Very long-term survival following resection for …... · 2015/01/24 1 Very long-term survival following resection for pancreatic cancer is not explained by commonly mutated genes:

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Very long-term survival following resection for pancreatic cancer is not explained by

commonly mutated genes: results of whole-exome sequencing analysis

Running title: Very long-term survival in pancreatic cancer

Marco Dal Molin*1, Ming Zhang*

2, Roeland F. de Wilde*

1, Niki A. Ottenhof

1, Neda Rezaee

3,

Christopher L. Wolfgang3, Amanda L. Blackford

2, Bert Vogelstein

2, Kenneth W. Kinzler

2,

Nickolas Papadopoulos2, Ralph H. Hruban

1, Anirban Maitra

1, and Laura D. Wood

1

Departments of 1Pathology,

2Oncology, and

3Surgery, Johns Hopkins University School of

Medicine, Baltimore, MD, USA.

To whom correspondence should be addressed:

Laura D. Wood, MD, PhD

CRB2 Room 345, 1550 Orleans Street

Baltimore, MD 21231

[email protected]

Research. on June 4, 2020. © 2015 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on January 26, 2015; DOI: 10.1158/1078-0432.CCR-14-2600

http://clincancerres.aacrjournals.org/

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Conflicts of Interest

Under agreements between the Johns Hopkins University, Genzyme, Exact Sciences, Inostics,

Qiagen, Invitrogen and Personal Genome Diagnostics, N.P., B.V., and K.W.K. are entitled to a

share of the royalties received by the University on sales of products related to genes and

technologies described in this manuscript. N.P., B.V., and K.W.K. are co-founders of Inostics

and Personal Genome Diagnostics are members of their Scientific Advisory Boards, and own

Inostics and Personal Genome Diagnostics stock, which is subject to certain restrictions under

Johns Hopkins University policy. L.D.W. is a paid consultant for Personal Genome Diagnostics.

R.H.H. receives royalty payments from Myriad Genetics for the PALB2 invention.




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Statement of Translational Relevance

Pancreatic cancer is a deadly disease in dire need of new clinical approaches. Although the vast

majority of patients with pancreatic cancer have a dismal prognosis, a very rare subset has long-

term survival after surgery. Knowledge of the factors that mediate long-term survival could aid

in the prognostication of patients with pancreatic cancer and provide insights into the underlying

biology of this deadly cancer. In this study, we analyzed the genomes of 35 patients with

pancreatic cancer who survived more than 10 years after surgery. We discovered that the somatic

mutation profiles in these patients were very similar to those of “garden variety” pancreatic

cancer, suggesting that somatic mutations are not the primary determinant of long-term survival

in this disease.




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Abstract

Purpose: The median survival following surgical resection of pancreatic ductal adenocarcinoma

(PDAC) is currently <20 months. However, survival ≥10 years is achieved by a small subset of

patients who are defined as very long-term survivors (VLTSs). The goal of this study was to

determine whether specific genetic alterations in resected PDACs determined very long-term

survival.

Experimental Design: We sequenced the exomes of 8 PDACs from patients who survived ≥10

years. Based on the results of the exomic analysis, targeted sequencing of selected genes was

performed in a series of 27 additional PDACs from VLTSs.

Results: KRAS mutations were identified in 33 of 35 (94%) cancers from VLTSs and

represented the most prevalent alteration in our cohort. TP53, SMAD4, and CDKN2A mutations

occurred in 69%, 26%, and 17%, respectively. Mutations in RNF43, which have been previously

associated with intraductal papillary mucinous neoplasms, were identified in 4 of the 35 cancers

(11%). Taken together, our data show no difference in somatic mutations in carcinomas from

VLTSs compared to available data from PDACs unselected for survival. Comparison of clinico-

pathological features between VLTSs and a matching control group demonstrated that younger

age, earlier stage, well/moderate grade of differentiation, and negative resection margins were

associated with VLTS. However, more advanced stage, poor grade or nodal disease did not

preclude long-term survival.

