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Reprint requests: Charlotte Vestrup Rift, MD, PhD, Department of Pathology, Copenhagen University Hospital Rigshospitalet, Blegdamsvej 9, DK-2100 Copenhagen Ø, Denmark.
Affiliations
Department of Pathology, Copenhagen University Hospital Rigshospitalet, Copenhagen, Denmark
Department of Pathology, Copenhagen University Hospital Rigshospitalet, Copenhagen, DenmarkGastroenterology Unit, Copenhagen University Hospital Herlev and Gentofte, Herlev, Denmark
Pancreatitis Centre East, Gastroenterology Unit, Copenhagen University Hospital Hvidovre, Hvidovre, DenmarkDepartment of Clinical Medicine, University of Copenhagen, Copenhagen, Denmark
Gastroenterology Unit, Copenhagen University Hospital Herlev and Gentofte, Herlev, DenmarkDepartment of Clinical Medicine, University of Copenhagen, Copenhagen, Denmark
Department of Pathology, Copenhagen University Hospital Rigshospitalet, Copenhagen, DenmarkDepartment of Clinical Medicine, University of Copenhagen, Copenhagen, Denmark
Department of Pathology, Copenhagen University Hospital Rigshospitalet, Copenhagen, DenmarkDepartment of Clinical Medicine, University of Copenhagen, Copenhagen, Denmark
Gastroenterology Unit, Copenhagen University Hospital Herlev and Gentofte, Herlev, DenmarkDepartment of Clinical Medicine, University of Copenhagen, Copenhagen, Denmark
Department of Pathology, Copenhagen University Hospital Rigshospitalet, Copenhagen, DenmarkDepartment of Clinical Medicine, University of Copenhagen, Copenhagen, Denmark
Recent advances have introduced molecular subtyping of pancreatic cystic lesions (PCLs) as a possible amendment to the diagnostic algorithm. The study evaluated the feasibility and diagnostic accuracy of molecular analysis and subtyping of PCLs using the recently introduced EUS-guided through-the-needle-biopsy (TTNB) sampling.
Methods
We prospectively included 101 patients in the study who presented with PCLs >15 mm in the largest cross-section. EUS-guided TTNB samples were obtained by a micro-biopsy forceps introduced through a 19-gauge needle. The TTNB samples were analyzed by next-generation sequencing (NGS) for point mutations in tumor suppressors and oncogenes using a 51-gene customized hotspot panel. Sensitivity and specificity were calculated with the histologic diagnosis as reference.
Results
After initial microscopic evaluation of the samples, 91 patients had residual TTNB samples available for NGS. Of these, 49 harbored mutations, most frequently in KRAS and GNAS, reflecting an excess frequency of intraductal papillary mucinous neoplasms (IPMNs) in the study population. A sensitivity and specificity of 83.7% (95% confidence interval [CI], 70.3-92.7) and 81.8% (95% CI, 48.2-97.7), respectively, were demonstrated for the diagnosis of a mucinous cyst and 87.2% (95% CI, 74.2-95.2) and 84.6% (95% CI, 54.5-98.1) for the diagnosis of an IPMN.
Conclusions
Thus, molecular analysis of TTNB samples by NGS has high sensitivity and specificity for diagnosing mucinous cysts and IPMNs. Although the procedure comes with a risk of adverse events of 9.9%, TTNB samples are a robust alternative to cyst fluid for a combined histologic and molecular diagnosis of PCLs. (Clinical trial registration number: NCT03578445.)
Pancreatic ductal adenocarcinoma can develop from pancreatic cystic lesions (PCLs), including intraductal papillary mucinous neoplasms (IPMNs) and mucinous cystic neoplasms.
A new micro-forceps, introduced through a 19-gauge needle, has proven useful for procurement of through-the-needle-biopsy (TTNB) samples that represent both the epithelial and stromal component of the cyst wall.
Diagnostic accuracy of EUS-guided through-the-needle-biopsies and simultaneously obtained fine needle aspiration for cytology from pancreatic cysts: a systematic review and meta-analysis.
We published a study regarding the adequacy of TTNB sampling for mutational analysis using next-generation sequencing (NGS) and a custom-made panel of frequently mutated oncogenes and tumor suppressor genes, with promising results.
