Real-time Genomic Characterization of Advanced Pancreatic Cancer to Enable Precision Medicine

Pancreatic ductal adenocarcinoma (PDAC) is the third-leading cause of cancer-related death in the United States and is projected to become the second leading cause by 2030 (1). Most patients present with advanced disease and die within 12 months of diagnosis (2, 3). Recent genomic studies of primary PDAC resection specimens have identified recurrent molecular alterations and genomic subtypes of the disease (4–12). Moreover, RNA analyses of PDAC cohorts have identified gene expression signatures with prognostic and biological relevance (7, 12–14). Although these molecular subtypes of PDAC may theoretically help guide precision medicine approaches, molecular characterization of PDAC in patients with advanced disease is not yet standard clinical practice. Biopsy-driven genomic studies have been challenging due to rapid disease progression and the small-volume and heterogeneous nature of biopsies that impede deep molecular characterization. Despite the fact that conventional therapies are often ineffective, the rate of enrollment of patients with PDAC onto clinical trials is extremely low (15). A proactive, standardized approach to acquire PDAC biopsy tissue and perform rapid turnaround molecular analysis is required to efficiently generate and utilize genomic information in patients with PDAC.

The PancSeq protocol was developed as an institutional review board (IRB)–approved multidisciplinary biopsy program to obtain tissue from core-needle biopsies or fine-needle aspirates in patients with metastatic or locally advanced PDAC for rapid turnaround genomic analysis. Between March 2015 and June 2017, 79 patients underwent biopsy on the PancSeq protocol (Supplementary Table S1; Supplementary Fig. S1). An initial pilot phase (n = 10 patients) was conducted in patients having clinically indicated biopsies to optimize workflow, tissue processing, nucleic acid extraction, whole- exome sequencing (WES), and RNA sequencing (RNA-seq) from small-volume needle biopsies (approximately 15–20 mg of tissue per biopsy). Subsequently, 69 additional patients underwent biopsy and WES was performed in a Clinical Laboratory Improvement Amendments (CLIA)–certified laboratory, with return of selected somatic and germline variants to referring clinicians. Patients had a median age of 64 years and most had metastatic disease (96%) and no prior therapy (70%; Supplementary Table S1). Most patients had a diagnosis of PDAC (95%), although several patients with less common histologies were enrolled. Biopsies were obtained from liver (n = 63), pancreas (n = 7), peritoneum (n = 6), lymph nodes (n = 2), and ovary (n = 1; Supplementary Table S2). Biopsies were performed percutaneously by interventional radiology (n = 72), with endoscopic ultrasound (n = 6), or intraoperatively (n = 1), and a median of 5 (range, 1–10) cores or biopsy specimens were collected per patient. A low rate of serious complications was observed, with only 1 patient having a self-limited hepatic subcapsular hematoma in the setting of a clinically indicated liver biopsy and therapeutic anticoagulation with an oral factor Xa inhibitor (rivaroxaban) for prior deep venous thrombosis.

From the formalin-fixed, paraffin-embedded core, tumor content was quantified by histopathology, and 92% (73/79) of cases had sufficient tumor content (≥5% tumor nuclei) to proceed with WES. ABSOLUTE purity (16) was examined in WES data, and successful mutation and copy-number calls could be made for all but two of these samples (Supplementary Table S2; Supplementary Fig. S1).

Consistent with prior genome sequencing studies of primary PDAC samples, samples from our cohort of patients with PDAC displayed low neoplastic cellularity, with a median cellularity by histologic assessment of 40%, and by WES using the ABSOLUTE algorithm of 34% (Supplementary Fig. S2A–S2B; ref. 16). Neoplastic cellularity estimates by histologic assessment and by the ABSOLUTE algorithm on WES data were significantly correlated (Pearson product–moment correlation = 0.386; 95% CI, 0.195–0.548; P = 0.00016; Supplementary Fig. S2A). This level of correlation is consistent with that observed in other studies (12, 16) and is affected by variability between biopsy specimens as well as differential sensitivity between the two methods for identification of neoplastic cellularity. For our biopsy samples, we performed deep WES with a median of mean target coverage of 191× in the tumor and 176× in the normal (Supplementary Table S2). Within the CLIA-certified phase of the study, analyzed results were available within an average of 39 days (range, 16–67) from the date of clinically indicated biopsies and 28 days (range, 15–51) for research-only biopsies (Supplementary Table S2). Additional time for return of results for clinically indicated biopsies was required, as these specimens were held in the pathology department until a formal histologic diagnosis was confirmed. A report of clinically relevant events was returned to the referring clinician detailing somatic mutations, small insertions/deletions, and copy-number alterations (CNA) as well as pathogenic/likely pathogenic germline alterations in a curated list of 81 PDAC-relevant genes (Supplementary Table S3). These genes were chosen based on somatic or germline clinical relevance, therapeutic actionability, and/or recurrent mutation across published PDAC genome-sequencing cohorts (4–12). Most of these genes (n = 69) were associated with clinical trial or off-label FDA-approved targeted therapies. Comprehensive analysis of all genes from WES data was simultaneously performed in the research setting.

