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Mutational Mosaics of Cell-Free DNA from Pancreatic Cyst Fluids

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Abstract

Background

Pancreatic cyst fluids (PCFs) enriched in tumor-derived DNA are a potential source of new biomarkers. The study aimed to analyze germinal variants and mutational profiles of cell-free (cf)DNA shed into the cavity of pancreatic cysts.

Methods

The study cohort consisted of 71 patients who underwent endoscopic ultrasound fine-needle aspiration of PCF. Five malignant cysts, 19 intraductal papillary mucinous neoplasms (IPMNs), 11 mucinous cystic neoplasms (MCNs), eight serous cystic neoplasms (SCNs), and 28 pseudocysts were identified. The sequencing of 409 genes included in Comprehensive Cancer Panel was performed using Ion Proton System. The mutation rate of the KRAS and GNAS canonical loci was additionally determined using digital PCR.

Results

The number of mutations detected with NGS varied from 0 to 22 per gene, and genes with the most mutations were: TP53, KRAS, PIK3CA, GNAS, ADGRA2, and APC. The frequencies of the majority of mutations did not differ between non-malignant cystic neoplasms and pseudocysts. NGS detected KRAS mutations in malignant cysts (60%), IPMNs (32%), MCNs (64%), SCNs (13%), and pseudocysts (14%), with GNAS mutations in 20%, 26%, 27%, 13%, and 21% of samples, respectively. Digital PCR-based testing increased KRAS (68%) and GNAS (52%) mutations detection level in IPMNs, but not other cyst types.

Conclusions

We demonstrate relatively high rates of somatic mutations of cancer-related genes, including KRAS and GNAS, in cfDNA isolated from PCFs irrespectively of the pancreatic cyst type. Further studies on molecular mechanisms of pancreatic cysts malignant transformation in relation to their mutational profiles are required.

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Abbreviations

NGS:

Next-generation sequencing

EUS-FNA:

Endoscopic ultrasound fine-needle aspiration

PCF:

Pancreatic cyst fluid

PCL:

Pancreatic cystic lesion

IPMN:

Intraductal papillary mucinous neoplasm

MCN:

Mucinous cystic neoplasm

SCN:

Serous cystic neoplasm

dPCR:

Digital PCR

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Funding

This work was supported by the National Science Centre [2012/05/B/NZ5/01539].

Author information

Authors and Affiliations

  1. Department of Gastroenterology, Hepatology and Clinical Oncology, Medical Center for Postgraduate Education, Roentgena 5, 02-781, Warsaw, Poland

    Agnieszka Paziewska, Marcin Polkowski & Jerzy Ostrowski

  2. Department of Genetics, Maria Sklodowska-Curie Institute-Cancer Center, Roentgena 5, 02-781, Warsaw, Poland

    Agnieszka Paziewska, Krzysztof Goryca, Jakub Karczmarski, Michalina Dabrowska, Michal Mikula & Jerzy Ostrowski

  3. Next Generation Sequencing Core Facility, Centre of New Technologies, University of Warsaw, 02-097, Warsaw, Poland

    Krzysztof Goryca

  4. Department of Endoscopy, Ministry of Internal Affairs Hospital, 70-362, Szczecin, Poland

    Anna Wiechowska-Kozlowska

Authors
  1. Agnieszka Paziewska
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Contributions

Conception and design of the study were done by JO, AP, MP, and MM. MP and AWK contributed to patients recruitment and clinical data compilation. AP, JK, and MD contributed to DNA isolation, Comprehensive Cancer Panel sequencing, and digital PCR; sequencing data analyses were done by KG; drafting of the manuscript was done by JO, MP, KG, and MM.

Corresponding author

Correspondence to Jerzy Ostrowski.

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Conflict of interest

The authors declare that they have no competing interests.

Ethical approval

The study protocol was approved by the Ethical Review Board at the Maria Sklodowska-Curie Institute-Cancer Center, Warsaw, Poland. The study was conducted according to the principles expressed in the Declaration of Helsinki and informed written consent was obtained from the participants.

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10620_2019_6043_MOESM1_ESM.xlsx

Non-silent exonic variants detected in assessed samples. CHROM, POSITION—genome coordinates of variant; REF/ALT—reference/alternative variant sequence; foundVariants—number of samples with alternative variant detected, p value Fisher—p value for comparison of alternative variant occurrence between IPMN + MNC + SCN and PC group (Fisher exact test); gene_variants—number of samples with alternative, non-silent variant detected at any site across the whole gene; p value Gene—p value for comparison of alternative variant occurrence across the whole gene IPMN + MNC + SCN and PC group (Fisher exact test); esp6500siv2_all—variant frequency according to National Heart, Lung, and Blood Institute GO Exome Sequencing Project; 1000g2015aug_eur—variant frequency in the 1000 Genomes Project database (European); SIFT/Polyphen2—prediction of variant impact on protein structure: B-benign, T-tolerated, D-deleterious; columns AA-CQ—fraction of reads supporting alternative variants for respective samples (XLSX 97 kb)

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Paziewska, A., Polkowski, M., Goryca, K. et al. Mutational Mosaics of Cell-Free DNA from Pancreatic Cyst Fluids. Dig Dis Sci 65, 2294–2301 (2020). https://doi.org/10.1007/s10620-019-06043-1

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  • DOI: https://doi.org/10.1007/s10620-019-06043-1

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