The Oncocytic Subtype Is Genetically Distinct from Other Pancreatic

Modern Pathology (2016) 29, 1058–1069

The oncocytic subtype is genetically distinct from other pancreatic intraductal papillary mucinous neoplasm subtypes Olca Basturk1,7, Marcus Tan2,7, Umesh Bhanot1, Peter Allen3, Volkan Adsay4, Sasinya N Scott1, Ronak Shah1, Michael F Berger1, Gokce Askan1, Esra Dikoglu5, Vaidehi Jobanputra5,6, Kazimierz O Wrzeszczynski5, Carlie Sigel1, Christine Iacobuzio-Donahue1 and David S Klimstra1

1Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, USA; 2Department of Surgery, University of South Alabama, Mobile, AL, USA; 3Department of Surgery, Memorial Sloan Kettering Cancer Center, New York, USA; 4Department of Pathology, Emory University, Atlanta, GA, USA; 5Department of Molecular Diagnostics, New York Genome Center, New York, USA and 6Department of Pathology, Columbia University Medical Center, New York, USA

In 2010, the World Health Organization reclassified the entity originally described as intraductal oncocytic papillary neoplasm as the ‘oncocytic subtype’ of intraductal papillary mucinous neoplasm. Although several key molecular alterations of other intraductal papillary mucinous neoplasm subtypes have been discovered, including common mutations in KRAS, GNAS,andRNF3, those of oncocytic subtype have not been well characterized. We analyzed 11 pancreatic ‘oncocytic subtype’ of intraductal papillary mucinous neoplasms. Nine pancreatic ‘oncocytic subtype’ of intraductal papillary mucinous neoplasms uniformly exhibited typical entity-defining morphology of arborizing papillae lined by layers of cells with oncocytic cytoplasm, prominent, nucleoli, and intraepithelial lumina. The remaining two were atypical. One lacked the arborizing papilla and had flat oncocytic epithelium only; the other one had focal oncocytic epithelium in a background of predominantly intestinal subtype intraductal papillary mucinous neoplasm. Different components of this case were analyzed separately. Formalin-fixed, paraffin- embedded specimens of all cases were microdissected and subjected to high-depth-targeted next-generation sequencing for a panel of 300 key cancer-associated genes in a platform that enabled the identification of sequence mutations, copy number alterations, and select structural rearrangements involving all targeted genes. Fresh frozen specimens of two cases were also subjected to whole-genome sequencing. For the nine typical pancreatic ‘oncocytic subtype’ of intraductal papillary mucinous neoplasms, the number of mutations per case, identified by next-generation sequencing, ranged from 1 to 10 (median = 4). None of these cases had KRAS or GNAS mutations and only one had both RNF43 and PIK3R1 mutations. ARHGAP26, ASXL1, EPHA8, and ERBB4 genes were somatically altered in more than one of these typical ‘oncocytic subtype’ of intraductal papillary mucinous neoplasms but not in the other two atypical ones. In the neoplasm with flat oncocytic epithelium, the only mutated gene was KRAS. All components of the intestinal subtype intraductal papillary mucinous neoplasms with focal oncocytic epithelium manifested TP53, GNAS,andRNF43 mutations. In conclusion, this study elucidates that ‘oncocytic subtype’ of intraductal papillary mucinous neoplasm is not only morphologically distinct but also genetically distinct from other intraductal papillary mucinous neoplasm subtypes. Considering that now its biologic behavior is also being found to be different than other intraductal papillary mucinous neoplasm subtypes, ‘oncocytic subtype’ of intraductal papillary mucinous neoplasm warrants being recognized separately. Modern Pathology (2016) 29, 1058–1069; doi:10.1038/modpathol.2016.98; published online 10 June 2016

