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Home , Pancreatoblastoma, Peritoneal mesothelioma, Renal oncocytoma

Cadiz, 13th April 2018

The Challenge of of Unknown Primary (CUP)

J. Klos MD Department of Pathology Stavanger University Hospital Norway

1 Conflict of interests

NONE Unknown Primary Tumor (Cancer of the Unknown Primary – CUP) Histologically confirmed metastatic cancer for which primary site cannot be identified after standard diagnostic approach.

• Accounts for 2-5% of diagnosed in the US • 7th or 8th most frequent cancer • 4th or 5th most common cause of cancer death in both sexes • The number of cases shows decreasing tendency …

5 - 15% of all presents as Cancer incidence in Norway 22185 new cases in 2000 population 4,5 mln 31651 new cases in 2014 population 5,1 mln 2534 new cases in 2014 in Rogaland county

Males (new cases in 2014) Females (new cases in 2014) • Prostate (4889) • Breast (3324) • Colon and rectum (2157) • Colon and rectum (2009) • Lung (1596) • Female genital (1687) • Urinary bladder (1034) • Lung (1423) • Hematopoietic (1377) • Hematopoietic (1172) • (1015) • Melanoma (988)

1055 cases (3,3%) tumors of unknown primary location in 2014

4 Unknown primary tumor

• ‘Undifferentiated’ (~5%) (, malignant lymphomas, , , germ cell tumours)

• Squamous cell carcinomas (~5%) (lung, head and neck, oesophagus, uterine cervix, …)

• (~90%) (, lung, colon and rectum, liver and , stomach, , ovary, prostate, breast …) 2/3 well differentiated and 1/3 poorly differentiated

5 Relevance of cancer type/origin

• Differences in prognosis • Differences in treatment regimes . carcinomas (breast, prostate, ovary, thyroid, . . .) . malignant lymphomas . sarcomas (GIST, synovial . . .) . germ cell tumours . neuroendocrine tumors

Pathology is an effective method of investigating in CUP.

6 Essentials for right diagnosis • Quality and quantity of the material • Quality of standard laboratory procedures • Quality of immunohistochemistry • Sufficient number of available antibodies • Pathologist with sufficient knowledge of morphological spectrum of tumors as well as variation in their immunophenotype and at least basic knowledge of immunohistochemistry!

7 How to approach the problem?

No “shot gun” immunohistochemistry! Step by step

• Review the slide - get your first morphological impression • Get additional information • Review the slide again - get your second morphological impression • Determine the basic panel defining the broad category of or go to the next step if needed • Determine advanced panel defining subtype/origin of neoplasm

Important to secure sufficient number of slides for additional stains at early stage of investigation!

9 Interpretation of Morphology + Clinical + Laboratory Data

Not sufficient! Sufficient Ancillary tests Final diagnosis Interpretation

Not sufficient! Sufficient More ancillary tests Final diagnosis Interpretation

Not sufficient! Sufficient Cautious description Final diagnosis stating the degree of uncertainness and/or expert consultation

10 Part I Approach to neoplasm with non-characteristic morphology (poorly differentiated)

11 Basic IHC panel which defines the line of differentiation of tumor cells based on reactivity with wide spectrum antibodies • CD45 • Pancytokeratin • S-100 • • SALL4/ OCT4/PLAP (if germinal cell tumor suspected) • CD30 (if CD30+ lymphoproliferative/ embryonal ca suspected) • GFAP (when CNS is the location)

12

“Wishful thinking” profiles

13 “Wishful thinking” profiles

CD45 PanCK S-100 Vim SALL4/ OCT4/ PLAP Hemato-lymphoid + - - - - tumors Epithelial tumors - + - - - Melanocytic & - - + + - neural tumors Mesenchymal - - - + - tumors Mesothelial tumors - + - + -

Germ cell tumors - + - + +

14 The real “life” is more complicated

15 Real “hard life” profiles

CD45 PanCK S-100 Vim SALL4/ OCT4/ PLAP Hemato-lymphoid +/(-) -/(+) -/(+) +/- -/(+) tumors Epithelial tumors - +/(-) - /+ -/+ - Melanocytic & - -/(+) + +/(-) - neural tumors Mesenchymal - -/(+) -/+ + - tumors Mesothelial tumors - + - +/(-) -

Germ cell tumors - +/- -/+ +/- +/-

16 CD45

Family of membrane bound glycoproteins with tyrosine phosphatase properties involved in signal transduction and cell activation • Present at cell membranes of lymphocytes, monocytes and blast cells except erythropoietic cells, plasma cells and mature megakaryocytes • Not found on cells of other lineages

• Membranous reactivity pattern

• The concentrated mAb clones 2B11+PD7/26 and X16/99 and RTU with above clones as well as mAb clone RP2/18 can give optimal results

