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ORIGINAL ARTICLE
Year : 2023 | Volume
: 22
| Issue : 4 | Page : 501-507
Clinicopathological and immunohistochemical characterization of gastrointestinal stromal tumour at four tertiary health centers in Nigeria using CD117, DOG1, and human epidermal growth factor receptor-2 biomarkers
Mumini Wemimo Rasheed1, Afolayan Enoch Abiodun1, Uchechukwu Brian Eziagu2, Najeem Adedamola Idowu3, Abdullahi Kabiru4, Taiwo Adeyemi Adegboye5, Waheed Akanni Oluogun6, Adekunle Adebayo Ayoade7
1 Department of Anatomic Pathology, University of Ilorin Teaching Hospital, Ilorin, Kwara State, Nigeria
2 Department of Histopathology, University of Uyo Teaching Hospital, Uyo, Akwa Ibom, Nigeria
3 Department of Surgery, Ladoke Akintola University of Technology, Ogbomosho, Oyo, Nigeria
4 Department of Histopathology, Usmanu Danfodiyo University Teaching Hospital, Usmanu Danfodiyo University, Sokoto, Nigeria
5 Department of Epidemiology and Community Health, University of Teaching Hospital, Ilorin, Kwara State, Nigeria
6 Department of Histopathology, Ladoke Akintola University of Technology, Osogho, Nigeria
7 Department of Morbid Anatomy and Histopathology, Ladoke Akintola University of Technology, Ogbomosho, Oyo, Nigeria
Correspondence Address:
Mumini Wemimo Rasheed
University of Ilorin Teaching Hospital, P M.B. 1459, Along Old Jeba Road, Oke-Ose, Ilorin, Kwara State
Nigeria
Source of Support: None, Conflict of Interest: None
CheckDOI: 10.4103/aam.aam_180_22
Abstract
Aims: Gastrointestinal stromal tumors (GISTs) are neoplastic lesions that primarily affect the digestive tract and develop from interstitial cells of Cajal. These lesions require histopathological and immunohistochemical characterization due to their malignant potential and personalized treatment. In this investigation, the sex, age, lesional sites of origin, histopathological types, the prevalence of human epidermal growth factor receptors (HER-2) expression, prognostic indices (based on tumor size and mitotic figures), expression of CD117 and DOG1, and characteristics of patients with GIST were all characterized. Materials and Methods: This was a retrospective cross-sectional analysis of GIST cases seen at four tertiary health-care centers in Nigeria over a 10-year period (2008–2017) and investigated utilizing histopathological and immunohistochemical (CD117, DOG1, and HER-2) methods. Results: In this investigation, there were twenty GIST cases. Notably, the majority (40%) of the cases had tumors with sizes between 7.0 and 8.0 cm; the stomach was the most frequent site (70%) and the spindle cell type of GIST was the most prevalent (80%) histopathological type. In addition, the stomach was significantly associated with GIST as an origin site (with a P = 0.001), and 100% and 50% of these tumors were immunoreactive with CD117 and DOG1, respectively. Conclusions: In our study, GISTs most frequently develop in the stomach, and CD117 and DOG1 are essential for correctly diagnosing these tumors. However, HER-2 immunoreactivity is a predictive marker of survival for personalized care.
