a 47-year - old postmenopausal woman presented with slowly increasing abdominal girth with a palpable abdominal mass and vaginal bleeding . at the age of 15
, she underwent abdominal surgery due to intussusception of the small bowel of unknown cause . during the preoperative evaluation
, colonoscopy identified eight hamartomatous polyps , nine hyperplastic polyps , and two tubulovillous adenomas in her large intestine .
she had abundant mucocutaneous melanin pigmentation around the lips . based on her clinical features and the histopathological diagnosis of hamartomatous polyps and mucocutaneous pigmentation ,
no other family members had clinical signs of pjs . on pelvic and abdominal ultrasonography and computed tomography ,
she was found to have huge bilateral ovarian cystic masses with ascites in the pelvic cavity . on exploratory laparotomy ,
the left ovarian mass was ruptured , and approximately 1,500 ml of ascitic fluid was identified .
the patient underwent hysterectomy , bilateral salpingo - oophorectomy , and small bowel polypectomy via small bowel enterostomy .
grossly , the left ovarian tumor was a multilocular cyst measuring 22206 cm ( fig .
1a ) , whereas the right ovarian tumor was an oligolocular cyst measuring 875 cm ( fig .
microscopically , both ovarian tumors showed cysts of varying sizes lined by a single layer of columnar epithelium , and the architecture of the glandular arrangement was characterized by clusters of small glands budding from surrounding a centrally located large duct - like structures , forming a lobular arrangement at lower magnification ( fig .
the uterine cervix , endometrium , and fallopian tubes were meticulously examined , but no histologic evidence of mucinous epithelial lesions or metaplasia was identified .
polyps of the small intestine showed a branching polypoid structure with crypts and villi of variable lengths and cystically dilated glands , which were divided by muscularis mucosa branching in various directions ( fig .
on combined alcian blue ( ph 2.5 ) and periodic acid - schiff after diastase ( dpas ) staining , the intracytoplasmic mucin in the epithelium of the ovarian tumor was negative for alcian blue ph 2.5 and positive for dpas .
the cytoplasm stained bright pink for combined alcian blue ph2.5/dpas , implying that the mucinous contents of the ovarian tumor were neutral ( fig .
1 g ) , in contrast to mucin in the normal endocervical mucosa , which was positive for both alcian blue ph 2.5 and dpas and showed a purple - violet color for combined alcian blue ph2.5/dpas ( fig .
we performed immunohistochemical staining using formalin - fixed , paraffin - embedded , 4-m - thick tissue sections with an optiview dab immunohistochemical detection kit ( roche diagnostics , mannheim , germany ) on a benchmark xt autoimmunostainer ( ventana medical system , tucson , az , usa ) .
the mucinous epithelium of the ovary showed diffuse immunoreactivity for muc6 ( novo , newcastle upon tyne , uk ) , moderately intense immunoreactivity for carbonic anhydrase - ix ( novus biologicals , littleton , co , usa ) , and focal immunoreactivity for muc5ac ( marker of foveolar - type mucin ; novo ) and hik1083 ( toyo 2chome , tokyo , japan ) ( fig .
d ) . the epithelium did not show immunoreactivity for p16 ( santa cruz biotechnology , santa cruz , ca , usa ) , estrogen receptor ( novo ) , progesterone receptor ( novo ) , or p53 ( oncogene , uniondale , ny , usa ) .
genomic dna was extracted from peripheral blood samples by a qiaamp dna blood kit ( qiagen gmbh , hilden , germany ) and from fresh frozen tissue of the left ovarian tumor by a quickgene dna tissue kit ( fujifilm life science , tokyo , japan ) .
the stk11 gene was amplified via polymerase chain reaction by using 10 sets of primers in intronic flanking regions containing all exons ( table 1 ) .
sequencing analysis was performed by a cycle sequencing kit ( thermo fisher scientific , waltham , ma , usa ) following the manufacturer s instructions .
multiple ligation - dependent probe amplification ( mlpa ) was performed to detect deletions and duplications in the stk11 gene by stk11 mlpa kit p101 ( mrc - holland , amsterdam , the netherlands ) . the peak height of the probes was analyzed using genemarker software v.1.7 ( softgenetics llc , state college , pa , usa ) . a peak ratio less than 0.65 was interpreted as a 
deletion, whereas a peak ratio greater than 1.35 was interpreted as a  duplication. sequencing analysis did not detect any mutation in the exons and exon - intron boundaries of the stk11 gene in the blood and ovarian tumor samples .
using mlpa analyses , however , peripheral blood leukocytes showed a germline heterozygous deletion mutation at exons 17 of the stk11 gene ( c.1-?_920+?del ) and the tumor tissue showed mosaic loss of heterozygosity due to the mutation ( heteroplasmy ) ( fig .
we performed immunohistochemical staining using formalin - fixed , paraffin - embedded , 4-m - thick tissue sections with an optiview dab immunohistochemical detection kit ( roche diagnostics , mannheim , germany ) on a benchmark xt autoimmunostainer ( ventana medical system , tucson , az , usa ) .