Genome-sequencing studies have elucidated the molecular landscape of archived primary PDAC tumors and have demonstrated distinct molecular subtypes of disease (6–12). In our cohort of 71 patients with PDAC, we identified significantly recurrent mutations in KRAS, TP53, CDKN2A, SMAD4, ARID1A, and TGFBR2—a collection of genes that were also recurrently mutated in primary PDAC tumors (refs. 7, 10, 12; Fig. 1). Moreover, we observed frequent mutations in additional tumor suppressor genes (e.g., RNF43) or oncogenes (e.g., BRAF, GNAS), as well as recurrent mutations in genes involved in DNA-damage repair (DDR) and chromatin modification (Fig. 1). Recurrent high-level amplifications were observed in several genomic loci, encompassing genes such as MYC, AKT2, and GATA6 (Fig. 1; Supplementary Fig. S3). Deletions were identified at numerous loci, including frequent homozygous deletions of CDKN2A and SMAD4 (Fig. 1; Supplementary Fig. S3).

Mutational signature analysis was performed using a Bayesian variant of the non-negative matrix factorization approach in a two-stage manner from the set of single-nucleotide variants (SNV) in our dataset, as previously described (SignatureAnalyzer; Supplementary Experimental Methods; refs. 17–20). First, we performed de novo signature discovery and our analytic pipeline identified three primary signatures: SigA that best resembled Catalogue of Somatic Mutations in Cancer (COSMIC) signature 3 with cosine similarity 0.87 [BRCA mutant signature suggestive of homologous recombination deficiency (HRD)]; SigB that best resembled COSMIC signature 1 with cosine similarity 0.96 (C>T transitions at CpG dinucleotides, Aging); and SigC that best resembled COSMIC signature 17 with cosine similarity 0.91 (etiology unknown; Supplementary Fig. S4A–S4B). In addition, we observed a relative elevation of C>G transversions and C>T transitions at TC[A/T] contexts in SigA corresponding to canonical hotspots of APOBEC mutagenesis (COSMIC signatures 2 and 13), suggesting that APOBEC signature is possibly operative in this cohort, but not cleanly separable due to a lack of mutations (17). Based on this de novo analysis, we concluded that four main mutational processes were likely active in these data (Aging/COSMIC1, BRCA/HRD/COSMIC3, APOBEC/COSMIC2+13, and COSMIC17). To better evaluate discrete contributions of these mutational processes in our data and to minimize signature contamination, we next performed a projection analysis to infer a signature activity across our biopsy cohort using the signature profiles of the five contributing COSMIC signatures: COSMIC1, 2, 3, 13, 17 (Fig. 2A; Supplementary Experimental Methods). Consistent with the de novo analysis, the inferred signature activity with these five COSMIC signatures reveals a clear contribution of the Aging/COSMIC1 signature in almost all patients and a notable activity of the HRD/COSMIC3 signatures in many patients (Fig. 2A).

To further investigate the HRD/COSMIC3 signature in these data, we integrated mutation and copy-number data from WES and gene-expression data from RNA-seq for a core set of known homologous recombination (HR) genes (BRCA1, BRCA2, PALB2, and RAD51C) that are known to be associated with the HRD/COSMIC3 signature (18) and categorized samples as “HRD altered” or “WT” (wild-type). We defined HRD-altered samples based on the presence of damaging germline and somatic mutations (null, truncating, and splice-site variants), homozygous deletions, or more than 2-fold downregulation of mRNA expression levels. We observed a significant enrichment of HRD/COSMIC3 signature mutations within HRD-altered samples (Fig. 2B; P < 0.000002 by one-tailed Wilcoxon rank-sum test). The increased occurrence of large deletions of up to 50 base pairs with overlapping microhomology is another characteristic of HR-deficient samples, and we indeed observed an increased incidence of such deletions (≥9 base pairs) in our HRD-altered samples (Fig. 2C; P < 0.00002 by one-tailed Wilcoxon rank-sum test). Eight of the top 14 samples with HRD/COSMIC3 signature activity harbored deleterious mutations or homozygous deletions in one of the four core HRD genes. Six of these eight HRD-altered samples had both germline and somatic events or a somatic alteration with coexisting loss of heterozygosity (LOH) in BRCA1 or BRCA2, supporting a “two-hit” hypothesis. Another sample (0400253) had both a p.Q750* nonsense and a p.F1016S missense mutation in PALB2. Furthermore, one sample harbored homozygous deletion of RAD51C. Two additional samples with HRD/COSMIC3 signature activity but without genomic alterations in the four core HRD genes displayed downregulation of RAD51C expression in the RNA-seq data, an event previously associated with HRD/COSMIC3 signature activity (18). Thus, a total of 10 of 14 samples with a high HRD/COSMIC3 signature activity could be explained by genomic alterations or downregulation of gene expression in BRCA1, BRCA2, PALB2, or RAD51C (Fig. 2B).

