Thermal liquid biopsy (TLB) focused on benign and premalignant pancreatic cyst diagnosis

Journal of Personalized Medicine Article Thermal Liquid Biopsy (TLB) Focused on Benign and Premalignant Pancreatic Cyst Diagnosis Sonia Hermoso-Durán1,2,† , Guillermo García-Rayado 1,3,4,†, Laura Ceballos-Laita 1,2 , Carlos Sostres 1,3,4, Sonia Vega 2, Judith Millastre 1,3, Oscar Sánchez-Gracia 5, Jorge L. Ojeda 6,Ángel Lanas 1,3,4,7 , Adrián Velázquez-Campoy 1,2,4,8,9,* and Olga Abian 1,2,4,8,10,*   Citation: Hermoso-Durán, S.; García-Rayado, G.; Ceballos-Laita, L.; Sostres, C.; Vega, S.; Millastre, J.; Sánchez-Gracia, O.; Ojeda, J.L.; Lanas, Á.; Velázquez-Campoy, A.; et al. Thermal Liquid Biopsy (TLB) Focused on Benign and Premalignant Pancreatic Cyst Diagnosis. J. Pers. Med. 2021,11, 25. https://doi.org/ 10.3390/jpm11010025 Received: 9 December 2020 Accepted: 29 December 2020 Published: 31 December 2020 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1Instituto de Investigación Sanitaria Aragón (IIS Aragón), 50009 Zaragoza, Spain; [email protected] (S.H.-D.); [email protected] (G.G.-R.); [email protected] (L.C.-L.); [email protected] (C.S.); millastr[email protected] (J.M.); [email protected] (Á.L.) 2Joint Units IQFR-CSIC-BIFI, and GBsC-CSIC-BIFI, Institute of Biocomputation and Physics of Complex Systems (BIFI), Universidad de Zaragoza, 50018 Zaragoza, Spain; [email protected] 3Servicio de Digestivo, Hospital Clínico Universitario Lozano Blesa (HCULB), 50009 Zaragoza, Spain 4Centro de Investigación Biomédica en Red en el Área Temática de Enfermedades Hepáticas y Digestivas (CIBERehd), 28029 Madrid, Spain 5SOTER BioAnalytics, Enrique Val, 50011 Zaragoza, Spain; oscar[email protected] 6Department of Statistical Methods, Universidad de Zaragoza, 50009 Zaragoza, Spain; [email protected] 7Department of Medicine, University of Zaragoza, 50009 Zaragoza, Spain 8Fundación ARAID, Gobierno de Aragón, 50009 Zaragoza, Spain 9Departamento de Bioquímica y Biología Molecular y Celular, Universidad de Zaragoza, 50009 Zaragoza, Spain 10 Instituto Aragonés de Ciencias de la Salud (IACS), 50009 Zaragoza, Spain *Correspondence: [email protected] (A.V.-C.); [email protected] (O.A.); Tel.: +34-976-762996 (A.V.-C.); +34-876-555417 (O.A.) † S.H.-D. and G.G.-R. contributed equally to this work and are both first authors of the manuscript. Abstract: Background: Current efforts in the identification of new biomarkers are directed towards an accurate differentiation between benign and premalignant cysts. Thermal Liquid Biopsy (TLB) has been previously applied to inflammatory and tumor diseases and could offer an interesting point of view in this type of pathology. Methods: In this work, twenty patients (12 males and 8 females, average ages 62) diagnosed with a pancreatic cyst benign (10) and premalignant (10) cyst lesions were recruited, and biological samples were obtained during the endoscopic ultrasonography procedure. Results: Proteomic content of cyst liquid samples was studied and several common proteins in the different groups were identified. TLB cyst liquid profiles reflected protein content. Also, TLB serum score was able to discriminate between healthy and cysts patients (71% sensitivity and 98% specificity) and between benign and premalignant cysts (75% sensitivity and 67% specificity). Conclusions: TLB analysis of plasmatic serum sample, a quick, simple and non-invasive technique that can be easily implemented, reports valuable information on the observed pancreatic lesion. These preliminary results set the basis for a larger study to refine TLB serum score and move closer to the clinical application of TLB providing useful information to the gastroenterologist during patient diagnosis. Keywords: pancreatic cysts; thermal liquid biopsy; differential scanning calorimetry; diagnosis; generalized linear models 1. Introduction During recent years, the detection of pancreatic cysts has become more frequent due to improvements in abdominal imaging techniques. The incidence of this pathology is approximately 2% in the adult population [ 1 ]. Computed tomography (CT) scans are reported detection between 1.2% and 2.6%, and magnetic resonance imaging (MRI) has even a higher detection capability, ranging between 13.5% and 19.9% [ 1 , 2 ]. The management of J. Pers. Med. 2021,11, 25. https://doi.org/10.3390/jpm11010025 https://www.mdpi.com/journal/jpm J. Pers. Med. 2021,11, 25 2 of 19 these incidentally detected pancreatic cysts is still a challenge, because, even though the risk of being malignant is low, the prognosis in case of pancreatic adenocarcinoma and intraductal papillary mucinous neoplasms- (IPMN)-related pancreatic adenocarcinoma is very poor and has not improved recently [ 3 ]. Thus, distinguishing between benign and malignant cysts is difficult, and very often requires surgical intervention with considerable morbidity and mortality. Since 2005, some guidelines have been published and updated: the American Society for Gastrointestinal Endoscopy [ 4 ], international consensus guidelines by the International Association of Pancreatology (Sendai guidelines) [ 5 ], the American College of Gastroenterology [ 6 ], and the International Association of Pancreatology (Fukuoka guidelines) [ 7 , 8 ]. More recently, The European Study Group on Cystic Tumors of the Pancreas published an update, replacing the 2013 European Consensus Statement Guidelines [9]. From a clinical point of view, the classification according to the prognosis of the lesion is: (1) cysts with malignant potential (mucinous), (2) cysts without malignant potential, and (3) malignancies. However, most studies provide a description of biomarkers for differentiating the mucinous and non-mucinous type of cysts. The non-mucinous group comprises serous cystadenomas (SCAs) (the most common type), pancreatic pseudocysts (PCs), and a variety of rare cysts (benign epithelial, lymphoepithelial, congenital, and squamoid cysts). Most are found incidentally and none of them represents a risk for becoming malignant [ 10 ]. On the contrary, the mucinous group, including mucinous cystic neoplasms (MCNs) and IPMNs, constitutes the majority of neoplastic premalignant cysts identified in the pancreas, and a precise diagnosis technique would be vital for the management of patients [ 11 ]. The development of techniques with greater pre-surgical diagnostic precision would make it possible to avoid the morbidity and mortality associated with a high-risk intervention when it is not strictly necessary, as well as reduce the healthcare overload derived from unnecessary outpatient follow-up in cystic lesions without the potential for malignancy. Cystic fluid markers become especially relevant when transabdominal ultrasonography, CT or MRI are inconclusive. In these cases, it is necessary to employ another risk predictor to indicate surgery as the most appropriate treatment given the estimated risk. The presence of amylase at high concentration in cyst fluid indicates that there is a communication between the cyst and the ductal system. This occurs in both pseudocysts and IPMN lesions. When amylase levels are lower than 250 U/L, communication with the conduct can be discarded, with a specificity of 98% [ 12 ]. However, amylase value alone is not enough to differentiate between mucinous/non-mucinous or MCN/IPMN, which is important from the point of view of patient management, in deciding whether surgical resection or cyst time-monitoring is recommended [ 13 ]. A previous episode of pancreatitis can be of help in distinguishing a pseudocyst from an IPMN lesion (occasionally related to pancreatitis) [14]. Carcinoembryonic antigen (CEA) is also employed as a biomarker. This is a set of highly related glycoproteins involved in cell adhesion, mucin being one of them. It can be used to distinguish between cysts with (MCNs and IPMNs) and without (SCAs and PCs) mucinous epithelium [ 10 ]. The major inconvenience of CEA is the absence of an appropriate cutoff value [ 12 ]. The current accepted value to classify a cyst as mucinous is CEA > 192 ng/mL [15]. In order to improve diagnosis, studies related to the identification of new biomarkers in cystic fluid or serum have been recently reported [16–21]. In 2007 Chaires et al. [ 22 ] described the application of differential scanning calorimetry (DSC) in diagnosis using plasma/serum samples from cancer patients. Since then, many studies have confirmed the potential clinical use of this technique, not only applied to plasma/serum [ 23 – 30 ], but also to other biological human samples such as cerebrospinal fluid [ 31 , 32 ]. In 2018 our group coined the name “thermal liquid biopsy” (TLB) for DSC applied to cancer diagnosis and cancer patient’s treatment monitoring [ 33 , 34 ]. The TLB thermogram reports the global denaturation profile for all the proteins present in the serum/plasma sample and the influence of potential interactions between blood plasma J. Pers. Med. 2021,11, 25 3 of 19 proteins and metabolites, therefore reflecting any alteration induced by a certain disease. As in the case of plasma or serum, cystic fluid is also composed of a mixture of proteins and TLB may also be applied as a clinical diagnosis tool. In this work, the potential of TLB as a clinical biomarker for cyst classification has been pursued. In this pilot study, 20 cyst fluid samples were analyzed and their TLB cyst profiles were obtained, with the purpose of finding a correlation between the TLB thermogram and the type of cyst. The proteomic analysis also allowed a description of the more abundant proteins in the cyst fluid, as well as the post-translational modifications present in those proteins. TLB was also applied to serum samples in some of the patients, and TLB serum profile differences between groups was studied to try to determine whether or not differences observed in cyst fluid TLB correlated with serum TLB. This would be extremely important because, in case cyst features are reflected in certain serum alterations, a simple and risk-free plasma/serum TLB analysis could be employed for cyst diagnosis/classification. Despite the low number of samples considered in this pilot study, different patterns in cyst fluid and plasma from patients with pathology could be observed. 