This study aimed to investigate the feasibility and diagnostic accuracy in terms of diagnostic yield, sensitivity, and specificity of NGS of TTNB samples in a larger prospective cohort.
Methods:
Patient cohort
From February 2018 until August 2019, we prospectively included patients referred to the regional multidisciplinary conference at Copenhagen University Hospital, Rigshospitalet, which serves more than 2.6 million people. The cohort was identical to that of our previously published study used to evaluate clinical and procedural parameters.
we enrolled patients with a PCL ≥15 mm and any PCL with high-risk stigmata or worrisome features. The included patients had an EUS performed with procurement of TTNB samples and cyst fluid for cytology and carcinoembryonic antigen analysis as previously described.
Excess cyst fluid after the preparation of cytology slides was prepared as a formalin-fixed paraffin-embedded cell block. As an additional inclusion criterion of this study, patients had to have available tissue after initial specimen processing and immunohistochemical (IHC) analysis according to the algorithm previously described by Rift et al.
The study was approved by the Regional Ethics Committee (study ID: H-17031060) and registered at ClinicalTrials.gov (NCT03578445).
Quality of the TTNB samples
Representative hematoxylin and eosin–stained slides of the TTNB samples were digitized using a Hamamatsu 360 nanozoomer slide scanner (Hamamatsu, Hamamatsu, Japan). The area and largest cross-section of each biopsy sample was measured in the Nanozoomer Digital Pathology viewer software (Hamamatsu, Hamamatsu, Japan) to evaluate relation between the size of TTNB sample and the cellularity of neoplastic cells.
Targeted NGS
TTNB samples, cell blocks, and surgical specimens were submitted for NGS. Two separate tissue blocks with regions of interest with >50% tumor cells were submitted from each resected specimen. A modified Ion AmpliSeq Cancer Hotspot Panel version 2 (Thermo Fisher, Waltham, Mass, USA) that covers the most prevalent hot spot mutations in 51 genes, including KRAS, GNAS, RNF43, CDKN2A, SMAD4, TP53, BRAF, PTEN, VHL, and PIK3CA (Supplementary material, available online at www.giejournal.org), was used. Details regarding DNA extraction and targeted NGS can be found in our previous study.
The investigator (L.C.M.) who performed the analysis and confirmed the clinically relevant variants was blinded to the clinicopathologic data of the TTNB samples.
Outcome measures
The primary endpoint was to determine the diagnostic yield of molecular analysis of TTNB samples as per intention to diagnose. This was done for molecular analysis of TTNB samples alone and for an integrated evaluation of both molecular analysis and previously described histopathology of TTNB samples.
Furthermore, we evaluated the sensitivity and specificity of the NGS analysis by comparison with the histology of the TTNB sample as a criterion standard. For these outcomes, the following categories were defined regarding the molecular findings: 1, diagnosis of neoplastic cyst not otherwise specified (presence of any genetic aberration); 2, mucinous cyst not otherwise specified (minimum KRAS or GNAS); 3, IPMN (KRAS and/or GNAS); and 4, serous cystic neoplasm (SCN) (VHL). Sensitivity and specificity were calculated for all categories, and diagnostic yield was calculated for categories 2 and a combination of categories 3 and 4, designated as specific cyst type. This compilation of categories 3 and 4 was done to compare diagnostic yield with our previously reported data.
When appropriate, continuous variables are presented as mean (range), median, or proportions and were compared by t test or Wilcoxon signed-rank test. Statistical significance was set at P < .05. Diagnostic yield was calculated per intention to diagnose. Sensitivity and specificity for the profile obtained by NGS were calculated from 2 × 2 contingency tables for TTNB samples with a final diagnosis of either neoplastic cyst, mucinous cyst, IPMN, or SCN, obtained by microscopic evaluation of the TTNB samples and thus excluding all nondiagnostic samples. For example, a case diagnosed as IPMN with pathogenic variants of KRAS and GNAS was determined to be true positive for a neoplastic cyst, a mucinous neoplasm, and IPMN and true negative for an SCN. Statistical software “R” was used for all analyses.
