Prospettive del “gene expression profiling” nella prevenzione, diagnosi e cura dei tumori

(1)

Aldo Scarpa

ARC-

NET

Centre for Applied Research on Cancer and Department of Pathology

University of Verona, Italy

La prospettive del molecular profiling

nella prevenzione diagnosi e cura dei tumori

(2)

Disclosures

• Advisory Boards / Honoraria / Speakers’ fee / Consultant for:

Astra-Zeneca, Celgene, Novartis, Roche

• Research Support / Grants from:

European Union FP7 grant no 602783 Ministry of University

and Research (FIRB RBAP10AHJB);

5X1000 grant n. 12182

Ministry of Health FIMP, J33G13000210001

Biobanche

(3)

Next-generation molecular technologies

(4)

4 hours - 1 hour Hands On

Sanger sequencing

Sequence reaction

PCR Primer design

350 bp per PCR (1 gene fragment) Product

Clean up

Scarpa

single gene fragment for one patient

4

(5)

Next generation sequencing

6 hours - 1 hour Hands On

Up to 1Mb per multiplex PCR (~500 genes)

Adaptors ligation Multiplex

PCR

Adapter clean-up

Primers Removal

Scarpa

Primer design

multiple genes amplified simultaneously

5

(6)

Starting DNA Clonal amplification of thousands copies Emulsion PCR amplification

- one DNA molecule per bead

Starting DNA Clonal amplification

of thousands copies Solid phase bridge PCR

- one DNA molecule per cluster

6 Next generation sequencing

(7)

0 10 20 30 40 50 60 70 80 90 100

CTNNB1 FGFR3 APC PIK3CA

V ar iant fre qu enc y

SPT27 SPT30

Allelic silent SNP Allelic silent

SNP Mutation

◼ Each cell has two alleles

◼ ß catenin mutation is heterozygous (one allele)

◼ We should find 40% of alleles mutated

Mutation

Scarpa

◼ 80% neoplastic cells

◼ ß catenin mutation

Solid pseudopapillary tumour

NGS data are quantitative

(8)

CTNNB1

CTNNB1 + PIK3CA Normal

20%

40%

Scarpa

NGS data are quantitative and reveal

clonal composition

(9)

Adaptors ligation Multiplex

PCR Primer design

Up to 1Mb per multiplex PCR

(~500 genes) Primers

Removal

Barcode = identifies the patient DNA

9 NGS permits the simultaneous analysis of multiple

genes

(10)

Colorectal cancer

190 PCR in one tube 46 genes, 930 mutations

20µl

Scarpa

KRAS BRAF EGFR TP53 PIK3CA CSF1R JAK2

NRAS PTPN11 ERBB2 SRC FGFR3 NPM1 CDKN2A

RET HNF1A SMAD4 GNAS PDGFRA MPL ABL1

PTEN FLT3 STK11 SMARCB1 KIT MET NOTCH1

FGFR2 RB1 JAK3 VHL KDR SMO

HRAS AKT1 ALK MLH1 FBXW7 ERBB4

ATM CDH1 IDH1 CTNNB1 APC FGFR1

10 NGS permits the simultaneous analysis of multiple

genes

(11)

Adaptors ligation Multiplex

PCR Primers Removal

Different Barcodes = multiple patients

Scarpa

Primer design

FFPE BLOOD

Genomic DNA

A B C D

A BC

D

11 NGS permits the simultaneous analysis of multiple

patients

(12)

Sample CRC1 CRC2 CRC3 CRC4 CRC5 CRC6 CRC7 CRC8 CRC9 Neoplastic

cells 90% 80% 85% 60% 40% 85% 55% 70% 50%

Subtype MSS MSS MSS MSS MSS MSS MSS MSS MSS

Variant type (frequency)

KRAS ^61Q/H

(23%)

12G/D (23%)

TP53 ^271E/K_(20%) ^245G/R_(73%) ^R209del_(46%) ^278P/S_(35%) ^175R/H_(22%)

KDR ^482C/R

(50%)

SMAD4 ^361R/H

(24%)

