New WHO Classification

Liver and Bile Duct Tumours

P. Schirmacher

Institute of Pathology

University Hospital Heidelberg

Changes/Aims in 5th Edition

• First volume of 5th edition; frontrunner effect

• Stringent formal criteria, harmonized terminology; evidence based claims (not casuistic)

• Completely rewritten; concise text, more tables, better figures

• Non-organ specific entities (haematolymphoidneoplasms, mesenchymal tumors, metastases andothers, hereditary tumor syndromes) taken out ofthe organ chapters, but not in NET

Pattern of HCC

• No significant link to pathogenesis, prognosis and therapy

• Diffuse (cirrhotomimetic) pattern generally underestimated by imaging

nodular massive

diffuse multifocal

HistologicalPattern

(Micro)trabecular

Solid

Macrotrabecular

Pseudoglandular

Macroscopic Pattern

Fat

Mallory hyalin

MDBs

Globular hyaline inclusions (α1AT?) Pale bodies

(fibrinogen?)

Glycogen

(clear cell change)

Copper Oncocyticchange

Bile

• No relevance for prognosis or therapy

• May support diagnosis (metastases)

Cytological Changes (Pattern)

Immunohistological Markers of Hepatocellular Differentiation

WHO-Classification of Tumours (5th ed)Digestive System Tumours

G1 G2 G3 G4

• Several grading schemes (Edmondson-Steiner (1954), AFIP (Nzeako et al., 1995), Burt et al., 2018); none of the grading approaches validated

• No internationally consented, harmonized grading• Prognostic relevance (survival after resection and transplantation)• Needle biopsy grading shows reasonable correlation with resection grading

The Grading Issue

Nzeako et al., 1995

Numeric Descriptive Global Criteria Remarks

I Welldifferentiated

Hepatocyticdifferentiation √Malignancy ?

Maximally mild nuclearatypia; ‚normal-like‘ cytoplasmic differentiation

Differential diagnosisDysplastic nodule, Adenoma

II Moderatelydifferentiated

Hepatocyticdifferentiation √Malignancy √

Moderate nuclear atypia; increased cytoplasmicbasophilia

III Poorlydifferentiated

Hepatocyticdifferentiation ?Malignancy √

Marked nuclear atypia Includes former grade IV, also anaplasia, sarcomatoid dediff.

Proposal: Pragmatic Grading Scheme

Molecular Subtyping of HCC?

Calderaro, J. et al, J. Hepatol. 2017

Open questions:• Unclear whether types or stages• Only stage I/II tumors• Relation/hierarchy of

pattern/changes N• not distinct but overlapping groups• Some defined subtypes missing

Fibrolamellar HCC

DNAJB1-PRKACA Translocation

Honeyman et al., Science, 2014

DNAJB1: Chaperon DNAJ homologue

PRKACA: catalytic domain of PKA

Chr. 19p13.12

• 24/24 FLC positive

• 7/7 FLC PRKACA overexpression

• 0/80 other primary liver tumors positive

• Multinational trial: 102/103 FLC positive

PGK (126 bp)

Graham et al., Mod Pathol, 2015, 2017

DD: FLC-like areasin conventionalHCCSolution:TL-testing

Chromophobe HCC

Wood et al., Mod Pathol 2013

chromophobe abrupt anaplasia

ALT-Phenotype (alternative lenghtening of telomeres)

pseudocysts

- Association with HBV (50%)

- Frequency: 5% of HCCs

- 100% ALT vs. 7% in unselected HCCs

Scirrhous HCC

Characteristics:

• Abundant intratumoral stroma; no capsule

• TSC1/TSC2 mutations; activatedTGFß signalling

• estimated frequency: 3-4%

• Mimicks ICC on imaging

• Prognosis better?

DD: ICCA/Metastasis; solution: Markers of hepatocellular and pancreato-biliary differentiation

Macrotrabecular massive (high AFP) HCC

Calderaro et al., J Hepatol 2017

Lymphocyte-rich HCC(syn: Lymphoepithelioma-like; lymphocyte-rich stroma)

• Better prognosis

• All EBV negative, except 1 (Si et al., 2004)

Emile et al., 2000, Si et al., 2004, Chen et al., 2007, Nemolato et al., 2008, Wie et al., 2015, Cacciato-Insilla et al., 2015, Wang et al., 2017

Homogenous subtype (immunoprofile; hot/cold?)DD: hepatic lymphoma/lymphoma infiltration of liver; solution: prove HCC-cells

Granulocyte-rich HCC(Syn: GCSF-producing)

• Diffuse granulocytic infiltration; sarcomatoid changes• GCSF production by tumor cells• GCSF, CRP, WBC elevated; fever• Resolves after resection

Yamamoto et al., 1999, Araki et al., 2007, Joshitaet al., 2010, Kohno et al., 2013, Nagata et al., 2016

DD: undifferentiated/rhabdoid carcinoma (INI-/SWI/SNF-deficient) ?