2. Materials and Methods 2.1. Subjects and Samples Cyst liquid and serum samples from patients with cystic lesions in the pancreas detected by transabdominal ultrasonography or CT or MRI were referred to the Department of Digestive Endoscopy at the Hospital Clínico Universitario Lozano Blesa (HCULB), Zaragoza, Spain, between January 2016 and September 2018. The procedure was in accordance with the recommendations of the local ethics committee and all patients gave their informed consent. Pancreatic cystic fluids were collected by EUS-guided fine needle aspiration (FNA). The EUS-FNA procedure was performed with an Olympus ® 140 curvilinear echo-endoscope. Boston Scientific TM Expect ® 19 or 22-gauge needles were used depending on the cystic endosonographic features. We noted the characteristics of the aspirated cystic fluid: volume, color, and viscosity. The majority of the fluid was examined by the same cytopathologist for every patient and the rest (at least 1 mL Eppendorf for each patient) was collected for detection of biochemical markers, DSC measurements, and proteomic studies described in this manuscript. The collected cystic fluid samples were then stored at −80 ◦C until they were prepared for analysis. Serum samples from healthy subjects as control group (HC) consisted of 85 serum samples from Spanish Caucasian subjects, apparently cancer-free, from the FISABIO (Fundación para el Fomento de la Investigacion Sanitaria y Biomedica de la Comunitat Valenciana) biobank with a homogeneous distribution, including gender (53% men and 47% women), with an average age of 45.2 ±14.2. 2.2. Thermal Liquid Biopsy (TLB) Profile Determination DSC thermograms were measured using a high-sensitivity differential scanning VPDSC microcalorimeter (MicroCal, Malvern-Panalytical, Malvern, UK). Cystic liquid samples, serum samples, and reference solutions were properly degassed and carefully loaded into the cells to avoid bubble formation. The baseline of the instrument was routinely recorded before the experiments. Experiments were performed in cystic liquid samples (diluted 1:10 in phosphate buffered saline, PBS) and serum samples (diluted 1:25 in PBS) at a scanning rate of 1 ◦ C/min. Thermograms were baseline-corrected and analyzed using software developed in our laboratory implemented in Origin 7 (OriginLab, Northampton, MA, USA). 2.3. Data Analysis We have developed a phenomenological model in which the TLB serum thermogram is deconvoluted into several individual transitions, modeling each individual transition by the logistic peak or Hubbert function [ 30 , 33 ]. This model has been successfully applied in the analysis of serum samples from melanoma and gastric and lung cancer patients [ 30 , 34 , 35 ]. J. Pers. Med. 2021,11, 25 4 of 19 From this multiparametric analysis, a TLB serum score (between 0 and 1) can be calculated reporting the level of alterations in plasma (TLB serum score < 0.5, absence of alterations; TLB serum score > 0.5, presence of alterations). The Kolmogorov-Smirnov test was performed to assess the normal distribution of the variables. Medians between two independent groups were compared with the Wilcoxon test, in non-normal distributions. Averages between two independent groups were compared with the t-test, in normal distributions. 2.4. Protein Sample Preparation and Protein Identification and Quantification by Mass Spectrometry Protein concentration: Measured by Bradford protein assay (Bio-Rad, Madrid, Spain) using purified bovine serum albumin (BSA) (10 mg/mL, New England BioLabs, EVRY cedex, France) in PBS as standard. Absorbance at 595 nm of two dilutions from each serum sample was measured in triplicate in a Synergy HT multimode microplate reader (BioTek Instruments, Winooski, VT, USA). In solution digestion: Samples were evaporated and resuspended in 10 µ L of denaturing buffer (6 M urea, 100 mM Tris buffer pH 7.8). Next, cysteines were reduced with 1.5 µ L DTT (200 mM) for 30 min at 37 ◦ C and alkylated with 6 µ L of iodoacetamide (200 mM) for 30 min in the dark. Unreacted iodoacetamide was consumed adding 6 µ L of the reducing agent (200 mM DTT) for 30 min at room temperature. Samples were diluted with 50 mM ammonium bicarbonate to a urea final concentration lower than 1 M. Trypsin digestion (Gold Trypsin, Promega, Madison, WI, USA) was carried out overnight at 37 ◦ C at a 1:20 enzyme/protein ratio. Reaction was stopped adding concentrated formic acid (Merck KGaA, Darmstadt, Germany). Samples were evaporated, resuspended in 2% acetonitrile (ACN), 0.1% formic acid, and filtered through 0.45 µm filters. Protein identification by LC-ESI-MS/MS: Protein identification was performed on a nano-LC 2D system (LC 425, Eksigent Ekspert TM, Dublin, CA, USA) coupled to a hybrid triple quadrupole/linear ion trap mass spectrometer (4000 QTRAP, Sciex, Foster City, CA, USA). On-line pre-concentration and desalting of samples was performed using a C18 trap cartridge (Luna ® 0.3 mm id, 20 mm, 5 µ m particle size, Phenomenex, CA, USA) at 10 µ L/min for 5 min. Peptide separation was performed using a C18 column (Gemini ® 0.3 mm id, 150 mm, 3 µ m particle size, Phenomenex, CA, USA), at 5 µ L/min of flow rate. Column was maintained at 35 ◦ C. The elution gradient was from 5 to 35% ACN (0.1% formic acid) in 90 min. The mass spectrometer was interfaced with an ESI source (Turbo V ™ ) using a 25 µ m ID hybrid electrode and