sign in sign up
<<
Home , Clear cell sarcoma, Renal oncocytoma

Immunhistokjemi for patologer 17.03.2021 Stavanger

Unknown primary tumor by immunohistochemistry

J. Klos MD Department of Pathology Stavanger University Hospital Norway 1 Conflict of interests

NONE

2 Unknown Primary Tumor ( 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 malignancies 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 …

Metastasis as a first presentation in 5 - 15% of

3 Unknown primary tumor

• (~80%) pancreas, , colon and rectum, liver and biliary tract, stomach, , ovary, prostate, breast … 2/3 well differentiated and 1/3 poorly differentiated

• Squamous cell and urothelial (~5-10%) • lung, head and neck, oesophagus, uterine cervix, …

• Nonepithelial (~10-15%) malignant lymphomas (10%), (6%), (1%), germ cell tumours (06%), (0,2%), other (2%)

4 Relevance of cancer type/origin

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

Pathology is an effective method of investigating in CUP

5 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!

6 How to approach the problem?

No “shot gun” immunohistochemistry!

7 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 considering transcription factors in the first line

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

8 Step by step

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

9 Part I Approach to neoplasm with non-characteristic morphology (panel for poorly differentiated tumors)

10 The most important factor in designing IHC panels is … user`s experience

11 Basic IHC panel defines the line of differentiation of tumor cells based on reactivity with wide spectrum antibodies

• CD45 • Pancytokeratin • S100/SOX10 • (non specific, but sometimes useful) • SALL4/ OCT4/PLAP (if germinal cell tumor suspected) • CD30 (if CD30+ lymphoproliferative / embryonal ca suspected) • GFAP (when CNS is the location) • SMA, CD34, MDM2, CDK4, … (if sarcoma suspected) • Synaptophysin /INSM1 for neuroendocrine tumors

12 Real “hard life” profiles

CD45 PanCK S100/ Vim SALL4/ OCT4/ SOX10 PLAP Hemato-lymphoid +/(-) -/(+) -/(+) +/- -/(+) tumors Epithelial tumors - +/(-) -/+ -/+ - Melanocytic & - -/(+) + +/(-) - neural tumors Mesenchymal - -/(+) -/+ + - tumors Mesothelial tumors - + - +/(-) -

Germ cell tumors - +/- -/+ +/- +/(-)

13 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.

14 CD45 in malignant

15 Case 1

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

16 Case 0

17 Vimentin CD45 Case 0 • PanCK negative • S-100 negative • CD45 weaker then normal lymphocytes Vimentin 18 Case 0 Diagnosis: Acute myeloid leukemia

CD34 MPO

CD99 TdT CD45 PanCK S-100/ Vimentin SOX10 Hematolymphoid tumors +/(-) -/(+) - (+) +/-

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-100 is positive in dendritic cell and histiocytic neoplasms • Vimentin is positive in ~70% of hematolymphoid neoplasms and is positive in many other tumors

20 Pancytokeratin (PanCK)

Cytokeratins (CK): 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 is seen in alcohol fixed tissues/cells

Concentrated mAbs: AE1/AE3 (CK1-8,10,14-16 and 19, but does not detect CK17 or CK18), AE1/AE3/5D3 and AE1/AE3/CAM5.2 (does not detect CK17) and mAb BS5 as well as RTU: AE1/AE3 and AE1/AE3/PCK26 may give optimal results. MNF116 stains CK 5, 6, 8, 17 and 19? - gave inferior performance last runs. 34βE12 stains CK1, 5, 10, 14 and ? CK19? - sensitive, but not appropriate for detection of CKHMW (use CK5 or CK5/6 instead).

21 Case 1 • Male 65 years • Investigated for anemia/pancytopenia • Smear from BM: massive with monotonous blast-like malignant cells. • Lymphoproliferative disease? • Trephine of BM

22 Case 1

23 Case 1

PANCK PANCK-/+

CD45- S-100- and Vimentin-

24 Case 1

CD34- TdT+

PAX5- CD99+

25 Case 1

EPCAM+ TTF1+

Synaptophysin+ CD56+ Case 1 Diagnosis: Metastatic small cell neuroendocrine (PANCK negative by IHC staining) probably from lung.

• Cytokeratin is absent by IHC staining ~10% of small cell carcinoma. • EP-CAM and CD56 are more often positive than cytokeratin. • EPCAM and EMA may be used in panel as secondary broad spectrum epithelial markers if knowing their limitations. • 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.

