Next‑generation sequencing to identify genetic mutations in pancreatic cancer using intraoperative peritoneal washing fluid
- Authors:
- Published online on: February 2, 2024 https://doi.org/10.3892/ol.2024.14272
- Article Number: 139
Abstract
Introduction
Pancreatic ductal adenocarcinoma (PDAC) is among the most aggressive cancers, whose 5-year survival rate was approximately 8% in 2020; this rate was 20-30% for patients who underwent curative resection (1–3). PDAC first recurs in sites with peritoneal dissemination. Intraoperative peritoneal washing fluid (IPWF) is routinely collected, and the washing cytology of IPWF (CY) is examined. CY-positive (CY+) status for PDAC is a well-known indicator of poor prognosis and a risk factor for peritoneal dissemination (4,5), which occurs in 20% of CY-negative (CY-) patients because of the insufficient sensitivity of CY due to the presence of many noncancerous cells including mesothelial cells and leukocytes (6). Therefore, a sensitive and reliable method for predicting the risk of peritoneal dissemination is needed.
Recently, various molecular techniques for detecting genomic mutations in patients with PDAC have been reported, including PCR-based methods and next-generation sequencing (NGS) (7–9). Specifically, liquid biopsies are promising tools for quantifying minimal residual disease, monitoring treatment response, and assessing the emergence of drug resistance in patients with PDAC (10). Moreover, deep NGS can be used to sequence a genomic region multiple times, thereby enabling the detection of genomic mutations with a variant allele frequency (VAF) of <1% (11,12). This method can detect tumor-derived DNA in various sample types, such as body cavity fluids, including pleural, pericardial, and peritoneal fluid (13–17), and micrometastases, which are too small to be observed in imaging tests. Tumor-derived DNA from these samples can be evaluated before extensive metastatic spread occurs. NGS analysis of tumor-derived DNA extracted from the peritoneal fluid or IPWF of patients with PDAC is not commonly performed (13–17). Additionally, genomic mutations in tumor-derived DNA have not been evaluated using NGS of IPWF samples from patients with PDAC who intend to undergo curative pancreatectomy. Therefore, in this study, we determined whether genomic mutations in tumor-derived DNA from IPWF samples of patients with PDAC who intend to undergo curative resection can be detected using NGS and if IPWF can be used for liquid biopsies.
Materials and methods
Patients and data collection
Twelve patients with PDAC who underwent surgical procedures between January 2020 and December 2021 were enrolled in this study. Patients who intended to undergo curative resection and with both FFPE and IPWF were included, and those with definitive distant metastasis by preoperative CT, MRI, or other imaging examination were excluded. According to the National Comprehensive Cancer Network guidelines, the patients with borderline resectable PDAC received GnP (intravenous infusion of 1,000 mg/m2 gemcitabine and 125 mg/m2 nab-paclitaxel, on days 1, 8, and 15 of each 28-day cycle) for 3 cycles followed by radiotherapy (45.0 Gy in 25 fractions) combined with S-1 (<1.25 m2, 60 mg; 1.25-1.5 m2, 80 mg; >1.5 m2, 100 mg), or modified FOLFIRINOX (intravenous infusion of 85 mg/m2 oxaliplatin, 150 mg/m2 irinotecan, 2,400 mg/m2 5-FU infusion over 46 h, no bolus 5-FU, every 2 weeks) as neoadjuvant treatment. Modified FOLFIRINOX was usually repeated for 6 to 8 cycles but the number of cycles depended on physicians' decisions or the patients' conditions. All patients were histologically diagnosed with PDAC using a standardized system consistent with WHO classification, 5th edition (18), and provided written informed consent to participate in the study. Samples were analyzed retrospectively to patient recruitment. This study was approved by the Human Experimentation Committee of Keio University Hospital (Tokyo, Japan; approval nos. 20120443 and 20170086) and performed in accordance with the tenets of the Declaration of Helsinki (2013).