Correspondence: Dr DS Klimstra, MD, Department of Pathology, ‘Oncocytic subtype’ of intraductal papillary muci- Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York 10065, USA. nous neoplasms of the pancreas was originally E-mail: [email protected] described as a separate variant of pancreatic intra- 6These authors contributed equally to this work. ductal neoplasms.1 However, the current (2010) This study was presented in part at the 103rd annual meeting of World Health Organization designated this neoplasm the United States and Canadian Academy of Pathology in Boston, as a subtype of intraductal papillary mucinous MA, in March 2015. Received 29 January 2016; revised 25 April 2016; accepted 25 neoplasm of the pancreas because oncocytic subtype April 2016; published online 10 June 2016 has some overlapping features with other subtypes of

www.modernpathology.org Molecular features of ‘oncocytic subtype' of IPMN O Basturk et al 1059 intraductal papillary mucinous neoplasm.2 For CTNNB1/β-catenin (4%), IDH1 (4%), STK11 (4%), example, they all present as an at least partially PTEN (4%), ATM (2%), CDH1 (2%), FGFR3 (2%), cystic pancreatic mass.3 The cystic appearance is and SRC (2%).17,24 due to dilation of the ducts by an intraductal In contrast, the literature on the molecular features neoplasm composed of tumor cells arranged in a of ‘oncocytic subtype’ of intraductal papillary muci- papillary pattern.1,4 These neoplasms also have nous neoplasm is fairly limited, partially due to less aggressive course than conventional pancreatic relative rarity of the neoplasm. Emerging studies ductal adenocarcinoma, even if there is an associated including case reports,31 a few cases studied together invasive carcinoma.1,3–10 In fact, the family of with other subtypes of intraductal papillary muci- intraductal papillary mucinous neoplasms may con- nous neoplasms,15,32,33 or studies analyzing selected tain ‘individuals’, which show overlapping features gene mutations, such as KRAS,34,35 have shown that between the four so far recognized subtypes. ‘oncocytic subtype’ of intraductal papillary muci- However, ‘oncocytic subtype’ of intraductal nous neoplasm generally lacks the mutations papillary mucinous neoplasm also has several commonly found in other subtypes of intraductal distinguishing pathologic characteristics such as papillary mucinous neoplasms. This raises the the complex arborizing papillae with delicate questions of whether ‘oncocytic subtype’ of intra- fibrovascular cores and distinctive intraepithelial ductal papillary mucinous neoplasm is genetically lumina formation, in addition to the oncocytic distinct from the other subtypes of intraductal nature of the cells, due to abundant intracytoplasmic papillary mucinous neoplasm and furthermore, mitochondria.1,3,4,9,11 In other organs, tumors that whether it should be considered as a separate are prone to accumulate abundant intracytoplasmic diagnostic entity?36 To further elucidate this ques- mitochondria in the exclusion of other organelles tion, we analyzed a series of ‘oncocytic subtype’ appear to have distinct pathogenesis and different of intraductal papillary mucinous neoplasms by biology than their non-oncocytic counterparts, with targeted next-generation sequencing, using a broad the best example being renal oncocytic neoplasms or panel of cancer-related genes, to investigate genes Hurthle cell tumors of the thyroid.12,13 Recently, the not previously assessed. studies have shown that despite being very complex lesions, ‘oncocytic subtype’ of intraductal papillary mucinous neoplasms are also actually either non- Materials and Methods invasive or minimally invasive, and although they may recur, their seldom lead to mortality of the With approval of the Institutional Review Board, the patient.1,9,10 surgical pathology databases of Memorial Sloan With the introduction of routine molecular Kettering Cancer Center and Emory University were searched for patients with a diagnosis of pancreatic genetic analyses, including next-generation sequenc- ‘ ’ ing,14–16 various molecular alterations have been oncocytic subtype of intraductal papillary muci- identified in other subtypes of intraductal papillary nous neoplasm between 1991 and 2013. Resections mucinous neoplasms. As in many ductal neoplasms of 11 pancreatic neoplasms were identified for which including pancreatic ductal adenocarcinomas for the slides and tissue blocks were available. The which KRAS mutation appears to be a nearly diagnoses were confirmed by the authors. Medical pre-requisite baseline change, KRAS mutations are records including pathology reports were reviewed the most common mutations and have been detected to obtain clinical data including age, gender, treat- in the majority of intraductal papillary mucinous ment modalities, and outcome. neoplasms (up to 100%).8,15,17–22 More interestingly, activating GNAS mutations at codon 201 have Targeted Next-Generation Sequencing been identified in approximately half (41–66%) of intraductal papillary mucinous neoplasms,15,16,23–26 Twenty 10-micron-thick sections were cut from particularly in the intestinal subtype.15,23,25 Inacti- formalin-fixed, paraffin-embedded tissue blocks con- vating mutations in the RNF43 gene, a likely tumor taining ‘oncocytic subtype’ of intraductal papillary suppressor and negative regulator of the Wnt signal- mucinous neoplasms. From these sections, areas of ing pathway,27 are also seen in up to 75% of interest were needle microdissected. For each intraductal papillary mucinous neoplasms.15,24 patient, extraction of DNA was performed on Mutations of PIK3CA are described in 3–11%.28–30 dissected tissue and where available on normal, Less common alterations involve CDKN2A/p16 (loss non-pancreatic tissue (stomach, spleen or duode- of expression in 18% of non-invasive intraductal num). Deep coverage, targeted next-generation papillary mucinous neoplasms and 53% of asso- sequencing was then performed on a panel ciated invasive carcinomas), SMAD4 (loss of expres- of 300 genes, including KRAS, GNAS, and RNF43, sion in 3% of non-invasive intraductal papillary listed in the supporting information (Supplementary mucinous neoplasms and 30% of associated invasive Information File 1), known to undergo somatic carcinomas), TP53 (10% of high-grade intraductal genomic alterations in cancer, as previously papillary mucinous neoplasms), BRAF (6% of high- described.37,38 Briefly, massively parallel sequencing grade intraductal papillary mucinous neoplasms), libraries (Kapa Biosystems, New England Biolabs)

