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TUMOR DNA ANALYSIS IN PERITONEAL FLUID TO DETECT AND MONITOR PERITONEAL CARCINOMATOSIS.
Muhammad Talha Nawaz
*5,1, Muhammad Talha Waheed
4, Kirsten Dennison
5,1,2, Bradon Mcdonald
1,5,2, Stephanie McGregor
3,2, Syed Nabeel Zafar
1,2, Mustafa Raoof
4, Muhammed Murtaza
1,5,21University of Wisconsin-Madison Department of Surgery, Madison, WI; 2University of Wisconsin-Madison Carbone Cancer Center, Madison, WI; 3University of Wisconsin-Madison Department of Pathology and Laboratory Medicine, Madison, WI; 4City of Hope Department of Surgery, Duarte, CA; 5Center for Human Genomics & Precision Medicine, University of Wisconsin-Madison, Madison, WI
Introduction:
Gastrointestinal (GI) cancers often metastasize to the peritoneum. Current diagnostic tools for peritoneal carcinomatosis (PC) show limited accuracy. Reliable biomarkers to detect and monitor treatment response for PC are urgently needed. While circulating tumor DNA in plasma is a promising cancer biomarker, contribution of tumor DNA from PC into blood is minimal. We hypothesize that peritoneal fluid tumor DNA (ptDNA) may be useful for PC detection and response monitoring. Currently, there are no established laboratory methods for ptDNA analysis. In this study, we evaluate the feasibility of two DNA extraction methods using peritoneal fluid (PF) samples.
Methods: We first evaluated the performance of two cell-free DNA extraction kits based on magnetic beads (MagMax) or silica columns (QIAamp) using commercially-obtained PF from two individuals, at 1 mL and 4 mL starting volumes. Based on these results, we evaluated the optimal method further using PF samples from 32 patients with PC from GI cancers (biliary tract, appendiceal, and colorectal primary) undergoing surgery or paracentesis at City of Hope National Medical Center. PF samples were collected prior to therapy. We extracted DNA using 4 mL inputs, eluted in 30 µL buffer, and assessed using electrophoresis (TapeStation) and fluorometry (Qubit).
Results: In commercially obtained samples, adequate DNA yield and quality was obtained using both kits. Compared to QIAamp, mean DNA concentration was slightly lower using MagMax (24.88 ng/mL [range 13.90 - 35.85] vs. 25.66 ng/mL [range: 16.08-35.25; p=0.905). Similarly, mean fragment length was also shorter using MagMax compared to QIAamp (287.5 bp [range 202–350] vs. 294.3 bp [range: 239–348; p=0.80). In contrast to our prior experience with plasma DNA, ptDNA was observed at higher concentrations (3.7-fold higher) and longer fragment lengths. Based on these results and processing considerations, we selected MagMax kit to evaluate patient PF samples. We observed similar fragment lengths (mean: 277.6 bp, range: 136–427), with concentrations ranging from 0.8 ng/mL to 105 ng/mL in PF (mean: 19.44 ng/mL).
Conclusions: Our results demonstrate the feasibility of ptDNA analysis using PF samples in patients with PC. Furthermore, the yield and quality of DNA extracted is adequate for downstream molecular analyses. We were able to detect differences in PF vs. plasma DNA and observe inter-patient variability potentially related to heterogeneity in PC burden. We are now using this optimal PF DNA extraction method to expand our study and evaluate detection and quantification of peritoneal tumor-specific DNA as a biomarker for PC detection and monitoring.
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