ATM ^410V/A

(50%)

604P/S (64%)

ERBB2 ^777V/L

(43%)

776G/V (39%)

CTNNB1 ^45S/F

(43%)

FBXW7 ^452E/Q

(56%)

FGFR3 ^384F/L_(61%)

MET 375N/S

(59%)

375N/S (45%)

CDH1 ^72D/N

(13%)

Scarpa

NGS permits the simultaneous analysis of multiple

genes in multiple patients

(13)

Emerging Molecular Taxonomy

Prevalence of Phenotypes

Molecular Heterogeneity

of cancers

(14)

Emerging Molecular Taxonomy

Prevalence of Phenotypes

Cowley MJ et al. J Hepatobiliary Pancreat Sci 2013 [Epub ahead of print] Copyright (2013) by permission of Wiley

Molecular Heterogeneity

of cancers

(15)

Reprinted by permission from Macmillan Publishers Ltd: Pao W & Hutchinson KE. Nat Med 2012;18(3):349-351 © (2012)

Same morphology, different cancers

Lung adenocarcinoma

15

(16)

Cancer Genome Atlas Research Network. Nature 2014;511(7511):543-550 © (2014) Creative Commons license

mutually exclusive molecular types

Lung adenocarcinoma

16

(17)

SMAD4 TGFBR2 ACVR2A TGFBR1

TGFb pathway: (40%)

KRAS

MAPK pathway: (95%)

Germline mutation ATM

BRCA2 BRCA1 PALB2 REV1L ZZZZ AAAA

Missense

Nonsense, Indel Homozygous deletion

DNA damage repair: (20%)

Scarpa, unpublished

Significantly mutated pathways in pancreas adenocarcinomas

17

(18)

RTK=90%

MAPK=60%

PI3K-mTOR=37%

DNA

repair=63%

Reprinted by permission from Macmillan Publishers Ltd: Ding L et al. Nature 2008;455(7216):1069-1075 © (2008)

Significantly mutated pathways in

lung adenocarcinoma

(19)

Cancer Genome Atlas Research Network. Nature 2013;489(7417):519-525 © (2013) Creative Commons license

RTK=53%

PI3K-mTOR=67%

Significantly mutated pathways in

lung squamous cell carcinomas

(20)

Colorectal cancer

The Cancer Genome Atlas Network. Nature 2012;487(7407):330-337 © (2012) Creative Commons license

20

(21)

Aldo Scarpa

ARC-

NET

Centre for Applied Research on Cancer and Department of Pathology

University of Verona, Italy

La prospettive del molecular profiling

nella prevenzione diagnosi e cura dei tumori

(22)

(23)

17 candidate genes with bi-allelic inactivation

protein truncating variants (PTV) plus LOH of normal allele

(24)

FAN1 NEK1

RHNO1

(25)

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

(26)

Review Article 2014

Pancreatic Adenocarcinoma

N Engl J Med 371(11):1039-1049 September 11, 2014

(27)

www.arc-net.it

Sequenziamento del genoma di tumori del pancreas

Il Centro di Ricerca ARC-NET è

capofila dell’iniziativa Italiana

(28)

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

(29)

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

Waddell N, et al – Nature 2015

14%

(30)

Ductal

adenocarcinoma

(31)

3/11 cases

Acinar cell

carcinoma

(32)

2/7; 28.6%

Intraductal papillary mucinous neoplasm

8/37; 21.6%

Ductal

adenocarcinoma

(33)

33 Scarpa et al. 2017, Nature

Neuroendocrine

tumours

(34)

The mutational landscape of pancreatic cancer

(35)

35 Which is important?

(36)

10 pathways

(37)

(38)

Multigene panels for pathway analysis

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 HR Repair

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

Molecular Diagnosis

1

(39)

DNA RNA

(40)

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

(41)

WP3- Progress and results

Morphological revision of 200 resected PDAC

(42)

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 of 200 patients

(43)