Abstract in French Résumé
Objectifs: Les tumeurs stromales gastro-intestinales (GIST) sont des lésions néoplasiques qui affectent principalement le tube digestif et se développent à partir des cellules interstitielles de Cajal. Ces lésions nécessitent une caractérisation histopathologique et immunohistochimique en raison de leur potentiel malin et d'un traitement personnalisé. Dans cette enquête, le sexe, l'âge, les sites d'origine des lésions, les types histopathologiques, la prévalence de l'expression des récepteurs du facteur de croissance épidermique humain (HER-2), les indices pronostiques (basés sur la taille de la tumeur et les chiffres mitotiques), l'expression de CD117 et DOG1, et les caractéristiques des patients atteints de GIST ont toutes été caractérisées. Matériels et méthodes: Il s'agissait d'une analyse transversale rétrospective de cas de GIST observés dans quatre centres de soins de santé tertiaires au Nigeria sur une période de 10 ans (2008-2017) et étudiée à l'aide d'analyses histopathologiques et immunohistochimiques (CD117, DOG1 et HER). 2) méthodes. Résultats: Dans cette enquête, il y a eu vingt cas de GIST. Notamment, la majorité (40 %) des cas présentaient des tumeurs mesurant entre 7,0 et 8,0 cm ; l'estomac était le site le plus fréquent (70 %) et le type de GIST à cellules fusiformes était le type histopathologique le plus répandu (80 %). De plus, l'estomac était significativement associé au GIST comme site d'origine (avec un P = 0,001) et 100 % et 50 % de ces tumeurs étaient immunoréactives avec CD117 et DOG1, respectivement. Conclusions: Dans notre étude, les GIST se développent le plus souvent dans l'estomac, et CD117 et DOG1 sont essentiels pour diagnostiquer correctement ces tumeurs. Cependant, l'immunoréactivité HER-2 est un marqueur prédictif de survie pour une prise en charge personnalisée.
Mots-clés: Biomarqueurs, tumeurs stromales gastro-intestinales, histopathologie, immunohistochimie
Keywords: Biomarkers, gastrointestinal stromal tumors, histopathology, immunohistochemistry
Introduction 
One percent of all malignant tumors of the gastrointestinal tract (GIT) are gastrointestinal stromal tumors (GIST).[1],[2] The majority (47%–60%) of these mesenchymal tumors of the GIT originate in the stomach and small intestine, with a small proportion, also occurring in other GIT organs.[3] There have been reports of extraintestinal GIST(s) in locations such as the vulvovaginal, rectovaginal septum, omentum, and retroperitoneum that have comparable immunohistochemical and clinical features.[4] GISTs resemble interstitial cells of Cajal (ICCs) genetically, immunophenotypically, and morphologically. The ICCs are the GIT's pacemaker cells, and they exhibit both smooth muscle and neuronal differentiation in their ultrastructure.
The ICCs control peristalsis.[5],[6] KIT (CD117) has been identified as a receptor tyrosine kinase that plays a role in the development and maintenance of ICC as well as a sensitive marker of GISTs, irrespective of the site; it is expressed in many as 95% of GISTs.[7] These biomarkers are needed for the diagnosis. It has been consistently demonstrated to originate from ICCs, which express the c-KIT proto-oncogene and have both myeloid and neuronal characteristics.[7]
GISTs express the proto-oncogene and stem cell growth factor, c-KIT (CD 117), particularly. It is a tyrosine kinase, and CD117 is one of its products.[1],[2] To distinguish GIST from other gastrointestinal mesenchymal tumors (GMT), the c-KIT mutation or platelet-derived growth factor receptor-alpha (PDGFRA), which is expressed in all GISTs but not in genuine smooth muscle and neural tumors, has become a crucial tool.[1] KIT and PDGFRA, which encode tyrosine kinase receptors, specifically include oncogenic mutations in about 80% and 8%–10% of the tumors, respectively.[8],[9] Targeted therapy has been built around these mutations, and some genotypes can predict how well patients would respond to treatment with the tyrosine kinase inhibitors like imatinib and sunitinib.[1] In patients with metastatic or advanced disease, the tumor responds to imatinib mesylate (STI-571, Gleevec), a selective tyrosine kinase inhibitor of c-KIT and PDGFRA. This drug provides therapeutic benefits in roughly 85% of patients with advanced GIST.[1],[2],[10]
The role of DOG1 in GIST tumor diagnosis was described as a protein that is constitutively expressed in GISTs irrespective of mutation status. It encodes a transmembrane protein, anoctamin associated with Ca+-dependent Cl-channel, and stains ICC. Protein kinase C theta (PKC-theta), a novel PKC isotype involved in T-cell activation, is strongly expressed in GIST and very specific. More importantly, DOG1 was said to be a reliable marker for the diagnosis of GIST tumors that are negative for c-KIT or CD117.[11]