Notably, we also observed 4 samples that did not have clear DNA alterations or mRNA downregulation of BRCA1, BRCA2, PALB2, or RAD51C but nevertheless had enrichment of the HRD/COSMIC3 mutational signature at a level equal to or greater than those samples in the HRD-altered class (Fig. 2B and C). We conducted a broader examination of genes involved in DDR, including those responsible for HR, nonhomologous end joining, base excision repair, nucleotide excision repair (NER), and DDR checkpoint responses. One patient with a high activity of the HRD/COSMIC3 signature had an ERCC2^N238S mutation in a conserved helicase domain of this key enzyme involved in NER. This ERCC2^N238S mutation corresponds to one of the recurrent mutational hotspots observed in patients with muscle-invasive urothelial carcinoma who achieved a complete response to neoadjuvant cisplatin-based therapy (21). ERCC2 mutations have been associated with a separate mutational signature that has a significant overlap with the HRD/COSMIC3 signature (20); thus, the apparent enrichment of the HRD/COSMIC3 signature for this patient with PDAC with an ERCC2^N238S mutation may be attributed to the presence of an ERCC2 mutational signature. Notably, this patient experienced a partial response to platinum-based FOLFIRINOX therapy (5-FU, leucovorin, irinotecan, and oxaliplatin), but progressed after 4 to 5 months of treatment. For the remaining three samples with unexplained HRD/COSMIC3 signature enrichment, we did not observe other mutations or copy-number events in recognized DDR genes. Thus, our data suggest that the HRD/COSMIC3 signature analysis in WES data may detect additional patients with HRD not identified solely by mutation profiling of specific genes known to be related to HR.

Identification of germline variants associated with PDAC can have important implications for treatment of the proband as well as for counseling and genetic screening of family members. Thus, in addition to somatic mutation and copy-number analysis, we simultaneously interrogated each patient's germline sequencing data for pathogenic/likely pathogenic variants in 81 genes (Supplementary Table S3). In total, we observed pathogenic/likely pathogenic germline variants in 18% (13/71) of patients (Table 1; Fig. 1). These patients were referred for genetic counseling and outreach to family members (Table 1).

Pathogenic/likely pathogenic germline variants were observed in several DDR genes, including BRCA1 (n = 2), BRCA2 (n = 3), and ATM (n = 3). An additional pathogenic CHEK2 deletion was observed through commercial testing in a patient who was referred due to a strong family history of malignancy (Table 1). Notably, our CLIA-certified WES platform was not validated for detection of exon-level CNAs in the germline at the time of this study. However, reexamination of the germline WES data confirmed the CHEK2 deletion seen on commercial testing.

All 5 patients with a germline mutation in BRCA1 or BRCA2 demonstrated evidence of enrichment in the genomic HRD/COSMIC3 signature (Fig. 2B), as well as LOH through somatic copy-number loss of the wild-type allele (Table 1). However, only 1 of 3 patients with a germline ATM mutation harbored a somatic alteration in the other allele. A somatic second hit was also not identified for patients with germline pathogenic/likely pathogenic mutations in BLM, FANCA, FANCL, or RAD50. Beyond the BRCA1/2-mutant cases, none of the patients harboring other germline DDR gene mutations showed evidence of HRD/COSMIC3 signature enrichment (Fig. 2B). The mean age at diagnosis did not significantly differ between cohorts with or without a pathogenic/likely pathogenic germline mutation (60.2 vs. 63.9 years, respectively; two-tailed t test, P = 0.14).

Recent gene expression studies have identified subtypes of PDAC with prognostic and biological relevance (7, 12–14). These studies converge on at least two major neoplastic subtypes of PDAC including a squamous/basal-like/quasimesenchymal subtype and a classic/pancreatic progenitor subtype. To investigate these neoplastic PDAC subsets within our metastatic biopsy cohort, we performed RNA-seq on a separate biopsy specimen from that used for WES. We achieved successful RNA extraction and sequencing from 80% (63/79) of patients (Supplementary Fig. S1). Consistent with our prior observations in primary PDAC specimens, we observed clear distinctions at the RNA level of the basal-like and classic subtypes (ref. 14; Fig. 3A). However, samples with low neoplastic cellularity were more difficult to classify with the basal-like and classic subtype gene sets, and several of these samples showed a strong association with gene expression from normal liver tissue (Fig. 3A and B; Supplementary Fig. S5A), suggesting that the biopsy may have captured primarily adjacent liver parenchyma rather than the target tumor lesion. Moreover, integrated analysis of tumor and normal gene expression enabled clustering of samples by tumor-specific subtype and site of biopsy as well as identification of outlier samples based on tumor type, atypical genetic lesions, or predominant contribution from adjacent normal or stromal gene expression (Supplementary Fig. S5B).

In addition to neoplastic subtypes of PDAC, two stromal subtypes have been identified: “normal” and “activated” stromal subtypes (14). To capture a single composite stroma signature, we generated a merged “stroma score” reflecting the combined total expression of the top 25 activated and top 25 normal expressed stroma genes. Notably, biopsies from metastatic liver lesions on average demonstrated lower stroma scores than those from other sites or compared with primary tumor resection specimens (Fig. 3C). Comparison of ABSOLUTE neoplastic cellularity derived from WES revealed a trend toward higher neoplastic cellularity for liver lesions (mean 0.39) versus pancreatic biopsies or resections (mean 0.30; two sample t test, P = 0.07; Supplementary Fig. S2B). Despite liver metastases showing on average lower stroma scores, many samples did show elevated scores at a level similar to those seen in primary resections or biopsies of other sites. Thus, the stroma score shows important differences according to site of biopsy but also significant interpatient variability within each biopsy site.

Genomic analyses of primary specimens have suggested that approximately 40% of patients with PDAC may harbor clinically relevant genomic events that could potentially affect treatment decisions (12). However, most patients with PDAC do not undergo timely genomic analysis to identify these events for clinical decision-making. Leveraging our rapid turnaround CLIA-certified sequencing program, we performed real-time assessment of genomic data for use in clinical decision-making (Supplementary Fig. S6A; Supplementary Table S4). Excluding common events in KRAS or CDKN2A, 48% (34/71) of patients within this cohort had cancers with at least one genomic alteration that could potentially confer eligibility for current clinical trials or support off-label usage of an agent approved for another indication (Supplementary Fig. S6A). Furthermore, 11% (8/71) of the patients had cancers with two or more such events, suggesting a potential basis for genotype-driven combination therapy trials.

