Genomic and Molecular Analyses Identify Molecular Subtypes of Pancreatic Cancer Recurrence.

Pancreatic cancer (PC) remains a highly lethal malignancy, and most patients with localized disease that undergo surgical resection still succumb to recurrent disease. Pattern of recurrence after pancreatectomy is heterogenous, with some studies illustrating that site of recurrence can be associated with prognosis. Another study suggested that tumors that develop local and distant recurrence can be regarded as a homogenous disease with similar outcomes. Here we investigate novel molecular determinants of recurrence pattern after pancreatectomy for PC.

Recurrence patterns were classified as liver, lung, local only, and other distant, whereas the no recurrence group was defined as those that did not develop any recurrence during the study period (minimum of 24 months of follow-up). Genomic, transcriptomic, immunohistochemical, and clinical data of primary resected tumor specimens from the Australian Pancreatic Cancer Genome Initiative (Australian contribution to the International Cancer Genome Consortium) PC cohort were used in the molecular analysis (n = 435). Full methods and description of cohort undergoing each analysis are available in the Supplementary Methods section.

Liver metastases after pancreatectomy (median survival, 16.0 months) was associated with significantly worse disease-specific survival than lung (29.7 months) and local recurrence (25.6 months; P < .001; Supplementary Figure 1a). Liver recurrence after pancreatectomy was associated with poor tumor differentiation (P < .001). Margin status (P = .217) and lymph node status did not predict recurrence patterns (P = .062).

Supplementary Figure 1

(A) Kaplan-Meier curve of disease-specific survival stratified by recurrence pattern in all 3 cohorts (total n = 1087). Only those with recurrent disease (n = 704) are shown. (B) Proportion of each recurrence pattern in patients with classical and squamous subtype. Squamous subtype enriched for liver recurrence (P < .001); classical subtype associated with lung recurrence (P = .007). Bar chart demonstrates the relative proportions of each molecular subtype in different recurrence patterns. Proportion is based on the frequency of each recurrence pattern per molecular subtype. (C) Proportion of gene mutations in each recurrence pattern. P value calculated using chi-square. (D) Table of significantly mutated gene analysis in recurrence patterns. A significant q value (<0.05) represents an association between mutation and recurrence pattern. Only BRAF (liver) and RNF43 (no recurrence) were significant in specific recurrence patterns. (E) Table of clinical, molecular, and pathologic features of RNF43 mutants in analyzed cohort.

Patients from the Australian Pancreatic Cancer Genome Initiative cohort were categorized based on transcriptional expression of the primary tumor as squamous (basal-like) or classical. Squamous tumors correlated with liver recurrence and short disease-free survival after pancreatectomy (P < .001; Figure 1a, Supplementary Figure 1b). Conversely, lung recurrence significantly correlated with the classical subtype (P = .007).

Figure 1

(A) Heatmap of RNA sequenced cases with disease recurrence pattern. Heatmap demonstrates recurrence pattern, molecular subtype, relative expression of key genes of PC, classical and squamous subtype lineage, and outcome. (B) KRAS allelic imbalance and whole genome doubling in recurrence patterns. Scale is based on proportion of overall cohort. Dark shade represents no whole genome doubling and light shade whole genome doubling for each recurrence pattern stratified by KRAS allelic status. P value calculated for KRAS imbalance in liver versus all other recurrence patterns using chi-square test. (C) Relevant molecular pathways enriched in specific recurrence patterns categorized by gene module defined by Bailey et al. Size of bar proportional to statistical weight, horizontal scale numerical for P. (D) Stromal signatures as defined by Puleo et al for each recurrence pattern. P calculated as analysis of variance between groups. (E) Heatmap of relative immune cell infiltration histoscore in different recurrence patterns. Histoscore represents cumulative score for all tissue microarray cores per patient.

The proportion of BRAF and RNF43 mutations were higher in the liver and no recurrence groups, respectively (Supplementary Figure 1c). These failed to be below the P < .05 significance level, likely due to insufficient power due to the infrequent mutations. Significantly mutated gene analyses identified RNF43 mutations (q < 0.001) to be associated with the no recurrence group and BRAF mutations (q = 0.020) to be associated with the liver recurrence group (Supplementary Figure 1d). No other gene mutation was significantly associated with a recurrence pattern. Only 1 RNF43 mutant was pathologically described as an intraductal papillary mucinous neoplasm (IPMN) with invasion (Supplementary Figure 1e). In those that developed no recurrence, almost all patients (90%) had balanced KRAS allelic status (Supplementary Figure 2a), whereas KRAS imbalance was a feature of primary tumors that developed liver recurrence (Figure 1b, Supplementary Figure 2a). KRAS wild type or mutation type did not associate with any recurrence pattern (Supplementary Figure 2a).