Steatohepatitic HCC

Characteristics:

• Histological features ofsteatohepatitis;

• 60% steatohepatitis in nontumorous liver (ETOH, metabolic syndrome)

• estimated frequency: 5-10%

• Activated IL-6/JAK/STAT signaling

HCC

DD: Steatohepatitis; solution: recognize/prove neoplastic nature

Steatohepatitic HCC– mod. Gomori

Clear Cell HCC

Characteristics:

• DD: metastasis (kidney)

• Unclear distinction from clear cellpattern (80%?)

• No specific molecular changes so far

• estimated frequency: 3-5%

• better prognosis?

DD: clear cell carcinoma of kidney metastasis (rarely other extrahepatic primariesSolution: non-clear cell areas/markers of hepatocytic differentiation

Distinct HCC Subtypes

Subtype Histology Molecular Clinical Frequency Prognosis Reference

Steatohepatitic Steatohepatitictumor appearance

IL-6/JAK/STAT activation

5-10 ~ Salomao et al., 2010

Clear cell >80% clear cellmorphology; glycogen

? Later recurrenceafter resection

3-7 ↑ Li et al., 2011

Macrotrabe-cular massive

Macrotrabeculargrowth >50%

High homogenousAFP expression; p53mut; FGF19amp

High AFP, poorprognosis, vascularinvasion

5 ↓ Calderaro et al., 2017

Chromophobe Light/clearcytoplasm, abrupt anaplasia

Alternative lengthening oftelomeres (ALT)

3 ~ Wood et al., 2013

Fibrolamellar Oxyphiliccytoplasm, lamellar fibrosis

DNAJB1:PRKACA Translocation

2 ~ Graham et al., 2015, 2018

Scirrhous Dense tumoralfibrosis, no capsule

TSC1/2 mutations; TGFß activation

Young (median 25), no primary liverdisease

2 ↑/~ Matsuura et al., 2005

Neutrophil-rich Diffuse neutrophils GCSF expression Elevated WBC, CRP, IL6

<1 ↓ Nagata et al., 2016

Lymphocyte-rich

Lymphocytes >> tumor cells

?; not consistentlyEBV-related

1-2 ↑ Emile et al., 2000; Nemolato et al., 2008

HCC ‚NOS‘

Defined subtypes (~ 20-30%): specific morpho-molecular phenotypes

Molecular Pattern

Reanalysis

?

Morpho-molecular HCC Classification

Macroscopic Pattern

Histologic Pattern

Cytologic Pattern

Development of HCC

premalignant lesion small HCC (<2 cm) classical HCC

early HCC

progressed HCC

Dysplastic

FocusDysplastic Nodule (DN)

vague

nodular

Nodule-in-

nodule HCC

G1

G2

International Consensus Group

for Hepatocellular Neoplasia (2009)

G2

G3

G3

advanced HCC

early HCC Progressed small HCC

low-grade high-grade

‚Matrix Diagnosis‘ – HCC and Precursors

• Gender: m > f

• Patient from endemic risk factor area?

• HCC-predisposing diseases: HBV, HCV, NASH, gen. haemochromatosis, alcohol abuse

• State of non-tumorous liver: chronichepatitis, cirrhosis

Molecular Alterations during HCC Development

WHO-Classification of Tumours (5th ed)Digestive System Tumours

Diagnostic Criteria for HCC Precursors and Malignant Transformation

WHO-Classification of Tumours (5th ed)Digestive System Tumours

Tatrai et al., Lab Invest 2006

RN regenerative

nodule

HCC Neoangiogenesis

Sometimes suggestive, but neither sufficiently specific nor sensitive

Lennerz et al., Am J Pathol 2011

HCC – Loss of Ductular Reaction

Benign vs. Malignant

Di Tommaso et al., J Hepatol, 2009

International Consensus Group; Hepatology 49 (2009) 658-664

Distinct: progressed! (12 mm)

Vaguely nodular: early HCC

Small HCCs

Features of Early and Small Progressed HCC

WHO-Classification of Tumours (5th ed)Digestive System Tumours

Focal Nodular Hyperplasia

• No neoplasia (abnormal vascular supply), reactive/ hyperplastic proliferative lesion; no malignant transformation