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that contain barcoded universal primers were gener- and a-tailing enzymes. The samples were then ated from 115 to 250 ng genomic DNA from the ligated with Biooscientific adapters and PCR ampli- tumor material and matched normal tissue. After fied using KAPA Hifi HotStart Master Mix (Kapa, library amplification and DNA quantification, Kapa Biosystems, Wilmington, MA, USA). The DNA equimolar pools were generated consisting of up to libraries were clustered onto flowcells using Illumi- 24 barcoded libraries. These DNA pools were na’s cBot and HiSeq Paired End Cluster Generation subjected to solution-phase hybrid capture with kits as per manufacturer protocol (Illumina, San synthetic biotinylated DNA probes (Nimblegen Diego, CA, USA). Sequencing was performed using SeqCap) targeting all protein-coding exons from the 2 × 150 Illumina HiSeqX platform with v2.5 chem- selected 300 cancer genes as well as introns known istry reagents. Genotyping was performed using to harbor recurrent translocation breakpoints. Genes HumanOmni2.5 M BeadChips (Illumina). were selected to include commonly implicated Whole-genome sequencing and data analysis was oncogenes, tumor suppressor genes, and members performed at the New York Genome Center (NYGC, of pathways deemed actionable by targeted thera- New York, NY, USA). Paired-end 2 × 150 bp reads pies. Each hybrid capture pool was sequenced to were aligned to the GRCh37 human reference using deep coverage in a single paired-end lane of an the Burrows-Wheeler Aligner (BWA aln v.0.7.8)42 Illumina flow cell. Subsequently, the sequencing and processed using the best-practices pipeline that data were deconvoluted to match all high-quality includes marking of duplicate reads by the use of barcoded reads with the corresponding tumor sam- Picard tools and realignment around indels and base ples, and genomic alterations (single-nucleotide recalibration via Genome Analysis Toolkit (GATK) sequence variants, small insertions/deletions, and ver. 2.7.4.43 We employ the following variant callers: DNA copy number alterations) were identified. For muTect v1.1.4,39 LoFreq v2.0.044 (single-nucleotide matched tumor/normal tissue pairs (n = 9), somatic variants only), Strelka v1.0.1345 (both single- single-nucleotide variants and insertions and nucleotide variants and indels), Pindel46 and deletions were called using MuTect and the Soma- Scalpel47 (indels only) and return the union of calls, ticIndelDetector tools in GATK, respectively.39,40 For filtered using the default filtering criteria as imple- unmatched tumors (n = 2), MuTect was run against a mented in each of the callers. Single-nucleotide pool of unrelated DNAs from normal formalin-fixed, variants and indels were annotated via snpEff, paraffin-embedded tissue blocks, and variants were snpSift,48 and GATK VariantAnnotator using filtered out if they were present in the 1000 Genomes annotation from ENSEMBL, COSMIC,49 Gene Ontol- project at a population frequency of 41%. All ogy, and 1000 Genomes. candidate mutations, insertions, and deletions were Structural variants, such as copy number variants reviewed manually using the Integrative Genomics as well as complex genomic rearrangements, were Viewer.41 detected by the use of multiple tools: NBIC-seq50 for copy number variants/structural variants calling, Delly,51 Crest,52 and BreakDancer53 for structural Whole-Genome Sequencing variant calling. We prioritize structural variants in the intersection of callers and structural variants for Fresh frozen tumor material and matched normal which we can find additional split-read evidence tissues of two cases (Cases 4 and 5) were also using SplazerS.54 Structural variants for which there subjected to whole-genome sequencing, which was is split-read support in the matched normal or that performed using Illumina paired-end chemistry on a are annotated as known germline variants (1000 HiSeqX sequencer, which yielded coverage of at Genomes call set, DGV) were removed as likely least 80 × for tumor samples and 40 × for normal remaining germline variants. The predicted sets of samples, with more than 95% of the target bases somatic structural variants were annotated with gene having at least 5 × coverage. overlap (RefSeq, Cancer Gene Census) including prediction of potential effect on genes (eg, disrup- Genomic DNA isolation. Tissue was extracted tive/exonic, intronic, intergenic, fusion candidate). using Qiagen AllPrep DNA/RNA Mini Kit. DNA In addition, copy number variants and loss of was quantified using the Qubit 2.0 Fluorometer, heterozygosity were also analyzed from the genotyp- Invitrogen, and quality was determined by using ing chip using Nexus (Biodiscovery) software. Agilent Bioanlyzer.