Supplementary Figure 2

(A) Table demonstrating frequency of KRAS allelic imbalance and whole genome doubling in recurrence patterns of PCAWG cohort. (B) Transcriptional modules defined as significant by Bailey et al in each recurrence group for patients with RNA sequencing data. Significant modules that are associated with recurrence patterns are highlighted with black arrow on left y-axis. Key processes and gene programs previously described by Bailey et al that define squamous and classical pancreatic (pancreatic progenitor, immunogenic, ADEX) subtypes are highlighted along right-hand y-axis. (C) Relative enrichment score of genes associated with recurrence pattern in each module that associated with recurrence pattern, from the APGI microarray cohort. Statistical significance levels are ∗≤.05, ∗∗≤.01, ∗∗∗≤.001, and ∗∗∗∗≤.0001 with the first given recurrence pattern as reference (for example, for green, yellow, and orange modules the reference is liver, for light yellow it is local, and for turquoise and grey 60 the reference is lung).

Transcriptional networks and gene programs that were identified as key features of the squamous subtype, significantly associated with liver recurrence (Figure 1a, Supplementary Figure 2b). Pathways that predispose to liver recurrence included cell cycle checkpoint activation, epidermal growth factor signaling, glycolysis, and hypoxia (Figure 1c, Supplementary Figure 2c).

Lung recurrence was strongly associated with the classical subtype and was enriched for transcriptional pathways regulating pluripotent stem cells and endoplasmic reticulum stress (Figure 1c).

Next, we investigated additional molecular differences between liver and lung recurrence, the most common metastatic sites in PC. In addition to enrichment in the gene programs described, liver recurrence was, relative to lung recurrence, enriched in pathways associated with innate immune response, interferon signaling, and antiviral response (Figure 1c).

Local recurrence was enriched for networks associated with neuronal signaling and neuron cell-cell interaction (Figure 1c). This may reflect the local infiltrative nature of these tumors into the peri-pancreatic nerve plexuses which predisposes to local recurrence despite negative resection margins.

Local and no recurrence groups were associated with transcriptional networks associated with immunogenic activation and infiltration (Figure 1c). By applying the stromal subtypes described by Puleo et al, enrichment of the structural vascularized stroma subtype (P = .020) was found in the local recurrence group (Figure 1d). Immunohistochemistry of the International Cancer Genome Consortium (ICGC) cohort showed that those that developed no recurrence were enriched for infiltration of CD3+ T cells (P = .009), and the local and no recurrence groups were enriched for CD163+ macrophages (P = .029; Figure 1e). The liver and other recurrence groups were relatively enriched for CD68+ macrophages (P = .034).

Here we demonstrate the impact of novel, previously undefined molecular features of the primary tumor on spatiotemporal recurrence patterns after pancreatectomy for PC. Liver recurrence is associated with significantly shorter disease-free and overall survival. TP63 expression, cell cycle checkpoint activation, epidermal growth factor signaling, glycolysis, and hypoxia are features of the squamous (basal-like) subtype and strongly associated with liver recurrence. When compared with lung recurrence, the liver recurrence group was enriched for inflammatory pathways likely driven by chronic inflammation secondary to genomic instability and constitutional STING (Stimulator of Interferon Genes) activation that is a feature of the squamous subtype.

Lung recurrence was associated with the classical subtype and longer disease-free and overall survival. In addition, local and no recurrence groups had very similar transcriptomic profiles with favorable immune signaling compared with those that develop distant metastases based on transcriptome. The no recurrence group was enriched for CD3+ immune cell infiltration. Quantification of immune cell infiltration alone may be insufficient to delineate specific stromal signaling and its influence of this on recurrence pattern and highlights the potential contribution of a cell autonomous signaling mechanism. The no recurrence group was enriched for RNF43 mutations, yet only 1 (lung recurrence group) of the RNF43 mutants histologically resembled IPMN with invasion and this does not explain the better prognosis associated with these mutations. This highlights the association of these mutations with primary PC out with the setting of transformed IPMN.

This study is limited by only having primary tumor samples available for analysis. Parallel molecular profiling of primary and metastatic tumors, accrued through multi-institutional studies, such as Precision-Panc, will allow more detailed analyses of pathways and processes that define and promote recurrent disease.

In summary, our results demonstrate that resected pancreatic cancers should not be considered to harbor the same “systemic” disease. Liver recurrence is the dominant spatiotemporal phenotype, with primary tumors enriched for specific molecular features that differ from those that develop lung or local recurrence. Delineating these processes, and their influence on priming the metastatic niche, dissemination, and seeding of tumor cells warrants further study to inform personalized adjuvant antimetastatic agents and surveillance strategies.