IMMUNOHISTOCHEMICAL DIFFERENTIAL DIAGNOSES IN UROLOGICAL PATHOLOGY: An Overview

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IMMUNOHISTOCHEMICAL DIFFERENTIAL DIAGNOSES IN
UROLOGICAL PATHOLOGYAn Overview

• Mark R. Wick, M.D.

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Diagnostic Urological Immunohistochemistry

• The diagnosis of urological disease is a
  microcosm of surgical pathology at large, but
  with some selected differential diagnoses that
  are unique to the urogenital tract
• As such, most immunohistochemical studies of
  urological lesions typically utilize antibody
  reagents that are common to diagnostic problems
  in other parts of the body
• For those reasons, accurate restricted
  morphological differential diagnosis is extremely
  important before application of immunostaining
  panels in urologic pathology

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DIAGNOSTIC IMMUNOHISTOLOGY CHOICE OF REAGENTS

• Choices of panels of antibodies are dependent
  upon the specific differential diagnostic
  entities under consideration
• Several antibodies appear in more than one panel,
  but their places in the relative sequences of
  interpretation (or the diagnoses that positivity
  yields) may differ from one setting to another
• New antibodies may be substituted for old ones or
  used to supplement existing reagents in all panels

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Useful Immunohistochemical Antibody Reagents in
Non-Hematopoietic Urologic Pathology

• -Monospecific keratins 7, 18, 19, 20 MoAb 34BE12
• -Vimentin -Desmin -MS Actin -Collagen IV
• -PSA -PSAP -PSMA -ERP/PRP
• -ARP -GCDFP-15 -CA-125 -CEA
• -CDX2 -TTF-1 -Thrombomodulin
• -EMA -p63 -p504S -PLAP
• -S100 -HMB45 -MART-1 -AMACR
• -CD10 -CD26 -CD30 -CD56
• -CD99 -CD117 -GATA3 -Inhibin
• -PAX8 -RCC -HEPPAR1 -NB84
• -Synaptophysin -CGA -FLI-1

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PROSTATIC NEOPLASMS
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Prostate-Specific Markers

• Prostatic specific antigen (PSA), Prostatic acid
  phosphatase (PSAP), and Prostate specific
  membrane antigen (PSMA) are the markers in
  current biochemical and immunohistochemical use,
  for identification of prostatic epithelial
  proliferations
• PSA PSAP antibodies are available from a
  variety of commercial sources monoclonal
  antibodies should be used diagnostically to avoid
  unwanted cross-reactivity seen with
  heteroantisera
• Prostate-specific membrane antigen (PSMA) is a
  750-residue integral membrane glycoprotein
  recognized by a monoclonal antibody (7E11) from
  Hybritech Co.

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How Specific Are Prostate-Specific Markers?

• PSA and PSAP have been reported rarely in
  non-prostatic neoplasms, such as primary breast
  carcinoma, primary salivary duct carcinoma, and
  pure neuroendocrine carcinomas of various sites
• PSMA is peculiarly expressed in the intratumoral
  endothelium of various neoplasms, but only by
  epithelial cells of the prostate and prostatic
  carcinomas

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P504S (Alpha-Methylacyl-CoA-Racemase AMACR)
Prostatic Tumors

• Cytoplasmic protein identified by cDNA library
  subtraction with microarray screening, from
  prostatic carcinoma
• AMACR functions in the beta-oxidation of
  branched-chain fatty acids
• Anti-P504S is marketed by Zeta Co., Sierra Madre,
  CA, USA (murine monoclonal antibody)
• Complete organ/tissue distribution not yet
  studied, but urothelium urothelial tumors are
  P504S-negative

P504S Metastatic Prostatic Carcinoma in Lymph
Node
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Jiang Z, et al. P504S-- a new molecular marker
for the detection of prostate carcinoma. Am J
Surg Pathol 2001 25 1397-1404.

• Expression of P504S in Prostatic Carcinoma
• Gleason Score No. Cases Positive No. with gt75
• of Positive Tumor Cells
• 5 2 2 2
• 6 80 80 77
• 7 32 32 28
• 8 to 10 23 23 19
• TOTALS 137 137 126 (92)

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Yang Y, et al. P504S in prostatic epithelial
proliferations. Am J Surg Pathol 2002, in press.

