Abstract
High-grade pancreatic intraepithelial neoplasia (HG-PanIN) is the major precursor of pancreatic ductal adenocarcinoma (PDAC) and is an ideal target for early detection. To characterize pure HG-PanIN, we analysed 23 isolated HG-PanIN lesions occurring in the absence of PDAC. Whole-exome sequencing of five of these HG-PanIN lesions revealed a median of 33 somatic mutations per lesion, with a total of 318 mutated genes. Targeted next-generation sequencing of 17 HG-PanIN lesions identified KRAS mutations in 94% of the lesions. CDKN2A alterations occurred in six HG-PanIN lesions, and RNF43 alterations in five. Mutations in TP53, GNAS, ARID1A, PIK3CA, and TGFBR2 were limited to one or two HG-PanINs. No non-synonymous mutations in SMAD4 were detected. Immunohistochemistry for p53 and SMAD4 proteins in 18 HG-PanINs confirmed the paucity of alterations in these genes, with aberrant p53 labelling noted only in three lesions, two of which were found to be wild type in sequencing analyses. Sixteen adjacent LG-PanIN lesions from ten patients were also sequenced using targeted sequencing. LG-PanIN harboured KRAS mutations in 94% of the lesions; mutations in CDKN2A, TP53, and SMAD4 were not identified. These results suggest that inactivation of TP53 and SMAD4 are late genetic alterations, predominantly occurring in invasive PDAC.
Keywords: pancreas, pancreatic intraepithelial neoplasia, HG-PanIN, pancreatic ductal adenocarcinoma, whole-exome sequencing, targeted next-generation sequencing, TP53, SMAD4, cancerization
Introduction
Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer death in the United States, with a 5-year survival rate of only 7.7% [1]. Approaches to detect curable disease are crucial to improve outcomes, and high-grade precursors are the ideal target lesions of early detection [2]. Pancreatic intraepithelial neoplasia (PanIN), the common precursor to PDAC, is classified based on the grade of dysplasia of the neoplastic epithelium [3]. While low-grade PanINs (LG-PanINs) are common and low risk, high-grade PanINs (HG-PanINs) are regarded as lesions immediately preceding invasive PDAC and thus are a primary target for intervention [4,5]. Because these lesions cannot be detected by standard clinical and radiological approaches, molecular approaches, such as mutational analysis of pancreatic juice, will be critical for the detection of HG-PanIN [6,7].
HG-PanIN lesions are rarely detected clinically, and knowledge about isolated HG-PanIN is limited. Most studies of HG-PanIN have been conducted using specimens that also harboured an invasive PDAC [8–14]. These studies are confounded by the fact that invasive carcinomas can grow back into and spread within the duct system (‘cancerization of the ducts’), histologically mimicking HG-PanIN [15]. Thus, instead of HG-PanIN, many previous studies may have analysed intraductal invasive cancer, raising concerns about the reliability of these data.
To gain knowledge about HG-PanIN in the absence of invasive carcinoma (isolated HG-PanIN), we conducted a multicentre study investigating its clinicopathological and molecular characteristics. We found that while KRAS and CDKN2A were frequently targeted, TP53 mutations were rare and SMAD4 mutations were absent in isolated HG-PanIN.
Materials and methods
Our study cohort included 23 isolated HG-PanINs from 21 patients; samples were retrieved from the database of the Department of Pathology of The Johns Hopkins Hospital or collected from the participating institutions after the approval of the Institutional Review Board. Neoplastic cells were isolated from formalin-fixed, paraffin-embedded (FFPE) tissue sections by laser capture microdissection (supplementary material, Figure S1). DNA was extracted and analysed by targeted next-generation sequencing of pancreatic cancer driver genes using Ion AmpliSeq library preparation on an IonTorrent Personal Genome Machine (17 HG-PanINs and 16 LG-PanINs) or by whole-exome sequencing using Agilent SureSelect library preparation on an Illumina HiSeq (five HG-PanINs). In addition, immunohistochemistry for p53 and SMAD4 protein was performed on FFPE sections (18 HG-PanINs). Additional details are provided in the Supplementary materials and methods.
