We profiled and compared nonmetastatic and metastatic primary PDAC tumors and identified a six-gene signature. Although this signature was not derived on the basis of outcomes, we show that it was prognostic in a true test set of resectable PDAC patients. Importantly, our six-gene signature was independently predictive of survival, stratifying patients with median survivals of 15 compared to 49 mo, outperforming current pathological staging criteria, suggesting that our signature will be a powerful prognostic tool for patients with localized PDAC.
PDAC continues to be a devastating disease with few long-term survivors. Surgery remains the standard therapy for patients diagnosed with resectable PDAC 
. Yet with a median survival only of less than 2 y after surgery, the attendant postoperative mortality rate of 2%–6% 
, and postoperative complication and hospital readmission rates of 59% 
, the decision for surgery should be made cautiously. Therefore, improved patient selection for therapy is necessary. For the majority of patients who cannot undergo surgery, gemcitabine chemotherapy remains the best option, yet only 5%–10% of patients respond to the treatment 
. Given the current therapeutic limitations, additional prognostic tools are needed to help a patient decide whether to have surgery, and/or neoadjuvant chemotherapy, or when to consider participation in a clinical trial.
Our analysis identified a surprisingly small number of genes with differential expression between early compared to late stage primary PDAC (Table S1
). This finding suggests that primary PDAC may be largely homogenous from a global gene expression standpoint. Nonetheless, the differences that we identified appear to be clinically and therefore biologically important. Our findings of molecular differences in resected primary PDAC tumors suggest that there is subtle biological variation in these tumors that influences outcome. A review of previous published studies did not identify differential expression of our six genes 
. This finding is not surprising, as previous studies examined differential gene expression changes between either normal pancreas or chronic pancreatitis and PDAC 
. Only one study has looked at gene expression changes between PDAC of different stages 
. Ours was the first, to our knowledge, to study molecular differences between nonmetastatic versus metastatic primary tumors and identify and validate a prognostic signature for PDAC.
Of the six genes identified in this study, most do not have an obvious role in carcinogenesis. Three of the six genes demonstrated significantly higher expression in the poor prognostic groups (SIGLEC11
; Table S2
, and SIGLEC-11
have not been studied in cancer. SIGLEC-11 is thought to be expressed by tissue macrophages and also the brain microglia 
. Interestingly, a missense mutation of SIGLEC-11
(S465A) was identified in the mutation discovery screen of the recent genome-wide sequencing of PDAC 
. NFKBIZ, also called IkappaB zeta, binds to the p50 subunit of nuclear factor (NF)-kappaB and is important for interleukin-6 (IL-6) induction and may be induced by IL-1 receptor and Toll-like receptors 
. Given the prevalence of chronic pancreatitis and high degree of stromal fibrosis, it is possible that NFKBIZ may play a role in PDAC and inflammation.
KLF6 is a transcription factor and its full length transcript is thought to be a tumor suppressor gene involved in prostate, lung, and ovarian carcinogenesis 
. However a splice variant KLF6-SV1 has been shown to have oncogenic properties. The oligonucleotide probes used in the Agilent whole human genome array and the antibody against KLF6 did not differentiate between the full-length and splice variant. In agreement with a previous study 
, we found that KLF6 protein expression was higher in tumors than normal pancreas. In addition we found that higher KLF6 expression was associated with worse survival. Hartel et al. further investigated KLF6-SV1 expression in their study using real-time PCR and demonstrated that the higher KLF6 expression seen in tissues was associated with a higher ratio of KLF6-SV1 compared to full-length KLF6. Therefore our findings that KLF6 expression is higher in tumors and is prognostic is likely in agreement with this study.
Only one patient in the UNC1 cohort was treated with neoadjuvant chemotherapy compared to 80% of NEB patients who were treated with palliative chemotherapy. Although there is a possibility that our signature may be reflective of gemcitabine treatment or perhaps resistance, as NEB patients died of metastatic disease despite gemcitabine treatment, the successful application of our six-gene signature on an independent test set of patients where only 3% of patients with localized PDAC were treated with neoadjuvant therapy suggests that it is a rigorous predictor of prognosis in previously untreated patients. We found no association between our six-gene signature and whether a patient received adjuvant chemotherapy. In addition, chemotherapy treatment in this cohort, either pre- or postoperative, did not demonstrate a survival advantage.
Another concern is the validity of our hypothesis that gene expression changes at different stages of primary PDAC development may occur and be important for prognosis. Our study is in agreement with Lowe and colleagues' findings that differential gene expression changes can be identified within primary PDAC 
. However, they did not address the prognostic value of their findings. Several studies have also suggested that gene expression changes in metastasis may be found in primary tumors. In a study of molecular differences between primary tumors and metastases, Golub and colleagues identified a gene expression signature of metastasis present that could be identified in primary tumors 
. In addition, studies in melanoma have suggested that metastatic cells may be found in the parent primary tumor 
. Finally studies in breast cancer have demonstrated that gene expression changes found in breast cancer cells with metastatic potential may be prognostic and predictive of patients who will develop metastasis 
. Our study is the first to demonstrate that molecular differences in metastatic PDAC can be identified at earlier stages, and that these differences are predictive of future behavior. Whether these molecular changes are biologically associated with metastatic potential will require further investigation.
We have applied our six-gene signature to an independent dataset of 67 patients, and have validated its prognostic value. In addition, we have validated the protein expression of KLF6 in a 50-patient TMA. Although not nearly as powerful a predictor of prognosis as our six-gene signature, we found that KLF6 expression was prognostic in our 50-patient TMA. Further validation studies will be needed to see if KLF6 alone may be a useful prognostic marker as others have shown 
. Our findings suggest that the prognostic value of KLF6 is strengthened in evaluating the six genes in their entirety.
Studies of patients with resectable PDAC demonstrate median survivals of up to 22 mo, equivalent to the median survival of patients in our training and testing cohorts 
. Our finding that our six-gene signature is able to stratify patients, with startling differences in survival, suggests that it may be used to select patients for therapies. For example, for patients who are at high operative risk, knowledge of a median survival of 49 compared to 15 mo, may be helpful in the operative decision-making process. Similarly, patients who have a poor prognosis based on the six-gene signature may be considered for neoadjuvant therapy. Currently, the minority of centers use neoadjuvant therapy as a standard of care, most instead reserve this for patients with locally advanced unresectable or borderline resectable tumors. Therefore the current decision-making process is based on anatomical considerations. Our prognostic signature may refine this paradigm such that neoadjuvant therapy is offered to patients on the basis of biological considerations, regardless of resectability, and may allow us to further study and maximize the benefits of neoadjuvant treatment. In addition, as new therapies are developed, it may help to determine whether patients may require more or less aggressive treatment. Finally, our findings that there are molecular differences associated with late-stage primary tumors, which translate into differences in prognosis, suggest that the six genes in this signature should be further studied for their potential as biomarkers, and some of these genes, or the pathways that they fall into, may represent new therapeutic targets.