• Expression of P504S in Prostatic Hyperplasias
  Carcinomas
• No. Cases Positive Negative
• UPH 20 0 20
• Atypical
• adenomatous
• hyperplasia 40 7 (17.5) 33
• Carcinomas 20 20 0

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34BE12-Keratin in Prostatic Proliferations

• The monoclonal antikeratin known as 34BE12 was
  developed by Gown Vogel in 1983, against CKs
  1,5,10, 14 it preferentially labels squamous
  epithelium, urothelium, basal cells of the
  prostate
• 34BE12 has been erroneously called keratin 903,
  using a commercial catalogue number rather than
  the actual clone designation
• This marker labels prostatic basal cells in
  hyperplasias of all forms and atypical
  adenomatous proliferations, but it is typically
  absent in adenocarcinoma

AAH
Well- Diff. ACA (Top) Hyperpl. Glands (Bottom)
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P504S (Alpha-Methylacyl-CoA-Racemase AMACR)
34BE12 inCombination in Prostatic Biopsies

• P504S can be used complementarily with
  34BE12-keratin to help to distinguish
  well-differentiated prostatic adenocarcinoma from
  atypical benign glandular proliferations
• Those 2 markers produce mirror image results
  with respect to one another

34BE12
P504S
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Transcription Factors as Immunohistological
Determinants (modified from A. Gown)
--Estrogen receptor protein in breast
carcinoma --Myogenin in striated muscle
neoplasms --Thyroid transcription factor-1 in
lung thyroid tumors --CDX-2 in intestinal
neoplasms --p63 in squamous and urothelial
tumors (and as a marker of basal cells) --GATA3
as a marker of urothelial, mammary,
squamous epithelium
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DiComo CJ, et al. p63 expression profiles in
human normal and tumor tissues. Clin Cancer Res
2002 8 494-501.

• A spectrum of human non-neoplastic and tumor
  tissues was studied immunohistologically and by
  reverse transcription-PCR using isoform-specific
  primers
• p63 expression was restricted to the nuclei of
  positive cells and tissues
• These were seen in stratified epithelia,
  including skin, esophagus, uterine cervix, and
  urinary bladder, as well as basal cells of
  prostate, breast, and bronchus
• p63 was predominantly localized to basal cell
  carcinoma, squamous cell carcinoma, transitional
  carcinoma, and thymoma among tumor tissues, and
  was not observed in adenocarcinomas, lymphomas,
  melanomas, germ cell tumors, endocrine tumors, or
  sarcomas

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p63 in Squamous Epithelium
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p63 Another Marker of Prostatic Basal Cells

• Comparison of the Basal Cell-Specific Markers,
  34betaE12 and p63, in the Diagnosis of Prostate
  Cancer.Shah RB, Zhou M, LeBlanc M, Snyder M,
  Rubin MA.Departments of Pathology and Urology,
  University of Michigan School of Medicine and
  Comprenhensive Cancer Center, Ann Arbor, Michigan
  48109, USA
• Am J Surg Pathol 2002 26 1161-1168.

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Shah RB, et al. Am J Surg Pathol 2002 26
1161-1168.p63 in Prostate Biopsies

• 94 cases studied prostatic needle biopsies
  TURP specimens
• None of 67 needle biopsy cases showing carcinoma
  was positive for p63 or 34BE12
• Of 108 needle biopsy cores studied overall, 45
  (41) showed more p63 labeling than 34BE12
  staining, and p63 was more intense

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p63 in Prostatic Hyperplasia
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Jiang Z Paradigm for Immunohistologic Evaluation
of Atypical Prostatic Glandular Lesions
Morphologically-atypical prostatic
glandular proliferation
34BE12 P63 P504S
34BE12- P63- P504S-
34BE12 P63 P504S-
34BE12- P63- P504S
GPUMP
Benign
Carcinoma
HGPIN or AAH
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Practical Differential Diagnoses Involving
Prostatic Carcinoma Immunohistochemistry

• Prostatic carcinoma vs. poorly-differentiated TCC
• Prostatic acinar carcinoma vs. pure
  neuroendocrine CA
• Prostatic carcinoma vs. poorly-differentiated
  colonic CA
• Prostatic carcinoma vs. Cowpers glands or
  seminal vesicle
• Prostatic carcinoma vs. nephrogenic metaplasia
  (adenoma)
• Prostatic acinar carcinoma vs. clear-cell
  adenocarcinoma
• Metastatic prostatic carcinoma vs. 1o or 2o
  salivary CA
• Metastatic prostatic carcinoma vs. 1o or 2o
  breast CA

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Selected Differential Diagnoses Involving
Prostatic Carcinoma

• Prostatic carcinoma vs. Intraprostatic TCC
• Prostate CK7 or 20 but not both PSA PSAP
  PSMA P504S CEA generally - Thrombomodulin-
  p63- GATA3-
• TCC Conjoint CK7 20 is most helpful PSA-
  PSAP- PSMA- P504S- CEA Thrombomodulin
  p63 GATA3
• Prostatic carcinoma vs. High-grade Colon CA
• Prostate PSA/PSAP/PSMA CEA CDX2 usually-
• Colon PSA/PSAP/PSMA- CEA CDX2 usually
• Prostatic acinar carcinoma vs. Nephrogenic
  metaplasia vs. Clear-cell CA of the urogenital
  tract
• Prostate PSA/PSAP/PSMA CA125- CEA usually -
  PAX8 -
• Nephrogenic metaplasia PSA/PSAP/PSMA- CA125-
  CEA- PAX8
• Clear cell CA PSA/PSAP/PSMA- CA125 CEA
  PAX8

