GENES, CHROMOSOMES & CANCER 54:353–360 (2015)
RESEARCH ARTICLES
Ovarian Microcystic Stromal Tumor: A Novel Extracolonic Tumor in Familial Adenomatous Polyposis Sang Hun Lee,1 Young Wha Koh,2 Hyun Jin Roh,1 Hee Jeong Cha,3 and Yong-Soon Kwon1* 1
Department of Obstetrics and Gynecology,University of Ulsan College of Medicine,Ulsan University Hospital,Ulsan,Korea Department of Pathology, Ajou University School of Medicine, Suwon,Korea 3 Department of Pathology,University of Ulsan College of Medicine,Ulsan University Hospital,Ulsan,Korea 2
Ovarian microcystic stromal tumor (MCST) is a very rare neoplasm; hence, its nomenclature was recently designated as “Distinctive morphologic and immunohistochemical features” in 2009. Its exact origin, etiological genetic alterations, and background are not yet clearly known. Familial adenomatous polyposis (FAP) is an autosomal dominant disease that leads to development of colorectal polyps via germ-line mutations of the APC gene on chromosome 5q2122. In this study, we report a 40-year-old female patient who had ovarian MCST and FAP. On sequencing the APC gene in ovarian MSCT, we detected a novel somatic mutation of the APC gene in exon 11, with a heterozygous deletion at nucleotide position c.1540delG (p.Ala514 Profs*9). Mutations of b-catenin (CTNNB1) and FOXL2 were not detected. Although one case demonstrating involvement of Wnt/b-catenin in ovarian MCST associated with FAP has been presented previously, no detailed C 2015 V information was provided. Thus, this is the ovarian MCST with a somatic mutation of APC in a patient with FAP. Wiley Periodicals, Inc.
INTRODUCTION
Familial adenomatous polyposis (FAP) is an autosomal dominant disease that is characterized by early development of hundreds or thousands of adenomatous polyps in the colorectum through germ-line mutation of the APC gene on 5q2122 (Powell et al., 1992; O’Sullivan et al., 1998). Although FAP accounts for less than 1% of colorectal cancers (CRCs), it is one of the best understood genetic diseases (Half et al., 2009). Cases of CRC and other extracolonic tumors showing APC gene alteration through a second somatic mutation in the wild-type APC allele have been repeatedly reported. However, only a small number of cases of extracolonic tumors, such as those in the thyroid, liver, bile ducts, and the central nervous system, have been reported because of low disease prevalence (Turcot et al., 1959; Komorowski et al., 1986; Hamilton et al., 1995; Giardiello et al., 1996; Cetta et al., 1998; Cetta et al., 2000; Maire et al., 2002; Thomas et al., 2003; Chetty et al., 2005; Gupta and Mazzara, 2005; Sudo et al., 2005; Aretz et al., 2006; Groen et al., 2008). APC, a classical tumor suppressor gene, has a central role in bcatenin degradation and is closely correlated with the Wnt signaling pathway. Wnt signaling is associated with the stability of a protein complex consisting of b-catenin and GSK3 (glycogen synthase C 2015 Wiley Periodicals, Inc. V
kinase 3). In the absence of Wnt signaling or the presence of wild-type APC protein, b-catenin is degraded in the normal postembryogenesis period (normal pattern). In the presence of Wnt signaling or the absence of APC, b-catenin target genes, including MYC, show altered gene expression. This alteration, in turn, leads to altered expression of the polyamine ornithine decarboxylase gene which is a proto-oncogene (Barth et al., 1997; Polakis, 2000; Fearnhead et al., 2001). Ovarian microcystic stromal tumor (MCST) is a novel entity whose nomenclature was recently designated by Irving and Young (2009) based on 16 reported cases. Although the molecular pathogenesis and background genetic alterations still remain unclear, Maeda et al. (2011) reported two cases of oncogenic somatic mutations of the b-catenin gene (CTNNB1) in exon 3 in ovarian MCST, and verified the morphologic similarity between ovarian MCST and pancreatic solid pseudopapillary neoplasms (SPNs). However, the study did not *Correspondence to: Yong-Soon; Department of Obstetrics and Gynecology, Ulsan University Hospital, University of Ulsan College of Medicine, 290-3 Joenha-dong, Dong-gu, Ulsan 682-714, Korea. E-mail: [email protected] Received 19 September 2014; Accepted 25 November 2014 DOI 10.1002/gcc.22233 Published online 28 March 2015 in Wiley Online Library (wileyonlinelibrary.com).
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Figure 1. Abdominal pelvis CT (with contrast enhancement) and MRI female pelvis (with contrast enhancement). APCT and MRI female pelvis with contrast enhancement showed an approximately 15-cm complex cystic mass with bridging vascular pedicles contiguous with the left gonadal vein and a clear cleavage plane between this mass and uterine serosa.
adequately explain the relevance of APC in these two cases. A case demonstrating the involvement of Wnt/ b-catenin alterations in ovarian MCST associated with FAP has already been described by Maeda et al. (2012) in another series, but it was presented only as an abstract and did not provide clear detailed information. Thus, this study is the first one on ovarian MCST showing somatic mutation of APC in a patient with FAP.