Results
Clinicopathological features of isolated HG-PanIN
Twenty-three HG-PanIN lesions were characterized from 21 patients (supplementary material, Table S1). The preoperative clinical findings included pancreatitis and irregular shape of the main pancreatic duct on imaging. Some of the HG-PanINs were identified incidentally in pancreata resected for other neoplasms, including neuroendocrine tumour/carcinoma and peri-ampullary cholangiocarcinoma. When HG-PanIN occurred with cholangiocarcinoma, we confirmed that both lesions harboured different mutations of KRAS using pyrosequencing and/or targeted sequencing.
Histologically, the extent of duct involvement by the HG-PanIN lesions varied (Figure 1 and supplementary material, Table S1). Of 23 lesions, 11 HG-PanIN lesions in nine patients showed extensive spread along the pancreatic duct and were clinically recognized due to pancreatitis or irregular shape of the main pancreatic duct on imaging. In contrast, ten HG-PanIN lesions in ten patients showed focal growth and were mostly discovered incidentally in pancreata resected for other lesions. Data were not available for two lesions. Importantly, all of the lesions, even those with extensive growth, met histological criteria for diagnosis of PanIN, not intraductal papillary mucinous neoplasm (IPMN), a distinct and larger precursor lesion [15]. LG-PanIN lesions were detected in all 21 patients.
Figure 1.
A representative isolated HG-PanIN. (A) The atypical proliferation spreads along the pancreatic duct. (B) Atypical cells showed cytological features of high-grade atypia, including irregular nuclear stratification, coarse chromatin, and prominent nucleoli.
Targeted sequencing of isolated HG-PanIN and associated LG-PanIN
Of 23 HG-PanIN lesions, sufficient lesional tissue for microdissection and sequencing was available in 20 lesions (Tables 1 and 2). Sufficient coverage for mutation analysis was obtained from 17 HG-PanIN lesions in 15 patients and 16 LG-PanIN lesions in ten patients (supplementary material, Tables S2, S3, and Figure S2).
Table 1.
Somatic mutations of isolated HG-PanINs identified by targeted sequencing
| Patient ID | Lesion designation | Gene | Mutation position (hg19)
|
Mutation type | Consequence | Variant allele frequency* | |
|---|---|---|---|---|---|---|---|
| Nucleotide (genomic) | Amino acid (protein) | ||||||
| PDS-2 | B-12 | KRAS | chr12:25398284C>T | p.G12D | Substitution | Missense | 84% (2025; 2416) |
| B-12 | RNF43 | chr17:56448310G>A | p.R113X | Substitution | Nonsense | 14% (154; 1098) | |
| B-13† | KRAS | chr12:25398284C>T | p.G12D | Substitution | Missense | 87% (635; 730) | |
| PDS-3 | B-14 | KRAS | chr12:25398285C>G | p.G12R | Substitution | Missense | 36% (953; 2641) |
| PDS-6 | B-5 | KRAS | chr12:25398285C>G | p.G12R | Substitution | Missense | 39% (663; 1707) |
| PDS-7 | B-6 | KRAS | chr12:25398284C>T | p.G12D | Substitution | Missense | 46% (1131; 2459) |
| B-6 | CDKN2A | chr9:21971108C>A | p.D84Y | Substitution | Missense | 88% (213; 243) | |
| B-6 | RNF43 | LOH‡ | |||||
| PDS-8 | B-7 | KRAS | chr12:25398284C>A | p.G12V | Substitution | Missense | 39% (168; 429) |
| B-7 | GNAS | chr20:57484421G>A | p.R201H | Substitution | Missense | 39% (152; 392) | |