IPWF
We performed routine CY immediately after laparotomy. First, 100 ml isotonic saline was introduced into the pelvic cavity. After gentle agitation, as much fluid as possible was collected using the suction tube from the pouch of Douglas. The sample was transferred to the laboratory approximately 10-20 min later and centrifuged at 1,580 × g for 2 min at 21°C. After resuspending the pellet, the fluid was dispensed to Cyto-Tek® 2500 (Sakura Finetek, Tokyo, Japan) and centrifuged at 700 × g for 4 min at 21°C. A pathologist examined the smears after conventional Papanicolaou and Giemsa staining. The CY results were provided to the surgeons before pancreatectomy, and normal or benign, indeterminate, and potentially malignant samples were defined as CY(−), whereas malignant samples were defined as CY(+). Another 50 ml of harvested peritoneal washing fluid was transferred to the laboratory immediately and centrifuged twice at 800 × g for 10 min at 4°C. The pellet was stored at −80°C until further use. The pellet was centrifuged at 10,000 × g for 5 min at 4°C prior to DNA extraction. In this study, we collected IPWF only during the surgery.
Preparation and extraction of tumor-derived DNA from IPWF and FFPE specimens
Resected specimens from the primary tumor were immediately fixed in 10% buffered formalin at 21°C and then embedded in paraffin within several days. Ten-micrometer-thick sections of tumor tissue were cut from each block and placed on the slide glass. The paraffinized sections were stained with hematoxylin and eosin, and an experienced pathologist identified the tumor lesions. The tumor lesions were dissected from the 10-µm sections, and fractions from five sections were collected in a sterile Eppendorf tube. Genomic DNA was extracted and purified from the stored pellet of peritoneal washing fluid and FFPE tissue samples using the QIAamp DNA FFPE Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The final elution volume was 30 µl. The concentration of the genomic DNA was measured using NanoDrop 2000c (Thermo Fisher Scientific, Waltham, MA, USA), and genomic DNA samples were stored at −80°C (7,8).
NGS
Genomic testing was performed in-house using the PleSSision testing platform (Keio University Hospital). Libraries were generated from 50 ng DNA per sample using the Human Comprehensive Cancer Panel, GeneRead DNAseq Panel PCR kit V2, GeneRead DNA Library I Core Kit, and GeneRead DNA Library I Amp Kit (Qiagen), and the library quality was assessed using Agilent High Sensitivity D1000 ScreenTape (Agilent Technologies, Santa Clara, CA, USA). Targeted amplicon exome sequencing was performed using a 160 cancer-related gene panel as described previously (19,20). The targeted regions of all 160 genes were specifically enriched using oligonucleotide probes. The enriched libraries were sequenced using a paired-end (150 bp ×2) sequencing method on the Next-Seq sequencing platform (Illumina, San Diego, CA, USA), resulting in a mean depth of 500. The sequencing data were analyzed using the GenomeJack bioinformatics pipeline (Mitsubishi Space Software Co., Ltd., Tokyo, Japan) (http://genomejack.net/) as described previously (20). First, the amino acid change in FFPE samples were checked, and then the same amino acid change in IPWF as FFPE samples were checked. VAF (%)/read depth value of 0 corresponds to a lack of mutations.
Follow-up
Patients were followed up every 3-6 months post-surgery as outpatients. Clinical examinations and laboratory investigations were performed at each hospital visit, and computed tomography scans were performed every 3-6 months. When considering patterns of recurrence, only the first site was documented herein. Locoregional recurrence was defined as recurrence in the remnant pancreas or the surgical bed, such as soft tissue along the celiac or superior mesenteric artery or around the pancreatojejunostomy site. Distant recurrence was stratified into three different categories, namely liver, lung, and peritoneal recurrence. Liver, lung, and peritoneal metastases were defined as a mass detected on computed tomography during the postoperative follow-up. Recurrence was determined by a multidisciplinary team comprising radiologists, hepatobiliary-pancreatic surgeons, and physicians. If recurrence was suspected after a radiological examination, histological confirmation was deemed not mandatory. Diagnostic criteria were based on the Japan Pancreatic Society General Rules for the Study of Pancreatic Cancer, 7th edition.