GIST is detectable by the presence of positive CD117 and DOG1 staining in a GMT.[1] Another crucial diagnostic marker, CD34, is seen in roughly 70% of GISTs and is linked to a malignant phenotype. It has been shown that a better prognosis correlates with the expression of CD44. Several countries around the world have documented and reported GIST; however, there are few studies from Africa in general and Nigeria in particular, where 72 cases have been reported so far in seven publications.[10],[11],[12],[13],[14],[15],[16],[17] At the Obafemi Awolowo University Teaching Hospitals Complex, Ile-Ife, Nigeria, patients that have histological diagnosis of GIST and confirmed with CD117 immunoreactivity have benefited from the ongoing Gleevec (imatinib mesylate) international patient-assistance program therapy.[16]
Human epidermal growth factor receptor-2 (HER-2)/neu is a gene that encodes a 185-kDa transmembrane glycoprotein with intrinsic tyrosine kinase activity. This protein is involved in signal transduction of numerous pathways, which in turn affect cell proliferation, survival, motility, and adhesion. HER-2/neu is located on chromosome 17q.[18] Breast cancers and gastric adenocarcinomas are just two of the many human malignancies, for which the HER-2/neu marker has been found to have predictive value.[19],[20] Thus, it appears that HER-2/neu and CD117 shared many of the same signaling pathways. Crosstalk between the two signaling pathways is also possible, but more research is required to determine whether the overexpression of HER-2/neu, which may be present in GIST, and the mutation of CD117 act independently or synergistically.[21] Upper GIT bleeding and other generalized GIT symptoms accompany the solitary submucosal nodule that characterizes GIST.[8] All races experience it frequently between the sixth and seventh decade of life, with men being more susceptible than women.[8] Histopathologically, it can be classified as epithelioid, spindle cell, or mixed type, and prognostic indices depend on tumor sizes such that tumors with size >5 cm with mitotic figures >5/50 high-power fields (HPF), or tumor sizes >10 cm with any mitosis, or regardless of size with >10/50 HPF indicate malignant behavior.[21],[22]
Therefore, this study aimed to identify the sex, age, lesional sites of origin, histopathological types, the prevalence of HER-2 expression with prognostic indices (based on tumor size and mitotic figures) correlation, and expression of CD117 and DOG1 in patients with GIST.
Materials and Methods Study design and participants
This was a retrospective survey of all cases of GISTs diagnosed over 10 years, from January 2008 to December 2017, at the Departments of Pathology, the University of Ilorin Teaching Hospital (UITH), Ilorin (South-West region); the Ladoke Akintola University of Technology (LAUTECH) Teaching Hospital, Osogbo (South-West Region); Federal Medical Centre (FMC), Birnin Kudu (North-Central Region); and the Usmanu Danfodiyo University Teaching Hospital (UDUTH), Sokoto (North-West Region).
Data collection
Both the formalin-fixed paraffin-embedded (FFPE) tissue blocks and the archival histopathology reports' age, sex, histopathological type, and diagnosis information were obtained from all the cases within the study period.
Inclusion criteria
All histologically diagnosed cases of GIST with a complete demographic profile such as age, sex, site of the specimen and immunohistochemistry (IHC) comprising CD117, DOG1, and HER-2 within the study period.
Exclusion criteria
Cases that do not meet inclusion criteria where the paraffin-wax embedded tissue blocks could not be found or where remnant tissues in the blocks are not representative.
Tissue processing and staining
FFPE tissue blocks were created from the specimens through fixation, dehydration, clearing, infiltration with paraffin wax, and embedding. These FFPE tissue blocks were sliced into thin sections (3 μm) and stained with hematoxylin and eosin (H and E). Subsequently, at the Obafemi Awolowo University Teaching Hospital in Ile Ife, immunohistochemical staining for these study cases was carried out under strict adherence to the manufacturer's instructions for monoclonal antibodies, such that these FFPE tissue blocks were freshly sliced into thin (3 μm) sections and stained with CD117, DOG1, and HER-2 immunohistochemical monoclonal antibodies.
Hematoxylin and eosin tissue preparation
Sections were cut at 5μm from the FFPE tissue blocks, floated in a 40°C water bath containing distilled water, and picked up onto clean glass slides. The sections were then passed through 2 changes of xylene for 5 min each and rehydrated in the graded concentration of alcohol. The tissue sections were stained with H and E following standard operation procedure for routine H and E staining procedures. The H and E sections were reviewed by classifying the tumor into histological types such as spindle cell type, epithelial cell type, and mixed type.
