In patients presenting with localized PDAC, resection is usually followed by adjuvant chemotherapy or chemo-radiotherapy.(26
) However, most patients treated in this fashion will eventually die of recurrent disease. For patients with locally advanced disease, no standard of care exists. Consequently, a number of centers have evaluated the usefulness of with neoadjuvant therapy to attempt to downsize/downstage tumors, improve rates of complete resection, and reduce local relapse rates.(27
) This approach provides the potential benefit of early treatment of local disease and allows for the identification of those patients with rapidly progressive metastatic disease (thereby avoiding futile surgery). A recent large population-based comparison demonstrated a survival benefit for the neoadjuvant approach.(28
) However, prospective randomized data comparing primary resection with neoadjuvant therapy are lacking.
In this study we initially evaluated the spatial expression of miR-10b, miR-21, miR-155, miR-196a and miR-210 in 10 resected PDAC samples, and determined that miR-10b was the most frequently overexpressed miRNA in the cancer cells within the tumor mass. We also demonstrated for the first time the spatial expression of miRNAs in EUS-FNA samples by ISH. The co-localization of miR-10b with CK19 in the same FNA samples enabled us to reproducibly assess miR-10b expression in the cancer cells within the tumor mass and the ductal cells in the normal pancreas. We were thus able to demonstrate that miR-10b expression was increased in the pancreatic cancer cells within the FNA samples by comparison with miR-10b levels in pancreatic ductal cells within benign lesions. Moreover, we determined that lower levels of miR-10b in the cancer cells were associated with response to neoadjuvant gemcitabine-based chemo-radiotherapy, surgical resectability, time to metastasis and overall survival.
The 5 year survival of PDAC patients treated with either upfront surgery or neoadjuvant chemo-radiotherapy is less than 30% despite complete resection.(4
) Although our study focused principally on patients who underwent neoadjuvant chemo-radiotherapy, it demonstrates for the first time that miR-10b expression in FNA samples can be used to delineate a subgroup of patients that will truly benefit from subsequent surgery. Thus, those patients whose cancers express low levels of miR-10b are predicted to have greater than 50% survival after 2 years. By contrast, high levels of miR-10b project poor outcome and early disease progression even after surgical resection. Our study design does not allow us to assess the potential role of characterizing miR-10b expression in patients who undergo primary surgical resection, but raises the possibility that such an analysis would also be useful in this group of patients.
It is noteworthy that miR-10b expression is elevated in several malignancies, including leukemia,(29
) hepatocellular carcinoma,(30
) and malignant glioma,(32
) underscoring the potential importance of this miRNA in human cancers. Interestingly, the significance of miRNAs in tumor metastasis was first described by Ma et al. who demonstrated high expression of miR-10b in the metastatic breast cancer cell lines.(19
) Moreover, silencing of miR-10b led to more than a 10-fold reduction in the invasive properties of these cells, whereas miR-10b overexpression induced a 4–5 fold increase in cell motility and invasiveness.(19
) In vivo
, miR-10b overexpression triggered a significant desmoplastic reaction and enhanced cell invasion. (33
) Conversely, the administration of a miR-10b antagonist to orthotopic breast cancer cell implantation in mice markedly suppressed the formation of distant lung metastases.(33
) Taken together, these reports underscore the importance of miR-10b in human malignancies and its potential to promote metastatic disease.
PDAC is a highly metastatic malignancy, and miR-10b has been linked to the pro-metastatic protein Twist1. (18
) miR-10b also exerts post-transcriptional inhibition of the target gene HOXD10,
) and represses the expression of genes that have significant role in modulating cell migration and remodeling the extracellular matrix.(19
) Recently, a cascade of activation has been described whereby the extracellular matrix component hyaluronan binds to CD44 and promotes c-Src kinase activation, which then activates the transcription factor Twist.(34
) Further analyses reveal that miR-10b is controlled by Twist and this process results in the reduction of a tumor suppressor protein HOXD10 leading to RhoA/RhoC up-regulation and Rho-kinase (ROK) activation promoting breast tumor cell invasion.(34
) CD44 was also found to be expressed in tumor stem cells that have the unique ability to act as tumor initiating cells. (35
) Additional targets for miR-10b were identified in esophageal cancer cells where miR-10b directly targets Kruppel-like factor 4(KLF4), promoting cell migration and invasion initiated by miR-10b.(20
) By contrast, Tiam1 (T lymphoma invasion and metastasis 1) was identified as an additional target gene for miR-10b causing inhibition of breast cancer cell migration.(36
) This finding underscores the importance of context specific expression of miR-10b in cancer cells. It will be important, therefore, to conduct studies with pancreatic cancer cell lines to examine the mechanisms of action of miR-10b in PDAC, which may provide additional evidence for considering miR-10b as a potential therapeutic target in PDAC.
EUS-FNA is commonly used for evaluating a pancreatic mass lesion, with 80% to 90% diagnostic sensitivity.(37
) However, false negative and equivocal results may diminish the utility of this procedure. Previous studies that have analyzed miRNA expression in FNA samples have used qRT-PCR, which does not provide information regarding the cell type present in the sample and the specific site of expression of a particular miRNA. For example, let-7a-1 precursor RNA expression was consistently detected at lower levels in PDAC FNA samples compared to normal pancreatic tissue, but the small number of patients studied did not allow for the assessment of its diagnostic or prognostic value.(38
) Similarly, the ratio of miR-196a to miR-217, as determined by qRT-PCR, was proposed to distinguish PDAC FNA samples from chronic pancreatitis and benign lesions.(13
) However, it is not apparent from either of these studies whether a minimum content of cancer cells in these FNA samples was required to obtain an accurate diagnosis. This is of particular importance in light of recent findings that indicate that the accuracy of prognostic value of EGFR and gemcitabine resistance-related genes in FNA samples from PDAC patients who received gemcitabine-based therapy was greatly improved when laser capture microdissection (LCM) was performed to enrich for cancer cell content prior to qRT-PCR analysis.(39
) Thus, the use of a combined ISH/IHC assay enables rapid and direct evaluation of changes of miRNA expression within the suspicious cells highlighted by CK19 staining and provides an innovative alternative approach to the use of the technically demanding LCM coupled with qRT-PCR analysis.
Our findings must be interpreted with caution due to the limitations common to retrospective studies. Nonetheless, the present data suggest that assessing miR-10b expression by this combined ISH/IHC assay in FNA samples from pancreatic lesions could serve as a diagnostic maker that could help distinguish malignant from benign pancreatic tissue, and that miR-10b expression could potentially serve as a prognostic marker in PDAC. Analysis of additional miRNAs and proteins in the same FNA sample may further enhance the diagnostic accuracy of the EUS-FNA procedure and inform therapeutic options. Moreover, the same technology could be used in assessing biopsy material from a variety of solid tumors. Therefore, prospective studies are needed to determine whether this approach may ultimately serve as important tool for clinicians to guide clinical decision making regarding neoadjuvant treatment and surgery.