PANCREATIC DUCTAL ADENOCARCINOMA

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Pancreatic cancer is the fourth leading cause of cancer death in the United States, with 5-year survival rates of less than 5%. Pancreatic ductal epithelial adenocarcinoma (PDA) accounts for more than 90% of the total pancreatic cancers. Currently, surgery is the only treatment that is partially effective for pancreatic cancer. Most patients with pancreatic cancer are diagnosed at an advanced stage and are therefore not candidates for surgical resection. Traditional chemotherapy and radiotherapy do not provide substantive palliation and improvement in survival for patients with non-resectable pancreatic cancer. Although a great deal of information has been added into our understanding of pancreatic cancer during the last decade, the exact mechanism of pancreatic cancer development is still unknown.   

GENETICS

Although the etiology of pancreatic cancer is still elusive, the last decades have witnessed significant advancements in understanding the genetic lesions of pancreatic cancer. Similar to the progression model for colon cancer, a genetic progression model for pancreatic ductal adenocarcinoma was proposed by Hruban et al. several years ago (Figure 1.1) ⁵⁹. In this model, normal duct epithelium progresses to infiltrating cancer through a series of histologically defined precursors (pancreatic intraepithelial neoplasia, PanINs). This has been supported by histological studies and clinical observation. Histologically, PanINs progress with a series of morphological alterations, which correspond to stages of increasingly dysplastic growth (Fig.1.2) ⁶⁰. The overexpression of HER-2/neu and point mutations in the KRAS gene can be detected at pancreatic duct lesions with minimal cytological and architectural atypia. Inactivation of the p16 gene occurs at an intermediate stage, and the inactivation of p53, DPC4, and BRCA2 occur relatively late in the development of pancreatic neoplasia.  Despite all the genetic knowledge we have, how these genetic lesions lead to development of pancreatic cancer is still poorly understood.

KRAS mutation (mostly G12V or G12V) is present in more than 85% of ductal carcinomas, and the p16 tumor-suppressor gene is inactivated in around 90% of pancreatic cancers ^60,61. Mouse models have convincingly shown that KRAS mutation is an initiating step in PDA pathogenesis and is also crucial for maintenance ^62,63. However, KRAS mutation is not tumor specific and is also detected at high frequencies in normal epithelium (19% to 38%) ^64,65 and in patients with chronic pancreatitis (18% to 62.5%) ^66,67.

Loss of p16INK4A function by mutation, deletion, or promoter hypermethylation occurs in 80% to 95% of pancreatic cancers ^68,69. INK4A loss is generally seen in moderately advanced lesions that show features of dysplasia. The 9q21 locus encodes two overlapping tumor suppressors—INK4A and ARF, and their respective protein products p16^INK4A and p19^ARF—via distinct first exons and alternative reading frames in shared downstream exons ⁷⁰. INK4A inhibits CDK4/6-mediated phosphorylation of RB and block cells from entering into the S (DNA synthesis) phase of the cell cycle. ARF stabilizes p53 by inhibiting its MDM2-dependent proteolysis. Since homozygous deletion of 9p21 is seen in about 40% of tumors, many pancreatic cancers sustain loss of INK4A and ARF tumor suppression pathways. INK4A has been indicated to play a central role as a PDA tumor suppressor ⁶⁰. Transgenic mouse studies have shown INK4A mutations cooperate with KRAS in the development of PDA and accelerate tumor progression in the setting of concurrent mutations in p53 ⁷¹. Moreover, ARF was shown to have an independent, cooperative function in PDA tumor suppression.

