Histopathology and Immunohis to chemical Examinations in Adenos quamous Carcinoma, Cancer of the Stomach - Case Study

1. Abstract Theauthorspresentacaseofgastricadenosquamouscarcinomain a62-year-oldfemalepatient.Amonthearlier,anadenocarcinoma infiltration was diagnosed during gastroscopy in the histopathological examination of specimens from the pyloric ulcer.

2. Introduction Gastriccancerisstillamajordiagnosticandtherapeuticproblem. In 2020, 768793 deaths were reported worldwide due to this cancer, which is the fourth most frequent cause of death due to neoplastic disease [1]. In Poland, this neoplasm is the fifth cause of death due to neoplastic diseases in men and eighth in women [2]. A definitive diagnosis of gastric tumours requires microscopic confirmation.Thevastmajorityofgastriccancersareadenocarcinomas,othertypesofthiscanceroccursporadically.Forexample, adenosquamouscarcinomaofthestomachaccountsfor0.2-0.25% of all cancers of this organ [3, 4]. Only a few case reports of this disease[5-7]andcomprehensivestudiesusinglargecaseseries[8- 12]werefoundintheanalyzedliterature.Itisalsoraretofindpublications on the usefulness of immunohistochemical tests for the diagnosis of adenosquamous carcinoma [7, 13, 14]. The authors decidedtosubmitforpublicationacaseofgastricadenosquamous carcinoma,diagnosedonthebasisofahistopathologicalexamina- tion using a panel of immunohistochemical tests.

