Background
Despite many recent advances in treatment of malignant disease, pancreatic cancer remains the most lethal common solid tumor, with an overall 5-year survival rate of less than 10% [
1]. The predominant histologic form of pancreatic cancer, ductal adenocarcinoma (PDAC), is biologically aggressive and often develops asymptomatically in the early course of the disease [
2]. Surgical resection is the only curative option available today. However, current imaging technology is suboptimal for identifying early-stage tumors or high-grade precancerous lesions, and no clinically reliable biomarker test is available for early disease detection [
3]. As a result, most patients diagnosed with PDAC present with a non-resectable advanced-stage disease and are left with only palliative treatment options. Thus, there is a strong need for progress in early detection and therapeutic approaches to improve patient outcomes in pancreatic cancer.
Deep sequencing (also known as next-generation sequencing or NGS) of circulating tumor DNA (ctDNA) in body fluids has emerged as a potential tool for cancer diagnostics and management [
4]. Detection of molecular alterations in ctDNA isolated from pancreatic juice may represent a useful clinical test in pancreatic cancer diagnostics [
5] as this fluid flows through the ductal system where most precursor lesions of malignant pancreatic tumors arise [
3]. Early disease detection based on ctDNA should also take into account that the somatic mutations of PDAC are likely to arise in a certain temporal order because these tumors are considered to develop from defined precursor lesions, the most common being pancreatic intraepithelial neoplasia (PanIN) [
6].
Exome sequencing reveals that many somatic mutations required for PDAC development, most frequently
KRAS and
TP53, are shared among moderate and high-grade PanINs and adjacent PDAC [
7]. Oncogenic
KRAS mutations are present in at least 90% of PDAC tumors [
8], and they are likely to arise from early mutational events that occur in the large majority of low-grade PanINs (PanIN-1) [
6]. Similarly, around 70% of PDAC cases harbor inactivating
TP53 mutations that arise in high-grade PanINs (PanIN-3) before they progress to invasive adenocarcinoma [
6,
8]. If these and other mutations commonly present in pancreatic cancer or high-grade dysplasia could be reliably detected in pancreatic juice, there might be a potential to identify individuals with early-stage pancreatic cancer or carcinoma in situ before these lesions become visible by imaging. This may provide a window for early medical intervention and a better chance for survival.
In most reports on mutation analysis in pancreatic juice, either none or only a small number of matched tissue specimens were analyzed in parallel [
9‐
14]. One study from 2008 reported similar mutation profiles between surgically collected pancreatic duct juice and tumor tissues from PDAC patients, but only three hotspot
KRAS mutations were analyzed [
9]. Information about concordance between tumor and juice samples with regard to
TP53 and other mutations associated with PDAC is generally scarce. Thus, it remains to be firmly established to which degree the mutations found in pancreatic juice reflect those present in the primary tumor.
In this study, our aim was to provide a better understanding of the clinical potential and challenges in early malignant disease detection by deep-sequencing-based mutational analysis of DNA isolated from pancreatic juice. We evaluated the concordance between KRAS and TP53 mutation profiles in PDAC tissue and pancreatic juice sampled from the distal dilated duct during resection of the primary tumor. We found that pancreatic juice DNA harbors a panorama of KRAS mutations, making any diagnostic evaluation based only on this gene of limited value.
Discussion
Here we have characterized the mutation patterns of KRAS and TP53 in matched pancreatic tumor and juice samples from 21 PDAC patients, using targeted deep sequencing with Sanger sequencing and PNA clamp assay as complementary methods. We identified multiple KRAS mutations in the juice DNA from almost all cases (95%). Most of the KRAS mutations in pancreatic juice were present at low frequencies (VAF < 3%) and were not seen in the primary tumor.
Previous mutational analyses have shown that
KRAS mutations are commonly detected in pancreatic juice sampled from patients with pancreatic cancer [
9,
13,
27] or from persons undergoing screening because they are considered high-risk subjects [
12,
14,
28,
29]. Our observation of multiple
KRAS mutations in most juice samples is consistent with an earlier report focusing on three hotspot
KRAS mutations of codon 12 in matched pancreatic juice and tumor specimens [
9]. Unlike that study, our method covered the full spectrum of known somatic
KRAS mutations, occurring in codons 12, 13, 59, 61, 117 and 146 [
30], and we demonstrate that the mutation detected in the primary tumor not necessarily was the predominating
KRAS variant in the patient’s juice sample. Moreover, when the mutation found in the tumor was absent from the juice, other
KRAS mutations were usually present.
Particularly illustrative in this regard is case #10 with a primary tumor that was
BRAF-positive and
KRAS-negative (Table
3). Still, 22.9% of the
KRAS exon 3 reads from the corresponding juice sample displayed the mutation Q61H, whereas
BRAF alterations were not detected. An oncogenic
BRAF mutation is reported to occur in 3% of PDAC cases and is most often mutually exclusive with the presence of a
KRAS mutation [
31]. This is in line with the finding that dysregulation in the RAS-RAF-MAPK signaling pathway is a key driver for PDAC [
32]. The absence of the
BRAF mutation in the juice of case #10 suggests that the fluid contained little DNA arising from the tumor, and that the
KRAS mutation may have its origin somewhere else, most likely in the tail region of the pancreas drained by the distal duct.