17 CD45 in malignant melanoma

18 CD45 PanCK S-100B Vimentin

Hematolymphoid tumours +/(-) -/(+) - (+) +/-

Hematolymphoid neoplasms which are most likely negative for CD45:

• Acute Lymphatic Leukemia/Lymphoblastic lymphoma • Plasmacytoma • Plasmablastic lymphoma* • ALCL* • HRS cells in classic Hodgkin lymphoma

* ALCL and plasmablastic lymphoma may show positivity for (CK8/18) - most often in form of paranuclear dots • S-100B is positive in dendritic cell and histiocytic neoplasms • Vimentin is positive in ~70% of hematolymphoid neoplasms and is positive in many other tumors 19 Case 1

• 50 yrs. • , growing tumor of testis • No relevant medical history • Orchidektomy

20 Case 1

21 Vimentin CD45 Case 1 • PanCK negative • S-100 negative • CD45 weaker then normal lymphocytes

Vimentin 22 Case 1 Diagnosis: Acute myeloid leukemia

CD34 MPO

CD99 TdT Pancytokeratin (PanCK)

Cytokeratins (CK): group of proteins of intermediary filament type important for intracellular transport and physical cell properties • 20 different CK identified (+ 34 trichokeratins) • PanCK antibody should react with as many CK types as possible including CK8/18 • Cytoplasmic reactivity pattern • Often (~70%) true positive in necrotic areas • More cytokeratin positivity in alcohol fixed tissues/cells Pancytokeratin = cocktails of concentrated mAb clones: AE1/AE3 (CK1-8,10,14-16 and 19, but does not detect CK17 or CK18), AE1/AE3/5D3 and AE1/AE3/CAM5.2 as well as RTU: AE1/AE3 and AE1/AE3/PCK26 may give optimal results. MNF116 stains CK 5, 6, 8, 17 and probably 19.

24 Different patterns of Cytokeratin staining

CK7 Kidney chromophobe CK18 in breast carcinoma

CK20 Merkel cell carcinoma CK17 Pancreas 25 CD45 PanCK S100 Vimentin

Epithelial tumors - +/(-) -/+ -/+ Co-expression of IF in carcinomas Carcinomas which most often Carcinomas which most often co-express Cytokeratin & Vimentin co-express Cytokeratin & S-100 Thyroid ca 100% Endometrial ca 75% Sarcomatoid ca >90% Breast ca 60% Myoepithelial ca >90% Salivary duct ca 60% Renal cell ca 60% Sweat gland ca 50% Endometrial ca 50% Ovarian ca 50% Ovarian ca 15-30% Renal cell ca 30% Lung ca 15% Thyroid ca 30% Salivary duct ca 10% Gastric ca 20% Breast 10% Lung ca 10%

Adrenocortical ca are in 80% CK- 26 Pancytokeratin positive non epithelial tumors • (Vimentin, Calretinin, WT1, Podoplanin, …) • Myoepithelial tumors (Vimentin, S-100, SMA, GFAP, Calp…) • Chordoma, Parachordoma (Vimentin, S-100, EMA, …) • Some myofibroblastic proliferations (Vimentin, Actin, …) • Sex cord stromal tumors (Vimentin, SF1, Inhibin, ...) • (biphasic growth pattern - Vimentin, …) • Sarcomas (synovial, epithelioid, , …- Vimentin, …) • Chorioid plexus tumors (GFAP, S-100, … ) • Aberrant expression in different tumors (i.e. lymphomas) • Occasional astrocytic tumors with true epithelial component only (Vimentin, GFAP, …) otherwise it`s cross-reactivity with AE1!

27 Aberrant CK expression in lymphoma

PANCK (CK8/18)+ in Plasmablastic lymphoma Case 2

• Male 65 yrs. • Investigated for anemia/pancytopenia • Smear from BM: massive with monotonous blast-like malignant cells. • Lymphoproliferating disease? • Trephine of BM

29 Case 2

30 PANCK PANCK+/-

CD45 S-100 and Vimentin CD34 TdT

PAX5 CD99 CD56 BER-EP4

Synaptophysin TTF133 Case 2

Diagnosis: Metastatic small cell carcinoma probably from lung

• Cytokeratin is absent by IHC staining in about 10% of small cell carcinoma • EPCAM and CD56 are more often positive than cytokeratin • TTF1 is absent in about 10% of small cell carcinoma of the lung • TTF1 is positive in 20-30% of extra pulmonary small cell carcinomas • In discrepant cases additional panel of antibodies should be applied.

34 SOX10

SOX10 is a transport protein between nucleus and a cytoplasm and acting as a transcriptional activator. Nuclear staining in virtually all cases of melanocytic nevi and malignant melanoma (reactivity may be focal in desmoplastic and spindle cell melanomas), schwannoma, neurofibroma, granular cell tumor, of tendons and aponeuroses. Majority of salivary gland tumors like: , , myoepithelioma, epithelial-myoepithelial and myoepithelial carcinoma, pleomorphic , canalicular adenoma as well as oligodendroglioma, astrocytoma and glioblastoma are positive.

