Adenocarcinoma of the unknown primary (ACUP) - NordiQC

Montreal July 10th 2010 

Adenocarcinoma of the 

unknown primary 

(ACUP) 

Mogens Vyberg 

Assoc. professor 

Scheme director 

Inst. of Pathology, Aalborg Hospital 

Aarhus University Hospital 

Denmark 

Tumours of the unknown primary: Occurrence 

3 - 30% of all cancer biopsies 

+ uncertain if primary or metastatic? 

• Adenocarcinomas (80-90%) 

• Squamous cell carcinoma (5-10%) 

• ‘Undifferentiated’ neoplasms (5-10%) 

- carcinomas 

- malignant lymphomas 

-sarcomas 

- malignant melanomas 

- germ cell tumours . . . . 

Workshop Diagnostic IHC 

July 10, 2010 

1 

Mogens Vyberg 1


& 42, liver adenocarcinoma of unknown primary 

& 42, liver adenocarcinoma of unknown primary 

Colon-rectum? 

Pancreas/biliary tract? 

Stomach? 

Lung? 

Ovary/endometrium? 

CDX2 

& 42, liver adenocarcinoma of unknown primary: colon-rectum 

CK20 

CK7 

Mogens Vyberg 2


&63, adenocarcinoma in uterine cervix – endometrial? 

CD45 

pigment 

&63, adenocarcinoma in uterine cervix – endometrial? 

CD45 

CEA VIM 

&63, adenocarcinoma in uterine cervix – rectal? 

CD45 

CK7 CK20 

pigment 

pigment 

Mogens Vyberg 3


&63, adenocarcinoma in uterine cervix – primary? 

CD45 

ER P16 


CD45 

MUC5AC MUC2 


CDX2 

CD45 

pigment 

pigment 

pigment 

Mogens Vyberg 4


&63, adenocarcinoma in uterine cervix – rectal biopsies 

CD45 

pigment 


CK20 CK7 


IHC confusing or misleading ! 

CDX2 

Mogens Vyberg 5


&48, suspicion of stomach cancer 



CK-PAN CD68 

Mogens Vyberg 6



&48, suspicion of stomach cancer – an artefact ! 

Confusing morphology “confirmed” by IHC 

Adenocarcinomas of the unknown primary: Planning 

Consider the likelihood of an origin 

Morphology 

Age and sex 

Clinical information, imaging . . . . 

CD34 

Select antibodies for a diagnostic algorithm: 

Primary and secondary antibody panels 

Turn-around-time 

Laboratory expenses 

Interpret the stains in a proper context 

Mogens Vyberg 7


“Narrow-spectrum” markers for adenocarcinomas I 

Cytokeratin subtypes 

“Breast markers” 

Estrogen receptor 

GCDFP-15 

Mammaglobin 

“Lung markers” 

Thyroid transcription factor-1 

Napsin A 

“GI-markers” 

CDX-2 protein 

CEA 

“Pancreas marker” 

SMAD4 

“Fem.genit.tract markers” 

CA125 

WT1 

“Narrow-spectrum” markers for adenocarcinomas II 

“Urinary tract markers” 

CD10 

GP200 

PAX2 

Prostate markers 

PSA 

Prostein 

Cytokeratin subtypes 

“Endocrine markers” 

Synaptophysin 

Chromogranin A 

Hormones 

Thyroglobulin 

Hepatocyte antigen 

Calretinin 

Inhibin . . . . . . 