III II

IV III

II

IV

A

Identification of Cancer Subtypes

B C

Genetic profile Patient

Xenografts Patient

cancer

Genetic profile

BIOBANK OF 175 PATIENT XENOGRAFTS (PDX)

to associate Morphology and Genetics

(44)

Cancer diagnostics tasks

4 Follow up Diagnosis 1

Prognosis 2

3 Predict drug efficacy

(45)

Multigene panels for pathway analysis

Panel Name

GENES

GNAS SETD2 SLIT2

CDH1 SMARCA4 SRGAP3

U2AF1 U2AF2

PDAC Periampullary

TGFß Pathway

SWI/SNF Chromatin

WNT

Spliceosome

DNA HR Repair

Molecular Diagnosis

1

(46)

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 15 3 8

PALB2 8 2 1 5

REV3L 15 3 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

FAM175

A 1 -

1 163 34 34 95

3 Predict drug efficacy

34/200 (17%)

(47)

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

Waddell N, et al – Nature 2015

(48)

Multigene panels

for pathway based molecular diagnostics

Panel Name

GENES

GNAS SETD2 SLIT2

CDH1 SMARCA4 SRGAP3

U2AF1 U2AF2

PDAC Periampullary

TGFß Pathway

SWI/SNF Chromatin

WNT

Spliceosome

DNA Damage Repair

Molecular Diagnosis

1

(49)

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

(50)

Multigene panels

for pathway based molecular diagnostics

Panel Name

basic TGFB Pathway SWI/SNF Chromatin Remodelling

WNT non canonical - Spliceosome

DNA DAMAGE REPAIR

GENES

GNAS SETD2 SLIT2

CDH1 SMARCA4 SRGAP3

U2AF1 U2AF2

PDAC Periampullary

TGFß Pathway

SWI/SNF Chromatin

WNT

Spliceosome

DNA Damage Repair

Molecular Diagnosis

1

(51)

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%)

(52)

DNA RNA

(53)

Oncotarget. 2016 Jan 12;7:1076-83.

CE-IVD kit - CH

HR1 BRCA NGS kit

(54)

Amplifications

Single nucleotide variations

Translocations Deletions

Technology advancement

(55)

2GL

4GL

6GL

7GL

8GL

9GL

10GL

11GL

13GL

14GL

15GL

16GL 12G

L

TML: 216.61; MSH6: Glu1322Ter;

POLE: Arg742Cys

5G L

TML: 336.36; MSH2:

Val684Ter

3G L

TML: 888.35 1GL

TML: 616.59; MLH1: Ser193Leu

Next generation histopathological diagnosis

(56)

Next generation histopathological diagnosis

(57)

Next generation histopathological diagnosis glioblastomas

MSI-

N T

MSI+

N

T

(58)

MSI-

N T

MSI+

N

T

(59)

Next generation histopathological diagnosis glioblastomas

2 G L

4 G L 6 G L 7 G L 8 G L 9 G L

1 0 G L 1 1 G L 1 3 G L 1 4 G L1 5 G L1 6 G L 1

2 G L

TML: 216.61;

MSH6: Glu1322Ter;

POLE: Arg742Cys

5 G L

TML: 336.36;

MSH2:

Val684Ter 3 G L

TML: 888.35 1

G L

TML: 616.59;

MLH1: Ser193Leu

(60)

Next generation histopathological diagnosis

Campbell et al. Cell 2017

(61)

200 µm

A

B

C

POLE inactivation

ultramutator phenotype

(62)

Assay design: defining content

Commercial and LDT cancer panels show poor concordance in gene selection, likely due to lack of objective methodologies

Panel No. of genes

Ion AmpliSeq comprehensive 409

FoundationOne CDx 309

Caris Molecular Intellegence 593

Illumina TSO500 523

Tempus xT 594

Dana Farber OncoPanel V3 447

MSK-IMPACT 468 468

MD Anderson v1 409

n=1,084 genes

159 45 57

60

74 223 122 344

Panel genes overlap

All 8 panels 7 of 8

6 of 8 5 of 8 4 of 8 3 of 8 2 of 8 1 of 8

UNPUBLISHED DATA PLEASE DO NOT

DISTRIBUTE

(63)