Conclusion: Our results suggest that in most patients somatic mutations in commonly mutated

genes are unlikely to be the primary determinant of very long-term survival following surgical

resection of PDAC.




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Introduction

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest solid human

malignancies. It is estimated that more than 46,000 new patients have been diagnosed with

PDAC in 2014, and only approximately 6% of those patients will survive 5 years (1). Such a

dismal prognosis is attributed to the late stage at which most patients are diagnosed, together

with the lack of effective systemic therapies to control the disease (2).

Surgical resection of PDAC at an early stage offers the best hope for improving survival

rates, but despite advances in pancreatic surgery, surgically resected patients have a median

survival <20months (2-4). Long-term survival after surgery, however, is achieved by a subset of

patients: up to 20% of all resected patients survive five years after their operation and

approximately 10% are still alive after ten years (5-17). Thus, long-term survival is uncommon

even among patients eligible for surgical resection.

The factors responsible for long-term survival of patients with PDAC are poorly

understood. Previous clinical studies focusing on 5-year and 10-year survivors have reported that

low stage of disease, negative surgical margins, and negative lymph nodes are predictors of a

more favorable prognosis (11, 13-16). Of note, these same studies also showed that positive

resection margins or tumor metastasis to lymph nodes did not preclude long-term survival, as 20-

40% of patients who survived at least five years after surgery had nodal disease and/or margin

positivity. These findings suggest that pathological staging is not the sole determinant of long-

term survival in patients with pancreatic cancer. Hence, the less aggressive phenotype observed

in a subset of pancreatic cancers may be dependent upon distinct genetic, epigenetic, or other

biological factors such as changes in the tumor microenvironment or enhanced immune response

to the cancer by the host.




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In order to determine if specific somatic genetic alterations in resected carcinomas are

associated with very long-term survival, we performed whole-exome sequencing of a series of

well-characterized, surgically resected PDACs obtained from a group of patients who survived at

least ten years after surgical resection.

Materials and Methods

Patients

The study was approved by the Institution Review Board (IRB) of The Johns Hopkins

Hospital. For the purpose of this study, very long-term survivors (VLTSs) were defined as

individuals who underwent surgical resection of an invasive ductal adenocarcinoma of the

pancreas and lived ten or more years following their surgery. Patients included in this study were

selected from all consecutive patients who underwent a pancreatic resection for invasive ductal

adenocarcinoma of the pancreas at The Johns Hopkins Hospital between 1989 and 2000.

An expert pancreatic pathologist carefully reviewed all of the available histological slides to

confirm the diagnosis. Variants of ductal adenocarcinoma, such as colloid carcinoma or

adenosquamous carcinomas, were excluded (18). Ductal adenocarcinomas arising from

intraductal papillary mucinous neoplasms (IPMNs) or mucinous cystic neoplasms (MCNs) were

also excluded from the analysis, as it has been shown that pancreatic cancer originating from

cystic precursor lesions may have a more favorable prognosis than conventional ductal

adenocarcinoma (19, 20). All cases for which microscope slides or tissue blocks were not

available were excluded from the analysis. Patients who had received chemotherapy and/or

radiotherapy prior to surgical resection were also excluded from the analysis, to avoid potential




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confounding effects of treatment-induced DNA damage. The exclusion criteria were applied to

all VLTSs included in our study (both discovery and validation sets).

Date of death or date of last follow-up (for patients who were still alive at the time the

study was initiated) was confirmed by querying the Johns Hopkins Electronic Patient Record and

the Social Security Death Index (SSDI). After accurate selection, thirty-seven patients that met

our inclusion criteria were identified. Among these, ten patients were selected based on the

availability of fresh-frozen neoplastic tissue with adequate cellularity for sequencing. Two

patients were subsequently excluded due to low tumor neoplastic cellularity and insufficient

DNA quality. Ultimately, PDACs from eight patients were available for whole-exome

sequencing (discovery set). Twenty-seven additional pancreatic cancers from VLTSs were

included in the validation set. Non-neoplastic tissue was available for each of the cases analyzed.