Results
Clinicopathologic characteristics of patients with successful procurement of TTNB samples for NGS
At the multidisciplinary conference, 521 patients were screened, 101 patients were included for the EUS-TTNB sampling procedure, 95 patients had successful procurement of TTNB samples, and 91 patients had biopsy samples available for NGS and were included in the study (Fig. 1). Failure of tissue procurement during EUS-TTNB sampling was because of small cyst size. Reasons for the exclusion of 420 patients are provided in our previously reported study.
Figure 1Study cohort. Of 101 eligible patients, 95 underwent successful EUS-guided through-the-needle-biopsy (TTNB) sampling procedure. Of these, 91 patients had tissue available for next-generation sequencing (NGS) after initial microscopic and immunohistochemical evaluation.
The study cohort had a mean age of 68 years with an almost equal distribution of men and women (Table 1). Most cysts were located at the head of the pancreas with a mean cyst size of 27.4 mm. The cysts were initially classified by microscopic examination and IHC subtyping of the TTNB sample as previously described
Size and quality of biopsy samples before targeted sequencing
After the initial evaluation, 172 TTNB samples from 91 patients were available, with a median number of 2 TTNB samples from each patient. The mean size of the TTNB samples was .61 mm2 (range, .029-2.62) in the largest area and 1.09 mm (range, .20-2.63) in the largest cross-section. Fourteen patients (15.4%) had only 1 TTNB sample available for analysis, whereas 66 (72.5%) and 11 (12.1%) patients had 2 or 3 TTNB samples, respectively.
The TTNB samples from each patient were divided into 3 groups based on their diagnostic eligibility by microscopic examination of hematoxylin and eosin–stained slides (Fig. 2): TTNB samples deemed sufficient for NGS based on the presence of >200 neoplastic epithelial cells per slide (n = 83), second best TTNB samples suitable for NGS based on a reduced number of cells of <200 per slide (n = 35), and TTNB samples with no neoplastic epithelial cells (n = 54). Comparison of the 3 groups revealed that group 1 with the most suitable biopsy samples in terms of cellularity of neoplastic cells also had the largest TTNB samples in cross-section (P = .0035) and area (P =.0011) (Supplementary Fig. 1, available online at www.giejournal.org).
Figure 2Examples of through-the-needle-biopsy samples with (A) >200 neoplastic cells, (B) <200 neoplastic cells, and (C) no neoplastic cells. (H&E stain, ×10 magnification; scale bar, 250 μm.)
All patients had at least 1 TTNB specimen submitted for NGS regardless of the microscopic eligibility. Of the 91 patients, 75 had a minimum of 1 biopsy sample adequate for NGS in terms of both cell and DNA quantity, and 16 were insufficient (Fig. 1). The NGS analysis was performed on the adequate TTNB samples after DNA extraction with a mean mapped read of 976,287 (range, 266,027-6,454,890), mean depth of ,825 (range, 662.5-13,186), and a mean uniformity of 91.6% (range, 77.5%-96.8%). Of the TTNB samples adequate for NGS, 49 had molecular aberrations and 26 were wild-type. An overview of the individual biopsy samples and the mutated genes is presented in Figure 3.
Figure 3Overview of patients with detected mutations stratified by cyst type. Each row represents a patient and each small square a biopsy sample. For example, patient 2 in the first row had an intraductal papillary mucinous neoplasm (IPMN) of the gastric subtype. The patient had 2 biopsy samples available for next-generation sequencing (NGS). However, only 1 biopsy sample was sufficient for analysis in terms of cellularity and thus was submitted for NGS. The NGS analysis revealed concomitant mutations in KRAS and GNAS. (Figure created with Biorender.com.)
Of the patients with molecular aberrations, 25 had 2 biopsy specimens with sufficient DNA submitted for NGS analysis. Of these, 13 patients had identical or shared variants between the first and second biopsy sample, including 3 patients with 2 wild-type TTNB samples, whereas 12 patients had different mutations in the 2 TTNB samples. Of the 56 IPMNs, 42 had mutations in KRAS and/or GNAS with a mutated allele frequency of 2% to 55% and 3% to 47%, respectively. One patient (patient 87, Fig. 3) had a nondiagnostic TTNB sample before sequencing but was diagnosed with an IPMN based on concomitant KRAS and GNAS mutations. Seven IPMN patients with mutations in KRAS and/or GNAS also had alterations in TP53, BRAF, PTEN, CDKN2A, PIK3CA, NOTCH1, APC, NRAS, ATM, and/or SMARCB1, all with lower or similar frequency as concomitant KRAS and/or GNAS.
