Peer review of: Genomic characterization of malignant progression in neoplastic pancreatic cysts (2020)

Reviewers' comments: 

 

Reviewer #1 (Remarks to the Author): 

 

The authors claim that their data “established that both IPMNs and MCNs are direct precursors of 
invasive pancreatic cancer“. This would be the most important conclusion of this manuscript and, if 
true, it is a significant observation. An important limitation as mentioned by the authors is the small 
number of patients. However, as the authors point out, this is the largest study of that short 
published to date. 

The authors’ central conclusion that IPMNs/MCNs are in lineage with invasive pancreatic cancer was 
argued only by point mutations and not rearrangements. Point mutation based lineage analysis 
suffers from two issues: 1. Some point mutations (such as commonly seen driver mutations i.e. 
KRAS_G12V) are not clonally specific as they can be shared among inter-patient samples and can 
appear in intra-patient samples by chance. 2. Even if LCM was used in some cases, there is a 
possibility from contamination from the invasive component as invasive cells migrate. Therefor, in 
this reviewer’s opinion, it would be helpful to alleviate these two concerns by some additional 
analysis and clarification. For example, what would happen if all specific point mutations that are 
found in cancers (or maybe pancreatic cancers) across individuals such as the KRAS (G12V) would be 
eliminated from the analysis? And what if all mutations that are found in more that one patient in 
the current study are not used in the analysis? Statistical analysis that shows the likelihood that a 
patient-specific mutation can be seen in random in intra-patient samples could augment their 
conclusion. Allelic frequency analysis of shared mutations can also be used to eliminate the 
possibility of contamination. 

The detection of many driver mutations in only pre-invasive neoplasms is also curious. It is true that 
subclones evolve on their own and could accumulate mutations that are different that the invasive 
component, but this is observed here in commonly seen driver genes in invasive pancreatic cancer. 
Furthermore, the authors found IPMN regions that shared no mutations with the invasive cancer, 
suggesting that they represented genetically independent clones. The authors also point out that 
there is notably higher degree of intra-patient similarity between invasive and non-invasive samples 
in copy-number space that the mutational space but did not give any biological explanation. Taken 
together, the information presented by the authors makes their claim that “IPMNs and MCNs are 
direct precursors of invasive pancreatic cancer” somewhat questionable but not indisputable. The 
manuscript might improve if some of the points mentioned above are clarified.   

The SMAD4 story is also perplexing and should be further clarified because, as presented in this 
manuscript, it will likely confuse the scientific community. SMAD4 alterations in both IPMNs and 
invasive cancers are found in this study. We know that in invasive carcinoma biallelic inactivation of 
SMAD4 is very common. This can happen by combinations of mutations and rearrangements. 
However, this study is not comprehensive as it lucks the SMAD4 alterations that are due to junctions 
or deletions. Therefor, the question of the possibility of SMAD4 biallelic inactivation in IPMN/MCNs 
has not been answered. Could it be possible that only monoallelic SMAD4 alterations are present in 

IPMN/MCN and it is the second hit on the other allele that turns them to invasive? Also there is a 
possibility that the detected SMAD4 mutations in pre-invasive neoplasia samples are due to 
contamination. This point should be mentioned as a limitation or further be argued using allelic 
frequencies. 

 

 

 

Reviewer #2 (Remarks to the Author): 

 

In this manuscript, Noe and colleagues have conducted whole exome sequencing on paired 
IPMNs/MCNs and associated invasive cancers from 18 patients. In addition they have conducted 
targeted multi region sequencing from additional non invasive foci of IPMNs/MCNs. Using this data, 
they have constructed an evolutionary cascade of IPMN/MCN progression to invasive cancer, 
including putative timelines of progression. 

 

The major findings of the study are as follows: 

A: IPMNs and MCNs are precursors of the associated pancreatic cancer. 

B: Non invasive IPMNs and MCNs in a single patient may contain heteregeneous driver mutations 
such as in KRAS or RNF43. 

C: Some IPMNs represent cancerization of the ducts by retrograde extension of the cancer. 

D: Some mutations in IPMNs/MCNs are early (KRAS, GNAS) and others are late (SMAD4, TGFBR2) 

E: IPMNs and MCNs do not progress overnight to pancreatic cancers and there is a timeline of 
progression in terms of several years. This represents a window of opportunity for early detection. 

 

Each and every one of these findings has been demonstrated previously by work from Johns Hopkins 
and Memorial Sloan Kettering investigators in the field. While I have no quibbles with the volume 
and rigor of work, I am unable to grasp the novelty of the findings in this manuscript. 

 

Minor point: I would be very cautious about 17% somatic mutation rate in ATM especially from 
archival material. ATM (a large gene) has a lot of dubious "mutations" in literature due to spurious 
variant calling.