was operated in the positive ion mode. MS source parameters were as follows: capillary voltage 5000 V, de-clustering potential (DP) 85 V and curtain and ion source gas (Nitrogen) 15 psi. Analyses were performed using an information dependent acquisition (IDA) method with the following steps: single enhanced mass spectra (EMS, 400–1400 m/z) from which the 5 most intense peaks were subjected to an enhanced product ion [EPI (MS/MS)] scan. Protein identification was carried out using the Mascot search engine (Matrix Science; London, UK) and the non-redundant SwissProt database (553,655 sequences; 198,177,566 residues). Search parameters were monoisotopic mass accuracy, peptide mass tolerance ± 0.5 Da, fragment mass tolerance ± 0.3 Da; one allowed missed cleavage; allowed fixed modification carbamido-methylation (Cys), and variable modification oxidation (Met). Positive identification was assigned with Mascot scores above the threshold level (p< 0.05), with at least two identified peptides with a score above homology Protein SDS electrophoresis: Samples mixed with NuPAGE LDS Sample buffer (Invitrogen), and heated at 95 ◦ C for 4 min, were analysed by sodium dodecyl sulphate– polyacrylamide gel electrophoresis (SDS–PAGE) using 10% acrylamide resolving gels and 4% acrylamide stacking gels (Bio-Rad). The gels were fixed with a mixture of ethanol, acetic acid, and deionized water (40:10:50) for 1 h. After washing in water for 5 min, the gels were stained with Coomassie Brilliant Blue R250 (0.1% in 25% methanol, 10% acetic acid) and de-stained by incubation in 30% acetic acid and 20% methanol. Molecular weights were estimated by comparison with the migration rates of standard proteins (Bio-Rad). J. Pers. Med. 2021,11, 25 5 of 19 3. Results 3.1. Clinical Sample Description Patients who underwent endoscopic ultrasonography procedure were included in this work. A total of 20 subjects, 60 and 40% men and women, respectively, with an average age of 62 ±13 years. Based on imaging and cytopathology, the pancreatic cysts were classified into different categories (Table 1). Table 1. Patient Description. Type of Cyst Non-Cyst Malignant Lesions Benign Pre-Malignant PC (n= 5) WOPN (n= 3) SC (n= 1) LYM (n= 1) IPMN (n= 7) MCN (n= 1) PDAC (n= 2) Total (n= 20) Age (years) * 63 ±10 62 ±10 72 ±0 52 ±0 72 ±13 42 ±0 40 ±2 62 ±13 Male/female % 80/20 67/33 100/0 0/100 71/29 0/100 0/100 60/40 * Average ± standard deviation (sd) PC = pseudocyst; WOPN = Walled-off pancreatic necrosis; IPMN = intraductal papillary mucinous neoplasm; SC= Serous Cyst; MCN= Mucinous Cystadenoma; LYM= lymphocele; PDAC= Pancreatic Ductal Adenocarcinoma. Clinical information of the samples is detailed in Table 2. All the cysts were between 2 and 15 cm in size and they were located in any region in the pancreas. According to clinical data (amylase and CEA concentrations), samples were divided in two groups: benign cysts (PC, WOPN and SC) and premalignant cysts (IPMN and MCN). There are two samples that turned out not to be cysts, but malignant lesions (PDAC). Table 2. Clinical Cyst Sample Description. Group Name Localization in the Pancreas Cyst Size (cm) Amylase (U/L) CEA (ng/mL) Final Clinical Diagnosis Benign Cysts PC1 Body 5.5 >11,000.0 9.23 Pseudocyst (In Acute Pancreatitis Context) PC2 Head 4.1 >11,000.0 64.2 Pseudocyst PC3 Head 3.5 5635.0 68.4 Pseudocyst PC4 Body 15.0 nd nd Pseudocyst (In Acute Pancreatitis Context) PC5 Head 3.0 >11,000.0 28.8 Pseudocyst (In Chronic Pancreatitis Context) WOPN1 Tail 6.4 >11,000.0 2.4 Walled-off pancreatic necrosis WOPN2 Head 10.0 >11,000.0 2.0 Walled-off pancreatic necrosis WOPN3 Body 4.0 >11,000.0 50.0 Walled-off pancreatic necrosis SC1 Body 5.0 41.0 0.7 Serous Cyst LYM Head 4.9 24.0 0.8 Lymphocele Pre-Malignant Cysts IPMN1 Body 2.6 >11,000.0 489.2 Branch duct IPMN IPMN2 Head, Body, Tail 2.0 162.0 1488.0 Main duct IPMN IPMN3 Head 2.3 >11,000.0 156.0 Branch duct IPMN IPMN4 Head 2.5 >11,000.0 556.0 Branch duct IPMN IPMN5 Isthmus 3.5 >11,000.0 225.0 Mixed Branch and Main duct IPMN IPMN 6 Head 3.0 10.0 392.0 Main Duct IPMN with pancreatic extension IPMN 7 Head, Body, Tail 3.5 4.0 >50,000.0 Main Duct IPMN with pancreatic extension MCN1 Body 3.3 3401.0 1617.0 Mucinous Cystadenoma Non-Cyst Malignant Lesions PDAC 1 Body, Tail 8.0 nd nd Pancreatic Ductal Adenocarcinoma PDAC 2 Head 0.5 >11,000.0 1192.0 Pancreatic Ductal Adenocarcinoma nd = not determined: Amylase < 250 U/L, communication with the conduct can be discarded; CEA > 192 ng/mL to classify a cyst as mucinous. Pancreatic pseudocysts (PC) are pockets of fluid, common sequelae of acute pancreatitis or chronic pancreatitis. PCs are important in terms of management and differentiation from other cystic processes or masses in this region. According to the updated Atlanta J. Pers. Med. 2021,11, 25 6 of 19 classification [ 36 ], there are two main groups of mature-well defined fluid collections associated with acute pancreatitis: A/Fluid collections in interstitial edematous pancreatitis (PC), and B/Fluid collections in necrotizing pancreatitis (WOPN). Both PC and WOPN were considered benign cysts. From our PC samples, PC 1 and 4 were in the context of acute pancreatitis, and PC 5 was in the context of chronic