More than 50% of pancreatic cancer cases have mutations in the P53 gene, which generally are missense alterations of the DNA-binding domain ⁶⁸. The p53 transcription factor is activated in response to DNA damage or oncogene activation. p53 mutation appears in later-stage PanINs that have acquired significant features of dysplasia ^72,73. SMAD4 is another important tumor suppressor gene that is frequently mutated or lost in pancreatic cancer. Loss of heterozygosity of Smad4 can be seen in around 90% of pancreatic cancers, and 50% of patients show complete loss of functional Smad4 proteins ^74,75. The mechanism by which SMAD4 loss contributes to tumorigenesis is likely to involve its central role in TGF-β-mediated growth inhibition. The biological role of the TGF-β pathway in human malignancy is complex, exerting both growth-inhibitory and growth-promoting effects depending on the cell type and cell context ⁷⁶. The loss of SMAD4 in PDA may have a primary role in modulating the interaction of the tumor with the microenvironment rather than in controlling the growth of the tumor cells themselves. There is recent evidence that SMAD4 deficiency may inhibit TGF-β-induced cell cycle arrest and cell migration, while not affecting EMT, thereby shifting the balance of TGF-β signaling from tumor suppression to tumor promotion ⁷⁷. Consistent with these observations, it appears that elevated TGF-β expression contributes to PDA progression ⁷⁸.

LKB1/STK11 mutation is a characteristic genetic alteration in the Peutz-Jeghers syndrome (PJS) which is a familial cancer syndrome associated with an increased incidence of PDA ^79,80. LKB1 is a tumor suppressor gene encoding a serine/threonine kinase that is involved in regulation of diverse processes, such as cell polarity and metabolism, and has been linked to specific signaling pathways including mTOR and AMPK ⁸¹. Heterozygous loss of LKB1 in mice induces intestinal polyps formation and highly invasive endometrial adenocarcinomas ^82,83. Inherited BRCA2 mutations are typically associated with familial breast and ovarian cancer syndrome, but also carry a significant risk for the development of pancreatic cancer. One study estimates that 17% of pancreatic cancers occurring in a familial setting harbor mutations in this gene ⁸⁴. BRCA2 is known to play a critical role in the maintenance of genomic stability by regulating homologous recombination-based DNA repair processes. Consequently, BRCA2 deficiency in normal cells results in the accumulation of procarcinogenic or lethal chromosomal aberrations ⁸⁵.

PATHWAYS 

Oncogenes and tumor suppressor genes are mutated or deregulated in pancreatic cancer. Mutational activation of RAS due to amino acid substitutions at positions 12, 13 or 61 results in oncogenic versions insensitive to GAP stimulation that persist in the GTP-bound state. Mutated KRAS is constitutively active and enhances tumorigenesis through its downstream RAF/MEK/ERK, PI3K/AKT, and RAL pathways. RAS mediates its cellular regulation effects through its downstream effectors such as Raf and PI3-Kinase ⁸⁶. Raf phosphorylates and activates the dual specificity serine/threonine and tyrosine kinase MEK, which in turn activates the extracellular signal-regulated (ERK) family of mitogen activated protein (MAP) kinases. The ERKs phosphorylate a variety of targets in the cytoplasm and the nucleus, and then regulate multiple functional endpoints through direct phosphorylation and/or regulation of gene transcription, including cellular proliferation, differentiation, motility and invasion. Stimulation of PI3-kinase leads to protection from apoptosis via activation of the serine/threonine kinase AKT and also cytoskeletal re-organization via regulation of Rho family GTPases. Oncogenic ras mutation has been considered a key contributor to the tumorgenesis of pancreatic cancer.

The epidermal growth factor receptor (EGFR) and its endogenous ligands, epidermal growth factor (EGF) are overexpressed in PDA^87,88.  When bound to its ligand, EGFR dimerises with each other or with other ErbB family members, which results in phosphorylation of tyrosine residues on the intracellular domain. The tyrosine phosphorylation attracts downstream effectors to bind to the cytoplasmic region of EGFR and become activated. The major downstream mediators of EGFR include RAS/MEK/ERK, PI3K/AKT, and the signal transducer and activator of transcription (STAT) family proteins which are involved in cancer adhesion, invasion, and cell survival ⁸⁹.