Illumina whole-genome sequencing and genotyping. DNA libraries were prepared using the KAPA Hyper Results Prep Kit (Kapa, Kapa Biosystems, Wilmington, MA, Clinicopathologic Features USA). For each sample library preparation, 100 ng of high molecular weight genomic DNA was fragmen- Eleven patients were identified. The clinicopatholo- ted using the Covaris LE220 system to an average gic features of the cases are summarized in Table 1. size of 350 bp. Fragmented samples were end Half of these patients were male and the mean age repaired and adenylated using Kapa’s end-repair was 60 years (range, 45–78). No patients received

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Table 1 Clinicopathologic features of the cases

Gender Male 50% Female 50%

Mean age 60 years

Type of resection Whipple 67% Distal pancreatectomy 33%

Tumor location Head 67% Tail 33%

Mean tumor size 5 cm

Histologic type Pure 91% Mixed 9%

Invasive component Yes 18% No 82%

Lymphovascular invasion No 100%

Perineural invasion ‘ ’ No 100% Figure 1 Macroscopically, typical pancreatic oncocytic subtype of intraductal papillary mucinous neoplasms were characterized Resection margin by tan, friable, and large papillary excrescences filling cystic Positive 29% structures. Negative 71%

Lymph node status No 100% invasive carcinoma, in the form of multiple micro- scopic foci of small micropapillary clusters of Median follow up 61 months oncocytic cells infiltrating the periductal stroma. The invasive component was too small for molecular Survival analysis. Alive 77% ‘ ’ Dead 23% Two oncocytic subtype of intraductal papillary mucinous neoplasms had atypical morphology. Case 10 (Table 2) was non-papillary and revealed a flat cyst-lining of neoplastic epithelial cells with neoadjuvant chemotherapy or chemoradiation. One oncocytic features (Figure 3). Case 11 (Table 2) patient with an associated invasive colloid carci- was predominantly intestinal subtype intraductal noma received adjuvant chemotherapy, but not papillary mucinous neoplasm with a distinct focus chemoradiation. The majority (67%) of tumors of oncocytic epithelium on H&E (Figure 4). Of note, a was located in the head and patients underwent previously performed MUC6 immunohistochemical pancreatoduodenectomy. Tumor size varied from stain was negative in the intestinal component and 1 to 10 cm (median, 5.5 cm). positive only at the base of the papillae of the Of 11 cases, 9 exhibited typical entity-defining oncocytic focus. This case also had an associated characteristics. Grossly, these tumors were charac- invasive colloid carcinoma (Figure 5). All compo- terized by large, soft tan friable nodules associated nents of this case (intestinal subtype intraductal with cystic spaces (Figure 1). In contrast to other papillary mucinous neoplasm, oncocytic, and inva- subtypes of intraductal papillary mucinous sive carcinoma) were analyzed separately. neoplasm, the intraductal location was difficult to The follow-up period ranged from 2 to 190 months recognize. Microscopically, the tumors were archi- (median, 61 months). Two of nine patients with tecturally complex, with arborizing papillae growing available follow-up died; the remaining seven (77%) into the lumens of massively dilated ducts. The were alive without disease. Case 