MATERIALS AND METHODS Case History
A 40-year-old woman, gravida 2 and para 2, with no relevant past medical history visited our hospital due to pain in the lower abdomen. Physical examination revealed a large palpable mass in the lower abdomen. Through transvaginal ultrasonography, a 15-cm heterogeneous mass with a predominantly solid and partially cystic appearance was observed in the left ovary. It was suspected to be an ovarian malignancy, rather than a uterine Genes, Chromosomes & Cancer DOI 10.1002/gcc
malignancy. The levels of tumor markers, such as carcinoembryonic antigen, CA19-9, CA 125, and AFP, were within the normal range. For further examination, abdominal and pelvic computed tomography (APCT), magnetic resonance imaging (MRI) of the pelvis, and 18-fluoro2-deoxy-D-glucose positron emission tomography/ computed tomography (18PET/CT) were performed. APCT and MRI with contrast enhancement showed an approximately 15-cm complex cystic mass with bridging vascular pedicles contiguous with the left gonadal vein and a clear cleavage plane between the mass and uterine serosa. These findings were suggestive of a malignancy in the left ovary. There were no remarkable findings in the right adnexa and pelvic lymph node enlargement was not observed. However, mild bilateral hydronephroses caused by the pelvic mass effect were detected (Fig. 1). On PET-CT, a large, heterogeneous, hypermetabolic, lobulated mass with internal necrosis was detected in the pelvic cavity (max SUV 4.3); but it was not clear whether the malignant tumor originated in the ovary or uterus (Fig. 2).
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Figure 2. 18-Fluoro-2-deoxy-D-glucose positron emission tomography/computed tomography (18PET/CT). 18PET/CT showed a large, heterogenous, hypermetabolic, lobulated mass with internal necrosis in the pelvic cavity (max SUV 4.3). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Based on the imaging findings of APCT, MRI pelvis, and 18PET/CT, we suspected ovarian cancer. Colonoscopy and esophagogastroduodenoscopy were performed for preoperative evaluation to rule out any other primary cancer metastasizing to the ovary. Interestingly, through colonoscopy up to the cecum, hundreds of polyps were found in the sigmoid colon, descending colon, transverse colon, and ascending colon. Histopathological findings in the colonic specimens revealed villotubular adenomas, except for one well-differentiated adenocarcinoma lesion on a background of villotubular adenoma. There was no remarkable finding on esophagogastroduodenoscopy. Genetic studies for FAP and pedigree analysis were performed to examine the possibility of double primary malignancies. We would like to clarify that an agreement signed by the patient was obtained before performing the examination for ethical considerations. This study was approved by the Institutional Review Board at the University of Ulsan Medical School. The genetic studies revealed a strong multigenerational history of colon cancer. The patient’s father had died of CRC in his mid-fifties and her brother had undergone an operation for colon cancer diagnosed in his mid-thirties. A collaborative operative procedure with sufficient preparation, involving the gynecology and surgery teams, was performed. Frozen section of the left ovarian mass demonstrated an ovarian stromal tumor. Left salpigno-oophorectomy, right
ovarian wedge resection, and total colectomy were performed. The patient remains stable without any complications for 9 months after the operation. Patient and Tissue Specimens
We reviewed the hematoxylin and eosin-stained sections of ovarian MCST, FAP, and colon adenocarcinoma that were surgically removed at the University of Ulsan Hospital. Pathological diagnosis was made by two of the authors (Y.W.K and H.J.C). The tumor samples (ovary, intestine, polyp, CRC) were fixed in 10% buffered formalin, processed routinely, and embedded in paraffin. Then 4-mm sections were stained with H&E, and histopathological diagnosis was confirmed by the authors, including at least two pathologists. Immunohistochemistry
All samples were fixed in formalin and embedded in paraffin. In all of the cases, full tissue sections, 4mm thick, were used for immunohistochemistry. Immunohistochemical staining was performed according to standard techniques on a Ventana Benchmark XT autostainer (Ventana Medical Systems Inc., Tucson, AZ). Appropriate positive and negative controls were included. The antibodies used in this study were CD 10 (Novocastra, Clone 56C6, 1:150 dilution), b-catenin (Novocastra, Clone 17C2, 1:400), HMB45 (DAKO, Clone HMB 451,1:1000 dilution), Placenta alkaline phosphatase (Neomarkers, Clone 8A9, 1:2 dilution), Vimentin Genes, Chromosomes & Cancer DOI 10.1002/gcc
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Figure 3. Histology of the ovarian MCST and colonic adenocarcinoma. (A) Low-power view of the MCST. The tumor showed microcysts and variably prominent solid cellular areas with intervening hyalinized fibrous bands. (B) The cells had moderate amounts of lightly eosinophilic cytoplasm and inconspicuous nucleoli. (C) Well-differentiated adenocarcinoma identified in a tubular adenoma. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
(ZYMED, Clone V9, 1:150 dilution), Ki-67 (ZYMED, Clone 7B11, 1:400 dilution), C-Kit (DAKO, Clone, 1:1000 dilution), Epithelial membrane antigen (ZYMED, Clone ZCE 113, 1:250 dilution), Estrogen receptor (Novocastra, Clone DAK Calret 1, 1:150 dilution), a-Inhibin (Biocare, Clone BC/R1, 1:75 dilution), and Cytokeratin (Novocastra, Clone 5D3 and LP 34, 1:300 dilution). Genomic DNA Preparation
