CARCINOMA OF UNKNOWN PRIMARY:

AN EXPLORATION OF TRANSCRIPTION
FACTOR IMMUNOHISTOCHEMISTRY
Jason L Hornick, MD, PhD
Director of Surgical Pathology
and Immunohistochemistry
Brigham and Women’s Hospital
Professor of Pathology
Harvard Medical School
Boston, MA, USA
 Poorly differentiated malignant neoplasms
S100 SALL4
Tumor types KRT CD45
SOX10 OCT4
Carcinoma ++ ‒ ‒ ‒
Mesothelioma ++ ‒ ‒ ‒
Melanoma ‒ ‒ ++ ‒
Lymphoma ‒ ++ ‒ ‒
Sarcoma ‒ ‒ ‒/+ ‒
Germ cell tumor +/‒ ‒ ‒ ++
 Carcinoma of unknown primary
Definition: histologically confirmed metastatic
carcinoma for which primary site cannot be
identified after standard diagnostic approach:
• Detailed history and physical examination
• Blood counts and biochemical analysis
• Urinalysis and stool occult blood test
• CT of thorax, abdomen, and pelvis
• Histologic review including IHC
Origin of primary tumors (autopsy)
Organ Incidence
Pancreas 20-25%
Lung 15-20%
Liver/biliary 10%
Colon/rectum 8%
Stomach 5%
Kidney 5%
Ovary <5%
Prostate <5%
Breast 2%
Other 1%
Histologic groups of carcinoma of unknown
primary

Histology Frequency
Well or moderately differentiated
60%
adenocarcinoma
Poorly differentiated adenocarcinoma
30%
or undifferentiated carcinoma

Squamous cell carcinoma 5%

Undifferentiated malignant neoplasm 5%

 Overview
• Keratins in carcinomas

• Lineage-restricted markers and primary

site determination

• Primary tumors of the liver

• Special circumstances and pitfalls

 Keratin family members in carcinomas

• Low-molecular-weight keratins (CK8, CK18, CAM5.2)

– Glandular epithelium, hepatocytes

• High-molecular-weight keratins (CK5, CK14, 34βE12)

– Squamous epithelium, urothelium, basal cells
Keratin family members in carcinomas:
CK7 and CK20

• CK7 wide distribution in epithelial cells

• CK20 restricted to lower GI tract epithelium,

umbrella cells of the urinary bladder, Merkel
cells
CK7 and CK20 – are they (still) useful?

Phenotype Primary sites

CK7– / CK20+ Colon/rectum

CK7+ / CK20+ Bladder, upper GI, pancreas

CK7– / CK20– Uncommon (prostate, HCC)

CK7+ / CK20– Nearly everything else

Metastatic colonic adenocarcinoma

CK20
Metastatic colonic adenocarcinoma

CK7
Metastatic colonic adenocarcinoma

CK20
 IHC for lineage/site specification:
cytoplasmic/membranous markers
Primary site Marker
Bladder Uroplakin
GCDFP-15
Breast
(prolactin-inducible protein)
Mammaglobin
Breast
(SCGB2A1/A2)
Colon/rectum Cadherin-17
Colon/rectum Villin
Lung Napsin A
Prostate Prostate-specific antigen
Prostate Prostatic acid phosphatase
Thyroid Thyroglobulin
 IHC for lineage/site specification:
cytoplasmic/membranous markers

• In general, expression is decreased (or entirely

absent) with poor differentiation – significant
impact on sensitivity

• Exception: CDH17
Breast carcinoma

GCDFP
Breast carcinoma

mammaglobin
 IHC for lineage/site specification:
nuclear transcription factors
• Insights gained from developmental and cell
biology research
• Transcription factors involved in patterning of
organ systems, lineage commitment
• Some are highly specific for particular cell type
or visceral organ
• Others show expression limited to several
tissue types
• Very helpful in determining primary site for CUP
 IHC for lineage/site specification:
nuclear transcription factors

• 25 years ago, very few were used in surgical

pathology practice
– TTF1, CDX2, MYOG

• In 2023, over 60 markers with potential

diagnostic applications are available
– Carcinomas, lymphomas, melanoma, germ
cell tumors, sarcomas
 IHC for lineage/site specification:
nuclear transcription factors
Transcription factor Primary site
CDX2 Colon/rectum, upper GI
GATA3 Breast, bladder
NKX3-1 Prostate
OCT4 Seminoma, embryonal carcinoma
PAX8 Thyroid, kidney, Müllerian
SALL4 Germ cell tumors
SATB2 Colon/rectum
SF1 Adrenal cortex
TTF1 (NKX2-1) Lung, thyroid
WT1 Müllerian, mesothelioma
 IHC for lineage/site specification:
nuclear transcription factors