KRAS mutations were observed in 90% (64/71) of patients in our cohort. Although this alteration has been used to enroll patients onto clinical trials of MAPK-directed therapy, these trials have demonstrated limited efficacy to date (22). However, in the subset of KRAS wild-type tumors, we observed mutations and CNAs in additional MAPK pathway activating genes, such as BRAF mutations (see below), a ROS1 translocation and high-level FGFR1 amplification (Fig. 1; Supplementary Fig. S6A). Across the entire cohort, we observed multiple other alterations that could warrant experimental therapies, including RNF43 mutation (e.g., porcupine inhibitor), AKT2 amplification (e.g., AKT inhibitor), TSC2 mutation (e.g., MTOR inhibitor), MYC amplification (e.g., bromodomain inhibitor), and CDK4 amplification (e.g., CDK4 inhibitor; Supplementary Fig. S6A). Moreover, sequencing identified other lesions that may contraindicate therapy with certain agents, such as RB1 mutations (n = 4 patients) and CDK4/6 inhibition.

A total of 24% (17/71) of patients enrolled on the PancSeq study were treated with an experimental agent, either through enrollment onto a clinical trial or through off-label use of an approved agent (Table 2). In particular, genomic information from WES dictated the choice of experimental agent in 15% (11/71) of cases. Moreover, 18% (13/71) of patients had a clinically relevant germline mutation necessitating referral for genetic counseling (Table 1). Thus, accounting for both new therapeutic options and genetic counseling indications, a total of 30% (21/71) of patients enrolled on the PancSeq protocol experienced a change in clinical management as a result of the obtained genomic data (Tables 1 and 2).

In 20% of patients, we observed germline and/or somatic alterations in one or more of the following DDR genes: BRCA1, BRCA2, PALB2, ATM, and CHEK2 (Fig. 1; Supplementary Fig. S6B). When considering mutations in a wider spectrum of genes across several DDR classes, as well as specific mutational signatures consistent with HRD, we identified a total of 44% (31/71) of patients displaying genomic evidence of potential DDR deficiency (Table 1; Supplementary Fig. S6B). DDR gene mutations may confer increased sensitivity to platinum chemotherapy (9). Moreover, BRCA1 or BRCA2 gene mutations have been reported to confer sensitivity to poly-ADP polymerase (PARP) inhibition in preclinical models and early clinical trials of PDAC (9, 23, 24). All 6 patients in the cohort with germline or somatic mutations in the BRCA1 or BRCA2 genes demonstrated evidence of the HRD/COSMIC3 mutational signature (Supplementary Fig. S6B). By contrast, none of the patients with mutations in ATM, ATR, or CHEK2 demonstrated enrichment of the HRD/COSMIC3 signature. All six patients with BRCA mutations received an oxaliplatin-based chemotherapy regimen (FOLFIRINOX or FOLFOX) in the first or second-line setting, and all demonstrated some degree of radiographic response to these regimens (Fig. 1; Supplementary Table S4). Two of these patients with a germline BRCA1 mutation were subsequently enrolled onto a randomized trial of the PARP inhibitor olaparib versus placebo for maintenance therapy after receipt of 4 to 6 months of FOLFIRINOX (NCT02184195). Two further patients with a BRCA2 mutation received off-label olaparib, including 1 patient with a germline mutation and 1 with a somatic mutation. Besides patients with these BRCA1/2 mutations, 2 other patients with DDR gene mutations were treated with a PARP inhibitor (Table 2). Eight of 10 patients with ATM, ATR or CHEK2 mutations were treated with an oxaliplatin-based chemotherapy regimen and 5 patients demonstrated clinical benefit as defined by partial response or stable disease at first radiographic follow-up scans.

As noted above, 2 patients with BRCA2 alterations were treated with off-label olaparib. The patient harboring a somatic BRCA2 mutation was a 45-year-old man who presented with jaundice and abdominal pain and was diagnosed with metastatic PDAC involving the liver and lymph nodes, with a serum CA19–9 of 3,592 U/mL on presentation. He underwent biopsy of a metastatic liver lesion which confirmed a poorly differentiated PDAC. Genome sequencing on the PancSeq protocol revealed KRAS (p.G12R), TP53 (p.M246fs), and somatic BRCA2 (p.LS298fs) mutations (Fig. 4A). He received first-line therapy with the platinum-containing regimen FOLFIRINOX and experienced normalization of serum CA19-9 levels (Fig. 4B) and a complete radiographic response to therapy within 5 months of treatment (Fig. 4C). FOLFIRINOX was discontinued after a total of 13 two-week cycles due to transaminitis and neuropathy. The patient chose to forego further cytotoxic chemotherapy and was initiated on off-label olaparib 300 mg twice daily as maintenance therapy. He remains free of radiographically detectable disease 28 months after diagnosis and 20 months after beginning olaparib (Fig. 4C).