27 CD45 PanCK S100/ Vimentin SOX10 Epithelial tumors - +/(-) -/+ -/+ Co-expression of IF in carcinomas

Carcinomas which most often co-express Carcinomas which most often co-express Cytokeratin & Vimentin 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- …..

28 Non epithelial tumors and PanCK

• Mesothelioma (Calretinin, Podoplanin, WT1, Vimentin, …) • Myoepithelial tumors (S-100, SMA, GFAP, Calponin, Vimentin, …) • Chordoma, Parachordoma (S-100, EMA, Vimentin, …) • Some myofibroblastic proliferations (Actin , Vimentin, …) • Sex cord stromal tumors (SF1, Inhibin, Vimentin, EMA-, ...) • (biphasic growth pattern - Vimentin, …) • Sarcomas (synovial, epithelioid, , …- Vimentin, …) • Chorioid plexus tumors (GFAP, S-100, … ) • Aberrant expression in different tumors (e.g. lymphomas, melanomas) • Occasional astrocytic tumors with true epithelial component (Vimentin, GFAP, …) otherwise it`s cross-reactivity with AE1!

29 Expression of epithelial markers (CKs and EMA/MUC1) in nonepithelial tumors

Expression of epithelial markers is not uncommon in hematolymphoid tumors: ALCL (majority of cases), Plasma cell tumors, NLPHL, DLBCL, TCHRLBCL, ALK+ LBCL, Plasmablastic lymphoma, FDS … Certain and tumors show also positivity: , , , , epithelioid hemangioma and angiosarcoma, perineurioma, myoepithelial tumors, epithelioid sarcoma, DSRCT, extrarenal rhabdoid tumor, osteosarcoma, chordoma …

EMA expression in ALK+ ALCL in pleural fluid Aberrant cytokeratin (CK8/18) expression in Plasmablastic lymphoma 30 S100

Member of a family of calcium binding proteins. Exact function is not known, but protein is involved in activation of protein kinases, 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, liposarcoma, chondrosarcoma, serous and endometrioid carcinoma, myoepithelial tumors, , papillary and follicular thyroid carcinoma, chordoma, rhabdomyosarcoma, …. • 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.

31 S-100 staining in epithelioid melanoma

32 SOX10 SOX10 is a transport protein between nucleus and cytoplasm and acting as a transcriptional activator involved in embryonic development. 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 and aponeuroses. Majority of myoepithelial and salivary gland tumors, 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 peripheral nerve sheath tumor (MPNST). All other epithelial and mesenchymal tumors are negative in more than 95%.

The mAb clones BC34 and BS7 and the rmAb clones EP268 and SP267 can give optimal staining. 33 SOX10 in cutaneous malignant melanoma

34 Immunoreactivity S100 vs. SOX10

• S100+ /SOX10- • S100+/SOX10+ • Adipocytic, chondroid tumors, chordoma, • Melanoma • Some adenocarcinomas ( • Nerve Sheath Tumors • Histiocytic/dendritic cell tumors • Some sarcomas: synovial sarcoma (15%), • Majority of myoepithelial carcinomas Ewing sarcoma (21%), rhabdomyosarcoma (24%), chondrosarcoma (75%), extraskeletal myxoid chondrosarcoma (45%), ….

• S100-/SOX10- • S100-/SOX10+ • Most carcinomas and sarcomas, • Mesothelioma, germ cell tumors • Some melanomas • Half of MPNST, but more often S100+ than • Some myoepithelial carcinomas SOX10+ • Neuroblastoma, paraganglioma, • Few MPNST pheochromocytoma - only sustentacular cells in are positive, but tumor cells are negative. CD45 PanCK S100/ Vimentin SOX10 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+/SOX10+ and Vimentin+ profile.

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

! Malignant melanoma may show also true positivity for a number of antibodies including cytokeratin, CD117, GATA3, CEA, EMA, Syp, …. ! E-Cadherin is positive in 40-80% of melanomas. ! Metastatic melanoma often looses expression of markers of melanogenesis.

36 Aberrant antigen expression in malignant melanoma

S-100 PANCK CD45

37 Metastatic CD117+ melanoma in liver

S-100+/ Vimentin+/ PanCK-/ CD45 ND

CD117 Melan A

38 Expression of melanogenesis markers (Melan A, HMB45)

In epithelial proliferations In mesenchymal proliferations • Adrenal cortical tumors, • PECOMA, • Sex cord stromal tumors, • Clear cell sarcoma, • Translocation renal cell • Melanotic schwanian carcinoma (all with t(6;11) tumor and rare with Xp11.2)

Translocation renal cell ca - MelanA Adult granulosa cell tumor - MelanA PECOMA - MelanA

39 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 is seen in tumors from effusions.