• In general, even poorly differentiated carcinomas

maintain (relatively) diffuse expression in most
cases – high sensitivity

• With increasing study, specificity decreases

• Beware of relying on a single diagnostic marker

 TTF1 (NKX2-1)
• Lineage-specific transcription factor long history
of use in diagnostic IHC
• Originally named for role in activating transcription
from thyroglobulin promoter
• Expressed in normal and neoplastic thyroid
follicular cells
• Most widely used application: ascribing lung origin
to primary and metastatic adenocarcinomas (and
supporting adenocarcinoma over squamous cell
carcinoma in poorly differentiated NSCLCA)
Lung adenocarcinoma

TTF1
Metastatic poorly differentiated lung adenocarcinoma

TTF1
Poorly differentiated (insular) thyroid carcinoma

TTF1
 WT1
• Wilms tumor 1
• Transcription factor plays diverse roles in
cancer depending upon tumor type and
biological context
• Expressed in malignant mesothelioma, serous
carcinoma
• Positive in nearly all serous carcinomas of the
ovary; uncommon in serous carcinomas of
the endometrium (variable results in different
studies)
Malignant mesothelioma

WT1
Metastatic mesothelioma

WT1
Metastatic serous carcinoma

WT1
 WT1
• Among carcinomas, nuclear WT1 relatively
specific for ovarian serous tumors

• Positive in <5% of breast, lung, gastric,

colorectal, urothelial carcinomas

• Cytoplasmic staining detected in subset of

other carcinomas and various other tumor
types – only nuclear staining should be
considered positive for ascribing site of origin
 CDX2
• Caudal-type homeobox transcription factor
involved in intestinal differentiation
• Nuclear expression in >90% colorectal
adenocarcinomas
• Somewhat lower sensitivity in high grade
and MMR-deficient carcinomas
• Widely used to support colorectal origin
• No significant loss of sensitivity in the
metastatic setting
Colonic adenocarcinoma

CDX2
Metastatic colonic adenocarcinoma

CDX2
 CDX2
• Also expressed in carcinomas from other
gastrointestinal primary sites associated with
intestinal differentiation
– Esophagus and stomach
– Pancreas and biliary tree

• Often more heterogeneous staining in tumors

from these other sites
• Particularly helpful in differential diagnosis
between primary (poorly cohesive) gastric
carcinoma and metastatic breast carcinoma
Gastric adenocarcinoma

CDX2
 CDX2

• Pitfall: CDX2 can also be positive in mucinous

adenocarcinomas from diverse anatomic sites
– Ovary
– Lung
– Pancreas
– Bladder
 GATA3
• Transcription factor originally recognized for role
in T-cell function
• Clinically useful as marker for breast or urothelial
origin
• Positive in >80% of breast and urothelial
carcinomas
• No significant loss of sensitivity in the metastatic
setting
• Comprehensive surveys revealed expression in
wide range of tumor types
Bladder urothelial carcinoma

GATA3
Breast carcinoma

GATA3
Expression of GATA3 in other tumors
Tumor type Frequency
Squamous cell carcinoma (skin) 80%
Squamous cell carcinoma (cervix) 33%
Squamous cell carcinoma (lung) 10%
Lung adenocarcinoma 5-10%
Skin adnexal carcinomas 80-100%
Mesothelioma 25-60%
Salivary gland tumors 20-50%
Pancreatic ductal adenocarcinoma 40%
Paraganglioma 80%
Choriocarcinoma 100%
Chromophobe renal cell carcinoma 50%
Potential value of GATA3 in differential diagnosis

Positive Negative
Metastatic lobular breast carcinoma Gastric signet-ring-cell carcinoma

Metastatic ductal breast carcinoma Lung/GI/ovarian adenocarcinoma

Urothelial carcinoma Prostatic adenocarcinoma
Squamous cell carcinoma of skin Squamous cell carcinoma of lung
Malignant mesothelioma Lung adenocarcinoma