pancreatitis. The WOPN cysts had the biggest size, between 3 and 15 cm. Both types of pancreatic collections (PC and WOPN) were amylase positive (above 250 U/L) and CEA negative (below 192 ng/mL). Serous cysts (SC) are benign neoplasms composed of numerous small cysts that are arrayed in a honeycomb-like formation and most individual cysts are typically <10 mm. Lymphocele (LYM), also known as cystic lymphangioma, is a rare disease. There are no typical clinical manifestations, and most patients were diagnosed incidentally during imaging or surgery. Therefore, diagnosis is challenging. Surgical resection is still considered as the most effective approach for lymphocele, and prognosis is favorable. In our study, SC and Lym were 5 cm in size, and amylase and CEA negative. Intraductal papillary mucinous neoplasms (IPMN) are epithelial pancreatic cystic tumors of mucin-producing cells that arise from the pancreatic ducts. They are most commonly seen in elderly patients, with sex distribution roughly balanced, a possible slight male predominance. IPMNs are slow growing tumors that have malignant potential and distinct variants have been described: main duct (IPMN 6 and 7), branch duct (IPMN 1, 3 and 4), and mixed branch and main duct (IPMN 2 and 5). Main duct IPMNs have a very high rate of malignancy (up to 70% in reported surgical series [ 8 ]); for this reason, the usual recommendation is surgical removal of the affected portion of the pancreas. Branch duct IPMNs are cystic neoplasms of the pancreas that have malignant potential and their management is challenging; the risk of surgery must be carefully weighed against the risk of malignancy when deciding on surgical removal or surveillance. This is the reason why great efforts are taken to distinguish mucinous cysts from other cyst lesions (specially, main duct IPMNs). All IPMNs are considered as premalignant cysts. They had the smallest size, between 2 and 3.5 cm. Four were amylase positive (above 250 U/L) and all were CEA positive (above 192 ng/mL or very closed in case of IPMN3 with 156 ng/mL). Mucinous Cystadenoma (MCN) is another type of mucinous cystic neoplasm of the pancreas, traditionally considered typical of middle age females. MCN1 was 3 cm in size, amylase and CEA positive. 3.2. Analysis of TLB from Cystic Liquid Samples TLB thermograms of 20 cystic fluid samples were obtained. Protein concentrations and dilutions could be considered, but in this case TLB curves were normalized according to their area under the curve values (AUC); therefore, signals from the different samples can be compared and uncertainties in protein concentration (inherent to colorimetric methods) are avoided. TLB cyst profiles clustered according to their clinical assessment (benign or premalignant nature) are represented in Figure 1. Regarding the benign cystic group, the WOPN group exhibited a very similar cyst thermogram profile with two peaks at 65 and 82 ◦ C. We can easily distinguish this group from the other benign cysts (Figure 2A). PC5 is the only PC lacking the 85 ◦ C peak, and it is the only PC in a chronic pancreatitis context. In the premalignant cyst group, branch duct IPMNs (IPMN1, IPMN3 and IPMN4) exhibited a similar profile (Figure 2C). Main duct IPMNs (IPMN6 and IPMN7) exhibited a single peak. J. Pers. Med. 2021,11, 25 7 of 19 J. Pers. Med. 2021, 11, x FOR PEER REVIEW 7 of 19 to their area under the curve values (AUC); therefore, signals from the different samples can be compared and uncertainties in protein concentration (inherent to colorimetric methods) are avoided. TLB cyst profiles clustered according to their clinical assessment (benign or premalignant nature) are represented in Figure 1. Figure 1. Individual TLB thermograms from cystic liquid samples. Samples were clustered according to their benign or premalignant nature. Regarding the benign cystic group, the WOPN group exhibited a very similar cyst thermogram profile with two peaks at 65 and 82 °C. We can easily distinguish this group from the other benign cysts (Figure 2A). PC5 is the only PC lacking the 85 °C peak, and it is the only PC in a chronic pancreatitis context. In the premalignant cyst group, branch duct IPMNs (IPMN1, IPMN3 and IPMN4) exhibited a similar profile (Figure 2C). Main duct IPMNs (IPMN6 and IPMN7) exhibited a single peak. 3.3. Analysis of Proteomic Signatures from Cystic Liquid Samples Proteins identified by LC-ESI-MS/MS (detailed in Table S1) were analyzed and clustered according to the benign or premalignant nature of the cyst (Figure 3). This first classification distinguishes 52 proteins common in the cyst groups, and 12 and 11 proteins present only in benign cysts and premalignant cysts, respectively. A deeper analysis according to different groups of cysts was performed. Figure 1. Individual TLB thermograms from cystic liquid samples. Samples were clustered according to their benign or premalignant nature. 3.3. Analysis of Proteomic Signatures from Cystic Liquid Samples Proteins identified by LC-ESI-MS/MS (detailed in Table S1) were analyzed and clustered according to the benign or premalignant nature of the cyst (Figure 3). This first classification distinguishes 52 proteins common in the cyst groups, and 12 and 11 proteins present only in benign cysts and premalignant cysts, respectively. A deeper analysis according to different groups of cysts was performed. 