Cave! SOX10 is expressed in about half of cases of basal-like and metaplastic triple- negative breast carcinomas and less than half of malignant nerve sheath tumor. Apart from the above all other epithelial and mesenchymal tumors are uniformly SOX10 negative.

The mAb clones BC34 and BS7 and the rmAb clones EP268 and SP267 can be used to obtain optimal staining S-100 Member of a family of calcium binding proteins. Exact function is not known, but protein is involved in activation of protein kinase, cell growth and proliferation, cell motility and adhesion.

• Present (often with co-expression of IF) in gliomas, neural and melanocytic tumors, sweat and salivary gland tumors, PNET, , chondrosarcoma, endometrioid carcinoma, myoepithelial tumors, chordoma, ….

• Cytoplasmic and nuclear reactivity pattern

• The concentrated mAb clone 4C4.9, pAb Z0311, pAb NCL-L- S100p as well as RTU pAb IR504, pAb GA504, pAb 760-2523 can give good or optimal results

36 S-100 staining in epithelioid melanoma

37 CD45 PanCK S100 Vimentin

Melanoma & - -/(+) + +/(-) schwannoma

This profile can be present in melanoma, neurogenic, lipogenic and myoepithelial tumors, but possibility of malignant melanoma should always be very seriously considered in cases with S100+/Vimentin+ profile.

The final diagnosis of malignant melanoma should be confirmed with other antibodies (MelanA, HMB-45, tyrosinase, MITF)

! Metastatic melanoma often looses expression of markers of melanogenesis. ! Malignant melanoma may show also true positivity for cytokeratin antibodies and other antibodies including CD117.

38 Biopsy from supraclavicular lymph node

39 Biopsy from supraclavicular lymph node

PANCK S-100

Vimentin

Metastatic malignant melanoma Melan A HMB45 40 Metastatic tumor in liver

S-100+/ Vimentin+/ PanCK-/ CD45 not done

CD117 Melan A 41 Aberrant antigen expression in malignant melanoma S-100 PANCK CD45

42 Vimentin

Vimentin – intermediary filament (IF) with structural function in the cytoplasm.

• Present in all cell types during early development, but replaced in later development by other IF types. In mature tissues present in mesenchymal cells, mesothelium, epithelia of mesodermal derivation, thyroid gland, Sertoli and granulosa cell tumours.

• Found in wide variety mesenchymal tumors, many lymphomas, gliomas, carcinomas of some organs and poorly differentiated carcinomas in general

• Cytoplasmic staining pattern • More Vimentin positivity in tumors from effusions

• The mAbs clones Vim 3B4 and V9 and the rmAb clone SP20 can give optimal staining

43 CD45 PanCK S100 Vimentin

Mesenchymal -/+ tumours - -/(+) +

Mesenchymal tumors which often Mesenchymal tumors which rarely co-express vimentin and cytokeratin: express vimentin: Angiosarcoma (epitheloid) Alveolar soft part sarcoma (25%) Leiomyosarcoma Epithelioid sarcoma Dendritic cell sarcoma Chordoma / Parachordoma DRSCT

44 CD45 PanCK S100 Vimentin

Malignant mesothelioma - + - +/(-)

In addition to malignant mesothelioma should vimentin positive carcinomas and cytokeratin positive sarcomas be included in differential diagnosis.

The final diagnosis of malignant mesothelioma should be confirmed with other immunostains (positive staining for Calretinin, Podoplanin (D2-40), HBME-1, WT-1, CK5/6, is seen in > 80-90%) or/and negative staining for Ber- EP4, E-cadherin, CEA, CK20, p63, … depending on considered differential diagnoses). In minority of cases expression of GATA3 (40%), RCC (20%) and in 5-10% PAX8, CD138, Synaptophysin/Chromogranin A, SMA, S-100, CD30, CD31 may cause diagnostic problems.

45 Case 3 Tumor under scapula in 73 yrs old man PanCK+ CK20 CK7+

CK5/6 EPCAM

EPCAM

Synaptophysin SMA PanCK+, Vimentin?, CD45?, S100? CK7+/CK20-, CK5/6-, TTF1-, PAX8-, CDX2-, P63-, SYP-, RCC-, AMACR-, PSA-, Prostein-, mCEA-, CA125-, SALL4-, CD31-, CD34-, SMA-, … EPCAM- = 26 Abs

WT-1 Podoplanin

Calretinin Calretinin

Infiltration from malignant epithelioid mesothelioma. Carcinomas with rare co-expression Cytokeratin & Vimentin • Breast ductal and • Gastrointestinal carcinoma • Lung adenocarcinoma and • Prostate carcinoma • Small cell carcinoma • Ovary mucinous tumors

