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Pathology – Research and Practice 210 (2014) 392–396

Contents lists available at ScienceDirect

Pathology – Research and Practice

jou rn al hom epage: www.elsev ier .com/ locate /prp

eaching cases

ultiple sporadic gastrointestinal stromal tumors concomitant withmpullary adenocarcinoma: A case report with KIT and PDGFRAutational analysis and miR-221/222 expression profile

tella Blandamuraa,1, Lara Alessandrinia,∗,1, Roberta Bertorelleb, Francesca Simonatoa,incenza Guzzardoa, Elisa Valentinia, Imerio Angrimanc, Ambrogio Fassinaa

Surgical Pathology and Cytopathology Unit, Department of Medicine, DIMED University of Padova, Padova, ItalyIstituto Oncologico Veneto, IRCCS, Padova, ItalyDepartment of Surgical and Gastroenterological Sciences, University of Padova, Padova, Italy

r t i c l e i n f o

rticle history:eceived 12 November 2013eceived in revised form 8 January 2014ccepted 23 January 2014

eywords:ISTs

a b s t r a c t

GISTs originating multifocally at different GI sites, in patients lacking familial syndromes, could be inter-preted as recurrent/metastatic disease. MiR-221/222 have recently been identified as regulators of KITexpression in GISTs. We report the first case of synchronous GISTs in the stomach and duodenum con-comitant with an ampullary adenocarcinoma. Different CD117 expression patterns could be related todifferent KIT mutational status in the two lesions: gastric GIST showed a dot-like pattern and lacked KITmutations; duodenal GIST had a strong membranous expression pattern, likely due to KIT exon 9 dupli-

utationolyclonalitydenocarcinomaiR-221/222

cation, which is associated with lower response to imatinib. MiR-221/222 were downregulated in GISTsas compared with normal tissue (p < 0.05) and expressed increased levels in the gastric GIST as comparedwith duodenal one (p < 0.05). Our data support an independent origin of the two GISTs. Determiningwhether these tumors are multiple primaries or recurrencies is helpful to predict their malignancy andto select proper treatment.

© 2014 Elsevier GmbH. All rights reserved.

ntroduction

Gastro-intestinal stromal tumors (GISTs) are the most commonesenchymal tumors of the gastrointestinal tract. They exhibit aide spectrum of clinical behaviors, from indolent and curable

o highly aggressive and fatal. GISTs arise mostly in the stomach60%), followed by the small intestine (35%) and rectum, esopha-us, omentum and mesentery (<5%) [14]. The main event in theathogenesis of these tumors is gain-of-function mutations of type

II receptor tyrosine kinases, in particular KIT or in related platelet-erived growth factor receptor-� (PDGFRA) [14]. Apart from theost frequent exon 11 mutations, 7–10% of GISTs present a muta-

ion in an extracellular domain encoded by exon 9, mainly in themall and large intestine tumors, but also sometimes in the stom-ch [6]. Consistent with their extensive functional overlap, KIT and

∗ Corresponding author at: Pathology Unit, Department of Medicine, DIMED Uni-ersity of Padova, Via Gabelli 61, 35121 Padova, Italy. Tel.: +39 049 8274287;ax: +39 0498272265.

E-mail address: boxlara@tin.it (L. Alessandrini).1 These authors contributed equally to this work.

ttp://dx.doi.org/10.1016/j.prp.2014.01.019344-0338/© 2014 Elsevier GmbH. All rights reserved.

PDGFRA mutations are mutually exclusive in GISTs [6]. GIST muta-tional status is a fundamental predictor of the response to a targetedtherapy, since KIT exon 9 mutated tumors show a lower responserate to imatinib and requiring higher dosages [7].

Multiple GISTs in other gastrointestinal (GI) sites are rare andmay occur within a setting of familial disorders (familial multipleGISTs, with germline mutations of KIT or PDGFRA gene) as part ofthe NF-1 syndrome, Carney’s triad, or sporadic multiple GISTs. Thelatter have only recently been described and must be differentiatedfrom recurrent or metastatic disease to allow both proper stag-ing and therapeutic patients management [1,9,10,12,17]. Amongreported series of sporadic GISTs, synchronous multifocal GISTs inboth duodenum and stomach are even rarer, with only three casesbeing described [1,9,16]. The synchronous occurrence of othermalignancies has been reported in 13–14% of patients with GISTs,with gastrointestinal carcinomas being the most frequent [2,18].