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Differential Diagnoses Involving Prostatic
Carcinoma for Which Immunohistochemistry is of
Limited or No Use

• Metastatic prostatic carcinoma vs. Primary or
  metastatic breast CA or salivary gland CA
• Because all 3 of these organ sites give rise to
  tumors with largely-overlapping immunophenotypes,
  they demonstrate potential sharing of PSA, PSAP,
  ERP, PRP, ARP, mammoglobiin, GCDFP-15
• The most helpful reactants are PSMA (limited to
  prostate) GATA3 (usually seen in breast
  salivary gland) CD10 (prostate) S100 protein
  (breast salivary gland but not prostate)
• Primary or metastatic prostatic carcinoma vs.
  Primary or metastatic neuroendocrine carcinoma
• Many prostatic carcinomas show occult
  neuroendocrine differentiation and thus share
  several endocrine determinants with pure
  neuroendocrine carcinomas (NECs) however,
  positivity for TTF-1 favors NEC, whereas PSMA
  favors prostatic carcinoma
• Acinar vs. large-duct carcinoma of the prostate
• They are probably the same entity

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ADULT RENAL NEOPLASMS
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ADULT RENAL NEOPLASMSPractical Differential
Diagnoses

• Conventional renal cell carcinoma (RCC) vs.
  Chromophobe carcinoma
• RCC vs. Oncocytoma
• RCC vs. Angiomyolipoma
• High-grade RCC vs. Anaplastic TCC
• RCC vs. Adrenocortical carcinoma
• Sarcomatoid RCC vs. True renal sarcoma
• Metastatic RCC versus
• Primary or metastatic lung carcinoma
• Primary or metastatic hepatocellular carcinoma
• Primary or metastatic melanoma
• Primary clear-cell thyroid carcinoma
• Primary or metastatic clear-cell Mullerian
  carcinoma
• Primary or metastatic germ cell tumor
• Gastrointestinal stromal tumor
• Malignant mesothelioma

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Melanoma, Clear Cell Sarcoma, Adrenocortical
CA. or Epithelioid MPNST
S100/HMB45/MART-1
--
--

PLAP
Seminoma
--
CD45
VIMENTIN
Clear Cell Non-Hodgkins Lymphoma


--
Clear-Cell Osteosarcoma or Chondrosarcoma
or GIST (CD117)
Clear-Cell Carcinoma or Clear-Cell Mesothelioma
(both EMA) or Clear-Cell HCC
or Non-Seminomatous Germ Cell Tumor (NSGCT) (both
EMA- NSGCT also PLAP)
KERATIN
--
--

Technically inferior specimen

BER-EP4/CD15/CEA/S100
Clear-Cell Carcinoma, NOS or Chordoma (S100 only)
IMMUNOHISTOCHEMICAL DIAGNOSIS OF MALIGNANT
CLEAR-CELL TUMORS
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Which Are the Renal-Cell Carcinoma-Selective
Antibodies?

• RCC antigen, PAX2, PAX8 are selective for
  renal epithelial neoplasms
• Protease digestion or microwave-mediated epitope
  retrieval is necessary for proper labeling of
  paraffin sections
• RCC labels clear-cell papillary variants of
  renal cell carcinoma, but not chromophobe
  carcinoma or oncocytoma
• PAX2 PAX8 are potentially common to all forms
  of renal epithelial neoplasia

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Clear-Cell Renal Cell Carcinoma vs.
Adrenocortical Carcinoma

• Renal Cell Carcinoma--
• Positive for Keratin, Vimentin (), CD10, RCC,
  BG8, EMA
• Negative for MART-1, Inhibin
• Adrenocortical Carcinoma--
• Positive for Vimentin, usually for MART-1
  Inhibin
• Negative for Keratin (in paraffin sections),
  CD10, RCC, BG8, EMA

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Sarcomatoid Renal Cell Carcinoma vs. True Renal
Sarcoma

• Sarcomatoid carcinoma is a final common pathway
  of clonal evolution, with or without divergent
  differentiation. Thus, sarcomatoid RCCs (SRCCs)
  show a marked diminution of reactivity for
  keratin and EMA, augmentation of vimentin
  labeling, and the possible appearance of
  heterologous markers such as desmin,
  muscle-specific actin, osteonectin
• True intraparenchymal renal sarcomas are
  extraordinarily rare, and represent diagnoses of
  ultimate exclusion
• Immunohistochemical diagnosis on small tumor
  biopsies is especially treacherous