Paraganglioma Other neuroendocrine tumors

Am J Surg Pathol. 38:13-22, 2014

 NKX3-1
• Homeobox transcription factor, androgen-dependent

• Expression limited to prostate

• Usually more diffusely positive than conventional

cytoplasmic markers

• Helpful to distinguish high grade prostatic

adenocarcinoma from urothelial carcinoma

• Helpful to suggest prostatic origin in metastatic

carcinoma
Prostatic adenocarcinoma

NKX3-1
 PAX8
• One of the most widely used lineage-specific
transcription factors in IHC approach to CUP

• Highly sensitive for carcinomas originating in

ovary, kidney, thyroid

• >90% serous, endometrioid, and clear cell

ovarian carcinomas positive for PAX8

• Much lower rate of expression in ovarian

mucinous adenocarcinomas
Expression of PAX8 in carcinomas

Primary site Positive cases

Renal 85-95%
Thyroid (papillary, follicular, poorly diff) 90-100%
Thyroid (anaplastic, medullary) 60-80%
Ovarian (non-mucinous) 90-100%
Ovarian (mucinous) 30-40%
Endometrial 90-100%
Cervical 90-100%
Renal cell carcinoma

PAX8
Metastatic serous carcinoma

PAX8
Metastatic serous carcinoma

PAX8
Metastatic serous carcinoma

TTF1
Poorly differentiated thyroid carcinoma

TTF1

PAX8
Metastatic lung adenocarcinoma

TTF1

PAX8
 Pan-PAX?
• Widely used polyclonal anti-PAX8 antibody not
specific for PAX8
• Cross-reacts with other PAX family members
(PAX5, PAX6, PAX3)
• Responsible for staining in B lymphocytes (PAX5)
• Responsible for staining in well-differentiated
neuroendocrine tumors of the pancreas (PAX6)
• PAX8-specific monoclonal antibodies available
 Polyclonal anti-PAX8 antibody

PAX8

PAX6

PAX5
 SF1
• Steroidogenic factor 1 (NR5A1)
• Recently investigated transcription factor
• Highly sensitive marker for sex cord-stromal
tumors
• Highly sensitive marker for adrenal cortical
carcinoma
• Particularly useful in distinguishing primary
adrenal cortical carcinoma from metastatic
renal cell carcinoma
Adrenal cortical carcinoma

SF1
Embryonic stem cell transcription factors
• Transcription factors involved in maintenance
of pluripotency
• Large body of literature on role in embryonic
stem cell biology
• Some of these transcription factors useful in
diagnostic IHC
• OCT4 (also known as OCT3/4) widely used in
clinical practice
• Other markers less often used
Expression of embryonic stem cell transcription
factors in germ cell tumors

Subtype SALL4 OCT4 NANOG SOX2

Seminoma ++++ ++++ ++++ –
Embryonal carcinoma ++++ ++++ ++++ ++++
Yolk sac tumor ++++ – – –
Teratoma – – – ++
Choriocarcinoma variable – – –
Seminoma

OCT4
Don’t forget to consider a
primary liver tumor!
 Hepatocellular carcinoma
• Alpha fetoprotein (AFP)
– Low sensitivity
• Polyclonal carcinoembryonic antigen (CEA)
– Bile canalicular pattern (other patterns not specific)
• Carbamoyl-phosphate synthase 1 (CPS1) =
Hep-Par 1 antibody
– Urea cycle enzyme
– Also expressed in 5-10% of adenocarcinomas of
diverse sites (most often of foregut origin)
• Arginase 1 (ARG1)
– Urea cycle enzyme
– Most sensitive and specific hepatocellular marker
Hepatocellular carcinoma

pCEA
Hepatocellular carcinoma

CPS1/Hep-Par1
Hepatocellular carcinoma

ARG1
 Intrahepatic cholangiocarcinoma
• Radiologic features:
– Large solitary mass +/- satellite lesions
– Biliary dilatation
– Hepatic capsular retraction
– Delayed enhancement
– Lobar/segmental atrophy
• Histology/immunophenotype essentially
indistinguishable from pancreatic ductal
adenocarcinoma
– CK7/CK19 positive
– Loss of SMAD4 (~50%)
• When you encounter a CK7+ AdCA in the liver,
consider cholangiocarcinoma
 Intrahepatic cholangiocarcinoma
Capsular retraction Biliary dilatation