MicroRNA (miRNA) are small, non-coding, regulatory RNAmolecules which play an important role in major cellular processes

(e.g., proliferation, differentiation, apoptosis), and their abnormalexpression may contribute to cancer development/progression.miRNA-expression profiling of human tumors has identified sig-natures associated with diagnosis, staging, progression, prognosis,

S. Blandamura et al. / Pathology – Research and Practice 210 (2014) 392–396 393

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ig. 1. Duodenal ampullary adenocarcinoma infiltrating the adipose peripancreatiriginal magnification 20× and (b) – original magnification 100×.

nd response to treatment. miRNA-221 and miRNA-222 were iden-ified as regulators of KIT expression in GISTs by Koeltz et al. [13],evealing new aspects in the molecular pathogenesis and futureherapy of these tumors. We report here the first case of a syn-hronous sporadic GIST in the stomach and duodenum associatedith ampullary adenocarcinoma, and highlight the fundamental

ole played by molecular analyses for correct staging and appropri-te therapy, and the different expression levels of miRNA-221/222n these neoplasms.

ase report

linical history

A 72-year-old Caucasian man, with negative family history,as referred to Padua Hospital in December 2012 with signifi-

ant weight loss (>20 kg) over the previous 12 months, causedy increasing vomiting. Computed tomography (CT) and magneticesonance imaging (MRI) of the abdomen revealed gastric ectasiaue to severe narrowing of the lumen of the third portion of theuodenum, with ectasia of the upper segments. The intrahepaticiliary tree and main ducts were dilated, and the pancreatic headppeared to be dyshomogeneous. Annular neoplastic stenosis of theuodenum was also confirmed by esophago-gastroduodenoscopy.aboratory data, including tumor markers, were unremarkable. Theamilial history was negative for gastrointestinal tumors or tumorsrom other sites. Duodenocephalopancreasectomy revealed a well-efined nodule in the wall of the first duodenal segment.

athologic findings

During gross examination of surgical specimens, as well as of anmpullary neoplastic mass (5 cm maximum diameter), two clearlyemarcated subserosal nodules were found, one in the gastric pos-erior wall and the other in the first segment of the duodenal wall,

easuring 1.3 and 1.4 cm respectively.Surgical specimens were sampled according to current pro-

ocols. Formalin-fixed, paraffin-embedded tissue samples werebtained, and 4-�m sections were stained with hematoxylin andosin. Five-micron sections were cut for immunohistochemicaletermination. When required, sections were pre-treated with

eat-mediated sodium citrate buffer antigen retrieval (10 mM,H 6.0) for 30 min in an automated system (Bond-MaX; Leica,ewcastle-upon-Tyne, UK), and the following primary antibodiesere used: CD117 (c-kit polyclonal antibody, 1:300 dilution; DAKO,

e and showing lymphovascular invasion (b). Hematoxylin and eosin (H&E): (a) –

Glostrup, Denmark), CD34 (monoclonal antibody, clone QBEND/10;1:10 dilution; Diagnostic BioSystems, Pleasanton, CA, USA), smoothmuscle actin (SMA) (monoclonal antibody, clone 1A4, 1:400 dilu-tion; DAKO), S100 (polyclonal antibody, 1:2000 dilution; DAKO).Color was developed with 3.3′-diaminobenzidine (DAB), and slideswere counterstained with Meyer’s hematoxylin.

Histological assessment of the duodenal ampullary lesionshowed a G2 ductal adenocarcinoma, infiltrating the pancreas headand adipose peripancreatic tissue. According to the pathologicalTNM staging system (7th edition), the tumor was staged as pT4 pN0(Fig. 1). The other two lesions in the stomach and duodenum werediagnosed as GISTs according to morphology and immunohisto-chemistry. Both were of spindle cell type, without necrosis and with0–1 mitoses/50 HPF. On the basis of their size, mitoses and loca-tion, both were considered at low risk of malignancy/recurrence[14]. CD117 showed a focal expression (dot-like pattern) in the gas-tric GIST, and strong diffuse staining (membranous pattern) in theduodenal GIST. CD34 was focally positive only in the gastric lesion(Fig. 2). Both SMA and S-100 were negative.

Molecular findings

Analysis of KIT and PDGFRA mutations

DNA was isolated from formalin-fixed, paraffin-embeddedtumor tissues with the QIAmp DNA mini-kit (Qiagen, Milan, Italy)according to the manufacturer’s instructions.