11 (with heteregen- neoplastic epithelial cells had abundant eosinophilic ous epithelium and an associated invasive colloid granular cytoplasm and nuclei with single promi- carcinoma) died of perioperative complications nent nucleoli. Intraepithelial and intracellular 2 months after the surgery, and Case 2 (typical lumina were also identified (Figure 2). Only one of ‘oncocytic subtype’ of intraductal papillary the nine typical ‘oncocytic subtype’ of intraductal mucinous neoplasm) died of unrelated causes after papillary mucinous neoplasm had an associated 190 months.

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Figure 2 Papillae of typical pancreatic ‘oncocytic subtype’ of intraductal papillary mucinous neoplasms were often very delicate and arborizing (a), and the cells revealed distinctive oncocytic appearance with voluminous acidophilic granular cytoplasm and single prominent nucleoli. (b) Multiple intracellular lumina were also present.

Molecular Features mutation at G12V). Case 11 (with heteregenous epithelium and an associated invasive colloid carci- Targeted next-generation sequencing. A total of 13 noma) manifested GNAS (missense mutation at tumor samples from 11 patients underwent targeted R201C and R201S), RNF43 (missense mutation at deep sequencing. The results of these genetic studies are summarized in Table 2, and details of M1I), and TP53 (missense mutations at R248W and the individual cases are described in the supporting S241F as well as nonsense mutation at W146) information (Supplementary Information File 2). mutations in all three components. Whereas, A total of 41 mutations were identified in the 11 PDGFRA (missense mutation at T230M) and ATRX cases, ranging 1–10 mutations per neoplasm (missense mutation at E1492G) mutations were (median = 4, Figure 6). In the nine typical ‘oncocytic exclusive to the intestinal component. subtype’ of intraductal papillary mucinous neoplasms, the following four genes were mutated in at least two neoplasms: ARHGAP26 (missense muta- Whole-genome sequencing. Fresh frozen tumor sam- tions at K592N or P4Q), ASXL1 (missense mutation ples from two patients (Cases 4 and 5) also underwent at V119L or a frameshift deletion at M341fs), EPHA8 whole-genome sequencing. Details of the individual (missense mutations at R375H or R384H), and cases are described in the supporting information ERBB4 (missense mutations at L939F or L1163M). (Supplementary Information Files 3 and 4). None of the nine typical ‘oncocytic subtype’ of Copy number analysis revealed that both samples intraductal papillary mucinous neoplasms had had multiple copy number gains and losses, the vast KRAS or GNAS mutations. One typical ‘oncocytic majority of which including a contiguous set of subtype’ of intraductal papillary mucinous neoplasm genes, in multiple chromosomes (Case 4: chromo- (Case 2) had RNF43 (frameshift/deletion at L61fs), somes 8, 12, 17, and 21; Case 5: chromosomes PIK3R1 (a missense mutation at P194T), and PIK3R3 1, 2, 3, 7, 8, 9, 13, 14, 15, and 22). Also both (missense mutation at R49Q) mutations. specimens had clonal loss of heterozygosity of an In case 10 (with flat oncocytic epithelium), the entire chromosome (Case 4: chromosome 12; Case 5: only somatic mutation found was KRAS (missense chromosome 20).