We extracted genomic DNA from whole blood using the Intron_G-DEXTM IIb Genomic DNA Extraction Kit (Intron, Seongnam, Korea). The ovarian MCST and colon tubular adenoma were microdissected from the paraffin-embedded tissue blocks and processed by ethanol/xylene dewaxing, followed by DNA extraction using a QIAampTM DNA mini kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. Mutation Analysis
Polymerase chain reaction (PCR) and sequencing analyses were performed using a standard protocol. In brief, PCR primer pairs were designed to Genes, Chromosomes & Cancer DOI 10.1002/gcc
amplify 48 DNA fragments that overlapped the entire coding sequence and exon/intron boundaries of APC (31 primer pairs), CTNNB1 (14 pairs), and FOXL2 (3 pairs). Primer sequences are available upon request. PCR amplifications were performed in a 20 ml reaction volume containing 100 ng genomic DNA, 2.5 mM MgCl2, 200 lM for each dNTP, 10 pmol for each primer, 53 BD (Nanohelix, Daejeon, Korea), and 5 U Taq DNA polymerase (Nanohelix, Daejeon, Korea). The following thermal profile was applied using a PTC-220 DYADTM thermal cycler (Bio-Rad, Hercules, CA): initial denaturation for 5 min at 95 C followed by 42 cycles at 95 C for 30 sec, 62 C for 40 sec, and 72 C for 45 sec, with a final extension at 72 C for 7 min. The PCR products were purified using exonuclease I and shrimp alkaline phosphatase (GE Healthcare, Uppsala, Sweden) and sequenced in both directions with an AB 3730xl genetic analyzer (Life Technologies, Foster city, CA), according to the manufacturer’s instructions. All of the variations detected were reviewed and confirmed by repeating the PCR and the sequencing analyses. All sequence variations were described according to the HGVS (Human Genome Variation Society) mutation nomenclature (den Dunnen and
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Figure 4. Immunohistochemical features of the ovarian MCST. Diffuse and strong positivity for (A) vimentin and (B) CD10, and negativity for (C) cytokeratin and (D) epithelial membrane antigen. (E) Diffuse and strong nuclear positivity for b-catenin. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Antonarakis, 2000). Two different in silico bioinformatics prediction programs were used to predict the pathogenic effect of the novel variant c.3796G>A of APC; PolyPhen2 (http://genetics. bwh.harvard.edu/pph2/) and SIFT (http://sift.jcvi. org/). RESULTS Gross Features of Ovarian MCST and FAP
The left ovarian tumor measured 15.0 3 15.0 3 9.0 cm in size. The surface of the ovary was smooth, although the tumor was confined to the ovary. The cut surface of the mass was grayish yellow, firm, and granular. Multifocal necrosis and
hemorrhage were noted. There were more than 200 polyps in the colectomy specimen.
Histologic Features of Ovarian MCST and Colorectal Adenocarcinoma
On routine histopathological examination, 27 slide sections of the ovarian tumor were reviewed. The tumor showed microcysts and variably prominent solid cellular areas with intervening hyalinized fibrous bands (Fig. 3A). The cells in the cellular areas had moderate amounts of lightly eosinophilic cytoplasm and inconspicuous nucleoli (Fig. 3B). There was no significant histologic finding on the right ovarian wedge biopsy. Adenocarcinoma was Genes, Chromosomes & Cancer DOI 10.1002/gcc
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Figure 5. The electropherogram of the APC gene. (A) Sequencing data of exon 15 revealed a constitutional heterozygous deletion at nucleotide position c.2376_2378 delGCAinsC (p.Lys792 Asnfs*28). (B) Sequencing data of exon 15 in a colonic adenomatous polyp revealed a heterozygous missense at nucleotide position c.3796G>A (p.Asp 1266Asn). (C) Sequencing data of exon 11 in the ovary with a hetero-
zygous frameshift at nucleotide position c.1540delG (p.Ala514 Profs*9). The top panel is the control sequence. The other panel is the corresponding tissue sequence. C indicates cysteine; D, aspartic acid; G, glycine; H, histamine; I, isoleucine; L, leucine; S, serine; Y, tyrosine. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
identified in the one of the tubular adenomas in the endoscopic mucosal resection specimen (Fig. 3C).
(CTNNB1) and FOXL2 in blood, colon polyp, or ovarian MCST.
Immunohistochemistry
The ovarian MCST displayed a diffuse and strong immunoreactivity for vimentin (Fig. 4A) and CD 10 (Fig. 4B). The tumor cells were negative for cytokeratin (Fig. 4C) and epithelial membrane antigen (Fig. 4D). Additionally, the tumor cells commonly showed diffuse and strong nuclear immunoreactivity for b-catenin (Fig. 4E). The tumor cells were negative for a-Inhibin, c-kit, HMB45, placental alkaline phosphatase, estrogen receptor, progesterone receptor, and calretinin. The Ki-67 labeling index was low (A mutation in a colon polyp (Fig. 5), a missense mutation resulting in a downstream stop codon (p.Asp 1266Asn); and (3) a somatic c.1540delG mutation in the ovarian MCST (Fig. 5), a frameshift mutation resulting in a downstream stop codon (p.Ala514 Profs*9). Mutation Analyses of b-Catenin (CTNNB1) and FOXL2 in Blood, Colon Polyp, and Ovarian MCST
Mutation analyses of b-catenin (CTNNB1) and FOXL2 revealed no mutations of b-catenin Genes, Chromosomes & Cancer DOI 10.1002/gcc
DISCUSSION
