IHC on cytology specimens from pancreas, liver and bile ducts

David F. Schaeffer

Department of Anatomical Pathology

Vancouver General Hospital

A ‘quick’ practical review

Disclosure

• No relevant financial relationship with commercial interest to disclose.

Multiple choice question

A single large hepatic mass is identified on screening ultrasound in a 44 year old woman with negative hepatitis serology. FNA shows malignant cells, that label focally positive with CK19 and CK7, while other areas are HepPar-1 positive. IDH-1 shows focal positivity. These features are most consistent with:

A. Fibrolamellar HCC

B. Metastatic gastroesophageal carcinoma

C. Combined Hepatocellular-Cholangiocarcinoma

D. Intrahepatic cholangiocarcinoma.

E. Bile duct hamartoma

Learning objectives

• Review specimen procurement in hepatobiliary and pancreatic (HPB) cytology.

• Discuss the addition of immunohistochemistry in increasing the diagnostic accuracy of fine needle aspiration specimens.

• Discuss diagnostic pitfalls in HPB cytology.

Hepatobiliary and pancreatic diseases

Public domainhttp://www.hopkinsmedicine.org/gastroenterology_hepatology/clinical_services/ad

vanced_endoscopy/endoscopic_retrograde_cholangiopancreatography.html

Endoscopic retrograde cholangiopancreatography (ERCP)

Endoscopic ultrasound (EUS)

http://trialx.com/curebyte/2012/10/20/images-related-to-endoscopic-ultrasound/

http://arabhealthmagazine.com/endoscopic-ultrasound/

1.5 cm

FNA specimens after EUS

Eltoum A et al. Arch Pathol Lab Med. 2012;136:447–453

Does IHC help in increasing accuracy?

Site n (%)

Pancreas 62 (73)

Stomach 8 (9)

Lymph nodes 6 (7)

Retroperitoneum 5 (6)

Bile duct 3 (4)

Duodenum 1 (1)

Total 85

EUS characteristics

Size of lesions [range] 4 cm [0.5 – 16]

Visible mass 98%

Needle size [22G] 82%

Needle passes [avg] 2.6 [1-4]

O’Connor et al. Cancer Cytology 2014 (submitted)

Cytology result

IHC

Follow-up

Category nBenign Malignant/

Neoplastic

Malignant 45 (53%) 20 (44%) 0 45 (100%)

Suspicious 8 (10%) 3 (38%) 0 6* (75%)

Atypical 6 (7%) 0 2 (33%) 4 (67%)

Benign 24 (28%) 0 16 (67%) 6* (25%)

Non Diagnostic 2 (2%) 0 0 2 (100%)

Total 85 (100%) 23 (27%)

Diagnostic yield and accuracy

*no follow up for 4 biopsies (5%)

Accuracy – Malignant / Neoplastic

Cytologic Diagnosis n=45 Excision/imaging confirmation n

Adenocarcinoma 34 (76%) Adenocarcinoma 34 (100%)

IPMN 1 (2%) (invasive) IPMN 1 (100%)

NET 3 (7%) NET (G1 (33%); G2 (66%)) 3 (100%)

GIST 4 (9%) GIST 4 (100%)

Lymphoma 2 (4%) High grade lymphoma 2(100%)

SPPT 1 (2%) SPPT 1(100%)

Cytology result Follow-up

Category n Dx n

Suspicious 8 (10%)

AdenoCa

Lymphoma

GIST

No f/u

3 (37%)

2 (25%)

1 (13%)

2 (25%)

Atypical 6 (7%)

AdenoCa

Benign

NET

SCA

2 (33%)

2 (33%)

1 (17%)

1 (17%)

Benign 24 (28%)

AdenoCa

Cystic neoplasm

GIST

No f/u

3 (13%)

2 (8%)

1 (4%)

2(8%)

Non Diagnostic 2 (2%)GIST

NET

12 (50%)

1 (50%)

Discrepant Follow-up Comparison studies

Study Accuracy Diagnostic yield

Without on site evaluation

Iglesias et al. 67% 87%

Kim et al. 46% 95%

Nguyen et al. 78% 97%

Our study 72% 98%

With on site evaluation

Iglesias et al. 91% 99%

Turner et al. 80% 98%

Eloubeidi et al. 83% 98%

Nguyen et al. Gastrointest Endosc 2009; 69: 1264-1270

Turner et al. Gastrointest Endosc 2012; 71: 91-98

Eloubeidi et al. Cancer Cytopathol 2003; 99: 285-292

Iglesias et al. Am J Gastroenterol 2011;106:1705-1710

Kim et al. J Clin Gastroenterol 2013;doi 10.11112

Critical adjunct information

Cytopathology is a triple test:

1. Clinical impression/information

2. Radiologic impression

3. Cytologic features

Endoscopy report:

� Demographics

� Hx of obstruction/stent

insertion

� Metabolic/hormonal

changes

� Weight loss

� Location of lesion

� Size

� Solid vs cystic

� If cystic -> association

with pancreatic duct

� Lymph nodes

Cytopathology is a quadruple test:

1. Clinical impression/information

2. Radiologic impression

3. Cytologic features

4. Immunohistochemical results

Diagnostic pitfalls

• HCC vs metastatic malignancies

• wd-HCC vs hepatic adenoma/FNH

• Cystic neoplasms of the pancreas

• IgG4 staining in the setting of pancreatitis

1. HCC vs. Metastatic malignancies

Hepatocellular carcinoma:

• Trabecular/acinar

• Sinusoidal endothelial cells

• Bile production

Cell block

HepPar-1IHC in HCC:

• HepPar-1

-> can be lost in poorly differentiated lesions

• pCEA/CD10

• AFP

-> beware of hepatoid (metastatic) carcinomata

CD10

1. HCC vs. Metastatic malignancies

Features that are associated with frequent misclassification of metastatic adenocarcinoma as HCC:

• Moderate amounts of granular cytoplasm

• Round nuclei with even chromatin

• Prominent trabeculae, bare nuclei and endothelial wrapping

Renshaw, A. A., J. Haja, et al. (2006). "Fine-needle aspirates of hepatocellular

carcinoma that are misclassified as adenocarcinoma: correlating cytologic

features and performance in the College of American Pathologists

Nongynecologic Cytology Program." Arch Pathol Lab Med 130(1): 19-22.