The DNA underwent polymerase chain reaction (PCR) treatmentwith primer pairs specific for KIT exons 9, 11, 13 and 17 and PDGFRAexons 12, 14 and 18. The amplified products were then sequencedwith a BigDye Terminator v1.1 Cycle Sequencing Kit and fluores-cent capillary electrophoresis (ABI PRISM 310 genetic analyses;Applied Biosystems, Foster City, CA, USA) with specific forward andreverse primers. Sequencing results were compared with the NCBIGeneBank KIT (Accession number X06182) and PDGFRA (Accessionnumber M21574) gene mRNA sequences. Mutation nomenclaturefollows the recommendations for descriptions of DNA sequencevariants (Human Genome Variation Society; http://hgvs.org).

The gastric GIST showed a PDGFRA exon 12 mutation consist-ing of a 15 bp deletion, leading to R558 I562del at protein level

(Fig. 3). The intestinal GIST revealed KIT exon 9 duplication of sixnucleotides, leading to Ala502 Tyr 503dup at protein level (Fig. 4).The KIT and PDGFRA genes, as expected, were wild type in adeno-carcinoma.

394 S. Blandamura et al. / Pathology – Research and Practice 210 (2014) 392–396

Fig. 2. H&E stainings, CD117 and CD34 immunohistochemical stainings for gastric GIST (left panels) and duodenal GIST (right panels). (a and d) Both gastric and duodenalGISTs were of spindle-cell type (H&E, original magnification 100×). (b) CD117 showed a focal expression (dot-like pattern) in the gastric GIST (original magnification 100×).( e) CDm ation

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c) CD34 was focally positive in the gastric lesion (original magnification 200×). (agnification 100×). (f) CD34 was negative in the duodenal GIST (original magnific

iRNAs analysis

Two-mm tissue cores were obtained from the paraffin blockf each specimen of tumor/normal tissue and deparaffinized withylene at 50 ◦C for 3 min. To detect and quantify mature hsa-miR221miR-221; primer sequence 5′-AGC TAC ATT GTC TGC TGG GTT TC-′), and hsa-miR222 (miR-222; primer sequence 5′-GAG CTA CATTG GCT ACT GGG T-3′), the NCodeTM miRNA qRT-PCR methodInvitrogen, Carlsbad, CA) was applied in a LightCycler 480 Real-

ime System (Roche Diagnostics, Mannheim, Germany). The resultsere normalized with the small nuclear RNA U6B (RNU6B; primer

equence: 5′-ACG CAA ATT CGT GAA GCG TT-3′; Invitrogen). All theeactions were run in triplicate, including non-template controls.

117 strong diffuse staining (membranous pattern) in the duodenal GIST (original100×).

Normalized expression was calculated using the comparative Ctmethod, and fold change was derived from the equation 2−��Ct foreach microRNA.

GISTs showed significantly lower levels of miRNA-221/222 thannormal tissue and adenocarcinoma (p < 0.05). Compared with theduodenal GIST, the gastric GIST expressed two-fold increased levelsof both miRNAs (p < 0.05).

Discussion

Detection of multifocal GISTs, apart from familial and syndromicconditions and irrespectively of the number, size or location of thelesions, is commonly viewed as a metastatic dissemination of a

S. Blandamura et al. / Pathology – Research and Practice 210 (2014) 392–396 395

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ig. 3. Sequencing analysis for PDGFRA in the gastric GIST which showed a PDGFRA

ingle primary GIST [1,9,10,12,17]. On this basis, patients with mul-ifocal GISTs are by default classified as advanced stage and treateds such. Therefore, determining whether GISTs represent recurren-ies of a single primary tumor or multiple synchronous tumors isssential for proper staging and patients management. Mutationalnalysis of lesions is thus mandatory to demonstrate the polyclonalrigin of the tumors – as in our case, which harbored a PDGFRAxon 12 mutation in the gastric GIST and a KIT exon 9 duplica-ion in the duodenal GIST. In multiple sporadic GISTs, discordantenotypes range between 60% and 100%, according to case series1,9,10,12,17].