The majority of clinically-informative data resides in structural variants (including CNAs)

The interpretable genomic space: variant type

Unpublished analysis of whole genome sequencing data with fusion gene prevalence form published literature 0%

20%

40%

60%

80%

100%

V aria n t class %

Mutation Structural variant Signature Fusion

UNPUBLISHED DATA PLEASE DO NOT

DISTRIBUTE

(64)

UNPUBLISHED DATA PLEASE DO NOT

DISTRIBUTE

(65)

The Clinical Cancer Genome assay reports on the vast majority of clinically-relevant genomic events

The Clinical Cancer Genome: reportable range

0%

20%

40%

60%

80%

100%

Variant %

Covered by the clinical cancer genome assay Under development (HRD signature)

Not amenable to DNA capture

UNPUBLISHED DATA

PLEASE DO NOT DISTRIBUTE

Unpublished analysis of whole genome sequencing data with fusion gene prevalence form published literature

UNPUBLISHED DATA PLEASE DO NOT

DISTRIBUTE

(66)

Anatomy

Physiology

Social Behavior

(67)

Cancer is a tissue

Pancreas Cancer

(68)

Stroma is composite and variable

Pancreas Cancer and its environment

(69)

Cancer cells

PDAC Subtypes – Microenvironment

(70)

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

(71)

PDAC Subtypes – Expression Profiles

Quasi Mesenchimal Classical Exocrine-like

Collisson et. al., Nat Med 2011 Daemen, et al., PNAS 2015 Mofitt, et. al., Nat. Gen. 2015 Noll, et al., Nat. Med. 2016 Bailey et. al., Nature, 2016

Worst Bad

Mesenchymal/stem cell/stromal

Epithelial and ribosomal

Overall survival

Gene signatures

Characteristics

Bad

Acinar-specific genes

Response No response

Gemcitabine

^{Not known} Therapy

Prognosis

Squamous PP _Immuno ADEX

Bailey Collison

(72)

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

(73)

(74)

Only 15% of patients is a surgery candidate

Pancreas Cancer

Diagnosis

1 Prognosis

2 Therapy

3 Surgical specimen

Cytology or Biopsy

(75)

matched

PDX Patient

Macrophage.Enrich Immune.Hot2.TAL1 Immune.Hot1.PDCD1 Immune.Dormant First Subtypes