Results
We performed NGS of genomic tumor-derived DNA from IPWF and formalin-fixed paraffin-embedded (FFPE) samples of 12 patients with PDAC who underwent surgical procedures between January 2020 and December 2021. The clinical characteristics of the patients are listed in Table I. Two patients (16.7%) had CY(+) status and one patient (8.3%) exhibited intraoperative macro peritoneal dissemination. Of the 12 patients, 11 underwent radical surgery for PDAC. Except for patient No. 4, participants had genetic mutations in the primary tumor, and except for patient No. 2 and 3, the participants had genetic mutations in ascites. Among the patients with genetic mutations in ascites, except for patient No. 4, genetic mutations corresponded to those in the primary lesion.
The gene alteration profiles and clinicopathological findings of the patients are shown in Tables II and III. Actionable gene alterations were detected in two patients (100.0%) with CY(+) status and eight (80.0%) of the ten patients with CY(−) status based on NGS of IPWF samples. Three patients (37.5%) among those with actionable gene mutations in DNA extracted from IPWF samples developed peritoneal dissemination after surgery within approximately 12 months. Detailed information regarding actionable gene alterations, concentration of tumor-derived DNA, and sequence read depth of IPWF and FFPE samples is presented in Table IV. According to the NGS analysis, the most common genomic mutation was in KRAS (9 patients, 75.0%), which was detected in tumor-derived DNA from FFPE samples, followed by mutations in TP53 (3 patients, 25.0%), SMAD4 (1 patient, 8.3%), and CDKN2A (1 patient, 8.3%). Table V lists the driver gene mutations identified in DNA extracted from IPWF and FFPE samples.
 | Table IV.Details of actionable gene alterations, concentration of tumor-derived DNA, and sequence read depth of IPWF and FFPE samples (n=12). |
| Table V.Detection, VAF and read depth of KRAS, TP53, SMAD4, and CDKN2A among tumor-derived DNA from IPWF and FFPE samples (n=12). |
Discussion
We used NGS to investigate genomic mutations in tumor-derived DNA obtained from IPWF samples. This is the first report on actionable gene alterations, concentration of tumor-derived DNA, and sequence read depth determined using NGS of tumor-derived DNA from IPWF and FFPE samples of patients with PDAC who intended to undergo curative pancreatectomy. A comparison of the CY status revealed that some CY(−) patients had genomic mutations, particularly KRAS mutations, in tumor-derived DNA extracted from IPWF samples and some patients exhibited peritoneal dissemination post-surgery. Our findings suggest that the genomic mutation status of tumor-derived DNA extracted from IPWF samples is a candidate PDAC biomarker for predicting the recurrence of early peritoneal dissemination.
Several studies (13–17) have reported the effectiveness of liquid biopsy from body cavity fluids, such as pleural, pericardial, and peritoneal fluid, for performing NGS. Thus, body cavity fluids are a promising source for genomic analysis and may help expand cytomolecular practices. Here, we focused on the peritoneal fluid, as previous studies have not used peritoneal washing fluid from patients with PDAC who intended to undergo radical surgery. Chiba et al (14) performed NGS to analyze the ascites fluid from 13 patients. Among them, 11 patients had CY (+) status, seven showed distant metastases, and they had genomic mutations in KRAS, TP53, GNAS, SMAD4, and CDKN2A. Bae et al (16) performed NGS to analyze the ascites fluid from 13 patients whose CY was malignant or suspected of being malignant [i.e., CY(+)]; 6 patients with stage IV pancreatic cancer exhibiting distant metastases harbored genomic mutations in KRAS, TP53, and CDH. The concentration of tumor-derived DNA extracted from ascites fluid may be high; hence, the VAF of the genomic mutations was higher than that in CY(+) patients in our study. Our results demonstrated that genomic mutations could be detected using NGS of tumor-derived DNA extracted from IPWF samples of patients with PDAC in earlier stages of the disease.