49 Tumors with CK+/Vim+/S100+

Frequent >50% Less frequent < 50%

Adenocarcinoma polymorphous low Adenocarcinoma endometrioid, grade Adult granulosa cell tumor Carcinoma adenoid cystic Carcinoma anaplastic, thyroid Carcinoma epithelial-myoepithelial Carcinoma large cell, lung Carcinoma follicular of thyroid Carcinoma papillary, thyroid Desmoplastic Small Round Cell Tumor Carcinoma myoepithelial Hemangioblastoma Carcinoma metaplastic breast Meningioma, secretory Chondroblastoma Peripheral Nerve Sheath Tumor Chordoma Juvenile granulosa cell tumor Rhabdoid tumor Parachordoma Sertoli-Leydig cell tumors ………….. Synovial sarcoma ….……

50 CD45 PanCK S100 Vimentin SALL4/ OCT4/ PLAP Germ cell +/- +/- tumours - -/+ +/(-) SALL4 - transcription factor interacting with NANOG and regulating expression of OCT4 • The first choice marker of germ cell tumours other than choriocarcinoma • Gives nuclear staining in gonadal and extragonadal germ cell tumours: seminoma, spermatocytic seminoma, embryonal carcinoma, gonadoblastoma, yolk sac tumour, choriocarcinoma (not all cases), majority of immature teratomas, some elements of metastatic and mediastinal mature teratomas. Reported positive also in some cases of ALL and AML. • The mAb clone 6E3 can give optimal results both in concentrated and RTU forms • Negative in monodermal and majority of mature teratomas • <7% of clear cell carcinoma may show focal, weak positivity 51 CD45 PanCK S100 Vimentin SALL4/ OCT4/ PLAP Germ cell +/- +/- tumours - -/+ +/(-)

OCT4 is positive in classic seminoma and embryonal carcinoma

PLAP is positive in 95% of seminomas (spermatocytic seminoma is often negative!) and with decreasing frequency in embryonal carcinomas, yolk sac tumors and choriocarcinomas Few cases of carcinomas of female genital tract, intestines, lung and breast are also reported PLAP+

Seminomas are in 10 % CK+ (spermatocytic seminoma often +), and Vimentin+ (in ~50% of classic seminomas) Embryonal carcinomas are 100% CK+, but Vimentin+ only in 20%

52 Case 4

• 38 yrs old man enlarged cervical nodes and mediastinal mass • No relevant clinical history • Core biopsy of cervical node CD45 PANCK+

S100 VIM5 OCT4 Podoplanin

SALL4

Metastatic seminoma in cervical lymph node Part II Approach to neoplasm with characteristic morphology (i.e. adenocarcinoma)

56 Advanced panels are used to define specific type of tumor or site of origin - various individually designed combinations of antibodies with narrow spectrum of reactivity

57 58 Combined expression of 7 & 20 CK7+ CK7- Colon (proximal) Colon and rectum CK20+ Ovary (mucinous) Merkel cell carcinoma Pancreas/ biliary tree Small cell ca of salivary gland Urothelial Stomach Breast Adrenal cortical CK20 - Endometrium Germ cell tumors Lung (non-small cell) Hepatocellular ca Ovary (non-mucinous) Lung (SqCC, some SCC) Thyroid Neuroendocrine Uterine cervix Renal cell ca Mesothelioma Prostate ca Synovial sarcoma

59 Distribution of combined expression CK7 & CK20 CK7+/CK20+ CK7+/CK20- CK7-/CK20+ CK7-/CK20- Tumor (no of cases) Colon& rectum adca (40) 10 0 75 15

Gastric adca (29) 38 17 35 10

Pancreas adca (23) 65 26 9 0

Lung adca. (57) 15 74 0 21

Ovary non muc. ca (19) 0 100 0 0

Breast ca (49) 15 84 1 0

Renal cell ca (38) 0 24 6 71

Prostate ca (13) 8 8 23 62

Hepatocellular ca (30) 7 17 0 77 Remember about expression cytokeratins in carcinoma

• Neuroendocrine carcinomas are often CK7-/CK20- • Squamous cell carcinomas are often CK7-/CK20- • Lobular breast carcinoma often cross-reacts with CKHMW (clone 34β12) • Combined positivity for CK5/6 and P63/P40 is characteristic for squamous cell carcinomas, transitional cell carcinomas and basaloid carcinomas, but myoepithelial tumors are often also positive.

61 Metastatic adenocarcinoma of the pancreas?

Cytokeratin 17 - basal type, complex epithelia, squamous and basaloid differentiation. Positive in ~80% of ductal pancreatic adca and ~60% of cholangiocellular ca, but not in gastric or colorectal carcinomas. Positive also in squamous cell ca, transitional cell ca, epithelial-myoepithelial ca.

