gastrointestinal stromal tumors ( gists ) are well recognized nonepithelial neoplasms of the gastrointestinal tract ( gi ) .
after decades of ultra structural , immunohistochemical , and genetic studies , it is now evident that most gists are believed to differentiate from cajal ( iccs ) pacemaker cells .
the cajal pacemaker cells are involved in gastrointestinal tract mobility and regulation of autonomous neuronal transmission .
other common sites include the jejunum and ileum ( 30% ) , duodenum ( 5% ) , colon and rectum ( 5% ) , and the esophagus ( 5% ) .
only seven gallbladder gists have ever been reported , and of these , four were malignant [ 39 ] .
these malignant gallbladder gists were all positive for cd 117 ( c - kit ) and cd34 or cd117 alone [ 47 ] .
we report a unique gallbladder gist which is negative for cd 117 and cd 34 but positive for platelet - derived growth factor receptor alpha ( pdgfra ) . apart from the extremely rare site of occurrence , gist of the gallbladder
remains a challenging topic and needs to be examined on the basis of histologic findings , clinical history , and molecular profile with the later having significant impact on the treatment strategy .
a 72-year - old woman was admitted to the first department of surgery , university of athens with symptoms suggestive of obstructive jaundice : fever and chills , dark urine , intermittent right upper quadrant ( ruq ) abdominal pain , and jaundice .
initial investigations showed a white cell count ( wcc ) of 11,556  cells / mm , total bilirubin / direct bilirubin ( tbil / dbil ) of 4.80/3.60  mg / dl , alkaline phosphatase ( alp ) 440  iu / l , and -glutamyl transpeptidase ( -gtp ) 210  iu / l .
ultrasound demonstrated a thickened gallbladder wall , bile stones , and moderate common bile duct ( cbd ) dilatation .
bile duct lithiasis was suspected and an endoscopic retrograde cholangiopancreatography ( ercp ) was performed .
this demonstrated an almost complete obstruction at the level of the cystic duct and common hepatic duct confluence .
however , the underlying pathology could not be identified , and an exploratory laparotomy was decided .
the mass was adherent to the duodenum involving the confluence of the cystic duct with the common hepatic duct and the retroduodenal part of the common bile duct .
the common bile duct and common hepatic were excised en bloc with the specimen ensuring free surgical margins along the extrahepatic billiary tree .
lymph node dissection was performed of the hepatoduodenal ligament , the hepatic artery lymph nodes , and the retropancreatic lymph nodes .
the enteric - billiary continuity was established through a retrocolic roux - en - y hepatico - jejunostomy .
the postoperative course was uneventful , and the patient was discharged ten days after operation .   in the pathological review ,
the gallbladder was dilated and measured 7.5  cm  3.5  cm in size . within the gallbladder
, there was a wide mass covering the entire wall , neck , and body , while the remainder of the mucosal layer was ulcerated .
microscopically , tumor cells infiltrated the muscle layer were spindle shaped in nature , arranged in a short fascicle pattern and immunopositive for pdgfra ( figures 1(a ) and 1(b ) ) .  
the cells demonstrated hypercromatic nuclei , severe pleomorphism and an increased mitotic rate of up to 50 mitosis/50 high power field ( hpf ) .

 genomic dna isolationten m sections were cut from the paraffin - embedded tissue block and treated with xylene / ethanol .
the samples were digested overnight at 37c using proteinase k. dna was extracted with phenol - chloroform , precipitated in ice - cold ethanol , redissolved in distilled water , and quantitated using a picodrop microliter spectrophotometer . 
 
ten m sections were cut from the paraffin - embedded tissue block and treated with xylene / ethanol .
the samples were digested overnight at 37c using proteinase k. dna was extracted with phenol - chloroform , precipitated in ice - cold ethanol , redissolved in distilled water , and quantitated using a picodrop microliter spectrophotometer . 

high - resolution melting analysisfor the detection of c - kit exon 9 , 11 , 13 , and 17 mutations as well as pdgfra mutations in exons 12 and 18 , a real - time polymerase chain reaction approach followed by high - resolution melting curve analysis has proved to be a rapid , highly sensitive , and efficient screening method .
pcr and hrm were consecutively performed on a lightcycler 480 ( roche diagnostics , gmbh , germany ) in one single run , and all reactions were repeated twice .
each reaction consisted of 20  ng of dna , 200  nmol / l of each primer , 10  l of lightcycler lc480 high resolution melting master ( roche ) , 3.5  mm mgcl2 and pcr - grade water adjusted to a total volume of 20  l . 
  for the detection of c - kit exon 9 , 11 , 13 , and 17 mutations as well as pdgfra mutations in exons 12 and 18 , a real - time polymerase chain reaction approach followed by high - resolution melting curve analysis
pcr and hrm were consecutively performed on a lightcycler 480 ( roche diagnostics , gmbh , germany ) in one single run , and all reactions were repeated twice .
each reaction consisted of 20  ng of dna , 200  nmol / l of each primer , 10  l of lightcycler lc480 high resolution melting master ( roche ) , 3.5  mm mgcl2 and pcr - grade water adjusted to a total volume of 20  l . 

sequencingpcr products with an abnormal hrm pattern were sequenced using the big dye terminator cycle sequencing kit ( applied biosystems , california , usa ) .
the sequencing products were analysed on an abi prism 310 genetic analyzer ( applied biosystems ) . 
 