Tumor suppressor genes p16, p53 and BRCA1 are commonly inactivated in PDA. The p16 gene binds and inhibits cyclin-dependent kinases (CDKs) CDK4/6, which sense of mitogenic signals and phosphorylate retinoblastoma (Rb). Phosphorylated Rb promotes the transcription of E2F-regulated genes and consequent entry into the S-phase ^90,91. Therefore, in pancreatic cancer, abrogation of p16 activity leads to uncontrolled CDK4/6 activity and enhanced Rb phosphorylation, and then more cells enter the S-phase. The other frequently mutated tumor suppressor, Smad4, has important effects on the tumor microenvironment and potentiation of invasion ^92,93. The Smad4 protein belongs to the Smad family of transcription factors and mediates signal transmission of the TGF-β superfamily of cytokines ⁹⁴. TGF-β ligands regulate cell growth, differentiation, and migration and have both tumor suppressor and tumor promoter functions. Loss of Smad4 seems to abolish TGF-β-mediated tumor suppressive functions, while maintaining some TGF-β-mediated tumor promoting functions ⁷⁷.

In addition, reactivation of developmental signaling, such as Notch and hedgehog pathways, has been demonstrated in pancreatic cancer. While playing an important role in directing decisions about the fate of cells in the development of the pancreas, the Notch pathway is also critical for pancreatic cancer initiation and invasion ^95,96. Binding of Notch receptors to their ligands, such as Delta, Serrate, and Lag-2, activates the Notch pathway and leads to proteolytic cleavage of Notch receptors. The cleaved product is the intracellular domain, which translocates to the nucleus and binds to the transcription factor CSL (RBP-Jκ/CBF in mammals; Suppressor of Hairless (Su(H)) in Drosophilia) inducing the transcription of a variety of target genes including the hairy enhancer of split (HES) family of transcriptional repressors. HES family members act to maintain cells in a precursor state. The pathway is turned off in the adult pancreas, but upregulated in pre-neoplastic lesions and in invasive pancreatic cancer ^97,98.

TREATMENT

The current treatment options of PDA include surgery, chemotherapy, radiation therapy, and symptom control. More than 80% of PDA patients are diagnosed at an advanced stage and are not suitable candidates for surgery, which is the only potential cure. PDA is highly resistant to traditional chemotherapy and radiation therapy. Few chemotherapeutic agents have been shown to have reproducible response rates of more than 10%. 5-Fluorouracil (5-FU) is an inhibitor of thymidylate synthetase, which is essential for synthesis of DNA nucleotides, and has been the most widely used in advanced pancreatic cancer, with a median survival of around 5–6 months and is better than the best supportive care ^99-101. The current preferred chemotherapeutic agent is gemcitabine, which showed an improved 1-year survival rate compared to 5-FU (18% vs. 2%). Gemcitabine also is less toxic and has higher clinical response when compared to 5-FU (24% vs. 5%) ¹⁰². Recent clinical trials suggested that a combination treatment of gemcitabine and other chemotherapeutics showed better survival than gemcitabine alone. The best combinations may be with capecitabine or platinum-based agents,  which induces less toxicity ^101,103. Target based drugs such as Erlotinib (an EGFR tyrosine kinase inhibitor) and cetuximab (antibody to EGFR) are also being explored to treat pancreatic cancer in combination with gemcitabine. 

Radiotherapy has been widely used for the treatment of pancreatic cancer. However, there is no evidence showing that radiation provides any additional beneficial effects ¹⁰¹. Results from clinical trials demonstrated that radiation induced strong toxicity and significantly reduced median survival when combined with gemcitabine ¹⁰¹.

Radical resection alone will result in a 5-year survival rate of only 10% owing to recurrence after surgery ¹⁰⁴. Nearly all patients develop metastatic disease, most commonly of the liver and peritoneum but also the lungs, and this may occur with or without local recurrence ^100,104. After pancreatic resection, adjuvant systemic chemotherapy is preferred and increases the 5-year survival from 9% to 12% with resection alone to 21% to 29% and 23% with either 5FU and folinic acid or gemcitabine, respectively ^105-107.