• F>M

• No significant bleeding risk

• Nonspecific symptoms, if at all

• Typically diagnosed by imaging (abnormal vascularisation)

• Single and multiple (‚FNH-syndrome‘)

Central scar, abnormal vessels, ductules, inflammatory infiltrates, transitional hepatocytesIn unclear cases: GS-IHC (map-like pattern)

Hepatocellular Adenoma

Hepatocellular Adenoma Subtyping

Nault et al., Gastroenterology 2017

New Subtype: shHCA

Prostaglandin-H2 D-Isomerase533 HCAs / 411 Patienten

Henriet et al., Hepatology 2017

New Marker: ASS1+ HCA

- Argininosuccinat Synthase 1 (ASS1)-IHC detects (all?) UHCA

- Association with bleeding risk (ca. 65%)

→ shHCA also ASS1+?

Malignant Transformation of HCA

Pilati et al ; Cancer Cell 25,,2014, 428 - 441

CK7

Hepar-1

Combined HCC-CC, ‚Classical Type‘

CK7

Hepar-1

EpCAM CD117

CK19AFP

Intermediate PLC

Subtyping of Mixed LC/Combined HCC/CC

Moeini, A. et al., J. Hepatol. 2017

CholangiolocellularCarcinoma –cHCC/CCA or CCA?

cHCC/CCA, Intermediate and UndifferentiatedPLC

Subtype Definition Frequency

Combined HCC/CCA Unequivocal hepatocyticand biliary differentiation

> 2,5%

Intermediate PLC tumor cell differentiationintermediate betweenhepatocytic and biliary

Very rare

UndifferentiatedPLC

Liver cancer withoutconvincing furtherdifferentiation

Very rare

Aishima et al., Am J Surg Pathol 2007

~ periductal infiltrating type (Sasaki et al., 1998)

~ mass-forming type (Sasaki et al., 1998)

Intrahepatic Cholangiocarcinoma Two Different Entities - Macroscopy

Intrahepatic Cholangiocarcinoma Two Different Entities - Histology

Liau et al., Mod Pathol, 2014

Large Duct Type – distal, periductal infiltrating

Small Duct Type – peripheral; mass forming

Histo-pattern; mucin productionIHC: NCAM, EMA, CRP?

Intrahepatic CholangiocarcinomaTwo different Entities

CCA- large duct type; distal/central, periductal-infiltrative

ICCA- small duct type; proximal/peripheral, primary mass forming

Anatomically extrahepatic (ECCA)

Anatomically intrahepatic (ICCA)

Premalignant Biliary Changes

Kappa-Values:

Reactive: 0.42

BilIN-1: 0.40

BilIN-2: 0.16

BilIN-3: 0.44

Zen et al., Mod Pathol 2007

BilIN-1

BilIN-2

BilIN-3

Low

High

CK7 PR

Biliary Intraepithelial Neoplasia (BilIN)

Mucinous Cystic Neoplasia (MCN)

Intraductal Papillary Neoplasia of the Bile Ducts (IPNB)

Intrahepatic CholangiocarcinomaTwo different Entities – Premalignant Lesions

CCA- large duct type; distal/central, periductal-infiltrative

ICCA- small duct type; proximal/peripheral, primary mass forming

Anatomically extrahepatic (ECCA)

Anatomically intrahepatic (ICCA)

BilINIPNBMCNAdenofibroma

?

Bridgewater et al., J Hepatol, 2014

Global Incidence of ICCA

Intrahepatic CholangiocarcinomaTwo different Entities – Etiology

CCA- large duct type; distal/central, periductal-infiltrative

ICCA- small duct type; proximal/peripheral, primary mass forming

Anatomically extrahepatic (ECCA)

Anatomically intrahepatic (ICCA)

FlukesPSCStones

HBV, HCVNASH, ASHFe, CuCirrhosis

Different Molecular Pattern of ICCA vs ECCA

Nakamura et al., Nature Genet, 2015

Graham et al., Hum Pathol, 2014

Goeppert, Rössler et al, 2018

Intrahepatic CholangiocarcinomaTwo different Entities – Molecular Pattern

CCA- large duct type; distal/central, periductal-infiltrative

ICCA- small duct type; proximal/peripheral, primary mass forming

Anatomically extrahepatic (ECCA)

Anatomically intrahepatic (ICCA)

RASPRKACA/B-TLELF3ARID1B

IDH1/2FGFR2-TLBRAF V600BAP-1ARID1A

Mutation ICC (%)

KRAS/NRAS 10-20

TP53 Mut 10-20

FGFR2 TL 20-30 *

IDH1/2 Mut 20-35 (*)

SMAD4 Mut 2-10

Her2/neu Mut 2-5 (*)

BRAF V600E 3-6 (*)

ARID1A 5-15

BAP1 5-15

PTEN 0-5

EGFR 2-3 (*)

BRCA1/2 3-4 (*)

NTRK-TL 2-3 (*)

ALK-TL 1-2 (*)

ROS1-TL 2-3 (*)

Frequency of druggable mutations in ICCA

60-80% ?