Within our CLIA-certified cohort, we discovered 2 patients with in-frame deletions in the BRAF oncogene (Fig. 5A). This class of deletions near the alphaC-helix region of the kinase domain has recently been shown to occur in KRAS wild-type PDAC and activates the protein to drive MAPK signaling (12, 25, 26). Furthermore, PDAC cell lines grown in vitro or as in vivo xenografts have been shown to be sensitive to MAPK inhibition, although this approach had not been tested in humans (26). To further define the frequency of this BRAF alteration in PDAC, we investigated a larger collection of samples (N = 406) profiled with a targeted genome sequencing panel at Dana-Farber Cancer Institute (27, 28) and found four KRAS wild-type tumors that harbored the same in-frame deletion (p.N486_P490del) and one additional KRAS wild-type tumor with a small, likely oncogenic in-frame insertion (p.T599dup). Thus, BRAF in-frame insertions or deletions occurred in approximately 10% of our patients with KRAS wild-type PDAC or 1% of all patients with PDAC (12, 25, 26).

In one case, a 66-year-old woman presented with dyspepsia and weight loss and was diagnosed with PDAC and liver metastases. She underwent first-line therapy with FOLFIRINOX but experienced progression of disease after only 5 two-week cycles. She underwent a biopsy and WES on the PancSeq protocol and then began second-line therapy with gemcitabine plus nab-paclitaxel that was discontinued after 6 four-week cycles due to disease progression (Fig. 5B). Sequencing revealed a BRAF in-frame deletion (p.N486-P490del) and TP53 mutation (p.V157G; Fig. 5A and B). Given that cell lines harboring this mutation were sensitive to MEK inhibitors but resistant to selective BRAF inhibitors in preclinical models (26), she was initiated on off-label treatment with the MEK1/2 inhibitor trametinib, which is FDA approved for use in BRAF^V600E-mutant melanoma (Fig. 5B; refs. 29–31). Within 4 weeks of initiating therapy, her serum CA19-9 had fallen from 36,000 to 8,100 U/mL, and the first restaging scan done 8 weeks after initiation of trametinib showed a partial response to therapy (Fig. 5C and D). Serial plasma samples were collected to measure the fractional abundance of BRAF- and TP53-mutant alleles in circulating cell-free DNA (cfDNA) by droplet-digital PCR (ddPCR; refs. 32, 33). cfDNA measurements for BRAF and TP53 alleles (two clonal alterations in this tumor) revealed a dramatic decline in response to trametinib therapy that mirrored the radiographic findings (Fig. 5E). After 5 months on trametinib therapy, a rise in the fractional abundance of the BRAF- and TP53-mutant alleles in cfDNA was observed. After 6 months of therapy, radiographic progression was identified. Evaluation of a more comprehensive panel of genes within cfDNA was pursued through a commercial test (Guardant360) at the time of progression and demonstrated the emergence of multiple subclonal mutations in the MAP2K2 (MEK2) gene (Fig. 5E). Notably, these mutations are homologous to MEK1 mutations that have been previously described to confer resistance to MEK inhibition in vitro in mutagenesis studies in BRAF-mutant melanoma (34). Retrospective analysis of the MEK2 mutations in serial plasma samples collected through the patient's treatment course revealed emergence of these mutant alelles in concert with increases in fractional abundance of mutant BRAF and TP53 alleles (Fig. 5E). Thus, these data likely reflect the emergence of heterogeneous polyclonal resistance mechanisms that evolved under the selective pressure of trametinib therapy.

Given the emergence of MAP2K2 resistance mutations while on trametinib therapy, the patient was subsequently treated with ulixertinib/BVD-523, an inhibitor of ERK1/2 (which signals downstream of MEK1/2), on a single-patient investigational new drug application. After treatment with ulixertinib/BVD-523 for 17 days, cfDNA analysis by ddPCR demonstrated a rapid decline in the fractional abundance of the BRAF- and TP53-mutant alleles (Fig. 5E). The MEK2 resistance alleles also rapidly decreased in absolute frequency and in relative abundance with respect to the overall tumor burden (as measured by the total MEK2/TP53 ratio; Fig. 5E). Unfortunately, the patient's functional status was quickly declining as she initiated therapy with ulixertinib/BVD-523, and this medication was stopped after 3 weeks of therapy. A last blood collection after cessation of ulixertinib/BVD-523 demonstrated rebound of the fractional abundance of the BRAF- and TP53-mutant alleles. The patient expired at home several weeks later with hospice services. This patient's clinical course suggests that a BRAF in-frame deletion may serve as an important biomarker for response to MAPK inhibition in patients with pancreatic cancer.

Many barriers to precision medicine exist for patients with PDAC, including low neoplastic cellularity of tumors and the aggressive nature of the disease that makes timely biopsy and genomic analysis difficult. We have established an integrated biopsy program for patients with advanced PDAC that enables CLIA-certified genomic profiling and rapid turnaround of WES data to referring clinicians. To overcome the challenges of low neoplastic cellularity, we have performed deep WES on small-volume tumor biopsies and germline DNA and have used analytic algorithms to accurately quantitate tumor DNA content and to identify mutations and CNAs. In addition, we have identified molecular driver alterations and clinically relevant subsets of disease in real time, during the course of the patient's illness. In an initial biopsy cohort of 79 patients, we obtained high-quality genomic information for 71 patients and identified potentially actionable somatic and germline alterations in 48% of cases. These include 26 patients harboring tumors with DDR gene mutations and 7 with KRAS wild-type tumors, including 2 with BRAF alterations and 1 with a ROS1 translocation. We have demonstrated how these data can affect clinical decision making, utilizing molecular information to treat multiple patients on clinical trials or with use of off-label targeted therapies. Nevertheless, further study will be necessary to confirm the rate and therapeutic implications of actionable alterations in a multi-institutional population of patients with PDAC. Overall, 30% (21/71) of enrolled patients experienced a change in clinical management as a result of genomic data, including 15% of patients for whom genomic information dictated the choice of an experimental agent and 18% of patients whose germline data warranted referral for genetic counseling. These numbers far exceed the average experience for patients with PDAC, and these data support the implementation of genomic evaluation as a standard clinical practice in patients with advanced PDAC.