In the non-IPMN group with an NGS result other than wild-type, 3 patients with unclassifiable TTNB samples before NGS analysis were deemed likely to have mucinous cysts based on the presence of aberrations in KRAS or GNAS. Two SCNs had detectable VHL mutations. Two patients were nonclassifiable based on the TTNB specimens alone but had alterations in PTEN and BRAF, respectively, and were thus reclassified as minimum neoplastic cysts.
Twenty-six patients had a wild-type result of the NGS analysis, 6 of whom were initially diagnosed by microscopic evaluation of the TTNB samples with low-grade gastric IPMNs, 7 with SCNs, 1 with a neuroendocrine tumor, and 2 with low-grade mucinous cystic neoplasms; the remaining 10 patients were unclassifiable by TTNB sampling. The 16 TTNB samples with inadequate quality of DNA were diagnosed as IPMNs (n = 9) of which 1 was an IPMN of pancreatobiliary subtype with high grade dysplasia, 2 were SCNs, and 5 were unclassifiable.
Analysis of cyst fluid
Twenty-two patients had additional cell blocks with neoplastic epithelial cells. The NGS analysis was performed with a mean mapped read of 1,052,632 (range, 302,051-2,428,376), mean depth of 3256 (range, 918-7571), and a mean uniformity of 92.4% (range, 89.9%-94.3%). Of these, the result of the NGS analysis was identical to the analysis of the corresponding TTNB samples in 6 cases, and 5 cases revealed shared genetic aberrations or a wild-type result (Supplementary Table 1, available online at www.giejournal.org). Additional mutations were identified in 5 cell blocks compared with TTNB samples, whereas 6 cases had inadequate DNA quality for the analysis.`
Surgical cohort
Thirteen patients underwent surgery within 3 to 221 days after the initial EUS procedure with procurement of TTNB samples (Supplementary Table 2, available online at www.giejournal.org). The NGS analysis was performed with a mean mapped read of 790,323 (range, 318,194-5,207,124), mean depth of 2413 (range, 906-16,250), and a mean uniformity of 93.4% (range, 72.7%-96.5%). Four patients who underwent surgery had an initial NGS result of the TTNB sample that was identical to the surgical specimen. Three patients had inconclusive or nondiagnostic TTNB samples by microscopic examination with a corresponding wild-type result of the NGS analysis. One patient (patient 3) was initially diagnosed with a low-grade IPMN on cytology, and the corresponding TTNB sample was inconclusive but was submitted to NGS, revealing concomitant KRAS and GNAS mutations in accordance with a diagnosis of IPMN. On examining the resected specimen, the diagnosis was changed to an SCN, and the NGS of the specimen revealed a VHL mutation in favor of this revised diagnosis. In the surrounding, otherwise normal-appearing background tissue of the specimen, several foci of low-grade pancreatic intraepithelial neoplasia were located, and thus contamination of the diagnostic samples was suspected. Macro-dissection and investigation of the separate foci of pancreatic intraepithelial neoplasia by NGS from the surgical specimen yielded a wild-type result.
Follow-up
According to the clinical guidelines, the remaining 88 patients who did not undergo surgery were enrolled in a clinical follow-up program.
Follow-up information was available for all patients for up to 3 years. All the IPMNs with mutations in TP53, BRAF, PTEN, PIK3CA, NOTCH1, APC, ATM, and/or SMARCB1 were classified as low grade based on their TTNB samples, and none has developed pancreatic cancer on follow-up. The patient with IPMN and concomitant alterations in KRAS, GNAS, and NRAS died of prostate cancer 10 months after the procedure. One patient with IPMN and concomitant KRAS/GNAS had an additional SMAD4 mutation detected in the cell block. This patient had no progression of the IPMN but died 2 years later of hepatocellular carcinoma. Another IPMN patient with concomitant alterations in KRAS/GNAS and CDKN2A declined surgery and is alive and well. Four other patients from the cohort died because of reasons other than pancreatic cancer, and the remaining patients are alive and well. No patients were lost to follow-up.