If in doubt: HepPar 1 +/- CD10

Cholangiocarcinoma

68 yr old woman:

• Known chronic HBV

• New liver lesion

Cell Block CK19

HepPar-1

Combined HCC-Cholangiocarcinoma

IDH1 in intrahepatic cholangiocarcinoma:

• IDH1 R132C mutation present in 30%

• Positive staining for IDH1

• Not yet verified in CytoLyte® preparations

Neuroendocrine tumours

Neuroendocrine tumours:

• Isolated (stripped) cells

• Eccentrically place nuclei

• Salt and pepper chromatin

IHC - NET:

• Synaptophysin

• Chromogranin A -> serum levels

• ki67

Synaptophysin Ki-67

Ki67 in FNA of NETs:

• Interpret with caution

• Sampling may over- or under call

• ‘Hot Spots’

Metastatic melanoma

76 yr old man:

• Known choroidal melanoma

• US showed suspicious new lesion

Melanoma IHC - CytoLyte®:

• HMB45

• Melan A

• S100 – may stain some CA

Varying success

BRAFV600E

Resection

2. wd-HCC vs. hepatic adenoma / FNH

If possible, advise to perform core needle biopsy!

Hepatic adenoma:

• Virtually impossible to separate from wd-HCC

• Advise against subclassification on FNA

• I do not perform beta-catenin

FNH – Glutamin synthetase:

• Marks hepatocytes in large anastomosing areas in a ‘map-like’ pattern.

• Not expressed in hepatocyte close to fibrotic bands

• Positive in wdHCC but in a diffuse pattern

HA - Glypican 3:

• High sensitivity for HCC

• Less helpful in wdHCC:

• Only 50% of wdHCCpositive

• HA and large regenerative nodules negative

• 43% of high-grade dysplastic nodules positive

Shafi zadeh N, et al. Utility and limitations of glypican-3expression for the diagnosis of hepatocellular carcinoma at both ends of the differentiation

spectrum. Mod Pathol. 2008;21:1011–1018.Bioulac-Sage P, et al. Over-expression ofglutamine synthetase in focal

nodular hyperplasia: a novel easy diagnostictool in surgical pathology. Liver Int. 2009;29(3):459–465.

FNH:

• Abundant normal-appearing hepatocytes

• Numerous ductal-type epithelium in sheets and clusters

3. Cystic neoplasms of the pancreas

Tumor Cytologic features

Serous cystadenoma Loose sheets and groups of single

monomorphic cuboidal/low columnar epithelial

cells; often proteinaceous background.

Mucinous Cystic neoplasm Sheets and groups of columnar epithelium

with variable degree of nuclear atypia and

crowding; variable amount of mucin in

background.

Solid Pseudopapillary tumor Fragments with fibrovascular cores lined by

bland cells with nuclear grooves and scant

cytoplasm.

Endocrine tumor Monomorphic, often discohesive,

monomorphic plasmacytoid cells with ‘salt

and pepper’ chromatin.

SPPTIHC - SPPT:

• Beta-catenin (nuclear)

• A1AT

• PR

• Racemase (P504s)

• Synapthophysin

• Chromogranin A

Beta-catenin

A1ATProgesterone Receptor

Cyst fluid analysis

• Kras based testing

• Proprietary systems

• Likely to supplement cytological assessment

• To be evaluated in conjunction with clinical info and cyst fluid marker assessment

4. IgG4 staining – pancreatitis

Autoimmune pancreatitis:

• Lymphoplasmacytic sclerosing pancreatitis

• Duct-destructive chronic pancreatitis

• Sclerosing chronic pancreatitis

54 year old woman with pancreas ‘mass’ on EUS.

? AIP

Do IgG4.

Periductal lymphoplasmacytic inflammationIgG4 – Type 1Storiform fibrosis

4. IgG4 staining – pancreatitis

54 year old woman with pancreas ‘mass’ on EUS.

? AIP

Do IgG4.

IgG4

Definitive diagnosis requires core needle biopsy.

?

4. AIP Diagnosis – Ampullary bx

IgG4 IgG4 + cells in Ampulla:

• >50/hpf - highly suggestive

• 10-50/hpf - equivocal

• < 10/hpf - unlikely

Moon Sh, Kim MH, Park D. et al. IgG4 immunostaining of

duodenal papillary biopsy specimens may be useful for

supporting a diagnosis of autoimmune pancreatitis. GastrointestEndosc. 2010 May;71(6):960-6.

Multiple choice question

A single large hepatic mass is identified on screening ultrasound in a 44 year old woman with negative hepatitis serology. FNA shows malignant cells, that label focally positive with CK19 and CK7, while other areas are HepPar-1 positive. IDH-1 shows focal positivity. These features are most consistent with:

A. Fibrolamellar HCC

B. Metastatic gastroesophageal carcinoma

C. Combined Hepatocellular-Cholangiocarcinoma

D. Intrahepatic cholangiocarcinoma.

E. Bile duct hamartoma

A. Fibrolamellar HCC

B. Metastatic gastroesophageal carcinoma

C. Combined Hepatocellular-Cholangiocarcinoma

D. Intrahepatic cholangiocarcinoma.

E. Bile duct hamartoma

Thank you!