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Table 2 Distinct mutations identified by next-generation sequencing

Case Gene Type of mutation Protein change

Case 1 (Typical IOPN) ARHGAP26 Missense mutation p.K592N EPHA8 Missense mutation p.R384H MLL2 Missense mutation p.G1281R PTPN11 Missense mutation p.F438V Case 2 (Typical IOPN) EPHA8 Missense mutation p.R375H EPHB1 Missense mutation p.P457L ERBB2 Missense mutation p.R47H JAK3 Missense mutation p.V718L PIK3R1 Missense mutation p.P194T PIK3R3 Missense mutation p.R49Q MLL Missense mutation p.P1840S NOTCH1 Missense mutation p.R2272H NOTCH1 Missense mutation p.N104S RNF43 Frameshift deletion p.L61fs Case 3 (Typical IOPN) ERBB4 Missense mutation p.L1163M EPHA10 Missense mutation p.A453V GLI3 Missense mutation p.S1137R RB1 Frameshift deletion p.T5fs Case 4 (Typical IOPN)a ARHGAP26 Missense mutation p.P4Q NTRK3 Missense mutation p.L629H RICTOR Missense mutation p.D1357Y Case 5 (Typical IOPN)a NKX2-1 Frameshift deletion 236_237GG4G TEK Missense mutation p.T401M Case 6 (Typical IOPN) ASXL1 Missense mutation p.V119L ABL2 Missense mutation p.S1000P NOTCH2 Missense mutation p.R1372W RET Missense mutation p.T244I RET Missense mutation p.K1011E Case 7 (Typical IOPN) PAX5 Nonsense mutation p.R377 Case 8 (Typical IOPN) ASXL1 Frameshift deletion p.M341fs Case 9 (Typical IOPN)b ERBB4 Missense mutation p.L939F KDM6A Frameshift deletion p.L416fs Case 10 (Intraductal neoplasm with flat oncocytic epithelium) KRAS Missense mutation p.G12V Case 11 (Predominantly intestinal subtype IPMN with a Intestinal subtype IPMN distinct focus of oncocytic epithelium)b TP53 Missense mutation p.S241F TP53 Missense mutation p.R248W TP53 Nonsense mutation p.W146 GNAS Missense mutation p.R844C RNF43 Missense mutation p.M1I Oncocytic epithelium ATRX Missense mutation p.E1492G PDGFRA Missense mutation p.T230M TP53 Missense mutation p.S241F TP53 Nonsense mutation p.W146 GNAS Missense mutation p.R844C RNF43 Missense mutation p.M1I Invasive colloid carcinoma TP53 Missense mutation p.S241F TP53 Missense mutation p.R248W GNAS Missense mutation p.R844C RNF43 Missense mutation p.M1I aCases 4 and 5 were also subjected to whole-genome sequencing. bCases 9 and 11 had an associated invasive carcinoma component. The invasive component of case 9 was too small for molecular analysis. Recurrent mutations are written in bold.

A total of 91 mutations within 87 genes were Discussion identified among the 2 cases, 40 mutations in Case 4 and 51 mutations in Case 5. Among the five gene Recent studies have helped to better characterize mutations in Cases 4 and 5 identified by targeted the histologic subtypes of intraductal papillary 15,24,32,55–60 next-generation sequencing, only TEK gene mutation mucinous neoplasm and have confirmed (missense mutations at T401M) was also detected by that they have differences in genetic progression patterns compared with conventional pancreatic whole-genome sequencing. Whole-genome sequen- – cing also failed to reveal mutations in any of the ductal adenocarcinoma.19,30,61 63 However, a well-recognized intraductal papillary mucinous neo- focused study of ‘oncocytic subtype’ of intraductal plasm or pancreatic ductal adenocarcinoma genes. papillary mucinous neoplasm has not been reported

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Figure 3 Case 10 lacked the arborizing papillae (a) but the neoplastic epithelial cells revealed oncocytic features (b).