| B-7 | RNF43 | chr17:56435161delC | p.G659Vfs | Deletion | Frameshift | 52% (351; 669) | |
| PDS-9 | B-31 | GNAS | chr20:57484421G>A | p.R201H | Substitution | Missense | 37% (17; 46) |
| PDS-10 | B-27 | KRAS | chr12:25398284C>A | p.G12V | Substitution | Missense | 47% (1483; 3135) |
| PDS-13 | B-32 | KRAS | chr12:25398284C>T | p.G12D | Substitution | Missense | 35% (122; 347) |
| PDS-CC2 | B-8 | KRAS | chr12:25398284C>T | p.G12D | Substitution | Missense | 40% (532; 1339) |
| B-8 | ARID1A | chr1:27100175_27100176insC | p.Q1327Afs | Insertion | Frameshift | 28% (598; 2142) | |
| PDS-16 | HGPN-1 | KRAS | chr12:25398284C>T | p.G12D | Substitution | Missense | 42% (830; 1971) |
| PDS-18 | HGPN-1 | KRAS | chr12:25398284C>A | p.G12V | Substitution | Missense | 86% (260; 304) |
| PDS-19 | HGPN-1 | KRAS | chr12:25398285C>G | p.G12R | Substitution | Missense | 39% (296; 753) |
| HGPN-1 | CDKN2A | chr9:21971036C>A | p.D108Y | Substitution | Missense | 71% (17; 24) | |
| HGPN-1 | TP53 | chr17:7574026C>A | p.G334V | Substitution | Missense | 64% (2273; 3551) | |
| PDS-20 | HGPN-2 | KRAS | chr12:25398284C>T | p.G12D | Substitution | Missense | 45% (944; 2087) |
| HGPN-2 | PIK3CA | chr3:178952085A>G | p.H1047R | Substitution | Missense | 43% (207; 481) | |
| HGPN-2 | RNF43 | LOH‡ | |||||
| HGPN-3† | KRAS | chr12:25398284C>T | p.G12D | Substitution | Missense | 37% (668; 1783) | |
| HGPN-3† | CDKN2A | chr9:21971028C>T | p.W110X | Substitution | Nonsense | 70% (85; 121) | |
| HGPN-3† | TGFBR2 | chr3:30691885_30691895del TGGTGAGACTT | p.G155Lfs | Deletion | Frameshift | 16% (8; 51) | |
| HGPN-3† | RNF43 | LOH‡ | |||||
| PDS-21 | HGPN-2 | KRAS | chr12:25398284C>T | p.G12D | Substitution | Missense | 55% (769; 1395) |
| PDS-CC4 | HGPN-1 | KRAS | chr12:25398284C>A | p.G12V | Substitution | Missense | 39% (252; 653) |
| HGPN-1 | TP53 | chr17:7577548C>T | p.G245S | Substitution | Missense | 48% (245; 514) | |
Numbers in parentheses indicate the following: (variant reads; total reads).
Two HG-PanIN lesions were separately microdissected and sequenced. In PDS-2, the degree of atypia differed between the two lesions; in PDS-20, the two lesions were spatially separate.
Loss of heterozygosity (LOH) of RNF43 was manifested by marked reduction of one of the heterozygous SNP signals (at rs115553539 and rs9652855 in PDS-7, and at rs3744093 in PDS-20).
Table 2.
Somatic mutations of LG-PanINs identified by targeted sequencing
| Case ID | Lesion designation | Gene | Mutation position (hg19)
|
Mutation type | Consequence | Variant allele frequency* | |
|---|---|---|---|---|---|---|---|
| Nucleotide (genomic) | Amino acid (protein) | ||||||
| PDS-3 | B-42 | KRAS | chr12:25398284C>A | p.G12V | Substitution | Missense | 30% (819; 2755) |
| PDS-6 | B-43 | KRAS | chr12:25398285C>G | p.G12R | Substitution | Missense | 40% (333; 837) |
| PDS-7 | B-36 | KRAS | chr12:25398285C>A | p.G12C | Substitution | Missense | 36% (1086; 2988) |
| B-45 | KRAS | chr12:25380275T>G | p.Q61H | Substitution | Missense | 17% (43; 254) | |
| B-45 | KRAS | chr12:25398284C>T | p.G12D | Substitution | Missense | 16% (24; 153) | |
| PDS-8 | B-34 | KRAS | chr12:25398284C>A | p.G12V | Substitution | Missense | 47% (181; 386) |