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Renal Angiomyolipoma Distinction from RCC

• Angiomyolipoma demonstrates specialized
  epithelioid perivascular-cell differentiation,
  and is consistently reactive with HMB-45, MART-1,
  anti-microophthalmia transcription factor
• Renal cell carcinoma is nonreactive with all of
  those reagents

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PEDIATRIC RENAL NEOPLASMS
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WILMS TUMORTypical Immunophenotype

• Reactive for
• CD26 (frozen sections only)
• Vimentin
• Keratin EMA (tubules only)
• CD10 (tubules only)
• WT-1 gene product
• Nonreactive for
• NB84
• CD99
• Synaptophysin
• FLI-1 protein
• HEP-PAR1
• Alpha-fetoprotein

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MESOBLASTIC NEPHROMAImmunophenotype

• Reactive for vimentin
• Usually reactive for
• Muscle specific actin
• Alpha-isoform (smooth muscle) actin
• Desmin
• Nonreactive for
• CD26
• Keratin
• EMA
• CD99
• CD10

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RENAL RHABDOID TUMORImmunophenotype

• Reactive for vimentin
• Often reactive for
• Keratin
• EMA
• Variably reactive for
• CD99
• WT-1
• Usually nonreactive for
• Synaptophysin
• CD26
• FLI-1
• NB84
• CD10

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CLEAR-CELL RENAL SARCOMA Immunophenotype

• Reactive for vimentin
• Variably reactive for WT-1
• Non-reactive for
• Keratin
• EMA
• CD99
• Synaptophysin
• NB84
• CD26
• CD10
• FLI-1 protein

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RENAL PNET Immunophenotype

• Reactive for CD99 FLI-1 protein
• Variably reactive for
• Vimentin
• NB84
• WT-1
• Synaptophysin
• Keratin
• Nonreactive for
• EMA
• CD26
• CD10
• HEP-PAR1

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TRANSITIONAL CELL CARCINOMAVARIANTS
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TRANSITIONAL CELL CARCINOMA OF THE PROSTATE
Distinction from Prostatic Adenocarcinoma

• Intraprostatic TCC
• Reactive for p63, GATA3, thrombomodulin,
  often for CEA, CA19-9, CK5/6 keratin may
  coexpress CK7 and CK20
• Negative for PSA, PSAP, P504S, PSMA, P504S
• Prostatic adenoCA
• Reactive for PSA, PSAP, P504S, PSMA
• Nonreactive for p63, GATA3, thrombomodulin,
  CA19-9
• Only rarely expresses CEA CK 5/6 may show CK 7
  or CK20 but not both

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HIGH-GRADE INTRARENAL TCC VS. RCC

• Intrarenal TCC--
• Reactive for thrombomodulin, GATA3, p63, and
  often for CK20, CA19-9, CEA, CD5/6
• Negative for RCC, PAX2, PAX8
• Renal cell carcinoma--
• Potentially reactive for RCC consistently
  labeled for PAX2 PAX8
• Nonreactive for p63, GATA3, thrombomodulin, CK20,
  CA19-9, CEA, and CK5/6

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SARCOMATOID TCC VS. TRUE SARCOMA

• Sarcomatoid TCC
• Reactive for keratin, and often for EMA
• Uncommonly demonstrates coexpression of desmin,
  muscle-specific actin, caldesmon, Myo-D1,
  myogenin, S100 protein, or osteonectin
• True urinary tract sarcoma
• Usually leiomyosarcoma
• Reactive for desmin, muscle-specific actin,
  alpha-isoform actin, caldesmon
• Uncommonly shows aberrant keratin-reactivity
• If mucosal carcinoma is present, the tumor is
  sarcomatoid carcinoma regardless of
  immunophenotype

Keratin
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SARCOMATOID TCC vs. PSCN

• BOTH proliferations share the capacity to
  coexpress vimentin, actin, desmin, and keratin
• Ki-67 indices are higher in sarcomatoid TCC, but
  there is too much overlap to use this in
  diagnosis
• p63 may also be helpful

P63 in STCC
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MALIGNANT GERM CELL TUMORS
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Malignant Germ Cell Tumors General Comments on
Immunophenotyping

• PLAP OCT3/4 are good screening markers for germ
  cell tumors, regardless of histologic subtype
• EMA CEA are absent in most MGCTs
• Keratin seen in only 10 of seminomas but is
  present in all other MGCTs
• Podoplanin CD30 are effective discriminants
  between seminoma embryonal carcinoma
• Alpha-fetoprotein correlates with the presence of
  yolk sac tumor
• B-hCG correlates with the presence of
  choriocarcinoma or other GCTs with isolated
  syncytial cells

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