IMMUNOHISTOCHEMISTRY FOR CARCINOMA OF UNKNOWN PRIMARY

Transcription

1 IMMUNOHISTOCHEMISTRY FOR CARCINOMA OF UNKNOWN PRIMARY Jason L Hornick, MD, PhD Director of Surgical Pathology Director of Immunohistochemistry Brigham and Women s Hospital Associate Professor of Pathology Harvard Medical School Boston, MA, USA 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 Carcinoma of unknown primary Account for 2-5% of malignancies diagnosed in the US 7 th or 8 th most frequent cancer 4 th or 5 th most common cause of cancer death in both sexes 31,000 new cases in the US in 2012 down from 45,000 new cases in 1995 Improved radiologic imaging Increasingly specific IHC markers Page 1 1

2 Origin of primary tumors (autopsy) Organ Incidence Pancreas 20-25% Lung 15-20% Colon/rectum 5-10% Liver/biliary 5-10% Stomach 5% Kidney 5% Ovary <5% Prostate <5% Breast 2% Other 1% Histologic groups of carcinoma of unknown primary Histology Well or moderately differentiated adenocarcinoma Poorly differentiated adenocarcinoma or undifferentiated carcinoma Frequency 60% 30% Squamous cell carcinoma 5% Undifferentiated malignant neoplasm 5% Overview Distribution of keratin family members in carcinomas Lineage-restricted markers and primary site determination Primary tumors of the liver and mimics Squamous cell carcinomas Primary site determination for metastatic neuroendocrine tumors Page 2 2

3 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 Page 3 3

4 Metastatic colonic adenocarcinoma CK20 Metastatic colonic adenocarcinoma CK7 Metastatic colonic adenocarcinoma CK20 Page 4 4

5 IHC for lineage/site specification: cytoplasmic/membranous markers Primary site Bladder Breast Breast Colon/rectum Lung Prostate Prostate Thyroid Marker Uroplakin GCDFP-15 (prolactin-inducible protein) Mammaglobin (SCGB2A1/A2) Villin Napsin A Prostate-specific antigen Prostatic acid phosphatase Thyroglobulin IHC for lineage/site specification: cytoplasmic/membranous markers In general, expression is decreased (or entirely absent) with poor differentiation significant impact on sensitivity Breast carcinoma GCDFP Page 5 5

6 Breast carcinoma GCDFP Breast carcinoma mammaglobin Expression of mammaglobin in other tumor types Tumor type Endometrial endometrioid adenocarcinoma Frequency 20-40% Skin adnexal carcinomas 20-40% Salivary gland neoplasms 20-50% Page 6 6

7 Prostatic adenocarcinoma PSA Lung adenocarcinoma Napsin A Napsin A Warning: napsin A is also positive in most papillary renal cell carcinomas Page 7 7

8 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 10 years ago, very few were used in surgical pathology practice TTF1, CDX2, MYOG In 2016, over 50 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 GATA3 NKX3-1 OCT4 PAX8 SALL4 SATB2 SF1 TTF1 (NKX2-1) WT1 Colon/rectum, upper GI Breast, bladder Prostate Seminoma, embryonal carcinoma Thyroid, kidney, Müllerian Germ cell tumors Colon/rectum Adrenal cortex Lung, thyroid Müllerian, mesothelioma Page 8 8

9 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 Page 9 9

10 Metastatic poorly differentiated lung adenocarcinoma TTF1 Poorly differentiated (insular) thyroid carcinoma TTF1 TTF1 Warning: TTF1 is also positive in a subset of endometrial adenocarcinomas (most common in grade 3 endometrioid and serous) Some variation in sensitivity and specificity based on particular clone Page 10 10

11 Expression of TTF1 in carcinomas I Primary site Positive cases Pulmonary (adenocarcinoma) 70-90% Pulmonary (squamous cell carcinoma) <10% Thyroid (all types) % Cholangiocarcinoma (extrahepatic) 5-25%* Endometrial 5-20%* Ovarian 5-30%* *These cases usually show expression in only a small fraction of tumor cells. Expression of TTF1 in carcinomas II Primary site Positive cases Gastric/esophageal <10% Cervical <5% Pancreatic <5% Breast <5% Urothelial <5% Colorectal <5% Hepatocellular <5% Salivary gland (adenoid cystic) 30-50% Salivary gland (other) <5% Adrenal cortical <5% TTF1: potential diagnostic pitfall Cytoplasmic staining relatively common Diffuse staining in normal hepatocytes and many hepatocellular carcinomas Likely represents cross-reactivity with CPS1 (antigen recognized by HepPar-1 antibody) Subset of adenocarcinomas from various sites (especially foregut) also show cytoplasmic staining Page 11 11