Moreover, we used tumor-derived DNA extracted from floating cells collected from peritoneal washing fluid. Chiba et al used tumor-derived DNA extracted from cell-free DNA (cfDNA) and showed that the cfDNA sample obtained from IPWF samples can be used to characterize the genetic profiles of tumor cells involved in peritoneal dissemination (14). They used cfDNA from IPWF samples of patients with PDAC who underwent curative pancreatectomy and performed NGS and droplet digital PCR (ddPCR). In contrast, we focused on floating cells collected from IPWF samples owing to their high concentration and stability. The concentration of tumor-derived DNA from IPWF samples is lower than that from peritoneal washing fluid samples, and cfDNA concentration from IPWF samples is also low. The concentration of tumor-derived DNA in floating cells from IPWF samples is typically higher than that of cfDNA (14), enabling the evaluation of the former in IPWF samples and obtaining a sufficient concentration of tumor-derived DNA with high quality and purity suitable for NGS. Hence, tumor-derived DNA from floating cells in IPWF may be clinically useful.
In the present study, the most frequently mutated gene was KRAS, which encodes a member of the RAS family of GTPases. Four driver genes, KRAS, TP53, SMAD4, and CDKN2A, were identified as representative cancer-related genes in PDAC, with KRAS being the most commonly mutated gene and associated with a poor prognosis (21,22). Although the VAF of these genes varied among patients, KRAS was the easiest gene to detect using inexpensive equipment (23). For detecting KRAS mutations, ddPCR is highly sensitive, cost-effective, and rapid, and requires only a small amount of DNA. Liquid biopsy using ddPCR for detecting KRAS mutations in patients with PDAC using FFPE or blood samples has been reported (24). Our NGS results revealed KRAS mutations in tumor-derived DNA from floating cells obtained from IPWF samples of patients with PDAC who intended to undergo curative pancreatectomy; however, it is an expensive analysis. Therefore, ddPCR analysis of KRAS mutations using tumor-derived DNA extracted from floating cells in IPWF samples is an innovative and direct strategy that is highly sensitive and cost-effective in clinical situations.
The present study was a case series and could not reveal whether genomic mutations, particularly KRAS mutations, identified in tumor-derived DNA from IPWF samples could predict survival rate, early recurrence, or treatment response. However, three (37.5%) of the eight patients who were CY(−) and harbored actionable gene mutations in their IPWF sample DNA developed peritoneal dissemination within approximately 1-year post-surgery. Thus, genomic mutations in tumor-derived DNA from IPWF samples may be useful biomarkers for predicting early recurrence of peritoneal dissemination and determining whether chemotherapy should be performed or continued. Recently, a phase I/II study in Japan evaluated the efficacy of adding intraperitoneal paclitaxel to the new treatment strategy for patients with PDAC and peritoneal dissemination (25); this treatment showed clinical efficacy with acceptable tolerability. Moreover, in this phase I/II study, a peritoneal access port was implanted in the lower abdomen of participants and multiple tests were made possible by the implantation of this port. Using this type of port, the status of ascites can be evaluated many times throughout the treatment course for PDAC. Further studies are needed to identify the efficacy of NGS for ascites multiple times.
In the present study, peritoneal washing fluid was evaluated via cytology to determine the treatment response and inclusion criteria for conversion surgery. Liquid biopsy is used to monitor minimal residual disease in various malignant diseases (26); therefore, if the genomic status is used to determine the treatment response or inclusion criteria for conversion surgery, it may be possible to prevent or inhibit the progression of peritoneal dissemination and thus, improve the survival rate of patients with PDAC. Moreover, for patients at high risk for early recurrence, adjuvant chemotherapy should be improved by administering modified-FOLFIRINOX or gemcitabine plus nab-paclitaxel (27,28). In the era of precision medicine, genomic mutations identified in tumor-derived DNA from IPWF samples can be used to identify patients at a high risk of early recurrence of peritoneal dissemination. Genomic mutations identified in tumor-derived DNA from blood samples or body cavity fluids may offer various treatment options based on the risk factor for the recurrence type.
In the present study, we used an amplicon-based NGS method, which has numerous advantages compared to hybridization capture methods (29). Amplicon-based approaches offer a simpler, faster workflow with high PCR specificity, allowing the enrichment of target gene regions using a low sample input. Genetic material from limited sample sources, such as IPWF, can be sequenced for biomarker discovery. Furthermore, amplicon-based approaches are useful for sequencing specific regions of interest, such as hotspots in KRAS, at high coverage depths. Here, the read depth of the KRAS mutation hotspot was approximately 6,400 although the mean depth was lower than 500.