to date. Our results show that typical ‘oncocytic Similarly, in a prior study, our group identified subtype’ of intraductal papillary mucinous neo- mutations in codons 12 and 13 of the KRAS gene in plasms do not harbor previously reported intraductal 18 of 26 (69%) intraductal papillary mucinous papillary mucinous neoplasm-related mutations neoplasm cases of other subtypes although there (KRAS, GNAS, PIK3CA, CDKN2A/p16, SMAD4, were no KRAS mutations in eight ‘oncocytic subtype’ TP53, etc.). Only one of our typical intraductal of intraductal papillary mucinous neoplasm cases.34 papillary mucinous neoplasms (Case 2) had an Patel et al.31 also found no activating point muta- RNF43 mutation, which is a different mutation than tions in codons 12 and 13 of the KRAS gene in a previously reported intraductal papillary mucinous single case of ‘oncocytic subtype’ of intraductal neoplasm-related RNF43 mutations. papillary mucinous neoplasm with associated inva- Our results provide support for the proposition ‘ ’ sive carcinoma. With possible evidence to the that oncocytic subtype of intraductal papillary contrary, Xiao et al.35 identified somatic KRAS gene mucinous neoplasm is a distinct entity with not mutations in codon 12 in 3 of 18 (17%) ‘oncocytic only different morphologic features and biologic subtype’ of intraductal papillary mucinous neo- behavior, but also a different genotype. The most plasms. However, the authors acknowledged they important genotypic difference from other subtypes ‘ ’ of intraductal papillary mucinous neoplasm is the included oncocytic subtype of intraductal papillary mucinous neoplasms exhibiting heterogeneous lack of KRAS mutations. KRAS gene mutations are 35 frequent in intraductal papillary mucinous neo- epithelium. Therefore, it is quite possible that the plasms without oncocytic differentiation. For exam- three cases they reported as KRAS mutated may have ple, Jang et al.22 investigated 37 intraductal papillary exhibited heterogeneous epithelium, for which the mucinous neoplasms and found mutations in codons oncocytic features were a morphologic variation 12 and 13 of the KRAS gene in 50% of pancreato- within a non-oncocytic intraductal papillary muci- biliary subtype, 36% of gastric subtype, and of 21% nous neoplasm. As evidenced by our Case 11, the of intestinal subtype. A review of relevant literature genotype of the intestinal and oncocytic components provided in their manuscript also shows that manifested similar mutations that are more typical KRAS gene mutations were reported in up to 100% of other intraductal papillary mucinous neoplasm of intraductal papillary mucinous neoplasms.22 subtypes.

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Figure 4 Case 11 was a predominantly intestinal subtype intraductal papillary mucinous neoplasm (a) with a distinct focus of oncocytic epithelium (b). Intestinal and oncocytic components were analyzed separately.

epithelium and an associated invasive colloid carcinoma manifested a GNAS mutation in all its components. GNAS gene mutations are common in other subtypes of intraductal papillary mucinous neoplasm, particularly in the intestinal subtype. Dal Molin et al.23 reported that 100% of intestinal subtype, 71% of pancreatobiliary subtype, and 51% of gastric subtype intraductal papillary mucinous neoplasms harbored a codon 201 GNAS mutation. The authors also analyzed two ‘oncocytic subtype’ of intraductal papillary mucinous neoplasm cases, which were found to be GNAS wild type,23 in accordance with our findings. Similarly, in a recent study, our group has identified frequent GNAS gene mutations in non-oncocytic intraductal papillary mucinous neoplasms.26 Of note, no GNAS mutations have been identified in other pancreatic cystic Figure 5 Case 11 also revealed an associated invasive carcinoma neoplasms; nor were they identified in pancreatic of colloid type characterized by pools of mucin that are partially ductal adenocarcinoma, suggesting that GNAS muta- lined by carcinoma cells. tions are specific for the non-oncocytic intraductal papillary mucinous neoplasm phenotype.15,23,25 The lack of involvement in ‘oncocytic subtype’ of We found further support of ‘oncocytic subtype’ of intraductal papillary mucinous neoplasms of the intraductal papillary mucinous neoplasm as an typical genetic underpinnings that occur in other entity distinct from other subtypes of intraductal subtypes of intraductal papillary mucinous neo- papillary mucinous neoplasm by the absence of plasm should not come as a surprise considering GNAS mutations. Only Case 11 with heteregenous the distinctive pathologic manifestations of this