pcr products with an abnormal hrm pattern were sequenced using the big dye terminator cycle sequencing kit ( applied biosystems , california , usa ) .
the sequencing products were analysed on an abi prism 310 genetic analyzer ( applied biosystems ) .
the specimen was analysed for the presence of activating mutations in exons 9 , 11 , 13 , and 17 of c - kit gene as well as exons 12 and 18 of pdgfra gene by performing high resolution melting analysis and sequencing .
a mutation at codon 824 coding for valine in exon 18 of pdgfra gene was detected by high - resolution melting analysis ( hrm ) and was further confirmed by sequencing as c. 2472 c >
this silent mutation has previously been described in 2 tumors from the central nervous system ( gliosarcomas ) as well as in soft - tissue tumors ( gists ) .
results were verified by a second independent pcr reaction and hrm analysis followed by sequencing .
an estimated five - to - six thousand new cases of gist are diagnosed annually with 10% to 30% of these being malignant .
most tumors are sporadic , affecting individuals in their 5th or 6th decade with some evidence indicating a male predominance .
gists can also present earlier and this is often seen in one of the rare  gist syndromes  , which include neurofibromatosis type 1 ( nf1 ) , the carney - stratakis dyad and familial gist syndrome .
these tumors may occur anywhere in the gastrointestinal tract but most commonly present in the stomach and small intestine .
they can also occur in the surrounding structures such as the peritoneum , omentum , liver , pancreas , ovaries , and uterus .
many gists are asymptomatic and are discovered incidentally ; however , over half of gastric gists present with signs of gi bleeding and anaemia with a smaller proportion presenting with abdominal pain or as an abdominal mass .
histology can often be varied , but gists are broadly divided into spindle , epithelioid , or mixed cell types . in general , the risk of malignancy is greater in epithelioid tumors than in spindle - celled neoplasms [ 11 , 12 ] . with the advent of immunohistology ,
previously , sarcomas , undifferentiated carcinomas , and melanomas would have featured heavily in the differential diagnosis ; however , these can largely be excluded with identification or absence of specific immunohistochemical markers .
the three most well - described immunohistochemical markers in gists are cd117 ( c - kit ) , pdgfra , and cd34 [ 13 , 14 ] .
kit is positive in over 95% of gists and only 58% of tumors are kit negative and pdgfra positive .
cd34 is a less sensitve marker for gists but is reported in up to 6070% of the tumors .
miettinen and lasota who have carried out the largest ever studies on gists define these tumors as generally kit - positive and kit or pdgfra mutation - driven mesenchymal tumors of the gi tract with a set of characteristic histologic features including spindle cell , epithelioid , and rarely pleomorphic morphology .
we present the case of a malignant gist of the gallbladder which demonstrated spindle cell morphology and was pdgfra positive and kit negative .
there have been a small number of gallbladder gists described in the literature , of which only four were malignant and all were kit positive . to our knowledge
the significance of the kit negative genotype has implications on the response to further management .
the identification of specific cellular markers has led to the development of effective targeted agents , namely , tyrosine kinase inhibitor ( tki ) therapy ( e.g. , imatinib ) .
most kit positive tumors are sensitive to imatinib ; however , the majority ( 80% ) of pdgfra mutations are resistant to treatment .
primary gists , as in the case here , have the potential for curative treatment with surgical resection .
overall 5-year survival rates for resectable gists have been shown to range from 46% to 78.5% ; however , predicting the recurrence rate of primary resectable gists has been very challenging [ 17 , 18 ] .
the survival rates from the reported gall bladder gists are mixed with only short - term followup noted in some of the cases .
although the mutation status is important , the current most important prognostic factor for gists is tumor size , mitotic count , and tumor location .
this scheme has evolved from studies initially outlined by the national institute of health ( nih ) and were greatly expanded on by the work of miettinen and lasota [ 13 , 14 ] .
the patient in this case had a very large tumor , with 50 mitosis/50 hpf , and was located in the gallbladder .
the first two findings alone place this gist in the high - risk group for recurrence .
this is likely compounded by the kit negative immunohistology which would potentially reduce the benefit of tyrosine kinase inhibitors treatment .
it is important to mention that the immunohistochemical examination does not provide information on the exon affected on the type of mutation both of which may be prognostically important .
this paper , presents a very unique case of gist , located in the gallbladder , negative for kit , and positive for pdgfra .
apart from this being a previously undocumented case , it highlights the challenges in establishing the diagnosis , prognosis , and most effective management for this unpredictable tumour .
 gastrointestinal stromal tumors ( gists ) compose the largest category of well - recognized nonepithelial neoplasms of the gastrointestinal tract ( gi ) 
 . gists of the gallbladder are extremely rare tumors . 
 only four malignant , two benign and one gist - like tumor of the gall bladder have ever been described . 
 the four malignant gists were all positive for cd 117 antigen ( c - kit ) . 
 we present for the first time a malignant gastrointestinal stromal tumor of the gallbladder , immunoreactive for platelet - derived growth factor receptor alpha ( pdgfra ) and negative for cd 117 antigen ( c - kit ) . 
1. Introduction
2. Case Report
3. Mutational Analysis of c-Kit and PDGFRA
4. Results
5. Discussion
6. Conclusion