DPC4 (SMAD4) - deleted in 40-50% pancreatic ca - negative staining supports strongly pancreatic origin. MUC1+/CK17+ positive markers for pancreatic ductal carcinomas, the ampullary carcinoma of pancreatobiliary origin, and with positive predictive values of 76%, 83%, and 58%, respectively. MUC2+/CDX2+ positive markers for the intestinal-type adenocarcinoma of duodenal papillary origin with a positive predictive value of 82%. CK19 should not be used alone to confirm pancreatic origin. It is not enough specific - reported in almost 100% of pancreatic and at least 150 other tumors including adenocarcinomas, squamous cell carcinomas, sarcomas. Synaptophysin Calcium binding membrane glycoprotein of presynaptic vesicles in all neurons and corresponding vesicles in all neuroendocrine cells. Positive staining is also seen in chorioid plexus , adrenal cortical cells, goblet cells and Paneth cells due to crossreactivity with closely related protein. Synaptophysin is more sensitive marker for the identification of neuronal differentiation and neuroendocrine tumors than Chromogranin A, however it is not as specific. It may be also used for the identification of adrenal cortical tumor. • The mAb clones 27G12, BS15, DAK-SYNAP, Snp88 and the rmAb clones MRQ-40 and SP11 could be used to obtain an optimal staining reaction. Chromogranin A

• A member of acid calcium-binding glycoprotein family closely associated with the matrix of dense-core neurosecretory granules in virtually all neuroendocrine (NE) cells and neurons.

• Present in large majority of epithelial neuroendocrine tumors, large majority of neuronal tumors as well as in some cases of multiphenotypic tumours (e.g., malignant rhabdoid tumor, desmoplastic small round cell tumour. Some tumors however being Chromogranin A negative show staining for Chromogranin B (i.e. hindgut ). Primitive neuro- ectodermal tumours (i.e. neuroblastoma) are usually described as CGA negative, but CGA may be detected focally with sensitive systems.

• Optimal results may be achieved with mAb clone LK2H10 in concentrated or RTU form. Case 5

• 67 yrs. male • Solitary tumor in the lung • Metastasis? Primary? • Needle biopsy from the tumor

Case 5 PANCK+, S100-, Vimentin-, CD45 not done, CK7+/CK20-, mCEA- CK5/6-, CK14-, CK5/6 and CK14

P40/P63 GATA3

Napsin A TTF1 Case 5 Preliminary diagnosis: Poorly differentiated carcinoma. Considered following: Primary lung carcinoma (large cell?) Metastatic urothelial carcinoma Other…

Phone call from the clinician! Patient was treated 11 yrs ago for grade 3 urothelial carcinoma in the bladder! Final diagnosis: Metastatic carcinoma consistent with spread from urothelial carcinoma Squamous cell ca vs. Urothelial ca Squamous cell Urothelial carcinoma Antibody carcinoma (except skin)

CK5 > 90% < 15 % Uroplakin-2 0 50- 60% CK20 < 5% > 60% GATA3 10- 30% > 75% CK7 < 40% > 90% CK14 > 80% < 30% S-100 (placental) < 10% > 80%

CK5/6 > 90% 50-60% (not significant)

m-CEA 40% 60% (not significant) P63 and its isoform P40 (ΔNp63) Transcription factor, member of p53 gene family, present in several isoforms, which regulate growth and development of epithelial organs. Mostly used for demonstration of myoepithelial/basal cells, but also in subtyping of carcinoma. When confirming the line of differentiation P63+ should be interpreted together with the staining for high molecular weight cytokeratin (CK5, CK5/6) !Cytokeratin 34BE12 can not be reliably used for confirmation of HMWCK since it shows cross-reactivity with low molecular weight CK19.

P40 expression seems to be more restricted to squamous, urothelial and myoepithelial cells. Absolute majority of cases show consistent expression of P40+/P63+ with P63 being slightly more often positive. Exceptions! Ewing sarcoma: positive for P40 as a rule, but for p63 only in 20%. Polymorphous low grade carcinoma and canalicular adenoma of salivary gland are positive for p63, but negative for p40.

P63 and its isoform P40

Only nuclear staining counts

P63 antibodies: mAbs clones 4A4 and DAK-p63 and rmAb DBR16.1 are all ca may give optimal results. Antibodies to P63 (clone 4A4 only?) give also positive staining in nuclei of transformed B- lymphocytes and cross-reacts with epitope in the cytoplasm of striated muscle.