We identified pathogenic/likely pathogenic germline mutations in 18% of patients in our cohort of patients with advanced PDAC. Analysis of germline variants was performed within a set of 81 genes included in our CLIA-certified analysis pipeline (Supplementary Table S3). Thus, this analysis may underestimate the true number of patients with PDAC with a hereditary pancreatic cancer predisposition. However, 18% is higher than the approximately 4% to 12% incidence of pathogenic germline variants that has been reported in recent primary tumor cohorts (12, 35, 36). This higher rate of germline variants may be due to the larger number of genes examined, different distribution of Ashkenazi Jewish patients (who are known to have higher rates of germline variants), the selected patient population who provided informed consent for research biopsy, and potential enrichment of germline variants in patients with advanced PDAC. Regarding the latter point, a similar observation of enrichment for germline mutations in DDR genes has been made in metastatic prostate cancer (37). Nevertheless, the frequency of germline mutations in patients with advanced PDAC will require further validation. Annotation of Ashkenazi Jewish descent was incomplete for patients included in the current study. However, only 3 of the 13 identified germline alterations were known to be Ashkenazi Jewish founder mutations.

Although all tumors with germline mutant BRCA1 and BRCA2 genes demonstrated somatic mutation or LOH of the second allele, only 2 of 9 (22%) of the other identified germline mutant genes demonstrated somatic mutation or LOH. The functional implications for loss or retention of the wild-type allele remain an important unanswered question that is worthy of further investigation. Given the prevalence of germline mutations in patients with PDAC, several groups now advocate for universal multigene germline testing for all patients, irrespective of family history or age at diagnosis (35, 38–40). The most effective approach to implementing universal germline testing as well as the optimal bioinformatic and functional frameworks for interpreting these data remain important to define.

As has been suggested in prior studies of primary PDAC resection specimens (7, 9, 12), we observed a striking prevalence of germline and somatic mutations in DDR genes in our cohort of patients with advanced PDAC. Beyond the 37% (26/71) of patients with DDR gene mutations or CNAs, 9 of whom had HRD/COSMIC3 signature enrichment, an additional 7% (5/71) of patients had enrichment of an HRD/COSMIC3 signature but no clear associated HR gene alteration. Two of these cases could be explained by downregulation of mRNA expression of RAD51C; however, three samples had HRD/COSMIC3 signature activity but no clear causal genetic or gene expression feature to explain the signature. Although the therapeutic implications of HRD/COSMIC3 signature enrichment without an identifiable mutation need further validation in preclinical models, this result suggests that more patients may have functional DDR deficiency than is detectable by DNA mutational profiling alone, thus arguing in favor of performing more global genomic characterization approaches such as WES in concert with integrative analyses of gene expression in patients with PDAC. Notably, whole-genome sequencing of PDAC has also suggested that chromosomally unstable tumors with a large number of structural variation events are associated with a BRCA mutational signature (9). We were unable to similarly evaluate the number of structural rearrangements in these tumors using WES, and additional studies will be needed to understand the association of DDR gene mutations, HRD/COSMIC3 mutational signature enrichment, and structural variation events.

In this study, 6 patients with DDR gene mutations were treated off-label or enrolled on clinical trials of PARP inhibitor therapy. Understanding the gene- and allele-specific differences for DDR genes conferring sensitivity to platinum chemotherapy, PARP inhibition, or other inhibitors of DDR checkpoints will be critical to enabling effective stratification of patients with PDAC onto efficacious therapies. As noted above, the functional implications of genomic correlates on therapeutic responsiveness, including somatic LOH versus retention of the wild-type allele or COSMIC3/HRD signature activity, also need further investigation in patient-derived models as well as in human clinical trials.

We and others have noted the importance of alternative oncogenic driver events in KRAS wild-type PDAC (10, 12, 41). Here, we demonstrate multiple oncogenic and targetable lesions that occur in KRAS wild-type tumors, including alterations in BRAF, ROS1, FGFR1, and other genes. In particular, we have identified in-frame deletions in BRAF in 2 patients in our cohort. These lesions have been reported to result in oncogenic activation (25, 26). We report the first human therapeutic experience with MAPK inhibition in a patient harboring a BRAF in-frame deletion. We demonstrate substantial clinical benefit in a patient who had a partial response to the MEK1/2 inhibitor trametinib (Fig. 5). Furthermore, by serial monitoring of driver alterations in plasma cfDNA through multiple lines of treatment, we provide molecular evidence for disease response and progression, as well as the emergence of heterogeneous MEK2 resistance alleles that correspond to radiographic progression of disease on trametinib. This patient was subsequently treated with an ERK inhibitor after progression on trametinib and showed a second decline in mutant BRAF and TP53 alleles within cfDNA and suppression of MAP2K2 resistance alleles, suggesting further evidence of response to MAPK inhibition. A second patient with rapidly progressive BRAF-mutant disease was also treated with off-label trametinib but failed to show a response. This heterogeneity of primary and secondary resistance mechanisms will necessitate effective combination therapy strategies with MAPK inhibition. These data suggest that a larger multicenter clinical trial with proper molecular correlates, including cfDNA monitoring of therapeutic response and resistance, should be developed to fully investigate the therapeutic efficacy of MAPK inhibition in patients with activating oncogenic BRAF deletions.

