Time-saving method for directly amplifying and capturing a minimal amount of pancreatic tumor-derived mutations from fine-needle aspirates using digital PCR

Abstract

It is challenging to secure a cytopathologic diagnosis using minute amounts of tumor fluids and tissue fragments. Hence, we developed a rapid, accurate, low-cost method for detecting tumor cell-derived DNA from limited amounts of specimens and samples with a low tumor cellularity, to detect KRAS mutations in pancreatic ductal carcinomas (PDA) using digital PCR (dPCR). The core invention is based on the suspension of tumor samples in pure water, which causes an osmotic burst; the crude suspension could be directly subjected to emulsion PCR in the platform. We examined the feasibility of this process using needle aspirates from surgically resected pancreatic tumor specimens (n = 12). We successfully amplified and detected mutant KRAS in 11 of 12 tumor samples harboring the mutation; the positive mutation frequency was as low as 0.8%. We used residual specimens from fine-needle aspiration/biopsy and needle flush processes (n = 10) for method validation. In 9 of 10 oncogenic KRAS pancreatic tumor samples, the "water-burst" method resulted in a positive mutation call. We describe a dPCR-based, super-sensitive screening protocol for determining KRAS mutation availability using tiny needle aspirates from PDAs processed using simple steps. This method might enable pathologists to secure a more accurate, minimally invasive diagnosis using minute tissue fragments. Abbreviations dPCR Digital PCR FNA Fine-needle aspiration PDA Pancreatic ductal adenocarcinoma It is challenging to acquire histological evidence regarding solid tumors non-invasively; this hamper clinical management decisions that need to be made at appropriate time points. Fine-needle aspiration (FNA) is a standard procedure for collecting tumor tissues; however, there are cases where inadequate sampling resulted in false negative results 1. This technical issue might be highlighted when tumors, including pancreatic ductal adenocarcinomas (PDAs), which have a low tumor cell content, are targeted. Because of the invasiveness of needle-assisted cytology and biopsy as well as the potential for tumor cell dissemination, albeit at a low incidence, the frequent repetition of the procedure is not generally recommended 2,3 .

Pathogenic CD8 T cells defined by longitudinal liver sampling in chronic hepatitis B patients starting antiviral therapy

“…FNAs have been used extensively to sample tumors, for example in the kidneys (Turner et al, 2020;Eszlinger et al, 2017;Ono et al, 2020;Franzén et al, 2019;Su et al, 2021;Eikrem et al, 2018), but have recently gained traction for the study of liver disease due to their better tolerability for patients compared to liver biopsies. A finer gauge needle is used for liver FNAs than what is used for core liver biopsies, which reduces discomfort and allows for frequent sampling.…”

Clinical implementation of single-cell RNA sequencing using liver fine needle aspirate tissue sampling and centralized processing captures compartment specific immuno-diversity

“…The OD450 measurement developed as a quantitative, sample sparing, approach to assess peripheral blood contamination reserves >95% of the FNA sample for scientific investigation. Some practical aspects alone, like assessing the degree of blood contamination, have potential use in current indications for FNAs, such as the cytological evaluation of tumor masses and cysts within organs (thyroid, breast, kidney, lymph node) [73][74][75][76][77][78] . The full post-sampling workflow could be applied to standard blood samples or to expand the use of FNAs in other tissues, such as kidney, where analysis of early inflammation in transplant recipients through longitudinal sampling could replace serial biopsies 79 .…”