the mucinous epithelium of the ovary showed diffuse immunoreactivity for muc6 ( novo , newcastle upon tyne , uk ) , moderately intense immunoreactivity for carbonic anhydrase - ix ( novus biologicals , littleton , co , usa ) , and focal immunoreactivity for muc5ac ( marker of foveolar - type mucin ; novo ) and hik1083 ( toyo 2chome , tokyo , japan ) ( fig .
d ) . the epithelium did not show immunoreactivity for p16 ( santa cruz biotechnology , santa cruz , ca , usa ) , estrogen receptor ( novo ) , progesterone receptor ( novo ) , or p53 ( oncogene , uniondale , ny , usa ) .
genomic dna was extracted from peripheral blood samples by a qiaamp dna blood kit ( qiagen gmbh , hilden , germany ) and from fresh frozen tissue of the left ovarian tumor by a quickgene dna tissue kit ( fujifilm life science , tokyo , japan ) .
the stk11 gene was amplified via polymerase chain reaction by using 10 sets of primers in intronic flanking regions containing all exons ( table 1 ) .
sequencing analysis was performed by a cycle sequencing kit ( thermo fisher scientific , waltham , ma , usa ) following the manufacturer s instructions .
multiple ligation - dependent probe amplification ( mlpa ) was performed to detect deletions and duplications in the stk11 gene by stk11 mlpa kit p101 ( mrc - holland , amsterdam , the netherlands ) .
the peak height of the probes was analyzed using genemarker software v.1.7 ( softgenetics llc , state college , pa , usa ) . a peak ratio less than 0.65 was interpreted as a 
deletion, whereas a peak ratio greater than 1.35 was interpreted as a  duplication. sequencing analysis did not detect any mutation in the exons and exon - intron boundaries of the stk11 gene in the blood and ovarian tumor samples .
using mlpa analyses , however , peripheral blood leukocytes showed a germline heterozygous deletion mutation at exons 17 of the stk11 gene ( c.1-?_920+?del ) and the tumor tissue showed mosaic loss of heterozygosity due to the mutation ( heteroplasmy ) ( fig .
minimal deviation adenocarcinoma ( an extremely well differentiated form of gastric type adenocarcinoma ) of the cervix and ovarian sex cord tumor with annular tubules are the most well - known tumors of the female genital organs associated with pjs , but association with ovarian mucinous tumors has rarely been described .
moreover , gastric phenotype of the mucinous epithelium in patients with pjs has not been clearly determined , and there is only one case briefly mentioning ovarian mucinous tumor with pyloric / gastric differentiation . in that case report ,
the pyloric gland differentiation involved the lesions of multiple organs , including the jejunum , urinary bladder , uterine cervix , fallopian tube , and ovary . in the extragenital organs , peutz - jeghers polyps in duodenum and jejunum have small areas of normal superficial gastric - type epithelium , suggesting that the proliferations of gastric - type mucinous epithelium are common features in any organs of the patients with pjs .
although the gastric subtype is very rare among mucinous tumors , it must be a distinct subtype of the ovarian mucinous tumors , which should be differentiated from the usual intestinal subtype .
legh in the uterine cervix is thought to be a precursor lesion of gastric - type cervical adenocarcinoma , which is often identified in patients with pjs .
therefore , the premalignant potential of an ovarian mucinous tumor resembling legh should be closely monitored . in our case , a germline heterozygous deletion mutation in stk11 ( c.1-?_920+?del ) ( exon17del ) and mosaic loss of heterozygosity
were detected in the left ovarian mucinous cystadenoma by mlpa assay , but direct sequencing did not detect the deletion of the large exon . in conclusion ,
our case combined with previously described cases suggests that stk11 gene mutations play an important role in the proliferation of pyloric gland - phenotype mucinous epithelium in various anatomical locations .
 we describe an ovarian mucinous neoplasm that histologically resembles lobular endocervical glandular hyperplasia ( legh ) containing pyloric gland type mucin in a patient with peutz - jeghers syndrome ( pjs ) . although ovarian mucinous tumors rarely occur in pjs patients , their pyloric gland phenotype has not been clearly determined . 
 the histopathologic features of the ovarian mucinous tumor were reminiscent of legh . 
 the cytoplasmic mucin was stained with periodic acid - schiff reaction after diastase treatment but was negative for alcian blue ph 2.5 , suggesting the presence of neutral mucin . 
 immunohistochemically , the epithelium expressed various gastric markers , including muc6 , hik1083 , and carbonic anhydrase - ix . 
 multiple ligation - dependent probe amplification detected a germline heterozygous deletion mutation at exons 17 of the stk11 gene ( c.1-?_920+?del ) in peripheral blood leukocytes and mosaic loss of heterozygosity in ovarian tumor tissue . considering that legh and/or gastric - type cervical adenocarcinoma can be found in patients with pjs carrying germline and/or somatic stk11 mutations , our case indicates that stk11 mutations have an important role in the proliferation of pyloric - phenotype mucinous epithelium at various anatomical locations . 
CASE REPORT
Immunohistochemistry
Mutation analysis
DISCUSSION