The standard treatment for localized, low-risk GISTs is a com-

lete surgical excision, without dissection of regional lymph nodes,hereas adjuvant treatment with tyrosine kinase inhibitor imati-ib mesylate is recommended in metastatic disease [4]. Imatinib

Fig. 4. Sequencing analysis for KIT in the duodenal GIST, which showed a KIT exon 9

12 mutation, consisting of a 15 bp deletion leading to R558 I562del at protein level.

is the standard treatment if complete surgery is not feasible [4].Since in our case both GISTs had been completely removed, therewas no reason to submit the patient to unnecessary treatment inorder to avoid that long-term success would be limited by the onsetof imatinib resistance via secondary mutations or clonal selection[4]. In addition, specific mutations are predictors of prognosis andresponse to therapy: KIT exon 9 mutations, as in our case, usu-ally show a lower response rate than those with exon KIT exon 11mutations, which require higher dosage of imatinib [7].

The various CD117 immunohistochemical staining patternsdetected in our study reflect different KIT mutational status inthe two lesions: the gastric GIST showed a dot-like pattern and

lacked KIT mutations; the duodenal GIST had a strong membranousexpression pattern, perhaps due to KIT exon 9 duplication, leadingto Ala502 Tyr 503dup at protein level. A similar finding, but with

duplication of six nucleotides, leading to Ala502 Tyr 503dup at protein level.

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different molecular underlying cause, was studied by Gasparottot al. [9] in a case of two synchronous GISTs of the stomach andmall intestine. Finally, higher levels of miRNA-221/222 were foundn CD117 negative tissues (adenocarcinoma, normal tissue) as com-ared with CD117-positive GISTs, and differences were also foundetween miRNA-221/222 and various patterns and intensities ofD117 expression in both GISTs. Our results match those of Koelzt al. [13], showing for the first time that miRNA-221/222, known toe dysregulated in many malignancies, can regulate KIT expression

n GISTs, shedding new light on the pathogenesis of these tumors.aller et al. [11] in their microarray analysis found that intestinalISTs had higher expression of miRNA-221/222 than gastric GISTs.owever, in their larger sample set analyzed by qRT-PCR, a multi-ariate analysis considering both anatomical location and mitoticount revealed a significant association of miRNA-221/222 expres-ion only with mitotic count. Interestingly, the authors found loweriRNA-221/222 expression in KIT and PDGFRA mutated GISTs as

ompared with wild type tumors [11]. In our case, both duodenalnd gastric GISTs showed significantly lower levels of miRNA-21/222 than (non-mutated) normal tissue and adenocarcinomas well.

Although miRNA-221/222 are not of diagnostic importance, ourase confirms that they may represent future therapeutic targets,s reported in recent studies [8,15].

Although the synchronous occurrence of GISTs and otherbdominal malignancies seems to be just a coincidence, the devel-pment of these tumors may involve common carcinogenic agents.ietary carcinogens, such as nitrosamines, are well-known risk

actors for the development of various kinds of gastrointesti-al adenocarcinomas and other cancers in humans. Experimentaltudies have shown that nitrosamines, such as N-methyl-N′-itro-N-nitrosoguanidine, can cause both adenocarcinomas and

eiomyosarcomas (when combined with acetyl salicylic acid ortress) of the digestive tract in rats [5]. Another explanation forhe synchronous occurrence of these two entities is represented by

etallothioneins (MT), proteins with increased affinity for heavyetal ions and associated with protection against DNA damage,

poptosis, cell survival, angiogenesis and oxidative stress. MTave been reported to be over-expressed in some tumors andown-regulated in others, such as gastric, colorectal, liver andentral nervous system tumors. Nuclear expression of such pro-eins was also detected in GISTs by Soo et al., suggesting that

T may be involved in GIST proliferation [16]. Therefore, a com-on dietary carcinogen may be linked to the development of both

pithelial and mesenchymal tumors of the gastrointestinal tract.imilarly, a common molecular pathway underlying the devel-pment of both GISTs and gastrointestinal adenocarcinomas maye hypothesized, e.g., the role of the KIT tyrosine kinase recep-or studied previously by Bellone et al. [3], who demonstratedxpression of both the KIT protein and its ligand – the stem cellactor – in a small proportion (10%) of colorectal adenomas andarcinomas.

The clinical importance of the case presented here relies on the

herapeutic implications of multiple GISTs, which probably requirepecific treatment and mutational analysis to define GIST poly-lonality in multiple sporadic cases. Detailed information on theelation of miRNA-221/222 expression with CD117 expression in

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h and Practice 210 (2014) 392–396

GISTs may be useful for the future development of therapeuticstrategies, and should be studied in larger series.

Conflict of interest

The authors declare that they have no conflict of interest.

Acknowledgement

The authors would like to thank Dr. Alessio Mussato for hisexpert technical assistance.

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