3. Case Presentation A62-year-old female patient was urgently admitted to the hospitalduetosudden,severeabdominalpain,mainlyintheepigastric region. On admission, the laboratory parameters are normal, and theabdominalX-rayiswithoutsignsofobstructionorperforation. Amonthearlier,sheunderwentanendoscopyoftheuppergastrointestinal tract.An ulcer was found within the pylorus and an adenocarcinoma infiltration was diagnosed in the histopathological examinationofthespecimenscollectedfromthisulcer.Duetothe features of peritonitis, a computer tomography of the abdominal cavity was performed, in which a perforated tumor of the pylorus was diagnosed and the patient qualified for urgent surgery. Alarge tumor of the pylorus was confirmed intraoperatively with a perforation hole on the posterior stomach wall. Subtotal gastric resection was performed with D2 lymphadenectomy, Bilroth II colonicanastomosis,andBraun’senzymeanastomosis.Postoperatively, good convalescence, decreasing inflammation indicators and no signs of anastomotic leak were observed. The patient was discharged home in good psychophysical and local condition. For histopathological examination (No. 1431/19 Department of Pathomorphology, MEDICAM Specialist Hospital in Gryfice), the material was obtained in three vessels. The first material was collectedfromlymphnodesintheareaoftheceliactrunkandthe second material from the lymph nodes in the area of the hepatic ligament.Thethirdmaterialisasewn-upfragmentofthestomach, which,afterbeingcutalongthegreatercurvature,haddimensions of 18 x 10 cm, with an adjoining omentum of 26 x 24 cm. In the mucosainthepyloricarea,anulceratedtumorwithperforationand 4 x 4.5 cm rolled rims was found, infiltrating the entire thickness of the stomach wall. The above macroscopic picture corresponds totype2ofadvancedgastriccancer(ulceratedtumorwithclearly delimited, raised margins undermining the mucosa) according to the Japanese Research Association for Gastric Cancer classification[15,16].Thetumorwaslocatedmacroscopically1.8cmfrom the distal line of the surgical incision. The material for histopathological examination was collected according to the recommendations of the Polish Society of Pathologists[16],takingintoaccountinthethirdmaterial:theboundaries of the surgical incision from the cardia and duodenum, the stomachwallwiththetumor,thesurroundinglymphnodes,thegastric omentum and the wall of the stomach outside the tumor. Routine histological examination of H&E stained specimens and immunohistochemistry with the use of the following markers: CK7, p63, p40, CK20, CD56, synaptophysin, chromogranin A, and Ki-67 were performed on the material under review, fixed in 4%bufferedformalinandembeddedinparaffin.Histopathological diagnosis was made on the basis of the 2019 WHO classification andtherecommendationsofthePolishSocietyofPathologists[3, 16]. The paraffin-embedded immunohistochemical materials were cut into4micrometersectionsandmountedonglassslideswithanadhesivecoating-SuperFrost®Plus(MenzelGläser®).Subsequently,thesectionsweremachinestained(BenchMarkULTRAIHC/ ISH System - Roche Diagnostics instrument) based on validated diagnostic protocols and reagent kits prepared by Roche. HER-2 protein was also determined in the neoplastic infiltration usingtherabbitantibodyVentanaPATHWAYHER-2/neu(4B5), sections for this study were also machine stained based on the diagnostic protocol and reagent kit prepared by Roche. Inthehistopathologicalexaminationofthematerialcollectedfrom the lymph nodes in the area of the visceral trunk, metastasis of tubular adenocarcinoma was present in one of the 4 lymph nodes withinfiltrationofthecapsuleandsurroundingadiposetissue.Reactive changes were found in the remaining 3 lymph nodes, free from tumor metastases. Inthehistopathologicalexaminationofthematerialcollectedfrom 3lymphnodesintheareaofthehepaticligament,notumormetas- tases were found, only reactive changes were present. Inthesurgicalmaterialfromsubtotalgastrectomy,adenosquamous carcinoma(WHO)wasfoundwithinthepylorictumor(Figure1). The squamous cell component constituted 30% of the neoplastic structure and the adenocarcinoma component 70% of its fabric, including 60% of the infiltration of tubular adenocarcinoma and 10% of mucinous adenocarcinoma.According to Lauren’s classification,itwastypeIlimitedstomachcancer.Thene oplasticinfiltratio ncoveredtheenti rethicknessof the stomachwal landspread to the surrounding adipose tissue without engaging the visceral surface of the sera.Angioinvasion was present. Infiltration along the nerves was not encountered. Neoplastic metastases with infiltration of the capsule and the surrounding fatty tissue were found in 16 out of 23 lymph nodes encountered. However, in 7 out of 23 lymph nodes only reactive inflammatory changes were found. In lymph node metastases, all threeprimarytumorformswerefound:tubularadenocarcinomain 7 nodes, mucinous adenocarcinoma in 3 nodes, adenosquamous carcinoma in 4 nodes (Figure 2) and squamous cell carcinoma in 2 nodes. No neoplastic infiltration (R0) was encountered in the proximal,distalandradialcutting lines.Apart from the tumor, no neoplasticinfiltration was foundinthesections taken from the gastricomentumand the wall of the stomach. According to the TNM classification of 2019 (3), the cancer advancement is: pT3 pN3b. Animmuno histochemical examination wasper for medon the material collected from the primarytumorandneoplasticmetastases. Cytoplasmic expression of CK7+ was found in the glandular and mucous components carcinoma of the stomach (Figure3).Nuclear expression of p63+ was found in the squamous cell componentof gastriccarcinoma (Figure4), and p40+wasals ofoundin the nucleiof neoplasti ccells in the squamous cell componentca rcinoma of thestomach(Figure5).Ontheotherhand,cytoplasmicexpression of CK20+wasfocalinmucinouscomponentofgastriccarcinoma. No CD56, synaptophysin or chromogranin expression was found in the neoplastic cells tested. The Ki-67+ proliferation index was 60% of the nuclei in the weaving of adenocarcinoma, about 50% of the nuclei in the weaving of mucinous adenocarcinoma, and 40%ofthenucleiintheweavingofthesquamouscellcarcinoma. It should be noted that the results of immunohistochemical tests were identical in the material collected from the primary tumor andinthematerialcollectedfromthelymphnodemetastases.The determination of the HER-2 protein in the cells of the neoplastic infiltration of the stomach wall was negative (score = 0). No significant microscopic changes or colonization of Helicobacter pylori were found in the gastric mucosa in the vicinity of the tumor (the slides were stained by histochemistry according to Giemsa and immunohistochemistry).