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

40 CD45 PanCK S100 Vimentin

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

Mesenchymal tumors which often Mesenchymal tumors which rarely co-express vimentin and express vimentin: cytokeratin: • Alveolar soft part sarcoma (0-25%) • Epithelioid sarcoma (80%) • DRSCT (>80%) • Dendritic cell sarcoma (>70%) • Synovial sarcoma (>60%) • Chordoma/Parachordoma (40%) • Angiosarcoma (epitheloid) (>30%) • Leiomyosarcoma (> 15-30%) . ….

41 CD45 PanCK S100 Vimentin

Malignant mesothelioma - + - +/(-)

In addition to malignant mesothelioma should vimentin positive carcinomas and cytokeratin positive sarcomas be included in differential diagnosis. Final diagnosis of mesothelioma has to be confirmed with the panel of Abs. Positive staining for Calretinin, Podoplanin (D2-40), HBME-1, WT-1, CK5/6 and negative staining for EPCAM, E-cadherin, CEA, CK20, p63/p40, … is seen in ~ 90% cases.

Negative nuclear BAP1 (50% cases) is a strong argument for mesothelioma.

Diagnostic problems may arise in cases expressing GATA3 (40%), RCC (20%) and in 5-10% PAX8, CD138, CD31, CD30, Synaptophysin/Chromogranin A, SMA …

42 Carcinomas with rare co-expression Cytokeratin & Vimentin

• Breast ductal and • Gastrointestinal carcinoma • Lung and • Prostate carcinoma • Small cell carcinoma • Endocervical carcinoma • Ovary mucinous tumors

43 Triple positive tumors (CK+/S100+/Vim+)

Frequent >50% Less frequent < 50%

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

44 Case 2

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

45 Case 2

CD45- PANCK-

46 S100- VIM- Case 2

Podoplanin+ OCT4+

SALL4+

Dg: Metastatic seminoma in cervical

47 CD45 PanCK S100 Vimentin SALL4/ OCT4/ PLAP Germ cell tumors - +/- -/+ +/- +/(-)

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 tumors: 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. • Negative in monodermal and majority of mature teratomas • <7% of clear cell carcinoma may show focal, weak positivity

The mAb clone 6E3 can give optimal results both in concentrated and RTU forms

48 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 is more often CK+), and Vimentin+ (in ~50% of classic seminomas). Embryonal carcinomas are 100% CK+, but Vimentin+ only in 20%.

49 Triple negative tumors PANCK-/CD45-/S100-(SOX10-)

• : p40, HMW-cytokeratin, EPCAM, EMA?, … • Poorly diff. neuroendocrine ca.: INSM1, Syp, Chromogr, … (?CD56). • Adrenal cortical carcinoma: SF1, Melan A (A103), Syp, Calret, Inhib • Sarcoma NOS: SMA, Desmin, CD34, …, MDM/CDK4 for dediff. liposarcoma) • Follicular dendritic cell sarcoma: CD21, CD23, CD35 • Acute Lymphoblastic Leukemia: PAX5, CD3, CD34, CD43 • ALK+ ALCL: ALK, CD30, p63 (negative prognostic factor • Plasma cell neoplasm (anaplastic): MUM1, CD138, CD79a, EBV EBER • Melanoma dedifferentiated: Melan A, HMB45, BRAF V600E, PRAME • Germ cell tumor: SALL4, (?PLAP) • Pheochromocytoma/Paraganglioma: GATA3, Syp, Chromogr, (?INSM1)

Modified from A. Bellizzi 2020

50 Part II Approach to neoplasm with characteristic morphology (differentiated tumor panel)

51 Advanced panels used to define specific type of tumor or site of origin - various individually designed combinations of antibodies with narrow spectrum of reactivity including transcription factors (oligospecific antibodies)

52 Combined expression of 7 & 20 in adenocarcinomas

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

53 True 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

54 Things that matter are not easy!

David Levithan Remember about expression of 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, basaloid carcinomas, and many myoepithelial tumors.

