cancers Systematic Review Circulating Tumor DNA Detection by Digital-Droplet PCR in Pancreatic Ductal Adenocarcinoma: A Systematic Review Marisol Huerta 1 , Susana Roselló 1,2 , Luis Sabater 3, Ana Ferrer 1,2, Noelia Tarazona 1,2, Desamparados Roda 1,2, Valentina Gambardella 1,2, Clara Alfaro-Cervelló 4 , Marina Garcés-Albir 3, Andrés Cervantes 1,2 and Maider Ibarrola-Villava 1,2,* 1 Department of Medical Oncology, INCLIVA Biomedical Research Institute, University of Valencia, 46010 Valencia, Spain; [email protected] (M.H.); [email protected] (S.R.); [email protected] (A.F.); [email protected] (N.T.); [email protected] (D.R.); [email protected] (V.G.); [email protected] (A.C.) 2 CIBERONC, Medical Oncology Unit, INCLIVA Biomedical Research Institute, University of Valencia, 46010 Valencia, Spain 3 Liver, Biliary and Pancreatic Unit, Department of Surgery, Hospital Clínico Universitario of Valencia, University of Valencia, INCLIVA Biomedical Research Institute, 46010 Valencia, Spain; [email protected] (L.S.); [email protected] (M.G.-A.) 4 Department of Pathology, INCLIVA Biomedical Research Institute, University of Valencia, 46010 Valencia, Spain; [email protected] * Correspondence: [email protected]; Tel.: +34-963-862-894 Simple Summary: Pancreatic cancer is a digestive tumor that is most difficult to treat and carries Citation: Huerta, M.; Roselló, S.; one of the worst prognoses. The anatomical location of the pancreas makes it very difficult to Sabater, L.; Ferrer, A.; Tarazona, N.; obtain enough tumor material to establish a molecular diagnosis, so knowing the biology of this Roda, D.; Gambardella, V.; tumor and implementing new targeted-therapies is still a pending issue. The use of liquid biopsy, a Alfaro-Cervelló, C.; Garcés-Albir, M.; blood sample test to detect circulating-tumor DNA fragments (ctDNA), is key to overcoming this Cervantes, A.; et al. Circulating difficulty and improving the evolution of this tumor. Liquid biopsies are equally representative of Tumor DNA Detection by the tissue from which they come and allow relevant molecular and diagnostic information to be Digital-Droplet PCR in Pancreatic obtained in a faster and less invasive way. One challenge related to ctDNA is the lack of consistency Ductal Adenocarcinoma: A in the study design. Moreover, ctDNA accounts for only a small percentage of the total cell-free Cancers 2021 13 Systematic Review. , , circulating DNA and prior knowledge about particular mutations is usually required. Thus, our 994. https://doi.org/10.3390/ aim was to understand the current role and future perspectives of ctDNA in pancreatic cancer using cancers13050994 digital-droplet PCR technology. Academic Editor: Giulia Siravegna Abstract: Pancreatic cancer (PC) is one of the most devastating malignant tumors, being the seventh Received: 14 January 2021 leading cause of cancer-related death worldwide. Researchers and clinicians are endeavoring to Accepted: 23 February 2021 develop strategies for the early detection of the disease and the improvement of treatment results. Published: 27 February 2021 Adequate biopsy is still challenging because of the pancreas’s poor anatomic location. Recently, circulating tumor DNA (ctDNA) could be identified as a liquid biopsy tool with huge potential as a Publisher’s Note: MDPI stays neutral non-invasive biomarker in early diagnosis, prognosis and management of PC. ctDNA is released from with regard to jurisdictional claims in apoptotic and necrotic cancer cells, as well as from living tumor cells and even circulating tumor cells, published maps and institutional affil- and it can reveal genetic and epigenetic alterations with tumor-specific and individual mutation and iations. methylation profiles. However, ctDNA sensibility remains a limitation and the accuracy of ctDNA as a biomarker for PC is relatively low and cannot be currently used as a screening or diagnostic tool. Increasing evidence suggests that ctDNA is an interesting biomarker for predictive or prognosis studies, evaluating minimal residual disease, longitudinal follow-up and treatment management. Copyright: © 2021 by the authors. Promising results have been published and therefore the objective of our review is to understand the Licensee MDPI, Basel, Switzerland. current role and the future perspectives of ctDNA in PC. This article is an open access article distributed under the terms and Keywords: ctDNA; digital-droplet PCR (ddPCR); pancreatic cancer; pancreatic ductal adenocarci- conditions of the Creative Commons noma (PDAC) Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Cancers 2021, 13, 994. https://doi.org/10.3390/cancers13050994 https://www.mdpi.com/journal/cancers Cancers 2021, 13, x 2 of 15 1. Introduction There is a great need to