3.3.1. Benign Cysts The WOPN cyst group exhibited a homogeneous proteomic profile (Table S1). 18 out of 41 (44%) proteins were shared by all cysts in this group (Figure 4A). The similarity in these samples was even higher, because 10 more proteins were common in WOPN1 and WOPN3. Low protein concentration in WOPN2 (Figure S1A) could prevent proper identification of more proteins in that sample. The most abundant proteins found in this group were globulins (macroglobulin and immunoglobulins), a type of protein related to immunological response as a consequence of an inflammation process. This is consistent with the nature of this specific type of cyst: walled-off pancreatic necrosis (WOPN) is a well-circumscribed area of necrosis which occurs as a late complication of acute pancreatitis, generally after four weeks from the initial episode. Singular proteins detected in WOPN1 and WOPN3 samples were S-100 proteins. They belong to the S100 protein family, having important roles in inflammation and may also be useful markers for gut inflammation [ 37 ]. Once secreted in the extracellular space, S100A9 acts as a chemo-attractant, recruiting J. Pers. Med. 2021,11, 25 8 of 19 further inflammatory cells and creating an inflammatory microenvironment that promotes tumor development [38]. J. Pers. Med. 2021, 11, x FOR PEER REVIEW 8 of 19 Figure 2. TLB thermograms from cystic liquid samples clustered according to their type. Average curves (colored lines) and standard deviations of curve values (grey) are represented: WOPNs (A), PCs (B), IPMNs (C) and IPMN7/MCNs (D). Figure 3. LC-ESI-MS/MS proteomic content of cystic samples. Common proteins were analyzed via Venn diagrams online tool (http://bioinformatics.psb.ugent.be/beg/tools/venn-diagrams). 12 and 11 proteins were detected only in benign cysts (blue) and premalignant cysts (pink), respectively, and 52 proteins appeared in both groups (intersection set). Table comprises the detailed information of the proteins in each set. 52 1112 PREMALIGNANT CYSTS BENIGN CYSTS Figure 2. TLB thermograms from cystic liquid samples clustered according to their type. Average curves (colored lines) and standard deviations of curve values (grey) are represented: WOPNs ( A ), PCs (B), IPMNs (C) and IPMN7/MCNs (D). J. Pers. Med. 2021, 11, x FOR PEER REVIEW 8 of 20 Figure 2. TLB thermograms from cystic liquid samples clustered according to their type. Average curves (colored lines) and standard deviations of curve values (grey) are represented: WOPNs (A), PCs (B), IPMNs (C) and IPMN7/MCNs (D). Figure 3. LC-ESI-MS/MS proteomic content of cystic samples. Common proteins were analyzed via Venn diagrams online tool (http://bioinformatics.psb.ugent.be/beg/tools/venn-diagrams). 12 and 11 proteins were detected only in benign cysts (blue) and premalignant cysts (pink), respectively, and 52 proteins appeared in both groups (intersection set). Table comprises the detailed information of the proteins in each set. 52 1112 PREMALIGNANT CYSTS BENIGN CYSTS Figure 3. LC-ESI-MS/MS proteomic content of cystic samples. Common proteins were analyzed via Venn diagrams online tool (http://bioinformatics.psb.ugent.be/beg/tools/venn-diagrams). 12 and 11 proteins were detected only in benign cysts (blue) and premalignant cysts (pink), respectively, and 52 proteins appeared in both groups (intersection set). Table comprises the detailed information of the proteins in each set. J. Pers. Med. 2021,11, 25 9 of 19 J. Pers. Med. 2021, 11, x FOR PEER REVIEW 10 of 19 Figure 4. LC-ESI-MS/MS proteomic content of cystic samples according to cystic types: (A) WOPN, (B) PC, (C) IPMN, and (D) MCN+IPMN. Common proteins were analyzed via Venn diagrams online tool. Colors code for different groups and numbers inside each set and shared sub-sets indicate the number of identified proteins. BENIGN CYSTS A B WOPN1,2,3 WOPN1,3 PC2,3, 4 ,5 P 0 27 6 3 - A l p ha-1-ac i d g l y co p rote i n 1 P01023-Alpha-2-mac roglobulin P01023-Alpha-2-mac roglobulin P04746-Panc reatic alpha-amylase P01024-Complement C3 P69905-Hemoglobin subunit alpha P68871-Hemoglobin subunit beta P02042-Hemoglobin subunit delta P02790-Hemopexin P00738-Haptoglobin P0DOX2-Immunoglobulin alpha-2 heavy chain P0DOX5-Immunoglobulin gamma-1 heavy chain P01876-Immunoglobulin heavy constant alpha 1 P01859-Immunoglobulin heavy constant gamma 2 P01860-Immunoglobulin heavy constant gamma 3 P01861-Immunoglobulin heavy constant gamma 4 P01834-Immunoglobulin kappa constant P02787-Serotransferrin P59 6 6 5 - Neutro p h i l de f ens i n 1 P02679-Fibrinogen gamma chain P55259-Pancreatic secretory granule membrane major glycoproteinGP2 P02100-Hemoglobin subunit epsilon P01857-I mmunoglobulin heavy constant gamma 1 P01871-Immunoglobulin heavy constant mu P0DOY2-I mmunoglobulin lambda constant 2 P16233-Pancreatic triacylglycerol lipase P05109-Protein S100-A8 P06702-Protein S100-A9 P 0 4 7 4 6 - Pancreat i c a l p ha-am y l ase P08861-Chymotrypsin-like elastase family member 3B P15086-Carboxypeptidase B P15085-Carboxypeptidase A1 P09093-Chymotrypsin-like elastase family member 