Clinico-pathological data from our cohort of very long-term survivors were retrieved

from the Surgical Pathology database. A separate group of 226 patients who underwent surgery

for pancreatic cancer during the years 1989-2000 was chosen as control to explore clinical and

pathological correlations with long-term survival. None of the controls had experienced very

long term survival. Furthermore, patients in the control group with survival <30 days after the

operation were excluded to rule out mortality related to surgical complications. Demographic

and clinico-pathological data were retrieved from a prospectively maintained surgical database;

in both the VLTSs and control groups, the staging of disease was reviewed and updated to

comply with the 7th

edition of the American Joint Committee on Cancer (AJCC) classification

(21).




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Sample acquisition/preparation

After pathology confirmation, each of the 8 fresh-frozen surgically resected carcinomas

was macrodissected to remove residual normal tissue and achieve a neoplastic cellularity of

>50%. Normal tissue was analyzed by frozen section to confirm that no neoplastic tissue was

present.

For each of the 27 cases included in the validation set, 20 slides were recut from

formalin-fixed paraffin-embedded (FFPE) blocks of representative tumor tissue. After

deparaffinization with xylene and H&E staining, slides were manually microdissected to enrich

tumor cellularity and avoid areas of non-neoplastic tissue. A cellularity ≥ 30% was achieved in

each of these 27 samples.

DNA was purified from the macrodissected frozen tumors using the AllPrep kit (Qiagen

Inc. Valencia, CA, cat. #80204) and from microdissected FFPE tumors with a Qiagen FFPE kit

(Qiagen Inc. Valencia, CA, cat. #56494).

Whole-exome Sequencing

We sequenced approximately 21,000 protein-coding genes (>37,000,000 base pairs of

coding sequence) in matched tumor and normal DNA. Genomic DNA libraries were prepared

and captured following Illumina’s (Illumina, San Diego, CA) suggested protocol. The Agilent

SureSelect paired end version 2.0 human exome kit was used to capture the coding sequences




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from individual libraries for each sample. The captured libraries were then sequenced using the

Illumina HiSeq Genome Analyzer (22, 23).

Sequencing reads were analyzed and aligned to human genome 18 (hg 18) using the

Eland algorithm in CASAVA 1.6 software (Illumina, San Diego, CA). The data were filtered for

quality, and alterations in the matched tumor and normal tissues were then compared to identify

tumor-specific somatic mutations as has been described (22, 23). A mismatched base was

identified as a somatic mutation only if the following conditions were met: a) it was identified by

five or more distinct pairs; b) it was identified in reads in both the forward and reverse

directions; c) the number of distinct tags containing a particular mismatched base was at least

15% of the total distinct tags; d) it was not present in any tags in the matched normal samples; e)

the matched normal sample had sufficient coverage to identify the mutation. In addition, for this

study, we only considered nonsynonymous mutations, which altered the protein sequence of the

encoded product. A subset of mutations was verified by visual inspection of the sequencing data.

In addition, 44 mutations were validated by conventional Sanger sequencing.

Targeted sequencing of the 27 additional PDACs was performed using SafeSeqS, an

approach in which template molecules are individually assessed via massively parallel

sequencing (24, 25). The mutational status of the following 9 genes (listed in alphabetical order)

was investigated: BRAF, CDKN2A, GNAS, KRAS, PIK3CA, RNF43, SMAD4, TP53, and VHL.

The validation panel included genes that were mutated in more than one sample in the exomic

analysis and genes known to be commonly mutated in cystic neoplasms of the pancreas (to test

whether some of our PDACs could have originated from a cystic precursor). The entire coding

sequence of CDKN2A, PIK3CA, RNF43, SMAD4, VHL, PIK3CA and TP53 was investigated.




