Patologia molecolare del carcinoma pancreatico

(1)

Aldo Scarpa

ARC-NET Centre for Applied Research on Cancer and Department of Pathology

University of Verona

Patologia molecolare del

carcinoma pancreatico

(2)

Ductal

adenocarcinoma Acinar cell

carcinoma

Neuroendocrine tumours

Intraductal papillary mucinous neoplasm

Serous cystic neoplasms

The Spectrum of Pancreatic Tumors

Mucinous cystic neoplasms

Solid pseudopapillary neoplasms

(3)

Ductal

adenocarcinoma Acinar cell

carcinoma

Neuroendocrine tumours

Intraductal papillary mucinous neoplasm

Serous cystic neoplasms

The Spectrum of Pancreatic Tumors

Mucinous cystic neoplasms

Solid pseudopapillary neoplasms

(4)

Genetics of ductal adenocarcinoma

1. What we know today

2. Information from sequencing

3. What we can do for the patient

(5)

Genetics of ductal adenocarcinoma

1. What we know today

2. Information from sequencing

3. What we can do for the patient

(6)

Emerging Molecular Taxonomy

(7)

The most important mutated genes in pancreatic ductal adenocarcinoma and its

variants

(8)

Objective ^: Sub-classify cancers using histopathology and molecules

Methods: Histopathological and molecular characterisation of primary cancers and model systems

Pancreatic Cancer

(9)

Genetics of ductal adenocarcinoma

1. What we know today

2. Information from sequencing

3. What we can do for the patient

(10)

The mutational landscape of pancreatic cancer

(11)

Stable

(<50 events)

Focal

(50-200, 50% on 1 Chr)

Unstable

(>200 widespread)

Scattered

(50 – 200 widespread)

30% 14%

20% 36%

Waddell N, et al – Nature 2015

There are 4 PDAC subtypes

Anatomy Whole genome sequence

of 100 PDAC

(12)

Anatomy

• 10 pathways

• 32 recurrently mutated genes

96%

78%

24%

14%

24%

25%

16%

15%

12%

47%

Driver Gene Analysis

Whole genome sequence of 457 PDAC

Bailey et al, Nature 2016

(13)

Genetics of ductal adenocarcinoma

1. What we know today

2. Information from sequencing

3. What we can do for the patient

(14)

Cancer diagnostics tasks

4 Follow up Diagnosis 1

Prognosis 2

3 Predict drug efficacy

(15)

Multigene panels

for pathway based molecular diagnostics

Panel Name

PDAC periampullary

basic

TGFB Pathway SWI/SNF Chromatin Remodelling

WNT non canonical - Spliceosome

DNA DAMAGE REPAIR

GENES

KRAS ACVR1A ARID2 ARHGAP4 BRCA1

BRAF ACVR1B ARID1B PRPF40A BRCA2

NRAS ACVR1C BAP1 PRPF40B PALB2

TP53 ACVR2A DPF1 RBM6 STK11

CDKN2A ACVR2B DPF2 RBM10 RPA1

PIK3CA TGFBR1 DPF3 RNF43 REV3L

APC TGFBR2 HLTF ROBO1 ATM

CTNNB1 SMAD1 KDM5C ROBO2

EGFR SMAD2 KDM6A ROBO3

ERBB2 SMAD3 KMT2C ROBO4

ERBB4 SMAD4 KMT2D SF1

FGFR3 SMAD5 MEF2C SF3A1

FLT3 SMAD9 PBRM1 SF3B1

GNAS SETD2 SLIT2

KDR/VEGFR2 SMARCA1 SRGAP1

FBXW7 SMARCA2 SRGAP2

CDH1 SMARCA4 SRGAP3

U2AF1 U2AF2

PDAC Periampullary

TGFß Pathway

SWI/SNF Chromatin

WNT

Spliceosome

DNA Damage Repair

ATM BRCA1 BRCA2 PALB2 REV3L STK11 RPA1 BARD1 BRIP1 PTEN RAD51B RAD51C RAD51D MRE11A NBN CHEK1 CHEK2 FAM175A

Molecular Diagnosis

1

(16)

Variable No %

Sex

Male 104 52

Female 96 48

Age

Mean 65

Median 67

Range 30 - 88

Overall Stage

IA 1 0.5

IB 2 1

IIA 26 13

IIB 159 80

III 2 1

IV 10 5

T stage

T1 1 0.5

T2 4 2

T3 193 97

T4 2 1

N stage

N0 31 16

N1 169 84

M stage

M0 189 95

M1 11 5

GRADE

1 12 6

2 117 59

3 68 34

X 3 1

Resection Margins

R0 121 60

R1 79 40

Tumour Site

Head 158 79

Body 18 9

Body-Tail 7 3

Tail 13 7

Istmus 2 1

Peri-ampullary 2 1

Cohort of 200 well annotated PDAC

(17)

CANCER TYPE SUBTYPE

Ductal adenocaricnoma (PDAC)