Satellite
 Intrahepatic cholangiocarcinoma
Capsular retraction
Satellite
Intrahepatic cholangiocarcinoma
Pancreatic ductal carcinoma

SMAD4
Metastatic cholangiocarcinoma

SMAD4
 Loss of SMAD4 in carcinomas
Primary site Loss of expression
Pancreatic/biliary (except peripheral
55%
intrahepatic cholangiocarcinoma)
Appendiceal 25%
Colorectal 20%
Esophageal 4%
Breast 3%
Gastric 2%
Ovarian (non-serous) 2%
Ovarian (serous) 0%
Endometrial 0%
Lung 0%
Hepatocellular 0%
Renal cell 0%
 Squamous cell carcinoma: P63 and P40
• Confirmation relatively straightforward
– P63 (also positive in urothelial and subset of
adenocarcinomas; ~20% lung AdCA)
– P40 (much more specific for squamous cell and
urothelial carcinomas)
• In general, IHC not helpful to determine primary site
(other than P16: HPV-associated SCC of
oropharynx, cervix, anal canal)
• In situ hybridization can be helpful (head and neck):
– EBER – nasopharyngeal carcinoma
– High-risk HPV – oropharyngeal carcinomas (tonsil, base
of tongue)
Lung squamous cell carcinoma

P40
Metastatic HPV-associated squamous cell carcinoma

P16
 Special circumstances and pitfalls I:
Keratin-positive soft tissue tumors
• Some soft tissue tumors are keratin positive
– Epithelioid sarcoma
– Synovial sarcoma (biphasic; rare cells in monophasic)
– Myoepithelial tumors
• Some sarcomas may show keratin expression
– Epithelioid angiosarcoma (30-50%)
– Epithelioid hemangioendothelioma (30%)
– Leiomyosarcoma (40%)
– Ewing sarcoma (20%)
Proximal-type Epithelioid Sarcoma

Pan-keratin
Proximal-type Epithelioid Sarcoma

SMARCB1/INI1
 Special circumstances and pitfalls II:
Thoracic SMARCA4-deficient undifferentiated tumor
 Special circumstances and pitfalls II:
Thoracic SMARCA4-deficient undifferentiated tumor
 Special circumstances and pitfalls II:
Thoracic SMARCA4-deficient undifferentiated tumor
• Present as mediastinal or lung mass, often with
disseminated metastasis
• Highly aggressive clinical course, dismal prognosis
• Genomic smoking signature; mutations characteristic
of lung carcinomas (KRAS, STK11)
• More likely undifferentiated/sarcomatoid carcinomas
• Nests and sheets of large cells, prominent nucleoli
• Variably positive for keratins, SALL4; usually SOX2
• Loss of SMARCA4 (BRG1) – defining feature
Thoracic SMARCA4-deficient Undifferentiated Tumor
Thoracic SMARCA4-deficient Undifferentiated Tumor

Pan-keratin
Thoracic SMARCA4-deficient Undifferentiated Tumor

SMARCA4
 Special circumstances and pitfalls III:
Undifferentiated/dedifferentiated melanomas

• Some metastatic (and more rarely primary) melanomas

negative for all conventional melanoma markers (S100,
SOX10, HMB-45, melan A)
• Undifferentiated melanoma: primary (or metastatic)
tumor negative for all melanoma markers
• Dedifferentiated melanoma: primary tumor expressed
melanoma markers, whereas metastasis negative
 Special circumstances and pitfalls III:
Undifferentiated/dedifferentiated melanomas
 Special circumstances and pitfalls III:
Undifferentiated/dedifferentiated melanomas
• Diagnosis extremely challenging
• Often diagnosed as “undifferentiated epithelioid
malignant neoplasm”
• Clues: anatomic site unusual for sarcoma
(especially axilla, neck), sometimes rhabdoid
• Identification of characteristic driver mutations
(BRAF, NRAS) allows for proper diagnosis
• Mutation-specific IHC also available
Undifferentiated Melanoma

BRAF V600E
 Practice points
• Review radiologic findings (could the biopsy be from
a primary liver tumor?)
• Pay close attention to histologic features – guide
judicious panel of IHC markers
• Increasing range of antibodies directed against
transcription factors becoming available
• Be aware of reported cross-reactivity (i.e., specificity)
to avoid misdiagnosis
• Be aware of diagnostic pitfalls
jhornick@bwh.harvard.edu
@JLHornick

THANK YOU!