In parallel to WES of DNA alterations, we also performed RNA-seq and demonstrated that RNA signatures of neoplastic PDAC subtypes are readily discernible from small-volume metastatic biopsies. The two primary neoplastic subtypes of PDAC have been shown to have prognostic importance and may correlate with chemosensitivity (14, 42); thus, identifying these subtypes and proactively incorporating this stratification into clinical trials remains an important translational priority. Detection of a high stromal signature was consistent across primary tumors and several metastatic sites but more variable in liver biopsy specimens, suggesting that the stromal makeup of liver lesions may be distinct from that of primary tumors. This observation has been made recently using an automated histologic approach (43) and will need rigorous follow-up with histologic and molecular approaches. Stromal fibroblasts have been proposed to play an important role in promoting PDAC progression and in blunting chemotherapeutic response (42, 44, 45); however, attempts to target fibroblasts in mouse models as well as clinical trials have yielded conflicting results (46–48). Additional trials of stroma-directed therapies are under way, including vitamin D receptor agonists to modulate stromal gene expression and improve chemosensitivity (42). Our data highlight the importance of biopsy and RNA-seq analysis to identify patients and even specific metastatic lesions that may respond to stroma-directed therapy.

Together with recent work from other PDAC referral centers (41, 49), this study demonstrates the feasibility and value of real-time genomic characterization of advanced PDAC and provides a path forward for treatment of patients with PDAC with molecularly defined therapy. To harness all potential therapeutic opportunities in this highly aggressive disease, we propose that biopsy-based genomic analysis early in a patient's treatment course should become standard of care for all patients with PDAC.

Investigators obtained informed, written consent for each patient enrolled to the PancSeq protocol (DF/HCC #14-408), and this study was performed in accordance with standard ethical guidelines approved by the Dana-Farber/Harvard Cancer Center IRB. DNA and RNA were extracted from tumor samples (and normal whole blood for germline DNA control), and WES was performed in a laboratory certified by CLIA (#22D2055652; refs. 12, 50). WES data were processed through the Broad Institute “Picard” pipeline (http://picard.sourceforge.net/), generating a BAM file for each sample. Mutation calling was performed using the MuTect algorithm (51). MutSigCV2 was used to determine significantly mutated genes (52). GISTIC2.0 was used to identify recurrent deletions and amplifications (53). ABSOLUTE (16) was used to determine purity, ploidy, and whole-genome doubling status using allelic copy-number data along with the allelic fraction of all somatic mutations as input. Annotated WES data were cross-referenced with a curated list of 81 PDAC-relevant genes, and variants in these genes were reviewed by a certified clinical geneticist. A report of clinically relevant germline and somatic events was returned to the referring clinician detailing somatic mutations, small insertions/deletions, and CNAs as well as pathogenic/likely pathogenic germline alterations. Only germline variants with a population frequency of <1% upon comparison with the ExAC database (http://exac.broadinstitute.org/) were retained for review as pathogenic/likely pathogenic. Mutational signature analysis was performed on the set of SNVs in our dataset using SignatureAnalyzer, as previously described (17–20). RNA-seq was performed on poly-A–selected mRNA at the Broad Institute, and gene-expression signatures were derived from Moffitt and colleagues (14). cfDNA analysis was performed by ddPCR as previously described (32, 33) or through a commercial assay (Guardant360; ref. 54). See also the Supplementary Experimental Methods for further description of experimental procedures. Data in this study have been deposited in dbGaP under accession number phs001652.v1.p1.

J.M. Cleary reports receiving a commercial research grant from Merck. G.I. Shapiro reports receiving commercial research grants from Lilly, Merck/EMD Serono, Sierra Oncology, Pfizer, and Merck and is a consultant/advisory board member for Pfizer, G1 Therapeutics, Lilly, Roche, and Merck/EMD Serono. R.B. Lanman has ownership interest (including stock, patents, etc.) in Guardant Health, Inc. M.B. Yurgelun reports receiving a commercial research grant from Myriad Genetic Laboratories, Inc. C.S. Fuchs is a consultant/advisory board member for CytomX, Sanofi, Eli Lilly, Merck, and Entrinsic Health. L.A. Garraway is Sr. Vice President at Eli Lilly and Company and has ownership interest (including stock, patents, etc.) in Tango Therapeutic and Foundation Medicine. B.E. Johnson reports receiving a commercial research grant from Toshiba and other commercial research support from Novartis. N. Wagle has ownership interest (including stock, patents, etc.) in Foundation Medicine. R.B. Corcoran reports receiving commercial research grants from AstraZeneca and Sanofi and is a consultant/advisory board member for Astex, Amgen, Roivant, Shire, Symphogen, Taiho, WarpDrive Bio, Avidity Biosciences, BMS, FOG Pharma, Genentech, LOXO Oncology, Merrimack, N-of-One, and Roche. B.M. Wolpin reports receiving a commercial research grant from Celgene. No potential conflicts of interest were disclosed by the other authors.