A small number of cases of extracolonic benign and malignant tumors in patients with FAP have been reported (Blaker et al., 2004; Groen et al., 2008). Gynecologic cases related to the ovary and associated with FAP have rarely been reported. One ovarian steroid cell tumor with biallelic APC inactivation in an FAP patient was reported in a study by Hu et al. (2012); this was the first study to suggest a somatic alteration of the APC gene (APC/ b-catenin) in an ovarian MCST patient with FAP. Ovarian MCST is a rare tumor, entity recently deliberated by Irving and Young (2009) based on 16 reported cases. The exact origin and genetic alterations of ovarian MCSTs have not yet been clearly identified. The characteristics of MCSTs are a combination of microcystic, macrocystic, and solid structures, presence of intervening thick fibrous stroma, growth of monotonous tumor cells with round-toovoid and generally simple nuclei with very fine chromatin, well-marked borders, and focal areas of congestive and hemorrhagic changes that can give an impression of a hemangiomatous lesion (Irving and Young, 2009; Maeda et al., 2011). Ovarian steroid cell tumors may be a differential diagnosis. Little is known about the pathogenesis of these tumors because of their rarity accounting for less than 0.1% of all ovarian tumors (Scully, 1979). Not much is known regarding their b-catenin status. However, in this study, we recognized that ovarian steroid cell tumors are clearly differential from ovarian MCST, in that ovarian steroid cell tumors do not display microcytic histologic features. Recognition of the importance of the APC gene began with genetic studies for FAP, an inherited disease with tendency to develop colon cancer (Powell
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et al., 1992; O’Sullivan et al., 1998). Under normal conditions, the APC gene suppresses the propagation of b-catenin by arbitrating its phosphorylation in the cell (Aberle et al., 1997). This function of the APC gene is disturbed when it loses the ability to control b-catenin regulatory activity, resulting in abnormal accumulation of b-catenin. Loss of the ability can be caused by either APC gene mutations or as a result of truncation of the b-catenin oncogene (Korinek et al., 1997; Morin et al., 1997). In this study, we did not detect any somatic mutations of the b-catenin gene, unlike in the study by Maeda et al. (2011). The latter group reported two cases of ovarian MCST with somatic mutations of the b-catenin gene at exon 3, implicating the Wnt/b-catenin gene (CTNNB1) pathway in ovarian MCST (Maeda et al., 2011). Some ovarian MCSTs are assumed to undergo other types of molecular change that inactivate the APC/b-catenin pathway. In sporadic and FAP-associated pancreatoblastomas, molecular alterations in the APC/b-catenin pathway were detected in 67% of pancreatoblastoms (six of nine), of which five tumors were caused by sporadic mutations of the b-catenin oncogene and one tumor was caused by biallelic APC inactivation without mutation of b-catenin oncogene in one patient with FAP (Abraham et al., 2001). The notable finding was that pancreatoblastomas, although sporadic, showed a predominance of b-catenin mutations, with APC mutation without b-catenin oncogene being demonstrated in only a patient with FAP. In our study, we detected a novel mutation, a heterozygous deletion at nucleotide position c.1540delG (p.Ala514 Profs*9), of the APC gene at exon 11 in the ovarian MSCT. This confirmed the role of APC in downregulating the level of bcatenin; the immunohistochemisty demonstrated a strong correlation between accumulation of bcatenin in the membrane, cytoplasm, and nucleus and APC gene alterations. Finally, we considered ovarian solid pseudopapillary tumors as a differential diagnosis for ovarian MCSTs, as from reported by Deshpande et al. (2010). According to our results, ovarian SPNs showed an aberrant nuclear accumulation of b-catenin. In conclusion, where a patient is diagnosed with ovarian MCST, the possibility of FAP should be considered, as ovarian MCST should be considered as one of the tumors associated with FAP. REFERENCES Aberle H, Bauer A, Stappert J, Kispert A, Kemler R. 1997. Betacatenin is a target for the ubiquitin-proteasome pathway. EMBO J 16:3797–3804.
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Abraham SC, Wu TT, Klimstra DS, Finn LS, Lee JH, Yeo CJ, Cameron JL, Hruban RH. 2001. Distinctive molecular genetic alterations in sporadic and familial adenomatous polyposisassociated pancreatoblastomas: Frequent alterations in the APC/ beta-catenin pathway and chromosome 11p. Am J Pathol 159: 1619–1627. Aretz S, Koch A, Uhlhaas S, Friedl W, Propping P, von Schweinitz D, Pietsch T. 2006. Should children at risk for familial adenomatous polyposis be screened for hepatoblastoma and children with apparently sporadic hepatoblastoma be screened for APC germline mutations? Pediatr Blood Cancer 47:811–818. Barth AI, Nathke IS, Nelson WJ. 1997. Cadherins, catenins and APC protein: Interplay between cytoskeletal complexes and signaling pathways. Curr Opin Cell Biol 9:683–690. Blaker H, Sutter C, Kadmon M, Otto HF, Von Knebel-Doeberitz M, Gebert J, Helmke BM. 2004. Analysis of somatic APC mutations in rare extracolonic tumors of patients with familial adenomatous polyposis coli. Genes Chromosomes Cancer 41: 93–98. Cetta F, Olschwang S, Petracci M, Montalto G, Baldi C, Zuckermann M, Mariani Costantini R, Fusco A. 1998. Genetic alterations in thyroid