Immunohistochemical classification of

the unknown primary tumour (UPT)

Part I

Prof. Mogens Vyberg

NordiQC
Institute of Pathology
Aalborg, Denmark
Tumours of unknown origin: Histology

Brain tumour - biopsy

Tumours of unknown origin: Immunohistochemistry

Pan-CK S-100

CD45 VIM
 IHC classification of the Unknown Primary Tumour

UPT: A tumour appearing in metastatic

setting without a histologically proven
primary tumour.
UPT pose an increasing challenge for the
pathologist - due to the progress in surgical
and oncological treatment possibilities.
 IHC classification of the Unknown Primary Tumour

New, relatively specific antibodies give the

pathologist more and better diagnostic tools.
But the diagnostic work also become more
complex in terms of planning, optimization of
protocols, interpretation of reaction patterns and
error trapping.
 IHC classification of the Unknown Primary Tumour

10 - 15% of cancers remain UPTs

+ ??% uncertain if primary or metastatic
- liver, lung, bone, lymph nodes, brain, peritoneum . . .
 ‘Undifferentiated’ neoplasms (5-10%)
- carcinomas, sarcomas, melanomas, germ cell tumours
- malignant lymphomas

• Adenocarcinomas (80-90%)
- lung, breast, prostate, colorectum, ovary, pancreas …

• Squamous cell carcinomas (5-10%)

- lung, esophagus, uterine cervix …
 IHC classification of the Unknown Primary Tumour

Differences in prognosis
Differences in treatment regimes
malignant lymphomas
carcinomas (breast, prostate, ovary . . .)
sarcomas (GIST, synovial sarcoma . . .)
germ cell tumours
Pathology tests cost effective
Pathology tests save patient discomfort
The patient’s ‘right to know’
The risk of hereditary cancer
 IHC classification of the Unknown Primary Tumour

 Most likely diagnoses

 Relevant differential diagnoses

 Optimal selection of antibodies for

a diagnostic algorithm
 Primary and secondary antibody panels
 Turn-around-time
 Laboratory expenses
 IHC classification of the Unknown Primary Tumour

Pathologist
- knowledge, acceptance, skill
Tumour material
- diagnostic markers
Antibodies available
- applic. in diagnostic algorithms GIST: S-100B Protease

Methods
- protocol:
sensitivity, specificity, reliability
- interpretation:
cut-off level for positivity
clinical relevance GIST: S-100B MWO
 IHC classification of the Unknown Primary Tumour
positive = any staining
Pathologist
- knowledge, acceptance, skill
Tumour material
- diagnostic markers
Antibodies available
- applic. in diagnostic algorithms CK 20
Methods negative = < 50%
- protocol: stained cells

sensitivity, specificity, reliability

- interpretation:
cut-off level for positivity
clinical relevance
CK 20
March 2008
Planning diagnostic immunohistochemistry

www.e-immunohistochemistry.info
Planning diagnostic immunohistochemistry
Planning diagnostic immunohistochemistry
my.statdxpathiq.com

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www.proteinatlas.org
Human protein Atlas – AE1/AE3
www.uptodate.com
Primary panel for the unknown primary tumour

”Real” CD45 CK S-100 VIM

Haemato-
lymphoid +/(-) -/(+) -/(+) +/(-)
neoplasms
Epithelial
neoplasms - +/(-) -/+ -/+
Mesothelial
neoplasms - + - +
Mesenchymal
and neuronal - -/(+) -/+ +
neoplasms
Non-neuronal
neuroepithelial - -/(+) + +
neoplasms
Germ cell
neoplasms - -/+ -/+ +
CD45 - Leucocyte common antigen (LCA)

• Transmembrane protein tyrosin phospatase essential for

haematopoietic signal transduction and cell activation
• Membrane associated component: 5 isotypes
• Intracellular component: one common type
CD45 - Leucocyte common antigen (LCA)

• Transmembrane protein tyrosin phospatase essential for

haematopoietic signal transduction and cell activation
• Membrane associated component: 5 isotypes
• Intracellular component: one common type
• Large majority of haematolymphoid cells
• Lost in maturing erythocytes, megakaryocytes and plasmacells
• ”Never” found in non-haematolymphoid cells
CD45 - Leucocyte common antigen (LCA)

Normal lymph node Malignant lymphoma

CD45 - Leucocyte common antigen (LCA)

 More than 90% of lymphomas are positive

 Negative on:
- some Acute Lymphoblastic Leukaemia/LBL
- plasma cell malignancies
- HR-S cells in classic Hodgkin Lymphoma
- some Anaplastic Large Cell Lymphoma (ALCL)
- ALK+ Large B-Cell Lymphoma
 Exceptionally positive in non haematol. tumours ?
CD45 - Leucocyte common antigen (LCA)
Liver Brain

Critical assay performance control

CD45 negative HRS cells in CD45 positive LH cells in NLPHL
classic Hodgkin Lymphoma

