Using methods in integrative genomics, we predicted an association between FABP-1 and pancreatic adenocarcinoma and pancreatic adenocarcinoma with diabetes. Compared to normal controls, we found a significant positive association between increased FABP-1 staining and PaC on FFPE-TMA, strengthened by the presence of diabetes.
FABP-1 is a cytosolic chaperone protein that is expressed in the liver, kidney, lung, and GI tract . The exact role of FABP-1 has yet to be elucidated. The association of FABP-1 with pancreatic adenocarcinoma and diabetes is of interest. Plausibility of FABP-1 as biomarker for cancer comes from previous literature suggesting an association with colon, gastric, and liver cancers. The literature is variable, but loss of FABP-1 expression in colonic tissue has correlated with progression from normal colonic tissue to adenoma to carcinoma . Negative FABP-1 expression in colon cancer metastases has been associated with decreased survival, and low FABP-1 expression in colon cancer has been associated with lymph node metastasis [26, 27]. FABP-1 gene expression has been identified as a marker of circulating colorectal tumor cells via RT-PCR . On the contrary, one study associated low FABP-1 expression in colon cancer with increased survival . FABP-1 is highly expressed in gastric intestinal metaplasia and a subset of gastric adenocarcinoma . In one study, half of 62 hepatocellular carcinoma samples contained L-FABP immunoreactive tumor cells . A single study of gene array analysis in a cohort of 21 patients revealed FABP-1 overexpression (16x) in PaC compared to chronic pancreatitis and normal pancreatic tissue, though no further testing was performed to validate this association . Another group using bioinformatics methods also identified FABP-1 as a candidate molecule potentially associated with PaC, though again, no further validation was performed .
Plausibility of FABP-1 as a biomarker for diabetes comes from animal and human data. FABP-1 null mice demonstrate age dependent weight gain, though may be protected from obesity and hepatic steatosis when fed a saturated fat diet [33–36]. In humans, high levels of FABP-1 in placental homogenates have been associated with gestational DM . Two genes with variants leading to Maturity Onset Diabetes of the Young (MODY), Hepatic nuclear factor (HNF)-4alpha and HNF-1alpha, are known to directly bind to the promoter region of FABP-1 . Effects of FABP-1 are modulated through PPAR-gamma, the molecular target for the thiazolidinedione class of diabetic medications . In humans, FABP-1 polymorphisms have been associated with increased levels of fasting triglycerides/LDL in females and urinary FABP-1 excretion is associated with hypertension and coronary artery disease risk factors [40, 41]. With its up-regulation in various forms of cancer and effects on metabolism, it is plausible that FABP-1 could be a biomarker for pancreatic cancer and pancreatic cancer-associated diabetes.
To our knowledge, this is the first study to attempt to validate the FABP-1 association for PaC and PaC with DM. The intent of our study was to examine whether a computational approach could identify proteins associated with a particular disease, as a preliminary step in biomarker discovery. As mentioned, we narrowed our search to proteins present in serum/urine because of the postulated presence of a circulating diabetogenic factor that exerts peripheral effects. Also, a diagnostic test would necessarily be a serum/urine assay.
Of note, FABP-1 has been in detected in animal and human serum/urine and commercial ELISA-kits are available for FABP-1 serum/urine detection [42–47]. Possible mechanisms for FABP-1 presence in the serum could involve cell lysis or cellular secretion. The discovery of a biomarker for PaC-associated DM would be of immense value for the early detection and treatment of PaC. Association by immunohistochemistry alone, however, does not necessarily translate to diagnostic utility nor causality. We have no evidence to prove that FABP-1 is responsible for the pathogenesis of diabetes. Future experiments will involve larger sets of both pathologic and serum samples, with closely phenotyped patient samples to further explore this association.
Global gene expression analysis in patients with PaC-associated DM is a complimentary and contributory approach to biomarker discovery using integrative bioinformatics. Gene expression analysis could confirm the FABP-1 overexpression we predict. There is no published literature that specifically investigates the gene expression of tumor tissue in PaC-associated DM. Future work will be important to fill that knowledge gap.
There are several limitations of our study. When considered a binary variable, FABP-1 staining was defined as a score ≥ 1. A more stringent cutoff for FABP-1 staining would change our reported associated with pancreatic cancer. However, we do feel that in this pilot study, grouping pancreatic cancer groups by the presence or absence of FABP-1 staining was reasonable, given that only one normal sample stained positive for FABP-1 and that the degree of FABP-1 expression is likely to vary between tumors. The extent of FABP-1 staining will be explored in future studies with more patients samples that are better phenotyped. In multivariate analyses, age was noted to be a confounder of FABP-1 staining, though it should again be noted that only one sample in the normal group stained with FABP-1 and this sample was from a patient that was younger than the average age of the staining population in the PaC group. Rather than a true confounder, the association between age and FABP-1 staining may be a result of selection bias from our convenience sample or a spurious association. Larger studies are needed to further explore this relationship.
There are limitations of the bioinformatics technique itself, including potential low specificity for a given biomarker, the assumption that differential gene expression is necessarily reflected in plasma or urine, and that we excluded proteins currently not known to be detectable in serum or urine. Biomarker discovery using this methodology is necessarily limited by quantity, quality, and availability of public microarray datasets for a disease. However, it should be noted that these limitations have not precluded successful discovery of biomarkers for other diseases .
Our data collection was retrospective and subject to idiosyncrasies in chart documentation. We could not differentiate pancreatic cancer-associated diabetes from pancreatic cancer coexisting with chronic diabetes. The PaC-DM group presented represents true PaC-associated DM cases as well as those in whom the DM was unrelated to PaC, in unknown proportions. This distinction can be elucidated in future serum FABP-1 studies with more detailed available clinical information.
Our sample was a nonrandom small convenience sample, which likely contributed to the lower than published prevalence of diabetes in PaC, the age difference between normal and cancer groups, and the fact that we were not adequately powered to run a matched analysis . However, one would expect nondifferential misclassification and a small sample size to skew results towards the null. The fact that a significant association between FABP-1 staining PaC and PaC with diabetes was found suggests that the true association is stronger. It would have been informative have more normal samples from diabetics to confirm lack of FABP-1 staining.
Finally, our normal samples were representative of histologically normal tissue that often did come from organs that had pancreatic pathology. We cannot rule out concerns of tumor microenvironment, though it should be noted that histologic normalcy is the basis for adequate surgical margins during tumor resection, and that this technique is standard practice in construction of microarrays and has yielded reproducible results for other types of cancer/normal tissue [22, 23]. Future studies will use a control group of patients without pancreatic disease. Only one pathologist scored immunohistochemistry; however, he has several years experience. Our study would have been strengthened by having at least two independent pathologists read and score FABP-1 staining.