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Analysis of KRAS, GNAS and BRAF was limited to the hotspot locations (KRAS exons 2 and 3;

GNAS exon 8 and BRAF exon 15).

A more detailed description of library preparation, exome capture, and the SafeSeqS

approach is provided in the Supplementary methods.

Statistical analyses

Continuous variables were presented as mean and standard deviation (SD) and compared

using the unpaired t-test. Categorical variables were compared using the Fisher’s exact test. A p-

value <0.05 was considered as statistically significant. Median survival was calculated using the

Kaplan-Meier method. All statistical analyses were performed using GraphPad Prism version

5.04 (GraphPad Software, La Jolla, California USA) and R version 3.1.1.

Results

The set of 35 PDACs from the VLTSs included tumors from 21 female (60%) and 14

male patients (40%) (Table 1). The average age at the time of surgical resection was 59.1 years.

Twenty-nine patients (83%) had undergone a Whipple procedure, four (11%) a distal

pancreatectomy with splenectomy and two (6%) a total pancreatectomy. Twenty-nine patients

(83%) had negative resection margins (R0 disease), whereas five patients (17%) had positive

margins (R1 disease). Twenty-one patients had a stage IIB disease (60%), four patients were

stage IIA (11%), four patients were stage IB (11%) and six patients were stage IA (17%). Data

on adjuvant therapy were available on 17 of the 35 patients (48.5%).




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Sequencing analysis

Whole exome sequencing was performed on 8 PDACs surgically resected from VLTSs.

A total of 50 MB of captured DNA was sequenced with an average depth of coverage of 122-

fold in the targeted region, and >93.6% of targeted bases were represent by at least 10 reads

(Supplementary Table 1). These carcinomas had a mean of 37.6 non-synonymous somatic

mutations and were all enriched for C:G-to-T:A transitions (78.7% of mutations). Three hundred

and one somatic mutations were identified in 274 genes in the 8 carcinomas (Supplementary

Table 2). Forty-four somatic mutations in 27 genes were confirmed with conventional Sanger

sequencing.

Six of eight carcinomas harbored KRAS mutations (75%) and 6 of 8 had TP53 mutations

(75%). Only one of the eight carcinomas harbored a mutation in the SMAD4 gene (12.5%). Two

mutations were identified in the CDKN2A gene (25%) and 3 carcinomas had mutations in the

RNF43 gene (37.5%) (Table 2).

The BRAF, CDKN2A, GNAS, KRAS, PIK3CA, RNF43, SMAD4, TP53 and VHL genes

were sequenced using Safe-SeqS in a panel of 27 additional surgically resected ductal

adenocarcinomas of the pancreas obtained from VLTSs. KRAS was the most commonly mutated

gene, as alterations were found in 27 of 27 (100%) of these validation cancers. Four of the 27

validation cancers harbored CDKN2A mutations (11%), eight harbored SMAD4 mutations (29%),

and 18 had TP53 mutations (68%). GNAS, RNF43 and BRAF were each found mutated in 1

sample (4%). No mutations were found in the PIK3CA and VHL genes (Supplementary Table 3).

When the results from the whole-exome and targeted sequencing were combined, KRAS

proved to be the most commonly altered gene, with activating mutations identified in 33 (94%)

of the 35 carcinomas. TP53 mutations were found in 24 (69%) of 35 cases, SMAD4 mutations in




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9 cases (26%), and CDKN2A mutations in 6 cases (17%). RNF43 mutations were identified in 4

(11%) of the carcinomas (Table 2).

Clinico-pathological correlations

Clinical and pathological characteristics of the cohort of 35 VLTSs were compared with a

control group of 226 surgically resected patients matched by years of surgery (1990-2000)

(Table 3). The VLTS group was significantly younger at the time of surgery (mean age 59.1 vs.