Sorry – another time 

CK: renal cell carcinoma 

Mogens Vyberg 8


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 

Low molecular weight CKs in (adeno-)carcinomas 

Carcinomas “always” 

MW-CK-positive, 

except some cases of 

Renal cell carcinoma 

Adrenal cortical carcinoma 

Small cell carcinoma 

Squamous cell carcinoma 

CK8: Adrenal cortical carcinoma CK8: Renal cell carcinoma 

CK: renal cell carcinoma 

LMW CKs in sarcomas 

Synovial sarcoma 

Angiosarcoma 

Mogens Vyberg 9


LMW CKs in malignant melanoma 

malignant melanoma 

CK subtypes paired 

• A CK class I and a CK class II - always paired 

Modified from: Lane & Alexander Semin Cancer Biol 1990, 1:165 

CK subtypes in carcinomas 

Neutral/Basic (B, class II) 

Acidic (A, class I) 

Squamous cell carcinoma 

Transitional cell tumour 

Malignant mesothelioma 

Adenocarcinoma: complex 

epith. (bronch., breast, prost.) 

Adenocarc.: biliary tract, 

pancr., endom., ovary 

Adenocarc.: stomach 

Adenocarc.: intestine 

Hepatocellular carcinoma 


Endocrine tumours: 

carcinoids 

- Merkel cell carcinoma 

- Thyroid carcinoma 

1 

10 

+ 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

4 

+ 

+ 

- 

- 

- 

- 

- 

- 

- 

- 

- 

++: almost always pos, +: usually pos., (+) less frequently pos., -: rarely or never pos. 

13 

+ 

+ 

- 

- 

- 

- 

- 

- 

- 

- 

- 

5 

++ 

+ 

++ 

(+) 

(+) 

- 

- 

- 

- 

- 

- 

14 

++ 

+ 

++ 

(+) 

(+) 

- 

- 

- 

- 

- 

- 

17 

+ 

+ 

+ 

(+) 

(+) 

- 

- 

- 

- 

- 

- 

19 

+ 

++ 

++ 

++ 

++ 

++ 

++ 

- 

+ 

+ 

+ 

7 

+ 

++ 

+ 

++ 

++ 

+ 

+ 

+ 

+ 

- 

++ 

20 

- 

++ 

- 

- 

(+) 

+ 

++ 

- 

- 

++ 

- 

8 

18 

+ 

++ 

++ 

++ 

++ 

++ 

++ 

++ 

++ 

++ 

++ 

Mogens Vyberg 10


CK subtypes in adenocarcinomas I 






carcinoids 



1 

10 

Squamous cell carcinoma (+) 

Transitional cell tumour - 



- 



- 


- 


- 


- 

- 

- 

- 

- 

4 

+ 

+ 

- 

- 

- 

- 

- 

- 

- 

- 

- 


- 

- 

- 

- 

- 

- 

- 

- 

- 

++ 

+ 

++ 

(+) 

(+) 

- 

- 

- 

- 

- 

- 

++ 

+ 

++ 

(+) 

(+) 

- 

- 

- 

- 

- 

- 

(+) 

+ 

+ 

(+) 

(+) 

- 

- 

- 

- 

- 

- 

(+) 

++ 

++ 

++ 

++ 

++ 

++ 

CK subtypes in adenocarcinomas (complex epithelia) 

+ 

+ 

CK5: Breast lob. carcinoma CK5: Breast duct. carc. 

CK subtypes in adenocarcinomas (complex epithelia) 

CK5: Prostate Breast lob. hyperplasia carcinoma CK5: prostate adenocarc. 

13 

5 

14 

17 

19 

- 

+ 

+ 

+ 

7 

(+) 

++ 

+ 

++ 

++ 

+ 

+ 

+ 

+ 

- 

++ 

20 

- 

++ 

- 

- 

(+) 

+ 

++ 

- 

- 

++ 

- 

8 

18 

(+) 

++ 

++ 

++ 

++ 

++ 

++ 

++ 

++ 

++ 

++ 

Mogens Vyberg 11


CK subtypes in adenocarcinomas II 






carcinoids 



1 

10 





- 



- 


- 


- 


- 

- 

- 

- 

- 

4 

+ 

+ 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

(+) 

(+) ! (+) ! (+) ! ++ ++ (+) ! ++ 

- - - ++ + + ++ 

- - - ++ + ++ ++ 


+ 

+ 

- 

++ 

+ 

++ 

- 

- 

- 

- 

++ 

+ 

++ 

(+) 