Immunohistochemical staining procedure
The FFPE tissue blocks were used for the immunohistochemical procedure. The IHC procedure used for this research was the avidin–biotin complex method also referred to as the avidin–biotin immunoperoxidase method.
Control
The control group was validated with positive and negative controls, both of which were supplied by the manufacturer and were included in each batch of sections during staining.
Below are the details of the IHC protocol.
Tissue preparation
Sections were cut at 4-μm thick and deparaffinized in xylene.
Rehydration
Sections were hydrated in graded alcohol (90%, 80%, and 70%) and then through the water.
Antigen retrieval
The heat method of antigen retrieval was used with a pressure cooker. The sections were immersed in 10 mM citric acid buffer, pH 6.0 heated in the pressure cooker for 10–20 min at 90°C, left to cool under tap water for 20 min, and washed in phosphate buffer saline (PBS).
Blocking of endogenous peroxidases
Endogenous peroxidases that were not easily denatured by fixation, but contribute to background staining (noise) were blocked by incubation in 3% hydrogen peroxidase (3 mml of OH in 100 ml of methanol), for 10 min at room temperature, followed by 5 min rinsing in PBS.
Immunohistochemical staining technique
The section was cut at 4micron, deparaffinized in xylene, and hydrated in graded alcohol, and a heat method of antigen retrieval was used. Endogenous peroxidase was blocked with hydrogen peroxide. Thereafter, the primary monoclonal antibodies comprising CD117, DOG1, and HER-2 were applied at a dilution of 1: 20 and incubated at room temperature for 60 min, wash again in PBS at room temperature, the primary antibody amplifier quanto was applied and incubated for 10 min. This was followed by another washing in PBS, and then horseradish peroxidase polymer quanto was applied and incubated for another 15 min. After further washing in PBS, the sections were exposed to commercially available 3, 3-diaminobenzidine tetrahydrochloride for 10 min at room temperature. The sections were again rinsed in deionized water and counterstained with hematoxylin and then wash in water for 2 min, followed by dehydration through increasing alcohol concentration and clearing with xylene. The sections were then mounted in distyrene tricresyl phosphate and xylene and covered slipped and dried for light microscopic examination.
Interpretation of immunostaining
The slides were examined for immunoreactivity on the cytoplasmic staining for CD117 while DOG1 staining pattern was observed on both cytoplasm and membrane. Next, Strong membrane immunoreactivity was considered positive for HER-2. Thus, complete membrane staining that appeared brownish for HER-2 was considered to be positive. However, immunostained negative tumor cells appeared bluish. Nonspecific binding/brown artifacts on cells and connective tissue were disregarded. HER-2 staining was interpreted based on the HercepTest test score which is itemized as follows.
No membrane staining in <10% of tumor cellsIncomplete faint membrane staining in >10% of tumor cellsA weak to moderate complete membrane staining in >10% of tumor cellsA strong complete membrane staining in >10% of tumor cells.Data analysis
The data were analyzed with the use of IBM SPSS version 23(2015 SPSS Inc, Illinois, USA), P < 0.05 was considered significant, and the outcomes were displayed in tables, graphs, gross images, and photomicrographs.
Ethical considerations
The ethical clearance was obtained with registration number NHREC/02/05/2022. The study was conducted according to the strict guidelines of the Helsinki declaration on biomedical research on human subjects; the patient's identity and personal health information remained confidential.
Results This hospital-based retrospective study involved 3 geopolitical zones in Nigeria comprising the Southwest; Ladoke Akintola University of Technology Teaching Hospital, Osogho, Northcentral; the University of Ilorin Teaching Hospital, Ilorin and 2 tertiary hospitals in the Northwest; Usmanu Danfodiyo University Teaching Hospital, Sokoto and Federal Medical Centre, Birnin Kudu, Jigawa. Although 23 cases were analyzed, due to missing information on age, sex, tumor size, and immunohistochemical confirmation, three cases did not have enough of this information to be included in this study. In view of the proportion of GISTs tumuor from different hospitals, the majority of cases are from UITH which accounted for 14/20 cases (70%) followed by 3/20 cases (15%) from UDUTH, 2/20 (10%) from LAUTECH, and the least 1 case (5%) from FMC Birnin Kudu.