carcinoma associated with familial adenomatous polyposis: Clinical implications and suggestions for early detection. World J Surg 22:1231–1236. Cetta F, Montalto G, Gori M, Curia MC, Cama A, Olschwang S. 2000. Germline mutations of the APC gene in patients with familial adenomatous polyposis-associated thyroid carcinoma: Results from a European cooperative study. J Clin Endocrinol Metab 85:286–292. Chetty R, Salahshor S, Bapat B, Berk T, Croitoru M, Gallinger S. 2005. Intraductal papillary mucinous neoplasm of the pancreas in a patient with attenuated familial adenomatous polyposis. J Clin Pathol 58:97–101. den Dunnen JT, Antonarakis SE. 2000. Mutation nomenclature extensions and suggestions to describe complex mutations: A discussion. Hum Mutat 15:7–12. Deshpande V, Oliva E, Young RH. 2010. Solid pseudopapillary neoplasm of the ovary: A report of 3 primary ovarian tumors resembling those of the pancreas. Am J Surg Pathol 34:1514– 1520. Fearnhead NS, Britton MP, Bodmer WF. 2001. The ABC of APC. Hum Mol Genet 10:721–733. Giardiello FM, Petersen GM, Brensinger JD, Luce MC, Cayouette MC, Bacon J, Booker SV, Hamilton SR. 1996. Hepatoblastoma and APC gene mutation in familial adenomatous polyposis. Gut 39:867–869. Groen EJ, Roos A, Muntinghe FL, Enting RH, de Vries J, Kleibeuker JH, Witjes MJ, Links TP, van Beek AP. 2008. Extra-intestinal manifestations of familial adenomatous polyposis. Ann Surg Oncol 15:2439–2450. Gupta C, Mazzara PF. 2005. High-grade pancreatic intraepithelial neoplasia in a patient with familial adenomatous polyposis. Arch Pathol Lab Med 129:1398–1400. Half E, Bercovich D, Rozen P. 2009. Familial adenomatous polyposis. Orphanet J Rare Dis 4:22. Hamilton SR, Liu B, Parsons RE, Papadopoulos N, Jen J, Powell SM, Krush AJ, Berk T, Cohen Z, Tetu B. 1995. The molecular basis of Turcot’s syndrome. N Engl J Med 332:839–847. Hu PJ, Knoepp SM, Wu R, Cho KR. 2012. Ovarian steroid cell tumor with biallelic adenomatous polyposis coli inactivation in a patient with familial adenomatous polyposis. Genes Chromosomes Cancer 51:283–289. Irving JA, Young RH. 2009. Microcystic stromal tumor of the ovary: Report of 16 cases of a hitherto uncharacterized distinctive ovarian neoplasm. Am J Surg Pathol 33:367–375. Komorowski RA, Tresp MG, Wilson SD. 1986. Pancreaticobiliary involvement in familial polyposis coli/Gardner’s syndrome. Dis Colon Rectum 29:55–58. Korinek V, Barker N, Morin PJ, van Wichen D, de Weger R, Kinzler KW, Vogelstein B, Clevers H. 1997. Constitutive transcriptional activation by a beta-catenin-Tcf complex in APC-/colon carcinoma. Science 275:1784–1787. Maeda D, Shibahara J, Sakuma T, Isobe M, Teshima S, Mori M, Oda K, Nakagawa S, Taketani Y, Ishikawa S, Fukayama M. 2011. Beta-catenin (CTNNB1) S33C mutation in ovarian microcystic stromal tumors. Am J Surg Pathol 35:1429–1440. Maeda D, Shibahara J, Sakuma T, Sueyoshi K, Sakata A, Noguchi M, Fukayama M. 2012. Microcystic stromal tumor is distinct ovarian neoplasm characterized by ß-catenin alteration. Mod Pathol 25:284–285.
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Maire F, Hammel P, Terris B, Olschwang S, O’Toole D, Sauvanet A, Palazzo L, Ponsot P, Laplane B, Levy P, Ruszniewski P. 2002. Intraductal papillary and mucinous pancreatic tumour: A new extracolonic tumour in familial adenomatous polyposis. Gut 51:446–449. Morin PJ, Sparks AB, Korinek V, Barker N, Clevers H, Vogelstein B, Kinzler KW. 1997. Activation of beta-catenin-Tcf signaling in colon cancer by mutations in beta-catenin or APC. Science 275:1787–1790. O’Sullivan MJ, McCarthy TV, Doyle CT. 1998. Familial adenomatous polyposis: From bedside to benchside. Am J Clin Pathol 109:521–526. Polakis P. 2000. Wnt signaling and cancer. Genes Dev 14:1837– 1851. Powell SM, Zilz N, Beazer-Barclay Y, Bryan TM, Hamilton SR, Thibodeau SN, Vogelstein B, Kinzler KW. 1992. APC muta-
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RESEARCH ARTICLES
Ovarian Microcystic Stromal Tumor: A Novel Extracolonic Tumor in Familial Adenomatous Polyposis Sang Hun Lee,1 Young Wha Koh,2 Hyun Jin Roh,1 Hee Jeong Cha,3 and Yong-Soon Kwon1* 1
Department of Obstetrics and Gynecology,University of Ulsan College of Medicine,Ulsan University Hospital,Ulsan,Korea Department of Pathology, Ajou University School of Medicine, Suwon,Korea 3 Department of Pathology,University of Ulsan College of Medicine,Ulsan University Hospital,Ulsan,Korea 2
Ovarian microcystic stromal tumor (MCST) is a very rare neoplasm; hence, its nomenclature was recently designated as “Distinctive morphologic and immunohistochemical features” in 2009. Its exact origin, etiological genetic alterations, and background are not yet clearly known. Familial adenomatous polyposis (FAP) is an autosomal dominant disease that leads to development of colorectal polyps via germ-line mutations of the APC gene on chromosome 5q2122. In this study, we report a 40-year-old female patient who had ovarian MCST and FAP. On sequencing the APC gene in ovarian MSCT, we detected a novel somatic mutation of the APC gene in exon 11, with a heterozygous deletion at nucleotide position c.1540delG (p.Ala514 Profs*9). Mutations of b-catenin (CTNNB1) and FOXL2 were not detected. Although one case demonstrating involvement of Wnt/b-catenin in ovarian MCST associated with FAP has been presented previously, no detailed C 2015 V information was provided. Thus, this is the ovarian MCST with a somatic mutation of APC in a patient with FAP. Wiley Periodicals, Inc.