- 

- 

- 

- 

(+) 

+ 

+ 

(+) 

- 

- 

- 

- 

(+) 

++ 

++ 

++ 

- 

+ 

+ 

+ 

(+) 

++ 

+ 

++ 

+ 

+ 

- 

++ 

CK subtypes in adenocarcinomas (pancreas/biliary tract) 

CK20: Gall bladder 

adenocarcinoma 

CK subtypes in adenocarcinomas (ovary) 

CK5: Serous ovarian 

carcinoma 

13 

CK17 

5 

14 

17 

19 

7 

20 

- 

++ 

- 

- 

- 

- 

++ 

- 

CK5: Biliary tract adenosquamous 

carcinoma 

8 

18 

(+) 

++ 

++ 

++ 

++ 

++ 

++ 

++ 

Mogens Vyberg 12


CK subtypes in adenocarcinomas III 







carcinoids 



1 

10 





- 



- 


- 


- 

- 

- 

- 

- 

- 

4 

+ 

+ 

- 

- 

- 

- 

- 

- 

- 

- 

- 


- 

- 

- 

- 

- 

- 

- 

- 

- 

++ 

+ 

++ 

(+) 

(+) 

- 

- 

- 

- 

- 

- 

++ 

+ 

(+) 

(+) 

- 

CK subtypes in adenocarcinomas (GI tract) 

CK20 

CK20 

13 

+ 

+ 

5 

14 

++ 

- 

- 

- 

- 

- 

CK7 

CK7 

Colon: Typical CK7/CK20 profile 

CK subtypes in adenocarcinomas (GI tract) 

CK7 

Colon: Deviant 

CK20 

17 

(+) 

+ 

+ 

(+) 

(+) 

- 

- 

- 

- 

- 

- 

19 

(+) 

++ 

++ 

++ 

++ 

++ 

++ 

- 

+ 

+ 

+ 

7 

(+) 

++ 

+ 

++ 

++ 

+ 

+ ! 

+ 

+ 

- 

++ 

20 

- 

++ 

- 

- 

(+) 

+ 

++ 

- 

- 

++ 

- 

8 

18 

(+) 

++ 

++ 

++ 

++ 

++ 

++ 

++ 

++ 

++ 

++ 

CK7 

CK20 

CK20 CK7 

Mogens Vyberg 13


CK subtypes in adenocarcinomas 






carcinoids 



1 

10 





- 



- 


- 


- 


- 

- 

- 

- 

- 

4 

+ 

+ 

- 

- 

- 

- 

- 

- 

- 

- 

- 


+ 

+ 

- 

- 

- 

- 

- 

- 

- 

- 


13 

- 

5 

++ 

+ 

++ 

(+) 

(+) 

- 

- 

- 

- 

- 

- 

14 

++ 

+ 

++ 

(+) 

(+) 

- 

- 

- 

- 

- 

- 

17 

(+) 

+ 

+ 

(+) 

(+) 

- 

- 

- 

- 

- 

- 

19 

(+) 

++ 

++ 

++ 

++ 

++ 

++ 

- 

+ 

+ 

+ 

7 

(+) 

++ 

+ 

++ 

++ 

+ 

+ 

+ 

+ 

- 

++ 

20 

- 

++ 

- 

- 

(+) 

+ 

++ 

- 

- 

++ 

- 

8 

18 

(+) 

++ 

++ 

++ 

++ 

++ 

++ 

++ 

++ 

++ 

++ 

CK7 

CK7 

HCC: typical HCC: deviant 

CK7 


Mallory body 

CK7: RCC CK7: RCC 

CK20 

Mogens Vyberg 14


CK subtypes in endocrine tumours 





Endocr. tumours: 

carcinoids 



1 

10 





- 



- 


- 


- 


- 

- 

- 

- 

- 

4 

+ 

+ 

- 

- 

- 

- 

- 

- 

- 

- 

- 


+ 

+ 

CK subtypes in endocrine tumours 

13 

- 

- 

- 

- 

- 

- 

- 

- 

- 

++ 

+ 

++ 

(+) 