CD45 weaker staining in B-CLL CD45 weak reactivity in ALCL

39
CD45 – NordiQC run 37 2013
www.nordiqc.org/Run-15/Assessment/
assessment-CD45.htm

Which is best?
CD45 – NordiQC run 37 2013
www.nordiqc.org/Run-15/Assessment/
assessment-CD45.htm

Optimal Insufficient
CD45 – NordiQC run 37 2013
www.nordiqc.org/Run-15/Assessment/
assessment-CD45.htm
B-CLL

56% of labs 18% of labs

Optimal Insufficient
CD45 - Leucocyte common antigen (LCA)

 CD45 RO ~ T-cells

Lymph node/Tonsil  CD45 RA ~ B-cells

CD45 - Leucocyte common antigen (LCA)
CD45 - Leucocyte common antigen (LCA)
CD45 - Leucocyte common antigen (LCA)
Pancreas tumour
Pancreas tumour

CD45
Pancreas tumour

CD163
Pancreas tumour: undifferentiated carcinoma

CK
Primary panel for the unknown primary tumour

CD45 CK S-100 VIM

Haemato-
lymphoid +/(-) -/(+) -/(+) +/(-)
neoplasms
Epithelial
neoplasms - +/(-) -/+ -/+
Mesothelial
neoplasms - + - +
Mesenchymal
and neuronal - -/(+) -/+ +
neoplasms
Non-neuronal
neuroepithelial - -/(+) + +
neoplasms
Germ cell
neoplasms - -/+ -/+ +
Cellular filaments

# Microfilaments: (6 nm)

# Intermediate filaments
(7- 11 nm)

# Microtubuli (23 nm)

Intermediate filaments

 Group of mainly cytoplasmic

filaments 7 – 11 nm in diameter
 Part of the cytoskeleton in
virtually all cells, creating as
meshwork and connecting nuclear
membrane with cell membrane
 Often associated with
microfilaments (6 nm) and
microtubules (23 nm)
 Important for mechanical
strength and cellular functions
Intermediate filaments – tetrameric units

Central core formed by

eight tetramers

Nagle, AJSP 1988, 12:4

Intermediate filaments - 5 classes

I acidic cytokeratins
II basic-neutral cytokeratins
III vimentin, desmin,
 glial fibrillary acidic protein,
 peripherin
IV neurofilament protein,
 α-internexin, nestin
V lamins
Cytokeratins as tonofilaments

Cytokeratin intermediate
filaments attached
to desmosomes

Drochmans et al.
J Cell Biol. 1978, 79:427
Cytokeratins in diagnostic pathology

 Cytokeratins (CKs) belong to the most fundamental

markers of epithelial differentiation
 CKs comprise a large family of subtypes. Different
cell types express different patterns of CK subtypes
 Cancers generally express CK patterns that at least
in part represent the pattern of the putative cell of
origin
 Metastases express CK patterns fairly concordant
with those of the primary tumours
Micrometastases identified by cytokeratin

HE CK
Carcinoma in frosen section identified by cytokeratin

HE-frozen CK-frozen
Cytokeratins in carcinomas with aberrant growth patterns

Sarcomatoid carcinoma CK LMW

Low molecular weight cytokeratins in carcinomas

 Carcinomas “always”
LMW-CK-positive,
except some cases of
Renal cell carcinoma
Adrenal cortical carcinoma
Small cell carcinoma

CK8: Adrenal cortical carcinoma CK8: Renal cell carcinoma

CK: renal cell carcinoma
Primary panel for the unknown primary tumour

CD45 CK S-100 VIM

Haemato-
lymphoid +/(-) -/(+) -/(+) +/(-)
neoplasms
Epithelial
neoplasms - +/(-) -/+ -/+
Mesothelial
neoplasms - + - +
Mesenchymal
and neuronal - -/(+) -/+ +
neoplasms
Non-neuronal
neuroepithelial - -/(+) + +
neoplasms
Germ cell
neoplasms - -/+ -/+ +
Cytokeratins in non-epithelial tumours

CK8 CD45

♀ 42 y, tumour infiltrating
retroperitoneum

Malignant lymphoma !
CD79a
Primary panel for the unknown primary tumour

CD45 CK S-100 VIM

Haemato-
lymphoid +/(-) -/(+) -/(+) +/(-)
neoplasms
Epithelial
neoplasms - +/(-) -/+ -/+
Mesothelial
neoplasms - + - +
Mesenchymal
and neuronal - -/(+) -/+ +
neoplasms
Non-neuronal
neuroepithelial - -/(+) + +
neoplasms
Germ cell
neoplasms - -/+ -/+ +
Cytokeratins in malignant mesothelioma

