Introduction
In the USA, there are approximately 56,770 people newly diagnosed with pancreatic cancer in 2019, with high mortality (~ 46,000 deaths) [
1‐
3] .Although the mortality from other types of gastrointestinal cancer, such as gastric or colorectal cancers, are declining over the past two decades, mortality from pancreatic cancer has not declined [
1]. It is estimated that pancreatic cancer will become the second cause of cancer death by 2030 [
2].
The majority of pancreatic cancers (80–90%) are classified as pancreatic ductal adenocarcinomas (PDACs) [
3]. One of the underlying reasons for high mortality associated with PDACs is that most patients have late-stage disease at the time of diagnosis. In fact, only 15–20% of patients are considered to be surgical candidates at the diagnosis [
4]. Furthermore, prognosis even among patients who were able to have surgery with negative margins remains poor (5-year survival rate was only 10–25% with the median survival between 10-20 months) [
5,
6].
Multi-agent systemic chemotherapies with regimens of 5-fluorouracil, leucovorin, irinotecan, and oxaliplatin (FOLFIRINOX) and gemcitabine plus nab-paclitaxel have shown improved survival over single-agent gemcitabine and have become standard treatment options for metastatic PDACs [
7,
8]. However, median progression-free survival (PFS) and overall survival (OS) remain dismal (PFS: FOLFIRINOX, 6.4 months; gemcitabine plus nab-paclitaxel, 5.5 months, gemcitabine alone, 3.3–3.7 months; OS: FOLFIRINOX, 11.1 months; gemcitabine plus nab-paclitaxel, 8.5 months, gemcitabine alone, 6.7–6.8 months) [
7,
8]. Therefore, along with the recent development in sequencing technology, a personalized, molecular-targeted approach for PDAC is becoming an active area of research [
9]. Several case studies showed a benefit of platinum-based chemotherapies or poly ADP-ribose polymerase (PARP) inhibitors for PDAC patients with
BRCA1/2 abnormalities, and the NCCN guidelines suggest consideration of platinum-based regimen as a first-line therapy for advanced-stage pancreatic cancer patients with
BRCA gene mutations [
10‐
14].
Although molecular analysis on tissue samples is generally attempted, its clinical utility is often diminished in pancreatic cancer due to the difficulty in obtaining tissue with adequate quality for comprehensive molecular testing [
15]. Furthermore, tumor heterogeneity may challenge small biopsies, particularly in metastatic disease with multiple tumors [
16]. In contrast, the utility of plasma-derived circulating tumor DNA (ctDNA) has recently been assessed in several tumor types [
17‐
21]. ctDNA has some advantages over tissue DNA analysis: (1) readily available, (2) less-invasive, (3) potential real-time monitoring of disease status or treatment response, and (4) may reflect shed DNA from multiple metastatic sites [
22‐
24]. On the other hand, the small amount of tumor DNA in the circulation results in limitations as well.
Herein, we assessed the genomic landscape of ctDNA in patients with PDAC, using clinical-grade next-generation sequencing (NGS). We investigated the clinical implications of the findings including concordance between tissue and blood DNA sequencing, relationship between ctDNA findings and survival, and potential as well as actual actionability, with the latter illustrated by a patient with multiple alterations affecting the MEK pathway whose tumor responded to the MEK inhibitor trametinib.
Discussion
Despite the recent development of aggressive chemotherapies, patients with pancreatic cancer, who are generally diagnosed with advanced stage disease, have a dismal outcome. Therefore, improvement in treatment strategies for this lethal malignancy based on a better understanding of its biology is urgently needed. Here, we investigated the landscape of ctDNA NGS in pancreatic cancer, its concordance with tissue DNA NGS, and the clinical implications of the findings.
We found that 70% of patients with PDAC had ≥ 1 characterized genomic alteration in ctDNA (Table
1). Importantly, among the 94 patients with advanced PDAC, 73% (
N = 69) had ≥ 1 theoretically actionable alteration by FDA-approved agents (on- or off-label) (Additional file
1: Table S2), opening the doors for precision matching trials [
35‐
37]. These observations may be especially pertinent because several approved agents (such as gemcitabine and erlotinib) have only a small impact on survival [
38]. The weak clinical impact may be due to lack of biomarker selection when prescribing treatment or existence of multiple oncogenic alterations. In fact, our data also showed that patients with advanced PDAC mostly had a unique pattern of molecular portfolios in ctDNA (even when the actual genomic alterations overlapped) and that more than half of them (
N = 48/94) had two or more alterations, suggesting the need for a deep understanding of the effect of abnormalities in specific gene loci [
39]. For instance,
KRAS is well known as the dominant oncogene in pancreatic cancer and its prevalence is generally over 90% [
40,
41]. Consistently, our data showed the prevalence of
KRAS alterations was 91% in tissue DNA NGS (Fig.