| B-34 | GNAS | chr20:57484421G>A | p.R201H | Substitution | Missense | 35% (11; 31) | |
| B-34 | RNF43 | chr17:56435161delC | p.G659Vfs | Deletion | Frameshift | 54% (174; 323) | |
| PDS-10 | B-35 | No mutations found | |||||
| B-47 | KRAS | chr12:25398285C>G | p.G12R | Substitution | Missense | 40% (966; 2420) | |
| B-47 | RNF43 | chr17:56492825insA | p.E39X | Insertion | Nonsense | 16% (17; 107) | |
| PDS-13 | B-37 | KRAS | chr12:25398285C>G | p.G12R | Substitution | Missense | 21% (452; 2187) |
| B-38 | KRAS | chr12:25398285C>G | p.G12R | Substitution | Missense | 33% (1601; 4917) | |
| B-39 | KRAS | chr12:25398285C>G | p.G12R | Substitution | Missense | 30% (508; 1714) | |
| PDS-CC2 | B-40 | KRAS | chr12:25398284C>T | p.G12D | Substitution | Missense | 37% (988; 2685) |
| B-41 | KRAS | chr12:25398284C>A | p.G12V | Substitution | Missense | 21% (764; 3671) | |
| B-41 | KRAS | chr12:25398284C>T | p.G12D | Substitution | Missense | 19% (713; 3671) | |
| PDS-16 | LGPN-1 | KRAS | chr12:25398284C>A | p.G12V | Substitution | Missense | 20% (925; 4656) |
| LGPN-1 | KRAS | chr12:25380276T>A | p.Q61L | Substitution | Missense | 11% (272; 2426) | |
| PDS-19 | LGPN-1 | KRAS | chr12:25398284C>A | p.G12V | Substitution | Missense | 35% (460; 1318) |
| LGPN-2 | KRAS | chr12:25398284C>A | p.G12V | Substitution | Missense | 26% (275; 1042) | |
| PDS-21 | LGPNc-1 | KRAS | chr12:25380275T>G | p.Q61H | Substitution | Missense | 37% (166; 446) |
| LGPNc-1 | GNAS | chr20:57484421G>A | p.R201H | Substitution | Missense | 32% (18; 56) | |
Numbers in parentheses indicate the following: (variant reads; total reads).
KRAS was the most frequently mutated gene in HG-PanIN, with oncogenic hotspot mutations in 16 of 17 HG-PanIN lesions (94%). RNF43 was altered in five HG-PanIN lesions from four patients, followed by CDKN2A with mutations in three HG-PanINs. GNAS and TP53 were each mutated in two HG-PanIN lesions, and PIK3CA, TGFBR2, and ARID1A in one HG-PanIN lesion each. Notably, no non-synonymous mutations in SMAD4 were detected. LG-PanIN also frequently harboured KRAS mutations, which were identified in 15 of 16 lesions (94%). In three LG-PanIN lesions, two different KRAS mutations were detected, indicating that two different clones of LG-PanIN arose in pancreatic ducts in a small area (supplementary material, Figure S3). Other mutations in LG-PanIN included RNF43 (n = 2) and GNAS (n = 2). No non-synonymous mutations of CDKN2A, TP53, or SMAD4 were detected in LG-PanINs.
Copy number analysis of gene loci of CDKN2A, TP53, and SMAD4 could be reliably performed in ten HG-PanINs and six LG-PanINs using the data from targeted next-generation sequencing. We identified loss of CDKN2A in three HG-PanINs from two patients. No copy number alterations in the assayed genes were identified in LG-PanINs.
HG-PanIN and adjacent LG-PanIN(s) were analysed by targeted sequencing in ten patients, including 16 pairs of HG-PanIN and LG-PanIN lesions. Interestingly, we found that 13 of 16 pairs of HG-PanIN and LG-PanIN fell into the group of molecularly independent, even though some were located on the same glass slide (Table 3 and supplementary material, Figure S3). There was only one pair of LG-PanIN and HG-PanIN that was ‘likely related’.
Table 3.