Among patients with genetic mutations in ascites, except for patient No. 4, genetic mutations corresponded to the mutations in the primary lesion, that is, we did not observe heterogeneity between FFPE and IPWF samples. Hashimoto et al (30) also revealed no heterogeneity between primary tumors and lymph node metastases in patients with PDAC. Baldus et al (31) also reported heterogeneity in KRAS between primary tumors and lymph node metastases in 31% of cases of colorectal adenocarcinoma. PDAC might be homogenous in terms of KRAS mutation and oncogenic activation of KRAS can be the first driver mutation in PDAC (30,32).
This study has some limitations. First, we conducted a retrospective study of a case series at a single institution and no statistically significant analysis was found in this study due to the small sample size because the cost of NGS of IPWF or some liquid samples is relatively high. This examination is not covered by the national health insurance program. A multi-center validation and prospective study is needed to investigate whether these genomic mutations identified in tumor-derived DNA from IPWF samples are candidate PDAC biomarkers for predicting survival rate, early recurrence, or treatment response and to obtain statistically significant data from the prospective study about these biomarkers in clinical situation. Our findings should help us take the first step to ensure that NGS for IPWF from the patients who intend to undergo curative resection becomes a common procedure if the cost issue is overcome. Second, in amplicon-based sequencing, the read depth of variants, except for hotspots in KRAS, may be low, leading to false-negative results. Thus, KRAS should be targeted because it is the most commonly mutated gene and may be useful as a pancreatic cancer biomarker (7,8). However, other genomic mutations in tumor-derived DNA from IPWF samples may also be suitable biomarkers for PDAC. For instance, mutations in TP53, SMAD4, and CDKN2A were detected at higher read coverages of hotspots in these genes. According to the restriction of the amplicon-based NGS, the detection rate of these genomic mutations differs. Considering the read coverage distribution, TP53, SMAD4, and CDKN2A are driver genes that are potential PDAC biomarkers. However, focusing only on KRAS may be cost-effective in clinical situations. Driver genes for PDAC can be evaluated using amplicon-based enrichment techniques if the read coverage of these genes is improved. Third, a few cases in which the primary tumor is negative for KRAS mutation but positive for cytology have been documented as NGS can identify genetic mutations other than those in KRAS and cytology can identify morphological changes in tumor cells by microscopic examination. This morphological change is due to nuclear and structural atypia, so the patient may have CY(+) even if the primary tumor is negative for KRAS mutation because there is a possibility that the primary tumor is positive for other genomic mutations, such as in TP53, SMAD4, or CDKN2A (8). Fourth, no specific measures were taken to address bias during this study. This study included consecutive patients who were required to meet inclusion criteria which demanded the availability of DNA extracted from both FFPE specimens and IPWF. Finally, we acknowledge that validation using an independent method can provide additional confidence in the results. However, given the high accuracy of modern NGS technologies and the additional cost and time associated with performing additional validation experiments, we opted to not perform independent validation in this study. We believe that the quality control measures and bioinformatics analyses employed provide sufficient certainty for the accuracy of our results.
In conclusion, we identified genomic mutations in tumor-derived DNA from IPWF samples of patients with PDAC who were CY(−) and intended to undergo curative resection. These may be candidate PDAC biomarkers for predicting the recurrence of early peritoneal dissemination. Further studies are warranted to determine the clinical relevance of these findings and investigate whether these genomic mutations are predictive clinical biomarkers of PDAC.
Acknowledgements
The authors would like to thank Mr. Kazumasa Fukuda (Keio University School of Medicine), for checking the manuscript.
Funding
Funding: No funding was received.
Availability of data and materials
The raw sequence data generated in the present study may be found in the Genome Sequence Archive (Genomics, Proteomics & Bioinformatics 2021) in National Genomics Data Center (Nucleic Acids Res 2022), China National Center for Bioinformation/Beijing Institute of Genomics, Chinese Academy of Sciences under accession number GSA-Human: HRA006272) or at the following URL: https://bigd.big.ac.cn/gsa-human/browse/HRA006272. The other data generated in the present study may be requested from the corresponding author.