(+) 

- 

- 

- 

- 

- 

- 

++ 

+ 

++ 

(+) 

(+) 

- 

- 

- 

- 

- 

- 

(+) 

+ 

+ 

(+) 

(+) 

- 

- 

- 

- 

- 

- 

(+) 

++ 

++ 

++ 

++ 

++ 

++ 

- 

+ 

+ 

+ 

(+) 

++ 

+ 

++ 

++ 

+ 

+ 

+ 

+ 

- 

++ 

- 

++ 

- 

- 

(+) 

+ 

++ 

CK20: Merkel cell carc. CK19: Thyr. papill. carc. 

Cytokeratins: Selection of antibodies 

optimal too diluted HIER Prot. 

CK5/6: clone D5/16 B4 

CK5: XM-26 

5 

14 

17 

19 

7 

20 

- 

- 

++ 

- 

8 

18 

(+) 

++ 

++ 

++ 

++ 

++ 

++ 

++ 

++ 

++ 

++ 

CK5/14/1/10/19?: 

34BE12 

Mogens Vyberg 15



CK-LMW optimal (8/18) performance too dilutedin 

3 NordiQC runs 

Clone Sufficient (optimal or good) 

C51 19 / 21 (91%) 

DC10 66 / 73 (90%) 

5D3 23 / 33 (70%) 

CAM5.2 29 / 66 (44%) 

35BH11 6 / 38 (16%) 

B22.1/B23.1 2 / 2 

optimal too diluted 

CK8/18: clones 5D3, TS1, 

C51, DC10, B22.1/B23.1 

RCC 

optimal too diluted 

CK8/18: clones 5D3, TS1, 

C51, DC10, B22.1/B23.1 

CK8 (7): CAM5.2 

CK8 (7): CAM5.2 

Mogens Vyberg 16


optimal 


Esophagus optimal 

CK19: clone A53-B/A2.26 CK19: clone RCK108 


Papillary optimal thyroid carcinoma 

After proteolysis as 

recommended by the vendor 

CK19: clone A53-B/A2.26 CK19: clone RCK108 

After proteolysis as 

recommended by the vendor 

Mogens Vyberg 17


Estrogen receptor alpha 

Estrogen sensitive tissues: 

Breast 

Ovarium 

Fallopian tube 

Endo- and myometrium 

Uterine cervix 

Thyroid gland 

Pituitary gland 

Pancreas 

Sweat gland 

Salivary gland 

Prostate 


Adenocarcinomas 

Breast carcinoma +/– 

Ovarian (non-muc.) +/– 

Endometrial carc. +/– 

Lung, kidney, endocrine . . . 

–/+ 

“Breast markers” 

ERα 

Sica et al. APLM 132,1889,2008 

Mogens Vyberg 18



Lung adenocarcinomas 

ERα 

Sica et al. APLM 132,1889,2008 

Estrogen receptor alpha: The influence of antibody selection 

92 lung adenocarcinomas 

ER+ (≥ 1% pos. cells) 

1D5: 8% 

6F11: 14% 

SP1: 27% 

Estrogen receptor alpha: EQA 

8 runs 2004 - 2010 

mAb 1D5 

mAb 6F11 

rmAb SP1 

Protocols 

244 

240 

247 

ERα 

Gomez-Fernandez et al. AIMM 2010;18:137 

false negative 

All protocol settings 

Sufficient 

62% 

76% 

85% 

Optimal 

18% 

39% 

Control 

61% 

57 

Mogens Vyberg 19



8 runs 2004 correct - 2010 


mAb 1D5 

mAb 6F11 

rmAb SP1 

Protocols 

127 

190 

231 


Optimal protocol settings 

Sufficient 

72% 

86% 

90% 

Optimal 

35% 

53% 

Control 

65% 

8 runs 2004 correct - 2010 


Control 

optimal insufficient 

GCDFP-15 (gross cystic disease fluid protein 15) 