1). Meanwhile, 9.1% (
N = 6/66) of patients with ctDNA and tissue DNA NGS had
KRAS wild type in both of the two tests (Table
2). Several previous reports have suggested unique targetable alterations even in tumors with
KRAS wild type, such as
EGFR exon 19 deletion and
ERBB2 amplification [
15,
42]. In our series, among the six patients whose ctDNA and tissue DNA NGS were both
KRAS wild type, all had at least one theoretically actionable alteration in both the two tests, including
EGFR amplification or
ERBB2 amplification.
Not unexpectedly, characterized alterations in ctDNA were more frequent in patients with advanced PDAC than in those with surgically resectable disease (median, 2 versus 0.5,
P = 0.04); median of maximum %ctDNA (0.4% versus 0%,
P = 0.02) and median of total %ctDNA (0.6% versus 0%,
P = 0.007) were also higher (Table
1). These findings are consistent with a previous report showing that ctDNA was more easily detectable in patients with metastatic cancer than those with localized diseases [
43,
44]. Higher tumor load presumably increases ctDNA shedding to blood.
Overall concordance rate between ctDNA and tissue DNA was 61% for
TP53 anomalies and 52% for
KRAS alterations (Table
2). In this series, the frequency of alterations in each gene was lower in ctDNA than in tissue DNA (Fig.
1). It should be noted that the sensitivity of ctDNA for tissue DNA in detecting alterations was lower, compared with that of tissue DNA for ctDNA (57% [29 of 51] versus 88% [29 of 33] for
TP53, and 47% [28 of 60] versus 100% [28 of 28] for
KRAS, respectively). Other studies have found similar results [
45‐
47]. Discordant cases that were positive in tissue and negative in ctDNA have been previously explained by low tumor load in surgically resectable cases [
43,
44]. In addition, detection of ctDNA can be affected by systemic treatment prior to blood draw [
48,
49]. In terms of spatial effects on concordance, we demonstrated that
KRAS concordance was significantly higher between ctDNA and metastatic sites than between ctDNA and primary tumor (72% versus 39%,
P = 0.01) (Table
2). Consistent with our observations, a prior study evaluating heterogeneity in ctDNA genomic profiling results in gastroesophageal cancers also reported that several targetable genomic alterations were 88% concordant between metastatic tissue and ctDNA even when primary tumor and metastatic sites had discordant results [
23]. The authors suggested that biomarker profiling of metastatic site tissue or ctDNA was potentially more effective in selection of therapy than interrogating primary sites. In fact, in this series, ctDNA
TP53 and
KRAS alteration concordance rates in the patients whose tissues were biopsied from metastatic sites were numerically higher than the rates in the patients whose tissues were biopsied from primary tumors (72% versus 54% for
TP53,
P = 0.20; 72% versus 39% for
KRAS,
P = 0.01) (Table
2). (The rate of tissue
TP53 alterations was 76% [19 of 25] for metastatic sites and 78% [32 of 41] for primary sites; for
KRAS alterations, it was 84% [21 of 25] versus 95% [39 of 41]; hence, there was no increased frequency of either alteration in tissue from metastatic sites.) It is conceivable that patients who have visible metastatic tumor that can be biopsied for sequencing may have higher tumor burden than those whose tissues for sequencing were available only from primary tumor, and this may explain the higher concordance rate with ctDNA. Further investigation is required [
47,
50,
51]. Somewhat surprisingly, there was no statistically significant difference in concordance when ctDNA and tissue sampling dates were ≤ 6 months versus > 6 months apart. To further assess these spatial and temporal effects on concordance, larger numbers of samples are required.
We also report that the total %ctDNA (dichotomized at median %ctDNA) was associated with patient survival (median OS from blood draw for ctDNA, 6.3 versus 11.7 months,
P = 0.001; median OS from advanced disease diagnosis: 10.8 versus 18.2 months,
P = 0.03) (Fig.