Diagnostic value of molecular analysis of TTNB samples
Diagnostic yield for detecting a mucinous cyst and specific cyst type by NGS alone was 44.5% and 27.7%, respectively (Table 2). When combining the diagnosis obtained by microscopic evaluation of the TTNB sample with the NGS result, the diagnostic yield rose to 73.3% and 70.3%, respectively, for the discrimination between mucinous and nonmucinous cysts and for detection of a specific cyst type. However, no statistical difference was found between these combined diagnostic yields and the originally reported diagnostic yields from our previous study.
The sensitivity and specificity of the additional NGS analysis were calculated in a subgroup of valid samples in a per-protocol approach (n = 60; Table 3). A sensitivity and specificity of 83.7% (95% confidence interval [CI], 70.3-92.7) and 81.8% (95% CI, 48.2-97.7), respectively, were demonstrated for the diagnosis of a mucinous cyst and 87.2% (95% CI, 74.2-95.2) and 84.6% (95% CI, 54.5-98.1) for the diagnosis of an IPMN.
Table 2Diagnostic yield of cytology and histology by TTNB sampling and NGS
Diagnostic yield
Cytology (%)
TTNB sampling (%)
NGS of TTNB samples (%)
TTNB sampling and NGS (%)
P value cytology vs TTNB sampling
P value cytology vs NGS of TTNB samples
P value TTNB sampling vs TTNB sample histology and NGS
In this study, we evaluated the utility and accuracy of TTNB samples for molecular analysis by NGS. Our cohort primarily consisted of gastric IPMNs, and in most of these, we identified concomitant mutations in KRAS and GNAS. We demonstrated a high diagnostic yield for the discrimination between mucinous and nonmucinous cysts when histology and NGS were combined. Furthermore, we found a high sensitivity and specificity of molecular analysis of TTNB samples for the diagnosis of mucinous cysts not otherwise specified and for IPMNs.
The sensitivity and specificity of NGS of TTNB samples did not reach as high a level as reported by Singhi et al.
However, it is important to keep in mind that although Singhi et al conducted a large-scale prospective study, they only included and reported patients with technical success of the EUS procedure in terms of available specimens submitted for molecular analysis. No diagnostic yield as per intention to diagnose was reported, and the size of the initial population of patients screened for inclusion in the study was also not reported, possibly introducing a significant selection bias. Furthermore, diagnostic yield for obtaining cyst fluid is reportedly low in previous studies comparing histology of TTNB samples with FNAs for cytology.
Diagnostic accuracy of EUS-guided through-the-needle-biopsies and simultaneously obtained fine needle aspiration for cytology from pancreatic cysts: a systematic review and meta-analysis.
The diagnostic subtyping is not limited to only determining the grade of dysplasia but can also stratify IPMNs into pancreatobiliary, intestinal, and gastric subtypes, of which the pancreatobiliary followed by the intestinal subtype has the highest risks of malignant transformation.
Subtype of intraductal papillary mucinous neoplasm of the pancreas is important to the development of metachronous high-risk lesions after pancreatectomy.
Data regarding subtype of IPMN are not included in the diagnostic algorithm for surgical interventions, but EUS-guided TTNB sampling may enable subtyping as an add-on to the algorithm. Altogether, this suggests that EUS-guided TTNB sampling may overall be a more robust choice of diagnostic modality.
Candidate biomarkers evaluated in this study are among other oncogenes such as TP53. Traditional sequencing studies of IPMNs and other pancreatic neoplasms have demonstrated the occurrence of TP53 in invasive areas of the lesion and in areas with low-grade dysplasia within the same lesion.
