuren,
`G.
`J., Zwartendijk,
`
`(kidney
`classification
`TNM
`(eds.), ThM Classification
`Berlin:
`Springer-Verlag,
`J. C., Huiskens-van
`J., Hoedemaeker,
`
`of
`1992.
`der Meij,
`P., and
`
`6 of 6
`
`OnCusp
`Ex. 1026
`
`