Accordingly, our observation of multiple, mostly low-abundance
KRAS mutations in pancreatic juice (Table
3), may be explained by the presence of several PanIN precursor lesions in the gland. Low-grade PanIN lesions are frequently present in healthy aged individuals [
33] and in PDAC patients [
34]. Over 90% of low-grade PanIN-1 lesions have already acquired a
KRAS mutation [
6], but obviously most do not progress to invasive cancer. Nevertheless, these lesions may shed DNA and contribute to the pool of cell-free DNA in the juice. In fact, the presence of more than one
KRAS mutation in each pancreatic juice sample has been reported from older healthy individuals and patients with pancreatic non-malignant abnormalities such as chronic pancreatitis and cysts [
9,
12]. These
KRAS mutations may dominate over the tumor-specific mutations, as demonstrated in our case series. This strongly suggests that the informative value of detecting
KRAS mutations in pancreatic juice DNA with the purpose of early pancreatic cancer detection or differential diagnostics is limited. It should be noted, though, that the presence of multiple
KRAS mutations in a pancreatic juice sample also might reflect clonal heterogeneity of the primary tumor [
8].
Detection of
TP53 mutations in combination with
KRAS in pancreatic juice could improve specificity for PDAC, because somatic alterations in
TP53 arise later during tumorigenesis and is generally present only in high-grade PanIN lesions [
6]. Such mutations are in general absent in juice samples from healthy individuals and chronic pancreatitis cases [
11]. Thus, with one exception, no juice sample in our series harbored more than a single
TP53 mutation. The striking difference between the
KRAS and
TP53 mutation distributions lends further support to the assumption that the majority of the multiple
KRAS mutations found in pancreatic juice DNA originate from low-grade PanIN lesions.
The detection rate of
TP53 mutations (29%) in the juice samples of our study is substantially lower than in other reports studying this biological material from PDAC patients (around 60%) [
11,
13,
14]. However, in those publications information of the
TP53 mutation status of the primary tumor was lacking for the majority of cases. Moreover, the pancreatic juice samples stemmed from the duodenal lumen of PDAC patients who had their tumors located in all regions of the pancreas [
11,
13,
14]. In contrast, the juice samples of our study were collected from the distal pancreatic duct where the fluid had accumulated due to obstruction imposed by the tumor located in the pancreatic head. This physical obstruction of the proximal pancreatic duct could possibly have favored the relative enrichment of DNA from the distal part of the pancreas rather than from the tumor. Consistently, we observed that the amount of ctDNA was low in those cases where a
TP53 mutation was detected only in the tumor (when using the percentage of the tumor-specific
KRAS mutation in pancreatic juice as a surrogate measure for ctDNA level). Because of their later occurrence during tumorigenesis,
TP53 mutations generally have a frequency that is lower than or similar to that of
KRAS during clonal expansion of the cancer [
35]. Thus, the scarcity of detected
TP53 mutations in the juice samples could partly be due to a ctDNA level below the detection limit and partly due to the fact that around 30% of PDAC cases lack
TP53 point mutations or small indels [
8].
Nevertheless, we found that TP53 mutations were absent from the majority of the juice samples, even when the tumor was positive. With reference to the mutation profile in the primary tumor, we suggest that analyzing TP53 mutations in combination with KRAS mutations in the juice might represent a more specific although, unfortunately, less sensitive test for PDAC detection.
Our study has several limitations. Firstly, the number of cases was limited and prevented us from investigating the relationship between mutation detection (or concentration) and clinico-pathological variables such as patient survival, cancer stage and tumor differentiation. Secondly, we employed a commercial deep sequencing panel that was constructed to cover 15 genes frequently mutated in various cancers (Additional file
2: Table S2). For PDAC, this panel covers only
KRAS and
TP53 among the frequently mutated genes in this cancer type. We detected a
BRAF mutation in one case, but otherwise the remaining 12 genes were negative for all specimens tested. Thirdly, the amplicon-based deep sequencing technology was not optimized to identify low-abundance mutations in the juice. The concentration of ctDNA in pancreatic juice can often be low (VAF < 3%), as shown in our study. This makes it challenging to reliably detect and distinguish low-frequency mutations from PCR artefacts and sequencing errors inherent in amplicon-based assays [
36].
For
KRAS exon 2, we circumvented this issue by using the highly sensitive PNA clamp technology to complement and independently identify low-abundance mutations. The results from the PNA clamp assay supported the deep sequencing results as all samples were positive except one (#17), which was negative with both techniques. Sanger sequencing of the PNA-clamped products also confirmed that multiple
KRAS mutations were indeed present in DNA from pancreatic juice. However, using complementary assays depending on mutation-specific probes (e.g. PNA clamp assays and droplet digital PCR) is impractical when a larger set of cancer-associated genes are to be screened. Strategies such as digital deep sequencing [
13,
14] and molecular barcoding [
37] should be implemented in order to better characterize the mutational load in pancreatic juice in future follow-up work. Moreover, sampling of duodenal fluid after secretin stimulation [
38] represents a less invasive procedure for obtaining pancreatic juice than sampling directly from the pancreatic duct and would therefore be the method of choice when screening pancreatic cancer high-risk patients. On the other hand, the tumor-specific DNA may then be more diluted, as duodenal juice also contains DNA (including bacterial DNA) and fluid from the duodenal lumen [
28].
Finally, we note that in the
KRAS- and
TP53-mutated primary tumor cases, the allele frequencies of both mutations tended to be similar (Fig.
1), supporting the view that the two mutations originated from the same tumor clone. We also found that some patients exhibited
KRAS and/or
TP53 mutations at an allele frequency of around 50% in the tumor (e.g. case #16). As stromal and other non-neoplastic cells will contribute significantly to the isolated DNA, this suggests an amplification event of the oncogenic
KRAS allele [
39] and deletion of the wild-type
TP53 allele [
8], respectively. Noteworthy, a subset of pancreatic cancers manifest genomic instability that leads to chromosomal alterations including the
KRAS and
TP53 loci [
40].