Prolactin induced 

glycoprotein of apocrine cells 


Breast +/– 

Sweat gland +/– 

Salivary gland +/– 

Lung –(+) 

Renal cell –(+) 

Ovary –(+) 

58 

59 

GCDFP-15 in sweat glands 

Mogens Vyberg 20


GCDFP-15 

Gastric biopsies, ♀ 43 y 

Breast carcinomas 

ER GCDFP-15 

Mammaglobin 

Mammary-specific member 

of the uteroglobin family 


Breast +/– 

Sweat gland +/– 

Salivary gland +/– 

Lung –/+ 

Stomach, biliary, 

thyroid, ovary, endom. 

–(+) 

Mammaglobin in breast 

Mogens Vyberg 21


Mammaglobin 

% 61, “metastatic adenocarcinoma “in the eye lid 


CK5 

Breast ductal carcinomas 

Mogens Vyberg 22



CK7 


GCDFP-15 

% 61, “metastatic adenocarcinoma “in the eye lid: Moll gland ca. 

MAMMAGLOBIN 

Mogens Vyberg 23


CDX-2 protein 

Drosophila caudal related 

homeobox gene 2 product: 

Nuclear transcription factor 

for intestinal differentiation 

Intestine 

all cell types incl. endocrine 

Intestinal metaplasia 

chronic gastritis 

Barrett’s esophagus 

Pancreas/bil.tract 

“GI markers” 

CDX-2 protein in adenocarcinoma 

Colorectum +(–) 

Mucinous ovar. +(–) 

Esoph./Stom. +/– 

Mucinous lung +/– 

Pancr./biliary –/+ 

Prostate –(+) 

Urothelial –(+) 

Endometrioid –(+) 

colon 

pancreas 

Cdx2 in colon adenocarc. 

Mogens Vyberg 24


CDX-2 protein in adenocarcinoma 

Colorectum +(–) 

Mucinous ovar. +(–) 

Esoph./Stom. +/– 

Mucinous lung +/– 

Pancr./biliary –/+ 


Urothelial –(+) 

Endometrioid –(+) 

Endometrioid carcinoma: ER & CDX-2 

CDX2 in endocrine tumours 

Foregut –(+) 

Stomach, lung 

Midgut + 

Hindgut –/+ 

Colon adenoc. medullary adenoc. 

Colon adenosquamous carc. 

ER CDX-2 

Ileum carcinoid 

Mogens Vyberg 25


CDX2: Selection of antibodies 

CDX2: Selection of antibodies 

DAK-CDX2 CDX2-88 

Pancreas 

“Lung markers” 

Colon ad. Muc.ad 

Mogens Vyberg 26



Nuclear gene regulating protein 

Thyroid gland: regulating thyroglobulin, thyroperoxidase 

and thyrotropin receptor. 

Lung: regulating surfactant proteins and 

Clara cell secretory protein. 

Thyroid follicles 

and C-cells 

Lung pneumocytes II 

and Clara cells 

Brain 

Pituitary 

Parathyroid 


Lung adenocarcinoma 

- large cell carcinoma 

- small cell carcinoma 

- carcinoid 

- squamous cell carcinoma 

Non-lung small cell carcinoma 

Thyroid carc. (non-anaplastic) 

Non-lung carcinomas 

Non-lung carcinoids 


% pos.: 

SCLC in liver, clone 8G7G3 

Normal lung 

SPT24 8G7G3/1 

80 70 

50 

90 

60 

15 

20-40 

100 

0 - 5 

~ 0 

40 

90 

20 

0 

? 