Relatedness of HG-PanIN and LG-PanIN
| Patient ID | HG-PanIN
|
LG-PanIN
|
Relationship between PanINs
|
|||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Lesion (HG1)
|
Lesion (HG2)
|
Lesion (LG1)
|
Lesion (LG2)
|
Lesion (LG3
) |
Histology | Mutation analysis | ||||||
| Gene | Alteration | Gene | Alteration | Gene | Alteration | Gene | Alteration | Gene | Alteration | |||
| Between HG-PanIN lesions | ||||||||||||
| PDS-2 | B-12 | B-13 | Contiguous (HG1 has more atypical features than HG2) | Likely related | ||||||||
| KRAS | p.G12D | KRAS | p.G12D | |||||||||
| CDKN2A | Deletion | CDKN2A | Deletion | |||||||||
| RNF43 | p.R113X | |||||||||||
| PDS-20 | HGPN-2 | HGPN-3 | Separate | Indeterminate | ||||||||
| KRAS | p.G12D | KRAS | p.G12D | |||||||||
| RNF43 | LOH | RNF43 | LOH | |||||||||
| CDKN2A | Deletion | CDKN2A* | p.W110X | |||||||||
| PIK3CA | p.H1047R | TGFBR2 | p.G155Lfs | |||||||||
| Between HG-PanIN and LG-PanIN(s) | ||||||||||||
| PDS-3 | B-14 | B-42 | Separate (on the same glass slide) | Independent | ||||||||
| KRAS | p.G12R | KRAS | p.G12V | |||||||||
| PDS-6 | B-5 | B-43 | Separate | Indeterminate | ||||||||
| KRAS | p.G12R | KRAS | p.G12R | |||||||||
| PDS-7 | B-6 | B-36 | B-45 | Separate (HG1 and LG1) | Independent (HG1 and LG1) | |||||||
| KRAS | p.G12D | KRAS | p.G12C | KRAS | p.Q61H | Separate (HG1 and LG2) | Independent/indeterminate (HG1 and LG2)† | |||||
| CDKN2A | p.D84Y | KRAS | p.G12D | |||||||||
| RNF43 | LOH | |||||||||||
| PDS-8 | B-7 | B-34 | Separate (on the same glass slide) | Likely related | ||||||||
| KRAS | p.G12V | KRAS | p.G12V | |||||||||
| GNAS | p.R201H | GNAS | p.R201H | |||||||||
| RNF43 | p.G659Vfs | RNF43 | p.G659Vfs | |||||||||
| PDS-10 | B-27 | B-35 | B-47 | Separate (HG1 and LG1, on the same glass slide) | Independent (HG1 and LG1) | |||||||
| KRAS | p.G12V | KRAS | Wild type | KRAS | p.G12R | |||||||
| RNF43 | p.E39X | Separate (HG1 and LG2) | Independent (HG1 and LG2) | |||||||||
| PDS-13 | B-32 | B-37 | B-38 | B-39 | Separate (HG1 and LG1, on the same glass slide) | Independent (HG1 and LG1) | ||||||
| KRAS | p.G12D | KRAS | p.G12R | KRAS | p.G12R | KRAS | p.G12R | |||||
| Separate (HG1 and LG2, on the same glass slide) | Independent (HG1 and LG2) | |||||||||||
| Separate (HG1 and LG3, on the same glass slide) | Independent (HG1 and LG3) | |||||||||||
| PDS-CC2 | B-8 | B-40 | B-41 | Contiguous (HG1 and LG1) | Indeterminate (HG1 and LG1) | |||||||
| KRAS | p.G12D | KRAS | p.G12D | KRAS | p.G12D | |||||||
| ARID1A | p.Q1327Afs | KRAS | p.G12V | Separate (HG1 and LG2, on the same glass slide) | Independent/indeterminate (HG1 and LG2)† | |||||||
| PDS-16 | HGPN-1 | LGPN-1 | Separate | Independent (HG1 and LG1)† | ||||||||
| KRAS | p.G12D | KRAS | p.G12V | |||||||||
| KRAS | p.Q61L | |||||||||||
| PDS-19 | HGPN-1 | LGPN-1 | LGPN-2 | Contiguous (HG1 and LG1) | Independent (HG1 and LG1) | |||||||
| KRAS | p.G12R | KRAS | p.G12V | KRAS | p.G12V | |||||||
| CDKN2A | p.D108Y | Separate (HG1 and LG2) | Independent (HG1 and LG2) | |||||||||
| TP53 | p.G334V | |||||||||||
| PDS-21 | HGPN-2 | LGPNc-1 | Contiguous | Independent | ||||||||
| KRAS | p.G12D | KRAS | p.Q61H | |||||||||
| GNAS | p.R201H | |||||||||||
Copy number alteration of CDKN2A could not be assessed in HG2 (HGPN-3).
