Recent advances in precision medicine for pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the leading causes of cancer mortality worldwide. Although advances in systemic chemotherapy for PDAC have improved survival outcomes for patients with the disease, chemoresistance is a major treatment issue for unselected PDAC patient populations. The existence of heterogeneity caused by a mixture of tumor cells and stromal cells produces chemoresistance and limits the targeted therapy of PDAC. Advances in precision medicine for PDACs according to the genetics and molecular biology of this disease may represent the next alternative approach to overcome the heterogeneity of different patients and improve survival outcomes for this poor prognostic disease. The genetic alteration of PDAC is characterized by four genes that are frequently mutated (KRAS, TP53, CDKN2A, and SMAD4). Furthermore, several genetic and molecular profiling studies have revealed that up to 25% of PDACs harbor actionable alterations. In particular, DNA repair dysfunction, including cases with BRCA mutations, is a causal element of sensitivity to platinum‐based anti‐cancer agents and poly‐ADP ribose polymerase (PARP) inhibitors. A deep understanding of the molecular and cellular crosstalk in the tumor microenvironment helps to establish scientifically rational treatment strategies for cancers that show specific molecular profiles. Here, we review recent advances in genetic analysis of PDACs and describe future perspectives in precision medicine according to molecular subtypes or actionable gene mutations for patients with PDAC. We believe the breakthroughs will soon emerge to fight this deadly disease.

undergone curative resection and chemoresistance for the current systemic chemotherapies (GnP and FOLFIRINOX) are major issues in the treatment of unselected PDAC patient populations.
Although molecular markers are often employed to effectively select patients for anti-cancer agents, only imaging modalities are applied to stage the disease and judge suitability for operative resection. Unfortunately, our knowledge of the genetic and biological backgrounds of this deadly disease has not yet been linked to a leap in patient survival. Knowledge obtained from the Human Genome Project, and subsequently The Cancer Genome Atlas, has yielded the landscape of precision medicine. The concept is that cancer patients can be sub-classified according to actionable driver mutations, which can be targeted by molecular-specific agents. Development of next-generation sequencing (NGS) has drastically progressed genomic sequencing technology and cleared actionable driver mutations for individual cancer patients.
Advances in precision medicine for PDACs according to genetics and molecular biology may be the next alternative approach to improve survival outcomes for this poor prognostic disease. PDACs have been divided into several molecular subtypes by recent advances in genetic analysis, [10][11][12][13][14][15][16][17] which is a precursor of precision medicine. Some molecular profiling studies have exhibited that up to 25% (range 12%-25%) of PDACs retained actionable molecular alterations. [10][11][12][13][14][15][16][17] Furthermore, the development of multigene panel assay has resulted in a fundamental change in the treatment of PDAC. Indeed, matching to appropriate molecular-specific treatments improves the OS of PDAC patients compared to that of those without actionable mutations or those who do not accept the molecular-specific therapy. 18 A better grasp of the genetics and molecular biology of PDAC accelerates the development of precision medicine.
Here, we review recent advances in genetic analysis of PDACs and describe future perspectives in precision medicine according to molecular subtypes or gene mutations for patients with PDAC.