56 P63 and its isoform P40 (ΔNp63)

Transcription factor, member of p53 gene family, present in several isoforms regulating growth and development of epithelial organs. Mostly used for demonstration of myoepithelial/basal cells, but also in subtyping of carcinoma. P63+ should be interpreted together with the staining for high molecular weight cytokeratin (CK5, CK5/6) when confirming the line of differentiation. ! 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 of salivary gland are positive for p63, but negative for p40. 57 Oligospecific antibodies

• 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 • Lymphoid tissue CD45, CD20, CD3 • Mesothelium: Calretinin, Podoplanin, WT-1, HBME-1 • Myoepithelium Combinations of antibodies • Prostate: PSA / PSMA / P501S / NKX3.1 / ERG (50%) • Sex cord stromal, adrenals: SF-1, Inhibin • Thyroid: TTF1, Thyroglobulin, PAX8 • ………..

58 Transcription factors may behave badly (sometimes!)

Once thought to be reliable are not as specific as once thought, with emerging studies showing aberrant and overlapping expression or loss of expression in neoplastic tissues.

• CDX2: in ovary (0-30%+), mucinous (50%) uterine endometrioid ca (<5%+), AML >90%+, testicular germ cell tumors ( YST ~100%+), ... • GATA3: variation between different locations (e.g. skin 80%+ vs. anus 10%+) • OCT4: adenocarcinoma lung (25%+), prostate (37%+), SqCC of esophagus (31%+), • PAX5: pulmonary small cell ca (33-83%), large cell NEC (27-73%), Ewing sarcoma (14%), rhabdomyosarcoma 67%, • PAX8: reacts also with NET (up to 65–100%), well–differentiated papillary mesothelioma (20%); malignant mesothelioma (peritoneal 12%; pleural 2%) • TTF1: reported in colon 5%, endometrium16-22%, breast 3%, gallbladder, prostate, ovary 1% etc. • SOX10: synovial sarcoma (0-7%+), rhabdomyosarcoma (7%+),

Lou S, Adeyi OA. Arch Pathol Lab Med 2019

59 TTF1 staining with different clones TTF1 positive primary colon adenocarcinoma

TTF1 clone 8G7G3 TTF1 clone SP-24

TTF-1 positive metastatic gallbladder carcinoma H12535/03

60 Case 3 • Woman 73 years • Palpable tumor in the left breast. • Mammography: suspected for malignancy (4). • 14 years ago operated because of the tumor in the ovary with diagnosis serous carcinoma. No signs of recurrence. • Core biopsy from the breast lesion.

61 Case 3

ER CK7

GATA3-/GCDFP15-/SYP-

PAX8 WT1

62 Case 3

Dg: Metastatic serous carcinoma of the ovary to the breast.

Antibody Ovary serous ca Breast ca NOS

PAX8 95% 0%

WT-1 85% 5%

CA125 80% 15%

GATA3 5% 80%

GCDFP15 2% 50%

Mammaglobin 10% >50%

Estrogen receptors 65% 70%

Her-2 overexpression <20% <15%

CK-7 >95% >90%

63 Case 4

• Man 57 years • No relevant medical history • 19 mm tumor in the left breast • Mammography: malignant (5) • Needle biopsy from the breast tumor

64 Case 4

65 Case 4

GATA3 GCDFP15

CK7 CK20

CDX2 CAD17

66 Case 4 Summary IHC: GATA3- /GCDF15- /S100- / CK17 Vim- /TTF1- / SYP- /SALL4- /CK7- /CK20+ / CDX2- /CAD17-/+ /CK17+. SATB2 not available. Dg: Metastatic adenocarcinoma in the breast - primary location in: pancreas?/biliary tract?/colon? Considered.

CAD17

Biopsy from ulcerated tumor in colon ascendens CK7 CK20

67 CDX2 / Cadherin 17/ SATB2

CDX2: transcription factor for intestinal epithelial cells. Nuclear positivity in adenocarcinomas with intestinal differentiation in different organs: colon, small intestine, appendix but also majority of cases of gastric, pulmonary mucinous, sino-nasal, ovarian mucinous, ampullary, bladder, mucinous urothelial-type carcinoma of prostatic urethra, urachal mucinous, intestinal type cervical adenocarcinoma, , midgut ). Used in combination with other antibodies incl. CK7 and CK20 to predict origin.

CAD17: membranous staining of of colon, rectum, 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, endometrioid adenocarcinoma, adenocarcinoma prostate.

SATB2: involved in regulation of transcription. Nuclear diffuse staining in 80-100% tumors of colon, rectum, appendix and ~20% papillary kidney ca. Focal staining in 10-20% adenocarcinomas of stomach, esophagus, pancreatic/biliary, lung, urothelial carcinoma, prostate and breast. Constant positivity in osteosarcoma.