Common type 173

Clear cell 3

Adenosquamous 3

Focal squamous 8 IPMN associated 5

Colloid 1

Periampullary 1

Acinar 2

Ampullary 1

Duodenal 2

Neuroendocrine (poor diff.) 1

TOTAL 200

Patient Cohort 200 patients

(18)

Multigene panels

for pathway based molecular diagnostics

Panel Name

basic

GENES

GNAS SETD2 SLIT2

CDH1 SMARCA4 SRGAP3

U2AF1 U2AF2

PDAC Periampullary

TGFß Pathway

SWI/SNF Chromatin

WNT

Spliceosome

DNA Damage Repair

Molecular Diagnosis

1

(19)

PDAC – Periampullary Basic panel

Gene Mutations Proportion

KRAS 188 94%

TP53 123 61%

SMAD4 30 15%

CDKN2A/p16 24 12%

GNAS 4 2%

APC 3 1%

PIK3CA 4 2%

Gene Mutations in 200 Cases

4 Follow up

Personal markers

(20)

Multigene panels

for pathway based molecular diagnostics

Panel Name

basic

GENES

GNAS SETD2 SLIT2

CDH1 SMARCA4 SRGAP3

U2AF1 U2AF2

PDAC Periampullary

TGFß Pathway

SWI/SNF Chromatin

WNT

Spliceosome

DNA Damage Repair

Molecular Diagnosis

1

(21)

Gene Mutations Pathogenic VUS SN P

ARID1A 12 7 5 -

ARID1B 9 3 3 3

ARID2 11 1 4 6

DPF1 1 - 1 -

DPF3 - - - -

HLTF 4 - 1 3

KDM5C 4 2 1 1

KDM6A 6 5 1 -

KMT2C

(MLL3) 36 9 9 18

KMT2D

(MLL2) 39 5 18 6

SETD2 9 1 5 3

SMARCA2 2 - 2 -

SMARCA4 12 2 7 3

PBRM1 7 3 4 -

BAP1 3

₁ ₂

155 39 63 53

Gene Mutations in 200 Cases

Histone Modifiers and Chromatin Remodelling

2 Prognosis

Sausen M et al. Nature Communications. 2015

39/200 (20%)

(22)

Multigene panels

for pathway based molecular diagnostics

Panel Name

basic

GENES

GNAS SETD2 SLIT2

CDH1 SMARCA4 SRGAP3

U2AF1 U2AF2

PDAC Periampullary

TGFß Pathway

SWI/SNF Chromatin

WNT

Spliceosome

DNA Damage Repair

Molecular Diagnosis

1

(23)

BRCA – Homologous Recombination Panel

Gene Mutations in 200 cases

Gene Variants Pathogenic Unknown SNP

ATM 40 3 7 12

BRCA1 24 2 2 11

BRCA2 25 14 3 8

PALB2 8 2 1 5

REV3L 15 2 9 5

STK11 4 2 3 -

RPA1 2 - - 2

BARD1 8 2 1 5

BRIP1 8 - 4 4

PTEN 2 - - 2

RAD51B 4 - - 4

RAD51C 3 - - 3

RAD51D 4 1 - 3

MRE11A 2 1 - 2

NBN 2 1 - 1

CHEK1 2 - 2

CHEK2 7 2 1 4

FAM175A 1 - 1

161 32 34 95

3 Predict drug efficacy

32/200 (16%)

(24)

HR gene mutations are associated with BRCA signature and response to platinum

Waddell N, et al – Nature 2015

(25)

Anatomy

Physiology

Social Behavior

(26)

Beyond anatomical lesions in DNA

• Physiology : RNA-Seq of primaries and xenografts to assess molecular subgroups

• Histopathological revisitation of cancer and its stromal environment

• Devise clinically applicable diagnostic methods for molecular subclassification using DNA panels immunohistochemistry and nanostring assays

• Devise clinical trials based on cancer subtypes

(27)

RNAseq of 96 cases

• 4 classes based on transcription factors and downstream targets

• Enriched with specific histological features

Bailey et al, Nature 2016

Physiology : Identification of PDAC Subtypes

(28)

Immunogenic Subtype

c a b

CD4+ve regulatory T cells

1 0 -1

GP6 GP7 GP8

1 0 -1

ADEX Immunogenic

Squamous Pancreatic Progenitor

APC co-stimulation APC co-inhibition Macrophages

T cell co-stim ulation

T cell co-inhibition B cells

CD8+ve T cells

Cytolytic activity

6.7 15.5 2.8 4.8 2.8 3.4 2.9 2.8 9.7 7.6 10.4 15.3 7.7 17.8 20.8 12 6.1 25.7 15.2 15.4 20.8 10 3 19.5 8.4 4.7

d

MACROPHAGE SIGNATURE

e

T CELL CO-INHIBITION SIGNATURE Signature

Score

Correlation

●●●

● ●

●

● GP6 GP7 GP8

Pval = 6.75E-11 Pval = 0.00625 Pval = 0.00945

-0.2 0 0.2 0.4

GP association (ME)