100 

0 - 5 

~ 0 

Matoso et al, AIMM 2010,18:142-149 

Mogens Vyberg 27



Napsin A 

liver 

lung carcinoid 

SPT24 8G7G3/1 

Aspartic proteinase 

Type II pneumocytes 

Proximal and convoluted 

renal tubules 

(Pancreatic acini and ducts) 

Napsin A 

Aspartic proteinase 

Type II pneumocytes 

Proximal and convoluted 

renal tubules 

(Pancreatic acini and ducts) 

Mogens Vyberg 28


Napsin A: lung adenocarcinoma ~ 80% 

Napsin A: Lung squamous carcinoma ??% 

Napsin A: Renal cell carcinoma 0 – 70% 

Mogens Vyberg 29


& 49 – Liver adenocarcinoma of unknown primary 

CK20 

& 49 – Liver adenocarcinoma of unknown primary: Lung 

TTF Napsin A 

SMAD4 

Ep-CAM 

Similar to Mothers Against Drosophila 4 = 

Deleted in pancreatic cancer-4 (DPC4) 

Nuclear transcription activator in all normal cells 

Deleted in ~ 50% of pancreatic carcinomas 

Deleted much less frequently in other carcinomas 

Marker of invasive growth (?) 

Mogens Vyberg 30


% 53 – liver with ACUP 

% 53 – liver with ACUP 

CDX2 SMAD4 

SMAD4 loss in pancreatic and ampullary carcinomas 

Ep-CAM 

Mogens Vyberg 31


“Glands” in pancreatic nerve 

SMAD4 loss in “glands” in panceatic nerve 

Carcinoembryonic antigen (CD66e) 

Adhesion molecule espc. 

associated with intestine 

Atrophic pancreas 

Mogens Vyberg 32


Carcinoembryonic antigen (CD66e) in adenocarcinomas 

Colorectal + 

Medull. thyroid + 

Pancreas/biliary tract +/– 

Stomach +/– 

Lung +/– 

Ovary, mucinous +/– 

Ovary, non-muc. –/+ 

Prostate – 

Kidney – 

Liver – 

Carcinoembryonic antigen 

metast. colon adenoc 

Medul. thyroid carc. Breast ductal carc. 

Pancreas carcinoma 

TTF 

Mogens Vyberg 33


Pancreas carcinoma 

CEA VIM 

Carcinoembryonic antigen – which antibody? 


Normal liver 

II-7 

TF3H8-1 

Mogens Vyberg 34



Mal. mesothelioma 

mAb II-7 pAb 

Cancer antigen 125 

Oncofetal glycoprotein. In fetal life associated with amnion, 

coelomic and Müllerian epithelium 

Mesothelial cells (visceral) 

Epithelial cells of 

Female genital tract 

Breast 

Pancreas / biliary tract 

Apocrine sweat glands 

Cancer antigen 125 in adenocarcinoma 

Serous ovarian + 

Endometrioid +/– 

Cervical +/– 

Pancreas/bil. +/– 

Lung –/+ 

Breast –/+ 

Colorectal –/+ 

Renal carcinoma –(+) 


Pancreas adenocarc. 

Mogens Vyberg 35


Cancer antigen 125 in lung adenocarcinoma 

Wilms’ tumour-1 (WT1) protein 

TTF CA125 

Transcription factor for development of the genitourinary system 

Mesothelium 

Ovary (surface / inclusion cysts) 


Female genital tract: stromal cells 

Bone marrow stem cells 


Wilms’ tumour-1 (WT1) protein in adenocarcinomas 

Serous carcinoma + 

Endometrioid carcinoma +/– 

Other carcinomas –(+) 

Serous carcinoma 

Mogens Vyberg 36


Wilms’ tumour-1 (WT1) protein in adenocarcinomas 


Mesothelioma 

Optimal Insufficient – too dilute Ab 

Aalborg Hospital 

Aalborg 

110 

111 

Mogens Vyberg 37


Around Aalborg 

112 

Mogens Vyberg 38

Adenocarcinoma of the unknown primary (ACUP) - NordiQC

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