68 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 (80%), cervical (30%), laryngeal (<20%), and pulmonary (~10%) • Mesothelioma (60%) • Salivary gland tumors (varying focal to diffuse even 100% in MASC) • Pancreatic ductal carcinomas (16-40%) • Chromophobe renal cell carcinoma (50%), renal (<20%), but other types of renal tumors only rarely positive. • Adenocarcinomas of lung, stomach, colon, endometrium, ovary, prostate <10% • Mesenchymal tumors are negative except pheochromocytoma, extra-adrenal paraganglioma and synovial sarcoma.

69 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 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 Panel for metastatic adenocarcinoma of the pancreas

Cytokeratin 17 - basal type, complex epithelia, squamous and basaloid differentiation. Positive in ~80% of ductal pancreatic adenocarcinoma 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 ~50% pancreatic ca, but also other cancers in the region - negative staining supports pancreatobiliary 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%. GATA3+ more common in pancreas adca than other gastrointestinal locations including cholangiocellular adenocarcinoma.

CK19 alone should not be used to confirm pancreatic origin. It is not enough specific - reported in almost 100% of pancreatic ductal carcinoma and at least 150 other tumors including adenocarcinomas, squamous cell carcinomas, sarcomas.

71 Urothelial carcinoma vs. squamous cell carcinoma

Urothelial carcinoma Squamous cell carcinoma • CK 5/6 + • CK5/6 + • P40/P63 + • P40/p63 + • GATA3 + • GATA3 -/+ clone & organ dependent • CK5 + 30% • CK5 + >95% • CK20 +/- (50%) • CK20 - (<5%) • Uroplakin III + (68%) • Uroplakin - (1%) • P16 + (50%) • P16 +/- (HPV) INSM1 Emerging neuroendocrine marker

• -associated 1 (INSM1) - a protein containing both a zinc finger DNA-binding domain and a putative prohormone domain. • Most sensitive and highly specific marker of neuroendocrine differentiation in normal and neoplastic cells so far. • !Parathyroid adenoma and adrenal cortical carcinoma are consistently negative for INSM1.

73 PRAME Emerging melanoma marker

PReferentially expressed Antigen in MElanoma - recognized by cytolytic T lymphocytes. Inhibits retinoic acid-induced differentiation, growth arrest, and apoptosis through interaction with the EZH2 polycomb group proteins It is not expressed in normal tissues, except testis, ovary, placenta, adrenals, and endometrium. Nuclear staining positive in ~95% malignant melanoma and ~10% melanocytic nevi. More sensitive than Melan A and HMB45 especially in metastatic lesions.

74 Mimics of metastatic carcinoma

• Hemangioblastoma, glioblastoma and meningioma with epithelial metaplasia, • Choroid plexus carcinomas, • Primary neuroendocrine carcinomas in unusual locations, • Special forms of sinonasal and salivary glandular adenocarcinoma, clear-cell thyroid carcinomas, • Unusual microscopic subtypes of pulmonary adenocarcinoma, epithelioid myomelanocytomas (“sugar tumors”), mesotheliomas, • Primary thymic carcinomas, • Endodermal choristomas of the interatrial myocardium, • Peripheral cholangiocarcinoma, • , • Adenocarcinomas of the urinary bladder, mucinous and “rhabdoid” • Tumors of the ovaries, • Adenocarcinomas of rete testis, • Interdigitating dendritic-cell sarcoma of lymph nodes, • Some sweat gland carcinomas, • Cutaneous Merkel cell carcinoma, • Primary dermal and subcutaneous melanoma, • Mucosal and visceral melanomas, • Epithelioid sarcoma, clear-cell sarcoma, • Adamantinoma of long .

Modified from Mark R. Wick 2018 75 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 precision therapy (NGS) • Prospective validating studies going on

76 Molecular tests in CUP

• Molecular methods applied to tumor tissue are able to predict the Tissue Of Origin, using gene expression profiling, gene microarrays, microRNA and DNA methylation analysis. • Several studies indicate that gene expression assays have high diagnostic accuracy for predicting tissue of origin in cancers of unknown primary. • The impact of these assays on patient management and patient survival remains uncertain and there is insufficient data to support its routine use at the moment. Further studies are going on.

Medical Policy Statement on Molecular Profiling for Cancers of Unknown Primary Origin AHS- M2065 – updated 9/8/20 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.

78 Thank you for your attention!

79