65.7, p=0.001). The mean tumor size was significantly smaller in the group of VLTSs than in the

control group (2.8 cm vs. 3.1 cm). Compared with the control group, VLTSs were more likely to

have stage IA-IB disease (p<0.001), well or moderately differentiated tumor grade (p=0.002),

and negative resection margins (p=0.011) (Table 3). The VLTSs also had a higher rate of

negative nodal status than the controls (p=0.036). The median survival for VLTSs and controls

was 196 months and 14 months, respectively. Of note, none of the VLTSs was known to

experience a tumor recurrence in the 10-year follow-up period, although we did not have this

data on all patients. None of the VLTSs had a family history of pancreatic cancer, although

several had personal histories of other tumor types, including breast, prostate, and lung cancers.

However, none of these patients had mutations suggestive of an inherited cancer predisposition

syndrome (for example, BRCA2 mutation in patient with breast and pancreatic cancer).

Discussion

The characterization of the coding sequences of pancreatic cancer has greatly advanced

our understanding of the genetic alterations that underpin this devastating disease (26). The

genetic landscape of PDAC is defined by four mutational “mountains” (KRAS, TP53, CDKN2A,




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SMAD4), which are thought to be the main drivers of carcinogenesis. In addition, numerous

other genes harbor mutations at much lower rates, most of which are considered of little

functional significance (passenger genes) (26).

More recent studies have improved the identification of driver events by integrating

sequencing data with data obtained from functional screens and animal models, or targeting

selected groups of patients, such as those with genetic predisposition to PDAC (27-29). These

studies have identified additional candidate driver genes that are potentially relevant in sporadic

(MLL3, USP9X, MAP2K4) and familial (BRCA2, PALB2, ATM) pancreatic cancer (27-31), but

which are each mutated in only a small fraction of the cancers.

The sequencing of cancers has helped facilitate the recognition of histologically

indiscernible molecular subgroups that might determine sensitivity to a specific therapy (32) or

have prognostic significance. For example, previous genetic analyses of surgically resected

PDACs have demonstrated that Smad4 protein loss correlates with patterns of metastatic spread

and worse prognosis (33-35). The patients whose pancreatic cancer had Smad4 loss were more

likely to die with widespread (in some cases thousands of) metastases, while the patients whose

pancreatic cancers had intact Smad4 were more likely to die with localized disease (33, 34).

We hypothesized that genetic analysis of a group of pancreatic cancers characterized by

unconventional clinical behavior, such as those from patients who survived ten years or longer

after surgery, could identify genetic determinants of long-term survival. Over 1700 surgical

resections for pancreatic cancer have been performed at The Johns Hopkins Hospital (5),

providing us with a unique patient population that includes a number of patients who survived

more than 10 years after their surgery. In an effort to define the genetic changes that characterize




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long-term survival, we applied whole-exome and targeted sequencing to a series of well-

characterized PDACs resected from very long term survivors (VLTSs).

To our surprise, we found no significant differences in the mutational profile of this

unique cohort of pancreatic cancers, compared to the mutational profile that has been previously

published by our group and others in “garden variety” PDAC (26, 27, 36). After merging the

discovery and validation sets, KRAS was confirmed as the most commonly mutated gene (94%)

in the PDACs from VLTSs, at a rate that is comparable to rates reported in literature. Similarly,

TP53, SMAD4 and CDKN2A were also commonly mutated at rates comparable to those

published in the literature for non-selected PDACs (Table 2). The overall prevalence of RNF43

mutations in our cohort was 11% (4 out of 35 cases). A similar prevalence (10%) was also

reported by the International Cancer Genome Consortium (ICGC), for a large cohort of

pancreatic cancers not selected based on long-term survival (37).