unveil the genetic landscape of pancreatic ductal adenocar‐ cinoma (PDAC), one of the most aggressive human malignancies. This tumor type, alt‐ hough not very frequent, has an increasing incidence and is the seventh leading cause of cancer‐related death in both men and women worldwide [1]. Indeed, it is predicted to become the second by 2030 [2]. Globally, 495,773 new cases and 466,003 deaths of pancre‐ atic cancer were reported in 2020 (GLOBOCAN, 4 February 2021 https://gco.iarc.fr/). The overall 5‐year survival rate is 9%, and the median survival is still shorter than 6 months, the lowest among all cancer types [3]. This poor survival is mostly attributed to the ab‐ Cancers 2021, 13, 994sence of effective screening methods, late diagnosis due to non‐specific symptoms, lack of 2 of 14 sensitive or specific biomarkers for early diagnosis, propensity for early metastatic spread, and the limited therapeutic advancements over the last years [4,5]. Consequently, the 1. Introduction prognosis of pancreatic cancer remains unimproved. The onset of symptoms and PDAC diagnosis is usuallyThere late, at is aan great advanced need to stage, unveil where the genetic most patients landscape have of metastatic pancreatic dis ductal‐ adenocarci- ease. noma (PDAC), one of the most aggressive human malignancies. This tumor type, although Cell‐free circulatingnot very frequent, tumor DNA has an (ctDNA) increasing is a incidence promising and non is‐invasive the seventh blood leading‐based cause of cancer- biomarker in cancerrelated management, death in both and men an and alternative women worldwide to traditional [1]. Indeed, blood‐based it is predicted protein to become the biomarkers [6,7].second ctDNA by has 2030 shown [2]. Globally, benefit in 495,773 early detection, new cases prognosis and 466,003 estimation, deaths oftreat pancreatic‐ cancer ment selection,were tumor reported dynamics in 2020 monitoring, (GLOBOCAN, minimal https://gco.iarc.fr/ residual disease (accesseddetection dateand 4tu February‐ 2021)). mor recurrenceThe during overall follow 5-year up survival [8–16]. ctDNA rate is 9%, is composed and the median of short survival segments is still of shorternucleic than 6 months, the lowest among all cancer types [3]. This poor survival is mostly attributed to the absence acids and reflects the genetic and epigenetic makeup of the tumor from which it origi‐ of effective screening methods, late diagnosis due to non-specific symptoms, lack of sensi- nates, making it a desirable and highly specific biomarker. ctDNA provides a better rep‐ tive or specific biomarkers for early diagnosis, propensity for early metastatic spread, and resentation of the molecular composition of a malignant disease than a single section from the limited therapeutic advancements over the last years [4,5]. Consequently, the prognosis a surgical tumor specimen or a tissue biopsy and, therefore, its clinical application in of pancreatic cancer remains unimproved. The onset of symptoms and PDAC diagnosis is PDAC is extremely important and interesting due to the difficulty of obtaining tumor tis‐ usually late, at an advanced stage, where most patients have metastatic disease. sues (Figure 1). Cell-free circulating tumor DNA (ctDNA) is a promising non-invasive blood-based One challenge related to ctDNA biology is a lack of consistency in the study design. biomarker in cancer management, and an alternative to traditional blood-based protein Thus, in this article, we review current knowledge regarding ctDNA for pancreatic cancer biomarkers [6,7]. ctDNA has shown benefit in early detection, prognosis estimation, using digital‐droplet PCR (ddPCR). The publications cited were selected from the Pub‐ treatment selection, tumor dynamics monitoring, minimal residual disease detection and Med database (http://www.ncbi.nlm.nih.gov/pubmed).tumor recurrence during follow up [8–16 Key]. search ctDNA terms, is composed or aliases, of used short segments of were ctDNA, cfDNA,nucleic liquid acids andbiopsy, reflects pancreatic the genetic cancer, and digital epigenetic PCR and makeup ddPCR. of the The tumor eligi‐ from which it bility criteria fororiginates, articles reviewed making it here a desirable included and original highly studies specific published biomarker. beyond ctDNA 2015 provides a better in patients withrepresentation a clinical and of histological the molecular diagnosis composition of pancreatic of a malignant adenocarcinoma disease than that a single section was molecularlyfrom characterized a surgical tumor in plasma specimen and serum or a tissue liquid biopsy biopsies and, by therefore, digital PCR its tech clinical‐ application nology. Studiesin published PDAC is extremely in a language important other andthan interesting