12 Hepatocellular carcinoma TTF1 Wilms tumor 1 WT1 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 Page 12 12

13 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 Page 13 13

14 CDX2 Caudal-type homeobox transcription factor involved in intestinal differentiation Nuclear expression in >90% colorectal adenocarcinomas Somewhat lower sensitivity in high grade and MSI-H carcinomas Widely used to support colorectal origin No significant loss of sensitivity in the metastatic setting Colonic adenocarcinoma CDX2 Metastatic colonic adenocarcinoma CDX2 Page 14 14

15 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 Warning: CDX2 can also be positive in mucinous adenocarcinomas from diverse anatomic sites Ovary Lung Pancreas Bladder Page 15 15

16 Expression of CDX2 in carcinomas Primary site Positive cases Colorectal and appendiceal % Gastroesophageal 40-60% Pancreatic/biliary 30-50% Ovarian (mucinous and endometrioid) 40-60% Ovarian (serous) <10% Endometrial 5-10% Pulmonary (mucinous) 70-80% Pulmonary (non-mucinous) <5% Bladder (adenocarcinoma) 30-50% Bladder (urothelial) <5% Prostatic <5% Breast <5% Renal <5% 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 More recent large surveys revealed expression in wide range of tumor types Bladder urothelial carcinoma GATA3 Page 16 16

17 Bladder urothelial carcinoma GATA3 Breast carcinoma GATA3 Breast carcinoma GATA3 Page 17 17

18 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 % 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 Urothelial carcinoma Squamous cell carcinoma of skin Malignant mesothelioma Paraganglioma Choriocarcinoma, yolk sac tumor Lung/GI/ovarian adenocarcinoma Prostatic adenocarcinoma Squamous cell carcinoma of lung Lung adenocarcinoma Other neuroendocrine tumors Embryonal carcinoma, seminoma Am J Surg Pathol. 38:13-22, 2014 NKX3-1 Homeobox transcription factor, androgendependent 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 Page 18 18

19 Prostatic adenocarcinoma NKX3-1 Expression of NKX3-1 in carcinomas Primary site Positive cases Prostatic % Breast (lobular) 15-30% Breast (ductal) <10% Bladder (urothelial and adenocarcinoma) <5% Pancreatic/biliary <5% Hepatocellular <5% Renal <5% Colorectal <5% Gastroesophageal <5% Pulmonary <5% Thyroid <5% SATB2 More recently described transcription factor expressed in colorectal/appendiceal epithelium Similarly high sensitivity and likely higher specificity than CDX2 Positive in 80-90% of primary and metastatic colorectal adenocarcinomas Higher sensitivity than CDX2 for medullary carcinomas Unlike CDX2, SATB2 rarely expressed in gastroesophageal and pancreaticobiliary adenocarcinomas Page 19 19

20 Colonic adenocarcinoma SATB2 Poorly differentiated colonic adenocarcinoma SATB2 Expression of SATB2 in carcinomas Primary site Positive cases Colorectal and appendiceal % Renal 25-35% Gastroesophageal 10-20%* Pancreatic/biliary 10-20%* Mullerian 10-20%* Pulmonary 10-20%* Bladder (urothelial) 10-20%* Prostatic 5-15%* Breast 5-15%* Thyroid <5%* *These cases show expression in only a small fraction of tumor cells. Page 20 20

21 PAX8 One of the most widely used lineagespecific 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 PAX2 largely supplanted by PAX8 Expression of PAX8 in carcinomas I Primary site Positive cases Renal 85-95% Thyroid (papillary, follicular, poorly diff) % Thyroid (anaplastic, medullary) 60-80% Ovarian (non-mucinous) % Ovarian (mucinous) 10-40% Endometrial % Cervical % Renal cell carcinoma PAX8 Page 21 21

22 Ovarian clear cell carcinoma PAX8 Metastatic serous carcinoma PAX8 Metastatic serous carcinoma PAX8 Page 22 22

23 Metastatic serous carcinoma TTF1 Papillary thyroid carcinoma PAX8 Poorly differentiated thyroid carcinoma TTF1 PAX8 Page 23 23

24 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 pancreatic well-differentiated neuroendocrine tumors (PAX6) PAX8-specific monoclonal antibodies available PAX8 PAX5 PAX6 Page 24 24