Two types of KRAS mutations were observed in the same LG-PanIN lesion, indicating that two different clones were present. When the LG-PanIN lesion contained both discordant and identical KRAS mutations, both correlation categories of ‘independent’ and ‘indeterminate’ were designated.
Whole-exome sequencing of five isolated HG-PanINs
We performed whole-exome sequencing of five HG-PanIN lesions that were also analysed by targeted sequencing. The total number of somatic mutations in isolated HG-PanINs ranged from 30 to 175 (median 33) (supplementary material, Table S4). All somatic mutations identified in targeted sequencing were again detected (supplementary material, Table S5 and Figure S2). No mutations were identified in TP53 or SMAD4.
Interestingly, one of the HG-PanIN lesions harboured 175 somatic mutations. This lesion harboured a somatic frameshift mutation, Q146Kfs of MLH1, likely resulting in the large number of somatic mutations in this lesion [16,17].
Copy number analysis of the whole-exome sequencing data revealed variations in 0 to 10 chromosomal loci (with a median of 5) (supplementary material, Table S6). Of these, deletion of the CDKN2A locus on chromosome 9 was detected in one HG-PanIN lesion. Other notable copy number variations included amplification of MYC in one lesion. The amplification of MYC was confirmed by fluorescence in situ hybridization (FISH) (supplementary material, Figure S3).
Immunolabelling for p53 and SMAD4
We performed immunolabelling for p53 and SMAD4 on HG-PanIN lesions (supplementary material, Figure S4). Aberrant expression of p53 was observed in 3 of 16 successfully labelled HG-PanINs, including two with diffuse nuclear labelling and one with lack of expression. Of note, only one of the three lesions with aberrant p53 expression had a somatic TP53 mutation, while the other two were wild type. SMAD4 immunolabelling was retained in all 17 HG-PanIN lesions successfully stained.
Discussion
HG-PanINs are a critical step in pancreatic tumourigenesis and potentially a key target of early detection approaches [6,7]. In order to understand this lesion, we used whole-exome and targeted DNA sequencing approaches to define the genetic alterations in HG-PanIN lesions. We overcame the confounding mimicker of HG-PanIN – cancerization of the ducts – by studying only HG-PanIN lesions from pancreata without invasive pancreatic cancer.
As expected from previous studies, we found that KRAS and CDKN2A are commonly targeted in PanIN lesions [16–19]. KRAS was mutated in 94% of the HG-PanIN lesions as well as 94% of the LG-PanIN lesions, consistent with the previous studies [11,13]. CDKN2A alterations (missense mutation and copy number loss) were also observed in HG-PanINs. In agreement with previous work, we did not find mutations or copy number loss of CDKN2A in LG-PanINs, suggesting that CDKN2A alteration may be a late event during PanIN tumourigenesis [20,21].
Remarkably, in contrast to studies of HG-PanINs from pancreata with invasive carcinoma, we identified TP53 mutations in only 2 of 17 HG-PanIN lesions (12%), and SMAD4 mutations were completely absent [9,10,12,14]. Immunohistochemistry for p53 and SMAD4 also rarely showed aberrant expression of these tumour suppressor genes. What might explain this discrepancy in TP53 and SMAD4 alterations in HG-PanIN compared with previous studies? The studies that reported frequent TP53 and SMAD4 alterations all analysed HG-PanIN in the setting of invasive PDAC – as such, it is possible that many of the ‘HG-PanINs’ in these studies were actually intraductal spread of invasive cancer (which frequently contains these tumour suppressor gene alterations) [15]. An alternative explanation for the discrepancy is that isolated HG-PanIN is biologically different from HG-PanIN associated with PDAC (which could be more advanced and perhaps more likely to have mutations in TP53 and SMAD4).
Other noteworthy alterations in isolated PanINs in our study included mutations in RNF43 and GNAS, which are frequent in IPMN. We cannot exclude the possibility that these HG-PanINs, if left in place, may have eventually grown into lesions large enough to meet size criteria for IPMNs [22]. Still, the presence of high-grade dysplasia argues that these are advanced lesions, not simply early pre-IPMNs.
In conclusion, we performed genetic analysis of isolated HG-PanIN lesions using next-generation sequencing. We confirmed KRAS and CDKN2A mutations in PanINs, but mutations of TP53 and SMAD4 were rarely found in isolated HG-PanIN. Our results suggest that inactivation of TP53 and SMAD4 is limited to, or predominantly occurs in, bona fide invasive carcinomas. These data will profoundly affect the interpretation of results from molecular screening approaches for pancreatic cancer.