The RNF43 gene, which encodes a protein with intrinsic U3 ubiquitin ligase activity, is

relatively understudied in pancreatic cancer (38). However, inactivating mutations in the RNF43

gene have been reported in intraductal papillary mucinous neoplasms (IPMNs) of the pancreas

(38, 39). It has been suggested that IPMN associated invasive carcinomas are less aggressive

than carcinomas that do not arise in association with an IPMN (19, 40). Origin in an IPMN, as

evidenced by the presence of RNF43 mutations, could therefore explain some of the VLTS in our

cohort. Although careful pathological re-evaluation of all cases included in our analysis showed

no evidence of IPMN, it is possible that in some instances the invasive carcinoma overgrew a

pre-existing non-invasive component, resulting in loss of the IPMN.




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Recent studies have shown that IPMNs commonly harbor activating GNAS mutation,

which are very specific for this tumor type (38, 39, 41, 42). GNAS was included in our validation

panel to verify whether some of the cancers had indeed originated from IPMNs. No GNAS

mutations were identified in the 8 carcinomas subjected to exome sequencing, and only one of

the 27 samples analyzed at targeted sequencing harbored a GNAS mutation (Supplementary

Table 2). Interestingly, that one sample did not harbor an RNF43 mutation. It should be noted

that the absence of GNAS mutations in the carcinomas from VLTSs might be the result of the

histologic inclusion criteria employed in this study. GNAS mutations are associated with

intestinal differentiation in IPMNs and intestinal-type IPMNs often give rise to colloid-type

invasive carcinomas, which were excluded from this study (39, 41, 42). Therefore, we may have

selected for IPMN-associated cancers that harbor RNF43 mutations but not GNAS mutations.

Theoretically, a specific genetic alteration may confer prolonged survival to patients with

PDAC by either rendering the cancer less aggressive or determining increased sensitivity to

therapies that target specific genetic abnormalities. The latter instance is exemplified by

alterations in the Fanconi anemia/BRCA2 pathways, which render cancer cells hypersensitive to

inter-strand crosslinking agents (43). A dramatic response to therapy with Mitomycin C and

other DNA-damaging agents has been occasionally reported in patients with metastatic

pancreatic cancer resistant to gemcitabine that harbored mutations in the BRCA2 or PALB2 genes

(44, 45). Our analysis did not reveal biallelic inactivation of the BRCA2 or PALB2 genes in any

of the 8 samples subjected to whole-exome sequencing, excluding the possibility that this could

have been a mechanism of very long-term survival in this portion of our VLTS cohort.

The comparison of clinico-pathological characteristics between the VLTSs and an

independent group of well-characterized, surgically resected pancreatic adenocarcinomas




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confirmed the results of previous clinical studies : VLTSs had more favorable features, such as

smaller and better differentiated tumors, lower stage of disease, and higher rate of negative

surgical margins. However, the majority of VLTSs in our cohort had cancer spread to lymph

nodes (66%); furthermore, a poorly differentiated cancer or positive resection margin were not

uncommon in the VLTS group (Table 3), suggesting that biological rather than clinical or

pathological factors are likely the main determinants of prognosis.

Although this study suggests that very long-term survival in patients with pancreatic

cancer is not dependent upon specific genetic alterations, it should be noted that our analyses

were limited to the exomes of these cancers. Other types of genetic changes, such as

chromosomal re-arrangements, translocations, large deletions and insertions, inversions,

chromothripsis, and intronic alterations, would have been missed by our approach, as would

epigenetic changes as well as changes in gene and microRNA expression. We therefore cannot

exclude the possibility that one of these other alterations is driving very long-term survival. In

addition, we cannot exclude the possibility that coding mutations were missed in our approach

(so called false negatives) due to variability in coverage or mutation calling. However, the

identification of somatic alterations in frequently mutated genes in PDAC at rates similar to

those previously described argues against significant false negatives in our analysis. Finally, we

did not examine the contribution of tumor microenvironment and host immune response, which

could also have been responsible for the improved survival.

Although only 35 VLTSs were analyzed in our study, this is a relatively large number,

given the exceptionally low number of patients with PDAC who survive 10 or more years. We

would have expected that, if a significant fraction of the VLTSs were dependent upon specific

alterations in coding DNA sequences, our analysis would have been able to appreciate them.