25 Expression of PAX8 in carcinomas II Primary site Positive cases Thymic (polyclonal antibody) 80-90% Pancreatic/biliary 5-10% Bladder (urothelial) 10-20% Gastric/esophageal 5-10% Colorectal/appendiceal <5% Hepatocellular <5% Breast <5% Pulmonary (adenocarcinoma) <5% Pulmonary (squamous cell carcinoma) 0-30% Head and neck (squamous cell carcinoma) <5% Adrenal cortical <5% Skin (squamous cell carcinoma) <5% SF1 Steroidogenic factor 1 (NR5A1) Recently investigated transcription factor Highly sensitive marker for sex cordstromal tumor Highly sensitive marker for adrenal cortical carcinoma Particularly useful in distinguishing primary adrenal cortical carcinoma from metastatic renal cell carcinoma Adrenal cortical carcinoma SF1 Page 25 25

26 ER and PR In addition to role as predictive biomarkers in breast cancer, can be useful to ascribe primary site in certain contexts Hormone receptor-positive breast carcinomas retain expression in metastatic foci in >90% of cases Most useful in patients with known history of hormone receptor-positive breast cancer and other known or suspected malignancies ER and PR Also commonly expressed in primary carcinomas of gynecologic tract Most ovarian serous carcinomas express ER Can be helpful to distinguish serous from clear cell carcinoma ER and PR ER consistently negative in colorectal, gastroesophageal, hepatocellular, pancreatic/biliary carcinomas ER positive (generally weak/focal) in small subset of lung adenocarcinomas PR less specific: positive in 30% of cholangiocarcinomas; positive in small subset of gastroesophageal, pancreatic, hepatocellular carcinomas Page 26 26

27 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 Page 27 27

28 Don t forget to consider a primary liver tumor! Hepatocellular carcinoma Alpha fetoprotein (AFP) Low sensitivity Polyclonal carcinoembryonic antigen (CEA) Bile canalicular pattern Carbamoyl-phosphate synthase 1 (CPS1) = Hep-Par 1 antibody Urea cycle enzyme Also expressed in 5-10% of adenocarcinomas of diverse sites Arginase 1 (ARG1) Urea cycle enzyme Appears to be most sensitive and specific Hepatocellular carcinoma pcea Page 28 28

29 Hepatocellular carcinoma CPS1/Hep-Par1 Hepatocellular carcinoma ARG1 Potential mimics of HCC Adrenal cortical carcinoma Inhibin Melan-A/MART1 (MLANA) A103 >> M2-7C10 SF1 (steroidogenic factor 1; NR5A1) Renal cell carcinoma RCC (renal cell carcinoma marker) low sensitivity in the metastatic setting PAX2 less sensitive than PAX8 PAX8 highly sensitive marker, also positive in thyroid and Müllerian carcinomas Poorly differentiated cholangiocarcinoma Page 29 29

30 Metastatic renal cell carcinoma RCC Metastatic renal cell carcinoma PAX8 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 Page 30 30

31 Intrahepatic cholangiocarcinoma Capsular retraction Biliary dilatation Satellite Intrahepatic cholangiocarcinoma Satellite Capsular retraction Intrahepatic cholangiocarcinoma Page 31 31

32 Intrahepatic cholangiocarcinoma Pancreatic ductal carcinoma SMAD4 Metastatic cholangiocarcinoma SMAD4 Page 32 32

33 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: HPVassociated 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 Page 33 33

34 Well-differentiated neuroendocrine tumors 10-20% of NET present as metastasis of unknown primary Midgut (ileum) >> pancreas >> other Determination of primary site increasingly important for well-differentiated NET Recent introduction of effective systemic therapies Different efficacies for midgut vs pancreatic origin Systemic therapy for metastatic welldifferentiated neuroendocrine tumors Agent Mechanism Midgut Efficacy Pancreas Octreotide Somatostatin analogue + + Interferon-α Immune activation + + Streptozocin Alkylating agent + Temozolomide Alkylating agent + Everolimus mtor inhibitor +/ + Sunitinib Tyrosine kinase inhibitor + Bellizzi Adv Anat Pathol 2013 Transcription factor expression in welldifferentiated neuroendocrine tumors Transcription factor CDX2 ISL1 PAX6 (ppax8) PDX1 TTF1 Primary sites Midgut (ileum), appendix >> pancreas Pancreas Pancreas, duodenum, rectum Pancreas, duodenum Lung Page 34 34

35 Transcription factor expression in welldifferentiated neuroendocrine tumors Transcription factor CDX2 ISL1 PAX6 (ppax8) PDX1 TTF1 Primary sites Midgut (ileum), appendix >> pancreas Pancreas Pancreas, duodenum, rectum Pancreas, duodenum Lung CDX2 expression in well-differentiated neuroendocrine tumors Primary site Positive Lung <5% Stomach 15-30% Duodenum 30-45% Pancreas 15-20% Jejunum/ileum 90% Appendix 90% Rectum 0-30% CDX2 expression in well-differentiated neuroendocrine tumors Primary site Positive Lung <5% Stomach 15-30% Duodenum 30-45% Pancreas 15-20% Jejunum/ileum 90% Appendix 90% Rectum 0-30% Page 35 35