A I S P A I S P A I S P

REACTOME_PD1_SIGNALING BIOCARTA_CTLA4_PATHWAY P.value = 0.01

0 0.5

Signature Score -0.5

P.value = 0.00843

A I S P A I S P

Bailey et al, Nature 2016

(29)

PDAC subtypes using colorectal cancer assinger

Scarpa, unpublished

(30)

Cancer is a tissue

Pancreas Cancer

(31)

Cancer Normal

Pancreas Cancer

(32)

Pancreas Cancer

Inter- and Intra- Patient Heterogeneity

(33)

Multigene panels

for pathway based molecular diagnostics

Panel Name

basic

GENES

GNAS SETD2 SLIT2

CDH1 SMARCA4 SRGAP3

U2AF1 U2AF2

PDAC Periampullary

TGFß Pathway

SWI/SNF Chromatin

WNT

Spliceosome

DNA Damage Repair

Molecular Diagnosis

1

(34)

Gene Mutations in 200 Cases

TGFß pathway

Gene Mutations Pathogenic VUS SNP

SMAD2 1 ¹ ^- ^-

SMAD3 12 4 1 7

SMAD4 46 30 8 8

SMAD5 2 - - 1

SMAD9 5 - 3 2

ACVR1 2 - - 2

ACVR1C 3 - 1 2

ACVR2A 8 5 3 -

ACVR1B 3 1 2

ACVR2B 4 1 2 1

TGFBR2 10 7 3 1

86 49 21 24

49/200 (25%)

(35)

Stroma is composite and variable

Pancreas Cancer and its environment

(36)

Dissecting the Stroma

Keratin Vimentin Fusion

(37)

VIM+

KER+

Stromal Cells

Tumor Cells

EMT

KRAS G12R 22%

TP53 R273H 39%

SMAD4 R361H 32%

DEP-ARRAY Silicon Biosystems

(38)

Dissecting the Stroma

TUMOR EMT

Recovery of homogeneous pools of cells

STROMAL

DEP-ARRAY

(39)

40% Tumor Cells: CNV assessment

Unsorted cells

(40)

Low-Pass Whole-Genome sequencing for CNV assessment confirm separation of S/T

DEPArray™ sorted Tumor cells

(41)

Balachandran et al. NATURE November 8, 2017

(42)

Neoantigen quality and immune infiltrate make the difference

Balachandran et al. NATURE November 8, 2017

(43)

Beyond anatomical lesions in DNA

• Physiology : RNA-Seq of primaries and xenografts to assess molecular subgroups

• Histopathological revisitation of cancer and its stromal environment

• Devise clinically applicable diagnostic methods for molecular subclassification using DNA panels, immunohistochemistry and nanostring assays

• Devise clinical trials based on cancer subtypes

(44)

Patologia molecolare del carcinoma pancreatico

Aldo Scarpa

ARC-NET Centre for Applied Research on Cancer and Department of Pathology

University of Verona

Patologia molecolare del

carcinoma pancreatico

Ductal

adenocarcinoma Acinar cell

carcinoma

Neuroendocrine tumours

Serous cystic neoplasms

The Spectrum of Pancreatic Tumors

Ductal

adenocarcinoma Acinar cell

carcinoma

Neuroendocrine tumours

Serous cystic neoplasms

The Spectrum of Pancreatic Tumors

Genetics of ductal adenocarcinoma

1. What we know today

2. Information from sequencing

3. What we can do for the patient

Genetics of ductal adenocarcinoma

1. What we know today

2. Information from sequencing

3. What we can do for the patient

Emerging Molecular Taxonomy

The most important mutated genes in pancreatic ductal adenocarcinoma and its

variants

Objective : Sub-classify cancers using histopathology and molecules

Methods: Histopathological and molecular characterisation of primary cancers and model systems

Pancreatic Cancer

Genetics of ductal adenocarcinoma

1. What we know today

2. Information from sequencing

3. What we can do for the patient

The mutational landscape of pancreatic cancer

Stable

Focal

Unstable

Scattered

30% 14%

20% 36%

Waddell N, et al – Nature 2015

There are 4 PDAC subtypes

Anatomy Whole genome sequence

of 100 PDAC

Anatomy

• 10 pathways

• 32 recurrently mutated genes

96%

78%

24%

14%

24%

25%

16%

15%

12%

47%

Driver Gene Analysis

Whole genome sequence of 457 PDAC

Bailey et al, Nature 2016

Genetics of ductal adenocarcinoma

1. What we know today

2. Information from sequencing

3. What we can do for the patient

Cancer diagnostics tasks

4 Follow up Diagnosis 1

Prognosis 2

3 Predict drug efficacy

Multigene panels

for pathway based molecular diagnostics

PDAC Periampullary

TGFß Pathway

SWI/SNF Chromatin

WNT

Spliceosome

DNA Damage Repair

Molecular Diagnosis

Objective ^: Sub-classify cancers using histopathology and molecules