Supplementary Material
Figure S1. Pre- and post-images of laser capture microdissection (LCM) of 17 HG-PanIN lesions sequenced
Figure S2. Representative results of somatic mutation analyses of targeted sequencing and whole-exome sequencing, visualized by Integrative Genomics Viewer (IGV)
Figure S3. A representative isolated HG-PanIN with associated multiple LG-PanINs (patient PDS-7)
Figure S4. Immunolabelling for p53 and SMAD4
Table S1. Clinicopathological characteristics of patients with isolated HG-PanIN
Table S2. Summary of targeted sequencing data for isolated HG-PanINs and matched normal samples
Table S3. Summary of targeted sequencing data for LG-PanINs
Table S4. Summary of whole-exome sequencing data for isolated HG-PanINs
Table S5. Somatic mutations of isolated HG-PanINs identified by whole-exome sequencing
Table S6. Copy number alterations of isolated HG-PanINs identified from the whole-exome sequencing data
Acknowledgments
We thank Dr James R Eshleman and Marija Debeljak for helpful discussions in data analysis of targeted sequencing. We acknowledge the following sources of funding: NIH/NCI P50 CA62924; NIH/NIDDK K08 DK107781; Sol Goldman Pancreatic Cancer Research Center; Buffone Family Gastrointestinal Cancer Research Fund; Kaya Tuncer Career Development Award in Gastrointestinal Cancer Prevention; AGA-Bernard Lee Schwartz Foundation Research Scholar Award in Pancreatic Cancer; Sidney Kimmel Foundation for Cancer Research Kimmel Scholar Award; AACR-Incyte Corporation Career Development Award for Pancreatic Cancer Research; Rolfe Pancreatic Cancer Foundation; Joseph C Monastra Foundation; The Gerald O Mann Charitable Foundation (Harriet and Allan Wulfstat, Trustees); Sigma Beta Sorority; Tampa Bay Fisheries Inc; Union for International Cancer Control (UICC) Yamagiwa-Yoshida Memorial International Cancer Study Grants; the Nijbakker-Morra Stichting; the Lisa Waller Hayes Foundation; and the Dutch Digestive Foundation (MLDS CDG 14–02).
Footnotes
Conflict of interest statement: LDW is a paid consultant for Personal Genome Diagnostics.
Author contributions statement
WH, RHH, and LDW conceived and designed the study. WH, LDW, and RHH provided a pathology review. WH, MEP, LAAB, NR, JAS, NY, TU, KH, AY, KH, YS, MS, JSS, AG, CLW, and LDW contributed to the materials and patients. JFG and MN provided support for DNA extraction experiments. WH and PC performed DNA sequencing experiments, with technical support from JY, KS, MS, and MGG. WH, RY, and YN performed FISH experiments. WH, PC, MN, JY, and LDW analysed and interpreted data of DNA sequencing. WH and LDW performed immunohistochemistry and the interpretation of results. WH and LDW generated tables and figures, and WH, RHH, and LDW wrote the manuscript. All authors participated in data interpretation, and critically revised and approved the final manuscript.
References
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Figure S1. Pre- and post-images of laser capture microdissection (LCM) of 17 HG-PanIN lesions sequenced
Figure S2. Representative results of somatic mutation analyses of targeted sequencing and whole-exome sequencing, visualized by Integrative Genomics Viewer (IGV)
Figure S3. A representative isolated HG-PanIN with associated multiple LG-PanINs (patient PDS-7)
Figure S4. Immunolabelling for p53 and SMAD4
Table S1. Clinicopathological characteristics of patients with isolated HG-PanIN
Table S2. Summary of targeted sequencing data for isolated HG-PanINs and matched normal samples
Table S3. Summary of targeted sequencing data for LG-PanINs
Table S4. Summary of whole-exome sequencing data for isolated HG-PanINs
Table S5. Somatic mutations of isolated HG-PanINs identified by whole-exome sequencing
Table S6. Copy number alterations of isolated HG-PanINs identified from the whole-exome sequencing data