| THE G ENOMI C L ANDSC APE OF PAN CRE ATI C C AN CER AND " B I G FOUR " M UTATI O N G E N E S
In 2008, the exome analysis of PDACs was completed. 19 The coding regions of >20 000 genes were sequenced, and an average of 63 genomic alterations per patient genome was discovered. These alterations consisted of 12 core signaling pathways and were detectable in the majority (from 67% to 100%) of PDACs. Among them, dysregulations in KRAS signaling, G1/S phase cell cycle transition, TGFβ signaling, integrin signaling, cell invasion, homophilic cell interaction, and small guanine triphosphate (GTPase)-dependent signaling were prominent. 19 The genetic landscape of PDACs is featured by four frequently mutated genes: KRAS, TP53, CDKN2A (p16), and SMAD4. 20 The four predominant gene mutations appear to occur sequentially as PanIN progresses ( Figure 1). KRAS mutations can be found even in normal pancreases and in PanIN1. In PDAC, the incidence of oncogenic KRAS mutation ranges from 88% to 100%. 12,16,17,19,21,22 Although the initial step for PDAC development remains to be elucidated, the oncogenic KRAS mutation is a key event, as evidenced by its presence in PanIN lesions 23,24 and the development of PanIN lesions in oncogenic KRAS-driven GEMMs. 25,26 The oncogenic KRAS mutation provokes the constitutively activated RAS protein and results in the dysregulated activation of proliferation and survival pathways.
In the clinical setting, cases with KRAS mutations displayed worse prognostic outcome with a median survival time of 17 months compared to 30 months for those without mutations. 27 In analysis of KRAS mutation type, codon G12D mutant was the most frequent (48%), followed by G12V (31%) and G12R (21%). 22 Intriguingly, 4% of PDACs exhibit multiple KRAS mutations, and these different KRAS mutations appeared in distinct cancer cells in a single tumor. 22 While G12D or G12V mutations are the most prevalent KRAS mutations in patients with PDAC, codon G13 and Q61 mutations have also been noted. 12,17,19,28 The point mutations in codon 12, 13,    in 75%-85% of PDAC cases. 20,34 SMAD4 encodes Smad4 protein, which is a transcription factor in TGFβ signaling pathway. 35 SMAD4 is inactivated in 43%-50% of PDAC cases. 11,20 It works with TGF-β1 as a tumor suppressor to regulate cell cycle arrest and apoptosis. 36 The loss of SMAD4 gene induces aberrant TGFβ signaling. PDAC patients with biallelic deletion of SMAD4 had more frequent metastasis compared to those with wild-type SMAD4. 37  genes. The increased number of altered genes was significantly associated with worse DFS and OS at autopsy. 38 Additionally, Hayashi et al 39 reported that PDAC patients with fewer mutations displayed a better prognostic outcome in 71 patients who underwent a radical operation followed by adjuvant chemotherapy. The existence of zero to two mutated genes was a predictor of a better OS. 39 Furthermore, genetic alterations of three genes (except KRAS), and thereby protein overexpression in PDAC tissues, are associated with malignant activity of PDAC. 40 In particular, loss of SMAD4 immunolabeling was an independent poor prognostic factor for OS and DFS in patients with resectable PDAC. 40 Intriguingly, all of the six patients who achieving 5-year survival displayed intact SMAD4 expression. Thus, the genetic status of the so-called "big four" mutation genes or their immunolabeling status is a prognostic biomarker in PDAC patients.
Unfortunately, there is still no available drug that can directly target the major four gene mutations in PDACs. The survival outcome of PDAC cases following surgical resection and standard medical treatment was remarkably better in the classical subtype than that in cases with the QM subtype; cases with the exocrine-like subtype showed an intermediate survival outcome between the two other subtypes. 13 Searching the clinical relevance of this classification using PDAC cell lines, the classical and QM subtypes offered differential reactions to gemcitabine and erlotinib.

| DNA damage repair pathways
DNA damage is a frequent event and must be immediately repaired to ensure the precise transfer of genetic information during cellular divi-  45 Although the risk of PDAC in carriers with a mutated BRCA1 gene is not fully elucidated, it is anticipated to be increased two to threefold over the general population. 46,47 In contrast, BRCA2 gene mutation was found in approximately 6% of the same cohort. 48 The risk of PDAC in carriers with a mutated BRCA2 gene is reported to be increased three to sixfold over the general population. 49,50 It is presently anticipated that 17%-25% of PDACs entertain germline or somatic DDR gene mutations such as BRCA1/2, PALB2, and ataxia telangiectasia mutated (ATM). 10 12,16,19,23,52 In a mouse model of PDAC, ATM deficiency accelerates genomic instability and metastatic ability. 53

| Platinum sensitivity
Platinum agents crosslink purine bases on DNA, thereby disturbing transcription and stopping replication, which lead to DSBs and apoptosis. 54 Consequently, mutations in HR genes display hypersensitivity to DNA crosslinking agents ( Figure 3). Indeed, PDAC cells with BRCA2, FANCC, or FANCG gene mutations exhibit hypersensitivity to DNA crosslinking agents such as cisplatin or mitomycin C in vitro and in vivo. 55 PDAC cases with impaired DNA repair pathways revealed better response to platinum-based chemotherapy and radiation therapy that induce DNA damage than those with normal DNA repair pathways. 16,56 It is noteworthy that structural variations in platinum agents, as has been observed for cisplatin and oxaliplatin, can be differences in DDR recognition. These differences in recognition influence the cytotoxicity of individual platinum agents. 57 Sporadic PDAC patients with BRCA1/2 mutation displayed worse survival after operation than those with wild-type BRCA. 58 On the other hand, platinum-based chemotherapy notably improved survival outcome in patients with BRCA1/2 mutations. 58 Consequently, the survival differences relative to wild-type patients were eliminated. 58 In other studies, patients with BRCA1/2 mutation displayed the enhanced response rates to platinum-based chemotherapy and improved survival outcome. 16

| PARP inhibitors
SSBs are the most frequent DNA damage. If they are not repaired efficiently, they develop into DSBs. 44,63 The base excision repair (BER) pathway is an important repair machinery for SSBs ( Figure 3).

CO N FLI C T S O F I NTE R E S T
The authors have no conflicts of interest to declare.

E TH I C A L S TATEM ENT
The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.