3A PC2,3,5 P 4 8 0 52 - C ar b ox y p e p t i dase A2 P08217-Chymotrypsin-like elastase family member 2A Q99895-Chymotrypsin-C P16233-Panc reatic triac ylgly cerol lipase P54317-Pancreatic lipase-related protein 2 P07477-Trypsin-1 PC3, 4 ,5 P 0 27 6 8 - A l b um i n P04745-Alpha-amylase 1A P68871-Hemoglobin subunit beta P05451-Lithostathine-1-alpha PRE-MALIGNANT CYSTS C D IPMN 1,2,3,4,6,7 IPMN 2,3,4 Branc h and Main Duc t + MCN1 Branc h and Main Duct but not in MCN1 Main Duc t + MCN1 P 0 27 6 8 - A l b um i n P04745-Alpha-amylase 1A P19961-Alpha-amylase 2B P04746-Pancreatic alphaamylase P15085-Carboxypeptidase A1 P48052-Carboxypeptidase A2 P15086-Carboxypeptidase B P08217-Chymotrypsin-lik e elastase family member 2A P09093-Chymotrypsin-lik e elastase family member 3A P08861-Chymotrypsin-lik e elastase family member 3B P04118-Colipase P17538-Chymotrypsinogen B Q99895-Chymotrypsin-C P68871-Hemoglobin subunit beta P 6 99 0 5 - Hemog l o b i n subunit alpha P02042-Hemoglobin subunit delta P02100-Hemoglobin subunit epsilon P69891-Hemoglobin subunit gamma-1 P01876I mmunoglobulin heavy constant alpha 1 P0DOX7I mmunoglobulin kappa light chainI GLC2_HUM P00995-Serine protease inhibitor Kazal-type 1 P16233-Pancreatic triac ylgly cerol lipase P61626-Lysozyme C P98088-Mucin-5AC Q9HC84-Mucin-5B P05451-Lithostathine-1alpha P07477-Trypsin-1 P 0 4 7 4 6 - Pancreat i c a l p haamylase P04745-Alpha-amylase 1A P19961-Alpha-amylase 2B P15086-Carboxypeptidase B P08217-Chymotrypsin-like elastase family member 2A P09093-Chymotrypsin-like elastase family member 3A P69905-Hemoglobin subunit alpha P 0 27 6 8 - A l b um i n P69905-Hemoglobin subunit alpha P69905-Hemoglobin subunit alpha P02042-Hemoglobin subunit delta P02100-Hemoglobin subunit epsilon P69891-Hemoglobin subunit gamma-1 P01876-Immunoglobulin heavy constant alpha 1 P61626-Lysozyme C P98088-Mucin-5AC Q9HC84-Mucin-5B P0DOY2-I mmunoglobulin lambda constant 2 P 0 4 7 4 5 - A l p ha-am y l ase 1A P19961-Alpha-amylase 2B P04746-Pancreatic alpha-amylase P15085-Carboxypeptidase A1 CBPA2_HUM P15086-Carboxypeptidase B P08217-Chymotrypsin-lik e elastase family member 2A P09093-Chymotrypsin-lik e elastase family member 3A P08861-Chymotrypsin-lik e elastase family member 3B P0DOX7-Immunoglobulin kappa light chain P00995-Serine protease inhibitor Kazal-type 1 P16233-Pancreatic triacylglycerol lipase P05451-Lithostathine-1-alpha P07477-Trypsin-1 P04118-Colipase P17538-Chymotrypsinogen B Q99895-Chymotrypsin-C P19 6 52 - A l p ha-1-ac i d glycoprotein 2 P02647-Apolipoprotein A-I P00915-Carbonic anhydrase 1 P01024-Complement C3 P59665-Neutrophil defensin 1 P02790-Hemopexin P00738-HaptoglobinI P0DOX5-Immunoglobulin gamma-1 heavy chain P01859-Immunoglobulin heavy constant gamma 2 P01860-Immunoglobulin heavy constant gamma 3 P01861-Immunoglobulin heavy constant gamma 4 P01871-Immunoglobulin heavy constant mu P01834-Immunoglobulin kappa constant Q08380-Galectin-3-binding protein P80188-Neutrophil gelatinaseassociated lipocalin Q6UX06-Olfactomedin-4 P01833-Polymeric immunoglobulin receptor P02787-Serotransferrin P02774-Vitamin D-binding protein IPMN 2,6,7 P 0 27 6 8 - A l b um i n P01876-Immunoglobulin heavy constant alpha 1 Figure 4. LC-ESI-MS/MS proteomic content of cystic samples according to cystic types: ( A ) WOPN, ( B ) PC, ( C ) IPMN, and ( D ) MCN+IPMN. Common proteins were analyzed via Venn diagrams online tool. Colors code for different groups and numbers inside each set and shared sub-sets indicate the number of identified proteins. J. Pers. Med. 2021,11, 25 16 of 19 multiparametric analysis [ 30 ] and, later, a TLB serum score [ 33 ] to manage and assess TLB thermograms according to a simple interpretation index (TLB serum score from 0 to 1) that could be implemented in diagnosis, easily allowing the stratification of the patients. We first compared the TLB serum thermograms from patients to thermograms from healthy subjects that were not suffering from the disease. We applied a general TLB serum score previously reported [ 33 ] and the results were statistically different when comparing healthy subjects with benign or malignant cyst patients, individually or together as a pooled group of patients (p-values lower than 0.05) (Figure 6). Specificity and sensitivity values (98% and 71%, respectively), as well as PPV and NPV values (83% and 98%, respectively), were quite high. TLB serum scores (from benign or premalignant cyst groups) were not statistically different when using the TLB general formula when comparing to healthy subjects. Therefore, on that basis it was not possible to distinguish between both types of cyst. Therefore, we focused our attention on specifically comparing both patients’ groups and obtaining a cyst-specific TLB serum score that could be applied to evaluate intra-group cyst patient variability. This new TLB serum score would strengthen the discrimination power, based on the specific parameters reflecting differences between cysts. The challenge is considerable, because a mono-variant analysis of cyst TLB parameters (Table S3) showed that none of the individual parameters was statistically different between the benign and premalignant cyst group. As we previously confirmed in our studies, the