P40 antibodies: mAb clone BC28 both in concentrated and RTU form and rmAb clone ZR8 in concentrated form may give optimal results. Organ/tissue “specific” antibodies (1) • Breast, salivary, sweat glands: GCDFP-15 / Mammaglobin • Breast, urothelial: GATA3 • Salivary gland, Brenner: GATA3 • Colon/rectum: Cadherin 17/ SATB2 /CDX-2 • Germ cell tumors: SALL4 / OCT-4 • GIST: CD117/ DOG1 • Kidney, endometrium: PAX8 • Ovary: PAX8, WT1 • Liver: Arginase 1 / Glypican 3 /Hepatocyte antigen • Lung, kidney: Napsin A • Lung, thyroid: TTF-1 • Mesothelium: Calretinin, Podoplanin, WT-1, HBME-1 • Myoepithelium Combinations of antibodies • Prostate: PSA / PSMA / P501S / NKX3.1 / ERG • Sex cord stromal, adrenals: SF-1, Inhibin • Thyroid: Thyroglobulin, PAX8 72 GCDFP15

Prolactin induced 15 kDa protein in breast secretory cells. Also found positive in secretory cells of normal apocrine/eccrine sweat glands, lacrimal, ceruminous, serous salivary, bronchial glands, prostate and seminal vesicles.

• Present in carcinomas of breast (10-100%): colloid and lipid rich (~100%), apocrine (~80%), pleomorphic lobular carcinoma (71%), Paget`s disease (~50-70% also extramammary), invasive micropapillary breast carcinoma (~50%) with less expression in metastatic setting (on average ~40%), but only in 5-12%% of triple negative breast ca.

• Present in many tumors of salivary glands, sweat glands and lacrimal glands.

• Few carcinomas in other organs are found to express this protein including ampulla Vateri (17%), of the ovary (1-10%), prostate (8%), lung (4%), colon and rectum (3%), pancreas (2%) and renal cell (2%).

73 74 GATA3

Transcription factor important in the differentiation of breast epithelia, urothelia, and subsets of T-lymphocytes. • >90% of primary and metastatic ductal and lobular carcinomas of the breast (but ~70% in triple negative breast ca), urothelial, cutaneous basal cell carcinomas, trophoblastic and endodermal sinus tumors as well as skin adnexal tumors

• Variable expression in squamous cell carcinomas: skin (81%), cervical (33%), laryngeal (16%), and pulmonary (12%) • Mesothelioma (58%) • Salivary gland tumors (43%) • Pancreatic ductal carcinomas (37%) • <10% in adenocarcinomas of lung, stomach, colon, endometrium, ovary, and prostate • Chromophobe renal cell carcinoma (51%), renal (17%), but other types of renal tumors only rarely positive. • Mesenchymal tumors are negative except pheochromocytoma, extra-adrenal paraganglioma and synovial sarcoma,

75 GCDF15 GATA3

Potential Discriminatory Value of GATA3 Immunohistochemistry in Tumor Diagnosis Examples of Contrasting Pairs of Tumors With Potential Morphologic Mimicry

Positive Tumors Negative Tumors • Metastatic lobular carcinoma of the breast Gastric/intestinal signet ring cell carcinoma • Metastatic ductal carcinoma of the breast Pulmonary/gastrointestinal/ovarian carcinoma • Urothelial carcinoma Prostate carcinoma • Squamous carcinoma of skin origin Squamous carcinoma of pulmonary origin • Malignant mesothelioma Pulmonary adenocarcinoma • Pancreatic adenocarcinoma Gastrointestinal adenocarcinoma/ • Chromophobe renal carcinoma Clear cell renal carcinoma • Endodermal sinus tumor, choriocarcinoma Embryonal carcinoma, seminoma • Paraganglioma Neuroendocrine carcinoma From Miettinen M et al. Am J Surg Pathol 2013 CDX2 /Cadherin 17/ SATB2 CDX2: transcription factor for intestinal epithelial cells. Nuclear positivity in adenocarcinomas with intestinal differentiation in different organs (gastric and small intestinal, pulmonary mucinous, sinonasal, ovarian mucinous, ampullary, bladder, mucinous urothelial-type carcinoma of prostatic urethra, urachal mucinous, intestinal type cervical adenocarcinoma, , midgut carcinoids are positive in most cases). Used in combination with other antibodies incl. CK7 and CK20 helps to predict origin

CAD17: membranous staining of epithelium of colon and rectum, epithelium of pancreatic ducts, less often epithelium of bile ducts, but not other cell types. Positive in >90% adenocarcinoma colon and rectum, ~80% -like carcinoma of colon and rectum, , ~60% adenocarcinoma of urinary bladder, adenocarcinoma of esophagus, ~40% adenocarcinoma of endocervix, stomach, ~15% adenocarcinoma of the pancreas, lung, <10% urothelial and hepatocellular carcinoma, adenocarcinoma endometrioid, adenocarcinoma prostate

SATB2: involved in regulation of transcription. Gives nuclear diffuse staining in the 80-100% tumors of colon, rectum, appendix and ~30% kidney. Focal staining reported in 10-20% adenocarcinomas of stomach, esophagus, pancreatic/biliary, lung, urothelial carcinoma, prostate and breast. Constant positivity in osteosarcoma reported. Case 6 • Man 57 years • No relevant medical history • 19 mm tumor in the left breast • Mammography: malignant (5) • Needle biopsy from the breast tumor