36 Metastatic well-differentiated neuroendocrine tumor from ileum CDX2 Metastatic well-differentiated neuroendocrine tumor from ileum CDX2 Metastatic well-differentiated neuroendocrine tumor from pancreas CDX2 Page 36 36

37 TTF1 expression in well-differentiated neuroendocrine tumors Primary site Positive Lung 30-70% Stomach <5% Duodenum <5% Pancreas <5% Jejunum/ileum <5% Appendix <5% Rectum <5% Lung carcinoid tumor TTF1 PAX6 expression in well-differentiated neuroendocrine tumors Primary site Positive Lung 0-10% Stomach 15% Duodenum 80% Pancreas 55-70% Jejunum/ileum 0% Appendix 15% Rectum 60% Page 37 37

38 PAX6 expression in well-differentiated neuroendocrine tumors Primary site Positive Lung 0-10% Stomach 15% Duodenum 80% Pancreas 55-70% Jejunum/ileum 0% Appendix 15% Rectum 60% Polyclonal PAX8 antibody PAX8 PAX6 PAX5 Pancreatic well-differentiated neuroendocrine tumor ppax8 Page 38 38

39 Metastatic well-differentiated neuroendocrine tumor from ileum ppax8 Pancreatic well-differentiated neuroendocrine tumor PAX6 Papillary thyroid carcinoma PAX6 Page 39 39

40 Lymph node PAX6 PDX1 Pancreatic and duodenal homeobox transcription factor Also known as insulin promoter factor 1 Necessary for pancreatic development Plays role in maturation of islet cells that secrete insulin (β-cells) and somatostatin (δ-cells) In adult pancreas, strong nuclear expression in islet cells, weak staining in centroacinar cells PDX1 expression in well-differentiated neuroendocrine tumors Primary site Positive Lung 0-5% Stomach 60% Duodenum 60% Pancreas 55-80% Jejunum/ileum 0% Appendix 40% Rectum 15% Page 40 40

41 PDX1 expression in well-differentiated neuroendocrine tumors Primary site Positive Lung 0-5% Stomach 60% Duodenum 60% Pancreas 55-80% Jejunum/ileum 0% Appendix 40% Rectum 15% Metastatic pancreatic well-differentiated neuroendocrine tumor PDX1 Metastatic pancreatic well-differentiated neuroendocrine tumor PDX1 Page 41 41

42 ISLET1 ISL1 Transcription factor with N-terminal LIM domains and C-terminal homeodomain Plays critical role in develop of pancreatic islets of Langerhans In adults, strong nuclear expression in islets Clinically used antibodies show cytoplasmic staining in perivascular smooth muscle ISL1 expression in well-differentiated neuroendocrine tumors Primary site Positive Lung 10-15% Stomach 0% Duodenum 90% Pancreas 70-85% Jejunum/ileum <5% Appendix 15-20% Rectum 90% ISL1 expression in well-differentiated neuroendocrine tumors Primary site Positive Lung 10-15% Stomach 0% Duodenum 90% Pancreas 70-85% Jejunum/ileum <5% Appendix 15-20% Rectum 90% Page 42 42

43 Pancreas ISL1 Pancreatic well-differentiated neuroendocrine tumor ISL1 Metastatic well-differentiated neuroendocrine tumor from ileum ISL1 Page 43 43

44 Small cell carcinoma Determination of primary site not important All patients treated with cisplatin/carboplatin + etoposide Localized Merkel cell carcinoma treated surgically TTF1 is not specific for lung in this setting TTF1 positive in >90% of small cell carcinoma of lung TTF1 positive in 30-50% of extrapulmonary small cell carcinoma TTF1 rarely positive in Merkel cell carcinoma Metastatic small cell carcinoma TTF1 Other transcription factors in small cell carcinoma Small cell carcinomas show marked transcription factor lineage infidelity! Common to detect expression of 5-10 (or more) different transcription factors in both pulmonary and extrapulmonary small cell carcinomas Important to be aware of this phenomenon to avoid misinterpretation Page 44 44

45 Transcription factor expression in small cell and Merkel cell carcinomas Courtesy of Dr. Andrew Bellizzi IHC for evaluation of metastatic WDNET of unknown primary Bellizzi Adv Anat Pathol 2013 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 for carcinomas and welldifferentiated neuroendocrine tumors Be aware of reported cross-reactivity (i.e., specificity) to avoid misdiagnosis Page 45 45