4. Discussion Theneoplasticinfiltrationofthepylorictumour,inourcase,con- sisted of a mixture of adenocarcinoma and squamous cell carcinomawithagradualtransitionbetweenthem.AccordingtoWHO 2019,adenosquamouscarcinomaofthestomachisaprimarycancerofthestomach,consistingofbothglandularandsquamouselements, with the squamous element accounting for ≥ 25% of the tumour [3]. In our case, the squamous component accounted for 30%, and therefore the neoplasm met the criteria for the histopathological diagnosis of adenosquamous carcinoma. In an endoscopic examination of the upper gastrointestinal tract performed before the surgery, adenocarcinoma was diagnosed in sections taken from the ulcer. This is because in our case, adenocarcinoma(70%ofweaving)dominatedintheneoplasticinfiltrate over squamous cell carcinoma (30% of weaving). Thus, biopsy diagnosis of adenocarcinoma or squamous cell carcinoma of the stomach does not exclude adenosquamous carcinoma [6, 9, 11]. Themostcommonsiteofgastricadenosquamouscarcinomaisthe lowerthird(45%ofcases),followedbytheupperandmiddleparts [3, 12]. In our case, the tumor infiltration was located in the pylorus, i.e. in the lower third of the stomach. Adenosquamous carcinoma of the stomach is usually associated withpoorpostoperativeprognosis(8,9,10).Thisneoplasmisusuallyfirstdiagnosedatahighstage.Thediameteroftheneoplastic infiltration is in most cases about 5 to 6 cm, and in 53% of cases, the neoplasm is first diagnosed at the pT4 stage according to the pTNM system.At the time of detection, as many as 86% of cases aremetastatictothelymphnodes.Lymphnodemetastasesmainly originatefromtheadenocarcinomacomponent,butthesquamous component or both may be evident in metastasis [3, 12]. In the case described by us, all the above-mentioned features indicate a bad prognosis. In the pyloric region of the stomach, an ulcerated tumor with perforation and 4 x 4.5 cm rolled edges was encountered.Neoplasticmetastaseswerefoundin17of30lymph nodesencountered.In11lymphnodesthemetastaseswereadenocarcinoma,in4adenosquamouscarcinomaandin2squamouscell carcinoma.Accordingtothe2019WHOclassification,thecancer stage was defined as: pT3 pN3b (3). The aggressiveness of this neoplasm is also evidenced by a case report of an early adenosquamous carcinoma with numerous metastases to the lymph nodes and rapid generalization of the neoplastic disease following surgery [7]. Inthedifferentialdiagnosisofgastrictumors,anantibodypanelis used to determine the histological type of cancer or gastric sarcoma[17].Anessentialcriterionforthediagnosisofadenosquamous carcinoma is confirmation of true differentiation of glandular or mucous cells by immunohistochemistry, and the squamous component should show all cytological and architectural features of squamouscellcarcinoma[7,13,14].Thepresenceofadenomatousormicrocysticlesionsinpuresquamouscellcarcinomaisnot sufficient for such a diagnosis (3). Inthepresentedcase,theimmunohistochemicaltestconfirmedthe diagnosis of adenosquamous carcinoma. Cytoplasmic expression ofCK7+wasfoundintheglandularandmucouscomponentscarcinomaofthestomach.Nuclearexpressionofp63+andp40+was found only in the squamous cell component of gastric carcinoma. Ontheotherhand,cytoplasmicexpressionofCK20+wasfocalin gastricmucinousadenocarcinomacomponent.NoCD56,synaptophysin or chromograninAexpression was found in the neoplastic cells tested. Inrecentyears,theTrastuzumabtherapyhasbeenintroducedinto thetreatmentmethodsofpatientswithgastriccancer.Thistherapy may be effective in patients with HER2 gene amplification associated with HER2 protein overexpression through immunohistochemistry. Unfortunately, in the available literature, and in our case,noimmunohistochemicalexpressionofHER2inadenosquamouscarcinomacellswasfound,whichlimitstheuseofeffective adjuvant chemotherapy in these patients [13, 16].

5. Conclusion For the correct histopathological diagnosis of gastric adenosquamouscarcinoma,itisnecessarytoperform,inadditiontotherou- tine histological examination, immunohistochemical tests for the components of this tumor


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Mehrdad Pahlevani. Histopathology and Immunohis to chemical Examinations in Adenos quamous Carcinoma, Cancer of the Stomach - Case Study. Annals of Clinical and Medical Case Reports 2022