INTRODUCTION
Familial adenomatous polyposis (FAP) is an autosomal dominant disease that is characterized by early development of hundreds or thousands of adenomatous polyps in the colorectum through germ-line mutation of the APC gene on 5q2122 (Powell et al., 1992; O’Sullivan et al., 1998). Although FAP accounts for less than 1% of colorectal cancers (CRCs), it is one of the best understood genetic diseases (Half et al., 2009). Cases of CRC and other extracolonic tumors showing APC gene alteration through a second somatic mutation in the wild-type APC allele have been repeatedly reported. However, only a small number of cases of extracolonic tumors, such as those in the thyroid, liver, bile ducts, and the central nervous system, have been reported because of low disease prevalence (Turcot et al., 1959; Komorowski et al., 1986; Hamilton et al., 1995; Giardiello et al., 1996; Cetta et al., 1998; Cetta et al., 2000; Maire et al., 2002; Thomas et al., 2003; Chetty et al., 2005; Gupta and Mazzara, 2005; Sudo et al., 2005; Aretz et al., 2006; Groen et al., 2008). APC, a classical tumor suppressor gene, has a central role in bcatenin degradation and is closely correlated with the Wnt signaling pathway. Wnt signaling is associated with the stability of a protein complex consisting of b-catenin and GSK3 (glycogen synthase C 2015 Wiley Periodicals, Inc. V
kinase 3). In the absence of Wnt signaling or the presence of wild-type APC protein, b-catenin is degraded in the normal postembryogenesis period (normal pattern). In the presence of Wnt signaling or the absence of APC, b-catenin target genes, including MYC, show altered gene expression. This alteration, in turn, leads to altered expression of the polyamine ornithine decarboxylase gene which is a proto-oncogene (Barth et al., 1997; Polakis, 2000; Fearnhead et al., 2001). Ovarian microcystic stromal tumor (MCST) is a novel entity whose nomenclature was recently designated by Irving and Young (2009) based on 16 reported cases. Although the molecular pathogenesis and background genetic alterations still remain unclear, Maeda et al. (2011) reported two cases of oncogenic somatic mutations of the b-catenin gene (CTNNB1) in exon 3 in ovarian MCST, and verified the morphologic similarity between ovarian MCST and pancreatic solid pseudopapillary neoplasms (SPNs). However, the study did not *Correspondence to: Yong-Soon; Department of Obstetrics and Gynecology, Ulsan University Hospital, University of Ulsan College of Medicine, 290-3 Joenha-dong, Dong-gu, Ulsan 682-714, Korea. E-mail: [email protected] Received 19 September 2014; Accepted 25 November 2014 DOI 10.1002/gcc.22233 Published online 28 March 2015 in Wiley Online Library (wileyonlinelibrary.com).
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Figure 1. Abdominal pelvis CT (with contrast enhancement) and MRI female pelvis (with contrast enhancement). APCT and MRI female pelvis with contrast enhancement showed an approximately 15-cm complex cystic mass with bridging vascular pedicles contiguous with the left gonadal vein and a clear cleavage plane between this mass and uterine serosa.
adequately explain the relevance of APC in these two cases. A case demonstrating the involvement of Wnt/ b-catenin alterations in ovarian MCST associated with FAP has already been described by Maeda et al. (2012) in another series, but it was presented only as an abstract and did not provide clear detailed information. Thus, this study is the first one on ovarian MCST showing somatic mutation of APC in a patient with FAP.
MATERIALS AND METHODS Case History
A 40-year-old woman, gravida 2 and para 2, with no relevant past medical history visited our hospital due to pain in the lower abdomen. Physical examination revealed a large palpable mass in the lower abdomen. Through transvaginal ultrasonography, a 15-cm heterogeneous mass with a predominantly solid and partially cystic appearance was observed in the left ovary. It was suspected to be an ovarian malignancy, rather than a uterine Genes, Chromosomes & Cancer DOI 10.1002/gcc
malignancy. The levels of tumor markers, such as carcinoembryonic antigen, CA19-9, CA 125, and AFP, were within the normal range. For further examination, abdominal and pelvic computed tomography (APCT), magnetic resonance imaging (MRI) of the pelvis, and 18-fluoro2-deoxy-D-glucose positron emission tomography/ computed tomography (18PET/CT) were performed. APCT and MRI with contrast enhancement showed an approximately 15-cm complex cystic mass with bridging vascular pedicles contiguous with the left gonadal vein and a clear cleavage plane between the mass and uterine serosa. These findings were suggestive of a malignancy in the left ovary. There were no remarkable findings in the right adnexa and pelvic lymph node enlargement was not observed. However, mild bilateral hydronephroses caused by the pelvic mass effect were detected (Fig. 1). On PET-CT, a large, heterogeneous, hypermetabolic, lobulated mass with internal necrosis was detected in the pelvic cavity (max SUV 4.3); but it was not clear whether the malignant tumor originated in the ovary or uterus (Fig. 2).
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Figure 2. 18-Fluoro-2-deoxy-D-glucose positron emission tomography/computed tomography (18PET/CT). 18PET/CT showed a large, heterogenous, hypermetabolic, lobulated mass with internal necrosis in the pelvic cavity (max SUV 4.3). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Based on the imaging findings of APCT, MRI pelvis, and 18PET/CT, we suspected ovarian cancer. Colonoscopy and esophagogastroduodenoscopy were performed for preoperative evaluation to rule out any other primary cancer metastasizing to the ovary. Interestingly, through colonoscopy up to the cecum, hundreds of polyps were found in the sigmoid colon, descending colon, transverse colon, and ascending colon. Histopathological findings in the colonic specimens revealed villotubular adenomas, except for one well-differentiated adenocarcinoma lesion on a background of villotubular adenoma. There was no remarkable finding on esophagogastroduodenoscopy. Genetic studies for FAP and pedigree analysis were performed to examine the possibility of double primary malignancies. We would like to clarify that an agreement signed by the patient was obtained before performing the examination for ethical considerations. This study was approved by the Institutional Review Board at the University of Ulsan Medical School. The genetic studies revealed a strong multigenerational history of colon cancer. The patient’s father had died of CRC in his mid-fifties and her brother had undergone an operation for colon cancer diagnosed in his mid-thirties. A collaborative operative procedure with sufficient preparation, involving the gynecology and surgery teams, was performed. Frozen section of the left ovarian mass demonstrated an ovarian stromal tumor. Left salpigno-oophorectomy, right