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‘oncocytic subtype’ of intraductal papillary mucinous neoplasms. The ARHGAP26 gene encodes Rho GTPase activating protein 26 (ARHGAP26), also known as GTPase regulator associated with focal adhesion kinase (GRAF), in humans. It is recognized as a tumor suppressor gene that binds to focal adhesion kinase. Mutations and deletions of the GRAF gene are strongly implicated in leukemia development.68–70 The GRAF gene has also recently been shown to be mutated in gastric cancer.71,72 The function of additional sex comb-like 1 (ASXL1) protein is not fully delineated,73 but it has been postulated that it may be involved in DNA and/or histone modification.74 Similar to ARH- GAP26, mutations in ASXL1 have been identified in myelodysplastic syndromes75 and other myeloid malignancies, like acute myeloid leukemia, chronic myelomonocytic leukemia, and myeloproliferative neoplasia.76 Both EPHA8 and ERBB4 genes are members of human receptor tyrosine kinase family. EPHA8 encodes ephrin type-A receptor 8 protein. EPH Figure 6 Number of mutations seen in each case analyzed. and EPH-related receptors have been implicated in mediating developmental events, particularly in the nervous system.77 Genetic studies suggest that EPHA8 tumor type. Also, it is becoming clear in other is involved in regulating cell adhesion78 and organs that oncocytic neoplasms characterized by apoptosis.79 ERBB4 encodes receptor tyrosine-protein abundant mitochondrial accumulation have differ- kinase erbB-4 enzyme, a member of the epidermal ent identity than their non-oncocytic counterparts of growth factor receptor subfamily. Mutations in this the respective organs.12,13 Similarly, the absence of gene have been associated with diverse cancers the genetic alterations that underlie pancreatic including esophagus,80 gallbladder,81 and colon.82 ductal adenocarcinoma may explain the incompar- In conclusion, although its intraductal nature and ably better clinical behavior of ‘oncocytic subtype’ of somewhat overlapping clinicopathologic features intraductal papillary mucinous neoplasms.1,9 More have led to classification of ‘oncocytic subtype’ of importantly, these differences suggest that pancrea- intraductal papillary mucinous neoplasms as a tic ‘oncocytic subtype’ of intraductal papillary variant of intraductal papillary mucinous neoplasm, mucinous neoplasm has distinct pathways of tumor our results suggest that, when defined strictly, progression, possibly through different underlying ‘oncocytic subtype’ of intraductal papillary mucinous mechanisms causing different genetic alterations. neoplasms are genetically distinct and thus should be In fact, the majority of the pancreatic cystic recognized separately. Further analysis of molecular neoplasms (intraductal papillary mucinous neo- alterations in biologically distinct pathway(s) includ- plasms, mucinous cystic neoplasms, serous cyst ing ARHGAP26, ASXL1, EPHA8, and ERBB4 genes adenomas and solid-pseudopapillary neoplasms) will likely shed new light on the mechanisms of are associated with recurrent mutations in intraductal tumor formation in the pancreas. components of ubiquitin-dependent pathways.15 However, ‘oncocytic subtype’ of intraductal papillary mucinous neoplasm does not seem to be Acknowledgments associated with ubiquitin-dependent pathways. In This work has been supported by a gift from the contrast, four specific genes (ARHGAP26, ASXL1, Farmer Family Foundation. We thank Ms. Dana EPHA8, and ERBB4) were found to be mutated by Haviland and Ms. Tanisha Daniel for their assistance targeted next-generateion sequencing in more than during manuscript preparation. one of our cases (due to smaller sampling of target regions with whole-genome sequencing, compared to targeted next-generation sequencing, whole- Disclosure/conflict of interest genome sequencing may not detect variants of low allelic frequency). Excluding reports of potential The authors declare no conflict of interest. involvement of the EPHA8 gene in pancreatic 64–67 carcinogenesis, these genes have not otherwise References been previously associated with intraductal papillary mucinous neoplasms. Therefore, they are worth 1 Adsay NV, Adair CF, Heffess CS et al. Intraductal further scrutiny as being more than mere epipheno- oncocytic papillary neoplasms of the pancreas. Am J mena for their potential role in tumorigenesis of Surg Pathol 1996;20:980–994.

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