The mean age was 52 ± 10.8, with the youngest patient being 46 and the oldest being 65. The fifth decade was the age group with the highest percentage of cases (45.0%; 9/20). The male-to-female ratio was 1:1.2, showing that this tumour was slightly more common in female patients [Table 1]. In addition, 15 cases were resected surgical specimens, and five cases were biopsies. The smallest tumor was 6.8 cm, while the largest tumor measured 14.0 cm, as per surgical specimens that were removed. The range of 8.39 ± 2.1 represented the tumor's largest proportion. This study found that the stomach predominated in 14/20 cases (70%), followed by the small intestine in 3/20 cases (15%), the retroperitoneum in 2/20 cases (10%), and the prostate in one case (5%). According to the histopathological subtypes, 16/20 (80.0%) of the patients had spindle cell variants, and one case of round cell variant was noted [Table 2].
Table 1: Sociodemographic characteristics of patients with gastrointestinal stromal tumor
Table 2: Clinicopathological charateristics of patients with gastrointestinal stromal tumorIn terms of immunohistochemical expression, all the histopathologically suspected cases were immunoreactive with CD117, while DOG1 was concurrently positive in 50% of the cases [Figure 1] and [Figure 2]. A case was tested using a monoclonal HER-2 antibody; however, the results were negative [Figure 1] and [Figure 2]. Given the correlation between tumor size, site, and histopathological type, spindle cell type predominated in most tumors with a size between 7.0 and 8.9 cm [Table 3].
Figure 1: Simple bar chart illustrating the frequency distribution of the respective immunostains (CD117, DOG1, and HER-2) in the GISTs cases. HER-2 = Human epidermal growth factor receptor-2, GIST = Gastrointestinal stromal tumor
Figure 2: (a) Histologic section of the spindle cell type of GIST tumor arranged in fascicles with interspersed fibrous tissue (H and E, ×400), (b) Histologic section of GIST tumor with immunoreactivity to CD117 (CD117 immunostain, ×400), and (c) Histologic section of GIST tumor with negative immunoreactivity with HER-2 (HER-2 immunostain, ×100). GIST = Gastrointestinal stromal tumor, H and E = Hematoxylin and eosin, HER-2 = Human epidermal growth factor receptor-2
Table 3: Association between tumor size, site with histopathological type Discussions The main objective of this study was to characterize the sex, age, lesional sites of origin, histopathological types, the prevalence of HER-2 expression with prognostic indices (based on tumor size and mitotic figures), expression of CD117 and DOG1, and in patients with GIST. Notably, in view of the demographic profile of the GISTs tumour, we found 45% of the cases within the 50-59 age bracket and had a tumour size of 7.0-8.9cm. The tumour size range from 6.5cm to 14.0cm. They may arise anywhere along the GIT tract, the most common site in this study was the stomach which accounted for 70% followed by the jejunum and ileum 15%, and the least was the prostate 5%. Based on the histological classification, a large proportion of this tumour were spindle cell variants which constituted 80% with a (P = 0.001). The most significant fact to emerge from this study regarding the immunohistochemical characterization of GISTs was that all the histologically suspected cases were immunoreactive with CD117(100%) and DOG1(50%). However, HER-2 was not immunoreactive in 10% of cases.
According to our study, patients with GISTs range in age from 30 to 77-year-old, with the peak age incidence occurring in 9/20 (45.0%) of the cases and the mean age at presentation being 52 ± 10.8 years. These results are consistent with past research done in Nigeria from Ibadan, Ife, and Enugu.[14],[15],[17],[21] In addition, developed countries also provided information on a comparable review.[8],[9],[10] On the other hand, a rise in the fifth decade was found in a study of GIST tumors in Lagos [Figure 3].[12] In addition, the male-to-female ratio in this study is 1:1.2, which is consistent with studies conducted in other parts of the country, such as Ibadan and Lagos, and a report of a similar nature that was published in Ghana.[15],[23] However, according to some scholars, the fourth decade was 10 years lower in Lagos and Qatar.[12],[24] In contrast to past results, systematic literature analysis of 13,550 GIST patients from population-based studies conducted in 19 different countries revealed that the peak age was in the middle of the sixth decade.[25] The lack of information from developing nations, socioeconomic inequalities, access to health-care facilities, and tumor biology may be to blame for the variances seen around the globe.