GATA3 GCDFP15

CK7 CK20

CDX2 CAD17 Case 6 CK17 Summary IHC: GATA3- /GCDF15- S100- /Vim- /TTF1- / SYP- /SALL4- / CK7- /CK20+ / CDX2-/CAD17-/+ CK17+ / SATB2 not available Dg: Metastatic adca in the breast – pancreas?/biliary tract? Less likely colon. CAD17

Biopsy from ulcerated tumor in colon ascendens

CK7 CK20 PAX8

A transcription factor expressed mainly in embryonic tissues as well as adult and neoplastic tissues of thyroid gland, kidney and organs of Müllerian system. Carcinoma of the ovary: serous and clear cell (+), while endometrioid (+/-) and mucinous (-/+); Renal cell carcinoma: clear cell, medullary, collecting duct, papillary types and renal are (+) while chromophobe and translocation types (+/-). (+) Thyroid carcinoma: papillary and follicular (+) but anaplastic and medullary type (+/-); Endometrioid carcinoma: of uterine corpus (+/-); Uterine cervix: adenocarinoma in situ and HSIL (+), adenocarcinoma +/-, but squamous cell ca (-/+); : duodenal and colonic +/-, ventricle, appendix and rectum (-/+), ileum and bronchus (-); Pancreas: neuroendocrine tumours (+/-) but ductal adenocarcinoma (-/+); Urothelial carcinoma: (-/+), but clear cell carcinoma of urinary bladder is (+) Mesothelioma: cystic and papillary of (+), but malignant pleural mesothelioma is (-) and malignant is (-/+); Parathyroid adenoma: (+); (+); Seminoma (?+/-)

+ >90%; +/- 50-90%; -/+ 10-50%; - <10%

Single cases of squamous cell carcinoma of the lung, uterine cervix as well as varying number of other tumours including pancreas adenocarcinoma, cholangiocarcinoma, neuroendocrine tumours and even GIST are reported positive. Positive staining of B-lymphocytes may be confusing.

82 Lymphocytes in ileum Clear cell ca of Kidney Clear cell ca of Kidney

Cervical ADCIS Endometroid carcinoma Endometroid carcinoma Wilm's Tumour 1 protein

Nuclear transcriptions factor regulating genes involved in development of urogenital tract and suppressing bcl-2 protein and regulating effect of cadherins and p53. Expressed in CD34+ stem cells, podocytes in kidney glomeruli, non- germinal cells in gonads and mesothelium.

Only nuclear staining is of diagnostic relevance ! True cytoplasmic positivity has so far no diagnostic use.

• Positivity in serous ovarian carcinoma, mesothelioma, sex cord stromal tumors (weak in Leydig cell tumors and thecomas), Wilm`s tumor, DSRCT (C-terminus), carcinomas of Fallopian tube, poorly differentiated carcinoma of endometrium, rhabdoid tumour, lymphoblastic lymphoma and Burkitt lymphoma …

• Optimal results may be achieved with mAbs clones 6F-H2, WT49 and the rmAb EP122 in concentrated or RTU form.

84 WT1 protein

Serous ovarian carcinoma Malignant mesothelioma Case 7

• Woman 73 yrs • Palpable tumor in the left breast • Mammography: suspected for (4) • 14 yrs ago operated with because of the tumor in the ovary with diagnosis serous carcinoma. No signs of recurrence. • Core biopsy from the breast lesion ER CK7

GATA3-/GCDFP15-/SYP-

PAX8 WT1 Case 7 Metastatic serous carcinoma of the ovary in the breast. Antibody Ovary serous ca Breast ca NOS

PAX8 95% 0% WT-1 85% 5% GATA3 5% 80% CA125 80% 15% GCDFP15 2% 50% Mammaglobin 10% >50% ER 65% 70% Her-2 overexpress. 20% 20% CK7 >95% 90% Prostate specific antibodies PSA: Protease, highly sensitive for prostatic epithelial cells. Positive in scattered cells in the prostatic urethra, periurethral glands in males and females, anal glands and urachus. Reactivity in cystitis cystica, cystitis glandularis, female and male breast carcinoma, as well as salivary and sweat gland tumours reported. • The mAb 35H9 and ER-PR8*, rmAb EP109 and pAb 0562 concentrated and RTU could give optimal staining (*RTU may need protocol modification for optimal results).