Conception and design: A.J. Aguirre, L.K. Brais, A. Da Silva, N.J. McCleary, S.G. Silverman, D.J. Welsch, R.J. Mayer, W.C. Hahn, R.B. Corcoran, S.L. Carter, B.M. Wolpin

Development of methodology: A.J. Aguirre, R.A. Moffitt, A.A. Ghazani, M. Hazar-Rethinam, L.K. Brais, D.A. Rubinson, R.B. Lanman, D.J. Welsch, L.A. Garraway, R.B. Corcoran, S.L. Carter, B.M. Wolpin

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): A.J. Aguirre, J.A. Nowak, L.K. Brais, D. Ragon, E. Reilly, A. Da Silva, B. Nadres, E.E. Van Seventer, H.A. Shahzade, J.P. St. Pierre, K.P. Burke, T. Clancy, J.M. Cleary, L.A. Doyle, K. Jajoo, N.J. McCleary, J.E. Murphy, K. Ng, A.K. Patel, K. Perez, D.A. Rubinson, M. Ryou, G.I. Shapiro, E. Sicinska, S.G. Silverman, R.J. Nagy, D.J. Welsch, M.B. Yurgelun, C.S. Fuchs, L.A. Garraway, J.L. Hornick, M.H. Kulke, R.J. Mayer, P.B. Shyn, N. Wagle, R.B. Corcoran, B.M. Wolpin

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): A.J. Aguirre, J.A. Nowak, N.D. Camarda, R.A. Moffitt, A.A. Ghazani, M. Hazar-Rethinam, S. Raghavan, J. Kim, D. Ragon, D. McCabe, A. Babic, H.A. Shahzade, T. Clancy, N.J. McCleary, K. Ng, M.H. Rosenthal, D.A. Rubinson, R.J. Nagy, R.B. Lanman, D.J. Welsch, M.B. Yurgelun, C.S. Fuchs, G. Getz, B.E. Johnson, J.W. Miller, D.A. Tuveson, J.J. Yeh, R.B. Corcoran, S.L. Carter, B.M. Wolpin

Writing, review, and/or revision of the manuscript: A.J. Aguirre, J.A. Nowak, N.D. Camarda, R.A. Moffitt, A.A. Ghazani, M. Hazar-Rethinam, S. Raghavan, J. Kim, L.K. Brais, A. Babic, A. Da Silva, T. Clancy, J.M. Cleary, K. Jajoo, N.J. McCleary, J.A. Meyerhardt, K. Ng, M.H. Rosenthal, G.I. Shapiro, S.G. Silverman, R.J. Nagy, R.B. Lanman, D. Knoerzer, M.B. Yurgelun, C.S. Fuchs, J.L. Hornick, B.E. Johnson, M.H. Kulke, R.J. Mayer, P.B. Shyn, D.A. Tuveson, J.J. Yeh, W.C. Hahn, R.B. Corcoran, S.L. Carter, B.M. Wolpin

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): A.J. Aguirre, N.D. Camarda, L.K. Brais, M.W. Welch, E. Reilly, K. Anderka, K. Helvie, J.P. St. Pierre, J.A. Meyerhardt, L.A. Garraway, J.L. Hornick, B.E. Johnson, M.H. Kulke, N. Wagle

Study supervision: A.J. Aguirre, L.K. Brais, N. Oliver, D.J. Welsch, C.S. Fuchs, W.C. Hahn, S.L. Carter, B.M. Wolpin

Other (tissue acquisition): L. Marini

Other (provided drug and review): D. Knoerzer

Other (performed biopsies): P.B. Shyn

We dedicate this work to the memory of our patients, including Dr. Andrew Tager, a beloved colleague, mentor, and friend whose courageous battle with pancreatic cancer continues to inspire our pursuit of improved diagnosis and treatment for this difficult disease.

We acknowledge primary research support from the Lustgarten Foundation (A.J. Aguirre, S. Raghavan, W.C. Hahn, D.A. Tuveson, and B.M. Wolpin), and the Dana-Farber Cancer Institute Hale Center for Pancreatic Cancer Research (A.J. Aguirre, S Raghavan, W.C. Hahn, and B.M. Wolpin), with additional support from Hope Funds for Cancer Research (A.J. Aguirre and S. Raghavan), the Doris Duke Charitable Foundation (A.J. Aguirre), the Conquer Cancer Foundation of ASCO Young Investigator Award (A.J. Aguirre and S. Raghavan), the Broman Fund for Pancreatic Cancer Research (K. Ng), National Institutes of Health National Cancer Institute K08 CA218420-01 (A.J. Aguirre), P50CA127003 (A.J. Aguirre, M.B. Yurgelun, W.C. Hahn, R.B. Corcoran, and B.M. Wolpin), U01 CA224146 (A.J. Aguirre and W.C. Hahn), U01 CA210171 (B.M. Wolpin), R01 CA199064 (J.J. Yeh), The Harvard Clinical and Translational Science Center UL1 TR001102 (A.J. Aguirre and S. Raghavan), the DFCI Medical Oncology Translational Research Project Award (M.B. Yurgelun), the Pancreatic Cancer Action Network (A.J. Aguirre and B.M. Wolpin), the Noble Effort Fund (B.M. Wolpin), the Peter R. Leavitt Family Fund (B.M. Wolpin), the Wexler Family Fund (B.M. Wolpin), and Promises for Purple (B.M. Wolpin).