CK8 CK5
Primary panel for the unknown primary tumour

CD45 CK S-100 VIM

Haemato-
lymphoid +/(-) -/(+) -/(+) +/(-)
neoplasms
Epithelial
neoplasms - +/(-) -/+ -/+
Mesothelial
neoplasms - + - +
Mesenchymal
and neuronal - -/(+) -/+ +
neoplasms
Non-neuronal
neuroepithelial - -/(+) + +
neoplasms
Germ cell
neoplasms - -/+ -/+ +
Cytokeratins in sarcomas

Synovial sarcoma angiosarcoma

Cytokeratins in non-epithelial tumours

Leiomyosarcoma
Primary panel for the unknown primary tumour

CD45 CK S-100 VIM

Haemato-
lymphoid +/(-) -/(+) -/(+) +/(-)
neoplasms
Epithelial
neoplasms - +/(-) -/+ -/+
Mesothelial
neoplasms - + - +
Mesenchymal
and neuronal - -/(+) -/+ +
neoplasms
Non-neuronal
neuroepithelial - -/(+) + +
neoplasms
Germ cell
neoplasms - -/+ -/+ +
Cytokeratins in malignant melanoma
CK
Primary panel for the unknown primary tumour

CD45 CK S-100 VIM

Haemato-
lymphoid +/(-) -/(+) -/(+) +/(-)
neoplasms
Epithelial
neoplasms - +/(-) -/+ -/+
Mesothelial
neoplasms - + - +
Mesenchymal
and neuronal - -/(+) -/+ +
neoplasms
Non-neuronal
neuroepithelial - -/(+) + +
neoplasms
Germ cell
neoplasms - -/+ -/+ +
Cytokeratins in germ cell tumours

CK8: Seminoma CK8: Embr. carcinoma

Cytokeratins: retrieval causing false negativity

HIER Proteolysis
AE1 detects CK8 after HIER only
AE1 does not detect CK18
AE3 does not detect CK8/CK18

SCLC CK: renal cell carcinoma

Proteolysis
Cytokeratins: retrieval causing false negativity

TP FN

HIER Proteolysis

RCC

TP CK: renal cell carcinoma FN

Primary panel for the unknown primary tumour

CD45 CK S-100 VIM

Haemato-
lymphoid +/(-) -/(+) -/(+) +/(-)
neoplasms
Epithelial
neoplasms - +/(-) -/+ -/+
Mesothelial
neoplasms - + - +
Mesenchymal
and neuronal - -/(+) -/+ +
neoplasms
Non-neuronal
neuroepithelial - -/(+) + +
neoplasms
Germ cell
neoplasms - -/+ -/+ +
S-100 protein

 Family of acid calcium binding proteins 9/13 kDa

 Located in nuclei, cytoplasm and cell membranes
 At least 10 α-chains and one β-chain creating
homo- and heterodimers

 S-100 β-chain mainly found in

 Melanocytes
 Glial cells
 Langerhans’ cells / interdigitating reticulum cells
 Fat cells
 Myoepithelial cells
 Polyclonal antibodies primarily detects the β-chain
S-100 protein

brain chondrocytes
S-100 protein

Tonsil
S-100 protein – pancreas
S-100 in malignant tumours

mal. melanoma breast carcinoma

S-100 protein
To HIER or not..

S100B Frozen IHC

Proteolytic HIER
Primary panel for the unknown primary tumour

”Real” CD45 CK S-100 VIM

Haemato-
lymphoid +/(-) -/(+) -/(+) +/(-)
neoplasms
Epithelial
neoplasms - +/(-) -/+ -/+
Mesothelial
neoplasms - + - +
Mesenchymal
and neuronal - -/(+) -/+ +
neoplasms
Non-neuronal
neuroepithelial - -/(+) + +
neoplasms
Germ cell
neoplasms - -/+ -/+ +
Vimentin

 Cytoplasmic intermediate filament, 57 kDa

 Present in all mesenchymal cells
 Present in early stages of all cells, replaced by other
intermediate filaments in most
non-mesenchymal cells
 Coexpressed with cytokeratin in some epithelia
 Endometrium, renal tubules, thyroid gland …
 Coexpressed with cytokeratin
in some non-epithelial cells
 Mesothelium
Vimentin in normal tissue

HE VIM
Normal brain
Vimentin in carcinomas

renal cell carcinoma endometrioid carcinoma

Vimentin in non-epithelial tumours

mal. melanoma mal. mesothelioma

Secondary panels for carcinoma identification/subclassification

 ”Breast markers”
 Cytokeratin subtypes
 ”Lung markers”
 Oncofetal proteins
 ”GI-markers”
 Transcription factors
 ”Fem.gen.tract markers”
 Neuroendocrine proteins
 ”Urinary tract markers”
 Hormone receptors  Prostate markers
 Secretory proteins  Squamous cell markers
 Cell adhesion molecules  ”Mesothelial markers”
...  NE cell markers
 ”Liver markers”
 ”Adrenal cortical markers”
 Germinal cell markers