Figure 3: Gross section of GIST tumor with solid (thick yellow arrows) to microcystic (thin red arrows), firm, grey-white cut surface, and focal areas of hemorrhage (thick blue arrows). GIST = Gastrointestinal stromal tumorIn addition, the number of GISTs reported in this index study would add up to the previously reported cases in the country.[12],[13],[14],[15],[16],[17],[23] Similar to this, different percentages of GIST tumors have been recorded in various parts of the world, with the lowest number being 39 instances and the highest number coming from a systematic evaluation of 13,550 cases from 19 different nations.[1],[25],[26],[27],[28],[29] The vast variety of cases recorded in different parts of the world may be related to the various researchers' selection criteria and the tumor's demographic characteristics. Overall, it can be seen that the spindle cell variant is the most commonly diagnosed histology type of GISTs in this study which is concordant with reviews from both within and other parts of the world.[12],[13],[14],[16],[17],[23],[24],[25],[26],[27] The stomach was the most often affected region for the majority of our patients with a spindle cell histopathological type. In addition, analyses of numerous studies conducted in Nigeria and other nations around the globe have revealed that the stomach is the most typical location for GIST tumors. The small intestine and other locations come next after this. Both domestic and foreign authors back up this position.[12],[13],[14],[15],[16],[17],[23],[24],[25],[26],[27],[28] In-depth research has been done on CD117's significance in the diagnosis of GIST tumors, and writers have confirmed it in relation to elucidating the mesenchymal tumor in the gastrointestinal tumor and other sites. Recently, it was found that DOG1, which is extensively expressed in GIST regardless of mutation, exists. It stains the ICC and encodes a transmembrane protein linked to calcium-dependent chloride channels.[11] A case of malignant spindle cell tumor was identified in this study as a malignant spindle cell tumor of unknown potential based on histomorphological analysis, but it was later determined to be GIST because of positivity with CD117.
The most frequent mesenchymal tumor in the retroperitoneum, GIT, and other uncommon sites is the GIST.[12],[13],[14],[23],[24],[25],[30] Therefore, it is essential to test the suspected case for important diagnostic immunohistochemical markers including CD117 and DOG1.
The primary reason for this study's limitations is that it is a retrospective study, which means that the preanalytical and analytical quality control variables in IHC studies are not under the investigators' direct control. Furthermore, the veracity of the historical data could not be directly controlled. Due to a lack of necessary tools, it was also impossible to perform molecular biology research on these lesions. The investigators' next step will be to perform a prospective survey in which they will have sufficient control over these IHC quality control variables. Further characterization of these lesions utilizing molecular biology methods like polymerase chain reaction, in situ hybridization investigations, and next-generation sequencing will be done by the investigators in collaboration with centers that have these cutting-edge facilities.
Conclusions GISTs are primarily found in people aged 50–59 years in the population we investigated, occurring slightly more often in women. They considerably develop in the stomach and have an average tumor size of 7.0–8.9 cm. CD117 is crucial for appropriately detecting GISTs.
Acknowledgments
The list of authors contributions, credits, and other information are as follows: RMW (conception and design of the work; the acquisition, analysis, and interpretation of data for the work; drafting the work and revising it critically for important intellectual content; final approval of the version to be published; and agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved), AEA (reviewing, editing, and critical appraisal for intellectual content), UBE (design of the work; interpretation of data for the work; drafting the work and revising it critically for important intellectual content; and final approval of the version to be published), NAI (reviewing and editing with intellectual capacity to make it acceptable for publication, KA (provision of research materials, reviewing, editing and critical appraisal for intellectual content), AAT (critical analysis of the raw data), and OWA (provision of research materials, reviewing and editing for the acceptability of the journal).
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
References 
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