Giacomo Puppa

Service de Pathologie CliniqueHôpitaux Universitaires de Genève

Molecular analysis value of pancreatic cyst fluid

to preoperative diagnosis of

pancreatic mucinous neoplasms

Introduction

• Prevalence of pancreatic cysts: 1.2% to 2.6% onabdominal CT and 15% of patients undergoingabdominal MRI.

• Mucinous cysts IPMNs MCNs, have the potential toprogress to invasive PDAC.

• Despite a multimodal approach to the assessment ofpancreatic cysts (clinical and radiological, EUS-FNA,cytopathological interpretation and cyst fluid analysis(CEA), the distinction of mucinous cysts from otherpancreatic cysts and those that will progress to PDACcan be challenging.

Singhi 2017

The terms Triage comes from the French verb trier,meaning to separate

volume

<0.5 cc

> 0.5 cc

CEA

Man, 63 yrs. fortuitous detectionof 23 mm cyst pancreatic head

CEA 8.3 µg/l

Case n° 1

IPMN* LGDNo study has shown a GNAS mutation in any patients with MCN*

Female, 68 yrs. 8 cm suspect body-tailsolido-cystic mass

CEA 13,419 µg/l

Case n° 2

Wild Type

GGT GAT c.35G>A

Gly Asp p.G12D

ADN:

Amino acid:

NGS

NGS (ION TORRENT)

Total reads: 1733 Reads G: 1692 Reads A: 41 = 2,4% de mutation

DNA control

Gly12 Arg

Gly12 Val

Gly12 Asp

Mutation

Gène de contrôle qualité ADN

Flu

ore

scen

ce

Cycles PCR

ER

VIMENTINPAN-CK

Female, 79 yrs. 8 cm suspect body pancreas pseudo-cyst

CEA not requested (pseudo-cyst)

Case n° 3

gastriccontamination

?????

IPMN????

KRAS exon2

GNAS exon8

IPMN LGD

Sensib. Specif.

Kadayifici 2016 56.3% 93%

Kadayifici 2017 46.3% 91%

Kindly provided by Marta Pitman.

Sen. spec. for mucinous nature

Sensib. Specif.

Pitman 2017 43.4 % 96.2%

Cytology alone

Sens. spec. for IPMNKRAS sens.

KRAS specif.

GNAS sens.

GNAS spec.

Khalid 2009 45% 96%

Nikiforova 2013 54% 100%

Singhi 2014 70% 98% 36% 100%

Kadayifici 2016 48,8% 100%

Kadayifici 2017 98.8%

Variability among studies in sensib./specificity

• Number of pts. In some study small.

• Substantial discordance in the rates of detection of KRAS and GNAS mutations between preoperative EUS-FNA and studies using postoperative pancreatic cyst fluid.

• Sanger sequencing vs. NGS:

≠ limit of detection mutant alleles (15-20% vs. 3-5%)

≠ amount of DNA required for analysis.

Kadayifici 2017

The Performance of the three tests together may achieve a better result for IPMN diagnosis than single tests, but can still miss 15% of IPM

Kadayifici 2017

Singhi 2014

Kadayifici 2016

Bournet 2016

KRAS GNAS

Malignant transformation

Rationale for the introduction of the NGS

• PDAC is fundamentally a genetic disease that arises from non-invasive precursor lesions through the accumulation of genetic alterations.

KRAS GNAS CDKN2A CTNNB1 RNF43 PIK3CA PTEN TP53 SMAD4

IPMN

MCN

Singhi 2017

EARLY HIGH GRADE, INVASION

HUG: Cancer hot spot panel V2

including, ~2,800 COSMIC (207 amplicons) mutations of 50 oncogenes and tumor suppressor genes:

ABL1 EGFR GNAS KRAS PTPN11AKT1 ERBB2 GNAQ MET RB1ALK ERBB4 HNF1A MLH1 RETAPC EZH2 HRAS MPL SMAD4ATM FBXW7 IDH1 NOTCH1 SMARCB1BRAF FGFR1 JAK2 NPM1 SMOCDH1 FGFR2 JAK3 NRAS SRCCDKN2A FGFR3 IDH2 PDGFRA STK11CSF1R FLT3 KDR PIK3CA TP53CTNNB1 GNA11 KIT PTEN VHL

https://tools.thermofisher.com/content/sfs/brochures/Ion-AmpliSeq-Cancer-Hotspot-Panel-Flyer.pdf

• A KRAS or GNAS mutation supported an IPMN withnonmucinous CEA in 71%.

• A KRAS mutation reclassified 19% of nonneoplasticcysts with nonmucinous CEA as mucinous.

• Seven cyst fluids (8%) had either a TP53 mutationor loss of CDKN2A or SMAD4 in addition to KRASand/or GNAS mutations; 5 of 7 (71%) were clinicallymalignant, and high-grade cytology was detected inall 5.

• Overall, CEA was more specific for a mucinousetiology (100%), but NGS was more sensitive (86%vs 57%).

Jones 2016

AGA Algor.

Sensib. 62% 100%

Specif. 79% 90%

PPV 57% 79%

NPV 82% 100%

Sequencing

Cytology

CEA

• Triage based on volume

• When < 0.5 ml favors CEA or sequencing

• The combination of cytology, CEA, and NGS had 90% sensit. and 88% specif. for mucinouslesions.

• GNAS mutation distinguish IPMN from MN

• NGS: sensit. 45 % and 100 % specif. for carcinoma = not perfect for grading