Prostein/P501S: localized to the Golgi complex in the cytoplasm of prostate epithelial cells and not found in any other normal tissue. Positive in > 90% of prostate adenocarcinoma including 20-30% small cell carcinoma, 10-20% adenocarcinoma, urachus or urinary bladder • The mAb 10-E3 could give optimal results NKX3.1: nuclear transcription factor that has a critical function in prostate development. Found in ureter, testis and bronchial glands. Sensitivity similar to PSA, but is present also in 5-30% breast carcinomas. • The rmAb clone EP356 and pAb CP422 are the most successful PSA P501S PSMA Case 8

• 85 yrs man with history of cutaneous malignant melanoma 1 year ago Clark 4 (5mm Breslow) and X- ray treated prostate adenocarcinoma (Gleason score =8) 9 years ago. • Stable PSA since then • 12 mm solitary lung lesion • Core biopsy from the lung lesion Metastatic melanoma? BRAF mutation? PDL1 status?

Case 7

PSA PSMA

TTF1 and Napsin A staining negative TTF-1

• Thyroid Transcription Factor-1 is a nuclear tissue specific transcription factor expressed in thyroid and lung but rarely in other organs.

• Positive in non-anaplastic carcinomas of the thyroid and carcinomas of the lung (small cell ca 90%, adeno ca 80%, large cell 40%, spindle cell 30%, mucinous 25% and squamous ca 5-10% as well as 35% pulmonary carcinoids, 50% neuroendocrine ca and atypical carcinoids). Additionally ~30% of small cell carcinomas arising in other organs are stained positive.

• Rare aberrant expression (more often with clone SP24) reported in pancreato- biliary carcinomas, ovarian serous, endometrial, endocervical and colonic carcinomas as well as some neuroendocrine carcinomas.

• Nuclear staining pattern (cytoplasmic granular staining of hepatocytes and carcinoids with clone 8G7G3 is considered non specific)

93 TTF-1 in lung adenocarcinoma

94 TTF1 different clones TTF1 positive primary colon adenocarcinoma

TTF1 clone 8G7G3 TTF1 clone SP-24

TTF-1 positive metastatic carcinoma H12535/03 Thyroglobulin Glycosylated protein providing iodination sites for thyroid hormones. Specific for thyroid differentiation regardless localization, but also reported in leukemic blasts. Cytoplasmic and extracellular (secretions) staining pattern.

Entrapped thyroid tissue in of thyroid 96 Other less “organ specific” antibodies

AFP- foetal protein. Positive in 70% or more: , , hepatoid adenocarcinoma, yolk sac tumor, .

CD30- Lymphocyte activation antigen. Positive in >90%: classic Hodgkin's lymphoma, ALCL, embryonal carcinoma. Some lymphomas of B- and T- lineage.

EPCAM (Ber-EP4 and MOC31)- epithelial cell adhesion molecule. Positive: most carcinomas, majority of chromophobe (80%) and papillary (70%) renal cell carcinoma, synovial sarcoma. Negative: mesothelioma, hepatocellular carcinoma, sarcomas and lymphomas.

ER & PgR- hormone receptors. Positive: carcinomas of breast, ovary and endometrium and a subset of other carcinomas. Not recommended to use alone to predict primary location of carcinomas (clone dependent positivity!!!)

RCC- glycoprotein in renal tubules. Positive: >80% of primary and 60% of metastatic clear cell and papillary renal cell carcinomas, but negative in majority of chromophobe carcinomas or all sarcomatoid carcinomas, collecting duct carcinomas and oncocytomas. A subset of non-renal adenocarcinomas (lung, uterus, parathyroid, ovary) and some reported positive. 97 Lymphoid lineage “specific” antibodies

• CD20 B-lymphocytes • CD79a B-lymphocytes & Plasma cells • PAX5 B-lymphocytes & Small cell neuroendocrine ca • CD138 Plasma cells & Epithelium, many carcinomas, few sarcomas • CD3 T-lymphocytes • CD5 T-lymphocytes & Thymoma, few carcinomas • TDT Lymphoblasts & Small cell neuroendocrine ca

98 Antibody algorithms

• Useful general approach • A good starting point for planning your panel • Many different models and qualities • Many exceptions in tumor phenotypes makes them difficult to relay on in 100% www.immunoquery.com Largest database - careful reading! http://e-immunohistochemistry.info Free access! http://www.ncbi.nlm.nih.gov/pubmed/ Free access! www.pathologyoutlines.com Free access! Books and articles (D.Dabbs “Diagnostic Immunohistochemistry”, … ) Internal consultations Free access!

99 Molecular profiling of CUP An adjunct to immunohistochemistry or a competitor?

• RT-PCR based commercially available tests

• Promising results from preliminary studies

• Expected value for choice of targeted therapy (NGS)

• Prospective validating studies going on CUP summary Immunohistochemistry is a complex multistep diagnostic laboratory procedure, where each step is important for final results and interpretation.

Be aware of possible pitfalls and pay attention to correct interpretation.

Apply individual approach to each case considering step by step procedure.

The interpretation of IHC should always correlate to morphology and clinical data. Be careful and reasonable !

Thank you for your attention!