2 and Additional file
1: Figure S2). Several studies previously reported that the presence of detectable ctDNA was associated with poor survival in pancreatic cancer [
52,
53] or that the presence of
KRAS alterations in ctDNA was a poor prognostic marker for OS in advanced PDAC [
52,
54,
55]. In our series, multivariate analysis showed that ≥1 prior therapy and higher total %ctDNA, the latter perhaps reflecting greater tumor burden or shedding potential, were independently associated with worse OS (for higher total %ctDNA, HR, 4.35; 95%CI, 1.85–10.24; multivariate
P = 0.001) (Table
3). Also, the presence of prior therapies (HR, 2.89; 95%CI, 1.51–5.55; multivariate
P = 0.001) may reflect refractory cases who may have poorer prognosis.
To date, accumulating evidence has shown that matching drugs to sequenced genomic alterations can be promising for patients with advanced cancer [
56‐
59]. However, we were only able to match eight patients to therapy based on ctDNA and only one (13%) showed salutary effects (Fig.
3). The patient is unusual in several ways. In general, it is known that single targeted agents have limited effects in pancreatic cancer [
31,
60,
61]. Tumor heterogeneity or the existence of co-alterations may mediate resistance to scripted monotherapies [
62]. However, our patient had multiple alterations that could activate the MEK pathway (
GNAS R201C,
KRAS G12D, and
NF1 D1976fs) [
29‐
33] and demonstrated a remarkable responsiveness to the MEK inhibitor trametinib, with a steep decline in CA19-9 and %ctDNA as well as improvement in symptoms and PET imaging after therapy showing only minimal uptake in the tumor (Fig.
3). Our observation differs from previous literature suggesting that MEK inhibitors lack substantial anti-tumor activity among patients with pancreatic cancer [
31,
63]. The salutary effects in our patient might be due to the multiple MEK pathway abnormalities harbored by his cancer.
Meanwhile, low target-drug matching rate in this series (8 of 94 patients with advanced PDAC) is a realistic challenge. The remaining 55 patients received unmatched conventional chemotherapies following the molecular profiling and 31 had no systemic chemotherapies following the molecular profiling (mostly due to clinical deterioration or continuation of the regimen prior to the ctDNA test). Also, many patients with pancreatic cancer have genomic alterations that are not considered easily druggable. Therefore, to further investigate the efficacy of matched targeted therapy approaches, improvement in drug or clinical trial access as well as ctDNA testing in patients with less advanced disease will be necessary.
In the NCCN guidelines (
https://www.nccn.org), testing for germline and somatic
BRCA1/2 alterations is recommended for selection of platinum-based chemotherapies or PARP inhibitors based on emerging data from several small studies [
11,
12,
64‐
66]. In our series, the prevalence of
BRCA1/2 abnormalities in ctDNA and tissue DNA NGS were 1.8% (
N = 2/112;
BRCA1 Splice Site SNV and
BRCA2 T3033 fs) and 3.0% (
N = 2/66;
BRCA1 truncation intron 16 and
BRCA2 A938fs*21), respectively (germline alterations were not captured). Several genomic alterations are rare and the number of patients who can benefit from targeting those individual abnormalities is small, but further study to investigate highly targetable biomarkers based on deep sequencing can be justified.
This study has several limitations. First, the ctDNA gene panel expanded with time, increasing from 54 to 73 genes (Additional file
1: Table S1). Therefore, a limitation of the study pertains to the fact that the sequencing panels were different and so not all genes sequenced in tissue were sequenced in ctDNA. Nonetheless, our tissue and ctDNA panels allowed the comparison of most of the commonly altered genes in pancreatic cancer using clinical-grade assays frequently utilized in patients. The discrepancy in the frequency of
CDKN2A/B loss between ctDNA and tissue (with lower frequency in ctDNA) probably results from the fact that its allelic loss was not captured in older panels of the ctDNA sequencing. Second, not all patients had both ctDNA and tissue DNA tests; therefore, future concordance analysis should be performed with larger numbers of patients. Moreover, further analysis with tissue DNA from both primary tissue and metastatic sites may help inform the issues related to intratumoral heterogeneity (though in many patients with pancreatic cancer, accessing biopsy sites can be challenging or dangerous). Third, analysis of the influence of systemic treatment on ctDNA alterations is not feasible in this study due to the lack of serial ctDNA testing per patient. Finally, additional studies are also needed to determine the impact of matching ctDNA alterations to therapy beyond the eight patients matched in the current investigation.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.