combination of all the parameters in a multiparametric-based single TLB serum score increased the discrimination ability. Despite the small patient sample cohort, it was possible to distinguish between benign and premalignant cysts. This specific TLB serum score (values from 0 to 1) were over 0.75 (according to Youden) in 6 out of 8 (75%) premalignant samples, while TLB serum scores were below 0.75 in 6 out of 6 (100%) benign samples. The diagnosis accuracy based in the area under the curve (AUC) was 0.875, a promising starting point for extending the study. Therefore, more serum samples from patients with pancreatic cysts should be included in a larger future study, but these preliminary results are promising and allow us to foresee that the TLB serum score could be applied routinely in the clinic as an additional complementary tool helping physicians in making better diagnostic decisions. 5. Conclusions TLB analysis can be applied to both plasmatic serum and cyst fluid as a sort of high information content tool. Despite the small number of samples in this pilot study, it represents a proof of concept for developing a useful technique for classifying and evaluating risk in pancreatic cysts based on liquid biopsy in both body fluids. A future, larger study, with a larger number of samples for each cyst category, could confirm whether TLB of plasma or cyst fluid could be a new diagnosis tool to differentiate between benign and premalignant pancreatic cysts to help clinician gastroenterologists in making decisions about disease management. In case of serum, TLB would represent a quick, low-risk, minimally invasive tool easily translated to clinical practice for diagnosis and patient monitoring. In addition, TLB is reasonably cheap for serum tests (an estimated cost of 100–200 € /$ per test, although with a higher cost for cyst fluid test), and could be performed with predefined frequency for patient surveillance. Supplementary Materials: The following are available online at https://www.mdpi.com/2075-442 6/11/1/25/s1, Figure S1: Electrophoresis analysis of proteomic profiles, Figure S2: LC-ESI-MS/MS proteomic content of cyst samples types identification, Figure S3: ROC curve illustrating the statistical performance of TLB serum score (healthy vs. cysts); Table S1: Detailed information of cyst proteomic profiles, Table S2: Mono-variant analysis of TLB parameters of healthy controls and cysts patients, Table S3: Mono-variant analysis of TLB parameters of benign and premalignant cysts patients. J. Pers. Med. 2021,11, 25 17 of 19 Author Contributions: Conceptualization, A.V.-C., and O.A.; methodology, S.H.-D., J.L.O., O.S.-G., A.V.-C., and O.A.; software, S.H.-D., J.L.O., O.S.-G., A.V.-C., and O.A.; validation, J.L.O., S.V., O.S.-G., A.V.-C., and O.A.; formal analysis, J.L.O., S.V., O.S.-G., O.A., and A.V.-C.; investigation, L.C, S.V., A.V.-C., and O.A.; resources, G.G.-R., J.L.O., O.S.-G., Á.L., C.S., A.V.-C., and O.A.; data curation, S.H.-D., G.G.-R., C.S., J.L.O., and O.A.; writing—original draft preparation, S.H.-D., L.C.-L., J.L.O., A.V.-C., and O.A.; writing—review and editing, S.H.-D., G.G.-R., L.C.-L., J.L.O., S.V., O.S.-G., Á.L., J.M., A.V.-C., and O.A.; visualization, J.L.O., L.C.-L., O.S.-G., A.V.-C., and O.A.; supervision, Á.L., A.V.-C., and O.A.; project administration, O.A. and A.V.-C.; funding acquisition, Á.L., O.A., and A.V.-C. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Spanish Ministry of Economy and Competitiveness and European ERDF Funds (MCIU/AEI/FEDER, EU) (BFU2016-78232-P to A.V.C.); Projects funded by Instituto de Salud Carlos III and co-funded by European Union (ESF, ”Investing in your future”): “PI15/00663 (FIS project to O.A)”, “PI18/00349 (FIS project to O.A. and Contract to LC)”, “FI19/00146 (PFIS contract for SHD)”, “CPII13/00017 (Miguel Servet Program to OA)”; Diputación General de Aragón (Protein Targets and Bioactive Compounds Group E45_17R to A.V.C. and Digestive Pathology Group B25_17R to O.A.); and the Centro de Investigación Biomédica en Red en Enfermedades Hepáticas y Digestivas (CIBERehd). Institutional Review Board Statement: The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of CEICA (PI16/0228). Informed Consent Statement: All subjects gave their informed consent for inclusion before they participated in the study. Data Availability Statement: The data presented in this study are available on request from the corresponding author. Acknowledgments: Proteomic analyses were performed in the Proteomics Platform of Servicios Científico Técnicos del CIBA (IACS-Universidad de Zaragoza), ProteoRed ISCIII member, Zaragoza, Spain. Conflicts of Interest: The authors declare no conflict of interest. References 1. De Jong, K.; Nio, C.Y.; Hermans, J.J.; Dijkgraaf, M.G.; Gouma, D.J.; van Eijck, C.H.; van Heel, E.; Klass, G.; Fockens, P.; Bruno, M.J. High prevalence of pancreatic cysts detected by screening magnetic resonance imaging examinations. Clin. Gastroenterol. Hepatol. 2010,8, 806–811. [CrossRef] [PubMed] 2. Megibow, A.J.; Baker, M.E.; Gore, R.M.; Taylor, A. The incidental pancreatic cyst. Radiol. Clin. N. Am. 2011 ,49, 349–359. [CrossRef] [PubMed] 3. 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