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Still, since we only performed whole exome sequencing on 8 VLTSs, it is possible that our

analysis missed an uncommon mutation that contributes to long-term survival. Considering the

diversity of possible genomic alterations, additional studies incorporating multiple mutation

detection approaches with additional samples as part of multi-institutional efforts are required to

validate our findings. Analyses that consider mutations on a pathway rather than individual gene

level may also identify determinants of long term survival not evident from our initial gene-

based analysis.

In summary, our results suggest that nonsynonymous somatic mutations in commonly

mutated genes are unlikely to be the primary determinant of very long-term survival following

the surgical resection of pancreatic cancer.

Grant Support

This study was supported by Blum-Kovler Foundation, NIH grant CA62924, Lustgarten

Foundation for Pancreatic Cancer Research, Sol Goldman Pancreatic Cancer Research Center,

and The Virginia and D. K. Ludwig Fund for Cancer Research.




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Tables

Table 1. Clinico-pathological data of 35 patients with pancreatic cancer who survived more than

10 years after surgery

Table 2. Prevalence of mutations among candidate driver genes in VLTSs

Table 3. Clinico-pathological characteristics of VLTSs and control PDAC patients

Supplementary Table 1: Summary of exome sequencing data for VLTSs

Supplementary Table 2. Somatic mutations identified in exome sequencing of VLTSs




21

Supplementary Table 3. Somatic mutations in targeted sequencing from Validation Set




Sample ID Set

Age at

Surgery

(years)

SexTumor

LocationType of Surgery

Tumor

size

(cm)

Grade Margin

statusTNM

Stage

(AJCC

7th Ed.)

Adjuvant therapy

VLTS-1 Disc 59 M Head Whipple 3.0 G2 R0 T3N1M0 IIB chemotherapy, radiotherapy, immunotherapy

VLTS-2 Disc 68 F Head Whipple 1.8 G2 R0 T1N0M0 IA chemotherapy, radiotherapy

VLTS-3 Disc 52 M Head Whipple 4.0 G3 R0 T2N1M0 IIB chemotherapy, radiotherapy

VLTS-4 Disc 59 F Head Whipple 3.0 G2 R0 T2N1M0 IIB chemotherapy, radiotherapy

VLTS-5 Disc 65 M Head Whipple 2.8 G1 R0 T2N0M0 IB unknown

VLTS-6 Disc 51 F Head Whipple 3.0 G2 R0 T2N1M0 IIB unknown

VLTS-7 Disc 38 F Head Whipple 1.7 G2 R1 T1N1M0 IIB unknown

VLTS-8 Disc 42 M Head Whipple 2.5 G2 R0 T2N1M0 IIB chemotherapy, radiotherapy

VLTS-9 Val 64 F Head Whipple 1.5 G2 R1 T1N1M0 IIB unknown

VLTS-10 Val 57 F Head Whipple 1.0 G2 R0 T1N0M0 IA unknown


VLTS-12 Val 66 M Head Whipple 1.0 G1 R0 T1N0M0 IA unknown

VLTS-13 Val 60 F Head Whipple 2.1 G1 R0 T2N0M0 IB unknown


VLTS-15 Val 44 M Head Whipple 3.0 G2 R1 T2N1M0 IIB unknown


VLTS-17 Val 62 M Head Whipple 3.0 G3 R0 T3N1M0 IIB chemotherapy, radiotherapy

VLTS-18 Val 80 M Tail Distal pancreatectomy 2.9 G3 R0 T3N1M0 IIB unknown

VLTS-19 Val 59 F Head-Body-Tail Total pancreatectomy 2.0 G2 R1 T3N1M0 IIB unknown

VLTS-20 Val 78 M Head Whipple 2.7 G2 R1 T3N0M0 IIA unknown

VLTS-21 Val 50 F Head Whipple 2.7 G2 R0 T3N1M0 IIB chemotherapy, radiotherapy, immunotherapy