ovarian wedge resection, and total colectomy were performed. The patient remains stable without any complications for 9 months after the operation. Patient and Tissue Specimens
We reviewed the hematoxylin and eosin-stained sections of ovarian MCST, FAP, and colon adenocarcinoma that were surgically removed at the University of Ulsan Hospital. Pathological diagnosis was made by two of the authors (Y.W.K and H.J.C). The tumor samples (ovary, intestine, polyp, CRC) were fixed in 10% buffered formalin, processed routinely, and embedded in paraffin. Then 4-mm sections were stained with H&E, and histopathological diagnosis was confirmed by the authors, including at least two pathologists. Immunohistochemistry
All samples were fixed in formalin and embedded in paraffin. In all of the cases, full tissue sections, 4mm thick, were used for immunohistochemistry. Immunohistochemical staining was performed according to standard techniques on a Ventana Benchmark XT autostainer (Ventana Medical Systems Inc., Tucson, AZ). Appropriate positive and negative controls were included. The antibodies used in this study were CD 10 (Novocastra, Clone 56C6, 1:150 dilution), b-catenin (Novocastra, Clone 17C2, 1:400), HMB45 (DAKO, Clone HMB 451,1:1000 dilution), Placenta alkaline phosphatase (Neomarkers, Clone 8A9, 1:2 dilution), Vimentin Genes, Chromosomes & Cancer DOI 10.1002/gcc
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Figure 3. Histology of the ovarian MCST and colonic adenocarcinoma. (A) Low-power view of the MCST. The tumor showed microcysts and variably prominent solid cellular areas with intervening hyalinized fibrous bands. (B) The cells had moderate amounts of lightly eosinophilic cytoplasm and inconspicuous nucleoli. (C) Well-differentiated adenocarcinoma identified in a tubular adenoma. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
(ZYMED, Clone V9, 1:150 dilution), Ki-67 (ZYMED, Clone 7B11, 1:400 dilution), C-Kit (DAKO, Clone, 1:1000 dilution), Epithelial membrane antigen (ZYMED, Clone ZCE 113, 1:250 dilution), Estrogen receptor (Novocastra, Clone DAK Calret 1, 1:150 dilution), a-Inhibin (Biocare, Clone BC/R1, 1:75 dilution), and Cytokeratin (Novocastra, Clone 5D3 and LP 34, 1:300 dilution). Genomic DNA Preparation
We extracted genomic DNA from whole blood using the Intron_G-DEXTM IIb Genomic DNA Extraction Kit (Intron, Seongnam, Korea). The ovarian MCST and colon tubular adenoma were microdissected from the paraffin-embedded tissue blocks and processed by ethanol/xylene dewaxing, followed by DNA extraction using a QIAampTM DNA mini kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. Mutation Analysis
Polymerase chain reaction (PCR) and sequencing analyses were performed using a standard protocol. In brief, PCR primer pairs were designed to Genes, Chromosomes & Cancer DOI 10.1002/gcc
amplify 48 DNA fragments that overlapped the entire coding sequence and exon/intron boundaries of APC (31 primer pairs), CTNNB1 (14 pairs), and FOXL2 (3 pairs). Primer sequences are available upon request. PCR amplifications were performed in a 20 ml reaction volume containing 100 ng genomic DNA, 2.5 mM MgCl2, 200 lM for each dNTP, 10 pmol for each primer, 53 BD (Nanohelix, Daejeon, Korea), and 5 U Taq DNA polymerase (Nanohelix, Daejeon, Korea). The following thermal profile was applied using a PTC-220 DYADTM thermal cycler (Bio-Rad, Hercules, CA): initial denaturation for 5 min at 95 C followed by 42 cycles at 95 C for 30 sec, 62 C for 40 sec, and 72 C for 45 sec, with a final extension at 72 C for 7 min. The PCR products were purified using exonuclease I and shrimp alkaline phosphatase (GE Healthcare, Uppsala, Sweden) and sequenced in both directions with an AB 3730xl genetic analyzer (Life Technologies, Foster city, CA), according to the manufacturer’s instructions. All of the variations detected were reviewed and confirmed by repeating the PCR and the sequencing analyses. All sequence variations were described according to the HGVS (Human Genome Variation Society) mutation nomenclature (den Dunnen and
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Figure 4. Immunohistochemical features of the ovarian MCST. Diffuse and strong positivity for (A) vimentin and (B) CD10, and negativity for (C) cytokeratin and (D) epithelial membrane antigen. (E) Diffuse and strong nuclear positivity for b-catenin. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Antonarakis, 2000). Two different in silico bioinformatics prediction programs were used to predict the pathogenic effect of the novel variant c.3796G>A of APC; PolyPhen2 (http://genetics. bwh.harvard.edu/pph2/) and SIFT (http://sift.jcvi. org/). RESULTS Gross Features of Ovarian MCST and FAP
The left ovarian tumor measured 15.0 3 15.0 3 9.0 cm in size. The surface of the ovary was smooth, although the tumor was confined to the ovary. The cut surface of the mass was grayish yellow, firm, and granular. Multifocal necrosis and
hemorrhage were noted. There were more than 200 polyps in the colectomy specimen.
Histologic Features of Ovarian MCST and Colorectal Adenocarcinoma
On routine histopathological examination, 27 slide sections of the ovarian tumor were reviewed. The tumor showed microcysts and variably prominent solid cellular areas with intervening hyalinized fibrous bands (Fig. 3A). The cells in the cellular areas had moderate amounts of lightly eosinophilic cytoplasm and inconspicuous nucleoli (Fig. 3B). There was no significant histologic finding on the right ovarian wedge biopsy. Adenocarcinoma was Genes, Chromosomes & Cancer DOI 10.1002/gcc
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Figure 5. The electropherogram of the APC gene. (A) Sequencing data of exon 15 revealed a constitutional heterozygous deletion at nucleotide position c.2376_2378 delGCAinsC (p.Lys792 Asnfs*28). (B) Sequencing data of exon 15 in a colonic adenomatous polyp revealed a heterozygous missense at nucleotide position c.3796G>A (p.Asp 1266Asn). (C) Sequencing data of exon 11 in the ovary with a hetero-
zygous frameshift at nucleotide position c.1540delG (p.Ala514 Profs*9). The top panel is the control sequence. The other panel is the corresponding tissue sequence. C indicates cysteine; D, aspartic acid; G, glycine; H, histamine; I, isoleucine; L, leucine; S, serine; Y, tyrosine. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
identified in the one of the tubular adenomas in the endoscopic mucosal resection specimen (Fig. 3C).