Thus, TP53 is considered a shared genetic driver between preinvasive and invasive areas in PCLs and may compensate for a patchy distribution of advanced neoplasia. However, in our study and in a previously published study regarding mutational analysis of cyst fluid from PCLs,
TP53 has also been demonstrated in cysts with low-grade dysplasia and without clinical suspicion of cancer. This may be considered a major limitation to the utility of TP53 as a biomarker for advanced neoplasia. However, detection of TP53 with no corresponding high-grade lesion may indicate a non-negligible potential of the PCL for progression toward malignancy and would thus warrant a resection of the lesion. Singhi et al
demonstrated high sensitivities and specificities for diagnoses of IPMN and mucinous cystic neoplasm with advanced neoplasia based on the frequency of “GNAS >55% or TP53/PIK3CA/PTEN frequency equal to KRAS/GNAS” (p. 2137). We were unable to reproduce these results. Clinical studies with sufficient follow-up time should aim to address whether genetic aberrations, known to be shared between areas of advanced and nonadvanced neoplasia, can be used as a diagnostic biomarkers.
A major concern regarding the molecular analysis of TTNB samples from PCLs is the genetic heterogeneity of the lesions. In theory, cyst fluid contains cells and DNA shed across the entire inside of the cyst in contrast to TTNB samples, representing focal points in the cyst wall. Using the same NGS panel as in this current study, we recently explored the genetic heterogeneity of the cyst wall of resected IPMNs available for biopsy sample procurement relative to other regions of lesions and demonstrated the adequacy of 1 random biopsy sample to obtain a genetic profile, conditioned by sufficient cellularity of the biopsy sample.
By analyzing multiple TTNB samples in this prospective clinical study, we also found identical and shared variants or a shared wild-type status in most TTNB samples and cell blocks available. Albeit overcoming genetic heterogeneity within a single lesion, a shortcoming is to detect molecular aberrations of a concomitant but unrelated lesion, as can be seen with contamination of SCNs with low-grade pancreatic intraepithelial neoplasia, thus emphasizing the importance of independent microscopic evaluation by a pathologist.
In our study, we evaluated the size and cellularity of the TTNB samples and found that the largest TTNB samples were superior in terms of cellularity, indicating that the bottleneck is primarily the number of cells and thus the quantity of DNA rather than spatial heterogeneity. The superiority of large biopsy specimens points to a potential diagnostic gain by performing a macroscopic on-site evaluation of the TTNB samples in future clinical studies.
Our study revealed a high diagnostic yield for microscopic evaluation of TTNB samples that further increased when adding NGS of the residual tissue, albeit not reaching statistical significance. EUS-guided TTNB sampling is an invasive procedure with a risk of adverse events. In the cohort presented in this study, the adverse event rate was 9.9% with a risk of postprocedure acute pancreatitis of 8.9% and procedure-associated intracystic bleeding of 3%, previously reported in Kovacevic et al.
found that age, number of forceps passes, total cyst aspiration, and IPMN diagnosis to be independent predictors of adverse events. Perioperative administration of Ringer's lactate solution and rectal nonsteroidal anti-inflammatory drugs may reduce the rate of adverse events with TTNB sampling.
Also, NGS is expensive and may not be widely available, thus limiting the clinical utility of incorporating the technique into the diagnostic algorithm. Further optimization of technical aspects of the EUS-guided TTNB sampling procedure and specimen processing in the pathology laboratory need to be done, which may increase the diagnostic yield, sensitivity, and specificity of NGS. This includes limiting the number of unstained slides used for IHC to preserve tissue for NGS and thus increase the rate of TTNB samples with adequate DNA. Not submitting SCNs for NGS may also be considered because they are easily detected by morphology and IHC staining of the TTNB samples and because of their benign nature no additional clinical relevance is gained by NGS. TTNB samples with high-grade dysplasia also do not need additional NGS. NGS may not be limited to TTNB samples, the role of microscopic evaluation of TTNB samples in conjunction with cyst fluid NGS, and other advanced techniques such as confocal endomicroscopy, and cyst fluid glucose measurements need to be further studied.
Limitations of our study are the relatively small sample size and the single-center design. Estimation of sample size was not done specifically for the impact of a molecular diagnosis by NGS but rather for the clinical impact of microscopic examination of TTNB samples as described in our previous study.
The original sample size was 96 patients, which we believe can be directly transferred to this study, because we previously demonstrated the adequacy of 1 TTNB sample per patient for a molecular diagnosis.
Furthermore, our study cohort primarily included low-grade lesions with a majority of IPMNs and a small surgical cohort. Sensitivity and specificity were calculated based on TTNB sample histology of adequate biopsy samples in a subset of the cohort and not based on surgical specimens. We did not procure cyst fluid for NGS analysis specifically because of small cyst size and concerns regarding adverse events associated with a prolonged procedure time. However, we did analyze the cell block prepared from the pellet after centrifugation and preparation of cytology slides.