Authors' contributions
YN, GS, KN, YM, MK, HN and YK conceived and designed the study. YN and GS drafted the manuscript. RT, EA, HY, YA, YH, SH and MT analyzed the data and critically revised the manuscript. KN and HN confirm the authenticity of all the raw data. All authors were involved in the data interpretation and drafting of the manuscript and have read and approved the final version of the manuscript.
Ethics approval and consent to participate
This study was approved by the Human Experimentation Committee of Keio University Hospital (Tokyo, Japan; approval nos. 20120443 and 20170086) and performed in accordance with the tenets of the Declaration of Helsinki (2013). All participants provided written informed consent to participate in the study. Samples were analyzed retrospectively to patient recruitment.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Glossary
Abbreviations
Abbreviations:
|
CY |
cytology of IPWF |
|
ddPCR |
droplet digital PCR |
|
FFPE |
formalin-fixed paraffin-embedded |
|
NGS |
next-generation sequencing |
|
PDAC |
pancreatic ductal adenocarcinoma |
|
IPWF |
intraoperative peritoneal washing fluid |
|
VAF |
variant allele frequency |
References
|
Siegel RL, Miller KD and Jemal A: Cancer statistics, 2020. CA Cancer J Clin. 70:7–30. 2020. View Article : Google Scholar : PubMed/NCBI | |
|
Ryan DP, Hong TS and Bardeesy N: Pancreatic adenocarcinoma. N Engl J Med. 371:1039–1049. 2014. View Article : Google Scholar : PubMed/NCBI | |
|
Neoptolemos JP, Kleeff J, Michl P, Costello E, Greenhalf W and Palmer DH: Therapeutic developments in pancreatic cancer: Current and future perspectives. Nat Rev Gastroenterol Hepatol. 15:333–348. 2018. View Article : Google Scholar : PubMed/NCBI | |
|
Tsuchida H, Fujii T, Mizuma M, Satoi S, Igarashi H, Eguchi H, Kuroki T, Shimizu Y, Tani M, Tanno S, et al: Prognostic importance of peritoneal washing cytology in patients with otherwise resectable pancreatic ductal adenocarcinoma who underwent pancreatectomy: A nationwide, cancer registry-based study from the Japan pancreas society. Surgery. 166:997–1003. 2019. View Article : Google Scholar : PubMed/NCBI | |
|
Tanaka M, Mihaljevic AL, Probst P, Heckler M, Klaiber U, Heger U, Büchler MW and Hackert T: Meta-analysis of recurrence pattern after resection for pancreatic cancer. Br J Surg. 106:1590–1601. 2019. View Article : Google Scholar : PubMed/NCBI | |
|
Satoi S, Murakami Y, Motoi F, Uemura K, Kawai M, Kurata M, Sho M, Matsumoto I, Yanagimoto H, Yamamoto T, et al: Reappraisal of peritoneal washing cytology in 984 patients with pancreatic ductal adenocarcinoma who underwent margin-negative resection. J Gastrointest Surg. 19:6–14. 2015. View Article : Google Scholar : PubMed/NCBI | |
|
Nakano Y, Kitago M, Matsuda S, Nakamura Y, Fujita Y, Imai S, Shinoda M, Yagi H, Abe Y, Hibi T, et al: KRAS mutations in cell-free DNA from preoperative and postoperative sera as a pancreatic cancer marker: a retrospective study. Br J Cancer. 118:662–669. 2018. View Article : Google Scholar : PubMed/NCBI | |
|
Yokose T, Kitago M, Matsuda S, Sasaki Y, Masugi Y, Nakamura Y, Shinoda M, Yagi H, Abe Y, Oshima G, et al: Combination of KRAS and SMAD4 mutations in formalin-fixed paraffin-embedded tissues as a biomarker for pancreatic cancer. Cancer Sci. 111:2174–2182. 2020. View Article : Google Scholar : PubMed/NCBI | |
|
