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We read with interest the paper by Singhi et al.1 The authors prospectively assessed using targeted next-generation sequencing (NGS) 626 pancreatic cyst fluid (PCF) samples from 595 patients. They found that compared with Sanger sequencing, cytology and CEA, preoperative NGS for KRAS/GNAS mutations is highly sensitive for intraductal papillary mucinous neoplasms (IPMNs) and specific for mucinous pancreatic cysts (PCs).
Moreover, alterations in TP53/PIK3/PTEN are significantly associated with advanced neoplasia facilitating decision-making for the management of these lesions.
Despite the evidence that NGS outperforms complementary techniques, we remain puzzled by several features related to the methodology used.
The Results section of the paper shows that the majority of the cases (93%) were satisfactory for molecular testing.
On the contrary, the amount of cyst fluid was insufficient for CEA analysis in 28% of cases, and 60% of specimens were either less than optimal (47%) or unsatisfactory (13%) for cytopathological diagnosis. The proposed primary reason for specimen inadequacy was absent-to-scant cellularity.
In this regard, the authors did not provide any information regarding how they performed the PCF triage.
Considering that PCF is often scant in cellular content, the multimodal approach (cytology, biochemical testing and molecular analysis) further reduces the available material for each technique.
Could the authors specify how they processed the specimens?
The use of NGS for classification is also impressive but the applicability is questionable. The authors mention that the samples had in average 6.93 ng/µL of DNA (median at 4.7).
However, the specification of the PancreSeq panel requires 10 ng/µL (http://mgp.upmc.com/Applications/mgp/Home/Test/PanSeq_details). What percentage of samples actually fit the requirements for an NGS PancreaSeq analysis? This is important when routine implementation of the assay is considered. Similarly, what percentage of samples that had a sequencing depth above 500 (another requirement of the test)?
Finally, some questions remain regarding the classification criteria used in table 4. What is the rationale for the threshold at 55% for GNAS mutations? Isn’t there a risk for overfitting given that no test cohort was available?
Contributors GP and YC wrote the letter. TAMK edited the text for important intellectual content.
Competing interests None declared.
Patient consent Not required.
Provenance and peer review Not commissioned; internally peer reviewed.
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