3/11 Kipp et al., Hum Pathol, 2011

IDH-Mutations in ICC

Grassian, Curr Opin Gastroenterol 2014

IDH-Mutations in ICC

• Better natural course

• Correlation with morphology(clear cell change)

• Entity-specific IDH-mutation spectrum

• Poor response to IDH-inhibition in ICC (high resistance rate)

• Option: broader/2nd generationinhibition

Harding et al., Cancer Discov, 2018

Mutation ICC (%)

KRAS/NRAS 10-20

TP53 Mut 10-20

FGFR2 TL 20-35

IDH1/2 Mut 20-35

SMAD4 Mut 2-10

Her2/neu Mut 2-5

BRAF V600E 3-6

ARID1A 5-15

BAP1 5-15

PTEN 0-5

BRCA1/2 3-4%

NTRK-TL 2-3

ALK-TL 1-2

ROS1-TL <1

TLs/Gene Fusions in ICCA

Moeini et al, CCR, 2016

Gene fusions are a particular featureof small duct ICC

Heterogenous

Tailing effect

NTRK Gene Fusions

• Large intronic regions1,2

• Low complexity sequence, GC rich

• Inconsistent break points and fusion partners

• Fusions occur in a tumour-agnostic fashion

• Is endogenously expressed in some tumour/tissue types

• What does this means for testing?

– DNA-only panels, even with intronic coverage, lack sensitivity of detection

– IHC may lack specificity

– RT-PCR is not comprehensive

– FISH requires at least 3 assays

Take home: RNA-based NGS is the preferred method for detecting NTRK gene fusions in tumors

NTRK, neurotrophic tyrosine receptor kinase1. Sigal D et al. J Natl Compr Canc Netw 2017;15:1317-22; 2. Gagan J, Van Allen EM. Genome Med 2015;7:80; 3. Vaishnavi A et al. Cancer Discov 2015;5:25-34

Conclusion• 5th edition with more stringent formal criteria, harmonized

terminology/definitions and evidence based claims

• Aiming for increased reproducibility (worldwide, different expertiselevels), clinical relevance/making meaningful trials possible and tumordocumentation/registries

• Completely rewritten and updated in all subchapters

• Non-organ-specific entities taken out of organ chapters; relevant for liverespecially in vascular tumors and metastases!

• Novel conclusive morpho-molecular (sub)classification of HCC and ICCA reflecting macro- and histomorphology, etiology, pathogenesis, and clinicalpicture; should guide further trials and therapeutic approaches

• ICCA exemplifies the potential conflict between anatomical/organ-basedand histological classification/typing

• Small duct ICCA offers a high frequency of translocations and potential therapeutic options; should be recognized as specific entity

Future• Further shapen morpho-molecular typing (more

data) especially in ICCA; data with new technologies(e.g. proteomic, methylation, digital imageanalysis/AI)

• Integration in clinical and diagnostic trials, tumorboards etc.

• Basis for further translational research approaches; more predictive markers

• Use it, spread the news, and please give feedback!

• Check the internet version

• Update planned for 2021 (internet)

Thank you!Authors of the liver chapter:

C Sempoux, P Bioulac-Sage, S Kakar, JC Nault, MS Torbenson, IOL Ng, YN Park, M Roncalli, M Sakamoto, R Saxena, A Quaglia, WM Tsui, Y Nakanuma, O Basturk, S Aishima, I Esposito, DS Klimstra, M Komuta, Y Zen, F Kondo, P Schirmacher

Authors of the bile duct chapter:

I Esposito, S Aishima, O Basturk, Y Nakanuma, DS Klimstra, JC Roa, NV Adsay, J Arola, WM Tsui, Y Zen, S La Rosa

Editors of chapters:

V Paradis, NY Park, M Fukayama, AK Lam, DS Klimstra, P Schirmacher

Ian Cree and the IARC Team