Sugimori M, Sugimori K, Tsuchiya H, Suzuki Y, Tsuyuki S, Kaneta Y, Hirotani A, Sanga K, Tozuka Y, Komiyama S, et al: Quantitative monitoring of circulating tumor DNA in patients with advanced pancreatic cancer undergoing chemotherapy. Cancer Sci. 111:266–278. 2020. View Article : Google Scholar : PubMed/NCBI | |
|
Nagai M, Sho M, Akahori T, Nakagawa K and Nakamura K: Application of liquid biopsy for surgical management of pancreatic cancer. Ann Gastroenterol Surg. 4:216–223. 2020. View Article : Google Scholar : PubMed/NCBI | |
|
Goldman D and Domschke K: Making sense of deep sequencing. Int J Neuropsychopharmacol. 17:1717–1725. 2014. View Article : Google Scholar : PubMed/NCBI | |
|
Deveson IW, Gong B, Lai K, LoCoco JS, Richmond TA, Schageman J, Zhang Z, Novoradovskaya N, Willey JC, Jones W, et al: Evaluating the analytical validity of circulating tumor DNA sequencing assays for precision oncology. Nat Biotechnol. 39:1115–1128. 2021. View Article : Google Scholar : PubMed/NCBI | |
|
Patel A, Hissong E, Rosado L, Burkhardt R, Cong L, Alperstein SA, Siddiqui MT, Parl HJ, Song W, Velu PD, et al: Next-generation sequencing of cell-free DNA extracted from pleural effusion supernatant: Applications and challenges. Front Med (Lausanne). 8:6623122021. View Article : Google Scholar : PubMed/NCBI | |
|
Chiba K, Hata T, Mizuma M, Matsuda K, Aoki S, Takadate T, Kawaguchi K, Nakagawa K, Morikawa T, Motoi F, et al: Impact of tumor-derived DNA testing in peritoneal lavage of pancreatic cancer patients with and without occult intra-abdominal metastases. Ann Surg Oncol. 29:2685–2697. 2022. View Article : Google Scholar : PubMed/NCBI | |
|
Yang SR, Mooney KL, Libiran P, Jones CD, Joshi R, Lau HD, Stehr H, Berry GJ, Zehnder JL, Long SR, et al: Targeted deep sequencing of cell-free DNA in serous body cavity fluids with malignant, suspicious, and benign cytology. Cancer Cytopathol. 128:43–56. 2020. View Article : Google Scholar : PubMed/NCBI | |
|
Bae GE, Kim SH, Choi MK, Kim JM and Yeo MK: Targeted sequencing of ascites and peritoneal washing fluid of patients with gastrointestinal cancers and their clinical applications and limitations. Front Oncol. 11:7127542021. View Article : Google Scholar : PubMed/NCBI | |
|
Mayo-de-Las-Casas C, Velasco A, Sanchez D, Martínez-Bueno A, Garzón-Ibáñez M, Gatius S, Ruiz-Miró M, Gonzalez-Tallada X, Llordella I, Tresserra F, et al: Detection of somatic mutations in peritoneal lavages and plasma of endometrial cancer patients: A proof-of-concept study. Int J Cancer. 147:277–284. 2020. View Article : Google Scholar : PubMed/NCBI | |
|
Compton CC and Henson DE: Protocol for the examination of specimens removed from patients with carcinoma of the exocrine pancreas: A basis for checklists. Cancer committee, college of American pathologists. Arch Pathol Lab Med. 121:1129–1136. 1997.PubMed/NCBI | |
|
Nakamura K, Aimono E, Tanishima S, Imai M, Nagatsuma AK, Hayashi H, Yoshimura Y, Nakayama K, Kyo S and Nishihara Hl: Intratumoral genomic heterogeneity may hinder precision medicine strategies in patients with serous ovarian carcinoma. Diagnostics (Basel). 10:2002020. View Article : Google Scholar : PubMed/NCBI | |
|
Tsumura K, Arai E, Tian Y, Shibuya A, Nishihara H, Yotani T, Yamada Y, Takahashi Y, Maeshima AM, Fujimoto H, et al: Establishment of permutation for cancer risk estimation in the urothelium based on genome-wide DNA methylation analysis. Carcinogenesis. 40:1308–1319. 2019. View Article : Google Scholar : PubMed/NCBI | |