This study also has several strengths. This is the first prospective study of the performance of molecular analysis of TTNB samples with thorough reporting of TTNB sampling and DNA quality. The interpretation of sequencing results was performed in a blinded fashion. Molecular analysis of multiple biopsy samples from the same patients was performed separately, which has not been described previously in the literature. In a subset, we correlated our findings with the molecular aberrations of the surgical specimens and cyst fluid prepared as cell block, thus shedding light on tumor heterogeneity of PCLs.
In conclusion, we demonstrated feasibility of TTNB samples for molecular analysis by NGS with high diagnostic accuracy in terms of sensitivity and specificity. NGS enables subtyping and potentially risk stratification of PCLs by detection of shared genetic drivers that predict the presence of nearby advanced neoplasia that may not be apparent by microscopic examination alone. However, submitting TTNB samples for molecular analysis by NGS is not sufficient as a stand-alone diagnostic tool as of yet but has a high diagnostic yield when combined with microscopic evaluation and subtyping by IHC. The advantage of EUS-guided TTNB sampling over EUS-guided FNA is the ability to perform detailed cyst subtyping and the high technical success rate of the procedure. Further improvement of tissue processing of TTNB samples for DNA extraction may have the potential to increase the diagnostic yield of the NGS analysis. However, the procedure comes with a risk of adverse events and thus should be offered to patients where the value of an exact diagnosis outweighs the risks. Future studies should focus on defining the subgroup of patients who would benefit from the EUS-guided TTNB sampling procedure in conjunction with other novel EUS techniques.
Supplementary material
Customized Ion AmpliSeq Cancer Hotspot Panel version 2 (Thermo Fisher, Waltham, Mass, USA) covers the most prevalent hot spot mutations in ABL1, AKT1, ALK, APC, ATM, BRAF, CDH1, CDKN2A, CSF1R, CTNNB1, EGFR, ERBB2, ERBB4, EZH2, FBXW7, FGFR1, FGFR2, FGFR3, FLT3, GNA11, GNAQ, GNAS, HNF1A, HRAS, IDH1, IDH2, JAK2, JAK3, KDR, KIT, KRAS, MET, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, SRC, STK11, TP53, and VHL. The panel was supplemented by additional regions in genes BRAF, CDKN2A, KRAS, TP53, SMAD4, GNAS, and RNF43 (as described in Amato E, Molin MD, Mafficini A, et al. Targeted next-generation sequencing of cancer genes dissects the molecular profiles of intraductal papillary neoplasms of the pancreas. J Pathol 2014;233:217-27).
Appendix
Supplementary Figure 1Box and whisker plots of the sizes of through-the-needle biopsy samples. Comparison of largest area (A) and cross-section (B) measured on digitized slides and compared by the Wilcoxon signed rank test according to groups: >200 neoplastic cells, <200 neoplastic cells, and no neoplastic cells.
Diagnostic accuracy of EUS-guided through-the-needle-biopsies and simultaneously obtained fine needle aspiration for cytology from pancreatic cysts: a systematic review and meta-analysis.
Subtype of intraductal papillary mucinous neoplasm of the pancreas is important to the development of metachronous high-risk lesions after pancreatectomy.
DISCLOSURE: The following author disclosed financial relationships: P. Vilmann: Consultant for MediGlobe GmbH. All other authors disclosed no financial relationships. Researchsupportfor this study was provided by Rigshospitalets Research Foundation, The Novo Nordisk Foundation, The Danish Cancer Society, The Danish Cancer Research Foundation, Neye Fonden, Holms Mindelegat, Harboefonden, Agnete Løvgreens legat, Carl & Ellen Hertz legat, Direktør Jakob Madsen og Hustru Olga Madsens fond, Frimodt Heineke fonden, Torben & Alice Frimodts fond, Else og Mogens Wedell–Wedellsborgs fond, Fabrikant Ejnar Willumsens Mindelegat, Aase & Ejnar Danielsens fond, AP Møllers fond, and Anita & Tage Therkelsens Fond.
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