global

- 3 0 3

Sample_C44_0003Sample_C44_0004Sample_C44_0011Sample_C44_0012Sample_C44_0014Sample_C44_0017Sample_B55_0011Sample_B55_0012X1102T_ATTACTCG.TATAGCCT_L001X1152T_ATTACTCG.CCTATCCT_L001X1335T_TCCGGAGA.TATAGCCT_L001X1771T_ATTACTCG.TAATCTTA_L002X1957T_GAGATTCC.TATAGCCT_L001X2069T_GAGATTCC.ATAGAGGC_L001X2145T_GAGATTCC.CCTATCCT_L001X2192T_ATTCAGAA.CCTATCCT_L001X2230T_GAATTCGT.TATAGCCT_L001X2342T_GAGATTCC.CAGGACGT_L002X2491T_ATTCAGAA.AGGCGAAG_L002X2632T_GAATTCGT.AGGCGAAG_L002X2651T_CTGAAGCT.ATAGAGGC_L003X2666T_CTGAAGCT.GGCTCTGA_L003X2967T_TAATGCGC.ATAGAGGC_L003X3188T_TAATGCGC.GGCTCTGA_L003Sample_C44_0001Sample_C44_0023Sample_C44_0024Sample_C44_0026Sample_C44_0028Sample_B55_0013X1349T_ATTACTCG.GGCTCTGA_L001X1392T_TCCGGAGA.CCTATCCT_L001X1464T_CGCTCATT.TATAGCCT_L001X1786T_TCCGGAGA.AGGCGAAG_L002X1846T_CGCTCATT.AGGCGAAG_L002X1060T_TAATGCGC.TAATCTTA_L004X2191T_ATTCAGAA.ATAGAGGC_L001X2322T_GAGATTCC.AGGCGAAG_L002X2496T_ATTCAGAA.TAATCTTA_L002X2515T_ATTCAGAA.CAGGACGT_L002X3283T_CTGAAGCT.AGGCGAAG_L004X3332T_CTGAAGCT.CAGGACGT_L004Sample_C44_0002Sample_C44_0006Sample_C44_0007Sample_C44_0008Sample_C44_0015Sample_C44_0018Sample_C44_0019Sample_C44_0021Sample_C44_0027Sample_B55_0014X1116T_ATTACTCG.ATAGAGGC_L001X1777T_ATTACTCG.CAGGACGT_L002X1778T_ATTACTCG.GTACTGAC_L002X1790T_TCCGGAGA.CAGGACGT_L002X1841T_TCCGGAGA.GTACTGAC_L002X2187T_ATTCAGAA.TATAGCCT_L001X2200T_ATTCAGAA.GGCTCTGA_L001X2323T_GAGATTCC.TAATCTTA_L002X2636T_GAATTCGT.CAGGACGT_L002X2661T_CTGAAGCT.CCTATCCT_L003X2816T_TAATGCGC.TATAGCCT_L003X3170T_TAATGCGC.CCTATCCT_L003X963T_CTGAAGCT.GTACTGAC_L004X980T_TAATGCGC.AGGCGAAG_L004Sample_C44_0005Sample_C44_0009Sample_C44_0010Sample_C44_0013Sample_C44_0016Sample_C44_0020Sample_C44_0022Sample_C44_0025Sample_C44_0029X1378T_TCCGGAGA.ATAGAGGC_L001X1462T_TCCGGAGA.GGCTCTGA_L001X1628T_CGCTCATT.CCTATCCT_L001X1762T_ATTACTCG.AGGCGAAG_L002X1855T_CGCTCATT.CAGGACGT_L002X2150T_GAGATTCC.GGCTCTGA_L001X2243T_GAATTCGT.CCTATCCT_L001X2453T_GAGATTCC.GTACTGAC_L002X2648T_CTGAAGCT.TATAGCCT_L003X3189T_CGGCTATG.TATAGCCT_L003

First Subtypes id

CCL13 COLEC12 CLEC5A CASP10 CXCL13 CSF3R SELE CCL19 CD1E FCER1A CCR5 CD2 CD3G ITK ICOS LY9 SLAMF1 GZMK KLRG1 HLA-DQA1 CD37 SPN TNFSF18 TAL1 LRRN3

Immune Microenviroment Specific Subtypes in 85 Patients

M a c r o p h a g e - E n r i c h I m m u n e - H o t 2 - T A L 1 I m m u n e - H o t 1 - P D C D 1 I m m u n e - D o r m a n t

Names

g l o b a l

- 1 0 1

Im mu ne -D orm an t Ma cro ph ag e-E nri ch Im mu ne -H ot1 -P DC D1 Im mu ne -H ot2 -T AL 1

Names i d

CLEC5A SELE

CXCL13 ICOS

C D 3 7

H L A - D Q A 1 FCER1A

SPN

CASP10 TAL1 CD1E CMA1 TNFSF18

RNAseq NanoString

A B C D

Immunassigner subtypes

(76)

Subtype genes 228 62

TUMOR

MICRODISSECTION DNA NNANOSTRING

QUALIFICATION

Transcriptome subtypes

FNAB and nanostring assay

(77)

1 marker 3 markers

Unpublished data

Pancreas cancer associated fibroblasts (CAF)

(78)

Gene expression profiling of lung atypical carcinoids and large cell neuroendocrine

carcinomas identifies three transcriptomic subtypes with specific genomic alterations

(79)

Gene expression profiling of lung atypical carcinoids and large cell neuroendocrine

carcinomas identifies three transcriptomic subtypes with specific genomic alterations

(80)

E.U. FP7 grant no 602783

5X1000 grant n. 12182

Ministry of Health FIMP, J33G13000210001

Ministry of University and Research (FIRB RBAP10AHJB);