|
Shin SH, Kim SC, Hong SM, Kim YH, Song KB, Park KM and Lee YJ: Genetic alterations of K-ras, p53, c-erbB-2, and DPC4 in pancreatic ductal adenocarcinoma and their correlation with patient survival. Pancreas. 42:216–222. 2013. View Article : Google Scholar : PubMed/NCBI | |
|
Schlitter AM, Segler A, Steiger K, Michalski CW, Jäger C, Konukiewitz B, Pfarr N, Endris V, Bettstetter M, Kong B, et al: Molecular, morphological and survival analysis of 177 resected pancreatic ductal adenocarcinomas (PDACs): Identification of prognostic subtypes. Sci Rep. 7:410642017. View Article : Google Scholar : PubMed/NCBI | |
|
Olmedillas-López S, García-Arranz M and García-Olmo D: Current and emerging applications of droplet digital PCR in oncology. Mol Diagn Ther. 21:493–510. 2017. View Article : Google Scholar : PubMed/NCBI | |
|
Huerta M, Roselló S, Sabater L, Ferrer A, Tarazona N, Roda D, Gambardella V, Alfaro-Cervelló C, Garcés-Albir M, Cervantes A and Ibarrola-Villava M: Circulating tumor DNA detection by digital-droplet PCR in pancreatic ductal adenocarcinoma: A systematic review. Cancers (Basel). 13:9942021. View Article : Google Scholar : PubMed/NCBI | |
|
Yamada S, Fujii T, Yamamoto T, Takami H, Yoshioka I, Yamaki S, Sonohara F, Shibuya K, Motoi F, Hirano S, et al: Phase I/II study of adding intraperitoneal paclitaxel in patients with pancreatic cancer and peritoneal metastasis. Br J Surg. 107:1811–1817. 2020. View Article : Google Scholar : PubMed/NCBI | |
|
Kilgour E, Rothwell DG, Brady G and Dive C: Liquid biopsy-based biomarkers of treatment response and resistance. Cancer Cell. 37:485–495. 2020. View Article : Google Scholar : PubMed/NCBI | |
|
Conroy T, Hammel P, Hebbar M, Ben Abdelghani M, Wei AC, Raoul JL, Choné L, Francois E, Artru P, Biagi JJ, et al: FOLFIRINOX or gemcitabine as adjuvant therapy for pancreatic cancer. N Engl J Med. 379:2395–2406. 2018. View Article : Google Scholar : PubMed/NCBI | |
|
Ueno M, Morinaga S, Hashimoto Y, Umemoto K, Sasahira N, Saiura A, Seyama Y, Honda G, Ioka T, Takahashi H, et al: Tolerability of nab-paclitaxel plus gemcitabine as adjuvant setting in Japanese patients with resected pancreatic cancer: Phase I study. Pancreas. 50:83–88. 2021. View Article : Google Scholar : PubMed/NCBI | |
|
Samorodnitsky E, Jewell BM, Hagopian R, Miya J, Wing MR, Lyon E, Damodaran S, Bhatt D, Reeser JW, Datta J and Roychowdhury S: Evaluation of hybridization capture versus amplicon-based methods for whole-exome sequencing. Hum Mutat. 36:903–914. 2015. View Article : Google Scholar : PubMed/NCBI | |
|
Hashimoto D, Arima K, Yokoyama N, Chikamoto A, Taki K, Inoue R, Kaida T, Higashi T, Nitta H, Ohmuraya M, et al: Heterogeneity of KRAS mutations in pancreatic ductal adenocarcinoma. Pancreas. 45:1111–1114. 2016. View Article : Google Scholar : PubMed/NCBI | |
|
Baldus SE, Schaefer KL, Engers R, Hartleb D, Stoecklein NH and Gabbert HE: Prevalence and heterogeneity of KRAS, BRAF, and PIK3CA mutations in primary colorectal adenocarcinomas and their corresponding metastases. Clin Cancer Res. 16:790–799. 2010. View Article : Google Scholar : PubMed/NCBI | |
|
Ryan DP, Hong TS and Bardeesy N: Pancreatic adenocarcinoma. N Engl J Med. 371:2140–2141. 2014. View Article : Google Scholar : PubMed/NCBI |