VLTS-23 Val 53 F Tail Distal pancreatectomy 3.0 G2 R1 T2N0M0 IB unknown


VLTS-25 Val 52 F Tail Distal pancreatectomy 6.0 G3 R0 T2N0M0 IB unknown

VLTS-26 Val 65 M Head Whipple 1.6 G2 R0 T3N0M0 IIA unknown


VLTS-28 Val 66 F Head Whipple 2.0 G2 R0 T3N0M0 IIA chemotherapy, radiotherapy

VLTS-29 Val 58 F Head Whipple 3.5 G3 R0 T3N1M0 IIB chemotherapy, radiotherapy


Table 1. Clinico-pathological data of 35 patients with pancreatic cancer who survived more than 10 years after surgery




VLTS-31 Val 41 F Head-Body-Tail Total pancreatectomy 1.5 G3 R0 T1N0M0 IA radiotherapy

VLTS-32 Val 78 M Head Whipple 6.0 G2 R0 T3N0M0 IIA radiotherapy


VLTS-34 Val 64 F Head Whipple 3.0 G3 R0 T2N1M0 IIB chemotherapy, radiotherapy

VLTS-35 Val 66 F Tail Distal pancreatectomy 5.0 G2 R0 T2N1M0 IIB chemotherapy, radiotherapy

Disc: Discovery; Val: Validation; G1: well-differentiated; G2: moderately differentiated; G3: poorly differentiated. R0:

negative resection margin; R1: microscopic positive margin




GeneDiscovery set (n=8)

N (%)

Validation set (n=27)

N (%)

Combined (n=35)

N (%)

KRAS 6 (75) 27 (100) 33 (94.3)

TP53 6 (75) 18 (63) 24 (68.6)

SMAD4 1 (12.5) 8 (29.6) 9 (25.7)

CDKN2A 2 (25) 4 (11.1) 6 (17.1)

RNF43 3 (37.5) 1 (3.7) 4 (11.4)

BRAF 1 (12.5) 1 (3.7) 2 (5.7)

GNAS 0 (0) 1 (3.7) 1 (2.8)

PIK3CA 0 (0) 0 (0) 0 (0)

VHL 0 (0) 0 (0) 0 (0)

Table 2. Prevalence of mutations among candidate driver genes in VLTSs




VLTS (n=35) Controls (n=226) p

Age in years* mean (+/- SD) 59.1 (10.72) 65.7 (11.19) 0.001

Gender, n (%)^F 21 (60) 105 (46)M 14 (40) 121 (54)

Tumor size (cm)* mean (+/- SD) 2.8 (1.18) 3.1 (1.61) 0.029

Tumor location, n (%)^Head 29 (83) 186 (82)

Body-tail 4 (11) 23 (10)Entire gland 2 (6) 17 (7)

Stage, n (%)^IA 6 (17) 4 (2)IB 4 (11) 7 (3)IIA 2 (6) 26 (12)IIB 23 (66) 179 (79)III 0 10 (4)

Grade, n (%)^Well/moderately differentiated 27 (77) 110 (49)

Poorly differentiated 8 (23) 116 (51)Margin status, n (%)^

R0 29 (86) 131 (58)R1 6 (14) 71 (31)R2 0 24 (11)

Nodal status, n (%)^N0 12 (34) 39 (17)N1 23 (66) 187 (83)

* unpaired t-test, two-tailed^Fisher's Exact test

0.036

0.001

Table 3. Clinico-pathological characteristics of VLTSs and control PDAC patients

0.149

0.912

<0.001

0.011




Published OnlineFirst January 26, 2015.Clin Cancer Res Marco Dal Molin, Ming Zhang, Roeland F de Wilde, et al. whole-exome sequencing analysiscancer is not explained by commonly mutated genes: results of Very long-term survival following resection for pancreatic

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