(CTNNB1) and FOXL2 in blood, colon polyp, or ovarian MCST.
Immunohistochemistry
The ovarian MCST displayed a diffuse and strong immunoreactivity for vimentin (Fig. 4A) and CD 10 (Fig. 4B). The tumor cells were negative for cytokeratin (Fig. 4C) and epithelial membrane antigen (Fig. 4D). Additionally, the tumor cells commonly showed diffuse and strong nuclear immunoreactivity for b-catenin (Fig. 4E). The tumor cells were negative for a-Inhibin, c-kit, HMB45, placental alkaline phosphatase, estrogen receptor, progesterone receptor, and calretinin. The Ki-67 labeling index was low (A mutation in a colon polyp (Fig. 5), a missense mutation resulting in a downstream stop codon (p.Asp 1266Asn); and (3) a somatic c.1540delG mutation in the ovarian MCST (Fig. 5), a frameshift mutation resulting in a downstream stop codon (p.Ala514 Profs*9). Mutation Analyses of b-Catenin (CTNNB1) and FOXL2 in Blood, Colon Polyp, and Ovarian MCST
Mutation analyses of b-catenin (CTNNB1) and FOXL2 revealed no mutations of b-catenin Genes, Chromosomes & Cancer DOI 10.1002/gcc
DISCUSSION
A small number of cases of extracolonic benign and malignant tumors in patients with FAP have been reported (Blaker et al., 2004; Groen et al., 2008). Gynecologic cases related to the ovary and associated with FAP have rarely been reported. One ovarian steroid cell tumor with biallelic APC inactivation in an FAP patient was reported in a study by Hu et al. (2012); this was the first study to suggest a somatic alteration of the APC gene (APC/ b-catenin) in an ovarian MCST patient with FAP. Ovarian MCST is a rare tumor, entity recently deliberated by Irving and Young (2009) based on 16 reported cases. The exact origin and genetic alterations of ovarian MCSTs have not yet been clearly identified. The characteristics of MCSTs are a combination of microcystic, macrocystic, and solid structures, presence of intervening thick fibrous stroma, growth of monotonous tumor cells with round-toovoid and generally simple nuclei with very fine chromatin, well-marked borders, and focal areas of congestive and hemorrhagic changes that can give an impression of a hemangiomatous lesion (Irving and Young, 2009; Maeda et al., 2011). Ovarian steroid cell tumors may be a differential diagnosis. Little is known about the pathogenesis of these tumors because of their rarity accounting for less than 0.1% of all ovarian tumors (Scully, 1979). Not much is known regarding their b-catenin status. However, in this study, we recognized that ovarian steroid cell tumors are clearly differential from ovarian MCST, in that ovarian steroid cell tumors do not display microcytic histologic features. Recognition of the importance of the APC gene began with genetic studies for FAP, an inherited disease with tendency to develop colon cancer (Powell
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et al., 1992; O’Sullivan et al., 1998). Under normal conditions, the APC gene suppresses the propagation of b-catenin by arbitrating its phosphorylation in the cell (Aberle et al., 1997). This function of the APC gene is disturbed when it loses the ability to control b-catenin regulatory activity, resulting in abnormal accumulation of b-catenin. Loss of the ability can be caused by either APC gene mutations or as a result of truncation of the b-catenin oncogene (Korinek et al., 1997; Morin et al., 1997). In this study, we did not detect any somatic mutations of the b-catenin gene, unlike in the study by Maeda et al. (2011). The latter group reported two cases of ovarian MCST with somatic mutations of the b-catenin gene at exon 3, implicating the Wnt/b-catenin gene (CTNNB1) pathway in ovarian MCST (Maeda et al., 2011). Some ovarian MCSTs are assumed to undergo other types of molecular change that inactivate the APC/b-catenin pathway. In sporadic and FAP-associated pancreatoblastomas, molecular alterations in the APC/b-catenin pathway were detected in 67% of pancreatoblastoms (six of nine), of which five tumors were caused by sporadic mutations of the b-catenin oncogene and one tumor was caused by biallelic APC inactivation without mutation of b-catenin oncogene in one patient with FAP (Abraham et al., 2001). The notable finding was that pancreatoblastomas, although sporadic, showed a predominance of b-catenin mutations, with APC mutation without b-catenin oncogene being demonstrated in only a patient with FAP. In our study, we detected a novel mutation, a heterozygous deletion at nucleotide position c.1540delG (p.Ala514 Profs*9), of the APC gene at exon 11 in the ovarian MSCT. This confirmed the role of APC in downregulating the level of bcatenin; the immunohistochemisty demonstrated a strong correlation between accumulation of bcatenin in the membrane, cytoplasm, and nucleus and APC gene alterations. Finally, we considered ovarian solid pseudopapillary tumors as a differential diagnosis for ovarian MCSTs, as from reported by Deshpande et al. (2010). According to our results, ovarian SPNs showed an aberrant nuclear accumulation of b-catenin. In conclusion, where a patient is diagnosed with ovarian MCST, the possibility of FAP should be considered, as ovarian MCST should be considered as one of the tumors associated with FAP. REFERENCES Aberle H, Bauer A, Stappert J, Kispert A, Kemler R. 1997. Betacatenin is a target for the ubiquitin-proteasome pathway. EMBO J 16:3797–3804.
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